Protoporphyrin IX Nanoparticle Carrier: Preparation, Optical Properties, and Singlet Oxygen Generation Liane M. Rossi,* Paulo R. Silva, Lucas L. R. Vono, Adjaci U. Fernandes, Dayane B. Tada, and Maurı ´cio S. Baptista Institute of Chemistry, UniVersity of Sa ˜o Paulo, USP, Sa ˜o Paulo 05508-000, SP, Brazil ReceiVed March 17, 2008. ReVised Manuscript ReceiVed August 14, 2008 The present study is focused on developing a nanoparticle carrier for the photosensitizer protoporphyrin IX for use in photodynamic therapy. The entrapment of protoporphyrin IX (Pp IX) in silica spheres was achieved by modification of Pp IX molecules with an organosilane reagent. The immobilized drug preserved its optical properties and the capacity to generate singlet oxygen, which was detected by a direct method from its characteristic phosphorescence decay curve at near-infrared and by a chemical method using 1,3-diphenylisobenzofuran to trap singlet oxygen. The lifetime of singlet oxygen when a suspension of Pp IX-loaded particles in acetonitrile was excited at 532 nm was determined as 52 µs, which is in good agreement with the value determined for methylene blue in acetonitrile solution under the same conditions. The Pp IX-loaded silica particles have an efficiency of singlet oxygen generation (η Δ ) higher than the quantum yield of free porphyrins. This high efficiency of singlet oxygen generation was attributed to changes on the monomer-dimer equilibrium after photosentisizer immobilization. Introduction Protoporphyrin IX (Pp IX) is a naturally occurring porphyrin constituent of hemoglobin, cytochrome c and other biologically relevant molecules. It is used as a drug in photodynamic therapy (PDT) but its direct application is limited due to its aggregation and low solubility in a physiological medium. PDT is based on the concept that photosensitizer molecules generate singlet oxygen upon irradiation, which acts as the primary cytotoxic agent responsible for irreversible damage of the treated tissues. 1 In contrast with other conventional medical treatments, in PDT it is unnecessary to release the loaded drug, but the diffusion of surrounding oxygen molecules and the release of the produced active oxygen species must be sufficient for a therapeutic effect. 2 The indirect application of Pp IX is made possible by δ-ami- nolevulinic acid in chloridric acid solution (ALA · HCl). ALA is a pro-drug, a precursor for all in vivo porphyrins, which is cycled by biosynthesis generating tissue Pp IX. 3 Modifications to the Pp IX molecule have been made to increase the solubility of this potential drug in a physiological medium and stimulate its use in PDT. An important strategy is to explore the reactivity of vinyl groups to functionalize Pp IX with polar groups. To this end hydroxyl groups have been inserted in the vinylic positions 3 1 and 8 1 to produce hematoporphyrin IX. The hematoporphyrin IX, in both acid and basic media, polymerizes to form a mixture of chromophores named Photofrin, which is the most used drug in PDT. Further functionalization of carboxylic groups has also been used for the preparation of several derivatives. 4,5 Another strategy is to functionalize the vinylic group by means of a Diels-Alder reaction, 6 transforming the porphyrinic ring into a chlorin ring. This process produces BPDM (Visudyne), another derivative of Pp IX which is found on the market for use in PDT. Some of these drugs are currently approved by the Food and Drug Administration for the treatment of superficial cancers. However, there are still many problems for the clinical application of existing photosensitizers, such as low purity, because they are a complex mixture of several partially unidentified porphyrins and have poor selectivity for tumor tissue, low extinction coefficients that require the administration of relatively large amounts of drug to obtain a satisfactory phototherapeutic response, and finally, high accumulation rate in skin, which induces a prolonged cutaneous light sensitivity lasting for up to weeks after PDT treatment, obliging patients to stay out of the sunlight to avoid a severe sunburn reaction. Thus, the formulation of an injectable Pp IX product with satisfactory stability in aqueous solutions and which is easily targeted to the desired site of action is of great interest. In recent years, an increasing number of researchers have considered the possibility of using nanoparticles in photodynamic therapy (PDT). 7-10 Successful loading of a chlorin derivative in silica nanoparticles has been reported to give nanoparticle platforms that only deliver singlet oxygen for PDT while conserving the photosensitizer effect. 11 It was shown that nanoparticles can accumulate at the tumor site due to enhanced endocytotic activity and leaky vasculature in the tumors. 12 Also, nanoparticles can be modified superficially using special targeting moieties (such as antibodies) for site-specific action. Encapsula- tion of drugs in silica nanoparticles is very promising because silica surfaces are easy to functionalize, nontoxic, chemically inert, and optically transparent. Besides protecting the drug from external interferences, the silica matrix should improve the drug * Corresponding author. Phone: +55 11 30912181. Fax: +55 11 38155579. E-mail: lrossi@iq.usp.br. (1) (a) Bonnett, R. Chem. Soc. ReV 1995, 24, 19. (b) Dougherty, T. J. Photochem. Photobiol. 1987, 45, 879. (2) Snyder, J. W.; Skovsen, E.; Lambert, J. D. C.; Ogilby, P. R. J. Am. Chem. Soc. 2005, 127, 14558. (3) Xiao, Z.; Tamimi, Y.; Brown, K.; Tulip, J.; Moore, R. Urol. Oncol. 2002, 7, 125. (4) Lottner, C.; Bart, K. C.; Bernhardt, G.; Brunner, H. J. Med. Chem. 2002, 45, 2064. (5) Bonnett, R.; Gabriel, M. Tetrahedron 2001, 57, 9513. (6) Savellano, M. D.; Hasan, T. Photochem. Photobiol. 2003, 77, 431. (7) DeRosa, M. C.; Crutchley, R. J. Coord. Chem. ReV. 2002, 351, 233–234. (8) Zeitouni, N. C.; Oseroff, A. R.; Shieh, S. Mol. Immunol. 2003, 39, 1133. (9) Wang, S.; Gao, R.; Zhao, F.; Selke, M. J. Mater. Chem. 2004, 14, 487. (10) Tada, D. B.; Vono, L. L. R.; Duarte, E. L.; Itri, R.; Kiyohara, P. K.; Baptista, M. S.; Rossi, L. M. Langmuir 2007, 23, 8194. (11) Yan, F.; Kopelman, R. Photochem. Photobiol. 2003, 78, 587. (12) (a) Konan, Y. N.; Gurny, R.; Alle ´mann, E. J. Photochem. Photobiol., B: Biol. 2002, 66, 89. (b) Roby, A.; Erdogan, S.; Torchilin, V. P. Eur. J. Pharm. Biopharm. 2006, 62, 235. 12534 Langmuir 2008, 24, 12534-12538 10.1021/la800840k CCC: $40.75  2008 American Chemical Society Published on Web 10/04/2008