Due to the specificity of their structure, protein systems are adapted to carry various ligands. The structure of many proteins potentially allows for two types of immobilization of a therapeutic agent, either on the outer surface of the protein or within the protein structure. The existence of two active sites in BSA’s structure, the so-called Sudlow I and II, was confirmed. The conducted research involved determining the effectiveness of BSA as a potential carrier of 5-fluorouracil (5FU). 5-fluorouracil is a broad-spectrum anticancer drug targeting solid tumors. The research was carried out to estimate the physicochemical properties of the system using complementary measurement techniques. The optimization of the complex formation conditions made it possible to obtain significant correlations between the form of the drug and the effective localization of the active substance in the structure of the protein molecule. The presence of two amino groups in the 5FU structure contributes to the deprotonation of the molecule at high pH values (pH > 8) and the transition to the anionic form (AN1 and AN3). To investigate the binding affinity of the tautomeric form with BSA, UV-vis absorption, fluorescence quenching, zeta potential, QCM-D, and CD spectroscopic studies were performed. The experimental research was supported by molecular dynamics (MD) simulations and molecular docking. The simulations confirm the potential location of 5FU tautomers inside the BSA structure and on its surface.

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.

In this study, special attention was paid to the correlation between the degree of ionization of the components and the effective formation of the complex under alkaline conditions. Using UV-Vis, ¹H NMR, and CD, structural changes of the drug depending on the pH were monitored. In the pH range of 9.0 to 10.0, the G4.0 PAMAM dendrimer can bind 1 to 10 DOX molecules, while the efficiency increases with the concentration of the drug relative to the carrier. The binding efficiency was described by the parameters of loading content (LC = 4.80–39.20%) and encapsulation efficiency (EE = 17.21–40.16%), whose values increased twofold or even fourfold depending on the conditions. The highest efficiency was obtained for G4.0PAMAM-DOX at a molar ratio of 1:24. Nevertheless, regardless of the conditions, the DLS study indicates system aggregation. Changes in the zeta potential confirm the immobilization of an average of two drug molecules on the dendrimer’s surface. Circular dichroism spectra analysis shows a stable dendrimer-drug complex for all the systems obtained. Since the doxorubicin molecule can simultaneously act as a therapeutic and an imaging agent, the theranostic properties of the PAMAM-DOX system have been demonstrated by the high fluorescence intensity observable on fluorescence microscopy.

The unique structure of G4.0 PAMAM dendrimers allows a drug to be enclosed in internal spaces or immobilized on the surface. In the conducted research, the conditions for the formation of the active G4.0 PAMAM complex with doxorubicin hydrochloride (DOX) were optimized. The physicochemical properties of the system were monitored using dynamic light scattering (DLS), circular dichroism (CD), and fluorescence spectroscopy. The Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D) method was chosen to determine the preferential conditions for the complex formation. The highest binding efficiency of the drug to the cationic dendrimer was observed under basic conditions when the DOX molecule was deprotonated. The decrease in the zeta potential of the complex confirms that DOX immobilizes through electrostatic interaction with the carrier’s surface amine groups. The binding constants were determined from the fluorescence quenching of the DOX molecule in the presence of G4.0 PAMAM. The two-fold way of binding doxorubicin in the structure of dendrimers was visible in the Isothermal calorimetry (ITC) isotherm. Fluorescence spectra and release curves identified the reversible binding of DOX to the nanocarrier. Among the selected cancer cells, the most promising anticancer activity of the G4.0-DOX complex was observed in A375 malignant melanoma cells. Moreover, the preferred intracellular location of the complexes concerning the free drug was found, which is essential from a therapeutic point of view.

Due to their unique structure, poly(amidoamine) (PAMAM) dendrimers can bind active ingredients in two ways: inside the structure or on their surface. The location of drug molecules significantly impacts the kinetics of active substance release and the mechanism of internalization into the cell. This study focuses on the effect of the protonation degree of the G4PAMAM dendrimer and the anticancer drug 5-fluorouracil (5FU) on the efficiency of complex formation. The most favorable conditions for constructing the G4PAMAM-5FU complex are a low degree of protonation of the dendrimer molecule with the drug simultaneously present in a deprotonated form. The fluorine components in the XPS spectra confirm the formation of the stable complex. Through SAXS and DLS methods, a decrease in the dendrimer’s molecular size resulting from protonation changes at alkaline conditions was demonstrated. The gradual closure of the dendrimer structure observed at high pH values makes it difficult for the 5FU molecules to migrate to the interior of the support structure, thereby promoting drug immobilization on the surface. The ¹H NMR and DOSY spectra indicate that electrostatic interactions determine the complex formation process. Through MD simulations, the localization profile and the number of 5FU molecules forming the complex were visualized on an atomic scale.