j. Soc. Cosmet. Chem., 41, 85-92 (January/February 1990) Enhancement of DNA repair of UV damage in mouse and human skin by liposomes containing a DNA repair enzyme DANIEL B. YAROSH, JEANNIE TSIMIS, and VIVIEN YEE, Applied Genetics Inc., 205 Buffalo Avenue, Freeport, NY 11520. Received November 26, 1989. Presented at the Annual Meeting of the Society of Cosmetic Chemists, New York, December 7-8, 1989. Synopsis The DNA repair enzyme T4 endonuclease V was encapsulated in liposomes to enhance repair of pyrimidine dimers formed in DNA by UV. Addition of the liposornes to human epidermal keratinocytes in culture increased the removal of DNA damage, as measured by loss of endonuclease-sensitive sites, and enhanced DNA repair synthesis, as measured by an increase in unscheduled DNA synthesis. Topical treatment of UV-irradiated SKH-1 hairless mice with liposomes increased the removal of pyrimidine dimers from epi- dermal DNA. The results demonstrate that topical application of liposornes containing T4 endonuclease V may enhance DNA repair when applied in lotion either before or after UV exposure. INTRODUCTION Skin cancer caused by ultraviolet (UV) radiation has reached epidemic proportions in the United States (1). The most dramatic evidence of the link between UV and skin cancer comes from the human genetic disease xeroderma pigmentosum (XP). XP pa- tients have a biochemical defect in the repair of DNA damage caused by UV that causes a failure to remove photoproducts from their UV-irradiated DNA, leading to persistent DNA damage in skin and malignant transformation. Patients with XP develop solar damage and pigmentation abnormalities in childhood, and malignancies arise on sun- exposed skin at rates 4,800 times the frequency in the general population (2). Their life expectancy is 30 years less than the average. Repair of UV-induced pyrimidine dimers in epidermal DNA is a critical determinant of the incidence of UV-induced skin cancer in man. Excision repair requires: (a) recogni- tion and incision of the DNA strand containing damage (b) excision of the lesion (c) resynthesis of DNA using the complementary strand as the template and (d) sealing the mended region by DNA ligase. When repair occurs in normal skin, the rate of removal of DNA damage is saturated at doses that produce erythema (3). The rate-limiting step is the first one: recognition and incision of dimers. We have begun a research program to enhance human DNA repair of UV damage by delivering purified recombinant DNA repair enzymes to epidermal cells using lipo- 85

86 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS somes. Liposomes are often included in cosmetics to hydrate skin and have been used topically to deliver drugs intercellularly (4). We recently described a method to deliver intracellularly encapsulated purified recom- binant T4 endonuclease V (endo V), a DNA repair enzyme specific for UV-induced pyrimidine dimers (5). Endo V initiates DNA repair by producing a single-stranded break in DNA at the site of damage. We described here our results using endo V liposomes on human epidermal keratinocytes and mouse skin in vivo. MATERIALS AND METHODS The preparation of liposomes containing T4 endonuclease V has been described (5), and the encapsulation efficiency was between 1 and 10%. The liposomes, here designated T4N5 liposomes (patent pending), were composed of phosphatidylcholine, phosphati- dylethanolamine, oleic acid, and cholesteryl hemisuccinate in a 2:2:1:5 molar ratio. The assay for encapsulated enzyme activity has been described (5), and the activity of encapsulated endo V was confirmed in each batch of liposomes. The assay for the pH sensitivity of liposomes is based on the quenching of the fluorescent probe 8-amino- naphthalene-l,3,-6-trisulfonic acid (ANTS) by a high concentration of p-xylene-bis- pyridinium bromide (DPX) entrappeal within the liposome (6). Leakage from the lipo- some diluted DPX relative to ANTS, the quenching was reduced, and fluorescence was increased, as measured with a Hoefer TK-100 fluorometer. All measurements were made in duplicate and averaged. Normal human epidermal keratinocytes and keratinocyte growth media were from Clonetics Corporation (San Diego), and all cell culturing was performed with antibi- otics under sterile conditions without microbial contamination. Unscheduled DNA synthesis (UDS) was measured by growing keratinocytes on glass coverslips and irra- diating them with either 10 or 25 J/m 2 or UV-C from a Philips G15T8 germicidal bulb, monitored by a UVX digital radiometer and UV-C probe. The cells were incu- bated with T4N5 liposomes and 10 •Ci/ml 3H-thymidine (60 Ci/mmol). After four hours the cells were chased with 0.1 mM cold thymidine for one hour, fixed, and coated with Kodak NTB nuclear track emulsion. After seven days the slips were developed with Kodak D-19 developer, and for each treatment grains over the nuclei of 25 lightly labeled cells were counted microscopically and averaged. Loss of endonuclease-sensitive sites (ESS) was measured by growing keratinocytes in 10-cm dishes, irradiating them with 20 J/m 2 of UV-C, and incubating them for 24 hours with media containing T4N5 liposomes. The DNA was purified from cells and treated either with or without T4 endonuclease V, which produced single-stranded breaks at the site of pyrimidine dimers. The average length of the DNA was determined by alkaline agarose gel electrophoresis, and the frequency of ESS, i.e. pyrimidine dimers, was calculated by comparing endo V treated and untreated samples (7). All ESS measurements were performed at least in duplicate and the data averaged. Six-to-eight-week-old female SKH-1 hairless mice (Charles River Labs) were irradiated unrestrained from above with UV-B from two FS40 bulbs monitored with the UVX radiometer and UV-B probe, and each mouse was treated with 0.25 g of T4N5 lipo- somes dispersed in Johnson's baby lotion immediately before use. After six hours the mice were sacrifled and the DNA extracted from epidermis directly over the spine. The