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. 2021 Mar 3:16:1775-1787.
doi: 10.2147/IJN.S287806. eCollection 2021.

A Carrier-Free Folate Receptor-Targeted Ursolic Acid/Methotrexate Nanodelivery System for Synergetic Anticancer Therapy

Jin-Shuai Lan #  1   2 , Yan-Hong Qin #  2 , Li Liu  2 , Rui-Feng Zeng  2 , Yang Yang  3 , Kai Wang  3 , Yue Ding  1   2 , Tong Zhang  1   2 , Rodney J Y Ho  4
Affiliations

A Carrier-Free Folate Receptor-Targeted Ursolic Acid/Methotrexate Nanodelivery System for Synergetic Anticancer Therapy

Jin-Shuai Lan et al. Int J Nanomedicine. .

Abstract

Purpose: To avoid undefined metabolic mechanisms and to eliminate potential side effects of traditional nanocarriers, new green carriers are urgently needed in cancer treatment. Carrier-free nanoparticles (NPs) based on ursolic acid (UA) have attracted significant attention, but the UA NPs targeting the folate receptor have never been explored. We designed a novel self-assembled UA-Methotrexate (MTX) NPs targeting the folate-receptor and its synergetic anticancer activity was studied in vitro and in vivo.

Methods: UA-MTX NPs were prepared using the solvent precipitation method. Characterization of the UA-MTX NPs preparation was performed using a size analyzer, transmission electron microscopy, and UV-vis spectrophotometry. The in vitro pH-responsive drug release capability of UA-MTX NPs was tested at different pH values. The UA-MTX NPs targeting of folates was determined by comparing the endocytosis rates of cell lines with low or overexpression of the folate receptor (A549 and MCF-7 cells). The cytotoxicity and cell apoptosis of UA-MTX NPs were also studied to determine the in vitro synergistic effects. Combination chemotherapy of UA-MTX NPs in vivo was evaluated using MCF-7 xenografted tumor models.

Results: Compared with free UA or MTX, the water solubility of UA-MTX NPs improved significantly. Drug-release from the UA-MTX NPs was faster at pH 5.0 than pH 7.4, suggesting MTX-UA NPs could rapidly release MTX in the acidic conditions of the tumor microenvironment. Confocal laser scanning microscopy revealed the excellent folate receptor targeting of UA-MTX NPs in MCF-7 cells. Cytotoxicity and cell apoptosis results demonstrated greater antiproliferative capacity of UA-MTX NPs than that of free drug in folate receptor overexpressing MCF-7 cells. Anticancer effects in vivo suggested MTX-UA NPs exhibited good biological safety and could enhance antitumor efficacy due to the combination therapy.

Conclusion: Our findings indicate that the UA-MTX NPs targeting folate-receptors is an efficient strategy for combination chemotherapy.

Keywords: anticancer; carrier free; methotrexate; targeted drug delivery; ursolic acid.

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Conflict of interest statement

The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
Schematic representation of the carrier-free nanoparticles (NPs) via co-assembly between UA and MTX.
Figure 2
Figure 2
Characterization of the MTX-UA NPs.(A) Diameter. (B) Zeta potential. (C) Tyndall effect. (D) TEM analysis at 200 nm. (E) TEM analysis at 100 nm. (F) UV absorption spectra of MTX, UA and MTX-UA NPs. (G) UV absorption of MTX-UA NPs in NaCl solution. (H) UV absorption spectra of MTX-UA NPs in SDS solution. (I) Fluorescence spectra of free R6G, UA-R6G NPs and MTX-UA-R6G NPs.
Figure 3
Figure 3
Stability and drug release of MTX-UA NPs. (A) Size of MTX-UA NPs in H2O, PBS solution and DMEM medium containing 10% FBS for 7 days. (B) Zeta potential of MTX-UA NPs in H2O, PBS solution and DMEM medium containing 10% FBS for 7 days. (C) Size of MTX-UA NPs in the solution at various pH values. (D) Cumulative drug release profiles of UA from MTX-UA NPs under different pH conditions. (E) Cumulative drug release profiles of MTX from MTX-UA NPs under different pH conditions. (F) Photo of pH response. ***p < 0.001.
Figure 4
Figure 4
In vitro experiments of cells uptake. (A) CLSM images of MCF-7 cells incubated with MTX-UA-R6G NPs and UA-R6G NPs following 1 h, 2 h, 3 h and 4 h. (B) CLSM images of A549 cells incubated with MTX-UA-R6G NPs and UA-R6G NPs following 1 h, 2 h, 3 h and 4 h. *** p < 0.001.
Figure 5
Figure 5
The synergistic effect of UA and MTX, and the cytotoxicity in vitro. The cell viability of (A) A549 cells and (B) MCF-7 cellls cultivated with UA, UA NPs, MTX/UA, MTX-UA NPs for 24 h in vitro. (C) IC50 value of UA, UA NPs, MTX/UA, MTX-UA NPs to MCF-7 cells and A549 cells (calculated by concentration of UA). (D) Cell viability (%) of UA, MTX and various fixed molar ratios of two drugs. (E) Combination index of the synergistic effects at various fixed molar ratio of UA and MTX. (F) and (G) Apoptosis rate of UA, UA NPs, MTX/UA and MTX-UA NPs against MCF-7 cell. *p < 0.05, ***p < 0.001.
Figure 6
Figure 6
In vivo anti-tumor effects of MTX-UA NPs on nude mice bearing MCF-7 tumors. (A) Body weight changes of mice. (B) Tumor growth curve. (C) Mean weight of tumors. (D) Images of the excised MCF-7 tumor tissues at 20 days post-implantation. (E) Representative histology of MCF-7 tumor tissue and other organs after the administration of 10% Tween 80, UA/MTX mixture and MTX/UA NPs. Nuclei were stained blue, whereas the extracellular matrix and cytoplasm were stained red in the H&E analysis. The scale bar is 100 μm. *p < 0.05, **p < 0.01, ***p < 0.001.
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