j. Soc. Cosmet. Chem., 39, 275-281 (September/October 1988) Studies of the penetration of native collagen, collagen alpha chains, and collagen cyanogen bromide peptides through hairless mouse skin in vitro SCOTT D. COAPMAN, J. LEON LICHTIN, ADEL SAKR, and JOHN R. SCHILTZ, Cosmetic Science Program, Division of Pharmaceutics and Drug Delivery Systems, College of Pharmacy, University of Cincinnati, Cincinnati, OH 45267 (S.D.C., J.L.L., A.S.), and Skin Care Discovery, Shulton Research Division, American Cyanamid Company, Clifton, NJ 07015 (J.R.S. ). Received June 2, 1988. Synopsis Experiments are reported here which assessed the ability for radioactively-labeled native collagen, collagen alpha 1(I) chain subunits, and cyanogen bromide peptides derived from alpha 1(I) chains to penetrate hairless mouse skin mounted in percutaneous absorption cells. Sixty to 70% of the applied dose of radioac- tive label from [3H]-propionylated collagen penetrated the epidermis of tape-stripped skin during a 48-hour period, whereas none of this material penetrated through control skin possessing intact stratum corneum. Dimethyl sulfoxide did not influence absorption in either type of skin preparation. In marked contrast, none of the radioactivity from [3H]-proline-labeled collagen alpha chains (synthesized by 3T6 mouse fibro- blasts) penetrated control or tape-stripped skin. These contradictory results likely result from epidermal- mediated removal of the labeled propionic acid from the collagen preparation, followed by diffusion of the label into the dermis. The evidence for this is based on the observation that all the radioactivity recovered in the receptor phase had a molecular weight less than 8,000 daltons, as shown by dialysis. Further experiments demonstrated that approximately 30% of the applied dose of [3H]-amino acid-labeled cy- anogen bromide collagen peptides penetrated tape-stripped skin, whereas control skin with intact stratum corneum did not allow passage. We conclude from these experiments that native type I collagen, type I collagen alpha chain subunits, or type I collagen cyanogen bromide peptides do not penetrate intact mouse skin, although limited penetra- tion of cyanogen bromidepeptMes does occur in skin lacking stratum corneum. Furthermore, ½y4•erL.me4•ts which purport to demonstrate penetration of native collagen using preparations labeled with radioactive ligands should be accompanied by demonstrations that the labeled material which penetrates into the dermis has been unchanged. INTRODUCTION Collagen proteins form the major fibrous structural component of vertebrate connective tissues. Although approximately 12 different molecular species of collagens have now been described (types I-XII, reference 1 for a review), the species which comprises 275

276 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS about 85% of the collagen of the dermis is type I. The native type I collagen molecule consists of three chains, each with a molecular weight of about 96,000 daltons. Two of these chains, termed alpha 1(I), are identical, while the third chain, termed alpha 2, has a different primary structure. Following the synthesis of collagen and assembly into extracellular fibers, changes begin to occur in the fibers which are caused by enzyme-cat- alyzed covalent crosslinking between chains. As skin ages and crosslinking increases, the collagen becomes less soluble in aqueous solvents, less able to bind to water, and tensile strength increases (2,6). Because younger skin contains a larger proportion of desirable soluble collagen, it is inevitable that cosmetic chemists (and marketers of cosmetic products) have added the soluble protein to topically-applied skincare products in an attempt to reverse some of the clinical signs of aging skin. Several studies have been reported to determine if collagen is capable of percutaneous absorption. Huc (3) studied the penetration into living rat skin of biosynthetically-pre- pared [14C]-labeled acid-soluble collagen and collagen tagged with [125]-iodine. Al- though the [14C]-collagen did not penetrate the stratum corneum, the iodine label was quickly absorbed. No evidence was presented to rule out the possibility that the iodine label was released from the collagen, and this small molecule subsequently penetrated the skin. Nagelschmidt and Struck (4) recovered traces of [•4C]-collagen in the epi- dermis following application to the stratum corneum, and none in the dermis. Tronnier reported clinical improvement in skin conditions exhibiting scars, keloids, and pruritus, and ascribed the improvements to the penetration of collagen (5). Chvapil and Eckmeyer, after reviewing published studies demonstrating passage of collagen across the stratum corneum, ascribed most of the findings to contamination of the tissue specimens during sectioning of the frozen tissue for autoradiography (6). He provided evidence for this by performing similar experiments in which extensive washing of the skin with aqueous solvents prior to tissue autoradiography prevented the "penetration" seen in unwashed preparations. The depth of penetration of topically-applied nonla- beled collagen was determined by Johnson (7) by measuring the hydroxyproline content of sequential tape strippings. He found that collagen penetrated only through the out- ermost layers of the stratum corneum. MATERIALS AND METHODS COLLAGEN SAMPLES N-[Propionate-2,3-3H] propionylated rat skin type I collagen was obtained from New England Nuclear Corporation (NET-660). This material was mixed with native lathy- ritic rat skin collagen to yield a native collagen mixture (7.9 mg/ml in phosphate-buff- ered saline solution). [3H]-Proline-labeled collagen alpha 1(I) chains were prepared from cultured 3T6 mouse embryonic fibroblasts (ATCC, #CCL 96). The cells were maintained in Eagle's Min- imal Essential Medium supplemented with 10% fetal calf serum, penicillin (100 U/ml), streptomycin (100 U/ml) and Fugizone (2.5 ug/ml) in a humid atmosphere of 5% CO2/95% air. To prepare labeled collagen, confluent cultures were incubated 48 hours in this medium containing 5 I-tCi/ml [3H]-proline (New England Nuclear Corpo- ration, NET-323), 100 I.tg/ml ascorbic acid (cofactor for collagen synthesis), and 125 I.tg/ml beta-aminopropionitrile fumarate (collagen crosslink inhibitor which allows for