Surface Functionalization of Porous Glass Networks: Effects on Bovine Serum Albumin and Porcine Insulin Immobilization Herman S. Mansur,* ,² Ze ´ lia P. Lobato, ‡ Rodrigo L. Ore ´ fice, ² Wander L. Vasconcelos, ² Cintia Oliveira, ² and Lucas J. Machado § Department of Metallurgy and Materials Engineering, Department of Veterinary Medicine, and Department of Internal Medicine, School of Medicine, Federal University of Minas Gerais, Rua Espirito Santo, 35/2 andar, Centro, Belo Horizonte, MG, Brazil Received September 29, 2000 Biomolecules can be immobilized in many different ways. They can also be entrapped or tightly adsorbed within porous gels, clays, membranes, resins, and several other materials, but it is crucial that they retain their active conformation after the incorporation procedure. Porous gel matrixes with functionalized surfaces offer unlimited possibilities to control the protein-substrate interaction behavior. In the present work, we have studied the adsorption and the relative stability of bovine serum albumin (BSA) and porcine insulin (PI) incorporated in gels of SiO 2 glass matrixes. The porous gel matrixes were obtained using tetramethoxysilane (TMOS)/methanol and functionalized with (3-mercaptopropyl)trimethoxysilane and (3- aminopropyl)triethoxysilane. The relative adsorption kinetics and stability of BSA and PI incorporated in glass networks were evaluated by immersion in phosphate buffer saline (PBS) and alkaline elution media for different periods of time. The kinetics of protein release from the gel matrix was monitored by UV- visible spectroscopy. A significantly larger PI release was observed compared to BSA in PBS solutions. We believe this is mainly associated with the difference on protein interactions with the modified surface, according to the characterization results of porosity, surface area, and contact angle of different functionalized gel matrixes. We could not observe any evidence of denaturation with either proteins after their desorption from gel matrixes using the ultraviolet spectroscopy technique. These results have also been confirmed with the strong bioactivity response from “in vivo” tests conducted in rats, where porous gels with PI incorporated were implanted, showing that released proteins retained their native conformation. 1. Introduction The adsorption of protein from solution onto solid surfaces is a fascinating and complex process playing a major role in biological systems. Surface-immobilized proteins have drawn the attention of the research community in the last 2 decades. The high efficiency presented by biological macromolecules in selecting chemical species has motivated the development of devices that combine synthetic materials with biological entities. Proteins can de immobilized in many different ways, but it is crucial that they retain their active conformation after the incorporation procedure. 1,2 There are three major methods for immobilizing biomolecules and cells. Two of them are physically based, physical adsorption and physical entrapment. The third method is based on covalent (chemical) attachment. 2 Thus, it is important to note that the term immobilization can refer either to a temporary or to a permanent localization of the biomolecule on or within a support. Biological macromolecules, such as proteins, have very particular chain configurations and conformations that promote high levels of specificity during chemical interac- tions. The immobilization of proteins onto surface-function- alized substrates has been one of the most promising areas in the bioengineering field as discussed by several authors. 3-8 The performance of artificial materials in contact with biological systems is to a large extend determined by surface interactions. Therefore considerable efforts have been made in the last years to improve surface compatibility of materials used for biological and biomedical applications. 9 It has an enormous potential for application as biomaterial implants, immunological kits, drug delivery systems, and biosensors. In the present work, we aimed to go one step further in the analysis of this phenomenon of protein adsorption/ desorption. We have studied the incorporation of bovine serum albumin (BSA) and porcine insulin (PI) into porous gel networks of SiO 2 and into gels of SiO 2 with function- alized surface. We chose to study the adsorption of bovine serum albumin because albumin is dominantly present in body fluids among other proteins. Insulin was chosen because of its activity of regulating the glucose concentration level found in the blood of most mammals. The relative chemical stability of BSA and PI to remain incorporated into the SiO 2 glass matrixes was evaluated using UV-vis spectroscopy. “In vitro” tests were used to characterize the protein- adsorption process onto the porous glass matrixes followed * To whom correspondence should be addressed. Tel: +55 31 238- 1843. Fax: +55 31 238-1815. E-mail: hmansur@demet.ufmg.br. ² Department of Metallurgy and Materials Engineering, Federal Univer- sity of Minas Gerais, Brazil. ‡ Department of Veterinary Medicine, Federal University of Minas Gerais, Brazil. § Department of Internal Medicine, School of Medicine, UFMG, Brazil. 789 Biomacromolecules 2000, 1, 789-797 10.1021/bm0056198 CCC: $19.00 © 2000 American Chemical Society Published on Web 11/03/2000