Carrier-Free Microspheres of an Anti-Cancer Drug Synthesized via a Sodium Catalyst for Controlled-Release Drug Delivery

Abstract

There are several challenges involved in the development of effective anti-cancer drugs, including accurate drug delivery without toxic side effects. Possible systemic toxicity and the rapid biodegradation of drug carriers are potential risks in the use of carriers for drug-delivery formulations. Therefore, the carrier-free drug delivery of an anti-cancer drug is desirable. Herein, 4-amino-2-benzyl-6-methylpyrimidine (ABMP) was synthesized via a new method using a sodium catalyst, and proved to be effective in inducing breast cancer cell (MDA-MB-231) apoptosis. Moreover, the transparent amorphous state solid of ABMP was demonstrated to have a slow-release property in phosphate buffer solution (PBS). Microspheres of ABMP were prepared with diameters in the range of 5-15 μm. The slow-release property of the ABMP microspheres indicated their potential use for controlled-release drug delivery. We believe that microspheres of ABMP have potential as a new kind of carrier-free anti-cancer drug delivery system.

Keywords: 4-amino-2-benzyl-6-methylpyrimidine; biomaterial; carrier-free; drug delivery.

Figure 1

Synthesis and characterization of 4-Amino-2-benzyl-6-methylpyrimidine (ABMP). (a) Chemical structure of ABMP synthesized from benzyl cyanide using sodium as a catalyst; (b) Fourier transform infrared (FT-IR) spectrum of ABMP; (c) mass spectrum of ABMP; and (d) ¹H NMR spectrum of ABMP.

Figure 2

Differential thermal analysis of ABMP; the melting point is 110.617 °C, and the boiling point is 407.768 °C.

Figure 3

Three-dimensional (3D) photoluminescence spectra of ABMP in ethanol with the concentration of the sample set to 1% (w/v).

Figure 4

Relative cell numbers determined by the absorbance (OD) value using CCK-8 at 24 and 48 h. Breast cancer cells (MDA-MB-231) were treated with ABMP at different concentrations, i.e., 0, 10, 25, 50, 75, 100, 125, and 150 μg/mL. The experiments were carried out in quadruplicate. Double asterisks (**) and double pounds (##) refer to statistical significance p < 0.01 compared with control groups. Double percent (%%) refers to a statistical significance of p < 0.01 between the OD values after seeding for 24 and 48 h.

Figure 5

Relative cell numbers determined by the OD value using CCK-8 at 24 h. L929, bone marrow stem cell (BMSC), and MDA-MB-231 cells were treated with ABMP at different concentrations, i.e., 0, 10, 25, 50, 75, 100, 125, and 150 μg/mL. The experiments were carried out in quadruplicate. Double asterisks (**) and double at (@@) refer to statistical significance p < 0.01 compared with control groups. Double pound (##) refers to a statistical significance of p < 0.01 between the OD values of L929 and MDA-MB-231 after seeding for 24 h; double percent (%%) refers to a statistical significance of p < 0.01 between the OD values of BMSC and MDA-MB-231 after seeding for 24 h.

Figure 6

Cell numbers and viability evaluated using calcein-AM/ propidium iodide (PI) staining after 48-h seeding. (a) Control; (b) 25 μg/mL; (c) 50 μg/mL; (d) 75 μg/mL; (e) 100 μg/mL; and (f) 125 μg/mL.

Figure 7

ABMP arrests MDA-MB-231 cells in G0/G1. Proliferating MDA-MB-231 cells were treated with various concentrations of ABMP for 24 h, fixed with ethanol, and stained with PI. Cell cycle distributions were analyzed by fluorescence-activated cell sorter (FACS) analysis.

Figure 8

Histograms from FACS analysis at each ABMP concentration. The FL1 channel was used to detect annexin V-FITC staining, and the FL2 channel was for PI staining. (a) Control; (b) 25 μg/mL; (c) 50 μg/mL; (d) 75 μg/mL; (e) percentage of early apoptotic cells stained with annexin-V-FITC at various concentrations of ABMP; (f) percentage of late apoptotic or necrotic cells stained with both annexin V-FITC and PI at various concentrations of ABMP.

Figure 9

Transparent amorphous state solid of ABMP. (a) Appearance of amorphous state solid ABMP (ass-ABMP); (b) filiform appearance of ass-ABMP when an outside force was employed; (c,d) deformation is still possible after stretching several times.

Figure 10

Slow-release curve of ass-ABMP in phosphate-buffered saline (PBS) solution. The test was executed three times under the same conditions.

Figure 11

Morphology of microspheres of ABMP under an optical microscope: (a) overall distribution in a low-power image (100 times); and (b) the detailed morphology in a high-power image (400 times).

Figure 12

Morphology of ABMP microspheres under an optical microscope after release for (a) 0 h; (b) 10 h; (c) 20 h; and (d) 40 h in PBS. The diameter of a typical particle is labeled in each picture.