Targeted Delivery of Arctigenin Using Sialic Acid Conjugate-Modified Liposomes for the Treatment of Breast Cancer

Abstract

Arctigenin (ATG) is a broad-spectrum antitumor drug with an excellent inhibitory effect on malignant tumors such as breast cancer, glioblastoma, liver cancer, and colon cancer. However, the clinical application of ATG is limited by its poor water solubility and quick hydrolysis in the liver, intestine, and plasma, which might hinder its application. Sialic acid (SA) recognizes selectin receptors overexpressed on the surface of tumor-associated macrophages. In this study, SA was conjugated with octadecylamine (ODA) to prepare SA-ODA, which was employed to prepare SA functionalized nanoliposomes (SA-Lip) to achieve breast cancer targeting. The formulations were finely optimized using the Box-Behnken design to achieve higher ATG loading. The size, ζ potential, entrapment efficiency, drug loading, and release behavior of ATG@SA-Lip were fully investigated in comparison with conventional ATG@Lip. The ATG@SA-Lip displayed more potent cytotoxicity and higher cellular internalization compared to ATG@Sol and ATG@Lip in both MCF7 and 4T1 cells. Notably, ATG@SA-Lip showed the lowest impact on the immune system. Our study demonstrates that SA-Lip has strong potential as a delivery system for the targeted delivery of ATG.

Keywords: antitumor in vivo; arctigenin; nanoliposomes; sialic acid receptor; targeted delivery.

Figure 1

Chemical structure of arctigenin.

Figure 2

Effect of formulation and process parameters on EE, DL, and Size: (a) drug-to-lipid mass ratio; (b) lipid-to-cholesterol mass ratio; (c) volume of aqueous phase; (d) hydration time; (e) ultrasound time; (f) hydration temperature.

Figure 3

Response surface plots for effects of drug-to-lipid mass ratio (X₁), volume of aqueous phase (X₂), and ultrasound time (X₃) on the score: (a,d) interaction between X₁ and X₂; (b,e) interaction between X₁ and X₃; (c,f) interaction between X₂ and X₃.

Figure 4

Characterization of (a,b) ATG@Lip and (d,e) ATG@SA-Lip. (a,d) Appearance under TEM (scale bar = 100 nm); (b,e) particle size by DLS; Cumulative release of ATG formulations at (c) pH 5.2 and (f) 7.4. Dates are represented as mean ± (SD) (n = 3).

Figure 5

Biocompatibility of nanoliposomes in vitro: (a) hemolytic effect of ATG@Lip and ATG@SA-Lip; (b) cytotoxicity of SA-Lip.

Figure 6

Cellular uptake of fluorescent probes: (a) cellular uptake of C-6 labeled formulations on 4T1 cells observed by fluorescence microscopy; (b) fluorescence in cell distribution detected by flow cytometry; (c) fluorescence intensity calculated with FlowJo Soft 7.6.1 (ns p > 0.05, *** p < 0.001 compared with free C6).

Figure 7

Cytotoxicity of ATG formulations: (a–c) viability of MCF-7, 4T1and LO2 cells analyzed by CCK-8 assay; (d) IC₅₀ of ATG formulations for indicated cell lines; (e,f) colony formation assay of 4T1 cells; (g) viability of 4T1 cells treated with formulations of 25 μg/mL ATG (ns p > 0.05, * p < 0.05, *** p < 0.001 compared with control).

Figure 8

Anticancer activity and toxicity of ATG formulations: (a) tumor volume; (b) weight of tumors (*** p < 0.001); (c) tumor suppression rate; (d) images of xenograft tumors obtained from mice with different treatments after 14 days; (e) body weight of murine model; (f) H&E and TUNEL analysis of tumors obtained from sacrificed mice at end of study (*** p < 0.001 compared with NC or ATG@Sol).

Figure 9

Immune organ index of mice after 13 days of administration: (a) spleen index; (b) thymus index. (*** p < 0.001 compared with normal group, # p < 0.05, ## p < 0.01, ### p < 0.001 compared with NC group).