Preparation of protease immobilized on modified polyamidoamin dendrimer nanoparticle and study of their activity and stability

Abstract

Dendrimers are a novel class of nonlinear polymers and due to their extensive applications in different fields, called versatile polymers. Polyamidoamine (PAMAM) dendrimers are one of the most important dendrimers that have many applications in nanobiotechnology and industry, such as drug delivery, gene delivery, MRI, enzyme immobilization and etc. The structure of these dendrimers, includes a central core, branching units and terminal functional groups. Positive charges on the surface of amine dendrimers at physiological pH can interact with negative charge on cell membrane leading to cell lysis and consequently cytotoxicity. Therefore, to prevent this interaction, the surface charge should be modified. One goal of this study, reducing of Mentioned cytotoxicity with convert of amine groups to aldehyde groups. The aldehyde-terminated dendrimers are usually prepared through the activation of amine-terminated dendrimers by glutaraldehyde. This method has two disadvantages of glutaraldehyde toxicity and cross-linking due to existence of two aldehyde functional groups on its both ends. Aminoacetaldehyde dimethylacetal (AADA), as a special reagent which is protected on one end and has a reactive amine group on the other end, that reacted with ester terminated PAMAM dendrimer. Another objective of this study, immobilization of trypsin enzyme on aldehyde terminated dendrimers and then improvement of functional properties of trypsin enzyme.

Expermental: In this research polyamidoamine dendrimers from G0.5 (with 4 ester terminal functional groups) to G2 (with 16 amine terminal functional groups) were synthesized. Then, G1.5 with (16 ester terminal functional groups) reacted with aminoacetaldehyde dimethyl acetal to convert ester terminal functional groups to acetal terminal functional groups. Finally,acetal terminal groups were hydrolyzed by TFA to aldehyde terminal groups. Immobilization of trypsin on G2 PAMAL dendrimer was performed through covalent method and then enzyme activity was measured with azocasein as a substrate. Finally temperature, pH and solvent effects were investigated on activity of immobilized enzyme and free enzyme.

Results:

The results showed that cytotoxicity of dendrimers with aldehyde-terminated groups is much lower than that of G1 and G2 PAMAM-NH2 dendrimers. Also the findings of this study showed that immobilization of trypsin not only resulted higher optimal temperature, but also increased the thermal stability of the immobilized enzyme in comparison to the free enzyme. The results of the study of pH effect on immobilized enzyme activity, an increase about 0.6 unit of optimum pH are showed ratio to free enzyme. And the findings of the study of solvent effect on immobilized enzyme and free enzyme activity showed that activity change, depend on solvent are different.

Conclusion:

Dendrimers with aldehyde-terminated groups could be used as novel and convenient carriers for drug delivery with low cytotoxic effect compared with the amine-terminated dendrimers. The results revealed that the same generations of the dendri-mers with aldehyde-terminated groups are far less toxic than the corresponding amine-terminated dendrimers. Also some of functional properties of immobilized enzyme were improved in comparsion to the free enzyme such as thermal stability, optimum pH and increase of activity in organic solvent immiscible with water.