Efficiency of calcium phosphate composite nanoparticles in targeting Ehrlich carcinoma cells transplanted in mice

Abstract

The present study aimed to investigate the mode of action of nano-CaPs in vivo as a therapy for solid tumor in mice. To achieve this goal, Ehrlich Ascites Carcinoma (EAC) was transplanted into 85 Swiss male albino mice. After nine days, the mice were divided into 9 groups. Groups 1 and 2 were allocated as the EAC control. Groups 3 and 4 were injected once intratumorally (IT) by nano-calcium phosphate (nano-CaP). Groups 5 and 6 received once intraperitoneal injection (IP) of nano-CaP. Groups 7, 8, and 9 received nano-CaP (IP) weekly. Blood samples and thigh skeletal muscle were collected after three weeks from groups 1, 3, 5, and 7 and after four weeks from groups 2, 4, 6, and 8. On the other hand, group 9 received nano-CaP (IP) for four weeks and lasted for three months to follow up the recurrence of tumor and to ensure the safety of muscle by histopathological analysis. Tumor growth was monitored twice a week throughout the experiment. DNA fragmentation of tumor cells was evaluated. In thigh tissue, noradrenaline, dopamine, serotonin (5HT), and gamma-aminobutyric acid (GABA) were measured. In serum, 8-Hydroxy-deoxyguanosine (8-OHDG), adenosine triphosphate (ATP), and vascular endothelial growth factor (VEGF) were analyzed. Histopathological and biochemical results showed a significant therapeutic effect of nano-CaP on implanted solid tumor and this effect was more pronounced in the animals treated IP for four weeks. This improvement was evident from the repair of fragmented DNA, the significant decrease of caspase-3, 8-OHDG, myosin, and VEGF, and the significant increase of neurotransmitters (NA, DA, 5HT, and GABA). Additionally, histopathological examination showed complete recovery of cancer cells in the thigh muscle after three months.

Keywords: 5HT, serotonin; 8-OHDG, 8-hydroxy-deoxyguanosine; ATP, adenosine triphosphate; Calcium phosphate (CaP) nanoparticles; DNA, deoxyribonucleic acid; EAC transplantation; EAC, Ehrlich Ascites Carcinoma; FAK, focal adhesion kinase; FTIR, Fourier transform infrared; GABA, gamma aminobutyric acid; IP, intraperitoneal; IT, intratumoral; MAPK, mitogen-activated protein kinase; Nano-CaP, nano calcium phosphate; Nanomedicine; Neurotransmitters; RIR, reference intensity ratio; SEM, scanning electron microscopy; Solid tumor; TEM, transmission electron microscope; VEGFR2, vascular endothelial growth factor receptor 2; XRD, X-ray diffraction.

Fig. 1

XRD patterns of the sample prepared calcined at 1000 °C.

Fig. 2

IR spectra of the sample prepared (a) dried at 100 °C and (b) calcined at 1000 °C.

Fig. 3

SEM of calcined sample prepared at 1000 °C.

Fig. 4

TEM micrograph analysis of synthesized materials with its diffraction pattern.

Fig. 5a

Morphological features of EAC tumor in different groups.

Fig. 5b

EAC tumor size in different groups.

Fig. 6

DNA fragmentation of implanted tumor. Lanes (1 & 2): Once IT after 3 and 4 weeks respectively. Lanes (3 & 4): once IP nano-CaP after 3 weeks. Lanes (5 and 6): once IP nano-CaP after 4 weeks and (7 and 8) weekly IP nano-CaP after 3 weeks. Lanes 9 and 10 weekly IP nano-CaP after 4 weeks and 11 and 12 represent EAC without treatment.

Fig. 7

(a) Serum caspase-3, b) OHDG, c) myosin, d) ATP, and e) VEGF levels in different groups.

Fig. 8

(a) Tissue NA, b) DA, c) serotonin, and d) GABA levels in different groups.

Fig. 9

(a–d) Skeletal muscle of mice bearing EAC, showed compact and aggregation of the tumor tissue cells spread within the muscular tissues. (e–h) Skeletal muscle of mice bearing EAC treated by single dose of nano-CaP after three weeks, showed the presence of necrosis in 20% of implanted cancer cells. (i) Skeletal muscle of solid tumor bearing mice treated weekly with nano-CaP after three weeks. (j) Skeletal muscle solid tumor bearing mice treated weekly with nano-CaP after four weeks. (k) Skeletal muscle of mice bearing solid tumor and injected once intratumorally by nano-CaP after three weeks. (l) Skeletal muscle of mice bearing EAC and injected once intratumorally by nano-CaP after four weeks. (m–o) Skeletal muscle of solid tumor bearing mice to follow-up of the treatment of nano-CaP after three months on cancer cells invasion in thigh skeletal muscle, and p-once injection of nano-CaP in solid tumor bearing mice achieved complete recovery of cancer cells after three months.

Fig. 9

(a–d) Skeletal muscle of mice bearing EAC, showed compact and aggregation of the tumor tissue cells spread within the muscular tissues. (e–h) Skeletal muscle of mice bearing EAC treated by single dose of nano-CaP after three weeks, showed the presence of necrosis in 20% of implanted cancer cells. (i) Skeletal muscle of solid tumor bearing mice treated weekly with nano-CaP after three weeks. (j) Skeletal muscle solid tumor bearing mice treated weekly with nano-CaP after four weeks. (k) Skeletal muscle of mice bearing solid tumor and injected once intratumorally by nano-CaP after three weeks. (l) Skeletal muscle of mice bearing EAC and injected once intratumorally by nano-CaP after four weeks. (m–o) Skeletal muscle of solid tumor bearing mice to follow-up of the treatment of nano-CaP after three months on cancer cells invasion in thigh skeletal muscle, and p-once injection of nano-CaP in solid tumor bearing mice achieved complete recovery of cancer cells after three months.