Selective Cellular Uptake and Druggability Efficacy through Functionalized Chitosan-Conjugated Polyamidoamine (PAMAM) Dendrimers

Abstract

Nanotechnology has ushered in significant advancements in drug design, revolutionizing the prevention, diagnosis, and treatment of various diseases. The strategic utilization of nanotechnology to enhance drug loading, delivery, and release has garnered increasing attention, leveraging the enhanced physical and chemical properties offered by these systems. Polyamidoamine (PAMAM) dendrimers have been pivotal in drug delivery, yet there is room for further enhancement. In this study, we conjugated PAMAM dendrimers with chitosan (CS) to augment cellular internalization in tumor cells. Specifically, doxorubicin (DOX) was initially loaded into PAMAM dendrimers to form DOX-loaded PAMAM (DOX@PAMAM) complexes via intermolecular forces. Subsequently, CS was linked onto the DOX-loaded PAMAM dendrimers to yield CS-conjugated PAMAM loaded with DOX (DOX@CS@PAMAM) through glutaraldehyde crosslinking via the Schiff base reaction. The resultant DOX@CS@PAMAM complexes were comprehensively characterized using Fourier-transform infrared (FTIR) spectroscopy, transmission electron microscopy (TEM), and dynamic light scattering (DLS). Notably, while the drug release profile of DOX@CS@PAMAM in acidic environments was inferior to that of DOX@PAMAM, DOX@CS@PAMAM demonstrated effective acid-responsive drug release, with a cumulative release of 70% within 25 h attributed to the imine linkage. Most importantly, DOX@CS@PAMAM exhibited significant selective cellular internalization rates and antitumor efficacy compared to DOX@PAMAM, as validated through cell viability assays, fluorescence imaging, and flow cytometry analysis. In summary, DOX@CS@PAMAM demonstrated superior antitumor effects compared to unconjugated PAMAM dendrimers, thereby broadening the scope of dendrimer-based nanomedicines with enhanced therapeutic efficacy and promising applications in cancer therapy.

Keywords: PAMAM; cellular internalization; chitosan; drug delivery; druggability.

Figure 1

The assembly, cellular internalization, and drug release behavior of DOX@CS@PAMAM.

Figure 2

The FTIR spectra of PAMAM, free DOX, DOX@PAMAM, CS, and DOX@CS@PAMAM.

Figure 3

TEM images of PAMAM (a), DOX@PAMAM (b), CS (c), and DOX@CS@DOX (d). Scale bar: 50 nm.

Figure 4

The DLS of PAMAM (a), DOX@PAMAM (b), CS (c), and DOX@CS@PAMAM (d).

Figure 5

The drug release profiles of DOX@PAMAM in acidic PBS solution (pH = 4.5) (a), DOX@CS@PAMAM in acidic PBS solution (pH = 4.5) (b), DOX@PAMAM in neutral PBS solution (pH = 7.4) (c), DOX@CS@PAMAM in neutral PBS solution (pH = 7.4) (d), DOX@PAMAM in alkaline PBS solution (pH = 9) (e), and DOX@CS@PAMAM in alkaline PBS solution (pH = 9) (f).

Figure 6

The biocompatibility of PAMAM, namely, unconjugated PAMAM (a), CS@PAMAM, namely, CS conjugated PAMAM (b) and the cytotoxicity (c) of DOX@PAMAM in a normal environment (i), DOX@CS@PAMAM in a normal environment (ii), DOX@PAMAM in an acidic environment (iii), DOX@CS@PAMAM in an acidic environment (iv), and free DOX in a normal environment (v) and in an acidic environment (vi).

Figure 7

Fluorescence imaging profile of Skov 3 cells that were incubated with free DOX, DOX@PAMAM, and DOX@CS@PAMAM in an acidic environment and a normal environment.

Figure 8

The representative flow cytometry profile of Skov 3 cells that were incubated with free DOX in an acidic environment (a) and a normal environment (b), DOX@PAMAM in an acidic environment (c) and a normal environment (d), and DOX@CS@PAMAM in an acidic environment (e) and a normal environment (f).