Prodigiosin/celecoxib-loaded into zein/sodium caseinate nanoparticles as a potential therapy for triple negative breast cancer

Abstract

Nowadays, breast cancer is considered one of the most upsetting malignancies among females. Encapsulation of celecoxib (CXB) and prodigiosin (PDG) into zein/sodium caseinate nanoparticles (NPs) produce homogenous and spherical nanoparticles with good encapsulation efficiencies (EE %) and bioavailability. In vitro cytotoxicity study conducted on human breast cancer MDA-MB-231 cell lines revealed that there was a significant decline in the IC50 for encapsulated drugs when compared to each drug alone or their free combination. In addition, results demonstrated that there is a synergism between CXB and PDG as their combination indices were 0.62251 and 0.15493, respectively. Moreover, results of scratch wound healing assay revealed enhanced antimigratory effect of free drugs and fabricated NPs in comparison to untreated cells. Furthermore, In vitro results manifested that formulated nanoparticles exhibited induction of apoptosis associated with reduced angiogenesis, proliferation, and inflammation. In conclusion, nanoencapsulation of multiple drugs into nanoparticles might be a promising approach to develop new therapies for the managing of triple negative breast cancer.

Figure 1

Characterization of the extracted bacterial prodigiosin (PDG). UV–VIS spectrum of PDG (a), different coloration of PDG at different pH values (b), purified PDG on thin layer chromatography (TLC) plate (c), Fourier transform infrared (FTIR) spectrum of PDG (d), high performance liquid chromatography (HPLC) spectrum of purified PDG (e).

Figure 2

Fourier transform infrared (FTIR) spectra of free prodigiosin (PDG), free celecoxib (CXB), sodium caseinate (Na CAS), zein/Na CAS NPs and CXB/PDG-loaded NPs (a), differential scanning calorimetry (DSC) thermogram of free CXB, zein/Na CAS NPs and CXB/PDG-loaded NPs (b).

Figure 3

Particle size of celecoxib/prodigiosin-loaded NPs (a), Zeta potential (b), transmission electron microscope (TEM) micrograph of celecoxib/prodigiosin-loaded NPs; scale bar is 200 nm (c).

Figure 4

High performance liquid chromatography (HPLC) spectrum of encapsulated celecoxib (CXB) (a), prodigiosin (PDG) (b), In vitro release profile of CXB in PBS at pH 7 (c). CXB in PBS; pH 7.4 and 5.4 with 50% ethanol (d), PDG in PBS; pH 7.4 and 5.4 with 50% ethanol (e).

Figure 5

In vitro blood hemolysis of celecoxib/prodigiosin-loaded NPs (CXB/PDG-loaded NPs). Black arrows represent non hemolyzed red blood cells (RBCs) and red arrows represent hemolyzed RBCs; Scale bar is 10 μm (a), % of released hemoglobin (b), an image showing incubated RBCs with different formulation concentrations (c). Results are expressed as mean ± SD.

Figure 6

Cellular viability upon treating cells with celecoxib/prodigiosin-loaded NPs (CXB/PDG-loaded NPs) in comparison to free combination or control groups (a), morphological changes of MDA-MB-231 cell line by treating with 5 μg/mL of free prodigiosin (PDG), free (CXB), free CXB/PDG, zein/Na CAS NPs and CXB/PDG-loaded NPs in comparison with control group (b). Black arrows represent cellular debris. Results are expressed as mean ± SD.

Figure 7

Scratch wound healing assay on MDA-MB-231 cell line at Zero time and after 24h upon incubation with 10 μg of zein/Na CAS NPs, free celecoxib (CXB), free prodigiosin (PDG), free CXB/PDG and CXB/PDG-loaded NPs (a), wound closure % of treated and untreated groups (b). Values are represented as mean ± SD, n = 3. Statistical analyses were done using one-way ANOVA followed by Post Hoc Test (Tukey). (a) P ≤ 0.05 vs control, (b) P ≤ 0.05 vs zein/Na CAS NPs, (c) P ≤ 0.05 vs free CXB, (d) P ≤ 0.05 vs free PDG, (e) P ≤ 0.05 vs free CXB/PDG and (f) P ≤ 0.05 vs CXB/PDG-loaded NPs.

Figure 8

Dot plots of ki-67 antibody analysis of control, zein/Na CAS NPs, and 10 μg of free celecoxib (CXB), free prodigiosin (PDG), free CXB/PDG and CXB/PDG-loaded NPs-treated MDA-MB-231 cell line (a). Results were normalized according to the negative control. Relative change in ki-67 positive cells measured by flow cytometry (b). Values were represented as mean ± SD and n = 3. Statistical analyses were done using one-way ANOVA followed by Post Hoc Test (Tukey). (a) P ≤ 0.05 vs control, (b) P ≤ 0.05 vs zein/Na CAS NPs, (c) P ≤ 0.05 vs free CXB, (d) P ≤ 0.05 vs free PDG, (e) P ≤ 0.05 vs free CXB/PDG and (f) P ≤ 0.05 vs CXB/PDG-loaded NPs.

Figure 9

Prostaglandin E2 (PGE2) in cell culture (a), vascular endothelial growth factor (VEGF) in cell culture (b) and caspase 3 activity in cell lysate in treated and untreated group (c). Data were expressed as mean ± SD and n = 3. Statistical analyses were done using one-way ANOVA followed by Post Hoc Test (Tukey). (a) P ≤ 0.05 vs control, (c) P ≤ 0.05 vs free CXB, (d) P ≤ 0.05 vs free PDG, (e) P ≤ 0.05 vs free CXB/PDG and f P ≤ 0.05 vs CXB/PDG-loaded NPs.