RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology Preparation of Caffeic Acid Phenethyl Ester-Incorporated Nanoparticles and Their Biological Activity HYO-YOUNG LEE, 1 YOUNG-IL JEONG, 2 EUN JIN KIM, 3 KYUNG DONG LEE, 4 SEON-HEE CHOI, 1 YUN JIN KIM, 5 DA HYE KIM, 6 KI-CHOON CHOI 7 1 Department of Medical Science, Pusan National University School of Medicine, Yangsan, Gyeongnam 626-770, Korea 2 Pusan National University Hospital, Pusan 602-739, Korea 3 R&D Center, M.I.Tech Company, Ltd. Pyeongtaek, Gyeonggi-do 451-864, Korea 4 Department of Oriental Medicine Materials, Dongsin University, Naju 520-714, Korea 5 Department of Family Medicine, Pusan National University Hospital, Pusan 602-739, Korea 6 Faculty of Life and Environmental Science, Shimane University, Matsue, Japan 7 Grassland and Forages Division, National Institute of Animal Science, Rural Development Administration, Chungnam 331-808, Korea Received 7 April 2014; revised 14 July 2014; accepted 21 October 2014 Published online 21 November 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24278 ABSTRACT: The aim of this study is to fabricate caffeic acid phenethyl ester (CAPE)-incorporated nanoparticles using methoxy poly(ethylene glycol)-b-poly(ε-caprolactone) (CE) copolymer and to study their antitumor activity against pulmonary metastasis model of CT26 colon carcinoma cells. CAPE-incorporated nanoparticles showed spherical shapes having small diameters less than 300 nm and CAPE was continuously released from CE nanoparticles over 4 days. CAPE-incorporated polymeric micelles properly inhibited proliferation and induced apoptosis of CT26 cells as well as CAPE itself. Furthermore, they showed similar anti-invasive and antimigrative effect against CT26 cells at in vitro compared with CAPE itself, indicating that CAPE-incorporated nanoparticles have at least equivalent anticarcinogenic activity against CT26 cells compared with CAPE itself. At pulmonary metastasis model of CT26 cells using nude mouse, CAPE-incorporated nanoparticles have superior antimetastatic efficacy against, that is, control treatment with pulmonary metastasis model showed significant increase of lung weight because of the metastasis of tumor cells, whereas CAPE or CAPE-incorporated nanoparticles properly inhibited metastasis of tumor cells. We suggest CAPE-incorporated nanoparticles as a promising candidate for antimetastatic chemotherapeutic agent. C  2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:144–154, 2015 Keywords: caffeic acid phenethyl ester; nanoparticles; metastasis; invasion; cancer chemotherapy; natural products; polymeric drug carrier INTRODUCTION Caffeic acid phenethyl ester (CAPE) is a component of propo- lis of honeybee hives. 1 Various in vitro biological effect of CAPE has been reported, such as suppression of acute immune responses, antioxidant, antimitogenic, anticarcinogenic, anti- inflammatory, and antiallergic effect. 2–6 CAPE has activities in suppression of carcinogenic incidence and various anticancer effect by inducing apoptosis pathways in cancer cells. 4 Espe- cially, CAPE is known to be a specific inhibitor of activation of nuclear transcription factor NF-6B. 7 CAPE was completely inhibited the activation of NF-6B by tumor necrosis factor in a dose- and time-dependent manner. Liao et al. 8 and Hwang et al. 9 described the inhibitory effect of CAPE against angio- genesis, tumor invasion, and metastasis against cancer cells. Furthermore, antibacterial and antiviral activity of CAPE has also been reported by several investigators. 10–13 Nanoparticles or colloidal carriers have been extensively in- vestigated in biomedical field. 14–16 Because of its small particle sizes, nanoparticles or colloidal carriers are regarded to an ideal vehicle for site-specific drug delivery and tumor targeting. 17 Correspondence to: Ki-Choon Choi (Telephone: +82-41-580-6752; Fax: +82- 41-580-6779; E-mail: choiwh@rda.go.kr); Kyung Dong Lee (Telephone: +82-61- 330-3261; Fax: +82-61-330-3261; E-mail: leekd@dsu.ac.kr); Young-Il Jeong (Tele- phone: +82-55-360-3873; Fax: +82-55-360-3879; E-mail: nanomed@naver.com) Journal of Pharmaceutical Sciences, Vol. 104, 144–154 (2015) C  2014 Wiley Periodicals, Inc. and the American Pharmacists Association Sakhrani and Padh 17 described the efficacy of nanoparticulate- based drug delivery, that is, nanoparticles is a promising vehicle for the site-specific delivery of anticancer drugs to the disease site, whereas systemic injection of drug induces systemic toxi- city and side effects because of its lack of target-specific affin- ity. Especially, nanoparticles are studied to amplify biological activity of antineoplastic drug and natural products. For ex- ample, tea polyphenol-loaded poly(lactide-co-glycolide) (PLGA) nanoparticles revealed 30-fold higher prevention advantages on the DNA damage than native tea polyphenols such as theaflavin and epigallocatechin-3-gallate. 18 Nanoparticles also enhanced anti-invasive efficacy of all-trans retinoic acid against CT26 colorectal cancer cells. 19 In other words, nanoparticles are appropriate vehicle for solubilization of lipophilic bioactive agents and to use as an intravenous injection of such com- pounds. As CAPE is a hydrophobic compound, nanoparticles can be used to solubilize CAPE into an aqueous solution and to amplify biological activity in an in vitro–in vivo system. 20,21 Liposomal formulation of CAPE was studied to solve solubility problem of it and tested in vivo tumor-bearing mice. 22 The aim of this study is to prepare CAPE-incorporated nanoparticles using poly(g-caprolactone)/poly(ethylene glycol) (CE) block copolymer and to investigate their biological activ- ity. Properties of CAPE-incorporated nanoparticles were stud- ied and their biological activity was studied in the terms of antioxidant activity, anti-invasive activity against CT26 col- orectal carcinoma cells, and antibacterial activity. 144 Lee et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:144–154, 2015