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Cite This: Anal. Chem. XXXX, XXX, XXX−XXX

Investigation of Cytotoxic Activity of Mitoxantrone at the Individual Cell Level by Using Ionic-Liquid-Tag-Enhanced Mass Spectrometry Fedor A. Kucherov,† Ksenia S. Egorova,† Alexandra V. Posvyatenko,‡,§ Dmitry B. Eremin,† and Valentine P. Ananikov*,† †

N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky prospect 47, Moscow, 119991 Russia Institute of Gene Biology, Russian Academy of Sciences, Vavilova str. 34/5, Moscow, 119334 Russia § Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology, Ministry of Health of Russian Federation, Samory Mashela str., Moscow, 117198 Russia ‡

S Supporting Information *

ABSTRACT: A novel mitoxantrone conjugate was synthesized by coupling mitoxantrone with ionic liquid tags, and cytotoxic behavior of the designed conjugate was studied in normal and cancer cell lines. The synthesized mitoxantrone conjugate was oil at physiological temperatures and demonstrated high aqueous solubility. Sensitivity of electrospray ionization mass spectrometry (ESI-MS) to the mitoxantrone conjugate was improved by an order of magnitude, in comparison with original mitoxantrone dihydrochloride. The observed ESI-MS signals were shifted to a “clearer” lower-mass region of the spectrum, which allowed investigation of the drug at the level of individual cells. The ionic liquid tags proposed in the present work consist of an easily available imidazolium salt residue and show a number of key advantages from the points of view of drug conjugate synthesis, drug delivery and analytic detection.

O

signals because of overlapping with high-intensity signals of numerous other substances present in the living cell.3,16 For example, imaging MS can be employed only in cases of high doses of drugs which significantly narrows its application.16 Therefore, the improved detection limit and signal resolution are key requirements for modern drug research at the level of individual tissues and cells. The above-mentioned issues have urged critical rethinking of several aspects of drug design. Most modern drugs are solid substances, and the well-known problems of pharmaceutics include low aqueous solubility and polymorphism. 18,19 Insufficient aqueous solubility hinders the achievement of active concentrations of a drug and also diminishes its signal intensity in mechanistic studies. This drawback can be alleviated by using active pharmaceutical ingredients (API) in the form of salts, and, indeed, about a half of modern drugs is applied as salts. However, liquid formulations are uncommon,20 and the polymorphism issue still impedes the development and manufacture of known and novel drugs. An efficient solution of these problems has been proposed recently by assessing drugs in the ionic liquid form. Ionic liquids are liquid salts with a tremendous potential to be finetuned.21,22 Thus, one can design an ionic liquid possessing

ver the last decades, studies on activity and behavior of various drugs usually have been dealing with “bulky” biological objects consisting of many cells. However, inherent heterogeneity of most cell populations and even cell cultures has become evident lately.1−6 A recent progress in research on microbiomes has also pointed out an important role of cell inhomogeneity.7,8 Therefore, quantitative and qualitative singlecell analysis has emerged as an important approach in biological and medical research.3,9 Considering high heterogeneity of malignant tumors, studies on biological activity of drugs at the individual cell level will provide an important advance and stimulate further progress in drug design and therapy. Indeed, investigations of drug mechanisms of action present a cutting-edge opportunity in this field. Modern approaches for single-cell characterization include sequencing (genomics and transcriptomics),10 nuclear magnetic resonance (NMR),11 capillary electrophoresis,12 Förster resonance energy transfer (FRET) spectroscopy,13 fluorescence microscopy,14 mass spectrometry (MS),3,15 and others.3 MS is considered among the most proficient methods for examining metabolomics at the single-cell level.3 Mass spectrometry imaging is currently used for investigating the distribution of drugs in living organisms, which has been shown to depend on the administration route and formulation.3,16,17 However, the identification of chemical compounds and their transformations at the level of tissues and single-cells requires overcoming two limitations: (1) insufficient signal intensity for spectrometric analysis and (2) masking of low-intensity target © XXXX American Chemical Society

Received: August 31, 2017 Accepted: November 21, 2017

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DOI: 10.1021/acs.analchem.7b03568 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry

conjugate signals were shifted to a “clearer” lower-mass region of the spectrum (Figure 1C). The improved ESI-MS detection allowed us to explore the possibility of studying the drug at the level of individual cells (Figure 1D).

various combinations of properties, such as high aqueous solubility, required dissolution rate, hydrophobic/hydrophilic balance, hydrogen bonding, etc.23,24 Moreover, ionic liquids are liquid at physiological temperatures and, therefore, should not suffer from polymorphism. The idea of turning common APIs into ionic liquids (ILs) has gained much attraction, and the API-IL concept has been developing actively.20,23,25−27 API-ILs can be classified as follows: (1) ILs containing API as anion or cation; (2) ILs, in which API is covalently linked to an IL moiety; and (3) ILs containing both covalently linked API and API as anion or cation.28 So far, several examples have been published, including API-ILs with tetracycline,29 ampicillin,30,31 lidocaine,32,33 salicylic acid,28,34,35 etodolac,33 ibuprofen,36,37 and other drugs.24 Benefits of ionic interactions have also been used in the shaping and functioning of various drug delivery systems.24,38−40 In the present study, we employ the API-IL strategy from a new analytical viewpoint for investigating the cytotoxic behavior of a novel ionic liquid mitoxantrone conjugate in normal and cancer cell lines. Mitoxantrone is an anthracenedione antineoplastic drug, which is used for treatment of various types of cancer, including breast and prostate cancers, leukemias and lymphomas.41−44 Mitoxantrone acts as an inhibitor of DNA topoisomerase II in mammalian cells and also interacts with other biological molecules, such as RNA, proteins and lipids.44 Key advantages of the approach developed in this work are shown in Figure 1. The new ionic liquid mitoxantrone conjugate retained the cytotoxic activity of the original drug; it was oil at physiological temperatures and readily soluble in water (Figure 1A). Sensitivity of electrospray ionization mass spectrometry (ESI-MS) to the mitoxantrone conjugate was by an order of magnitude higher as compared to the original mitoxantrone dihydrochloride (Figure 1B), and the

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EXPERIMENTAL SECTION All chemical reagents were purchased from commercial suppliers and were tested by NMR before use. All reactions were performed in oven-dried (150 °C) glassware under argon atmosphere unless stated otherwise. 1H, 13C, 19F, and 31P NMR spectra were recorded using NMR spectrometers Bruker DRX500 and Bruker Avance 400 with the residual solvent peak as an internal standard. The NMR spectra were processed using TopSpin 1.3. Mass spectra were measured on a highresolution time-of-flight Bruker maXis instrument using electrospray ionization (ESI-MS). Measurements were performed in the positive ion mode, interface capillary voltage at 4.5 kV, effective scan range at m/z 100−1200, external calibration (ESI-L Low Concentration Tuning Mix, Agilent Technologies), direct syringe injection at flow rate of 3 μL × min−1, nitrogen as dry gas at 4 L × min−1, interface temperature at 180 °C. The spectra were processed using Bruker Data Analysis 4.0 software package. 3-(10-Carboxydecyl)-1-methylimidazolium Bromide (IL-Br).45

11-Bromoundecanoic acid (5.4 g, 20.3 mmol) and Nmethylimidazol (8 mL, 0.1 mol) was dissolved in 50 mL of ethanol; the solution was heated at 120 °C for 16 h in a pressure vessel. The volatiles were evaporated under reduced pressure, and the residue was recrystallized from the ethyl acetate/ethanol mixture. The target compound was obtained as white crystals, mp 134 °C, 5.4 g (77%). 3-(10-Carboxydecyl)-1-methylimidazolium Tetrafluoroborate (IL-BF4).46

3-(10-Carboxydecyl)-1-methylimidazolium bromide (200 mg, 0.58 mmol) was dissolved in 4 mL of water, and sodium tetrafluoroborate (316 mg, 2.9 mmol) was added. The mixture was stirred at 25 °C for 16 h, then the precipitate was filtered off, washed with water (2 × 1.5 mL) and dried under reduced pressure. The target compound was obtained as white solid, mp 95 °C, 123 mg (64%). 3-(10-Carboxydecyl)-1-methylimidazolium Hexafluorophosphate (IL-PF6).46

The solution of potassium hexafluorophosphate (1.32 g, 7.2 mmol) in water (11 mL) was added dropwise to a water solution (7 mL) of 3-(10-carboxydecyl)-1-methylimidazolium bromide (500 mg, 1.44 mmol) at 5 °C. The reaction was performed in a polypropylene flask at 25 °C for 4 h, then the precipitate was filtered off, washed with water (2 × 2 mL) and dried under reduced pressure. The target compound was obtained as white solid, mp 75 °C, 450 mg (76%).

Figure 1. Key results of the present study: (A) synthesis of ionic liquid mitoxantrone conjugate with high solubility, retained biological activity, and lack of polymorphism; (B) significant improvement of ESI-MS drug detection limit; (C) shift of drug signals to “clearer” observation area in mass spectra; and (D) possibility of investigating drug behavior at the level of individual cells by means of mass spectrometry. B


Article

Analytical Chemistry 3,3′-(((((((5,8-Dihydroxy-9,10-dioxo-9,10-dihydroanthracene-1,4-diyl)bis(azanediyl)) Bis(Ethane-2,1-diyl))bis((11-(1-methyl-1H-imidazol-3-ium-3-yl)undecanoyl)azanediyl)) Bis(Ethane-2,1-diyl))bis(oxy))bis(11-oxoundecane-11,1-diyl))bis(1-methyl-1H-imidazol-3-ium) Tetrakis(Tetrafluoroborate) (MX-IL-BF4).

5% CO2. The DMEM/F-12 (1:1) medium with 2.5 mM Lglutamine and 1.5 mM HEPES (HyClone, USA), and DMEM and RPMI-1640 medium without L-glutamine were used for 3215 LS, and CaCo-2 and Colo 320HSR, respectively. The media were supplemented with 10% fetal bovine serum (HyClone, USA), 100 units × mL−1 penicillin (OAO Sintez, Russia), and 100 μg × mL−1 streptomycin (OAO Biokhimik, Russia). L-glutamine (HyClone, USA) was added to DMEM and RPMI-1640 to the final concentration of 2 mM. MTS Assay. The MTS assay was used for evaluation of cytotoxicity of ILs, as previously described.47 The choice of the 24-h period of the incubation was governed by the availability of the corresponding data in the literature. Before the test, cells were passaged into 96-well flat-bottomed plates, 10000 3215 LS or Colo320 cells per well, and 6000 CaCo-2 cells per well. The utmost wells were filled with 200 μL phosphate-buffered saline (HyClone, USA). Cells were cultivated until reaching 70% monolayer (40−42 h in the case of 3215LS and Colo320, or 64−66 h for CaCo-2) and then were incubated for 24 h with mitoxantrone dihydrochloride or ILs in the concentration range from 400−6000 μM to 25 nM−150 μM, ten points in total. The same substance concentrations were applied to an empty plate to allow an adjustment for the IL influence on optical density. All test points were measured in 3−4 replicas. 1% Triton X-100 (Sigma-Aldrich) in the culture medium was used as a positive control, and the medium was used as a negative one. After the incubation, 20 μL of the MTS (3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) reagent (CellTiter 96 AQueous One Solution Cell Proliferation Assay, Promega, USA) were added into each well, and the plates were incubated for additional 4 h. Optical density was measured at 492 and 650 nm using Original Multiskan EX (Lab Systems, USA), and the values obtained at 650 nm were subtracted from those obtained at 492 nm to exclude background absorption. Statistical processing of the obtained data was carried out using Microsoft Excel 2010 (Microsoft) and Prism 5 (GraphPad); IC50 (half maximal inhibitory concentration) was calculated for each IL (data are expressed as the mean and its 95% confidence interval). Mass spectrometric detection of mitoxantrone or mitoxantrone conjugate (MX-IL-BF4) in cells. 3215 LS cells or CaCo-2 cells were cultured in 24-well plates (Corning Inc., USA), as described previously (100000 cells × mL−1, 1 mL per well). Upon reaching 70% monolayer, the cells were incubated with 20 μM mitoxantrone dihydrochloride or MXIL-BF4 for 1 h; the pure growth medium was used as control. Then the medium, which contained floated dead cells, was removed and saved for further analysis, whereas the cells were washed with PBS thrice. After that, 0.5 mL of 0.05% trypsin (HyClone, USA) was added to the cells for 3 min. Then 0.5 mL of the growth medium was added to the wells, the cells were resuspended thoroughly, were transferred to plastic microcentrifuge tubes (Costar, Corning Inc., USA) and were centrifuged for 4−5 min (1000 rpm, 4 °C). The pellets were resuspended in PBS thrice. All the washings were saved for subsequent usage. The cells were counted by using Gorjaev’s chamber, and samples were prepared in a fixed volume of acetonitrile by serial dilutions (1000 or 100 cells per 100 μL of acetonitrile (Fischer Chemicals, Switzerland); see Table 1 for 3215 LS cells and Table S-1 for CaCo-2 cells). The medium after the incubation with the target substances was centrifuged for 15 min (2000 rpm, 4 °C), and the supernatant was saved for MS study, whereas the pellet, which contained dead cells, was

For the functionalization, several coupling reagents have been tested (HBTU, TBTU, CDI, and EDC). It was found that ILBF4 and TBTU provided the best result, while in the other cases, the reaction proceeded incompletely or with poor selectivity. Apparently, the combination of identical counterions in the ionic liquid and in the coupling reagent excluded the occurrence of any ion exchange side-reactions. Usage of the HBTU/IL-PF6 pair provided the conjugate of insufficient purity. TBTU (124 mg, 0.38 mmol) and TEA (65 μL, 0.38 mmol) were added to a stirred solution of 1-methyl-3(10carboxydecyl)imidazolium tetrafluoroborate (68 mg, 0.19 mmol) in DMSO (1.2 mL). The mixture was stirred at room temperature for 15 min, and then 12 mg (0.023 mmol) of mitoxantrone (1,4-dihydroxy-5,8-bis[2-(2-hydroxyethylamino)ethylamino]-anthracene-9,10-dione) dihydrochloride were added. The reaction was stirred at 25 °C for 16 h; the volatiles were evaporated under reduced pressure without heating, then 5 mL of water was added to the residue, and the reaction vessel was kept in the ultrasonic bath for 1 h. The mixture was filtered through a pad of Celite and washed with water (2 × 5 mL); the oily residue was removed from the Celite with acetone (2 × 5 mL), and the combined organic solutions were evaporated and dried in high vacuum. The target compound was obtained as deep blue oil (38 mg, 92%). Mass Spectrometry Analysis of Detection Limit. Detection limit analysis was performed with two series of standard solutions. For mitoxantrone, 1.1 mg (2.5 × 10−3 mol) were dissolved in 25 mL of the 1:1 MeOH: H2O mixture in a volumetric flask giving the 1 × 10−4 M solution. Solutions from 1 × 10−4 to 1 × 10−18 M were prepared by serial dilution (100 μL of the concentrated solution in 900 μL of the solvent mixture). For MX-IL-BF4, 2.8 mg (1.56 × 10−3 mol) were dissolved in the same manner, giving 6.26 × 10−5 M. The 1 × 10−5 M solution was prepared by adding 162 μL of the initial solution to 838 μL of the solvent mixture. Further series were also prepared by serial dilution, as described above. Cell Cultures. 3215 LS cells (human fibroblasts; courtesy of E. Kopantsev, M. M. Shemyakin, and Yu. A. Ovchinnikov Institute of Bioorganic Chemistry RAS), Colo 320HSR cells (human colon carcinoma; The Russian Cell Culture Collection (RCCC), the Institute of Cytology RAS, St. Petersburg) and CaCo-2 (human colorectal adenocarcinoma, ATCC; purchased from the Institute of Cytology RAS, St. Petersburg, Russia) were cultured in clear plastic TC-treated dishes or multiple-well plates (Corning Inc., USA) in a HeraCell 150 incubator (Thermo Electron Corp., USA) at 37 °C, 95% humidity, and C


Article


molecules possess poor solubility in the hydrophobic membrane core, and the rate of their passive transport into the cell is presumably governed by relatively infrequent events of flip-flop.48 Though the size of a molecule can impact its passive transport, this influence is generally less important than lipophilicity of the drug.49 Thus, in our work, we selected an imidazolium ionic liquid containing ten methylene units as an appropriate tag, because such an alkyl linker should have no critical influence on the intercalation of mitoxantrone into DNA and should not hinder the passage of the conjugate through the cell membrane.44 Moreover, imidazolium ILs with long alkyl chains have been shown to destabilize biological membranes via penetrating into the lipid bilayer and disturbing its structure.50−52 Stepwise synthesis of the IL tags and conjugate studied in this work is shown in Figure 2. First, 3-(10-carboxydecyl)-1methylimidazolium bromide (IL-Br) was synthesized from Nmethylimidazole and 11-bromoundecanoic acid; this ionic liquid was subsequently used to obtain 3-(10-carboxydecyl)-1methylimidazolium tetrafluoroborate (IL-BF4) and 3-(10carboxydecyl)-1-methylimidazolium hexafluorophosphate (ILPF6) (Figure 2A). Mitoxantrone was introduced into IL-BF4 via an amide bond to obtain the resulting API-IL (MX-IL-BF4) (Figure 2B). Structures and purity of the synthesized ionic liquids were confirmed by 1H, 13C, 19F, and 31P NMR spectroscopy and ESI-MS. The preparation of MX-IL-BF4 is, to our knowledge, reported here for the first time. NMR and mass spectra of the synthesized ionic liquids are provided in the Supporting Information (Figures S-1−S-14). Of note, the regular ionic liquids IL-Br, IL-BF4, and IL-PF6 were solid at room temperature (mp 75−134 °C), whereas MX-IL-BF4 was oil. It should be noted that converting a solid substance into oil makes it possible to avoid the problem of polymorphism, which is characteristic for many common drugs. Cytotoxicity of Ionic Liquid Mitoxantrone Conjugate. Mitoxantrone is supposed to induce cytotoxicity via poisoning topoisomerase II, which therefore renders actively proliferating cancer cells more sensitive to this drug.44 We employed the MTS assay to assess the efficiency of the synthesized MX-ILBF4 in comparison with the pure drug (mitoxantrone dihydrochloride, MX·2HCl) and intermediate ionic liquids

Table 1. Samples for MS Detection of Mitoxantrone or MXIL-BF4 in 3215 LS Cellsa sample/substance 1000 living cells 1000 dead/ damaged cells 100 dead/ damaged cells total fraction of living cells total fraction of dead/damaged cells cell medium after incubation a

control

MX·2HCl

MX-IL-BF4

1000L 1000D

1000L_MX 1000D_MX

1000L_IL 1000D_IL

100D

100D_MX

100D_IL

LC (∼105 000 cells) DC (∼60 000 cells)

LC_MX (∼160 000 cells) DC_MX (∼90 000 cells)

LC_IL (∼105 000 cells) DC_IL (∼85 000 cells)

CM

CM_MX

CM_IL

Cell amounts for a representative experiment are shown.

resuspended in the corresponding washings for minimizing cell losses and then was centrifuged for 15 min (2000 rpm, 4 °C). The final pellet of dead/damaged cells was resuspended in PBS; the cells were counted by using Gorjaev’s chamber, and samples were prepared in a fixed volume of acetonitrile by serial dilutions (1000 or 100 cells per 100 μL acetonitrile (Fischer Chemicals, Switzerland); see Table 1). The dead/damaged and living cells remaining after the preparation of the dilutions were centrifuged for 15 min (2000 rpm, 4 °C), and the pellets were resuspended in 100 μL acetonitrile (see Table 1). The medium samples were diluted 10 times and measured after thorough centrifugation. The cell fractions were treated in an ultrasonic bath and were measured directly without washing between samples in a series of increasing concentrations with acetonitrile as a blank experiment. To enhance the sensitivity and to separate unfavorable ions, a desired region of masses was extracted by a triple quadrupole system before time-of-flight detection. m/z 443448 and m/z 358362 regions were chosen for mitoxantrone and MX-IL-BF4, respectively.

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RESULTS AND DISCUSSION Synthesis of Ionic Liquid Mitoxantrone Conjugate. Mitoxantrone is supposed to penetrate the cell membrane passively via the flip-flop mechanism.48,49 Complex amphiphilic

Figure 2. Synthesis of ionic liquid components (A) and ionic liquid mitoxantrone conjugate (B). D


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Analytical Chemistry Table 2. Cytotoxicity of Studied Substances Towards Colo 320HSR, CaCo-2, and 3215 LS Cell Linesa 24-h IC50 (μM)a 1. 2. 3. 4. 5. 6. 7. 8. a

−1

substance

Mw, (g·mol )

Colo 320HSR

CaCo-2

3215 LS

MX·2HCl MX-IL-BF4 IL-BF4 IL-PF6 IL-Br [C4MIM][BF4] [C4MIM][Cl] [C4MIM][PF6]

517.4040 1789.2145 354.1966 412.3572 347.2970 226.02 174.67 284.18

5.2 (2.4−8.0) 11.2 (6.6−15.7) >500 >500 >1000 b b b

7.1 (1.7−12.5) 17.0 (3.3−30.7) >350 >1000 >1000 >400047 >1900047 b

17.4 (9.0−25.7) 39.2 (28.7−49.6) >350 >350 >1000 >1000028 >1500028 >1000028

95% confidence intervals are shown. bNot tested.

selective activity of both MX·2HCl and MX-IL-BF4 can be achieved in particular cancerous cells. Improvement of Detection Limit Using Ionic Liquid Mitoxantrone Conjugate. In order to reveal potential advantages of employing the ionic liquid mitoxantrone conjugate in the studies on cytotoxic behavior, we estimated its detection limit by mass spectrometry. Notably, ESI-MS was by an order of magnitude more sensitive to MX-IL-BF4 than to the original mitoxantrone dihydrochloride. The detection limit of the former was 1 × 10−8 M, as compared to 1 × 10−7 M for the latter (see Table 3 and Figures S-15 − S-19). MX-IL-BF4

IL-BF4, IL-PF6, and IL-Br. For this purpose, we used the Colo 320HSR and CaCo-2 (colon adenocarcinoma) cell lines, together with 3215 LS (normal human fibroblasts). The results are shown in Table 2 (expressed as 24-h IC50, half maximal inhibitory concentration after 24 h exposure). According to our data, the resulting mitoxantrone-based conjugate (MX-IL-BF4) demonstrated slightly lower cytotoxic activity than mitoxantrone dihydrochloride (entries 1 and 2 in Table 2). Nevertheless, considering the confidence intervals, we suppose the IC50 values of both compounds comparable for the cell lines investigated. As expected, IL-BF4, IL-PF6 and IL-Br (entries 3−5 in Table 2), as well as the common commercial ionic liquids 1-butyl-3-methylimidazolium tetrafluoroborate ([C4MIM][BF4]), 1-butyl-3-methylimidazolium hexafluorophosphate ([C4MIM][PF6]), and 1-butyl-3-methylimidazolium chloride ([C4MIM][Cl]) (entries 6−8 in Table 2), did not demonstrate significant cytotoxicity in the concentration range studied.28,47 It should be noted that a mitoxantrone analogue bearing a methylene unit instead of the distal amino groups did not induce DNA cross-linking in tumor cells suggesting the importance of these groups for the drug action.53 These amino groups are supposed to participate in the formation of adducts between DNA and formaldehyde-activated mitoxantrone.44 However, in our work, substitution of the distal amino groups of mitoxantrone with the carboxylic groups of ionic liquid did not lead to significant disturbance of its activity. Contrariwise, the addition of four decyl chains and imidazolium cores to the mitoxantrone molecule resulted in the MX-IL-BF4 conjugate with preserved cytotoxic activity thus demonstrating good compatibility of the API-IL approach with the drug action. We suppose that though the introduction of the mitoxantrone group into the IL may reduce the activity of the former, the IL part of the conjugate can facilitate its penetration into the cell, thus counterpoising the reduction. According to the MS data, the conjugate acted as a single moiety in CaCo-2 cells: no ionizable products of its degradation were observed in the spectra. Interestingly, comparison of 24-h IC50 of the studied substances obtained in Colo 320HSR and in 3215 LS (human fibroblasts) suggested certain differences between them. Thus, MX·2HCl and MX-IL-BF4 manifested lower cytotoxicity in normal fibroblasts (entries 1 and 2 in Table 2), whereas IL-BF4, IL-PF6, and IL-Br displayed relatively low cytotoxicity regardless of the cell line origin (entries 3−5 in Table 2). Similar observations were made for [C4MIM][BF4], [C4MIM][PF6], and [C4MIM][Cl] both in CaCo-2 and 3215 LS cells (entries 6−8 in Table 2).28,47 These results imply that

Table 3. ESI-MS Detection Limit of Mitoxantrone Dihydrochloride and Ionic Liquid Mitoxantrone Conjugatea

a

Excellent, good, and fair correspond to signal visibility in the experimentally recorded ESI-MS spectra as evidenced from the signalto-noise ratio. None: Signal was not detected.

produced signals of excellent intensity at 1 × 10−5 and 1 × 10−6 M, and was seen distinctly at 1 × 10−8 (Figure S-15). The original mitoxantrone produced excellent signals only at 1 × 10−5, whereas at 1 × 10−8 M, the corresponding signals disappeared completely (Figure S-15). Different mechanisms involved in the formation of charged species are responsible for the observed enhancement of the detection limit. In the case of the original mitoxantrone, the charge acquirement in solution is governed by the acid/base equilibrium. The amino groups are most preferable for the proton attachment, but the dissociation is enabled due to the low strength of an acid being formed (Figure 3A). Only the ion bearing the charge (+1) was detected in the experimental ESIMS spectra under studied conditions, whereas none of the multiply charged ions (+2 ... +4) were observed (Figure 3A). In contrast to mitoxantrone, the charge of the MX-IL-BF4 conjugate is well-defined in solution due to the ionic nature of the molecular framework. Dissociation of the anion (BF4−) occurs easily, and the overall charge (+4) of the mitoxantrone conjugate is readily achieved. Dissociation of positively charged tags is not possible from the MX-IL-BF4 conjugate due to the covalent linkages between the ionic liquid tags and the drug E


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MS Detection of Mitoxantrone and Its Ionic Liquid Conjugate in Cells. Because of typical heterogeneity of cell populations, single-cell analysis finds numerous practical applications in various fields of biology and medicine. To estimate the number of cells in which the drug could be detected, we conducted an ESI-MS study in human fibroblasts and CaCo-2 cells, which were incubated with 20 μM of MX· 2HCl or MX-IL-BF4 for 1 h. The results are shown in Figures 5 and S-20−S-27 (calculated theoretical spectra of MX-IL-BF4 and mitoxantrone dihydrochloride are given for comparison in Figure 5A and Figure 5F, respectively). All the experiments were repeated independently several times in order to establish the reproducibility of the approach. A significant part of MX-IL-BF4 was found in the fraction of dead or damaged cells, where its high-intensity signals were observed (Figure 5D). Remarkably, the signals were evident in the sample containing about 100 dead/damaged cells (Figure 5C). The conjugate was also present in the fraction of living cells (Figure 5B) and in the medium, in which the cells were incubated (Figure 5E). In contrast, low-intensity signals of mitoxantrone dihydrochloride were seen only in the medium (Figure 5J), but were not detectable in any fraction of dead/ damaged or living cells (Figure 5G−I; see also Figure S-21). First, the signals of MX-IL-BF4 were present in the “clearer” region of lower masses in the spectra which contained less peaks of irrelevant compounds, as compared to the signals of MX·2HCl (Figures S-20−S-21, S-24, and S-25). Second, the detection limit enhancement due to the presence of charged tags played an important role in the analysis. To test the reliability of the developed approach, the remaining living cells were also studied. Apparently, upon accumulating the mitoxantrone conjugate, human fibroblasts detached from the plate, and only those containing small quantities of the drug and retaining the contact with the surface could be found in the samples of living cells. Thus, there was no signals of the MX-IL-BF4 conjugate in the sample containing ∼1000 living cells of 3215 LS (Figure S-20). Similar results were obtained in CaCo-2 cells (Figures S-24− S-27). The signals of the MX-IL-BF4 conjugate were seen in the sample containing about 1000 dead/damaged cells, as well as in the fraction of dead or damaged cells, in the fraction of living

Figure 3. Charge of mitoxantrone and MX-IL-BF4 conjugate in solution. (A) In the case of mitoxantrone, only the moiety bearing the charge (+1) was detected in the ESI-MS spectrum. (B) In the case of the MX-IL-BF4 conjugate, the charge (+4) was provided by covalent bonding of four charged tags.

molecule (Figure 3B). High strength of covalent bonds renders the (+4) charge an inherent molecular property of the synthesized conjugate. Since mass-spectrometric measurements detect m/z ratio, the signals of MX-IL-BF4 were shifted to a lower mass region due to the multiple charge (+4) on the API-IL cation, in contrast to mitoxantrone, which possessed only the charge (+1). Thus, the former were observed in the region of 360.2− 361.3 m/z, whereas the latter were found in the region of 445.2−447.3 m/z (Figure 4). Therefore, in addition to the sensitivity improvement, the synthesized drug conjugate demonstrated a signal shift to a lower-mass region in the spectrum. These important advantages inspired us to try spotting the presence of the drug at the level of individual cells.

Figure 4. Shift of ESI-MS signals of MX-IL-BF4 to the region of lower masses, as compared to MX·2HCl (the asterisk denotes the erucamide contaminant signal). F


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Figure 5. ESI-MS spectra of human fibroblasts treated with MX-IL-BF4 conjugate (B−E) and original mitoxantrone dihydrochloride (G−J): theoretically calculated spectra (A, F); fraction of living cells (B, G); ∼100 dead/damaged cells (C, H); fraction of dead/damaged cells (D, I); medium after incubation (E, J).

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cells and in the medium (Figure S-24). In contrast, the signals of mitoxantrone dihydrochloride were seen only in the fraction of dead cells and in the medium (Figure S-25).

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.7b03568. NMR and mass spectra of the synthesized compounds; mass spectra for determination of ESI-MS detection limit of mitoxantrone and its ionic liquid conjugate; mass spectra for detection of mitoxantrone and its ionic liquid conjugate in cells. (PDF)

CONCLUSIONS

The application of the charged tags provided by the API-IL strategy led to the design of the MX-IL-BF4 drug, which was oil at room temperature and therefore was supposedly resistant to the polymorphism issue. The synthesized mitoxantrone conjugate substantially retained its cytotoxic activity in the ionic liquid form. The ESI-MS sensitivity to MX-IL-BF4 was by an order of magnitude higher as compared to mitoxantrone dihydrochloride. Moreover, the MX-IL-BF4 signals were shifted to a “clearer” lower-mass region of the spectrum. Thus, the intrinsically encoded charged structure ensured the improved ESI-MS detection limit. The ESI-MS study confirmed penetration and accumulation of MX-IL-BF4 in the cells. The present study demonstrates an excellent potential of the API-ILs approach to incorporate charged ionic liquid tags in drug development platforms and opens new prospects in medical analytics. The detection of MX-IL-BF4 in the sample containing as few as ∼100 cells implies the possibility of using ionic liquid modifications of common drugs not only for refining their bioavailability and efficiency, but also for easy tracking of their accumulation, translocation and transformation in an organism.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Dmitry B. Eremin: 0000-0003-2946-5293 Valentine P. Ananikov: 0000-0002-6447-557X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Russian Science Foundation (RSF grant 14-13-01030). REFERENCES

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