Preparation of folic acid-conjugated, doxorubicin-loaded, magnetic bovine serum albumin nanospheres and their antitumor effects in vitro and in vivo

Abstract

Background: This study aimed to generate targeted folic acid-conjugated, doxorubicin-loaded, magnetic iron oxide bovine serum albumin nanospheres (FA-DOX-BSA MNPs) that lower the side effects and improve the therapeutic effect of antitumor drugs when combined with hyperthermia and targeting therapy. A new nanodrug using magnetic nanospheres for heating and addition of the folate receptor with cancer cell specificity was prepared. The characteristics of these nanospheres and their antitumor effects in nasopharyngeal carcinoma were explored.

Methods: FA-DOX-BSA MNPs comprising encapsulated magnetic iron oxide nanoparticles were prepared using a desolvation cross-linking method. Activated folic acid (N-hydroxysuccinimide ester of folic acid) was conjugated to the surface of albumin nanospheres via amino groups.

Results: Folic acid was successfully expressed on the surface of the nanospheres. Electron microscopy revealed that the FA-DOX-BSA MNPs were nearly spherical and uniform in size, with an average diameter of 180 nm. The nanomaterial could deliver doxorubicin at clinically relevant doses with an entrapment efficiency of 80%. An increasing temperature test revealed that incorporation of magnetic iron oxide into nanospheres could achieve a satisfactory heat treatment temperature at a significantly lower dose when placed in a high-frequency alternating magnetic field. FA-DOX-BSA MNPs showed greater inhibition of tumors than in the absence of folic acid in vitro and in vivo. Compared with chemotherapy alone, hyperthermia combined with chemotherapy was more effective against tumor cells.

Conclusion: Folic acid-conjugated bovine serum albumin nanospheres composed of mixed doxorubicin and magnetic iron oxide cores can enable controlled and targeted delivery of anticancer drugs and may offer a promising alternative to targeted doxorubicin therapy for nasopharyngeal carcinoma.

Keywords: KB cells; bovine serum albumin; doxorubicin; folic acid.

Figure 1

Morphology of nanoparticles by transmission electron microscopy. Note: The particles were approximately round in shape and their size was about 100 nm in diameter. Fe₃O₄ could be seen in the particles with a hydrodynamic size of 20 nm.

Figure 2

(A) Analysis of nanoparticle size and size distribution by dynamic light scattering. The main size distribution is 150 nm–172 nm. (B) Hydrodynamic size of FA-DOX-BSA MNP by dynamic light scattering in PBS or RPMI 1640 medium supplemented with 10% fetal bovine serum. Abbreviations: FA, folic acid; DOX, doxorubicin; BSA, bovine serum albumin; MNPs, magnetic nanoparticles; PBS, phosphate-buffered saline; RPMI, Roswell Park Memorial Institute.

Figure 3

Heating curve for FA-DOX-BSA MNPs. Note: When exposed to a high-frequency AMF (f =20 kHz; I =20 A), the nanospheres in different iron content rapidly reached the temperature required for heat treatment (41°~44°C) within 30 minutes, which remained stable after 30 minutes. Abbreviations: FA, folic acid; DOX, doxorubicin; BSA, bovine serum albumin; MNPs, magnetic nanoparticles.

Figure 4

Cumulative in vitro profile of doxorubicin release from FA-DOX-BSA MNPs. Note: Doxorubicin released from FA-DOX-BSA MNPs was evaluated using the dynamic dialysis method in different pH phosphate-buffered saline solutions (pH 5.4, 7.4, and 9.0) at 37°C and in phosphate-buffered saline solutions (pH 7.4) at 43°C. Abbreviations: FA, folic acid; DOX, doxorubicin; BSA, bovine serum albumin; MNPs, magnetic nanoparticles.

Figure 5

Ultraviolet spectra of FA-NHS, DOX-BSA MNPs, and FA-DOX-BSA MNPs. Note: The maximum absorption wavelength of FA-NHS appeared between 280 nm and 300 nm (dark gray arrow), which was consistent with the absorption wavelength of FA-DOX-BSA MNPs (light gray arrow). Abbreviations: OD, optical density; FA, folic acid; DOX, doxorubicin; BSA, bovine serum albumin; MNPs, magnetic nanoparticles; NHS, n-hydroxysuccinimide.

Figure 6

Apoptotic cell contents as determined by flow cytometry. Notes: Apoptosis existed in every treatment group. Fluorescence percentage is included in the flow cytometer scatter diagrams. Abbreviations: FA, folic acid; DOX, doxorubicin; BSA, bovine serum albumin; MNPs, magnetic nanoparticles; NPs, nanoparticles; PI, propidium iodide.

Figure 7

Histopathological changes in NPC xenograft tumors (stained with hematoxylin-eosin, 40×). Notes: (A) Negative control group. (B) DOX-BSA NP group. Inflammation can be observed in the tissues. (C) BSA MNP group with heating. A large amount of inflammatory cell infiltration and some spotty necrosis were noted. Brown magnetic materials were deposited in the tissue. (D) DOX-BSA MNP group with heating. Similar to (C), brown magnetic materials, inflammatory cell infiltration, and spotty necrosis are seen in the tissue and cells, albeit to a greater degree. (E) FA-DOX-BSA MNP group with heating. The effect in this group was the most different compared with the other groups. Massive necrosis with marked cell disintegration is evident throughout the image. Abbreviations: T, tumor tissue; N, necrosis; M, materials of Fe₃O₄; FA, folic acid; DOX, doxorubicin; BSA, bovine serum albumin; MNP, magnetic nanoparticles; NP, nanoparticles; NPC, nasopharyngeal carcinoma.