Preparation and Characterization of Protein Nanoparticles from Human Serum Albumin (HSA) as a novel drug delivery system

The aim of present study was to prepare human serum albumin (HSA) nanoparticle for drug delivery. These nanoparticles were prepared by desolvation method. The body distribution of colloidal drug delivery systems was mainly influenced by two physicochemical properties namely particle size and surface characteristics. The influence of several factors on the fabrication process including the pH value, HSA concentration [mg/ml], glutaraldehyde concentration [%], agitation speed [rpm] and the ratio of organic solvent/HSA solution were investigated herein. The smallest size of nanoparticles was 69 nm and the largest size was 424 nm which is suitable for drug delivery. The crosslinker concentration [%] and agitation speed [rpm] showed that have less effect on produced nanoparticle size. For characterizing the nanoparticle sample atomic force microscopy (AFM) and scanning electron microscopy (SEM) was employed whilst size distribution was measured by photon correlation spectroscopy (PCS). It was shown that the produced nanoparticles were almost formed by a smooth surface. Based on these characteristics, HSA nanoparticles were good enough to be candidate for drug delivery. The mechanistic of the optimum conditions for preparing protein nanoparticle as well as their characterization are discussed. Introduction In recent years the improvement of drug therapy in terms of a more controlled body distribution to reduce side effects was focused. Different new drug carrier systems in the micro and nanometer size range were generated to overcome these problems [1]. Nanoparticle sizes are defined from 10 to 1000 nm. When the drug is encapsulated by the nanoparticles, the products are called nanocapsules, if the medicine is uniformly coated on the exterior of the nanoparticle then is called nanosphere [2]. Conventional drug therapy requires periodic doses of therapeutic agents. For most drugs conventional methods of drug administration are effective, but some drugs are unstable or toxic and have narrow therapeutic ranges [3]. Some drugs also possess solubility problem. In such cases a method of continuous administration of therapeutic agents is desirable. To overcome these problems, controlled drug delivery systems were introduced. The principal advantage of this technology is that the carrier polymer matrix systems allow much less active agents to be used for the desire of activity [4]. Polymeric nanoparticles have attractive physicochemical properties such as size, surface potential, hydrophilic-hydrophobic balance etc. and for this reason they have been recognized as potential drug carriers for bioactive ingredients such as anticancer drugs, vaccines, oligonucleotides, peptides, etc [5]. Although various biodegradable nanoparticles of natural polymers such as starch, chitosan, liposomes etc. are largely in use as drug carriers in controlled drug delivery technology, however, albumin nanoparticles represent a promising carrier system for controlled drug delivery. According to the literature, albumin nanoparticles have been selected for many researches topics due to its properties [6]. Among these, human serum albumin (HSA) is a promising material. HSA is the most abundant protein in human PDF created with pdfFactory Pro trial version www.pdffactory.com 2 blood plasma. HSA (molecular weight of 65 KDa) belongs to a multigene family of proteins [7] and is the major soluble protein of the circulating system with a blood concentration about50 mg.ml. HSA consists of 585 amino acids containing 35 cyctein residues which build 17 disulfide bridges [8]. In this work the effect of different imperative factors on the fabrication of HSA nanoarticle with desolvation method are studied. The main goal is to concider these factors and optimizes the particle size and size distribution of HSA nanoparticles as drug delivry vehicle. Experimental Materials HSA (fraction V, purity 96-99%) and glutaraldehyde solution were commercially supplied by Sigma Aldrich. Ethanol and other reagents were purchased from Merck (Germany). Preparation of HSA Nanoparticles HSA nanoparticles were prepared by a desolvation technique as described previously [21]. In principle, between 50 and 200 mg HSA was dissolved in 2 ml of 10 mM NaCl solution, then titrated to pH 7.5-9 and under constant stirring desolvation of HSA solution was achieved by drop wise addition of ethanol. Ethanol was added drop wise until the solution become turbid. After the desolvation process, 8% glutaraldehyde in water was added to induce particle cross linking. Cross linking process was performed under stirring of the suspension over a time period of 24 hr. The resulting nanoparticles were purified by five cycles of centrifugation (25000×g, 10min) and redispersion of the pellet to the original volume in 10 mM NaCl at pH values of 7.5-9 respectively. Each redispersion step was performed in an ultrasonication bath. Results and Discussion The impact of pH value, HSA concentration, glutaraldehyde concentration [%], agitation speed and the ratio of organic solvent/HSA solution on the nanoparticle size were studied. The pH value of the HSA solution prior to ethanol addition influenced the resulting particle size. The effect of pH on nanoparticle size was conducted and shown in figure1. As the pH increased the particle size was decreased. Figure.1. Influence of the pH value on the diameter of HSA nanoparticles The influence of the HSA concentration on particle diameter of resulting samples is shown in figure2. Figure.2. Influence of the HAS concentration on the diameter of HSA nanoparticles 0 50 100 150 200 250 300 350 400 450 7.5 8 8.5 9 pH value P ar tic le s iz e( nm ) 20 30 40 50 60 70 80 90 100 110