Toxicity Evaluation of Choline Ionic Liquid-Based Nanocarriers of Pharmaceutical Agent For Lung Treatment With Extra Antitumor Activity

In vitro cytotoxicity evaluation of linear copolymer (LC) containing choline ionic liquid units and its conjugates with an antibacterial drug in anionic form, i.e. p-aminosalicylate (LC_PAS), clavulanate (LC_CLV), or piperacillin (LC_PIP) was carried out. These systems were tested against normal: human bronchial epithelial cells (BEAS-2B), and cancers: adenocarcinoma human alveolar basal epithelial cells (A549), and human non-small cell lung carcinoma cell line (H1299). Cells viability, after linear copolymer LC and their conjugates addition for 72 hours, was measured at concentrations range of 3.125 ‐ 100 μg/mL. The MTT test allowed the designation of IC 50 index, which was higher for BEAS-2B, and signicantly lower in the case of cancer cell lines. The cytometric analyzes, i.e. Annexin-V FITC apoptosis assay and cell cycle analysis as well as gene expression measurements for interleukins IL6 and IL8 were carried out, and showed pro-inammatory activity of tested compounds towards cancer cells, while it was not observed against normal cell line. In summary, the anticancer property of linear copolymers with antibacterial drugs was shown. which


Introduction
Polymer-drug conjugates are a common form of drug delivery systems, which are designed to improve drug solubility, control, and targeting therapy 1 . The other advantages have been reported for adjustment of drug release kinetics pro le by the strength of chemical bonding as the conjugation sites and the type/length of the linker connecting drug with the polymer matrix. The presence of reactive carboxylic or hydroxyl and amine groups in polyelectrolytes is convenient for conjugation reactions 2,3 . The speci c type of conjugates are corresponding to the ionic polymers with structures of complexes that use noncovalent bonds, which gain much consideration because of a variety of actions, e.g. electrical conductivity 4 , electrochemical 5 , anti-electrostatic 6 , energetic 7 , and optical 8 properties, as well as herbicidal 9 , or biological activity 10,11 . The last property of ionic conjugates can be generated by ionic conjugation of the pharmaceutical compounds to provide drug delivery polymer systems. The main requirements for polymeric carriers are related to biocompatibility and non-toxicity. Therefore, among the vehicles, the ionic conjugates can be especially distinguished, notably those based on ionic liquids (ILs), due to their unique bene ts, including the concept of green chemistry and their ability to ion exchange, which is a strategic way for the introduction of the active pharmaceutical ingredients (API) as the counterions [11][12][13] . Some of ILs are the well-known antibiotics (ampicillin, penicillin G), anti-in ammatory (pravadoline, ibuprofen, naproxen, salicylate), antiviral (tri uoridine), or antifungal (clioquinol) compounds, whereas the others have been used to obtain pharmaceutical precursors, such as lactam, pyrazolone, thiazole, imidazole, and thiazolidine 14 . Generally, API-Ils display enhanced solubility in water or dissolution rates of pharmaceutics 15 increasing their bioavailability 16 when compared with the original APIs. For medical purposes, it is highly desirable when the IL polymer matrix offers biological activity, namely antioxidant, anti-tumoral and antimicrobial activity 13 as it has been proved for tetrabutylammonium-, phosphonium-, imidazolium-, pyridinium-, piperidinium-, pyrrolidinium-or cholinebased cations [17][18][19][20][21] . Especially, the polymerizable ILs, e.g. 1-(4-vinylbenzyl)-3-methyl imidazolium and 1-(4-vinylbenzyl)-4-(dimethylamino)-pyridinium hexa uorophosphate 22 , 2,2-diallyl-1,1,3,3 tetraethylguanidinium chloride 23 , vinyl benzyl trimethylammonium chloride 24 , [2-(methacryloyloxy)ethyl]trimethylammonium chloride is also known as choline methacrylate [25][26][27] seem to be attractive in the polymer synthesis for drug delivery. The latter one has been also applied to obtain the graft copolymers with polyIL side chains carrying API anions with anti-in ammatory and anti-coagulant properties 28,29 .
Depending on the type of IL or API used, the obtained systems can be used in miscellaneous therapies.
Due to the deterioration of the general immune system in the body as a result of a disease caused by various pathogens, the weakened organs are particularly vulnerable to cancer [30][31][32] . For this reason, the drug delivery systems with extra anticancer activity are highly desirable. Recently, the ILs with ampicillin 16 , taurine 33 , or p-aminosalicylate and clavulanate 34 have been investigated as potential systems with additional antitumor activity.
Lung cancer as a common and deadly disease is often caused by various external factors, such as pollution of the environment or smoking, as well as by health factors and history of untreated lung diseases 35,36 . Therefore, in this paper, we report the cytotoxicity evaluation against cancer cell lines, i.e. adenocarcinoma human alveolar basal epithelial cells (A549) and human non-small cell lung carcinoma cell line (H1299), and normal one, namely human bronchial epithelial cells (BEAS-2B), of the ionic drugpolymer carrier. The previously synthesized well-de ned linear copolymers of (2-trimethylammonium)ethyl methacrylate and methyl methacrylate (LC) have been investigated successfully by their ability to self-assembling and anion exchange 37 . The biological activity of the ionic copolymers has been generated by the formation of its conjugates with antibacterial drugs in ionic form, i.e. p-aminosalicylate, (PAS), clavulanate (CLV), piperacillin (PIP), which are used in lung diseases treatment, especially tuberculosis. Satisfactory physicochemical properties of the nanocarriers and their drug release facility are su cient for improvement of the system characteristics with the cytotoxicity evaluation, including colorimetric tests with the use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and cytometric analyses by ow cytometry, i.e. apoptosis assay and cell cycle analysis, to con rm their bene cial application as drug delivery systems.

Results And Discussion
Amphiphilic linear copolymer (LC) based on [2(methacryloyloxy)ethyl]trimethyl-ammonium chloride as the ionic liquid and methyl methacrylate as non-ionic component, i.e. P(TMAMA-co-MMA) was received as a nanocarrier for delivery of ionic pharmaceutical compounds with antibacterial activity, i.e. paminosalicylate (PAS), clavulanate (CLV) and piperacillin (PIP). The previous studies on physicochemical characterization, as well as the drug introduction, and release process have proved that the polymer obtained by ATRP at 40 C, where the ratio of TMAMA/MMA units was equal to 25/75, is a promising drug vehicle 37 . The polymer ability to self-organization was con rmed by critical micelle concentration (CMC = 0.055 mg/mL), whereas the formed nanoparticles including those with therapeutic counterions reached sizes ranging in 170290 nm. The characteristics of conjugate systems varied with drug type and content are presented in Table 1.
The chloride-containing poly(ionic liquid) and its conjugates with PAS , CLV , and PIP were screened for their in vitro cytotoxic activity PAS is an inhibitor of mycobactin biosynthesis and used as a second-line antituberculosis drug, usually applied to support other agents 38 . CLV is a compound that has a similar structure to penicillin and exhibits inhibitory properties of βlactamase. It is usually used in combination with other drugs such as β-lactam antibiotics (amoxicillin). Penicillinase enzyme, which attacks the βlactam antibiotic, is blocked by β-lactamase inhibitor, i.e. CLV. The action of CLV is thoroughly described in 39 . PIP is a β-lactam antibiotic with a high activity degree in comparison to other penicillin derivatives. It is a drug with a broad spectrum of action 40 . These agents (PAS, CLV, and PIP) are generally used in respiratory tract infections that are caused by various microorganisms, which may promote cancer.
Therefore, an additional effect on early-stage or yet undetected cancer is highly anticipated.
The drug delivery systems were tested against a human bronchial epithelial (BEAS-2B) to exclude the poisonous effect on normal cell lines, and a contrary panel of human cancer cell lines, namely adenocarcinoma human alveolar basal epithelial cells (A549) and human non-small cell lung carcinoma cell line (H1299) to evaluate the anticancer activity as an additional property of the given drug nanocarriers, which can be used in the treatment of lung disease with co-existing cancer. Table 1 Characteristics of linear TMAMA copolymer (LC) and its conjugates with PAS, CLV, and PIP anions 37  LC: P(TMAMA-co-MMA) with the average molecular weight M n = 46900 g/mol, and dispersity index Ð = 1.74; DP n is the polymerization degree; F TMAMA is the content of TMAMA units; D h is hydrodynamic diameter by dynamic light scattering (DLS); WCA is water contact angle via goniometry using the sessile drop method; DC is drug content evaluated by UV-Vis.
The cells con uence was live-observed (from the MTT plates) and expressed as the percentage of the culture covered the bottom of the well (96-well plates) after 72h of incubation with the compound (LC and its conjugates at a concentration of 100 µg/mL, presented as % of control, Figure 1). It was reported that the polymer with Cl and CLV counterions caused an increase of the con uence in comparison to untreated control (CTR) of normal and cancer cells. Especially in the case of BEAS-2B (LC: 171 %; LC_CLV: 175% of the control). The conjugates with PAS and PIP induced the decrease of con uence, but these systems provoked a smaller decline in BEAS-2B cells con uence compared to the CTR (~60% decrease) than the cancer cells, where the con uence decreased up to 8-30% of CTR. Microscopic images of cells treated by tested systems are shown in Figures 1-4.
The colorimetric MTT test was carried out to evaluate the IC 50 index as the value of a compound concentration corresponding to a 50% growth inhibition. This parameter was determined only for compounds, for which a linear relationship between cell viability and the concentration was observed. The IC 50 index was not determined in the case of chloride-based LC, which did not cause the proliferation of 50% of the normal or cancer cells population. However, it was con rmed for the conjugates with PAS, CLV, or PIP, which selectively affected cells (Table 2). In the case of BEAS-2B cells, the IC 50 index was higher than 100 µg/mL, which means that only a high concentration of these compounds would have a toxic effect on normal cells. In turn, these systems have a higher toxic effect on cancerous cells, due to the lower IC 50 values (49-73 µg/mL). The lowest IC 50 was detected for LC_CLV in the treatment of A549 and H1299 cell lines, and the LC_PIP treated H1299 cell line, indicating the most toxic effect.   Figure 9). The acute and late phase in ammation marker IL6 in BEAS-2B showed on the physiological feature of epithelial cells from the respiratory system -the protection through the in ammation process against damaging agents and pathogens 42 . The IL8 gene expression in BEAS-2B cells was elevated above the control level, but similar for tested copolymers and relatively low ( Figure 9).
In cancer A549 and H1299 cells after 72 h of incubation, the marker gene IL6 for the late phase of acute in ammation was not expressed (Figure 10 and 11), similar to the low control level. Although the IL8 gene was activated, the cancer cell lines responded with different elevated expression levels. The A549 cells were more sensitive to stimulation of in ammatory cytokines production observed on the transcriptional level. The expression level in A549 cells after 72 h of incubation with LC resulted in lowered level in comparison to the untreated control, whereas LC_PAS was almost 4-times higher ( Figure  10). The LC_CLV and LC_PIP did not much in uence the pro-in ammatory gens expression.
Modi cation of copolymers and addition to the linear form (LC) additional drugs (LC_PAS, LC_CLV, and LC_PIP) showed increased potential in in ammatory pathway activation, especially in cancer cells. Both IL6 and IL8 cytokines are important chemokines for TNF α (tumor necrosis factor α) pathway regulation 43 and tested copolymers overstimulated IL8 expression better than LC in H1299 cells (Figure 11). In the more aggressive type of lung cancers, such as adenocarcinoma H1299, IL8 activation promotes also in ammation burst accelerating cell death (pathway activated also in epithelial cells of the pulmonary system upon viral and microbial pathogen infection) 44 . Considering possibilities to stimulation of such a mechanism of action, it could be a promising application for copolymers as antimicrobial and anticancer drugs in the future.

Conclusions
Cytotoxicity evaluation of nanocarriers based on choline linear copolymer (LC) and its ionic conjugates with PAS, CLV, and PIP (LC_PAS, LC_CLV, LC_PIP) as respiratory disease treatment systems, was carried. Due to the purpose of these agents, studies have been conducted against normal BEAS-2B cell lines. However the weakened immune system or often latent, early-stage form of tumor, is the cause of cancer promotion and development, so the extra antitumor activity of such systems is desirable. Therefore the toxicity against cancer cell lines, i.e. A549 and H1299 were also indicated.
Test MTT, cytometric analyses (apoptosis assay and cell cycle analysis), and gene expression measurements for interleukins IL6 and IL8 demonstrated the selectivity of action, wherein the systems: LC, and conjugates with PAS and PIP, did not harm BEAS-2B cell line, while they induced the proliferation of cancer cells. The most cytotoxic activity was shown by the LC_CLV system.
The mechanism of action pro le towards tested cell lines of investigated compounds is desirable. The

Characterization
Live Cell Analyzer (JuLI™ Br; NanoEnTek Inc., Seoul, Korea) was used to measure con uence, cell density, and viability monitoring analysis. Cell viability assay was performed by measuring the absorbance of the MTT reaction product (formazan) at 570 nm in a microplate reader (Epoch, BioTek, Winooski, VT, USA). The results of apoptosis and cell cycle analysis were obtained with the use of an Aria III ow cytometer (Becton Dickinson; Franklin Lakes, NJ, USA).

Cell culture
The cells were grown in a DMEM-F12 medium in sterile culture bottles (75 cm 2 of culture area) with 10% (v/v) FBS addition and kept at 37 C in the incubator with humidi ed atmosphere with 5% CO 2 . For the MTT tests, the cultures were plated in 96-well plates. For apoptosis and cell cycle assays the cell cultures were plated in 6-well plates.

Flow cytometry
10,000 cells were placed into 6-well plates in 2 mL of medium for 24 h. After treatment, each well was lled with 2 mL of polymer or conjugate sample. The incubation was carried out for 72 h. After that, the solution from the wells was divided into three parts.
The rst part was placed into sterile vials and centrifuged for 3 min (0.6 × g, RT). 50 µL of cold Annexin-V labeling buffer and 2.5 µL of FITC-labeled Annexin-V antibody were added to the pellet. Suspended cells were incubated for 30 min in darkness. Then, 250 µL of Annexin-V labeling buffer was added. Samples were tested immediately after preparation using an Aria III ow cytometer.
The second part of the solution was centrifuged, and then the supernatant was removed. After the addition of 250 µL of hypotonic buffer (hypotonic buffer comprised from PI 100 µg/mL in PBS; 5 mg/L of citric acid; 1:9 Triton-X solution; RNase 100 µg/mL in PBS from Sigma, Poznań, Poland) the samples were incubated for 15 min in cold and dark area. The DNA states were determined by uorescence measurements via BD FACS Aria™ III sorter (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) using PE con guration.

RT-qPCR genes expressions for pro-in ammatory interleukins IL6 and IL8
The third part of the cells solution was collected by trypsinization. From the pellet, RNA was extracted using a phenol-chloroform method, with the procedure from the Total RNA Isolation kit (A&A Biotechnology). The RNA concentration and quality were estimated by UV-Vis spectrophotometer (Nanodrop, Thermo sher). The commercial set of reagents (Real-Time 2xPCR Master Mix SYBR A; A&A Biotechnology) and following Genomed pairs: IL6 (AGATCACCTAGTCCACCCCC) and (GTTCTGCCAAACCAGCCTTG); IL8 (GGTGCAGTTTTGCCAAGGAG) and IL-8 reverse (ACCAAGGCACAGTGGAACAA); reference RPL41 (ACGGTGCAACAAGCTAGCGG) and (TCCTGCGTTGGGATTCCGTG) was used to gene expression assays for pro-in ammatory (IL6, IL8). The thermocycler CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad) was used to execute the quantitative PCR reaction, followed by reverse transcription (NG dART RT kit, EURx). For qRT-PCR reaction the following thermal pro le was used: I) 50°C, 2 min, II) 95°C, 4 min, III) 54 cycles ended with uorescence reading: 95°C, 45 s.; 52,3°C, 30 s.; IV) 72°C, 5 min, V) melting curve in the temperature range 5292 °C (every 0,5°C at 5 s), VI) incubation carried at 4°C. The thermal pro le of the reaction was described previously in 45       Typical dot plots of cell populations after treatment with LC, LC_PAS, LC_CLV, LC_PIP compared to control untreated cells by ow cytometry in BEAS-2B, A549, and H1299 cell lines.   Relative gene expression level, for pro-in ammatory IL6 and IL8 cytokines in BEAS-2B cells after 72 h of incubation [ ], results from 3 repeats with +/-5% error bar. Cells were treated with a dose of 0 (CTR) or 50 μg/mL; LC-CLV -not analyzed.

Figure 10
Relative gene expression level, for pro-in ammatory IL6 and IL8 cytokines in A549 cells after 72 h of incubation [ ], results from 3 repeats with +/-5% error bar. Cells were treated with a dose of 0 (CTR) or 50 μg/mL; LC-CLV -not analyzed.

Figure 11
Relative gene expression level, for pro-in ammatory IL6 and IL8 cytokines in H1299 cells after 72 h of incubation [ ], results from 3 repeats with +/-5% error bar. Cells were treated with a dose of 0 (CTR) or 50 μg/mL; LC-CLV -not analyzed.

Supplementary Files
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