J. Microbiol. Biotechnol. (2011), 21(1), 28–36 doi: 10.4014/jmb.1007.07041 First published online 19 November 2010 Amphotericin B Aggregation Inhibition with Novel Nanoparticles Prepared with Poly(ε-caprolactone)/Poly(N,N-dimethylamino-2-ethyl methacrylate) Diblock Copolymer Shim, Yong-Ho 1,2 , You-Chan Kim 3 , Hong-Joo Lee 4 , Francois Bougard 1 , Philippe Dubois 1 , Ki-Choon Choi 5 , Chung-Wook Chung 6 , Dae Hwan Kang 6 * , and Young-Il Jeong 6 * Laboratory of Polymeric and Composite Materials, Center for Innovation and Research in Materials and Polymers (CIRMAP), University of Mons-Hainaut, Place du Parc 20, Mons, 7000, Belgium Korea- Europe Technology Center, Seoul 158-072, Korea Department of Food Science and Biotechnology, Sungkyunkwan University, Suwon 440-746, Korea Gwangju Development Institute, Gwangju 506-042, Korea Grassland and Forages Research Center, National Institute of Animal Science, Rural Development Administration, Chungnam 331-808, Korea National Research and Development Center for Hepatobiliary Disease, Pusan National University Yangsan Hospital, Gyeongnam 626-770, Korea Received: July 20, 2010 / Revised: October 6, 2010 / Accepted: October 25, 2010 Diblock copolymers composed of poly(ε-caprolactone) (PCL) and poly(N,N-dimethylamino-2-ethyl methacrylate) (PDMAEMA), or methoxy polyethylene glycol(PEG), were synthesized via a combination of ring-opening polymerization and atom-transfer radical polymerization in order to prepare polymeric nanoparticles as an antifungal drug carrier. Amphotericin B (AmB), a natural antibiotic, was incorporated into the polymeric nanoparticles. The physical properties of AmB-incorporated polymeric nanoparticles with PCL-b-PDMAEMA and PCL-b-PEG were studied in relation to morphology and particle size. In the aggregation state study, AmB-incorporated PCL-b- PDMAEMA nanoparticles exhibited a monomeric state pattern of free AmB, whereas AmB-incorporated PCL-b- PEG nanoparticles displayed an aggregated pattern. In in vitro hemolysis tests with human red blood cells, AmB- incorporated PCL-b-PDMAEMA nanoparticles were seen to be 10 times less cytotoxic than free AmB (5 µg/ml). In addition, an improved antifungal activity of AmB- incorporated polymeric nanoparticles was observed through antifungal activity tests using Candida albicans, whereas polymeric nanoparticles themselves were seen not to affect activity. Finally, in vitro AmB release studies were conducted, proving the potential of AmB-incorporated PCL-b-PDMAEMA nanoparticles as a new formulation candidate for AmB. Keywords: Amphotericin B, infectious disease, nanoparticle, hemolysis, Candida albicans Amphotericin B (AmB), an amphoteric macrocyclic natural antibiotic, is known to bind strongly to sterol components, such as ergosterol, in susceptible fungal cell membranes and to induce changes in permeability that can substantially induce lethal cell injury [10, 12, 24]. Since AmB has a broad antifungal spectrum activity, it is one of the first considerations for standard antibiotic therapy when faced with life-threatening fungal infections, such as visceral leishmaniasis and mucocutaneous leishmaniasis, amongst others [3]. Although there are well-known side effects of AmB, such as nephrotoxicity, which limit its clinical usage, AmB is also a useful antibiotic for the treatment of systemic fungal infections [7]. Other major drawbacks of AmB include poor aqueous solubility and its amphiphilic properties [2, 6]. Owing to its amphiphilic nature, AmB can easily be aggregated in an aqueous solution in a micellar form, and it is known that the toxic side effects of AmB are closely related to its aggregated form [2]. For this reason, AmB should be completely solubilized in an aqueous solution in a monomeric state to make it therapeutically active and less cytotoxic to the human body. Commercially available Fungizone R , a colloidal dispersion of AmB in sodium deoxycholate, has acute *Corresponding author D.H.K. Phone: +82-55-360-3870; Fax: +82-55-360-3879; E-mail: sulsulpul@yahoo.co.kr Y.-I.J. Phone: +82-55-360-3873; Fax: +82-55-360-3879; E-mail: nanomed@naver.com