Processing and immobilization of enzyme Ribonuclease A through laser irradiation
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J. Roqueta Consejo Superior de Investigaciones Científicas, Centre d’Investigacions en Nanociència i Nanotecnologia (CSICCIN2), Campus UAB, 08193 Bellaterra, Spain
A. Pérez del Pino Consejo Superior de Investigaciones Científicas, Institut de Ciència de Materials de Barcelona (CSIC-ICMAB), Campus UAB, 08193 Bellaterra, Spain
M. Moussaoui and M. V. Nogués Departament de Bioquímica i Biologia Molecular, Facultat de Biociències, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain
E. Györgya) National Institute for Lasers, Plasma and Radiations Physics, 77125 Bucharest, Romania; and Consejo Superior de Investigaciones Científicas, Centre d’Investigacions en Nanociència i Nanotecnologia (CSIC-CIN2), Campus UAB, 08193 Bellaterra, Spain (Received 7 July 2010; accepted 12 October 2010)
We report the processing and immobilization of enzyme Ribonuclease A (RNase A) onto SiO2 glass collectors using the matrix assisted pulsed laser evaporation (MAPLE) technique. The experiments were performed inside a stainless steel irradiation chamber. A pulsed UV KrF* (k 5 248 nm, sFWHM 25 ns, m 5 10 Hz) excimer laser source was used for the irradiations. The laser fluence was varied in the range 0.4–0.7 J/cm2. The morphology of the obtained films was investigated by atomic force microscopy (AFM) and their structure and composition by Fourier transform infrared spectroscopy (FTIR). The FTIR spectra of the films obtained from composite MAPLE targets consisting of 1% (w/v) RNase A in Hepes-KOH 10 mM pH 7.5 buffer exhibit the same bands as the spectrum of the initial, nonirradiated material. The enzymatic activity of the obtained structures was analyzed using synthetic substrate polycytidylic acid (poly(C)). The poly (C) cleavage by the immobilized enzyme and the products of formation were analyzed by means of reverse phase high performance liquid chromatography (HPLC).
I. INTRODUCTION
Bovine pancreatic Ribonuclease A (RNase A E.C. 3.1.27.5) is a well known endonuclease with a molecular weight of 13.7 6 0.3 kDa that cleaves 39,59-phosphodiester linkages of single-stranded RNA when the base of the nucleotide in the 39 position is a pyrimidine.1–4 It is know that some RNases, belonging to the RNase A family, have cytotoxic and/or cytostatic effects on cancer cells.5–7 Therefore, the immobilization of these RNases on solid three-dimensional biocompatible and bioresorbable matrix materials could represent new solutions in cancer therapy by controlled drug release.8–10 The process, known as surface-mediated drug delivery, implies the release of therapeutic cargo from the surface of a biomaterial, typically an implant, to the surrounding cells, a)
Address all correspondence to this author. e-mail: [email protected] DOI: 10.1557/jmr.2010.55 J. Mater. Res., Vol. 26, No. 6, Mar 28, 2011
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organs, and tissues.11 Among the advantages of this process are the spatial control of release of the therapeutic molecules and a more efficient delivery due to the much higher local concentration of d
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