Abstract
This paper describes the production, purification, and immobilization of l-asparaginase II (ASNase II) in chitosan nanoparticles (CSNPs). ASNase II is an effective antineoplastic agent, used in the acute lymphoblastic leukemia chemotherapy. Cloned ASNase II gene (ansB) in pAED4 plasmid was transformed into Escherichia coli BL21pLysS (DE3) competent cells and expressed under optimal conditions. The lyophilized enzyme was loaded into CSNPs by ionotropic gelation method. In order to get optimal entrapment efficiency, CSNP preparation, chitosan/tripolyphosphate (CS/TPP) ratio, and protein loading were investigated. ASNase II loading into CSNPs was confirmed by Fourier transform infrared (FTIR) spectroscopy, and morphological observation was carried out by transmission electron microscopy. Three absolute CS/TPP ratios were studied. Entrapment efficiency and loading capacity increased with increasing CS and TPP concentration. The best ratio was applied for obtaining optimal ASNase II-loaded CSNPs with the highest entrapment efficiency. Size, zeta potential, entrapment efficiency, and loading capacity of the optimal ASNase II-CSNPs were 340 ± 12 nm, 21.2 ± 3 mV, 76.2% and 47.6%, respectively. The immobilized enzyme showed an increased in vitro half-life in comparison with the free enzyme. The pH and thermostability of the immobilized enzyme was comparable with the free enzyme. This study leads to a better understanding of how to prepare CSNPs, how to achieve high encapsulation efficiency for a high molecular weight protein, and how to prolong the release of protein from CSNPs. A conceptual understanding of biological responses to ASNase II-loaded CSNPs is needed for the development of novel methods of drug delivery.
Highlights
L-Asparaginase II (ASNase II) is an enzyme that is widely used for the treatment of hematopoietic diseases such as acute lymphoblastic leukemia
Half-life determination of the free and immobilized ASNase II The solutions of Tris–HCl (0.1 M, pH = 8.5), DDWglycerol (5%), and phosphatebuffered saline (PBS)-glycerol (5%) were considered for measuring the half-life of the free and immobilized enzyme
Production and purification of ASNase II As mentioned above, protein expression was carried out under conditions that were previously optimized in our laboratory
Summary
L-Asparaginase II (ASNase II) is an enzyme that is widely used for the treatment of hematopoietic diseases such as acute lymphoblastic leukemia. Candidate carrier biopolymers should exhibit chemical and physical stability, biological compatibility, high purity, homogeneous molecular weight (MW) distribution, and adequate functional groups for binding to biomolecules with high loading capacity They exhibit several drug loading mechanisms including electrostatic attractions, hydrophobic interactions, and covalent binding. One should bear in mind that covalent coupling of enzymes to polymers may result in conformational alterations, pharmacokinetic modifications, and a significant decrease in enzymatic activity. Examples of such biopolymer nanoparticles that ASNase II has already been incorporated in are liposomes [7], poly (D,L-lactide-co-glycolide) (PLGA) [8], and hydrogelmagnetic nanoparticles [9]
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