Preparation Of Self-assembled Micron-sized Particles Of Elastin-like Polypeptides For Delivery Of Lysozyme

Elastin-like polypeptides (ELPs) are temperature sensitive biopolymerscomposed of a Val-Pro-Gly-Xaa-Gly pentapeptide repeat that derived from astructural motif found in mammalian elastin. Due to their reversible phase transitioncharacteristic, excellent bio-compatatable, easy to produce by means of geneticengineering, ELPs have been widely used for a number of biomedical applications,such as drug delivery, and tissue engineering. Here, we used the [KV8F]n as thetarget, three aspects including the production process opitimization, the factorsinfluencing the elastin-like polypeptides self-assembled into micron-sized particlesand its molecular mechanism and the delivery of lysozyme by ELPs were studied.Firstly, using Gln to tentatively identify the concentration of each amino acid, weanalyzed the influence of amino acids on the production of elastin-like polypeptides[KV8F]40. The results showed Thr, Arg and Cys could improve the production at theconcentration of1.5mmol/L. Among them, Thr was the best; it could improve theproduction from82.4mg/L to126.9mg/L, with an improvement of54%. And then,we designed the response surface analysis with three factors and three levels tooptimize the cultivation medium of ELPs40. The experimental results fitted thetheoretical result very well, which was103.1mg/L and100.3mg/L, respectively.However, the production was less than that of single factor. By means of interactionanalysis, we found Thr and Cys inhibit each other, while others didn’t haveinteraction.Secondly, in this paper, using the ELPs [KV8F]n as the target, we took asystematical research on the factors with the dynamic light scattering. Our resultsshowed the particle size increased and the uniform of particles decreased with theincrease of the molecular weight. The analysis of size variation in self-assembledELPs in response to changes in salt concentration indicated that the size increasedwith increasing the salt concentration, and the concentration was above0.4mol/L theopposite response was observed. At these conditions, the particles are micron-sized and larger than1.1μm. However, when the fusions containing the same ELPs andxylanase or1,3-propanediol oxidoreductase, the size of the self-assembled ELPsparticles decreased dramatically, which was only about1/10relative to that of the freeELPs. We proposed that the solvent accessible charged area of the enzymes couldinteract with the ELPs, the sterical hindrance of the enzymes prevent the aggregationof the ELPs. This might be the most important parameter in altering the particle sizesharply.Finally, using the purified ELP140embedding lysozyme, we determined theembedding rate under the different conditions to explore the optimum conditions, anddetected the particle size of the pure ELPs and the embedding lysozyme of ELPs tofind the embedding way. The results showed that when the concentration of ELP140was30μM, the embedding rate was highest, up to23.24%. When the ratio ofELPs/Lysozyme was higher, the embedding rate was higher. The result of scanningparticle showed that there was no obviously changed, indicated that when to form theself-assembly microspheres, the lysozyme was encased into the microspheres.