ASP-Enzymosomes with Saccharomyces cerevisiae Asparaginase II Expressed in Pichia pastoris: Formulation Design and In Vitro Studies of a Potential Antileukemic Drug

Luciana F C Girão,Margarida Ferreira-Silva,Manuela Colla Carvalheiro,Maria Antonieta Ferrara,Jonas Perales,Surza L G Da Rocha,Elba P S Bon,M Bárbara F Martins,M Luísa Corvo

doi:10.3390/ijms222011120

Abstract

The bacterial enzyme asparaginase is the main treatment option for acute lymphoblastic leukemia. However, it causes side effects, such as immunological reactions, and presents undesirable glutaminase activity. As an alternative, we have been studying asparaginase II from Saccharomyces cerevisiae, coded by ASP3 gene, which was cloned and expressed in Pichia pastoris. The recombinant asparaginase (ASP) presented antileukemic activity and a glutaminase activity 100 times lower in comparison to its asparaginase activity. In this work, we describe the development of a delivery system for ASP via its covalent attachment to functionalized polyethylene glycol (PEG) polymer chains in the outer surface of liposomes (ASP-enzymosomes). This new delivery system demonstrated antiproliferative activity against K562 (chronic myeloid leukemia) and Jurkat (acute lymphocytic leukemia) cell lines similar to that of ASP. The antiproliferative response of the ASP-enzymosomes against the Jurkat cells suggests equivalence to that of the free Escherichia coli commercial asparaginase (Aginasa®). Moreover, the ASP-enzymosomes were stable at 4 °C with no significant loss of activity within 4 days and retained 82% activity up to 37 days. Therefore, ASP-enzymosomes are a promising antileukemic drug.

Highlights

Therapeutic enzymes have been sourced, from crude plant and animal extracts, as a digestive aid since the end of the 19th century [1]
Superoxide dismutases (SODs) was covalently linked at the distal end of polyethylene glycol (PEG) in the outer membrane of the PEG-liposomes (SOD-enzymosomes), exposing the enzyme on the liposomal surface, displaying enzymatic activity in an intact form without the need for liposomal disruption
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Summary

Introduction

Therapeutic enzymes have been sourced, from crude plant and animal extracts, as a digestive aid since the end of the 19th century [1] Since their use, production, and formulation have greatly advanced, covering several applications in which highly purified recombinant proteins have been mostly used either in their native form, chemically modified, conjugated to polymers, or nanoformulated to increase stability and effectiveness upon several therapeutic conditions. Their use, production, and formulation have greatly advanced, covering several applications in which highly purified recombinant proteins have been mostly used either in their native form, chemically modified, conjugated to polymers, or nanoformulated to increase stability and effectiveness upon several therapeutic conditions Drug delivery systems, such as polymer conjugates, liposomes, and nanoparticles, have demonstrated the capacity to overcome limitations of conventional treatments, reducing immunogenicity, prolonging plasma half-life, and enhancing protein stability. It is noteworthy that the techniques to perform the liposomal encapsulation of therapeutic enzymes have greatly evolved with upscaling capacities—a glass-capillary microfluidic technique has been proposed for the preparation of SOD Liposomes [10]

Methods

Results

Conclusion