Desolvation process and surface characterisation of protein nanoparticles
Introduction
The major advantage of colloidal drug carrier systems is the possibility of drug targeting by a modified body distribution (Kreuter, 1983) as well as the enhancement of the cellular uptake (Schäfer et al., 1992) of a number of substances. As a result undesired toxic side effects of the free drug can be avoided, for instance with methotrexate (Narayani and Rao, 1993).
Among these colloidal systems those based on proteins may be very promising, since they are biodegradable and non-antigenic (Rubino et al., 1993), relatively easy to prepare and their size distribution can be monitored easily (MacAdam et al., 1997). Because of the defined primary structure of proteins the protein-based nanoparticles may offer various possibilities for surface modification and covalent drug attachment. A rational development of a protein based colloidal drug carrier systems requires a systematic characterisation of the particles.
In the field of microparticles a great deal of work has already been performed. The extend of reaction of functional groups with glutaraldehyde in model compounds and proteins was already studied by Habeeb and Hiramoto (1968). Similar studies were performed with human serum albumin (HSA) microparticles prepared in biphasic systems. A complete and systematic study regarding the influence of HSA concentration, emulsification time and power, stirring rate, heat stabilisation temperature, and the type of the non-aqueous phase was carried out by Gallo et al. (1984). The influence of polycondensation pH (Edwards-Levy et al., 1993), cross-linking reaction time (Edwards-Levy et al., 1994), and cross-linker concentration (Andry et al., 1996) on the content of free amino groups on the capsule surface of HSA microcapsules cross-linked using terephthaloyl chloride was analysed by reaction with 2,4,6-trinitrobenzenesulfonic acid (TNBS). The number of modified carboxylic and hydroxylic groups was investigated using Fourier Transform Infrared Spectroscopic (FTIR) studies (Levy et al., 1994, Levy et al., 1995). MacAdam et al., (1997) determined the surface carboxylic and amino groups of albumin microspheres by covalent binding of radiolabelled markers. Several turbidity ratio tests were performed to evaluate the efficiency of cross-linking with glutaraldehyde (Rubino et al., 1993, Chen et al., 1994, Lin et al., 1994).
However, to date a systematic characterisation of protein nanoparticles is lacking. The objective of the present study is to evaluate the desolvation of HSA for the preparation of nanoparticles by investigation of particle size and the percentage of protein still dissolved in the reaction mixture as a function of the amount of added desolvating agent. Further investigations were focused on the influence of various glutaraldehyde concentrations and heat denaturing conditions on the number of available amino groups at the surface of HSA and gelatin nanoparticles. The amount of free amino groups also may be considered as a measure for the degree of cross-linking since lysyl is the only residue in the albumin molecule which is modified in the presence of glutaraldehyde (Rubino et al., 1993). Cross-linking in turn affects biodegradability and drug release from the carrier system (Lee et al., 1981).
Section snippets
Reagents and chemicals
Human serum albumin (Fraction V ), glutaraldehyde 8%, 2,4,6-trinitrobenzenesulfonic acid (TNBS) 5% aqueous solution, gelatin type A from porcine skin (175 Bloom), and gelatin type B from bovine skin (75 Bloom) were obtained from Sigma (Steinheim, Germany). Formaldehyde solution 35% was purchased from Merck (Darmstadt, Germany), and the BCA protein assay reagent was obtained from Pierce (Rockford, Illinois, USA). All reagents were of analytical grade and used as received.
Instruments
An Eppendorf Thermomixer
Results and discussion
The desolvation of HSA or gelatin with organic solvents followed by cross-linking with glutaraldehyde is a commonly used method for the preparation of protein nanoparticles (Marty et al., 1978). However, up to now a systematic characterisation of the desolvation process for the preparation of nanoparticles is still lacking.
The desolvation process of HSA nanoparticles was evaluated concerning the particle size and the quantity of protein still dissolved in the aqueous phase after desolvation in
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