Editor’s note: While Cosmetics & Toiletries acknowledges that animal testing is a sensitive issue to many readers worldwide, it is important to note that in China, where these authors conducted their research, it is currently legally required. All animal experiments involved in this study were performed in accordance with Regulations for the Administration of Affairs Concerning Experimental Animals of China.
UV radiation can be absorbed by different chromophores in the skin, and absorption of UV photons by these chromophores results in different photochemical reactions and secondary interactions involving reactive oxygen species (ROS), which result in harmful effects.1 UV radiation is comprised of 98% UVA (320–400 nm) and 2% UVB (290–320 nm).2 UVB radiation is mainly responsible for the most severe damage: acute damage such as sunburn and long-term damage, including cancer. It has a direct impact on cell DNA and proteins. Unlike UVB, UVA radiation is not directly absorbed by biological targets, but can still dramatically impair cell and tissue functions.3, 4
An ideal sunscreen should provide uniform UVB/UVA protection, and remain unchanged after UV irradiation.5 Another consideration is safety; this is a fundamental point in the field of cosmetics. Skin presents a significant barrier for most substances applied. Typically, one wants UV filters to stay on the surface or to not permeate lower than the upper epidermis; certainly not to the vascular dermis.
In the present study, two photosensitive compounds, i.e., 4-cholesterocarbonyl-4’-(N,N’-diethylaminobutyloxy) azobenzene (ACB) and 4-cholesterocarbonyl-4’-(N,N,N-triethylamine butyloxyl bromide) azobenzene (CAB), were synthesized. The phototoxicity of the two compounds was first evaluated in animal models. The cytotoxicity after combined either ACB or CAB into liposomes was then compared with NIH3T3 cells. In addition, their skin permeation ability was assessed. Finally, ACB, the safer of the two, was studied in greater depth for its stability under UV and visible light irradiation, as well as its UVA and UVB protective efficacy.
Materials: Egg phosphatidylcholine (PC) and cholesterol (CHOL) were purchaseda. The commercial sunscreenb 4-tert-butyl-4’-methoxydibenzoylmethane—also known as avobenzone (C20H22O3)—also was obtained. The cream substrate used was provided gratisc, and water-soluble CdTe630 quantum dots (QDs) were purchasedd. Finally, ACB (C48H71N3O3, Mw 737) and CAB (C50H76N3O3+, Mw 766) were synthesized.6–8
Phototoxicity: A phototoxicity assay was performed with guinea pigs, according to Hygienic Standard for Cosmetics.9 The sample was prepared as 1 mg/mL in distilled water for CAB, and 1 mg/mL in refined oil for ACB; 0.05% of 8-methoxypsoralene was used as the positive control.
Liposome preparation: Two types of liposomes were evaluated for different purposes: one for UV protection and another for skin permeation, which was loaded with QDs. The former included traditional liposome (PC-liposome), avobenzone-combined liposome (avobenzone-liposome), ACB-combined liposome (ACB-liposome) and CAB-combined liposome (CAB-liposome). The latter included QDs-loaded PC liposome (PC-QDs); QDs-loaded ACB liposome (ACB-QDs); and QDs-loaded CAB liposome (CAB-QDs). The preparation of liposomes was carried out using a standard sonication method under nitrogen, as described previously.10, 11
Cytotoxicity of blank liposomes: NIH3T3 cells were cultured in DMEM supplemented with 10% fetal calf serum. Then cells were seeded on the 96-well plates at a density of 5 × 104/mL with a humidified condition at 37°C and 5% CO2 for 24 hr. The culture medium was then replaced with medium containing serial dilutions of PC, ACB and CAB in various liposomes. The concentration of cholesterol in PC-liposome group was maintained the same as other groups. After the 24 hr incubation, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was conducted by a microplate readerf at 490 nm. The higher absorbance indicates a higher cell vitality.
Photo-isomerization of ACB in liposomes: ACB-liposome was irradiated by a 400 W high-pressure Hg lampg attached with a bandpass filter (λT = 275–400 nm) to obtain complete isomerization. The recovery of ACB was carried out using a fluorescent lamp. The spectrum was measured using a UV–Vis spectrophotometerh.
UVA/UVB protection of ACB-liposome in cream substrate: The ACB-liposome and cream substrate was applied at an amount of 20 µL cm-2 (~ 0.06 mg ACB) and 2 mg cm-2, respectively, onto tapesj (1 cm × 4 cm), which were then dried for 30 min at 37°C. The UVB protection effect was evaluated according to the QB/T 2410-1998 standard method.12 Briefly, the average absorbances at 280 nm, 290 nm, 300 nm, 310 nm and 320 nm were measured using ultraviolet UV spectrophotometer and calculated as Ai. The average absorbance Ai was repeated five times and recorded as Ā. When Ā ≥ 0.5, the cream could be considered to provide UVB protection. The UVA protective effect was assessed by the critical wavelength method.13
In vivo UV protection: In vivo, the UV protection efficacy was evaluated both with SD rats (150-200 g) and BALB/c nu/nu mice (five to six weeks). Four test sites, 1-cm2 per site, were selected on the back of shaved rats or nude mice (Figure 1). The four samples—including avobenzone-liposome, ACB-liposome, PC-liposome and avobenzone solution, containing 5% (v/v) glycerol—were spread uniformly on the four testing sites, respectively. The samples were allowed to dry for 15 min before UV irradiationg. The skin was irradiated for 5 hr or 3.5 hr at a distance of 15 cm from the light source. In order to ensure that UV absorption was the same, the applied volumes of avobenzone-liposome and ACB-liposome were determined according to their UV absorption at 360 nm.
In vivo permeation: For permeation studies, the treatment of BALB/c nu/nu mice was the same as described for UV testing. Four areas on the back were selected and naked QDs, PC-QDs, ACB-QDs and CAB-QDs in 5% glycerol (v/v) were applied at a QD concentration of 20 nmol cm-2. Mice (n = 3 per group) were sacrificed at 0, 2, 4, 8, 12 and 24 hr by injection, and the dorsal skins were rinsed three times with wet gauze. Skin samples were embedded in TEK OCTk, frozen at −20°C, and sectioned at 5 µm by cryostatm. The sections were examined using a fluorescence microscopen.
Phototoxicity and cytotoxicity: Dermal irritation studies revealed that both ACB and CAB, in their pure compound state, did not show phototoxic properties when applied topically (data not shown). The cytotoxicity of blank PC-liposome, ACB-liposome and CAB-liposome to NIH3T3 cell lines was compared, and for neutral ACB-liposome, no significant cytotoxicity was observed. This was the case even when the concentration of ACB was increased to 1.2 μmol/mL; i.e., the cytotoxicity of ACB-liposome was similar to the neutral PC-liposome. Although positive CAB-liposome showed some toxicity compared with ACB-liposome, it is safer than the commercial positive liposome N-(1-(2, 3-dioleoyloxy) propyl-N,N,N-trimethylammonium mesylatep. It is clear that ACB was better than CAB from a safety point of view. Therefore, the authors then investigated the possible application of ACB as a sunscreen.
ACB-liposome isomerization and stability under irradiation: As noted, photostability is a key parameter for effectiveness in commercial sunscreen products, and as shown in Figure 2, ACB in liposomes underwent reversible trans-cis isomerization—and this recovery process could be repeated at least 10 times. Unlike the commonly used UVA filter avobenzone,14 the azobenzene structure in ACB is stable and does not decompose under light irradiation. The transmission electron micrographs of ACB-liposome before and after 10 cycles of periodic UV-Vis irradiation showed that the structure of ACB-liposome also maintained its original form (see Figure 3). Therefore, periodic UV and visible light irradiation could not affect the photo-isomerization or structure of ACB-liposome.
ACB-liposome UVA/UVB protection with cream substrate: The critical wavelength method, first established by Diffey in 1994, is based on instrumental measurements to evaluate UVA protection.13 The critical wavelength (λc) is the wavelength at which the area under the spectrum from 290 nm to λc is 90% of the integral of the absorbance spectrum from 290 nm to 400 nm. As shown in Figure 4a, the total area of the spectrum from 290 nm to 400 nm is 75.299 nm * Abs; thus, 90% of this area is 67.769 nm * Abs. According to Figure 4b and Figure 4c, the area of the spectrum from 290 nm to 390 nm and from 290 nm to 389 nm is 68.434 nm * Abs and 67.706 nm * Abs, respectively.
So the λc of the ACB-liposome, when mixed with the cream substrate, was between 389 nm and 390 nm, i.e., λc > 370 nm. This satisfies the UVA measurement regulations of the U.S. Food and Drug Administration that allow only products with λc > 370 nm to be labeled as “broad spectrum.”15 For UVB, the average absorbance Ā of the ACB-liposome, when mixed with the cream substrate, was between 0.5 and 1.0, indicating this formulation provided UVB protection according to the QB/T 2410-1998.
UV protection in vivo: Erythema formation measurements were used to evaluate the UV protection levels of sunscreens in vivo.16 The efficacy of ACB-liposome in animal models is shown in Figure 5 and Figure 6, respectively. As seen in Figure 5a, after UV irradiation for 5 hr, the surrounding area for the PC-liposome showed much more obvious erythema than did the ACB-liposome, avobenzone-liposome or avobenzone. Figure 5b showed that the same erythema was not so obvious one day after irradiation. In Figure 5c, two days after UV irradiation, erythema continued to become even more inconspicuous, although festering became apparent. Three days after UV illumination, festering was not observed with the ACB-liposome or avobenzone-liposome, while skin coated with PC-liposome and avobenzone showed serious festering (see Figure 5d); thus, both ACB and avobenzone showed UV protection.
In Figure 6a, after UV irradiation for 3.5 hr, the total skin area became red but the area treated with PC-liposome and avobenzone seemed more obvious. Four days after UV irradiation, erythema was not obvious, as shown in Figure 6b, but slight skin ambustion could be observed in the areas without any coating or those coated with PC-liposome (see Figure 6c). Six days after UV irradiation, no skin ambustion was observed in the areas coated with ACB-liposome, avobenzone-liposome and avobenzone, while the skin coated with PC-liposome showed festering as serious as the area without any coating (see Figure 6d). Moreover, skin peeling could be observed in the areas coated with avobenzone-liposome and avobenzone. Therefore, both ACB and avobenzone displayed UV protection, but ACB proved to be much more effective.
In vivo permeation: As described, the permeation ability of the three kinds of liposomes into nude mouse dorsal skin was evaluated by loading QDs as fluorescent probes. As seen in Figure 7 and Figure 8, onaked QDs and QDs-loaded neutral liposomes, i.e., PC-QDs and ACB-QDs, broke through the barrier of the stratum corneum but stayed in the upper epidermis; they did not reach the dermis even after 12 hr. On the contrary, the cationic liposome CAB-QDs not only penetrated the barrier of the stratum corneum, they also gradually reached the deeper epidermis, close to dermis, after 8 hr (see Figure 8d), and near the vascular dermis after 12 hr. The authors propose that the cationic liposome could bind to negative stratum corneum, leading to an enhanced penetration into and through the skin.11, 17 Since the neutral ACB-liposome stayed on the skin surface and did not penetrate into the vascular dermis, it was considered appropriate for sunscreens.
As noted, the photostability of sunscreens is a key parameter since a sunscreen that loses its capacity to block UV radiation provides a clear risk of damage to the user. And as mentioned, the ACB-liposome not only protected skin from damage caused by UV irradiation but also had good stability both morphologically and chemically; i.e., UV irradiation did not decompose ACB. One might imagine that when ACB-liposome treated skin is exposed to UV light outdoors, the chemistry changes from trans to cis by absorbing UV light. Then as soon as they move indoors, it returns to the trans form and recovers its UV-absorbing ability again when the user steps out.
According to the present study, this process can be repeated at least 10 times, suggesting the potential for users to save time and cost. In addition, as an active sunscreen ingredient, the stability and photo-isomerization of ACB-liposome was evaluated when mixed with other cosmetic additives such as propylene glycol and glycerol. Results showed that in the presence of a mixture of 20% propylene glycol and 5% glycerol, the repeated photo-isomerization upon UV irradiation was the same as it was for the liposomal emulsion without additives.18 What’s more, when cosmetic actives such as ascorbic acid and catalase when incorporated into the ACB-liposome, their release could be controlled by UV irradiation.10 As a conclusion, this technology is promising for the development of reusable and multifunctional sunscreen formulations.
- P Kullavanijaya and HW Lim, Photoprotection, J Am Acad Dermatol 52 937-958 (2005)
- M Wharton, M Geary, N O’Connor and B Murphy, A rapid high performance liquid chromatographic (HPLC) method for the simultaneous determination of seven UV filters found in sunscreen and cosmetics, Int J Cosmetic Sci 33 164-170 (2011)
- D Moyal, A Chardon and N Kollias, UVA protection efficacy of sunscreens can be determined by the persistent pigment darkening (PPD) method (part 2), Photodermatol Photo 16 250-255 (2000)
- RM Lavker, GF Gerberick, D Veres, CJ Irwin and KH Kaidbey, Cumulative effects from repeated exposures to suberythemal doses of UVB and UVA in human skin, J Am Acad Dermatol 32 53-62 (1995)
- U Osterwalder and B Herzog, The long way towards the ideal sunscreen—Where we stand and what still needs to be done, Photoch Photobio Sci 9 470-481 (2010)
- XM Liu, B Yang, YL Wang and JY Wang, New nanoscale pulsatile drug delivery system, Chem Mater 17 2792-2795 (2005)
- XM Liu, B Yang, YL Wang and JY Wang, Photoisomerizable cholesterol derivatives as photo-trigger of liposomes: Effect of lipid polarity, temperature, incorporation ratio and cholesterol, Biochim Biophys Acta 1720 28-34 (2005)
- B Yang, SY Geng, XM Liu, JT Wang, YK Chen, YL Wang andb JY Wang, Positively charged cholesterol derivative combined with liposomes as an efficient drug delivery system, in vitro and in vivo study, Soft Matter 8 518-525 (2012)
- Skin Phototoxicity Test, version 2007, issued by Ministry of Health of China, (The Cosmetics Directive of the Council European Communities, 76/768/EEC, and amendments until 21 November 2005) wenku.baidu.com/view/7bfed324192e45361066f5e5.html (in Chinese) (Accessed Mar 10, 2014)
- YQ Dai, G Qin, SY Geng, B Yang, Q Xu and JY Wang, Photo-responsive release of ascorbic acid and catalase in CDBA-liposome for commercial application as a sunscreen cosmetic, Rsc Adv 2 3340-3346 (2012)
- G Qin, SY Geng, LP Wang, YQ Dai, B Yang and JY Wang, Charge influence of liposome on transdermal delivery efficacy, Soft Matter 9 5649-5656 (2013)
- QB/2410-1998, Standard method, China National Light Industry (in Chinese)
- BL Diffey, A method for broad spectrum classification of sunscreens, Int J Cosmetic Sci 16 47-52 (1994)
- W Schwack and T Rudolph, Photochemistry of dibenzoyl methane UVA filters Part 1, J Photochem Photobiol B 28 229–234 (1995)
- U.S. Food and Drug Administration, Labeling and effectiveness testing, Sunscreen drug products for over-the-counter human use, www.gpo.gov/fdsys/pkg/FR-2011-06-17/ pdf/2011-14766.pdf (Jun 17, 2011; no longer available)
- M Egawa and H Iwaki, In vivo evaluation of the protective capacity of sunscreen by monitoring urocanic acid isomer in the stratum corneum using Raman spectroscopy, Skin Res Technol 14 410-417 (2008)
- N Dragicevic-Curic, S Grafe, B Gitter, S Winter and A Fahr, Surface charged temoporfin-loaded flexible vesicles: In vitro skin penetration studies and stability, In J Pharm 384 100-108 (2010)
- Q Xu, SY Geng, Y Dai, G Qin and JY Wang, CDBA-liposome as an effective sunscreen with longer UV protection and longer shelf life, J Photoch Photobio B 129 78-86 (2013)