Potential of the Bioinspired CaCO3 Microspheres Loaded with

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Article Cite This: Chem. Res. Toxicol. 2018, 31, 629−636

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Potential of the Bioinspired CaCO3 Microspheres Loaded with Tetracycline in Inducing Differential Cytotoxic Effects toward Noncancerous and Cancer Cells: A Cytogenetic Toxicity Assessment Using CHO Cells in Vitro Kalpana Javvaji,†,∥ Gousia Begum,‡,∥ Shruti S. Deshpande,† Rohit K. Rana,*,‡ and Sunil Misra*,† Applied Biology Division, Genetic Toxicology Lab and ‡Nanomaterials Laboratory, Inorganic and Physical Chemistry Division, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500 007, India

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S Supporting Information *

ABSTRACT: Calcium carbonate (CaCO3)-based materials as feasible pH-sensitive drug carriers, which can actively dissolve in an acidic microenvironment of cancer cells, are finding increasing importance. This has drawn our interest in the development of a bioinspired polypeptide- mediated method to design calcium carbonate microspheres loaded with tetracycline (CaCO3-TC) with an aim to explore its safe application in cancer therapeutics. Its therapeutic application in cancer patients essentially demands its safety information on the normal cells. Herein our study presents the in vitro genetic toxicological information on CaCO3-TC using noncancerous mammalian CHO cells in comparison to bare TC at three different concentrations (100, 200, and 300 μM) selected based on the cytotoxicity data (MTT). Assessment of various end points like chromosome aberrations, micronucleus, mitotic index and effects on cell cycle distribution after 24 h post-treatment demonstrates a significant reduction in clastogenic (P < 0.001), aneugenic potential (P < 0.05), and nonmitotoxic nature of CaCO3-TC than that of bare TC. Noticeably, as inferred from the FACS analysis on cancer cells, G2/M phase accumulation in breast cancer cells (MDA-MB-231), and at G1 phase in cervical cancer cells (HeLa) reveal its potential anticancer property. On the other hand, the genotoxicity studies illustrate protective effects of CaCO3-TC on noncancerous cells. While the pH-dependent dissolution property of the CaCO3 matrix encasing tetracycline results in higher toxicity on cancer cells, the near neutral pH in the case of normal cells prevents complete dissolution of CaCO3 thereby not allowing the encapsulated TC to adequately interact with the cells. Therefore, thus assembled CaCO3 spheres not only provide a way for facile encapsulation of tetracycline under mild conditions but also result in an effective matrix for differential toxicity toward normal and cancer cells justifying its clinical development as a novel targetspecific drug in therapeutic applications for metastatic cancers.

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INTRODUCTION Metastasis is considered as the major cause of death from cancer1 because it involves a series of steps which follows even after surgical resection in cancer patients, where few tumor cells from primary site successfully migrate and colonize in distant organs or tissues for secondary growth.2,3 Understanding the metastasis uncovers various factors involving interactions between metastatic tumor cells and host factors like MMP’s signaling,4 low oxygen levels, and acidic pH which support the progression and survival of cancer cells in a given tumor microenvironment.5 Tetracycline (TC) came into light, a while ago as a potential antimetastatic drug6,7 which acts by effectively inhibiting matrix metalloproteinases (MMPs) that promote cell differentiation, proliferation, and metastasis/migration of cancer cells (adhesion and dispersion) in tumor microenvironments (TME).8,9 In this regard, extensive studies on TC and many © 2018 American Chemical Society

chemically modified TC’s were done and reported to be potently/effectively antimetastatic in cancer progression.10 However, an accomplishment of a successful formulation depends not only on identifying a potential drug in the current cancer research but also on an efficient drug carrier which can act according to environmental differences associated with cancer cells and normal cells, by necessarily avoiding unwanted DNA damages in normal cells. This selective delivery into intracellular systems results in an essential bioavailability required for improved therapeutic action at the cancer site.11Acidic pH is one other feasible factor on which drug carriers are designed in current cancer research because cancer cells maintain low pH (4.5−5.5) which aggravates metastasis in TME.12,13 Therefore, many of the in vitro and in vivo studies Received: May 22, 2018 Published: June 20, 2018 629

DOI: 10.1021/acs.chemrestox.8b00131 Chem. Res. Toxicol. 2018, 31, 629−636

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Chemical Research in Toxicology

spectroscopic analysis was carried on Varian Cary 5000 spectrophotometer. In Vitro Cytotoxicity and Genotoxicity Studies. Cytotoxicity Assay (MTT). Noncancerous mammalian cells, CHO (Chinese hamster ovary cells) were collected from Centre for Cellular and Molecular Biology (CSIR-CCMB), Hyderabad, India. Cancer cell lines MDA MB 231-Breast cancer (ATCC HTB26) and HeLa cervical cancer (ATCC CCL2) were obtained from ATCC (Bethesda, MD, USA) and maintained in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a 5% CO2 incubator. Cultured cells were loaded into 96well culture plate and allowed to attach properly. Test compounds of different concentrations ranging from 1 to 50 μM were added in triplicates and incubated for 24 h. Then, 3-(4,5-dimethyl-2-thiazolyl)2,5-diphenyl-2H-tetrazolium bromide (MTT) (0.5 mg/mL) was added and incubated for 3 h. To dissolve the insoluble formazan crystals, 100 μL DMSO was added to each well. Finally, the absorbance of the plates was measured using a Synergy H1MultiMode Plate Reader, USA, and IC50 values were determined. Cell Cycle Analysis. The in vitro cell cycle analysis was performed on CHO, MDA-MB-231, and HeLa as per our earlier published work.19 Three different concentrations of each test chemicals (CaCO3-TC and TC) were exposed to cell lines (CHO: 100, 200, and 300 μM; MDA-MB 231: 25, 50, and 75 μM; HeLa: 50, 75, and 100 μM) to understand the cell distribution at different phases of cell cycle. These concentrations were selected based on the cytotoxicity data (MTT) on noncancerous and cancer cells, respectively. To describe briefly, the treated cells were harvested and washed twice, centrifuged at 2000 rpm for 5 min, and fixed overnight in 70% ethanol at −20 °C. Then cells were washed thoroughly, centrifuged with 1× PBS and stained with propidium iodide (PI) solution supplemented with RNase and Triton-X for 45 min at 37 °C. After staining, the cells were washed with 1× PBS to remove the excess unbound PI and subjected for cell cycle analysis using BD FACS Canto, USA. Genotoxicity Assays. Genotoxicity effects of CaCO3-TC and TC on CHO were assessed using chromosome aberration (CA) test, mitotic index (MI), and micronucleus (MN) analysis according to our earlier published work.19 Dimethyl sulfoxide (DMSO) and mitomycin C were taken as negative and positive controls, respectively, for comparison. Chromosome Aberration Test. DNA damaging ability of CaCO3TC and TC in CHO cells was assessed by performing CA test. CHO cells were cultured in DMEM medium, treated with three test concentrations of TC (100, 200, and 300 μM) and CaCO3-TC (100, 200, and 300 μM) separately, and incubated for 24 h. Prior to harvesting, colchicine (0.02%) was added and incubated for 40−45 min to arrest the cells at metaphase. Then cells were harvested followed by hypotonic treatment for 20−25 min at 37 °C. The cell pellet was collected and fixed in 3:1(v/v) methanol/acetic acid. The same procedure was repeated twice, and chromosome slides were prepared using flame drying method. At least 100 well-spread metaphase plates in triplicates were screened for the calculation of structural damages like chromatid and chromosome types of aberrations. Mitotic Index. Mitotic index analysis was done to assess the effects of CaCO3-TC and TC on the rate of cell division by calculating the percentage of mitotically dividing cells. The same procedure performed in the CA test was followed in preparing the slides for MI. For this study, at least 2000 cells in triplicates, per each concentration of CaCO3-TC and TC, were analyzed to calculate the number of dividing cells. Micronucleus Analysis. Cells were cultured and treated with different concentrations of CaCO3 -TC and TC along with cytochalasin-B to observe the MN in the binucleated cells. The cells were incubated for 24 h and then harvested, centrifuged, and hypotonically treated with sodium citrate (0.9%) for 10 min. Cells were smeared onto clean wiped slides and stained with Giemsa. At least 2000 cells were examined in triplicates to assess the percentage of MN for each concentration.

focusing on drug carriers that can dissolve actively at low pH and neutralize the acidic cancer microenvironment to alkaline have emphasized CaCO3 nanoparticles as efficient drug carriers that can cease further proliferation of cancer cells.14,15 Much of the scientific evidence now makes it clear about the antimetastatic properties of tetracycline and the role of CaCO3 nanoparticles in increasing the bioavailability of TC in TME both in in vitro and in vivo models.6,15 Recently, we have also reported on the synthesis of TC loaded CaCO3 microspheres which showed excellent in vitro drug release along with its possible use in optical imaging and antibacterial properties.16 The cell viability test revealed a remarkable decrease in percentage of dead cells on treatment with CaCO3TC than TC tested in CHO cells. The results suggested that CaCO3 matrix loaded with TC has reduced the toxicity levels significantly with an IC50 value = 241.21 μM compared to bare TC (IC50 = 140.87 μM). The cytotoxicity efficacy of CaCO3TC and TC on different cancer cell lines showed CaCO3-TC to be potently cytotoxic on MDA-MB 231 (103.36 μM) and HeLa (130.79 μM) than the bare TC in MDA-MB 231 (313.6 μM) and HeLa (198.4 μM). The excellent antimetastastic properties and remarkable selective cytotoxicity of TC observed on various cancer cells motivated us to further investigate the DNA damaging efficacies of these CaCO3-TC in normal mammalian cells. Particularly, the severe normal cell toxicity of most of the anticancer drugs has been a limiting factor, and in addition it may lead to recurrence of secondary cancers.17,18 Herein we report detailed cytogenetic toxicity investigation carried out using CHO cells in vitro. This further insight on DNA damages on normal cells presents important toxicological information essential for future developmental process of CaCO3-TC as a novel target-specific drug formulation in the application of metastatic cancers.

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EXPERIMENTAL SECTION

Materials. Penta(L-lysine hydrobromide) (penta(L-lys), 1−5 kDa) (25988-63-0), poly(α,β)-DL-aspartic acid sodium salt (poly(L-asp), 2− 11 kDa) (94525-01-6), tetracycline (64-75-5), calcium chloride (CaCl2) (10043-52-4), penicillin (69-57-8), streptomycin (3810-740), MTT (298-93-1), trypsin (9002-07-7), L-glutamine (56-85-9), absolute ethanol (64-17-5), methanol (67-56-1), and bromothymol blue (76-59-5) were procured from Sigma-Aldrich. Ammonium carbonate (NH4)2CO3 (506-87-6) was obtained from Qualigen Fine Chemicals, India. Glacial acetic acid (64-19-7), mitomycin C (13614-98-7), and colchicine (64-86-8) were obtained from Hi media, India. Triton X (9002-93-1) was procured from Genetixbiotech, India. All the solutions were prepared using deionized water (18.2 MΩ, Millipore water purification system). Synthesis of Tetracycline Loaded CaCO3 Microspheres (CaCO3-TC). The synthesis of TC loaded CaCO3 microstructures was performed as reported earlier.16 Typically, 200 μL of poly(L-asp) (2 mg/mL) was added to 200 μL of penta(L-lys) (2 mg/mL) resulting in a turbid solution. To this mixture 250 μL of TC solution in 0.01 M HCl was added 250 μL of CaCl2 (0.05 M) and then 250 μL of (NH4)2CO3 (0.05 M). This mixture was then aged for 30 min at room temperature. The precipitate was centrifuged and washed 3 times with deionized water and dried at room temperature. The TC loading efficiency in the CaCO3 microspheres was determined from UV−vis spectroscopy. Material Characterizations. Field emission scanning electron microscopic (FE-SEM) analyses of the prepared materials were performed by using JEOL-7610F instrument. Dynamic light scattering (DLS) measurements to determine the particle size was done using a Malvern Zetasizer (Nano-ZS) instrument equipped with the detector at 173° angle and with a 668 nm laser source. The UV−vis 630

DOI: 10.1021/acs.chemrestox.8b00131 Chem. Res. Toxicol. 2018, 31, 629−636

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Figure 1. (a, b) FE-SEM images of CaCO3-TC at different magnifications. (c) Size distribution of the particles in colloidal form obtained by DLS measurement. (d) EDS elemental mapping of CaCO3-TC. Statistical Analysis. All the data were analyzed with Student’s t test, followed by “One-way ANOVA” with Dunnett’s multiple comparison test using Graph Pad Prism version 6.04 for Windows, Graph Pad Software, San Diego, CA.

TC. As shown previously, this bioinspired mineralization results in both vaterite and amorphous phase of CaCO3 confirmed by X-ray diffraction (XRD) and Fourier transform infrared spectroscopic (FT-IR) studies.16 From UV−vis spectroscopy the TC loading in the microstructures was found to be 55.5 wt % (Figure S1a and b). Thus, prepared CaCO 3 -TC was further utilized to investigate for their in vitro cytogenetic toxic effects on CHO cells. The cytotoxic potency was confirmed using an enzymebased method, MTT assay, which relies on the mitochondrial dehydrogenase activity of viable cells. The assay is based on the reduction of MTT (water-soluble yellow colored tetrazolium dye) to purple colored formazan crystals, which are then analyzed spectrophotometrically after dissolution in DMSO.20 A significant increase in the cytotoxic potency of CaCO3-TC on HeLa and MDA-MB 231 and decreased efficacy on CHO cells than bare TC were observed. The potent cytotoxic action of TC on these tested cancer cells could be due to its higher intracellular concentration, which in turn would impair the metabolic activity by directly interacting with mitochondria inside the cells.21 The results of cytotoxicity in CHO cells demonstrated 50% cell death with CaCO3-TC at 241.21 μM and TC at 140.87 μM (Table S1). This differential cytotoxic potential shown by CaCO3-TC microspheres both on noncancerous and cancer cells might be because of the pHdependent controlled dissolution of CaCO3 matrix. The microenvironment pH of HeLa and CHO cells was determined by using bromothymol blue as pH indicator, which changes its color from bluish green to yellow as the

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RESULTS AND DISCUSSION In a bioinspired approach, a combination of anionic polypeptide with cationic peptide oligomer was utilized to mineralize CaCO3, while it also simultaneously facilitated the encapsulation of TC in situ under extremely mild reaction conditions.16 Typically, negatively charged poly(L-asp) forms spherical aggregates in the presence of penta(L-lys) as the countercation in water. These peptide-aggregates in successive steps allow the encapsulation of TC and the mineralization of CaCO3 from CaCl2 and (NH4)2CO3 to generate microsphere structures loaded with the drug. Especially, the mild synthesis condition is conducive for the in situ encapsulation of sensitive compounds like the drugs. The morphological characterization of TC loaded CaCO3 microstructures was carried out by electron microscopy. The field emission scanning electron microscopy (FE-SEM) analysis illustrated the spherical morphology of the synthesized material with sizes ranging from 400 to 900 nm diameters (Figure 1a and b). Analysis by dynamic light scattering (DLS) also confirmed the size distribution of the particles in a similar range (Figure 1c). From energy dispersive X-ray spectroscopic (EDS) elemental analysis, the presence of calcium, carbon, oxygen, and nitrogen could be established as shown in Figure 1d. The existence of nitrogen peak clearly indicates the presence of peptides and 631

DOI: 10.1021/acs.chemrestox.8b00131 Chem. Res. Toxicol. 2018, 31, 629−636

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Chemical Research in Toxicology

Table 1. Percentage of Cells Distributed in Different Phases of Cell Cycle (Flow Cytometry Analysis) in CHO, MDA-MB-231, and HeLa Cells Treated with Different Concentrations of TC and CaCO3-TCa control CHO

CaCO3-TC 100

1.9 46 25.3 26.4

subG1 G1 S G2/M

control MDA-MB-231

HeLa

5.8 54.8 18.2 21.3

CaCO3-TC 300

1.8 66.9 5.1 25.3 CaCO3-TC 50

2.2 68.4 6.8 22.2 CaCO3-TC 75

control

6.2 49.6 15.3 29 CaCO3-TC 50

4.6 53.6 15.3 26.5 CaCO3-TC 75

6.7 46.6 16.6 30.1 CaCO3-TC 100

5.9 40.3 13.6 40.2

5.8 47.2 12.3 34.8

5.9 46.9 11.2 35.9

6.6 50.4 10.9 30.9

subG1 G1 S G2/M subG1 G1 S G2/M

CaCO3-TC 200

3.3 61.8 10.6 22.6 CaCO3-TC 25

TC 100 1.8 63 15.5 19.7 TC 25 5.8 51.8 18.7 23.6 TC 50 6.7 43.4 9.9 40

TC 200 1.9 66.9 9.5 21.7 TC 50

TC 300 2.6 72.1 9.1 16.2 TC 75

5.8 51.4 16.6 26.2 TC 75

5.9 50.7 18.1 25.2 TC 100

4.3 49.9 10.1 35.6

7.4 52.3 11.9 28.4

The number next to the sample code (TC and CaCO3-TC) indicates concentration with respect to TC in μM. Control- untreated.

a

Table 2. Frequency of Chromosomal Aberrations Induced by TC and CaCO3-TC after 24 h Post-Treatment in CHO Cell Line sample DMSO TC

CaCO3-TCb

mitomycin C

dose (μM) 100 200 300 100 200 300 2.5

% of aberrant metaphases (M ± SEM)a 8.63 13.97 11.51 18.09 8.90 10.24 7.46 25.93

± ± ± ± ± ± ± ±

% of aberrations including gaps (M ± SEM)a

0.857 0.357c 0.35 (ns)e 0.94d 0.94c 1.24c 1.12d 1.10d

9.95 23.17 27.9 33.88 13.55 14.09 11.98 44.16

± ± ± ± ± ± ± ±

1.69 1.84d 0.55d 1.48d 1.48d 2.25d 1.47d 1.63d

% of aberrations excluding gaps (M ± SEM)a 4.96 11.23 16.00 20.01 8.90 8.023 9.03 26.12

± ± ± ± ± ± ± ±

0.99 1.29c 0.57d 0.95d 0.95d 0.66d 0.84d 0.95d

a

M, mean; SEM, standard error of the mean; Dunnett’s multiple comparison test. bConcentration with respect to TC present in CaCO3-TC composite. cSignificance level: p < 0.01. dSignificance level: p < 0.001. ens: nonsignificant.

cells stop proliferating and get blocked by G1 checkpoint in cell cycle. A large number of G2/M arrest of cells was also noticed in MDA-MB-231 cells which could be due to the induced DNA damages or due to the possibility of overexpression of p53 gene in breast cancer cells.27 The cytotoxicity and FACS studies revealed that CaCO3-TC has higher toxic effects on cancer cells compared to noncancerous cells. Based on the cytotoxicity data (MTT) (Table S1), three different concentrations of CaCO3-TC (100, 200, and 300 μM) and TC (100, 200, and 300 μM) were selected. Thus, treated CHO cells were then investigated for the possible cytogenetic toxicity at 24 h of post-treatment period by analyzing the CA, induction of MN, and changes in the MI. To gain comprehensive understanding on the DNA damaging effect of CaCO3-TC, CA test was performed using CHO cells. Generally, in vitro CA test identifies the agents that cause potential genotoxic hazards in normal mammalian cells.28 In the vehicle control (DMSO treated cells), 8.63% of aberrant metaphases with 9.95% of aberrations including gaps and 4.96% of aberrations excluding gaps were observed, whereas cells treated with mitomycin C (2.5 μM) showed an increase in percentage of aberrant metaphases 25.93% with aberrations including gaps 44.3% and excluding gaps 27.37% which were statistically significant at level (p < 0.001) than the vehicle control (Tables 2 and S2; Figure 2). The structural DNA damages recorded by each test concentration of bare TC in noncancerous cells compared to the vehicle control revealed a dose dependent increase in clastogenic nature of TC (Tables

environment varies from neutral to acidic pH. The bromothymol blue-treated HeLa cells under bright-field microscopy appeared yellow in color indicative of their acidic pH environment (Figure S2).22 On the other hand, the color for the treated CHO cells was bluish green due to the neutral pH of their cellular environment. Therefore, in case of the acidic microenvironment of cancer cells (HeLa and MDA-MB 231), the effective dissolution of CaCO3 matrix would allow the released TC to interact with the cell.23 On the other hand, in the case of normal cells (pH ∼ 7.4), the incomplete dissolution of CaCO3 would prevent the direct mitochondrial interaction of the encapsulated TC.16,24 The cytotoxic analysis thus demonstrated potent action of CaCO3-TC on cancer cells with essential protective action on the normal cells. The effects of CaCO3-TC and TC on cell cycle distribution studies (FACS) were also assessed on both CHO and cancer cells. Cell cycle analysis is a method that distinguishes cells in different phases of the cell cycle employing fluorescence activated cell sorting mechanism usually with PI.25 The results on all the three cell lines are represented in the Table 1, which clearly depicted a distinct mode of action on cancer and noncancerous cells by CaCO3-TC and TC. Both these samples showed a clear G1 arrest on CHO and HeLa cells whereas G2/ M in the case of MDA-MB cells (Figures S3−S5). It is earlier reported that TC shows immense influence on the mammalian mitochondria by inhibiting the mitochondrial protein synthesis and lowering the membrane potential.26 Consequently, when large inhibition of mitochondrial protein synthesis occurs, the 632

DOI: 10.1021/acs.chemrestox.8b00131 Chem. Res. Toxicol. 2018, 31, 629−636

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TC may be due to unrepaired lesions of G1 and G2 phases.41 In contrast, CaCO3-TC showed a remarkable decrease in the induction of both chromatid and chromosome type of aberrations. This again supports the role of CaCO3 casing around TC, which minimizes the direct interaction of TC with CHO cells resulting in a significantly less DNA damage. Mitotic index was examined to determine the percentage of cells undergoing mitosis (from prophase to telophase).42 Upon treatment with DMSO, CHO cells exhibited 9.21% percentage of MI. Whereas the cells treated with mitomycin C (2.5 μM) exhibited a significant decrease in cell division rate in CHO cells, presenting 3.87% of MI which was statistically significant (p < 0.001) when compared to DMSO (Table 3 and Figure 3).

Figure 2. In vitro genotoxicity evaluation of TC and CaCO3-TC in comparison to control DMSO: Total number of aberrant metaphases; chromosome aberrations (including gaps) and chromosome aberrations (excluding gaps) with different concentrations (100, 200, 300 μM) of TC and CaCO3-TC and 2.5 μM mitomycin C as positive control in CHO cells after 24 h post-treatment. **, *** indicate a significant difference between the different concentrations of TC with control at p < 0.01, p < 0.001, respectively, and ns indicates nonsignificant variation.

Table 3. Effect of TC and CaCO3-TC on MI after 24-hr Post-Treatment in CHO Cells sample DMSO tetracycline

2 and S2; Figure 2). All the three test concentrations showed a significant increase in percentage of aberrant metaphases, aberrations including and excluding gaps (p < 0.001) than DMSO. Mostly chromatid and chromosome types of aberrations were observed by TC. Among all the tested concentrations of TC, TC-300 induced highest toxicity and hence was used as the positive control to assess the clastogenic effects of CaCO3-TC. It was observed that CaCO3-TC microspheres revealed a remarkable decrease in chromosome aberrations at all the three concentrations, which were statistically significant at level (p < 0.001) than that of TC300 (Tables 2 and S2; Figure 2). Chromosome breakages in normal cells trigger several human disorders and often gain tendency to develop certain types of malignancies.29−31 Earlier studies on TC made confounding reports on its genotoxic properties stating TC as a nongenotoxic agent in an in vitro model32 and later as a genotoxic agent.33,34 However, our studies revealed the genotoxic nature of TC on CHO cells showing a dosedependent increase in the total number of aberrant metaphases, aberrations including and excluding (gaps) compared to the vehicle control group (p < 0.001). TC is a bifunctional alkylating agent which mostly acts on DNA by interstrand cross-linking.35 Bifunctional alkylation of DNA with cross-linking has been reported to be more profoundly cytotoxic than any monofunctional alkylating agent.36,37 Bifunctional alkylating agents have two reactive alkyl groups and therefore can react twice with DNA and interrupt the normal pairing of base pairs in DNA strand. This cross-linking prevents DNA replication and transcription leading to DNA damage and cell death.38 In our examination of CA analysis, we have observed high percentage of chromosome aberrations with bare TC than CaCO3-TC in comparison to control group, DMSO. As bare TC is readily available for CHO cells, it might have caused high DNA damage. Noticeably both chromosome and chromatid type aberrations were found high in TC suggesting it as an S-phase-dependent compound.39,40 The CaCO3 matrix around TC might have limited the possibility of high DNA damage by virtue of the delayed release of TC at neutral pH. Nevertheless, the higher number of chromatid and chromosome type of aberrations (gaps and breaks) found with

CaCO3-TCb

mitomycin C

dose (μM)

total number of cells

total number of dividing cells observed

100 200 300 100 200 300 2.5

3059 3165 3120 3299 3096 3036 3091 4685

282 163 153 133 258 250 244 180

percentage of MI (M ± SEM)a 9.21 5.15 4.90 4.03 8.23 8.25 7.97 3.87

± ± ± ± ± ± ± ±

0.033 0.133c 0.654 0.149c 0.28c 0.31c 0.11c 0.22c

a

M, mean; SEM, standard error of the mean; Dunnett’s multiple comparison test. bConcentration with respect to TC present in CaCO3-TC composite. cSignificance level: p < 0.001.

Figure 3. Changes in MI: TC and CaCO3-TC taken at different concentrations (100, 200, 300 μM) and 2.5 μM mitomycin C as positive control in CHO cells after 24 h post-treatment. *, **, *** indicate a significant difference between the different concentrations of TC with control at p < 0.05, p < 0.01, p < 0.001, respectively, and ns indicates nonsignificant variation.

Similarly, all three concentrations of TC reduced the rate of cell division in a dose-wise manner which was statistically significant compared to vehicle control (p < 0.001). However, CaCO3-TC at all the tested concentrations displayed a significant increase (p < 0.001) in the percentage of dividing cells than that of TC-300 (Table 3 and Figure 3). As depicted in the results, TC revealed a significant decrease in the MI of CHO cells compared to the vehicle control group (p < 0.001). Usually any reduction in the number of cells reaching M-phase directly depends on the arrest of cells at a particular phase of the cell cycle due to DNA damage and repair in G1, S, and G2 phases.43 This resultant reduction of MI in TC treated cells also correlated with the FACS study that higher cell population was observed at G1 phase (Table 1). 633

DOI: 10.1021/acs.chemrestox.8b00131 Chem. Res. Toxicol. 2018, 31, 629−636

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Chemical Research in Toxicology Table 4. Effect of TC and CaCO3-TC on Micronuclei after 24-hr Post-Treatment in CHO Cells sample DMSO tetracycline

CaCO3-TCb

mitomycin C

dose (μM) 100 200 300 100 200 300 2.5

total number binucleated cells observed number of binucleated cells observed with MN percentage of micronuclei (M ± SEM)a 6039 6132 6259 7135 6147 6355 6395 6046

11 11 13 27 5 2 4 52

1.821 1.793 2.077 3.786 2.63 0.92 1.79 8.563

± ± ± ± ± ± ± ±

0.166 0.799 (ns)e 1.08 (ns)e 1.23 (ns)e 1.235 (ns)e 0.673c 0.154c 0.676d

a

M, mean; SEM, standard error of the mean; Dunnett’s multiple comparison test. bConcentration with respect to TC present in CaCO3-TC composite. cSignificance level: p < 0.05. dSignificance level: p < 0.001. ens: nonsignificant.

presence of TC may be due to lagging of acentric chromosome fragments during anaphase of the mitotic stage.46 Whereas with CaCO3-TC, a significant reduction of MN was noticed in comparison to TC-300 which also correlates with CA results implying that CaCO3-TC protects the CHO cells from the clastogenic and aneugenic effects of TC. To understand the genotoxic effects on cancer cells, both MI and MN induction potentials of CaCO3-TC on HeLa cells were investigated. As observed, there was a dose-dependent decrease in the number of dividing cells at all three concentrations of CaCO3-TC compared to bare TC (Table S3 and Figure S6). Although at lower and intermediate concentrations of CaCO3-TC (25 and 75 μM), the rate of dividing cells was lowered, and at the highest concentration (100 μM), the percentage of dividing cells of HeLa cells reduced significantly compared to bare TC (P < 0.01). Similarly, the MN induction potential of CaCO3-TC at all the three concentrations was found to be higher in HeLa cells, but it was significant at 100 μM of CaCO3-TC (Table S4 and Figure S7). These results from genotoxicity tests demonstrated a remarkable decrease in the normal cell toxicity by CaCO3TC compared to bare TC, resulting in lesser clastogenic, aneugenic, and cytotoxic effects on CHO cells. Therefore, the effect of CaCO3 casing of TC, which provides a way to limit its toxicity on noncancerous cells, while displaying the desired toxicity on cancer cells, further validates its future use in cancer therapy.

Hence it can be postulated that the damaged cells at G1 passing through S and G2 phases might have taken more time in reaching the mitotic phase. But in the case of CaCO3-TC treated CHO cells, the MI was not affected as the percentage of dividing cells was found to be similar to vehicle control group. So the MI study revealed that CaCO3-TC is nonmitotoxic in nature and does not interfere in normal cell division. The micronucleus test is a useful cytogenetic end point to assess both the clastogenic and aneugenic property of an agent in vitro and in vivo.17,44 A micronucleus can arise either due to a whole lagging chromosome (aneugenic event) or by an acentric fragment formed after a chromosome break (clastogenic events) during anaphase of the cell cycle by a toxic substrate; thereby it confirms both aneugenicity and clastogenicity of a test substance.45 The frequency of micronuclei induced by TC increased than that in control cells (DMSO), but the whole induction was found to be statistically nonsignificant (Table 4 and Figure 4). In contrary,

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CONCLUSIONS

Encapsulation of TC in CaCO3 (CaCO3-TC) was demonstrated to exhibit cytotoxicity in cancer cells with a protective effect on the normal mammalian cells, than on bare TC. Notably, CaCO3-TC caused 50% cell death in cancer cells at half of its concentration that was exposed to normal cells. Cytogenetic toxicity studies on CHO cells also revealed a remarkable decrease in clastogenic, aneugenic, and mitotoxic potential of CaCO 3-TC compared to bare TC. This appreciable minimization of toxicity by CaCO3-TC might be because of its pH sensitivity that naturally delays the dissolution of the CaCO3 matrix and further slows down the release of TC for action due to the near neutral pH environment in normal cells. This distinguishable behavior elucidates that the CaCO3 casing around TC might have prevented the direct interaction of TC with noncancerous cells, resulting in a significantly less genetic damage. Whereas, due to acidic pH condition in cancer cells, these microspheres may be actively releasing TC into TME, thereby effectively ceasing further proliferation of cancer cells. Hence, these engineered CaCO3-TC microspheres justify further investigation as a

Figure 4. Percentage induction of MN/1000 cells: TC and CaCO3TC taken at different concentrations (100, 200, 300 μM) and 2.5 μM mitomycin C as positive control in CHO cells after 24 h posttreatment. *, *** indicate a significant difference between the different concentrations of TC with control at p < 0.05, p < 0.001, respectively, and ns indicates nonsignificant variation.

CaCO3-TC revealed a significant decrease in induction of micronuclei than TC-300. Both of the doses of CaCO3-TC at 200 μM (0.92%) and 300 μM (1.79%) exhibited a statistically significant decrease (p < 0.05) in percentage of MN in comparison to TC-300 (Table 4 and Figure 4). In our studies MN induced by TC in CHO cells was found statistically nonsignificant, yet revealed a dose-wise increase in induction of micronuclei. This induction of MN in the 634

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novel advanced target-specific drug formulation offering a safe delivery of the drug into cancer cells for effective control and simultaneously preventing severe unwanted toxicity to the normal cells.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.8b00131. UV−vis spectra, cytotoxicity data (FACS and MTT assay), optical microscopic images of bromothymol blue treated HeLa and CHO cells, frequency of chromosomal aberrations induced by TC and CaCO3-TC in CHO cell line, changes in the mitotic index and frequency of micronucleus formation induced by CaCO3-TC and TC in HeLa cells. (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +91-40-27191367 *E-mail: [email protected]. Phone: +91-40-27191387. ORCID

Rohit K. Rana: 0000-0002-1754-240X Author Contributions ∥

These authors contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS G.B. thanks to DST, New Delhi, India (SB/FT/CS-111/2014) for the young scientist fellowship. S.M. is thankful to CSIR, New Delhi, India for supporting the RSP-4054 project. K.J. and S.D. thank CSIR, New Delhi, India for fellowships.

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REFERENCES

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