Thymoquinone nanoparticles protect against cisplatin-induced nephrotoxicity in Ehrlich carcinoma model without compromising cisplatin anti-cancer efficacy

2.1 Preparation and characterization of nanoparticles thymoquinone (NP THY)

NP THY was synthesized by double emulsion methods of thymoquinone encapsulted into PLGA and PVA natural polymers and Pluronic 127 non anionic surfactant (Feczkó et al., 2008; Alam et al., 2012). In brief, 60 mg PLGA, 20 mg Pluronic and 30 mg THY were dissolved in 0.1 ml DMSO under stirring for 30 min. The entire solution was then emulsified with 0.7 ml 2% PVA under prob sonication for an additional 90 s. forming the first nano emulsion. Then, the emulsion was sonicated for 60 s with 0.2 ml 1% chitosan forming the second emulsion. NP THY was washed twice with deionized water using centrifugation (14,800×g, 4 °C, 60 min). The precipitate was dispersed in 1 ml of chitosan.

2.2 Characterization of NP THY

The size distribution and zeta potential of NP THY in aqueous dispersions was determined using Dynamic Light Scattering (DLS) and Electrophoretic Light Scattering (ELS) techniques using a Malvern zeta sizer (Malvern Instrumentation Co, Westborough, MA, USA). Ten µl of the prepared NP were resuspended in 1 ml of water. THY concentration was determined spectroscopically by establishing standards calibration curve.

2.3 Encapsulation efficiency (EE) and loading ratio (LR)

The EE of NP THY as determined by analyzing the THY encapsulated in the NP compared to the amount of THY fed initially. After lyophilization, weighed nano-encapsulated powder was dispersed in 3 ml of DMSO for 30 min. The amount of THY in the DMSO was determined spectroscopically by calibration curve. The EE was calculated according to equation (1):

The LR was determined by measuring the amount of encapsulated THY and the weight of whole NP according to equation (2):

$\text{LR}=\frac{\text{Thymoquninone}\text{amount}\text{encapsulated}}{\text{Total}\text{weight}\text{of}\text{formulation}}\times 100$ (2)

2.4 Animals and drugs

Eight weeks adult female Swiss albino mice (n = 20) weighing between 20 and 25 g were reared at the animal house unit, King Fahd Medical Research Centre, King Abdulaziz University (KAU), Jeddah, Saudi Arabia. The animals were treated in a very humane way and all handling of animals were done according to the set rules put forward by the ethical committee at KAU university. The experimental design was approved by the Unit of Biomedical Ethics Research Committee, Faculty of Medicine, King Abdulaziz University (KAU), Jeddah, Saudi Arabia (Refernce No. 331.19). Before the experiment, animals will be acclimatized for 1 week with the new surrounding fed commercial diet and allows access to water ad libitum. CISP of Mylan Institutional LLC, Rockford, IL, USA, was used in this study.

2.5 Induction of Ehrlich solid carcinoma

ESC was induced in the mice using Ehrlich ascites carcinoma (EAC) cells purchased from the National Cancer Institute, Cairo, Egypt. The viability of ESC cells was measured using the trypan blue exclusion assay, and it was found to be 98 percent. On day zero 2.5 × 10⁶ viable EAC cells (0.2 ml PBS/mouse) were injected intramuscularly in the left thigh of each mouse to induce a solid tumour (Aldubayan et al., 2019; Mohamed et al., 2020). The tumor grew in 100% of the mice, with a palpable solid tumor mass within 10 days of implantation. The mice holding the tumour were divided into 3 equal groups (n = 5) besides a group of non-tumours bearing mice served as the control group. The tumour groups were designed as follows: tumour group, tumour + CISP (3.5 mg/kg), and tumour + CISP + NP THY (3 mg/kg). The dose of CISP were according to a study published by (Elkhawaga et al., 2019). CISP was intraperitoneally injected as a single dose while NP THY was orally administered daily for 2 weeks. Both the control and the tumour groups were orally injected daily with PBS.

2.6 Samples collection and storage

Two weeks after CISP, CISP + NP THY, and PBS injection, the mice were ether anesthetized and blood was withdrawn via cardiac puncture, centrifuged at 3,000 rpm to separate the serum. Serum samples were collected and stored at −80 °C. The mice were then sacrificed, and the tumour samples and kidney samples were excised and weighed and were formalin-fixed for histopathology. Parts of the kidney samples were kept frozen at −80 °C for assessment of the markers of oxidative stress, antioxidants, and inflammation.

2.7 Determination of serum kidney function markers

Serum concentrations of urea, uric acid, creatinine, and cystatin C were determined using ELISA kits of MyBioSource (USA) catalogue No. MBS2600001, MBS7606443, MBS749827, and MBS763996, respectively according to the manufacturer's instructions.

2.8 Determination of kidney tissue oxidative stress/antioxidants markers

Kidney tissue concentrations of oxidative stress marker, malondialdehyde (MDA), and the antioxidants markers, reduced glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT) were determined using ELISA kits of MyBioSource (USA) catalogue No. MBS268427, MBS265966, MBS036924, and MBS726781, respectively according to the manufacturer's instructions.

2.9 Determination of kidney inflammatory markers

Kidney tissue concentrations of inflammatory markers, tumour necrosis factor alfa (TNF-α), interleukin-1beta (IL-1β), and nuclear factor kappa beta (NF-kB) were determined using ELISA kits of MyBioSource (USA) catalogue No. MBS2507393, MBS825017, and MBS268833, respectively according to the manufacturer's instructions.

2.10 Histopathological investigation

The preparation of haematoxylin and eosin (H & E) stained sections of the collected tumour and kidney samples was carried out as previously described by (Suvarna et al., 2018). The sections were investigated and photographed by a blind pathologist for the pathological findings.

2.11 Statistical presentation of data

Data was presented as mean ± SD (n = 5). Comparison among groups were carried out using ANOVA followed by Tukey HSD post-hoc test, using GraphPad Prism version 5. The level of significance was settled as p ≤ 0.05.

3.1 Preparation and physicochemical characterization

Results of Z-average particle size and zeta potential were 210.9 nm and +32.8 mV, respectively (Fig. 1).

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Fig. 1

Nanoencapsulation of thymoquinone (THY) into PLGA-PEG nanoparticles.

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3.2 Entrapment efficiency (EE) and drug loading capacity

The results showed that EE was 96% which reflects the success of the preparation method to prevent loss of the active drug. The drug loading ratio was 10.2% measured by HPLC-UV.

3.3 Effect of NP THY on-serum kidney function markers measured during treatment with CISP in Ehrlich solid carcinoma mice model

Treatment of mice bearing Ehrlich solid tumour with CISP significantly increased kidney function markers (urea, uric acid, creatinine, and cystatin C) compared to both the control and Ehrlich tumour groups. The mice treated with NP THY together with CISP showed improvement in kidney function as significantly decreased urea, uric acid, creatinine, and cystatin C was observed compared with the CISP group (Fig. 2). These results showed that adding NP THY to CISP protects the kidney against CISP-induced toxicity.

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Fig. 2

Effect of nanoparticle thymoquinone (NP THY) on serum kidney function markers [A] urea, [B] uric acid, [C] creatinine, and [D] cystatin C measured during treatment with cisplatin (CISP) in Ehrlich solid carcinoma mice model. Results are expressed as mean ± SD (n = 5). ^asignificant difference against the control group. ^bsignificant difference against Ehrlich tumor group. ^csignificant difference against CISP group. ***p ≤ 0.001 and **p ≤ 0.01.

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3.4 Effect of NP THY on-kidney tissue oxidative stress/antioxidant markers measured during treatment with CISP in Ehrlich solid carcinoma mice model

Treatment of mice bearing Ehrlich solid tumour with CISP significantly increased kidney tissue oxidative stress marker (MDA) compared to both the control and Ehrlich tumour groups. The mice treated with NP THY together with CISP showed a significantly decreased MDA concentration compared with the CISP group (Fig. 3A). Treatment of mice bearing Ehrlich solid tumour with CISP significantly reduced kidney tissue antioxidants markers (GSH, SOD, and CAT) compared to both the control and Ehrlich tumour groups. The mice treated with NP THY together with CISP showed significantly increased GSH, SOD, and CAT concentrations compared with the CISP group (Fig. 3B, C, and D). These results indicated that adding NP THY to CISP enhances the antioxidant protection against CISP-induced oxidative stress in the kidney tissue.

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Fig. 3

Effect of nanoparticle thymoquinone (NP THY) on kidney tissue oxidative stress marker [A] MDA and antioxidant markers [B] GSH, [C] SOD, and [D] CAT measured during treatment with cisplatin (CISP) in Ehrlich solid carcinoma mice model. Results are expressed as mean ± SD (n = 5). ^asignificant difference against the control group. ^bsignificant difference against Ehrlich tumour group. ^csignificant difference against CISP group. ***p ≤ 0.001.

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3.5 Effect of NP THY on-kidney tissue inflammatory markers measured during treatment with CISP in Ehrlich solid carcinoma mice model

Treatment of mice bearing Ehrlich solid tumour with CISP significantly increased kidney tissue inflammatory markers (TNF-α, IL-1β, and NF-kB) compared to both the control and Ehrlich tumour groups. The mice treated with NP THY together with CISP showed significantly decreased TNF-α, IL-1β, and NF-kB concentrations compared with the CISP group (Fig. 4). These results indicated that adding NP THY to CISP prevents CISP-induced inflammatory reaction in the kidney tissue.

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Fig. 4

Effect of nanoparticle thymoquinone (NP THY) on kidney tissue inflammatory marker [A] TNF-α, [B] IL-1β, and [C] NFkB measured during treatment with cisplatin (CISP) in Ehrlich solid carcinoma mice model. Results are expressed as mean ± SD (n = 5). ^asignificant difference against the control group. ^bsignificant difference against Ehrlich tumor group. ^csignificant difference against CISP group. ***p ≤ 0.001.

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3.6 Effect of CISP alone and combined with NP THY on-kidney tissues pathology in Ehrlich solid carcinoma mice model

Treatment of mice bearing Ehrlich solid tumour with CISP caused marked disorganization of renal parenchyma with deformity and atrophy of renal corpuscle and glomeruli. CISP also induced marked degenerative changes (swollen cells, unstained cytoplasm, and deformed small nuclei) in the kidney tubule lining epithelium. In the sections of CISP treated mice, the infiltrated neoplastic cells looked unviable and degenerated. The mice treated with NP THY together with CISP showed marked preservation of kidney parenchyma structure with slight deformity of some renal corpuscles. The kidney tubules showed healthy lining epithelium like that of the control sections with slight luminal dilation in the cortical region. The infiltrated neoplastic cells also showed features of degenerative involution. These results showed that adding NP THY to CISP markedly ameliorates CISP-induced kidney histopathological changes in Ehrlich solid carcinoma model in mice (Fig. 5).

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Fig. 5

Effect of cisplatin (CISP) alone and combined with nanoparticle thymoquinone (NP THY) on kidney tissues pathology in Ehrlich solid carcinoma mice model. Sections in mice kidney cortex and medulla show renal corpuscles (white arrows), glomerular capillaries (black star) and kidney tubules (black arrows) stained by H&E. [A and E]: Control sections show normal renal corpuscle and glomerular capillaries (white arrow). Kidney cortical and medullary tubules show also normal narrow lumina and lining epithelium (black arrows). [B and F]: Ehrlich sections show aggregation of infiltrating viable neoplastic cells (white star), mild decrease in renal corpuscle (white arrow) and glomerular size (white arrow) and dilation of tubular lumina (black arrows). [C and G]: CISP sections show marked disorganization of renal parenchyma with deformity and atrophy of renal corpuscle (white arrow) and glomeruli (black star). Kidney tubule lining epithelium (Black arrows) showed marked degenerative hydropic changes (swollen cells, unstained cytoplasm, and deformed small nuclei). Neoplastic cells (white star) near blood vessels (BV) looked unviable and degenerated (dark pyknotic nuclei). [D and H]: CISP + NP THY sections show marked preservation of kidney parenchyma structure against CISP toxicity, slight deformity of some renal corpuscles. Kidney tubules show healthy lining epithelium similar to that of the control sections with slight luminal dilation in the cortical region (black arrows). Neoplastic cells near blood vessels (white star) show also features of degenerative involution.

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3.7 Effect of adding NP THY to CISP on the antitumor activity of CISP in Ehrlich solid carcinoma mice model

Treatment of mice bearing Ehrlich solid tumour with CISP significantly decreased tumour weight compared to Ehrlich tumour group. The mice treated with NP THY together with CISP showed also a significantly decreased tumour weight compared to Ehrlich tumour group. There was no significant difference observed between the tumour weight observed in the mice treated with CISP alone and those treated with CISP combined with NP THY (Fig. 6).

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Fig. 6

Effect of cisplatin (CISP) alone and combined with nanoparticle thymoquinone (NP THY) on Ehrlich solid carcinoma tumour weight. Results are expressed as mean ± SD (n = 5). ^asignificant difference against Ehrlich tumour group. ***p ≤ 0.001.

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These results were confirmed by the histopathological findings observed when examining the H & E-stained tumour sections of the various study groups. The tumor sections show a highly vascular bed and massive proliferating neoplastic cell infiltration with pleomorphic vesicular active nuclei and numerous mitotic figures. CISP treatment-induced decreased both tumor vascularity and neoplastic cell density. In sections of CISP treatment wide regions of tumor necrosis and small degenerated pyknotic nuclei were observed. with NP THY together with CISP produced more decrease in proliferating cell density, wider regions of tumor necrosis, degeneration of neoplastic cells, and fragmented darkly stained nuclei (Fig. 7).

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Fig. 7

Effect of cisplatin (CISP) alone and combined with nanoparticle thymoquinone (NP THY) on Ehrlich solid carcinoma tumour pathology. Sections are H & E-stained low power (A, C, and E x40) and high power (B, D, and F x600). [A and B]: Tumour sections show highly vascular bed. Notice the massive proliferating neoplastic cells infiltrating the muscles which have pleomorphic vesicular active nuclei with numerous mitotic figures (arrows). [C and D]: CISP sections show decreased tumor vascularity (star). Neoplastic cells markedly decreased in density with wide regions of tumor necrosis (pink stained area) and small degenerated pyknotic nuclei (arrows). [E and F]: CISP + NP THY sections show more decrease in proliferating cell density. More wider regions of tumor necrosis, degeneration of neoplastic cells become more evident with fragmented darkly stained nuclei (arrows).

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3.8 Effect of NP THY on body weight trend and percent survival observed during treatment with CISP in Ehrlich solid carcinoma mice model

Treatment of mice bearing Ehrlich solid tumour with CISP alone and combined with NP THY was not observed with any improvement in body weight throughout the experimental period. However, CISP alone and when added with NP THY increased survival percent tumor along the experimental period (Fig. 8).

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Fig. 8

Effect of nanoparticle thymoquinone (NP THY) on [A] body weight trend and [B] percent survival observed during treatment with cisplatin (CISP) in Ehrlich solid carcinoma mice model.

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