Unveiling the potential anticancer activity of Spirulina maxima extract-nanoemulsion through in vitro and in vivo studies

Abstract

Being the second leading cause of death globally, cancer has been a long-standing and rapidly evolving focus of biomedical research and practice in the world. Recently, there has been growing interest in cyanobacteria. This focus is particularly evident in developing innovative anticancer treatments to reduce reliance on traditional chemotherapy. This study investigates the anticancer potential of the Spirulina maxima extract nanoemulsion (SMNE) technique to improve the delivery, stability, and solubility of the S. maxima extract (SME). SMNE, prepared in three concentrations (SMNEC1, SMNEC2, SMNEC3), was characterized and confirmed to successfully load SME into silica-coated nanoparticles. Cytotoxicity tests on HepG2 and MCF-7 cell lines revealed a significant reduction in cell viability after 48-hour SMNE treatment, with IC50 values of 1488 µg/mL and 1721.936 µg/mL, respectively. SMNE also demonstrated efficacy in inhibiting tumor growth in mice with Ehrlich ascites carcinoma, normalizing alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, and reducing oxidative stress markers such as catalase (CAT) and malondialdehyde (MDA). Histopathological examination showed that SMNEC3-treated groups had almost normal liver architecture. Additionally, SMNE downregulated oncogenic miR-221-3p and miR-222-3p, activating cancer suppression genes p27 and PTEN. The study concludes that SMNE, with its anti-inflammatory and antioxidant properties and ability to modulate key miRNAs, enhances SME delivery and shows promise as an effective cancer treatment.

Keywords: EAC model; Gallic acid; HepG2 cells; MCF-7 cells; miR-221-3p; miR-222-3p.

Fig. 1

Transmission electron microscopy (TEM) images at two distinct magnifications of the three different concentrations of S. maxima extract nanoemulsion (SMNE): (a,b) represent concentration 1 (SMNEC1), (c,d) represent concentration 2 (SMNEC2), and (e,f) represent concentration 3 (SMNEC3) with respective concentrations of 3225 µg/mL, 6451 µg/mL, and 9677 µg/mL.

Fig. 2

Average particle size of (a) silica nanoemulsion (SNE), and S. maxima extract nanoemulsion (SMNE) with (b) concentration 1 (SMNEC1), (c) concentration 2 (SMNEC2), and (d) concentration 3 (SMNEC3) with respective concentrations of 3225 µg/mL, 6451 µg/mL, and 9677 µg/mL.

Fig. 3

Zeta potential of (a) silica nanoemulsion (SNE), and S. maxima extract nanoemulsion (SMNE) with (b) concentration 1 (SMNEC1), (c) concentration 2 (SMNEC2), and (d) concentration 3 (SMNEC3) with respective concentrations of 3225 µg/mL, 6451 µg/mL, and 9677 µg/mL.

Fig. 4

Effect of (a) S. maxima nanoemulsion (SMNE) and (b) Doxorubicin (DOX) on the viability of HepG2 and MCF-7 cell lines. The dose-response curves for SMNE on (c) HepG2 and (d) MCF-7 cells and for DOX on (e) HepG2 and (f) MCF-7 cells are shown.

Fig. 5

Initial and final body weights (BW) of mice subjected to various treatments. The values are expressed as mean ± standard error of the mean. The percentage values displayed above the gray bars represent the increase in body weight for each group. Normal: Mice without treatment; EAC: Ehrlich ascites-bearing mice without treatment; EAC + DOX: EAC-bearing mice treated with doxorubicin; EAC + SME: EAC-bearing mice with S. maxima extract treatment; EAC + SNE: EAC-bearing mice treated with SNE; EAC + SMNEC1: EAC-bearing mice treated with S. maxima extract nanoemulsion (SMNE) at 3225 µg/mL; EAC + SMNEC2: EAC-bearing mice treated with SMNE at 6451 µg/mL; EAC + SMNEC3: EAC-bearing mice treated with SMNE at 9677 µg/mL.

Fig. 6

Serum levels of (a) ALT and (b) AST in all studied groups. Comparisons among groups were analyzed using one-way ANOVA followed by the least significant difference test. Values are presented as the mean ± standard error of the mean (n = 8). Different letters in the same histogram indicate significant differences (p < 0.05). While the same letters show non-significant differences (p > 0.05). Normal: Mice without treatment; EAC: Ehrlich ascites-bearing mice without treatment; EAC + DOX: EAC-bearing mice treated with doxorubicin; EAC + SME: EAC-bearing mice with S. maxima extract treatment; EAC + SNE: EAC-bearing mice treated with silica nanoemulsion (SNE); EAC + SMNEC1: EAC-bearing mice treated with S. maxima extract nanoemulsion (SMNE) at 3225 µg/mL; EAC + SMNEC2: EAC-bearing mice treated with SMNE at 6451 µg/mL; EAC + SMNEC3: EAC-bearing mice treated with SMNE at 9677 µg/mL.

Fig. 7

Liver tissue content of (a) catalase (CAT) and (b) malondialdehyde (MDA) activity in all studied groups. Comparisons among groups were analyzed using one-way ANOVA followed by a Least Significant Difference test. Values are presented as the mean ± standard error of the mean (n = 8). Different letters in the same histogram indicate significant differences (p < 0.05), whereas the same letters denote insignificance (p > 0.05). Normal: Mice without treatment; EAC: Ehrlich ascites-bearing mice without treatment; EAC + DOX: EAC-bearing mice treated with doxorubicin; EAC + SME: EAC-bearing mice with S. maxima extract treatment; EAC + SNE: EAC-bearing mice treated with silica nanoemulsion (SNE); EAC + SMNEC1: EAC-bearing mice treated with S. maxima extract nanoemulsion (SMNE) at 3225 µg/mL; EAC + SMNEC2: EAC-bearing mice treated with SMNE at 6451 µg/mL; EAC + SMNEC3: EAC-bearing mice treated with SMNE at 9677 µg/mL.

Fig. 8

Relative expression level of miR-221-3p and miR-222-3p in liver tissues of all groups, measured by quantitative polymerase chain reaction (qPCR). Normal: Mice without treatment; EAC: Ehrlich ascites-bearing mice without treatment; EAC + DOX: EAC-bearing mice treated with doxorubicin; EAC + SME: EAC-bearing mice with S. maxima extract treatment; EAC + SNE: EAC-bearing mice treated with silica nanoemulsion (SNE); EAC + SMNEC1: EAC-bearing mice treated with S. maxima extract nanoemulsion (SMNE) at 3225 µg/mL; EAC + SMNEC2: EAC-bearing mice treated with SMNE at 6451 µg/mL; EAC + SMNEC3: EAC-bearing mice treated with SMNE at 9677 µg/mL.

Fig. 9

Photomicrographs of liver sections of mice stained with hematoxylin and eosin. (a) The Normal group exhibits normal hepatic architecture with hepatocytes radiating from the central vein (CV), blood sinusoids (S), and rounded vesicular nuclei (N). (b) The EAC group demonstrates a distorted normal architecture ranging from mild to moderate degree, with degeneration (arrowhead) and multiple patchy areas of inflammatory cell infiltrates, incomplete fibrous band formation, and connective tissue around the central vein (CV) and portal tract. Congestion of the central vein and pyknotic nuclei (P) were also observed. (c) The EAC + DOX group shows histopathological changes similar to the EAC group but with a higher degree of incomplete fibrous band formation and inflammatory cell infiltrates. (d) The EAC + SME group reveals liver architecture ranging from normal to mild degeneration (arrowhead), mild congestion of the central vein (CV), minimal inflammatory cell infiltrates (arrow), normal nuclei (N), and blood sinusoids (S). (e) The EAC + SNE group displays moderate histological and degeneration changes (arrowhead), noticeable congestion of the central vein (CV), inflammatory cell infiltrates (arrow), interstitial haemorrhage (Hg) and pyknotic nuclei (P). (f) The EAC + SMNEC1 group exhibits liver architecture ranging from normal to mild degeneration changes (arrowhead), mild congestion of the central vein (CV), mild interstitial haemorrhage (Hg), normal nuclei (N) and blood sinusoids (S). (g) The EAC + SMNEC2 group shows the nearly normal hepatic structure, with mild degeneration (arrowhead), central vein (CV), blood sinusoids (S), normal nuclei (N), and minimal inflammatory cell infiltrates (arrow). (h) The EAC + SMNEC3 group reveals an almost normal hepatic structure with hepatocytes radiating from the central vein (CV), blood sinusoids (S), normal nuclei (N), and a few numbers of inflammatory cells (arrow). Normal: Mice without treatment; EAC: Ehrlich ascites-bearing mice without treatment; EAC + DOX: EAC-bearing mice treated with doxorubicin; EAC + SME: EAC-bearing mice with S. maxima extract treatment; EAC + SNE: EAC-bearing mice treated with silica nanoemulsion (SNE); EAC + SMNEC1: EAC-bearing mice treated with S. maxima extract nanoemulsion (SMNE) at 3225 µg/mL; EAC + SMNEC2: EAC-bearing mice treated with SMNE at 6451 µg/mL; EAC + SMNEC3: EAC-bearing mice treated with SMNE at 9677 µg/mL.

Fig. 10

Graphical representation for the preparation and characterization of Spirulina maxima nanoemulsion (SMNE) with anticancer evaluation through in vitro (HepG2, MCF-7) and in vivo (biochemical, molecular, and histopathological) studies.

Fig. 11

Timeline of conducting in vivo study.

Fig. 12

Schematic presentation of mice groups of the in vivo study.