Naturally occurring sunscreen: An investigation into the effectiveness of squid ink melanin as a UV blocker, compared to current physical and synthetic UV protectors

Naturally occurring sunscreen: An investigation into the effectiveness of squid ink melanin as a UV blocker, compared to current physical and synthetic UV protectors

Arwen McGloin

Menai High School

There is limited research looking into the effectiveness of melanin in squid ink as a UV protection agent based on the photoprotective properties of the biopolymer within the skin. This project investigated the hypothesis that a melanin-based sunscreen is an effective alternative to current harmful synthetic and mineral sunscreens. The study compared how a simple melanin-based sunscreen derived from squid ink performed against commercial sunscreen by assessing the R (Red) values of pig skin samples when exposed to UV light, then looked at a more sophisticated nano-melanin based approach with melanin nanoparticles. It was found that there is no statistical significance between commercial sunscreens and the squid ink melanin and nanomelanin creams, where P=0.598 and P=0.214 respectively, indicating that there may be an effectiveness of the melanin-based creams as UV protection agents, however, there was also no significant erythema visible on the control skin sample as was expected.

Figure 1 – Melanin nanoparticles under a zetasizer – an instrument which was used to measure the particle size of the melanin nanoparticles.

Literature review

Ultraviolet radiation (UVR) is a well understood carcinogen that humans are overexposed to due to solar radiation (Yardman-Frank & Fisher, 2020). The Sun’s UV radiation is split into three subdivisions: UV-A, UV-B and UV-C, with UV-C having the shortest wavelength, and is therefore the most penetrative and consequently damaging to human cells. Skin melanoma was the third most common cancer by incidence for both males and females in 2020 (Sung, et al., 2021) likely due to the high ambient exposure to UVR in Australia correlated with the population’s large percentage of fair-skinned individuals (Gordon, et al., 2022). Photoprotection in black skin is significantly greater than fair skin as a result of the distribution of melanosomes, the organelles which produce melanin (Brenner & Hearing, 2007); this is supported by the contrasting rise in incidence of melanomas among white populations, which increases by 5-8% annually, and the static nature of melanoma incidence within black populations (Brenner & Hearing, 2007).

Current active ingredients utilised in sun protections pose threats to the aquatic environment, as well as being harmful to human health (Mohiuddin, 2019). Over 6,000 tonnes of sunscreen pollute the oceans annually, resulting in toxic levels of potentially harmful synthetic chemicals including oxybenzone, octocrylene and homosalate, which are of the most concern (Mohiuddin, 2019). Research exists on the endocrine disrupting properties of homosalate but is not yet conclusive. Furthermore, studies are currently being conducted to investigate other direct health concerns, and the possibility that homosalate exposure may mediate the absorption of pesticides into the body (Mohiuddin, 2019). Octocrylene is a serious concern to the contamination of aquatic environments, contaminating food chains spanning from algae to humans, and can reach concentrations of parts per million (Downs, Cruz, & Remengesau Jr., 2022). Moreover, even low concentrations of parts per trillion are damaging to corals, inducing tissue necrosis and disrupting homeostatic systems within the organism (Downs, Cruz, & Remengesau Jr., 2022). Octocrylene is a known endocrine disruptor in fish, similar to oxybenzone, (Downs, Cruz, & Remengesau Jr., 2022) and is thought to be the cause of developmental gene transcription issues in the liver and the brain (Ruszkiewicz, et al., 2017). Oxybenzone has been more widely investigated, leading to its ban from all cosmetic products in Hawaii in 2018 (Glanz, Kwong, Avelis, & Cassel, 2022), as there is strong evidence supporting the harmful properties of the chemical toward corals, with high concentrations causing coral bleaching and deformities in the larval phase (Downs, Cruz, & Remengesau Jr., 2022). Multiple studies support the developmental issues, reproductive abnormalities and birth defects (Huo, et al., 2015) effected by dermal contact with oxybenzone during pregnancy (Santamaria, et al., 2020).

Alternatively, many UV protection cosmetics contain mineral active ingredients, of which the most common compounds are zinc oxide (ZnO) and titanium dioxide (TiO2). TiO2 is an effective sunscreen due to its high refractive index and is most effective at scattering the UVR UV-B range, compared to ZnO which has a peak absorption in the UV-A band (Ruszkiewicz, et al., 2017) meaning their combination produces broad spectrum UV protection. Mineral sunscreens are generally nanoparticle compositions to address many of the limitations of conventional sunscreen formulations by increasing the cosmetic appeal and achieving deeper penetration of the active ingredients into the skin, thus, providing a greater and longer protection against UVR (Mohiuddin, 2019). Furthermore, nanoparticles overcome the short retention of active ingredients on outer layers of skin and improve the UVR protection efficiency, thus enabling lower concentrations of the active ingredients, increasing the safety of the products (Santos, et al., 2022). There is limited research into the dangers of nanoparticles in sunscreens: there are concerns about unknown effects of increased particle penetration, and the free radicals generated by nanosized zinc and titanium when exposed to UVR (Nasir, Wang, & Friedman, 2014). Free radicals may cause significant damage to DNA and RNA, and consequently, proteins and fats within cells. In addition, the use of nanoparticles in aerosol sunscreens is highly dangerous, as the particles can interfere with respiration and cause pulmonary dysfunction (Nasir, Wang, & Friedman, 2014). Despite these concerns, the Nanodermatology Society released a statement in 2013 concluding that whilst the use of nanoparticles in creams and liquid or aqueous dermatological products is safe, use in aerosols is dangerous, supported by the American Academy of Dermatology in 2018 (Jansen, Osterwalder, Wang, Burnett, & Lim, 2013).

Given the potential environmental and health issues arising from synthetic, and nonnanosized chemicals as active ingredients in sunscreens, it seems appropriate to investigate safer alternatives. One possibility could be melanin, a natural and biodegradable substance that can maintain the form of nanoparticles, while providing effective protection from UV radiation.

Melanin is a natural biopolymer found in the skin of most mammals, and a wide variety of organisms. It is the dominant photoprotective defence against UVR by absorbing the rays or dispersing them as heat (Mohania, et al., 2017). When found within the skin of animals, melanin reduces the percolation of UV light through the epidermis by absorbing and filtering the UV radiation (Brenner & Hearing, 2007), furthermore, the distribution and type of melanin is responsible for the pigmentation of skin, leading to the determination of the skin’s sensitivity to UV (Maresca, Flori, & Picardo, 2015). Melanin is a promising alternative to mineral active ingredients as melanin contains stable free radicals, thus eliminating the danger of UV-induced free radicals that relate to ZnO and TiO2 nanoparticles (Brenner & Hearing, 2007). Epidemiological studies suggest a strong connection between the photoprotective role of melanin and the prevention of UV-induced skin cancers, as melanin has a sun protective factor (SPF) of 4, meaning that 50-75% of UV rays are absorbed (Brenner & Hearing, 2007).

A few recent papers have explored the viability of melanin as a sun protection agent; however, an efficient source of melanin is still being investigated. One suggestion is obtaining melanin from squid ink which comprises 10-12% melanin (Abidin, Sulmartiwi, & Saputra, 2021). Although there is limited research into squid ink melanin, it appears to be a potential source for the raw material. A study conducted by Rahmasari et al. investigated the significance of squid ink powder lotions on the area of an erythema and supports the idea that squid ink is an effective photoprotective formulation, with a 3% cream performing best compared to a 1% and 2% lotion (Rahmasari, et al., 2021). Therefore, an investigation into the effectiveness of squid ink sunscreen, and a melanin nanoparticle derived from squid ink is a viable option for the study.

Scientific research question

Is squid ink melanin an effective active ingredient in blocking UVR to reduce erythema, compared to current commercial physical and synthetic UV protectors?

Are nanosized melanin particles derived from squid ink an effective UV blocking agent?

Scientific hypothesis

There will be no change in the overall RGB values of pig skin samples after exposure to UV light when protected by the melanin cream, as melanin is a known biological photoprotection agent (Brenner & Hearing, 2007, Mohania, et al., 2017, Rahmasari, et al., 2021). Furthermore, when coated in melanin nanoparticle cream, there will also be no change in the RGB values of the pig skin sample as the nanoisation of melanin will not decrease the efficiency of its photoprotection (Santos, et al., 2022, Jansen, Osterwalder, Wang, Burnett, & Lim, 2013).

Methodology

This project consists of two experiments designed to explore the hypothesis that a melanin-based skin protection offers a plausible and beneficial alternative to commercial sunscreens. The first experiment compares commercial mineral and synthetic sunscreens alongside melanin wild type cells, measuring their ability to block UV radiation. The second extends this comparison through the creation of melanin-based nanoparticles to allow a direct comparison with the wild type cells.

Both experiments used pig skin as it was an effective model of human skin (MonteiroRiviere & Riviere, 2005), and was an ethical option for the study.

In Experiment 1 the pig skin was cut into thirty 2.5 x 4 x 0.3cm samples. These were split into six equal groups. Two groups remained untreated, with one not receiving any UV exposure, and acting as the two controls for the experiment. The other four groups were treated with 0.2g of the separate cream samples, measured on a scale and spread evenly over the skin sample using a spatula. Cream samples 5 and 6, as seen in table 1, were commercially sourced, whereas the sample group 4 was created from aqueous cream and squid ink. The treatments are as follows:

Table 1: Sunscreens to be applied to each of the groups of pig skin samples.

Before the pig skin samples were treated with the creams, an image of each sample was recorded with the Colorimeter App to determine the RGB 1 (Red Green Blue) values of the sample. This image was taken from 10cm above the skin. The RGB values were then measured again after the application of the creams from the same distance. Then the skin samples were placed 13cm under a UVP Mini UV Lamp with 4 watts and an intensity of 176Wm-2, on the short wavelength setting of 254nm for 30 minutes. Based on the relationship between the UV intensity (IUV) and the UV index (UVI), UVI = (I_UV - 35.5)/15.1 the UV index of the lamp is 9.3 (Sánchez-Pérez, 2019). The colour of the skin was then measured and recorded again in RGB values by the Colorimeter App. Then the cream samples were wiped off the skin with a clean paper towel, and RGB values were measured and recorded again.

The second experiment followed the same procedure as experiment 1, but with three groups of pig skin. Groups 1 and 2 were the same as in table one, while the third group replaced the squid ink with a cream sample of 1% of melanin nanoparticles (NPs).

The nanoparticles were made from melanin pellets, extracted from the squid ink in the procedure that follows in figure 2 (Thi, et al., 2022).

Figure 2 - Production of melanin derived from squid ink; ‘residue’ refers to the solid produced by separation due to centrifuging the mixture.

The melanin nanoparticles were produced from melanin pellets that were dissolved in 0.5M sodium hydroxide at 37°C in a waterbath, then stirred in a centrifuge at 400rpm for 3 hours. Then the pH of the solution was adjusted to a pH of 7 using hydrochloric acid. The size of the melanin nanoparticles was then measured using a Zetasizer accessed at the University of Technology Sydney which measured the diameter of the nanoparticles using Dynamic Light Scattering (Stetefeld, McKenna, & Patel, 2016) to ensure there were melanin nanoparticles present in the solution.

Results

The results were simplified into table 2 and 3 from the raw data set collected, this enabled the R (Red) values of the RGB measurements to be considered separately in the analysis of the results, as there were insufficient statistical tests to consider the G and B values simultaneously. Student’s t-tests were performed on the R values from table 2 and table 3 to infer the statistical significance of the data collected and to test the hypothesis.

The student’s t-test compares the means of the R values of two of the active ingredients, assessing if the difference in the means is greater than would be expected due to random chance. The t-test generates a p value which represents the probability that the difference in the means was due to random chance.

The uncertainty of the measurements is ±1 for the RGB values as it is a digital measurement from the Colorimeter App.

R values after exposure to UV light.

Table 2 –R values after exposure to UV light and the active cream was wiped off.

Average R values for each active ingredient before UV and cream, and after UV and cream removed.

Table 3 – A comparison between the average R values of the pig skin samples before exposure to UV and the cream was applied, and after exposure to UV and the cream was wiped off.

Table 4 – The R values of the pig skin samples were taken for all the active ingredients before exposure to the active ingredient and the UV lamp, and then the average was taken. The average of the data set was compared in the paired t-test above against the R values of the skin after exposure to UV and the active ingredient was wiped off of the sample. The difference between the groups is statistically insignificant as P(T<=t) two-tail = 0.821 (P<0.05). This suggests that there were possible inaccuracies within the study causing the skin to not burn as expected.

Table 5 – The R values taken after the pig skin samples were exposed to UV from the control 2 (no active ingredients but under UV) and the synthetic samples were compared, the t-test suggests that there is no significance between the active ingredients P(T<=t) two-tail = 0.457 (P<0.05). This implies that the UV lamp did not affect either of the samples, or that the time of UV exposure was not sufficient.

Table 6 – The recorded R values of control 2 and the nanoparticles were compared using a t-test enabling the conclusion that the means are statistically insignificant with P(T<=t) two-tail = 0.214 (P<0.05), suggesting that there were limitations within the study that restricted the samples exposure to UV.

The remaining data comparisons were all statistically insignificant, shown by the following p values:

Table 7 – Remaining p values.

One significant result of the investigation was the synthesis of melanin nanoparticles derived from squid ink. The median size of the nanoparticles made was 287.7 nm, with the concentration being 1.6E11 particles /ml. The mean size of the nanoparticles synthesised in the study by Thi Le Na et al. was 200 ±18 nm suggesting that as a component of this investigation, melanin nanoparticles were successfully created from squid ink.

Null Hypothesis (1): There is no statistical significance between the R values of the squid ink melanin cream and the control groups.

Alternative Hypothesis N1 (1): There is a statistical significance between the R values of the squid ink melanin cream and the control groups.

Null Hypothesis (2): There is no statistical significance between the melanin nanoparticle UV protection agent and the control groups.

Alternative Hypothesis N1 (2): There is a statistical significance between the melanin nanoparticle UV protection agent and the control groups.

Discussion

The student t-tests conducted show no statistical significance between any of the variables. The lack of significance between the commercial sunscreens and the melanin and nanomelanin creams could be an indication that both creams guard against UVR at a similar level as the commercial sunscreens. Although, this cannot be concluded from the research as there was also no statistical significance between the R values of the skin samples applied with the active ingredients and the control groups of skin, indicating that there were limitations to the study such as the exposure time for the skin samples, and the effect of different lighting on the RGB value measurements. Based on the obtained results there is a definitive scope to expand the study.

As the UV index of the UV lamp was 9.3, a clear erythema should have been visible following 30 minutes of UV exposure, as the burn time for an exposure level of 9.3 is between 15 and 30 minutes, depending on the skin type (Sánchez-Pérez, 2019). The lack of significant reddening could be due to the high SPF value of the applied photoprotection agents, however, the lack of change within the control 2 group suggests that aspects of the study need revisiting.

LIMITATIONS AND FURTHER DIRECTIONS

A small sample size of five pig skin samples, due to restricted time and resources, limits the reliability of the results observed and the statistical tests conducted, relating to the effectiveness of melanin and nanomelanin as photoprotection agents. A larger sample size would be beneficial in increasing the reliability of data collected and enable a more accurate assessment of the consistency of the results. Future investigations should obtain a larger sample of results to ensure consistency, and therefore, reliability of the research.

The samples of pig skin were not homogenous in colour causing minor inaccuracies in the measurement of the paired RGB values, comparing the skin before and after UV exposure, as it was difficult to precisely position the camera with the target spot over the same area each time. This issue also limited the reliability of the paired t-test in table 3 causing the statistical analysis of the results to be less accurate. In future this could be corrected by employing more accurate equipment and technologies to average out the colour of the entire skin sample instead of limiting the measurement to the target size provided by the Colorimeter App.

The lack of appropriate facilities may have impacted the validity and accuracy of the study, specifically the inability to control and maintain the lighting in the room, and the lighting when measuring the RGB values of the skin samples. The lighting changes throughout the study could have been due to the time of day, or the brightness of the day that the investigation was being conducted. Further investigations should employ other technologies or techniques to ensure that external lighting is a controlled variable to increase the validity and accuracy of the experiment.

The time limitations caused progressively older samples of pig skin that were stored in the freezer to be used, due to the inability to expose more than five samples to the UV lamp at one time and to only be able to do one 30minute exposure successively limited the scope of the study. Thus, producing inaccurate and unreliable results to test the proposed hypothesis.

The analysis of the results was restricted to the comparison of two data sets, thus preventing the combined analysis of all the RGB values of each pig skin sample after UV exposure, compared to only analysing the R values of the data collected. This limits the statistical accuracy of the student’s t-tests performed, and in future investigations this inaccuracy should be curbed by utilising more appropriate statistical tests to analyse the results obtained.

No conclusion can be drawn on the effectiveness of squid ink melanin and nanomelanin derived from squid ink as UV protection agents from this investigation, however, the statistical insignificance between the commercial sunscreens and melanin-based creams warrants further research. Furthermore, previous studies have investigated and shown the role of melanin as a photoprotection agent within skin (Brenner & Hearing, 2007, Mohania, et al., 2017), suggesting that it would also be effective externally against UVR. Additionally, nanoparticles of ZnO and TiO2 are already used in commercial mineral sunscreens (Ruszkiewicz, et al., 2017), providing increased and prolonged protection due to deeper penetration into the skin (Mohiuddin, 2019) over conventional sunscreens, indicating that further research into melanin nanoparticles would be beneficial as it could provide a more efficient photoprotection agent, and be a safer alternative to current chemical sunscreens.

Conclusion

There is no clear connection between a change in R values and the effectiveness of melanin, and nanomelanin as photoprotection agents as P=0.598 and P=0.214 respectively, causing both null hypothesis 1 and 2 to be accepted. Despite the lack of significance between the R values of current sunscreens, and the melanin-based creams, which suggests a possible effectiveness, there was no significance between the control 2 and any of the samples, indicating that there were experimental limitations within the study, including the inability to perform the experiment in vivo due to ethical restrictions. Therefore, there is a definite scope to expand into future investigation, studying the effectiveness of melanin as a UV protection agent in vivo, and the further efficiency and cosmetic benefits of nanosized particles.

Acknowledgments

I would like to thank Dr Ying Zhu at UTS for allowing access to a zetasizer for measuring my nanoparticles. I would also like to thank my teacher, Ann Hanna, for extensive guidance throughout my project. And I would like to extend my appreciation to my dad, Professor David McGloin, for continued feedback concerning my scientific report.

Reference list

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Appendices

RISK ASSESSMENT

RGB ANALYSIS

The Colorimeter App measures the RGB values of the pig skin based on the area of focus as seen in figure 3, the app then records the RGB values of each image as seen in figure 4.

The red values were used in the analysis of the results to determine the change in the darkness of red in the skin samples before and after UV exposure. As the RGB values get closer to 0,0,0 they are getting closer to black and therefore lower numbers are darker, and closer to 255,255,255 is getting closer to white and therefore lighter.

Figure 3 – Target area in the Colorimeter App.

Figure 4 – The recorded RGB values on the colourimeter App.

ACTIVE INGREDIENT CONCENTRATIONS

The group 4 melanin cream sample was made to a concentration of 1% squid ink by weighing out 100g of aqueous cream in a beaker and adding 1g of squid ink. This was then poured onto a clean chopping board, and evenly mixed into the aqueous cream with a pharmacy spatula. The melanin cream was then stored in a beaker with a cover to protect the sample from external factors.

The melanin nanoparticle cream was made in a similar way to the melanin cream, by weighing 20g of aqueous cream into a beaker and adding 0.2g of melanin NPs. This was also mixed evenly using a pharmacy spatula on a clean board to create a 1% concentration of melanin NPs, and then used immediately.