CLytA-DAAO Chimeric Enzyme Bound to Magnetic Nanoparticles. A New Therapeutical Approach for Cancer Patients?

María Fuentes-Baile,Miguel Saceda,Jesús M Sanz,Álvaro Rodríguez-Lescure,Camino De Juan Romero,Víctor M Barberá,Daniel Bello-Gil,Elizabeth Pérez-Valenciano,Pilar García-Morales,Cristina Alenda

doi:10.3390/ijms22031477

Abstract

D-amino acid oxidase (DAAO) is an enzyme that catalyzes the oxidation of D-amino acids generating H2O2. The enzymatic chimera formed by DAAO bound to the choline-binding domain of N-acetylmuramoyl-L-alanine amidase (CLytA) induces cytotoxicity in several pancreatic and colorectal carcinoma and glioblastoma cell models. In the current work, we determined whether the effect of CLytA-DAAO immobilized in magnetic nanoparticles, gold nanoparticles, and alginate capsules offered some advantages as compared to the free CLytA-DAAO. Results indicate that the immobilization of CLytA-DAAO in magnetic nanoparticles increases the stability of the enzyme, extending its time of action. Besides, we compared the effect induced by CLytA-DAAO with the direct addition of hydrogen peroxide, demonstrating that the progressive generation of reactive oxygen species by CLytA-DAAO is more effective in inducing cytotoxicity than the direct addition of H2O2. Furthermore, a pilot study has been initiated in biopsies obtained from pancreatic and colorectal carcinoma and glioblastoma patients to evaluate the expression of the main genes involved in resistance to CLytA-DAAO cytotoxicity. Based on our findings, we propose that CLytA-DAAO immobilized in magnetic nanoparticles could be effective in a high percentage of patients and, therefore, be used as an anti-cancer therapy for pancreatic and colorectal carcinoma and glioblastoma.

Highlights

Increasing knowledge of protein immobilization systems has provided the opportunity to increase the usage of enzymes to several areas and expand it to multiple biotechnological processes, since immobilization increases the half-life and reduces protein degradation [1].The nature of the immobilization material used plays a fundamental role and determines whether the immobilized protein can reach its full therapeutic potential
Immobilization in gold nanoparticles (GNPs) and in alginate capsules significantly decreased the cell death induced by the treatment (Figure 1A), suggesting that these methods of CLytADAAO immobilization are less effective stabilizing the chimeric enzyme than magnetic nanoparticles (MNPs)
We had previously shown that the CLytA-D-amino acid oxidase (DAAO)-bound to MNPs together with D-Ala treatment induces a higher percentage of cells in subG1 phase than the free enzyme in several pancreatic carcinoma, colorectal carcinoma, and glioblastoma cell lines [11]

Summary

Introduction

Increasing knowledge of protein immobilization systems has provided the opportunity to increase the usage of enzymes to several areas and expand it to multiple biotechnological processes, since immobilization increases the half-life and reduces protein degradation [1].The nature of the immobilization material used plays a fundamental role and determines whether the immobilized protein can reach its full therapeutic potential. The immobilization matrix should be made of affordable and available materials and should provide a wide surface area and a high density of functional groups to facilitate enzyme immobilization. They should guarantee an adequate diffusion of the substrates and products involved in the enzymatic reaction [2]. Enzyme immobilization in nanoparticles reduces the limitations in the diffusion of substrates and products and increases the functional surface area and the loading capacity [3], which has a direct impact on the activity of the immobilized enzyme. These characteristics, along with their low toxicity and high biocompatibility and biodegradability, have resulted in the increased use of nanoparticles in biomedical research like in diagnostic tests, drug delivery systems, personalized enzyme therapies, and tissue regeneration [4,5]

Methods

Results

Conclusion