Folic acid-functionalized and acetyl-terminated dendrimers as nanovectors for co-delivery of sorafenib and 5-fluorouracil

Molecular dynamics (MD) simulations were employed to investigate the simultaneous association of sorafenib (SF) and 5-fluorouracil (5-FU) with generation 4 (G4) acetyl-terminated poly(amidoamine) (PAMAM) dendrimers conjugated with folic acid (G4ACE-FA). Simulations were conducted under physiological (pH 7.4) and acidic (pH < 5) conditions, representing the environments of healthy and cancerous cells, respectively. The average radius of gyration (R_g) of G4ACE-FA was determined to be approximately 1.85 ± 0.01 nm and 2.31 ± 0.03 nm under physiological and acidic conditions, respectively. Drug loading did not exert a significant influence on the size and conformational compactness of G4ACE-FA at both neutral and low pH. However, a discernible increase in dendrimer size was observed upon simultaneous encapsulation and/or conjugation of both drug molecules. The relaxation times of G4ACE-FA were calculated to be 10.2 ns and 9.6 ns at neutral and low pH, respectively, indicating comparable equilibrium rates under both pH environments. The incorporation of small 5-FU molecules did not demonstrably alter the dendrimer's microstructure. The observed doubling of the relaxation time under acidic conditions can be attributed to the relatively compact structure of the dendrimer at neutral pH and the continuous intrastructural rearrangements occurring at acidic pH. The prolonged relaxation time observed in the G4ACE-FA:5-FU:SF complex is attributed to competitive interactions between 5-FU and SF molecules during simultaneous encapsulation by the dendrimer. Analysis of the unloaded and loaded structures of G4ACE-FA under varying pH conditions revealed a densely packed conformation at neutral pH and a more open, sponge-like structure at low pH. The solvent-accessible surface area (SASA) of the dendrimer was assessed at both pH conditions. At neutral pH, SASA values were approximately 124.0 ± 2.8 nm², 127.5 ± 2.6 nm², 131.3 ± 2.6 nm², and 133.3 ± 2.6 nm² for unloaded G4ACE-FA and the G4ACE-FA:5-FU, G4ACE-FA:SF, and G4ACE-FA:5-FU:SF complexes, respectively. Drug incorporation had a minimal effect on SASA at neutral pH. At low pH, the corresponding values were 198.2 ± 4.7 nm², 195.8 ± 4.8 nm², 212.5 ± 6.1 nm², and 215.4 ± 4.2 nm². These findings suggest that 5-FU encapsulation resulted in minimal changes to the dendrimer's surface exposure to the solvent, potentially due to its small size. In contrast, SF interaction led to a more pronounced increase in SASA, indicating structural expansion to accommodate SF conjugation. The equilibrium stoichiometry of the G4ACE-FA:5-FU complex was determined to be 1:11 and 1:3 at neutral and low pH, respectively. Similarly, the G4ACE-FA:SF complex exhibited equilibrium stoichiometries of 1:10 and 1:4 at neutral and low pH. The G4ACE-FA:5-FU:SF complex displayed stoichiometries of 1:11:10 at neutral pH and 1:3:3 at low pH. Collectively, these findings suggest that G4ACE-FA holds promise as a versatile nanovector capable of tightly binding drug molecules at neutral pH and facilitating their release within tumor cells, thereby enabling targeted drug delivery. Furthermore, the co-loading of 5-FU and SF did not compromise the loading capacity of G4ACE-FA. At neutral pH, 5-FU molecules were distributed evenly across the dendrimer surface and within its cavities, with 6 molecules encapsulated internally and 5 conjugated on the surface. At low pH, all bound 5-FU molecules were located at the dendrimer periphery. Similarly, at neutral pH, SF molecules were found both internally (6 molecules) and on the surface (4 molecules). At low pH, 2 SF molecules were found on the surface and 2 were internally complexed. The preferred binding sites of 5-FU and SF remained largely unchanged when co-loaded onto the dendrimer. This suggests that co-delivery of 5-FU and SF using G4ACE-FA could be a promising strategy for enhancing the therapeutic efficacy of these chemotherapeutic agents.