Poly(amidoamine) Dendrimers as Nanocarriers for 5-Fluorouracil: Effectiveness of Complex Formation and Cytotoxicity Studies

Abstract

Two generations of positively charged poly(amidoamine) dendrimers (PAMAMs) were selected for study as potential carriers for the anticancer drug 5-fluorouracil (5FU), a drug primarily used in the treatment of colorectal cancer. Analytical techniques, such as UV-Vis spectrophotometry, NMR Spectroscopy and Laser Doppler Velocimetry (LDV), have shown that the most critical factor determining the formation of a PAMAM–5FU complex is the starting components’ protonation degree. The tests confirmed the system’s ability to attach about 20 5FU molecules per one dendrimer molecule for the G4PAMAM dendrimer and about 25 molecules for the G6PAMAM dendrimer, which gives a system yield of 16% for the fourth generation and 5% for sixth generation dendrimers. Additionally, using the QCM-D method, the adsorption efficiency and the number of drug molecules immobilized in the dendrimer structure were determined. A new aspect in our study was the determination of the change in zeta potential (ζ) induced by the immobilization of 5FU molecules on the dendrimer’s outer shell and the importance of this effect in the direct contact of the carrier with cells. Cytotoxicity tests (resazurin reduction and MTS tests) showed no toxicity of dendrimers against mouse fibroblast cells (L929) and a significant decrease in cell viability in the case of four human malignant cell lines: malignant melanoma (A375), glioblastoma (SNB-19), prostate cancer (Du-145) and colon adenocarcinoma (HT-29) during incubation with PAMAM–5FU complexes. The purpose of our work was to investigate the correlation between the physicochemical properties of the carrier and active substance and the system efficiency and optimizing conditions for the formation of an efficient system based on PAMAM dendrimers as nanocarriers for 5-fluorouracil. An additional aspect was to identify potential application properties of the complexes, as demonstrated by cytotoxicity tests.

Effect of Ionization Degree of Poly(amidoamine) Dendrimer and 5-Fluorouracil on the Efficiency of Complex Formation—A Theoretical and Experimental Approach

“…In good agreement with the literature [37], these NMR findings are considered evidence for the host-guest interaction and, principally, the proximity of the guest molecule to the dendrimer surface, since only the chemical shifts of the terminal methylene protons of G4PAMAM were sensibly affected. This is in good agreement with the results of our previous study, where we demonstrated the effect of 5FU molecules on the zeta potential of the G4PAMAM dendrimer [15]. The higher drug attachment efficiency for the higher dendrimer/5FU molar ratio (1:100) resulted in a reduction in the zeta potential of the system value of approximately 22%.…”

“…Our previous studies, summarized in Figure 1, have shown that under properly optimized conditions, the G4PAMAM dendrimer binds approximately 20 molecules of 5FU, acting as a non-toxic and effective nanocarrier, in agreement with the literature [12,15]. While, so far, attention has been mainly focused on the binding efficiency of 5FU to the PAMAM or PEGylated PAMAM, there is a lack of studies investigating the localization of 5FU in the dendrimer structure.…”

“…The higher drug attachment efficiency for the higher dendrimer/5FU molar ratio (1:100) resulted in a reduction in the zeta potential of the system value of approximately 22%. The change in the system's zeta potential towards a more negative one indicates that the immobilization of the 5FU molecules neutralizes the positive charge from the amino groups present on the surface of the dendrimer molecule [15].…”

“…One of the nanocarriers most widely studied in the literature is the fourthgeneration dendrimer (G4PAMAM). This is due mainly to its high drug retention ability, which is usually better than other higher-generation dendrimers (>G6PAMAM) [13][14][15]. Moreover, G4PAMAM dendrimers have an accessible flexible structure compared to the most recent ones [3,16].…”

“…Finally, we complemented the experimental work with molecular dynamics (MD) simulations for a better characterization of the nature of PAMAM-5FU interactions at the atomic level. The initial evaluation of cytotoxicity of dendrimers and their complexes with 5FU against mouse fibroblasts (L929), glioblastoma (SNB-19), prostate cancer (Du-145), and colon adenocarcinoma (HT-29) determined via metabolic activity assay of the cells cultured for 7 days in the presence of G4PAMAM dendrimers, G4-5FU complexes, and 5FU, with concentration corresponding to its concentration in complex [15]. The initial evaluation of cytotoxicity of dendrimers and their complexes with 5FU against mouse fibroblasts (L929), glioblastoma (SNB-19), prostate cancer (Du-145), and colon adenocarcinoma (HT-29) determined via metabolic activity assay of the cells cultured for 7 days in the presence of G4PAMAM dendrimers, G4-5FU complexes, and 5FU, with concentration corresponding to its concentration in complex [15].…”