Published: August 02, 2011 r2011 American Chemical Society 3186 dx.doi.org/10.1021/bm200679w | Biomacromolecules 2011, 12, 3186–3193 ARTICLE pubs.acs.org/Biomac In Vitro Release of Lysozyme from Gelatin Microspheres: Effect of Cross-linking Agents and Thermoreversible Gel as Suspending Medium Pradip Hiwale, † Sandrina Lampis, † Gabriele Conti, ‡ Carla Caddeo, § Sergio Murgia,* ,† Anna M. Fadda, § and Maura Monduzzi* ,† † Dipartimento di Scienze Chimiche, Universit  a di Cagliari, CNBS and CSGI, Cittadella Universitaria, s.s. 554 bivio Sestu, 09042 Monserrato (CA), Italy ‡ Dipartimento di Citomorfologia, Universit  a di Cagliari, Cittadella Universitaria, s.s. 554 bivio Sestu, 09042 Monserrato (CA), Italy § Dipartimento Farmaco Chimico Tecnologico, Universit  a di Cagliari and CNBS Via Ospedale 72, 09100 Cagliari, Italy 1. INTRODUCTION Nanostructured materials and nanotechnologies represent emerging tasks for nanomedicine applications. Thus, a major objective of formulation chemistry is to improve bioavailability, stability, and convenience to the patient. Indeed, a growing effort in the discovery of innovative therapies has led to an increasing demand for drug delivery vehicles whose capability should not be limited to a simple drug encapsulating and transporting affair. Protecting and selectively releasing the drug and, in particular, overcoming biological barriers that prevent the drug to reach the receptor represent novel, strict requirements. Actually, most drug delivery systems belong to the colloidal domain. Therefore, the awareness of the hydrophobicÀ hydrophilic intermolecular interactions along with the thorough knowledge of surfactant self-assembly is essential in engineering systems capable of achieving a well-defined biological target. In addition, a sustained drug release also at very long-term is of paramount importance in the development of modern drug delivery systems such as depot systems that can be very useful in the chronic therapeutical treatments. A variety of innovative drug delivery formulations have been proposed in the recent years. 1 They embrace microsphere hydrogels based on polysaccharides, emulsions and microemulsions, liposomes, micelles, lipid nanoparticles, as well as colloidal dispersions where liquid crystals may alternatively represent both the dispersed (cubosome and hexosomes) 2 or the continuous phase. 3À5 New materials, including nanoporous silica and polylactide nanoparticles, have been designed to accomplish the pressing demand for drug delivery systems with enhanced performance. 6 Among the different types of drug delivery systems being proposed recently, microspheres composed of biodegradable polymers for controlled release applications are extensively studied. 7 Gelatin is one of the common natural polymers used in the fabrication of particulate drug delivery systems such as microspheres. 8À10 It is a well-known natural polymer derived from collagen that possesses good biodegradability and bio- compatibility. 8,11,12 Moreover, it has been extensively used in various contemporary pharmaceutical dosage forms, and it is included on the FDA list of inactive ingredients. 13 Gelatin exhibits polyion complexation properties that can be utilized in Received: May 18, 2011 Revised: June 28, 2011 ABSTRACT: This study was aimed to characterize the microstructure and the performance of gelatin microspheres (GMs) cross-linked by two different cross- linkers viz. D-glucose and glutaraldehyde. New formulations were obtained, suspending the GMs in a thermoreversible Pluronic F127 (PF127) liquid- crystalline gel. Lysozyme was used as a model biomacromolecular drug to evaluate release features. Both types of cross-linked GMs were prepared by thermal gelation method. The lysozyme-loaded microspheres were characterized by scanning electron microscopy (SEM) for size distribution, shape, and surface texture. SEM revealed that both types of lysozyme-loaded GMs were spherical in shape and that the surface of glutaraldehyde cross-linked GMs was smoother than that of the glucose cross-linked GMs. The degree of cross-linking of microspheres was investigated using ATR-FTIR technique. The prepared GMs were suspended in 20% w/v aqueous PF127 gel for which the usual solÀgel transition temperature of 22 °C did not change in the presence of GMs, as indicated by rheological measurements. SAXS study of the PF127 gel confirmed the occurrence of a discrete cubic liquid-crystalline phase of the Fm3m type whose lattice parameter slightly decreased as a result of GMs addition. The in vitro release of lysozyme from both types of cross-linked GMs was successfully controlled when they were suspended in PF127 gel, thus suggesting the potential use of this new combined formulation as a drug-depot system.