Fig 1.
Fluorescence images of estrogen receptor-α (ERα) and erythropoietin receptor (EpoR) in LA7 cells.
A—DAPI stain (blue, arrow); B—ERα (green) (arrow); C—EpoR (red, red and yellow arrows). LA7 cells show moderate intensity of ERα expression in the nucleus (white arrow) while EpoR is mainly located around the nucleus (red arrow) and occasionally in the nucleus (yellow arrow). (200×).
Fig 2.
The concentration-response curve for treated LA7 cells at (A) 24, (B) 48, and (C) 72 h. The LA7 proliferation inhibition induced by TAMNLC and EPO-TAMNLC was greater 48 and 72 h than 24 h (black arrows). Each point is the average of three independent values. EPO = erythropoietin; TAM = tamoxifen; DMSO = dimethyl sulfoxide. EPO-TAMNLC = tamoxifen-loaded erythropoietin-coated nanostructured lipid carrier; TAMNLC = tamoxifen-loaded nanostructured lipid carrier.
Fig 3.
Effect of EPO–TAMNLC on the viability on LA7 as determined by MTT assay with parameters.
(A) Growth inhibition concentration, (B) total growth inhibition concentration, and (C) lethal concentration. Each point represents the mean ± std dev. *significantly difference between both EPO+TAM and TAM (p<0.05). TAM = tamoxifen; EPO = erythropoietin; EPO-TAMNLC = tamoxifen-loaded erythropoietin-coated nanostructured lipid carrier; TAMNLC = tamoxifen-loaded nanostructured lipid carrier. EPO-TAMNLC and TAMNLC caused greater reductions in GI50 and TGI values at 48 and 72 h than either EPO + TAM or TAM treatments.
Fig 4.
Viability of LA7 and MCF-10A cells treated with 20 μM EPO-TAMNLC, TAMNLC and TAM after 72 h.
The viability of treated non-tumorigenic MCF–10A was 10-fold higher than the tumorigenic LA7 cells. Each point represents the mean ± std dev. TAM = tamoxifen, EPO-TAMNLC = tamoxifen-loaded erythropoietin-coated nanostructured lipid carrier; TAMNLC = tamoxifen-loaded nanostructured lipid carrier.
Fig 5.
Flow cytometric analysis of LA7 cells treated with EPO-TAMNLC and TAMNLC at 24 h.
(A) Negative control, (B) NLC (carrier), (C1) 5, (C2) 10, (C3) 20 μM TAMNLC, and (D1) 5, (D2) 10, (D3) 20 μM EPO-TAMNLC. Cells were stained with FITC–conjugated Annexin V and PI. For each box, lower left quadrant = viable cells; lower right = early apoptotic cells; upper right = late apoptotic cells, and upper left = necrotic cells. The apoptotic effects of EPO-TAMNLC and TAMNLC increased with increase in treatment concentrations. More cells were in early than late apoptosis. Values are mean ± std dev. *For each treatment concentration, significantly difference were in comparison with negative control (p<0.05). NLC = nanostructured lipid carrier; EPO-TAMNLC = tamoxifen-loaded erythropoietin-coated NLC; TAMNLC = tamoxifen-loaded NLC.
Fig 6.
Flow cytometric analysis of LA7 cells treated with EPO-TAMNLC and TAMNLC at 48 h.
(A) Negative control, (B) NLC (carrier), (C1) 5, (C2) 10, (C3) 20 μM TAMNLC, and (D1) 5, (D2) 10, (D3) 20 μM EPO-TAMNLC. Cells were stained with FITC-conjugated Annexin V and PI. For each box, lower left quadrant = viable cells; lower right = early apoptotic cells; upper right = late apoptotic cells, and upper left = necrotic cells. The apoptotic effects of EPO-TAMNLC and TAMNLC increased with increase in treatment concentrations. At high treatment concentrations more cells were in late than early apoptosis. Values are mean ± std dev. *For each treatment concentration, significantly differences were in comparison with negative control and NLC treatment (p<0.05). **For each treatment concentration, significantly difference in comparison with EPO–TAMNLC treatment. NLC = nanostructured lipid carrier; EPO-TAMNLC = tamoxifen-loaded erythropoietin-coated NLC; TAMNLC = tamoxifen-loaded NLC.
Fig 7.
Cell cycle profile of treated LA7 at 24 h.
(A) Negative control, (B) NLC (carrier), (C1) 5, (C2) 10, (C3) 20 μM TAMNLC, and (D1) 5, (D2) 10, and (D3) 20 μM EPO-TAMNLC. The population of LA7 cells at G0/G1 phase increased after EPO-TAMNLC and TAMNLC treatment. Values are mean ± std dev. *For each treatment concentration, significantly differences were in comparison with negative control (p<0.05). NLC = nanostructured lipid carrier; EPO-TAMNLC = tamoxifen-loaded erythropoietin-coated NLC; TAMNLC = tamoxifen-loaded NLC.
Fig 8.
Cell cycle profile of treated LA7 at 48 h.
(A) Negative control, (B) NLC (carrier), (C1) 5, (C2) 10, (C3) 20 μM TAMNLC, and (D1) 5, (D2) 10, and (D3) 20 μM EPO-TAMNLC. The LA7 cell population at G0/G1 and sub-G0/G1 phase generally increased after EPO-TAMNLC and TAMNLC treatments. However, at 20 μM, EPO-TAMNLC caused a shift of cell population from G0/G1 to sub-G0/G1 phase suggesting increase in apoptotic effect. Values are mean ± std dev. *For each treatment concentration, significantly differences were in comparison with negative control (p<0.05). NLC = nanostructured lipid carrier; EPO-TAMNLC = tamoxifen-loaded erythropoietin-coated NLC; TAMNLC = tamoxifen-loaded NLC.
Fig 9.
Body weight of rats treated with NLC, TAMNLC and EPO-TAMNLC.
No significant difference (p>0.05) among groups. Values are mean with std dev. error bars.
Table 1.
Hematological parameters in rats treated with tamoxifen–loaded erythropoietin–coated nanostructured lipid carrier, tamoxifen–loaded nanostructured lipid carrier and nanostructured lipid carrier.
Table 2.
Renal function parameters of rats treated with tamoxifen-loaded erythropoietin-coated nanostructured lipid carrier, tamoxifen-loaded nanostructured lipid carrier, and nanostructured lipid carrier.
Table 3.
Liver function parameters of rats treated with nanostructured lipid carrier, tamoxifen–loaded nanostructured lipid carrier and tamoxifen–loaded erythropoietin–coated nanostructured lipid carrier.
Fig 10.
Representative images of (A) bone marrow, (B) kidney, (C) liver, and (D) spleen tissues of rats treated with EPO-TAMNLC and TAMNLC. No abnormality was observed in the tissues (400×).
Fig 11.
LA7 cell-induced mammary gland tumor in the rat.
The tumors are nodular and greyish white in appearance, and well-vascularised which is evident by the thick and engorged blood vessels (yellow arrows).
Fig 12.
Mammary gland tumor size in rats after treatment with nanostructured lipid carrier, tamoxifen-loaded nanostructured lipid carrier and tamoxifen-loaded erythropoietin-coated nanostructured lipid carrier.
EPO-AMNLC and TAMNLC had significantly inhibited growth of rat mammary gland in time–dependent manner. NLC = Nanostructured lipid carrier; EPO–TAMNLC = Tamoxifen–loaded erythropoietin–coated NLC and TAMNLC = Tamoxifen–loaded NLC.
Fig 13.
Mammary gland tumor tissue from treated rats.
(A) tumor control, (B) intravenous 5.0 mg kg-1 BW NLC, (C) oral 2.0 mg/kg BW TAM in corn oil, and intravenous (D) 1.25, (E) 2.5 and (F) 5.0 mg kg-1 BW EPO-TAMNLC and (G) 1.25, (H) 2.5, and (I) 5.0 mg kg-1 BW TAMNLC The tissues show hyperchromatic tumor cells with abnormal mitotic figures (brown arrows), tumor degenerative area with necrotic tissues (yellow arrows), marked infiltration of mononuclear inflammatory cells, particularly lymphocytes, together with severe vascular congestion (white arrows). NLC = nanostructured lipid carrier; EPO-TAMNLC = Tamoxifen-loaded erythropoietin-coated nanostructured lipid carrier; TAMNLC = Tamoxifen-loaded nanostructured lipid carrier.