Dendrimer Platforms for Targeted Doxorubicin Delivery-Physicochemical Properties in Context of Biological Responses

Abstract

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.

Keywords: DDS; PAMAM dendrimers; dendrimer-doxorubicin interactions; doxorubicin; drug delivery systems.

Figure 1

(a) CD spectra of aggregated forms of doxorubicin in water solution and its dependence on concentration (blue line—c = 0.5 mg/mL, pink line—c = 1.0 mg/mL). (b) Fluorescence spectra of doxorubicin and its dependence on pH (c = 2 μg/mL, H₂O).

Figure 2

(a) Change in electrophoretic mobility (μ_e) and (b) zeta potential (ζ) of aqueous solution of aggregated forms of doxorubicin as a function of pH with determined value of isoelectric point (iep) (c_DOX = 0.5 mg/mL).

Figure 3

(a) Schematic representation of the G4.0-DOX bilayer formation on the Au surface (created with BioRender.com). (b) The mass of the adsorbed G4.0 PAMAM/DOX bilayers (Γ_QCM-D) on the gold surface and its dependence on DOX pH, monitored by QCM-D (G4.0/DOX molar ratio 1:6, c_G4.0 = 17.6 μM, pH_G4.0 = 10.0, water). The gray color corresponds to the G4.0PAMAM monolayer, and the blue color corresponds to the DOX layer. The Sauerbrey model was used in the pH = 7.5–8.5 (light gray and light blue bars), and the Voigt model for pH = 9.0–10.0 (dark gray and dark blue bars). (c) The mass of the adsorbed G4.0 PAMAM/DOX bilayers on the gold surface and its dependence on DOX pH and concentration, monitored by QCM-D (c_G4.0 = 17.6 μM, pH_G4.0 = 10.0, water); with the exception of G4.0-DOX 1:3 pH = 9.0, the Sauerbrey and Voigt model was applied and compared to all the layers. (d) A comparison of the adsorbed masses by the QCM-D and MP-SPR methods. (e) The percentage of water in the QCM-D layers for the G4.0 PAMAM and DOX layers for the various pH conditions of the DOX solution (c_G4.0 = 0.25 mg/mL, pH_G4.0 = 10.0, c_DOX = 0.06 mg/mL, H₂O).

Figure 4

Fluorescence emission spectra of G4.0 PAMAM-DOX systems presented for: (a) G4.0-DOX at pH 7.5; (b) G4.0-DOX at pH 9.0; (c) G4.0-DOX at pH 9.5 and LOG((F0-F)/F) versus LOG[G4.0] plots.

Figure 5

Change in the zeta potential (ζ) of the G4.0 PAMAM dendrimer depending on the ionic strength and solvent (a) and complexes with doxorubicin before and after dialysis (b) with the determined values of isoelectric point (iep) (H₂O, molar ratio 1:6, pH 9.5).

Figure 6

The cytotoxic effect of G4.0 PAMAM nanocarriers against human cancer (A375, NCL-H23) and immortalized cells (HaCaT) as a model of the normal cell line, evaluated by the MTT assay after 24 and 72 h.

Figure 7

Internalization of G4.0 PAMAM-DOX and free DOX by confocal microscopy in A375 and HaCaT cells: intravital analysis after 24 h (a) and analysis of fixed cells after 24 and 72 h (b). DOX- green color; nuclei- DAPI blue color.

Figure 8

(a) Release profile of doxorubicin from the complex compared to the DOX of the same concentration (c = 38 μM) into the PBS buffer at pH 7.4. Triangle—final complex at pH 7.5; cross—final complex after reducing to pH = 4.0. (b) Change in the electrolytic conductivity of the PBS buffer during the release assay. (c) The fluorescence spectra of the G4.0-DOX complexes at the molar ratios of 1:12 and 1:24, their changes after dilution in water at pH 7.5 and pH 4.0 and comparison with the reference DOX solution at the same pH and concentration (c = 5 μM).