Evaluation of Cisplatin Efficacy on HepG2 and E. coli Cells under Acidic Conditions

Background: Cisplatin (Cispt) is a common anticancer drug for the treatment of several malignancies, including hepatocarcinoma. However, this drug suffers from instability in aqueous solutions. The study aimed to evaluate cisplatin efficacy on HepG2 and E. coli cells under an acidic condition. Methods: Acidic Cispt was prepared via incubation in acidic condition (pH=2) for a month duration. The chemical structure of the acidic Cispt was evaluated by using Fourier Transform Infrared Spectroscopy (FTIR) method. The cytotoxicity of the standard and acidic Cispt were then determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and minimum inhibitory concentration (MIC) assays on HepG2 and E. coli cells, respectively. Results: After preparing of acidic Cispt, its chemical structure was determined by FTIR method. In addition, cytotoxicity effects of Cispt in the standard and acidic forms were calculated 58 ± 2.9 and 65 ± 3.25 µM, respectively. MIC results also confirmed the results of MTT assay. MIC results for the standard and acidic Cispt were estimated 9.5 ± 0.47 and 9.8 ± 0.49 µM, respectively. Conclusion: Preparing Cispt in acidic condition not only did not degrade the drug, but also kept the potency of the drug approximately equal to parent drug. Regarding the instability issues of Cispt, keeping Cispt in acidic condition could be a promising approach to preserve its efficacy.


Introduction
prepared in sodium chloride infusion. To prevent potential precipitation, the drug keeps at room temperature. In addition, chemical stability of the drug depends upon pH and sunlight. The preferable pH for maintaining the chemical structure of Cispt ranges between 3.5 and 5.5. Furthermore, the drug is somewhat sensitive to light (Sewell, 2010).
This study aimed to evaluate cisplatin efficacy on HepG2 and E. coli cells under an acidic condition.

Preparation of acidic Cispt
To prepare acidic Cispt, 2 mg of Cispt in powder form (standard Cispt) was dissolved in 1 mL HCl 0.01 N. After one month, solvent phase was evaporated and Acidic Cispt in was obtained again.

Evaluating the acidic Cispt by using FTIR method
For this purpose, the acidic Cispt was dried in room temperature, mixed with bromide potassium and then pressed to prepare the tablet. Next, the tablet was analyzed by FTIR (Nicolet 740SX FTIR spectrophotometer with an MCT-B detector (USA)).

Determination of MIC
MIC is the lowest concentration of an antimicrobial agent capable to halt the growth of a microorganism (Kashani et al., 2012;Hosseini et al., 2016). For this purpose, 100 μL Luria Broth bacteria culture medium was introduced in 96-well plates. Acidic Cispt, standard Cispt and bacterial suspension were then added. Next, the plate was incubated at 37°C for 24 h. The bacterial growth was determined as absorbance raise by Elisa reader (Bio Tek Instruments, VT, USA) at 600 nm. The experiment repeated three times (Panghal et al., 2011;Ferdosian et al. 2015).

In vitro cytotoxicity of the Cispt
The cytotoxicity of the standard and acidic Cispt was determined by MTT assay in HepG2 cell line on 96-well plates. Cells were seeded in dilution rate of 1×104 for each well in DMEM containing 10% Fetal Bovine Serum (FBS), 2 mM glutamine, and antibiotics (60 mg/L penicillin G, 100 mg/L streptomycin, 50 µL/L amphotericin B) (37 o C, 5% CO 2 ). They were allowed to attach for 48 h. The culture media were then removed and cells were treated with the standard and acidic Cispt at the concentrations of 160, 80, 40, 20 and 0 µM. After 24 h incubation, the drug containing culture was removed and 100 µL MTT solutions (0.5 mg/mL, pH 7.4) was added to each well and incubated for 3 h at 37 o C. Later, MTT was discarded and 100 µL isopropanol 100% was added to dissolve the formazan crystals. Next, absorbance was read at 540 nm using spectrophotometric method. Finally, IC50 was measured by statistical package Pharm-PCS software, and cell viability (V) was calculated as in Eq. 1 (Sanganeria et al., 2015).

Statistical analysis
The results of MIC assay were analyzed by using Excel program and are expressed as mean with standard deviation. In addition, the results of MTT assay were analyzed by one-way ANOVA and T-test and the significance level was set at p < 0.05 (Lotfi et al. 2016;Piroozmand et al. 2017). For this purpose, SPSS version 19.00 software (SPSS, Chicago, IL, USA) was used.

Preparation of acidic Cispt
Cispt powder was effortlessly derived from the acidic solution. From the morphological point of view, Cispt kept the color and solidity as parent compound. The difference results between standard and acidic Cispt in MTT assay was not statistically significant (p = 0.15). The results showed that the acidic Cispt preserved its chemical structure. Also, chemical bonds of acidic Cispt were approximately equal to those of standard Cispt.
Results of the study indicated the stability of the drug for 14 days in all storage containers: polypropylene bags (100.1 ± 0.5%), glass bottles (99.7 ± 0.3%), polyethylene bags (98.9 ± 1.0%) and PVC bags (104.6 ± 1.0%). Rochard et al., (1992) prepared the concentration of 0.5 and 0.9 mg/ml of Cispt and located into ambulatory infusion pumps constructed from ethylene-vinyl acetate at 22°C and 35°C for 28 days. The results of the study confirmed the stability of Cispt in the both concentrations.
In the present study, we prepared Cispt in acidic solution at pH 2. Acidic Cispt was provided in the concentration of 2 mg/mL without difficulty. Cispt in acidic solution kept the appearance perfectly. FTIR results also confirmed that the chemical structure of Cispt was remained intact. The efficacy of Cispt was evaluated with MTT and MIC assays. Cytotoxicity results confirmed Cispt kept the efficacy under acidic conditions after one month. MIC results also demonstrated acidic Cispt has potency as compare as parent compound. This study is highlighted with the simplicity of evaluation methods for examine the drug efficacy which is reported for first time. Comparison the efficacy of acidic solution derived Cispt with the typical form of Cispt in sodium chloride infusion could be suggested as future work. Finally, the results of the study indicated that acidic conditions at pH 2 not only did not have detrimental effects on the stability of Cispt but also kept its efficacy approximately compared to parent Cispt.
In conclusion, Cispt is one of the frequently used anticancer drug for various malignancies. However, Cispt instability has considered as a drawback for the clinical use. Enhancing the stability of Cispt could improve the efficacy of the drug. So, as a solution, in this research, Cispt was incubated in acidic conditions. MTT and MIC techniques were used to evaluate the cytotoxicity and bactericidal effects of Cispt, respectively. FTIR method was used to determine the chemical structure of the drug as well. The study showed that keeping Cispt in acidic condition could be regarded for preserving the Cispt potency against degradation and moreover keeping the functionality of the drug for therapeutic purposes.

Compliance with Ethical Standards
This article does not contain any studies with human participants or animals performed by any of the authors.

Conflict of interest
None declared.
Evaluating the acidic Cispt by using FTIR FTIR results showed Cispt preserved its chemical structure (Figure 1). Platinum and ammonium bond was appeared in 463 cm −1 while platinum and chloride was appeared in 338 cm and 362 cm −1 . In addition, tensional trembles of ammonium groups emerged in 1,550 cm −1 , 1,680 cm −1 , 3,300 cm −1 , and 3,500 cm −1 (Shahmabadi et al., 2014). As Figure 1 indicated, chemical bonds of Cispt were preserved and did not changed in acidic Cispt.

Determination of MIC
The lowest concentration inhibitory of the standard and acidic Cispt was evaluated through MIC assay on E. coli strain. The results for the standard and acidic Cispt were measured 9.5 ± 0.47 and 9.8 ± 0.49 µM, respectively (p = 0.59).

In vitro cytotoxicity of the drug
The cell viability of the standard and acidic Cispt on the HepG2 cell line after 24 h incubation and cytotoxicity of the various concentrations of Cispt was investigated via MTT assay on HepG2 cell lines. IC50 of the standard and acidic Cispt were estimated at 58 ± 2.9 and 65 ± 3.25 μM, respectively (p = 0.15).

Discussion
Cispt in water solution that is free of Cl¯ ions becomes quickly decomposed, because of substitution reaction between one or both the chlorine atoms of the drug and water molecule. This consequently results in reduction of the drug concentration by 30-35% just after 4 hours (Allwood et al., 2002). Zieske et al., (1991) study confirmed that Cispt degradation depended upon pH values. The results showed that nearly 0.04% and 0.21% of the drug was decomposed per week at pH 4.3 and 6.3, respectively. In their study, high-performance liquid chromatography technique along with pH measurement were used to determine the stability of Cispt (Zieske et al., 1991). In another study, Hrubigkol et al. evaluated the stability of Cispt solutions in the concentrations of 1.0 and 1.6 mg/ml deposited in plastic infusion bags for two weeks. They found that temperature had no effect on the rate of the drug degradation. However, they observed that Cispt concentration was proportional with the solution temperature (25°C < 1%, 37°C < 3%, 60°C -8.2%) (Hrubiško et al., 1992). In contrast, Mitter et al. study demonstrated that temperature has the significant effect on the Cispt stability (Mittner et al., 1998). In this study, Cispt in the concentration of 0.1 mg/ml was prepared and kept in room temperature and refrigerator for 7 days. The drug loss rate kept in room temperature was significantly higher than the drug stored in refrigerator (84.5 ± 1.5% in room temperature compared to 97.4 ± 0.9% in refrigerator) (Mittner et al., 1998).
Also, It has been demonstrated that the drug did not interact with the packages, infusion bags or other elements used for preparing the final product of Cispt (Cubells et al., 1993). Cubells et al., (1993) evaluated a solution of the drug with the concentration of 0.166 mg/ml, protected it from the light, and stored at room temperature (30°C).