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. 2025 Mar 7;15(6):415.
doi: 10.3390/nano15060415.

Folic Acid-Conjugated Magnetic Oleoyl-Chitosan Nanoparticles for Controlled Release of Doxorubicin in Cancer Therapy

Banendu Sunder Dash  1 , Yi-Chian Lai  1 , Jyh-Ping Chen  1   2   3   4
Affiliations

Folic Acid-Conjugated Magnetic Oleoyl-Chitosan Nanoparticles for Controlled Release of Doxorubicin in Cancer Therapy

Banendu Sunder Dash et al. Nanomaterials (Basel). .

Abstract

To develop an efficient drug delivery system, we co-entrapped superparamagnetic Fe3O4 and the chemotherapeutic drug doxorubicin (DOX) in oleoyl-chitosan (OC) to prepare DOX-entrapped magnetic OC (DOX-MOC) nanoparticles (NPs) through ionic gelation of OC with sodium tripolyphosphate (TPP). The NPs provide magnetically targeted delivery of DOX in cancer therapy. Using folic acid (FA)-grafted OC, FA-conjugated DOX-entrapped magnetic OC (FA-DOX-MOC) NPs were prepared similarly for FA-mediated active targeting of cancer cells with overexpressed folate receptors. Considering DOX loading and release, the best conditions for preparing DOX-MOC NPs were an OC:TPP mass ratio = 1:4 and OC concentration = 0.2%. These spherical NPs had a particle size of ~250 nm, 87.9% Fe3O4 content, 53.1 emu/g saturation magnetization, 83.1% drug encapsulation efficacy, and 2.81% drug loading efficiency. FA did not significantly change the physico-chemical characteristics of FA-DOX-MOC compared to DOX-MOC, and both NPs showed pH-dependent drug release behaviors, with much faster release of DOX at acidic pH values found in endosomes. However, FA could enhance the intracellular uptake of the NPs and DOX accumulation in the nucleus. This active targeting effect led to significantly higher cytotoxicity towards U87 cancer cells. These results suggest that FA-DOX-MOC NPs can efficiently deliver DOX for controlled drug release in cancer therapy.

Keywords: chitosan; doxorubicin; folic acid; iron oxide; magnetic nanoparticles; oleic acid.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in study design, collection, analyses, or interpretation of the data, manuscript writing, or decision to publish the results.

Figures

Scheme 1
Scheme 1
A schematic representation of the preparation process of doxorubicin (DOX)-loaded folic acid (FA)-conjugated magnetic oleoyl-chitosan (OC) (DOX-FA-MOC) nanoparticles.
Figure 1
Figure 1
The effects of OC:TPP mass ratio on drug release in vitro at pH 5.5 (A) and 7.4 (B). OC concentration = 0.1%.
Figure 2
Figure 2
The effects of OC concentration on drug release in vitro at pH 5.5 (A) and 7.4 (B). OC:TPP mass ratio = 1:4.
Figure 3
Figure 3
The drug release curves at pH 5.5 (A) and 7.4 (B) for FA-DOX-MOC and DOX-MOC NPs.
Figure 4
Figure 4
The transmission electron microscope (TEM) images and particle size distribution of Fe3O4 in MOC, DOX-MOC, and FA-DOX-MOC nanoparticles (bar = 100 nm). The particle size distribution is shown below the TEM image, determined by counting the size of discrete particles for Fe3O4, while it was determined by dynamic light scattering (DLS) for MOC, DOX-MOC, and FA-DOX-MOC NPs. The insert in the TEM image of FA-DOX-MOC is the selected area electron diffraction (SAED) pattern of the dark Fe3O4 nanoparticles.
Figure 5
Figure 5
Fourier-transform infrared (FTIR) spectroscopy (A,B), X-ray diffraction (XRD) (C), and superconducting quantum interference device (SQUID) (D) analyses.
Figure 6
Figure 6
The thermogravimetric analysis (TGA) curves (A) and derivative thermogravimetric (DTG) curves (B,C).
Figure 7
Figure 7
The intracellular uptake of FITC-labeled MOC or FA-MOC by U87 cells was examined by confocal microscopy. The cell nuclei were labeled with DAPI to show blue fluorescence. The green fluorescence is the FITC-labeled MOC or FA-MOC. Bar = 50 μm. The FA-block group used excess free FA to treat U87 cells for one hour before adding FITC-labeled FA-MOC.
Figure 8
Figure 8
The localization of intracellular DOX by confocal microscopy 3 h after contacting U87 cells with DOX-MOC or FA-DOX-MC NPs. The cell nuclei were labeled with DAPI to show blue fluorescence. The red fluorescence is DOX. Bar = 20 μm.
Figure 9
Figure 9
(A) The biocompatibility of MOC NPs and FA-MOC NPs (concentration = 83.3 μg/mL) was determined at different cell culture times. (B) The in vitro cytotoxicity of DOX and FA-DOX-MOC NPs (concentration of DOX = 25 μg/mL) was determined at 24 h cell culture time. α p < 0.05 compared with DOX; β p < 0.05 compared with DOX-MOC. (C) The effect of DOX concentration on the in vitro cytotoxicity of DOX, DOX-MOC, and FA-DOX-MOC was determined at 24 h cell culture time. The dash line is 50% cell viability for calculating IC50.
Figure 10
Figure 10
The Live/Dead staining of U87 cells after cell culture with FA-MOC, DOX (25 μg/mL), FA-DOX-MOC (25 μg/mL DOX). The FA-DOX-MOC (magnetic targeted) group included cell culture with FA-DOX-MOC (25 μg/mL DOX) in the presence of a magnetic field created by placing a magnet at the bottom of the well. Bar = 100 μm. Live cells emit green fluorescence and dead cells emit red fluorescence.
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