Apigenin Loaded Lipoid-PLGA-TPGS Nanoparticles for Colon Cancer Therapy: Characterization, Sustained Release, Cytotoxicity, and Apoptosis Pathways

Abstract

Colon cancer (CC) is one of major causes of mortality and affects the socio-economic status world-wide. Therefore, developing a novel and efficient delivery system is needed for CC management. Thus, in the present study, lipid polymer hybrid nanoparticles of apigenin (LPHyNPs) was prepared and characterized on various parameters such as particle size (234.80 ± 12.28 nm), PDI (0.11 ± 0.04), zeta potential (−5.15 ± 0.70 mV), EE (55.18 ± 3.61%), etc. Additionally, the DSC, XRD, and FT-IR analysis determined drug entrapment and affinity with the selected excipient, demonstrating a promising drug affinity with the lipid polymer. Morphological analysis via SEM and TEM exhibited spherical NPs with a dark color core, which indicated drug entrapment inside the core. In vitro release study showed significant (p < 0.05) sustained release of AGN from LPHyNPs than AGN suspension. Further, the therapeutic efficacy in terms of apoptosis and cell cycle arrest of developed LPHyNPs against CC was estimated by performing flow cytometry and comparing its effectiveness with blank LPHyNPs and AGN suspension, which exhibited remarkable outcomes in favor of LPHyNPs. Moreover, the mechanism behind the anticancer attribute was further explored by estimating gene expression of various signaling molecules such as Bcl-2, BAX, NF-κB, and mTOR that were involved in carcinogenic pathways, which indicated significant (p < 0.05) results for LPHyNPs. Moreover, to strengthen the anticancer potential of LPHyNPs against chemoresistance, the expression of JNK and MDR-1 genes was estimated. Outcomes showed that their expression level reduced appreciably when compared to blank LPHyNPs and AGN suspension. Hence, it can be concluded that developed LPHyNPs could be an efficient therapeutic system for managing CC.

Keywords: apigenin; apoptosis; colon cancer; hybrid nanoparticle; mTOR; sustained release.

Figure 1

(a,b) Demonstrate particle size of blank LPHyNPs and drug-loaded LPHyNPs, and (c,d) show the zeta potential of blank LPHyNPs and drug-loaded LPHyNPs, respectively.

Figure 2

DSC thermogram of (a) AGN, (b) LPHyNP, (c) PLGA, (d) trehalose, (e) SPC-3, and (f) TPGS.

Figure 3

XRD diffractograms of (a) AGN, (b) LPHyNP, (c) PLGA, (d) SPC-3, (e) trehalose, and (f) TPGS.

Figure 4

FT-IR spectra of (a) AGN, (b) LPHyNP, (c) PLGA, (d) SPC-3, and (e) TPGS.

Figure 5

Morphological studies of LPHyNPs (a) SEM image, (b) TEM image, (c) size histogram of TEM.

Figure 6

In vitro release of AGN from LPHyNP and suspension of AGN in PBS containing SLS (1%) as a solubilizer by dialysis bag method.

Figure 7

Showing the outcome of MTT assay for blank LPHyNPs, AGN suspension, and LPHyNPs. Values are presented as mean ± SD where p < 0.001) was represented by ***.

Figure 8

Figure 8 showing the outcome of cell cycle analysis using flow cytometry where effect of different groups such as (A) control, (B) Blank LPHyNPs, (C) apigenin and (D) LPHyNPs were evaluated. Values are presented as mean ± SD where p < 0.05 and p < 0.001 was represented by * and ***. ‘ns’ demonstrated non-significance differences between results.

Figure 9

Showing the pro-apoptotic effect of various drug formulations such as control, Blank LPHyNPs, apigenin and LPHyNPs against colon cancer cell lines using flow cytometry. Values are presented as mean ± SD where p < 0.05 and p < 0.001 was represented by * and ***. ‘ns’ demonstrated non-significance differences between results.

Figure 10

Showing the the outcome of RT-PCR on various formulations such as blank LPHyNPs, AGN suspension, and LPHyNPs. (A) represent mRNA expression of Bcl-2, (B) represent mRNA expression of BAX, (C) represent mRNA expression of NF-kB, (D) represent mRNA expression of mTOR, (E) represent mTNs expression of MDR1 and (F) represent mRNA expression of JNK. Values are presented as mean ± SD where * (p < 0.05: p < 0.01 and p < 0.01) was represented by *, **, and ***. ‘ns’ demonstrated non-significance differences between results.