Synthesis and Characterization of PEGylated Calcium Phosphate Nanoparticles for Oral Insulin Delivery Rukmani Ramachandran, 1 Willi Paul, 2 Chandra P. Sharma 2 1 School of Chemical and Biotechnology, SASTRA Deemed University, Thanjavur 613402, Tamil Nadu, India 2 Division of Biosurface Technology, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram 695012, Kerala, India Received 1 November 2007; revised 22 July 2008; accepted 10 August 2008 Published online 22 October 2008 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.b.31241 Abstract: The inconvenience of subcutaneous insulin delivery leads to low patient compliance with the dosage regimens. The most desirable form of administration seems to be through the oral route. This work investigates the utility of PEGylated calcium phosphate nanoparticles as oral carriers for insulin. Calcium phosphate nanoparticles (CaP) with an average particle size of 47.9 nm (D50) were synthesized and surface modified by conjugating it with poly(ethylene glycol) (PEG). These modified nanoparticles were having a near zero zeta potential. Protection of insulin from the gastric environment has been achieved by coating the nanoparticles with a pH sensitive polymer that will dissolve in the mildly alkaline pH environment of the intestine. The release profiles of coated nanoparticles exhibited negligible release in acidic (gastric) pH, i.e., only 2% for CaP and 6.5% for PEGylated CaP. However, a sustained release of insulin was observed at neutral (intestinal) pH for over 8 h. The conformation of the released insulin, studied using circular dichroism, was unaltered when compared with native insulin. The released insulin was also stable as it was studied using dynamic light scattering. Radioimmunoassay was performed and the immunoreactivity of the released insulin was found to be intact. These results suggest PEGylated calcium phosphate nanoparticles as an excellent carrier system for insulin toward the development of an oral insulin delivery system. ' 2008 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 88B: 41–48, 2009 Keywords: calcium phosphate; nanoparticles; PEGylation; oral insulin INTRODUCTION Delivery through subcutaneous injections has been the mainstay of insulin therapy since its discovery. Injections suffer from the draw backs of being painful and inconven- ient to the patient and have shown inconsistent pharmaco- kinetics. 1 Numerous attempts have been made to use alternate routes so as to improve patient convenience. 2,3 The recent approval by United States Food and Drug Administration (US-FDA) of two nonconventional insulin delivery systems (buccal delivery through Oral-lyn and pul- monary delivery by Exubera) comes as a relief to many di- abetic patients. However, the most desirable form of administration of insulin seems to be through the oral route. Also, absorption through the intestine into the portal circulation mimics the physiological secretion of insulin. Particulate delivery systems can be designed in such a way so as to overcome most of the problems of oral delivery of proteins. Enteric coatings can be used to protect the drug in the gastric medium. Calcium phosphate microspheres have been studied for the delivery of insulin to the intestine. 4–7 This formulation decreased the elevated glucose level in di- abetic rats; however, the hypoglycemic effect was visible only for a period of 6–8 h. Ceramics have been widely used in the purification process of proteins and therefore it could be expected that the interaction between the protein and the ceramic material will not adversely affect the struc- ture or activity of the protein. Particles with a size of less that 100 nm are generally termed nanoparticles. The impor- tant technological advantages of nanoparticles as drug car- riers are its high stability (i.e., long shelf life), high carrier capacity (i.e., many drug molecules can be incorporated in the particle matrix), feasibility of incorporation of both hydrophilic and hydrophobic substances, and feasibility of variable routes of administration, including oral administra- tion and inhalation. These carriers can also be designed to enable controlled (sustained) drug release from the matrix. It has been demonstrated that delivering insulin via nano- Correspondence to: C. P. Sharma (e-mail: sharmacp@sctimst.ac.in) ' 2008 Wiley Periodicals, Inc. 41