Formulation Optimization for the Nanoparticles-in-Microsphere Hybrid Oral Delivery Systems Using Factorial Design Mayank D. Bhavsar, Sandip B. Tiwari, and Mansoor M. Amiji Department of Pharmaceutical Sciences, School of Pharmacy, Northeastern University, Boston, MA 02115 ABSTRACT The objective of the present study was to optimize the processing parameters for nanoparticles-in-microsphere oral delivery system (NiMOS) and to mathematically relate the process parameters with the selected attribute (particle size) of the hybrid system. Fluorescein Isothiocyanate (FITC) labeled gelatin nanoparticles were encapsulated in poly(epsilon caprolactone) (PCL) micropshere and this system was optimized for particle size using a full 3 3 factorial design. Particles with size less than 10 micrometer were processed based on the results obtained from factorial design. These particles can be used for the oral delivery of macromolecules such as peptide, proteins, and other therapeutics. Keywords: nanoparticle, microsphere, factorial design, response surface plot, oral delivery INTRODUCTION Advancements made in biotechnology and molecular engineering has afforded the use of polymeric systems as oral delivery devices for therapeutic/antigenic proteins and other macromolecules [1-3]. Despite the progress of knowledge in this field, present limitations of nanoparticles as transmucosal macromolecular delivery systems include their instability in contact with the gastrointestinal fluids and their limited interaction and transport across mucosal barriers [4]. Several researchers have suggested the use of microspheres prepared using biodegradable polymers to enhance the gastrointestinal stability of the encapsulated proteins and peptides. In order to enhance the targetability of the microspheres to the intestinal mucosae, designing of the microspheres below the size of 10 µm has been reported [5–9]. In this study, we have developed the formulation of gelatin nanoparticles encapsulated in PCL microspheres as a potential delivery system which can be used for intestinal mucosal delivery of therapeutic or antigenic proteins. The encapsulation of nanoparticles in microspheres was conceived with the intention of making these nanoparticles more stable when in contact with physiological fluids. The idea behind this concept was that the microsphere shell would hinder protein/enzyme adsorption, thereby avoiding the harsh environment to which the particles are exposed until they reach the absorbing epithelium. Factorial design based on response surface method was adopted to optimize particle size of NiMOS encapsulating, a model system, FITC labeled gelatin nanoparticles. A 3 3 full factorial design was employed to evaluate the combined effect of the selected variables on the size (in microns) of the prepared NiMOS. EXPERIMENTAL METHODS Preparation of FITC-labeled gelatin nanoparticles For the preparation of FITC-labeled gelatin, gelatin was dissolved in borate buffer (pH 8.5) at 37 o C. In a separate beaker FITC was dissolved in borate buffer (pH 8.5). The above solutions were mixed and incubated for 3 h at room temperature. The mixture was then extensively dialyzed against distilled water to remove any free FITC molecules. Nanoparticles of FITC-labeled gelatin were prepared by desolvation and controlled precipitation as previously described [10]. Preparation of NiMOS NiMOS were prepared using the “double emulsion like” technique. Briefly, FITC-labeled gelatin nanoparticles were suspended in distilled water and homogenized with PCL in dichloromethane using a Silverson lab mixture (Model L4RT-A, Silverson Lab Machines, Bucks, England) until formation of a stable emulsion like system. The resulting system was homogenized with poly(vinyl alcohol) solution in distilled deionized water. This dispersion was then magnetically stirred at room temperature to allow the dichloromethane to evaporate and harden the microspheres. The formed microspheres were collected by centrifugation, washed with distilled deionized water to remove PVA and lyophilized. Particle size analysis Freshly prepared suspension of FITC-labeled gelatin nanoparticles was analyzed for particle size and size distribution by light scattering method using 90Plus particle size analyzer, Brookhaven Corp. (Holtsville, NY). Particle size analysis was carried out at a scattering angle of 90 o and a temperature of 25 o C. Similarly, freshly prepared NiMOS were characterized for particle size and size distribution using NSTI-Nanotech 2006, www.nsti.org, ISBN 0-9767985-7-3 Vol. 2, 2006 366