Azacitidine Omega-3 Self-Assemblies: Synthesis, Characterization, and Potent Applications for Myelodysplastic Syndromes

Abstract

5-Azacitidine, a cytidine analogue used as a hypomethylating agent, is one of the main drugs for the treatment of myelodysplastic syndromes (MDSs) and acute myeloid leukemia (AML) in the elderly. However, after administration, it exhibits several limitations, including restricted diffusion and cellular internalization due to its hydrophilicity, and a rapid enzymatic degradation by adenosine deaminase. The aim of this study was to improve the drug cell diffusion and protect it from metabolic degradation via the synthesis of amphiphilic prodrugs and their potential self-assembly. Azacitidine was conjugated to two different omega-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The carboxylic acid group of the omega-3 fatty acids was effectively conjugated to the amine group of the azacitidine base, yielding two amphiphilic prodrugs. Nanoprecipitation of the obtained prodrugs was performed and self-assemblies were successfully obtained for both prodrugs, with a mean diameter of 190 nm, a polydispersity index below 0.2 and a positive zeta potential. The formation of self-assemblies was confirmed using pyrene as a fluorescent dye, and the critical aggregation concentrations were determined: 400 µM for AzaEPA and 688 µM for AzaDHA. Additionally, the stability of the obtained self-assemblies was studied and after 5 days their final stable arrangement was reached. Additionally, cryo-TEM revealed that the self-assemblies attain a multilamellar vesicle supramolecular structure. Moreover, the obtained self-assemblies presented promising cytotoxicity on a leukemia human cell line, having a low IC₅₀ value, comparable to that of free azacitidine.

Keywords: PUFAylation; azacitidine; docosahexaenoic acid; eicosapentaenoic acid; myelodysplastic syndromes; nanomedicine.

Figure 1

Hydrolysis of azacitidine. In the presence of water, a fast and reversible hydrolysis occurs, producing N-formylribosylguanylurea, followed by a second slow irreversible hydrolysis, producing ribosylguanylurea.

Figure 2

Formulation of self-assemblies via nanoprecipitation. The conjugates are dissolved in an organic solvent, here acetone, then added drop-wise to a stirring aqueous medium, allowing for the spontaneous formation of self-assemblies. The organic solvent is then evaporated using a rotary evaporator, thus yielding an opalescent aqueous suspension.

Figure 3

Synthesis of N4-azacitidine DHA (AzaDHA, 1) and N4-azacitidine EPA (AzaEPA, 2).

Figure 4

Fourier-transform infrared spectroscopy (FTIR) curves and wave numbers of interest between 4000 cm⁻¹ and 600 cm⁻¹ of azacitidine (green), EPA (purple), DHA (pink), AzaEPA (black) and AzaDHA (blue).

Figure 5

Boltzmann-type sigmoid data (red) obtained for the AzaEPA and AzaDHA suspensions in water, showing the CAC value, corresponding to the first sharp decrease point. Graph tangents (blue) are plotted, the tangent’s intersection with the graph (green) determines the CAC.

Figure 6

Cryo-TEM images of the formed self-assemblies: (A) AzaEPA, (B) AzaDHA.

Figure 7

Cytotoxicity studies of the self-assemblies compared to the free azacitidine and fatty acids after 24 h of treatment.