Study On Self-assembled Nanoparticles Of Hyaluronic Acid As Delivery System For L-asparaginase

Protein and polypeptide medicine are bio-macromolecules, and they are fragile and lack of stability when they are applied in clinic. For this reason, the carriers and transportation systems are necessary for protein products and polypeptide drugs. Drug carriers have a lot of advantages in improving the efficacy of drugs, such as enhancing the solubility of protein/polypeptide in the water, controlled releasing and avoiding the side-effect brought by initial burst release. Hyaluronic acid (HA) is a nature mucopolysaccharide with large molecular weight (105-107 Da), which exists in body fluid and tissues widely. Because HA has many crucial biological functions, such as good biodegradability and lower immunogenicity, it has been an ideal material applied in drug sustained releasing carrier. Currently, asparaginase showed a dramatically effect on the treatment of acute lymphoblastic leukemia (ALL) and other hematopoietic malignant tumors. Asparaginase is a tetramer enzyme which can induce the conversion from asparaginase to aspartic acid and ammonia. However, being a heterologous protein, asparaginase can lose their activation easily and be cleared quickly when used clinically, so repeated injections are needed to maintain therapeutical level, which lead to immune response reaction. So we can increase the half-life period of enzyme in blood circulation by embedding asparaginase to carriers.The HA modified by deoxycholate (DOCA) were prepared by active ester method, and HD with different substitution degrees were prepared by means of controlling the addition of DOCA. The increasing of content of DOCA-NH2 in HD led to the increasing of average MW and degree of substitution. Self-assembly nanoparticle suspension were manufactured by ultrasonic emulsification, and the particle size and size distribution were detected by dynamic light scattering. It was found that the particle size changed when prepared by HD with different degree of substitution. Zeta potential was an important indicator for the surface charge, the results showed that the Zeta potential of nanoparticle suspension prepared by HD with different degree of substitution showed little differences and the potential were all negative. The stability tests determined the stability of self-assembly HD nanoparticles in different attenuation, storage, pH, ion strength, lyophobic condition.In this paper, we entrapped the asparaginase into HD nanoparticles and examined the drug loading capacity and encapsulation efficiency. The results exhibited that the addition of asparaginase could cause increasing of drug loading capacity and decreasing of encapsulation efficiency. We also examined how the ionic strength impact drug loading capacity of HD nanoparticles, and the results indicated that there was hydrophobic interaction between nanoparticles and enzyme. The results of blood compatibility test and cytotoxicity test reflected that the material used in this experiment was nontoxicity and antitumor activity.The influences of temperature and pH reaction on the enzymatic activity were also discovered in this paper. The recovery of modificated enzyme was 83.10%. We also studied enzyme kinetics and Michaelis constant, and found that the HD nanoparticle could increase the affinity for the substrate and the enzymatic activity. In vitro releasing experiment showed that degree of substitution and slow-release effect are positive correlation.