In Vitro Cytotoxicity Evaluation of Highly Absorbent Foam Dressings Based on Silver Zirconium Phosphate via IC 50 Value

In this study, we, for the first time, tried to apply IC50 values (inhibitory concentration estimated to affect the endpoint in question by 50%) in the MTT colorimetric assay to investigate the cytotoxic effects of highly absorbent foam dressings based on silver zirconium phosphate, a newly nanobased matrix. Our results showed that silver released from dressings based on silver zirconium phosphate attributed mainly to highly cytotoxic to L929 cells cultured with MEM containing 10% fetal bovine serum. In addition, we have also compared the IC50 values among different dilutions of AgNO3 solution, silver based dressing extracts and material reference control (ZDEC) extracts using the optimized MTT assay, along with characterizing the silver content in the dressing extracts using atomic absorption spectroscopy. Results have shown that the IC50 values of AgNO3 solution, silver based dressing extracts and ZDEC extracts are 3.5 μg/mL, 3.8 μg/mL and 8.4%, respectively. And there exist some good agreements between qualitative and quantitative evaluation method as well. In conclusion, our study has led to the view that the IC50 value is a promising quantitative index for screening cytotoxicity with regard to silver based dressings.


Introduction
In recent decades, the silver ion is commonly used in functional dressings as topical antimicrobial agents [1]- [3].Though historical use of silver can be looked back to hundred years ago [4], as heavy metal ions, it has been observed that silver released from dressings may possess cytotoxic effects.Such severe side effects, no doubt, have extremely restricted its clinical applications.
To date, the matrix used for silver-bearing, which is of importance to silver release has been well developed, including zeolite silver, SiO 2 silver and the zirconium phosphate silver.Among these, the zirconium phosphate silver has been widely used in functional dressings for its nano-based matrix and good performance.However, cytotoxicity is still the major side effect that should be taken into account in safety control of these dressings [5].In this regard, optimized approaches by which to determine the cytotoxicity of these silver based dressings have generated considerable interest.
In vitro cytotoxic test is an important approach to screen the potential human health hazards in biomaterials and medical devices.To illustrate, several qualitative and quantitative methods are proposed to evaluate the cytotoxic results.Among these, the MTT assay is a sensitive, quantitative, and reproducible testing method which based on the measurement of the viability of cells via metabolic activity [6] [7].Similar to qualitative evaluation method, for materials with potential cytotoxicity, we also need a defined value to evaluate the cytotoxic effects by calculating the quantitative results we obtained.This promoted us to develop a possible value and apply it in the quantitative evaluation of the cytotoxic results.So far, accumulating evidence has shown that IC 50 value, an inhibitory concentration, which reduces the maximum possible viability of tested cells by 50% is a promising value in the cytotoxic assay.Interestingly, IC 50 value is a significantly toxic value which is calculated through concentration dependent inhibition curves using a known computer program [8].In addition, some researchers have used this value to evaluate the cytotoxicity of reference biomaterials [9] [10].
Though the IC 50 value has been well established in cytotoxic test such as V79 colony assay [11], there is little information available on its application in cytotoxicity mediated by medical device yet.In the present study, based on characterizing precisely the silver release by atomic absorption spectroscopy (AAS), we try to compare the cytotoxicity of silver-based dressing extracts, AgNO 3 solution and ZDEC extracts in order to illustrate the relationship between silver release and cytotoxicity of these dressings based on silver zirconium phosphate, and then to investigate the possibility of quantitatively evaluating MTT cytotoxic results with IC 50 value.

Materials
Silver-based foam dressings and corresponding control dressings only without silver was obtained from commercial sources.Silver nitrate (CAS Number, 7761-88-8), MTT stock solutions and isopropanol solutions were purchased from Sigma Company.The ZDEC polyurethanes and high-density polyethylene are purchased from the Food and Drug Safety Center, Hatano Research Institute, Japan.L929 cell lines were acquired from the American Type Culture Collection.Media and fetal calf serum used in cell cultures were purchased from Hangzhou Sijiqing Company, China.

Extracts of Dressing and Control Preparation
Silver-based dressings were aseptically cut into 5 cm × 6 cm size.And then perform the absorbent test compatible with the European reference of Test methods for primary wound dressings EN 13726-1:2002 and the absorbent capacity was calculated by the following formula: absorbent capacity = (W b − W a )/W a , where: W a is the original weight of the dressings; W b is the weight after fully soaked with 0.9% NaCl solution.Then the extract was conducted with MEM culture containing 10% FCS at a ratio of 3 cm 2 /mL under the condition of 72 h at 37˚C with 60 rpm horizontal vibration in accordance with ISO 10993-12:2012.Subsequently, extracts were used for silver content determination and then being diluted into six concentrations for the cytotoxicity assay.Control dressing was prepared in just the same way as above.The ZDEC polyurethanes and high-density polyethylene extracts were prepared with MEM culture containing 10% FCS at a ratio of 0.1 g/mL under the condition of 24 h at 37˚C in accordance with ISO 10993-12:2012, The ZDEC polyurethanes extracts were diluted 20%, 10%, 8%, 6%, 4% and 2% to calculate IC 50 value.

Preparation of AgNO3 Solution
In brief, AgNO 3 powder was weighed and dissolved by demonized water to a final concentration of 1 mg/mL.Solution is sterilized by sterile filtration using syringe filters (pore size ≤ 0.22 μm).After analyzed by AAS for the spike recovery, the primary AgNO 3 solution was then serial diluted two-fold with MEM culture containing 10% FCS starting from 64 μg/mL to 0.5 μg/mL, totally 8 dilutions used for the cytotoxicity assay.

Silver Characterization in Dressing Extract and AgNO3 Solution
Atomic absorption spectroscopy (AAS) is a spectroanalytical method for quantitative determination of silvers in silver-based dressing extracts and AgNO 3 solutions.Briefly, 1 mL extract of dressings were digested by 5 mL HNO 3 and 2 mL H 2 O 2 and dissolve all of the silver present.Then, solutions were diluted with demonized water at 1:10 ratio and were determined by AAS (Thermo Electron Corporation, iCE 3500) under the instruction.Sensitivity of AAS is 50 ppb.The silver content of dressing extract was subsequently determined and expressed in µg/mL.

Cytotoxicity Assay
Briefly, 100 μl cell suspension at 1 × 10 5 cells/mL was prepared and seeded in the designated 96-well plate.After 24 h incubation to form a half-confluent monolayer, aspirate culture medium from the cells, then add 100 μl of treatment medium with the appropriate concentration, including 8 different concentrations of the test extract and AgNO 3 solutions, 100% the positive and negative control extract, vehicle control and blank.In another test, the ZDEC polyurethanes extracts were diluted 20%, 10%, 8%, 6%, 4% and 2% to calculate IC 50 value.After incubating cells for 48 h, carefully observe and record changes in the morphology of the cells due to cytotoxic effects and then remove the culture medium from the plates, and add 50 μl of the MTT solution to each indicate well and the plates are further incubated for 2 h in the incubator at 37˚C.Then the MTT solution is decanted and 100 μl of isopropanol is added into each well.Vibrate the plate for 30 seconds and subsequently transfer it to a microplate reader equipped with a 570 nm filter to read the absorbance.

Statistical Analysis
The results were reported in mean ± SD.The significance of differences between different groups and controls was assessed by the Student's t-test using origin8 software.P < 0.05 was regarded as significant.

Absorbent Capacity of Highly Absorbent Foam Dressings Based on Silver Zirconium Phosphate
To testify the absorbent capacity of dressings based on silver zirconium phosphate, we used the indicated method compatible with the European reference of Test methods for primary wound dressings EN 13726-1:2002 to calculate the absorptive efficiency.Results have revealed that the average absorbent capacity of dressings is 11.3 and the highest absorbency is 0.93 mL/cm 2 , which demonstrated that these dressings had high absorbent performance (Table 1).Also, these results suggest that in the extraction process, the samples should be presoaked with 28 mL of MEM containing 10% FCS to obtain a ratio of 3 cm 2 /mL in the subsequent extract procedure.

Silver Content in the Dressing Extract and AgNO3 Solution
Silver contents of silver-based dressings are of importance to the following cytotoxic study.Therefore, we now used AAS to analyze the silver contents in silver-based dressing extracts and AgNO 3 solution, respectively.Results have revealed that the total silver content in the MEM containing 10% FCS extract was 0.625 mg and the AgNO 3 spike recovery was 112%, as demonstrated in Figure 1.

Cytotoxicity of the Dressing Extract and AgNO3 Solution
The cytotoxicity of dressing extracts and AgNO 3 solution is determined by the MTT method and the cytotoxic

Discussion
During recent years, varieties of silver-based dressings are available on the burn care domain partly because of its broad antibacterial effect [12].Meanwhile, accumulated evidences suggest that silver ions released from these dressings may give rise to cytotoxicity [4].To date, silver release and its cytotoxicity for silver-based dressings contain multiple factors, involving at least silver existential state, silver contents, silver release rate and mode and it is clear that highly absorbent capacity of silver-based dressings may possess obvious silver release and perform cytotoxic effect on monolayer cells [13].In this investigation, we have determined the absorptive capacity of silver-based dressings.As showed in Table 1, the dressing we evaluated has highly absorbent capacity, which has the absorbent capacity of 11.3 and the highest absorbency of the tested sample is 0.93 mL/cm 2 .Surely, this provided the feasibility for the following experiments.
Recent observations indicated that the silver release from the corresponding dressings mainly depended on the test fluid we used [14]- [16] and in particular when the dressings were presoaked with fetal bovine serum, the amount of silver released into culture medium was significantly enhanced [13].In this work, we also analyzed and compared silver release in different vehicles including MEM supplemented with 10% fetal bovine serum, human tissue simulation liquid, physiological saline and 5% glucose.Our results indicated that there are significant differences of silver release in different media, which is at least in part, in agreement with these conclusions (data not shown).Due to the general applicability and their widespread use in evaluating a large range of devices and materials, in vitro cytotoxic tests using MTT assay has been widely adopted since it was developed [17] [18].In this study, we firstly analyze the silver content in the dressing extracts with AAS (see Figure 1) and then try to take an optimized protocol to investigate the cytotoxic potentials of AgNO 3 solution, silver-based dressing extracts and SPU-ZDEC extracts by MTT assay and evaluate the results using IC 50 value, a qualitative index used to evaluate toxic effect of compounds.As showed in Figure 2, the IC 50 values are 3.5 µg/mL, 3.8 µg/mL and 8.4%, respectively.Interestingly, the results of AgNO 3 solution and silver-based dressings show good correlation depending on the silver concentration.Of note, we also compare the results between quantitative and qualitative evaluation methods, as presented in Figure 3 and Table 2, which have shown that these two methods also possessed a good agreement with respect to silver release.

Conclusion
In conclusion, our results demonstrate that there are good correlations between silver release and cytotoxicity of highly absorbent foam dressings based on silver zirconium phosphate, which also indicates good agreements between quantitative evaluation and qualitative morphological evaluation.Importantly, we confirmed that it is possible to apply the IC 50 value in quantitative evaluation of optimized MTT method and it is also a promising method for screening cytotoxicity of silver based dressings.Further studies will focus on elucidating the precise molecular mechanism of silver based dressings mediated cytotoxicity in order to fulfill their safely clinical applications in future.

Figure 1 .
Figure 1.Silver contents were determined by AAS in MEM supplemented with 10% FCS after extracted for 72 h under the condition of 37˚C.Simultaneously, another 1.0 mg AgNO 3 solution was analyzed by AAS to validate the AgNO 3 spike recovery.

Figure 2 .
Figure 2.After silver contents were determined by AAS, toxic effects of AgNO 3 solution,silver based dressing extracts and SPU-ZDEC extracts on L929 cells were measured by MTT assay at indicated concentrations after 24 h and 48 h incubation.(a) The OD value variation of different doses of AgNO 3 solution ranging from 0.5 µg/mL to 64 µg/mL; (b) The OD value variation of different dilutions of silver from the silver based dressing extracts ranging from 0.1 µg/mL to 13 µg/mL; (c) The OD value variation of indicated dilutions of SPU-ZDEC extracts from 20% to 2% dilution of 100% extracts; (d) The IC 50 value of AgNO 3 solution, silver based dressing extracts and SPU-ZDEC extracts calculated by indicatingprogram, respectively.

Figure 3 .
Figure 3.Comparison of cellular sensitivity among indicated concentrations of silver based dressing extracts, AgNO 3 solution, ZDEC extracts, HDPE extracts, dressing control extracts and vehicle controls.L929 cells were tested according to the optimized protocol of ISO 10993-5:2009 with the corresponding test materials using MEM containing 10% FCS and the cellmorphology was taken at48h after incubation (×100).

Table 1 .
Absorbent capacity of high absorbent foam dressings.
potential was expressed as the IC 50 value, an inhibitory concentration, which reduces the maximum possible viability of tested cells by 50%.As showed in Figure2, the IC 50 values of AgNO 3 solution, silver-based-dressing extracts and SPU-ZDEC extracts are 3.5 µg/mL, 3.8 µg/mL and 8.4%, respectively.These results have shown a proper cytotoxic potential of extracts of dressings based on silver zirconium phosphate.Figure3has demonstrated the cell morphology of different groups under a microscope.And also in Table2, qualitative results also illustrated that there were good correlations between quantitative and qualitative evaluation methods of AgNO 3 solution, silver-based dressing extracts and SPU-ZDEC extracts.