3.1. Transmission electron microscopy characterization (TEM)
The average diameter of the tested complex particles was determined to be 29.02 ± 5.02 nm as shown in Fig. 2. The complex particles are present in Nano size i.e., presents in a diameter between 1 and 100 nm in size that exhibit new or enhanced size-dependent properties compared with larger particles of the same material with many advantages such as: Increased bioavailability, dose proportionality, decreased toxicity, smaller dosage form (i.e., smaller tablet), stable dosage forms of drugs which are either unstable or have unacceptably low bioavailability in non-Nano particulate dosage forms, increased active agent surface area results in a faster dissolution of the active agent in an aqueous environment, such as the human body, faster dissolution generally equates with greater bioavailability, smaller drug doses, less toxicity and reduction in fed/fasted variability [31].
3.2. In vitro studies:
3.2.1. Evaluation of the cytotoxic effect of the tested complex on HepG-2 cell line by SRB assay:
Cytotoxicity results indicated that the tested complex NPs had IC50 = 3.87 µg/ml demonstrated potent cytotoxicity against HepG-2 cancer cell line, whereas the standard drug (Vinblastine Sulfate) had IC50 = 3.27 µg/ml.
The microscopic histograms showed the chemotherapeutic activity of the tested complex NPs. There is decreasing in the number of available cells. Most of the remaining observed degeneration changes in the form of irregulatory cell membrane opaque and not well formed chromatin regulated of swalling cytoplasm, other showed optatic change in the formed of chrunked cells and increase eosinophilia cells, and picknitoic nucleus as shown in figures (3–6). The obtained data indicated the surviving fraction ratio against HepG-2 tumor cell line increasing with the decrease of the concentration in the range of the tested concentrations. This can be explained as some metal ions bind to DNA. It seems that, changing the anion and the nature of the metal ion has effect on the biological behavior, due to alter binding ability of DNA binding, so testing of different complexes is very interesting from this point of view. Chemotherapeutic activity of the complex may be attributed to the central metal atom which was explained by Tweedy's chelation theory [32]. Also, the positive charge of the metal increases the acidity of coordinated ligand that bears protons, leading to stronger hydrogen bonds which enhance the biological activity [33]. Moreover, metal has been suggested to facilitate oxidated tissue injury through a free-radical mediated pathway analogous to the Fenton reaction [34].
3.3. In vivo studies:
3.3.1. Biochemical analyses:
The results of the present study [Table 1] recorded that the HCC rats treated with DENA and CCl4 resulted in a significant increase in serum AFP level compared to the control level, indicating the development of HCC in rats. This elevation in AFP was accompanied by the elevation of serum ALT, AST activities and albumin, bilirubin concentrations. The results in rats treated with the tested metal complex were comparable to results in the control rats group in most of the estimated parameters. However, the administration of the tested metal complex to the HCC rats was associated with a significant improvement in all the tested parameters where the treatment succeeded in reducing the elevation level of AFP, ALT, AST and bilirubin in serum and increase in albumin concentration.
Table (1). Statistical analysis (ANOVA) for liver and kidney function tests in the different groups treated with Cu complex NPs
Parameters
|
Control
|
Cu NPs
|
DENA
|
Cu NPs + DENA
|
AFP (ng/ml)
|
0.944 ± 0.048ab
|
0.934 ± 0.036ab
|
9.720 ± 0.303c
|
1.731 ± 0.665d
|
AST (U/l)
|
146.900 ± 11.930ab
|
140.500 ± 6.347ab
|
233.040 ± 39.160c
|
162.140 ± 21.904d
|
ALT (U/l)
|
51.140 ± 5.335abd
|
54.800 ± 4.700abd
|
193.096 ± 24.512c
|
69.574 ± 7.542 abd
|
Alb (g/dl)
|
3.979 ± 0.434ab
|
4.100 ± 0.316ab
|
2.037 ± 0.212c
|
3.768 ± 0.185d
|
ALP (U/l)
|
146.740 ± 21.221ad
|
129.980 ± 16.192b
|
262.980 ± 35.715c
|
141.060 ± 12.262ad
|
T.Bilirubin (mg/dl)
|
0.496 ± 0.111ab
|
0.510 ± 0.104ab
|
2.250 ± 0.415c
|
0.610 ± 0.215d
|
Creatinine (mg/dl)
|
0.508 ± 0.019ad
|
0.555 ± 0.026bc
|
0.568 ± 0.023bc
|
0.490 ± 0.043ad
|
ANOVA: analysis of variance; DEN: diethylnitrosamine; AFP: alpha fetoprotein; AST: aspartate aminotransferase; ALT: alanine aminotransferase; Alb: albumin; ALP: alkaline phosphatase; T.Biliribin: Total bilirubin; SD: standard deviation. Each value is represented as mean ± SD. Data with different superscripts are significantly different at p ≤ 0.05, aSignificance versus control group, bSignificance versus group treated with Cu NPs after 12weeks. cSignificance versus DEN group. dSignificance versus group treated with Cu NPs + DEN after 12 weeks. |
Moreover, the administration of the tested metal complex to HCC rats succeeded in restoring oxidative stress through decreases in MDA level and induced a significant improvement in the antioxidant biomarkers by the observed increase in GSH, TAC and SOD. [Table 2]
Table (2). Statistical analysis (ANOVA) for liver antioxidant levels and oxidative stress marker in the different groups treated with Cu complex NPs
Parameters
|
Control
|
Cu NPs
|
DENA
|
Cu NPs + DENA
|
MDA (nmol/L)
|
4.1940 ± 0.889ab
|
4.072 ± 0.671ab
|
7.972 ± 0.918c
|
5.114 ± 0.558d
|
GSH (U/L)
|
2101.540 ± 74.023a
|
2087.100 ± 59.252b
|
1112.000 ± 61.710c
|
1921.908 ± 66.017d
|
SOD (U/g)
|
866.240 ± 21.242ab
|
867.860 ± 29.046ab
|
407.140 ± 19.996c
|
791.731 ± 20.776d
|
TAC (mmol/L)
|
6.796 ± 0.695a
|
6.292 ± 0.809bd
|
2.596 ± 0.581c
|
6.188 ± 0.664bd
|
ANOVA: analysis of variance; DEN: diethylnitrosamine; MDA: malondialdehyde; GSH: glutathione; SOD: superoxide dismutase; TAC: total antioxidants capacity; SD: standard deviation. Each value is represented as mean ± SD. Data with different superscripts are significantly different at p ≤ 0.05. aSignificance versus control. bSignificance versus group treated with Cu NPs after 12 weeks, cSignificance versus DEN group, dSignificance versus group treated with Cu NPs + DEN after 12 weeks. |
3.3.2. Hematological studies:
Determination of hemoglobin (Hb), red blood corpuscles count (RBCs), total leucocytes count (TLC) and platelets count (PLTs) occurs. The data represented in table (3) indicated that Hb, RBCs and PLTs were significantly decreased in DENA rats than control animals whereas TLCs increased i.e., show leukocytosis. Treatment of DENA-induced group with Cu complex NPs showed a significant increase in Hb, RBCs and platelets levels and make decreasing in leucocytes count i.e., amelioration in hematological parameters was shown in DEN animals subjected to the treatment by both Cu complex NPs toward control animals in comparison to DENA group. It should be noted that, in comparison to the cases treated with a commercial drug, Doxorubicin which induced a significant alterations in hematologic parameters causing leucopenia, erythrocytopenia and thrombocytosis, which were less pronounced or absent in the groups treated Cu and Ag complexes NPs [35].
Table (3). Statistical analysis (ANOVA) for hematological tests in the different groups treated by Cu complex NPs
Parameters
|
Control
|
Cu NPs
|
DEN
|
Cu NPs + DEN
|
Hb (g/dl)
|
15.29 ± 1.547ab
|
15.102 ± 0.87ab
|
12.021 ± 0.588c
|
13.982 ± 1.148d
|
RBCs (X 106/cmm)
|
5.071 ± 0.813ab
|
4.992 ± 0.924abc
|
3.811 ± 0.51c
|
4.764 ± 1.025bc
|
TLC (X 103/cmm)
|
8.210 ± 1.554a
|
9.922 ± 1.716bd
|
12.83 ± 1.241c
|
10.122 ± 0.991bd
|
PLTs (X 103/cmm)
|
522.119 ± 42.712ab
|
509.441 ± 28.461ab
|
188.81 ± 17.792c
|
422.283 ± 31.22d
|
ANOVA: analysis of variance; Hb: Hemoglobin concentration; RBCs: Red blood corpuscles count; T.L.C: Total leucocytic cont; PLT: Platelets count. Each value is represented as mean ± SD. Data with different superscripts are significantly different at p ≤ 0.05, aSignificance versus control group, bSignificance versus group treated with Cu NPs after 12 weeks, cSignificance versus DEN group, fSignificance versus group treated with Cu NPs + DEN after 12 weeks.
3.3.3. Histopathological studies
There was no histopathological alteration, and the normal histological structure of the central vein and surrounding hepatocytes in the parenchyma was observed in control group and in normal healthy rats administrated with the tested complex NPs showing no evidence of side effects on normal liver cells as shown in figure (7) A&B and figure (8) A&B. Fine fibroblastic cell proliferation dividing the degenerated, necrotic, and dysplastic hepatocytes into nodules was seen in liver of DENA animals as shown in Figure (9) A&B. After 12 weeks of Cu complex NPs treatment, DENA rats hepatocytes appeared in normal histological arrangement pattern with very few mononuclear cells infiltrating the hepatic sinosoids with no signs of dysplasia as shown in figures (10) A&B.
3.3.4. Immunohistochemical investigation
The detection of apoptosis by the determination of caspase 3 activity in liver tissue by special immunohistochemical stains takes place. Immunohistochemical expression of CPP32 showed positive nuclear immunostaining (brown staining) in the apoptotic cells of HCC. The results showing that aptoptosis occurs in HCC liver sections by a ratio of 90% after 12 weeks of treatment by Cu complex NPs.