Chitosan Nanoparticle-Loaded Mannitol Microspheres: Structure and Surface Characterization Ana Grenha, ² Begon ˜ a Seijo, ² Carmen Serra, ‡ and Carmen Remun ˜a ´ n-Lo ´ pez* ,² Department of Pharmacy and Pharmaceutical Technology, University of Santiago de Compostela, Faculty of Pharmacy, Campus Sur, 15782 Santiago de Compostela, Spain, and Center for Scientific and Technological Support to Research, University of Vigo, E-36310, Vigo, Spain Received November 29, 2006; Revised Manuscript Received April 10, 2007 In this work, we aimed to characterize the surface and the internal structure of mannitol microspheres containing chitosan/tripolyphosphate nanoparticles, which were prepared by spray-drying. These microspheres were recently proposed as valuable candidates to transport therapeutic protein-loaded nanoparticles to the lungs owing to their favorable aerodynamic properties. To observe the distribution of chitosan nanoparticles and mannitol in the microspheres, specific characterization techniques, such as confocal laser scanning microscopy, X-ray photoelectron spectroscopy, and time-of-flight secondary ion mass spectrometry, were used. Results showed that mannitol is distributed in the whole particle and nanoparticles are homogeneously mixed with mannitol. Moreover, both components were detected in the microsphere surface, mannitol being present to a higher extent, which is in agreement with the theoretical mannitol/nanoparticle ratio of microspheres (80/20). Therefore, this work confirmed that chitosan nanoparticles were successfully encapsulated in mannitol microspheres, providing a homogeneous distribution of the nanoparticles and, hence, of the nanoencapsulated therapeutic macromolecule. Introduction Pulmonary systemic administration of therapeutic macromol- ecules is receiving increased attention due to several advantages such as the large lung surface available for absorption, the high blood flux and thin alveolar-vascular epithelium, the low proteolytic activity compared to other mucosal routes, and the possibility to avoid the first-pass effect. 1-3 In fact, several therapeutic macromolecules such as insulin, 4 parathyroid hor- mone, 5 and leuprolide, 6 have been described to be adequately absorbed through the lung epithelium. The requisite for a reliable and specific delivery to the lung is the use of powder carrier systems exhibiting adequate aerodynamic properties to reach the desired area. In this sense, microspheres have been extensively investigated, since they can be tailored to appropriate morphological and aerodynamic properties. 7 Nanoparticles have also been proposed as delivery systems for proteins and peptides to the lung epithelium 8-12 due to their ability to delay or avoid mucociliary clearance and macrophagic capture. 13,14 However, they present some limitations for this purpose, considering their reduced dimensions and mass, which make lung deposition a difficult issue, potentially exposing them to exhalation. 2,15-17 Furthermore, stability concerns due to the nanoparticle formula- tion as aqueous suspensions should also be taken into account. Our group has developed a new ionotropic gelation nanotech- nology that is extremely mild and rapid and allows the production of chitosan-based nanoparticles. 18 These nanopar- ticles have been shown to possess an excellent capacity for protein entrapment and for improvement of mucosal peptide absorption through several epithelia such as the nasal 19 and intestinal. 20,21 Taking into account the above-mentioned limita- tions presented by the colloidal carriers for pulmonary admin- istration, we proposed in a previous work the microencapsulation of protein-loaded chitosan nanoparticles using the carbohydrate mannitol as an attempt to improve their aerosolization to the lungs and to ensure their intact delivery at the drug absorption site, so that these restrictions could be solved. 12 The obtained microspheres presented adequate aerodynamic properties for pulmonary delivery, 12 and it was recently demonstrated that they are biocompatible with two human cell lines representative of the respiratory bronchial (Calu-3) and alveolar (A549) epithelia, respectively. 22 Furthermore, the physicochemical properties of the microencapsulated nanopar- ticles and the release profile of insulin were shown to not be negatively affected by the spray-drying process, and nanopar- ticles could be easily recovered upon incubation of the micro- spheres in an aqueous medium. 12 However, in that work we did not address the question of the microsphere structure and, more specifically, the nanoparticle distribution within the microspheres. It is of great interest to analyze and visualize the spatial distribution of the involved structures to confirm whether or not nanoparticles are homogeneously encapsulated in the microspheres and, hence, whether or not the nanoentrapped therapeutic protein is homogeneously distributed within the aerosolized powder. This will obviously influence the aerosol powder reproducibility and efficacy. In this manner, techniques such as confocal laser scanning microscopy (CLSM), X-ray photoelectron spectroscopy (XPS), and static time-of-flight secondary ion mass spectrometry (TOF-SIMS) should, alto- gether, provide information on the accurate characterization of the microspheres’ internal and external structure. The main advantage of CLSM is the ability to provide visualization of images parallel to the sample surface at both internal and external levels, at multiple depths, without any mechanical sectioning. Nevertheless, the technique itself is not very precise in providing information on the most superficial composition of the microspheres. The surface region of a biomaterial, a * Author to whom correspondence should be addressed. Phone: 0034 981 563100 Ext. 15405. Fax: 0034 981 547148. E-mail: ffcarelo@usc.es. ² University of Santiago de Compostela. ‡ University of Vigo. 2072 Biomacromolecules 2007, 8, 2072-2079 10.1021/bm061131g CCC: $37.00 © 2007 American Chemical Society Published on Web 06/22/2007