Unveiling the therapeutic potential of cabozantinib-loaded poly D,L-lactic-co-glycolic acid and polysarcosine nanoparticles in inducing apoptosis and cytotoxicity in human HepG2 hepatocellular carcinoma cell lines and in vivo anti-tumor activity in SCID female mice

Abstract

Introduction: The study aimed to develop a nano-based drug delivery system for the treatment of hepatocellular carcinoma (HCC), a type of liver cancer that accounts for 90% of all liver malignancies. The study focused on the use of cabozantinib (CNB), a potent multikinase inhibitor that targets the VEGF receptor 2, as the chemotherapeutic drug. We developed CNB-loaded nanoparticles made from Poly D, L-lactic-co-glycolic acid, and Polysarcosine (CNB-PLGA-PSar-NPs) for use in human HepG2 cell lines.

Methods: By O/W solvent evaporation method, the polymeric nanoparticles were prepared. The various techniques, such as photon correlation spectroscopy, scanning electron microscopy, and transmission electron microscopy were used, to determine the formulation's particle size, zeta potential, and morphology. SYBR Green/ROX qPCR Master Mix and RT-PCR equipment used to measure liver cancer cell line and tissue mRNA expression and MTT assay to test HepG2 cell cytotoxicity. Cell cycle arrest analysis, annexin V assay, and ZE5 Cell Analyzer apoptosis assay were also performed.

Results: The results of the study showed that the particle diameters were 192.0 ± 3.67 nm with 0.128 PDI and -24.18 ± 3.34 mV zeta potential. The antiproliferative and proapoptotic effects of CNB-PLGA-PSar-NPs were evaluated using MTT and flow cytometry (FCM). The IC50 value of CNB-PLGA-PSar-NPs was 45.67 µg/mL, 34.73 µg/mL, and 21.56 µg/mL for 24, 48, and 72 h, respectively. The study also found that 11.20% and 36.77% of CNB-PLGA-PSar-NPs-treated cells were apoptotic at 60 µg/mL and 80 µg/mL, respectively, suggesting that the nanoparticles were effective in inducing apoptosis in the cancer cells. It can also conclude that, CNB-PLGA-PSar-NPs inhibit human HepG2 hepatocellular carcinoma cells and kill them by upregulating the tumour suppressor genes MT1F, MT1X, and downregulating MTTP, APOA4. Further in vivo antitumor activity was well reported in SCID female mice.

Discussion: Overall, this study suggests that the CNB-PLGA-PSar-NPs are a promising drug delivery system for the treatment of HCC, and further research is needed to investigate their potential in clinical treatment.

Keywords: HepG2; annexin V staining; apoptosis analysis; cabozantinib; flow cytometry; polysarcosine.

Figure 1

Physical characterization of the CNB-PLGA-PSar-NPs: (A) Size of the NPs. (B1) Zeta potential of the NPs in water; (B2) Zeta potential of the NPs in Phosphate buffer solution (pH 7.4).(C) Field emission scanning electron microscopy (FE-SEM) images of the NPs. (D) Transmission electron microscopy (TEM) of the NPs (E) Atomic force microscopy image of NPs in 3D (F) Atomic force microscopy image of NPs in 2D.

Figure 2

(A) FTIR data of Cabozantinib (CNB), PLGA, PSar, PLGA-PSar-NPs, CNB-PLGA-PSar-NPs. (B) In vitro release kinetics of CNB from CNB-PLGA-PSar-NPs in PBS at pH 5.0 and 7.4 and data are presented as mean ± SD (n= 3).

Figure 3

Effect of different concentrations of CNB-loaded CNB-PLGA-PSar-NPs and free CNB on the viability of hepatocellular carcinoma cell line (HepG2) cells after 24, 48, and 72 h incubation. The figure shows that CNB-loaded CNB-PLGA-PSar-NPs are more anti-proliferative when compared with CNB (*p < 0.05). The cell viability was determined by MTT assay. These experiments are performed thrice mean ± SD; n= 3, *p < 0.05, **p < 0.01, where CNB group is considered as control.

Figure 4

Carbozanitib (CNB)-induced apoptosis and DNA damage in hepatocellular carcinoma cell line (HepG2) cells as examined by DAPI staining. HepG2 cells exposed to different concentrations of CNB-loaded CNB-PLGA-PSar-NPs and free CNB for 48 h and apoptosis was determined by DAPI staining by fluorescence microscopy. (A) represented the control (untreated), CNB 60 μg/mL, and CNB 80 μg/mL, respectively. (B) represented the control (untreated), CNB-PLGA-PSar-NPs 60 μg/mL, and CNB-PLGA-PSar-NPs 80 μg/mL, respectively. (C) Mean fluorescence intensity of HepG2 cells at 48hr when treated with 60 and 80 μg/mL of CNB. (D) Mean fluorescence intensity of HepG2 cells at 48hr when treated with 60 and 80 μg/mL of CNB-PLGA-PSar-NPs. Data are presented as mean ± SD. The graph represents data from two independent experiments. p<0.05, student t-test.

Figure 5

Flow cytometry was used to analyse cell cycle distribution and DNA content in HepG2 cells treated by CNB-loaded CNB-PLGA-PSar-NPs and free CNB for 48 h (A, B) The cells were stained with PI and the cell cycle stage was defined by DNA content. (C, D) The percentages of cell populations in the G0/G1, S, and G2/M phases was determined by gating during the flow cytometry experiment for control, free CNB and CNB-loaded CNB-PLGA-PSar-NPs at 60 µg/mL & 80 µg/mL concentration. These experiments are performed thrice mean ± SD; n= 3, **p <0.05, where untreated group is considered as control.

Figure 6

Flow cytometry analysis of HepG2 cells treated with different concentrations of CNB loaded CNB-PLGA-PSar-NPs and free CNB for 48 h, followed by staining with Annexin V-FITC and propidium iodide (PI). Results are representative of three independent experiments.

Figure 7

The expression levels of MT1F, MT1X, APOA4, MT1X, MTTP genes in relative to the reference gene; Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in HepG2 cancer cell line treated with the CNB-loaded CNB-PLGA-PSar-NPs and free CNB. *P< 0.05, **P< 0.01 and ***P< 0.001 vs. control was considered significant.

Figure 8

(A). Amount of LDH release after treating with different concentration (10 µg/mL,50 µg/mL,100µg/mL) of Cabozantinib (CNB), PLGA-PSar-encapsulated Cabozantinib nanoparticles (CNB-PLGA-PSar-NPs), and placebo (PLGA-PSar-NPs) nanoparticles against PBS (posative control) Triton X 100(negative control) at 8th hour. (B). Microsopic images of human erytocytic membrane after incorporation of CNB, PLGA-PSar-NPs, CNB-PLGA-PSar-NPs in the presence of posative and negative control.

Figure 9

(A) Tumor regression study in HePG2 cells Xenograft model showing changes in relative tumour volume with time (days) of different groups of SCID (severe combined immunodeficiency disorder) mice (n=6) treated with Cabozantinib (CNB), CNB-PLGA-PSar-NPs. equivalent concentration 20 mg/kg, I.V.) (B) Isolation of tumours after 21 days of studies (C) Comparative in vivo biodistribution of Cabozantinib (CNB), CNB-PLGA-PSar-NPs in rectum, heart, colon, small intestine, stomach, brain endothelium, liver, and kidney after i.v. administration of 20 mg/kg. i.v. bolus dose; values are represented as mean± SD (n=6) (****) highly significance; p<0.00001, (***) moderately significance<0.0001, (**)partial significance; p<0.001, (*) less significance< 0.05, (#) statistically non-significance difference from CNB (p>0.05; all the formulations were compared against controlled group(CNB).

Figure 10

Concentration-time profiles in plasma of Cabozantinib (CNB) after i.v. bolus administration of CNB suspension and CNB loaded polymeric nanoparticles (CNB-PLGA-PSar-NPs) at 20 mg/kg to 6 male albino Wister rats (200–250 g) (n=3). The reverse phase HPLC method was used to figure out how much CNB was in the plasma. Put in an HPLC chromatogram showing the amount of CNB in the plasma. The CNB concentration in plasma was determined using reverse phase HPLC. Incorporate the plasma CNB concentration as an HPLC chromatogram.

Figure 11

Immunohistochemical study for the Hep Par 1 expression by (A) control (PBS buffer treated), (B) free CNB, and (C) CNB-PLGA-PSar-NPs treated tumours. (D) The quantitative analysis of IHC results. Statistical comparisons were made using t-test; * partial significance; p < 0.001; ** moderately significance < 0.000. The treatment was compared with control (PBS buffer treated).