- Email: [email protected]
Antimicrobial properties of benzalkonium chloride derived polymerizable deep eutectic solvent
Journal Pre-proofs Antimicrobial properties of benzalkonium chloride derived polymerizable deep eutectic solvent Jing Wang, Jing Xue, Xiaoqing Dong, Qingsong Yu, Sheila N. Baker, Ming Wang, Haofei Huang PII: DOI: Reference:
S0378-5173(19)31066-X https://doi.org/10.1016/j.ijpharm.2019.119005 IJP 119005
To appear in:
International Journal of Pharmaceutics
Received Date: Revised Date: Accepted Date:
16 July 2019 24 November 2019 26 December 2019
Please cite this article as: J. Wang, J. Xue, X. Dong, Q. Yu, S.N. Baker, M. Wang, H. Huang, Antimicrobial properties of benzalkonium chloride derived polymerizable deep eutectic solvent, International Journal of Pharmaceutics (2019), doi: https://doi.org/10.1016/j.ijpharm.2019.119005
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier B.V.
Antimicrobial properties of benzalkonium chloride derived polymerizable deep eutectic solvent Jing Wanga,b,*, Jing Xuea, Xiaoqing Dongb, Qingsong Yub, Sheila N. Bakerc, Ming Wanga, Haofei Huanga
a
School of Chemistry and Chemical Engineering, Shandong University of Technology, Zibo, Shandong, China
b
Department of Mechanical & Aerospace Engineering, University of Missouri, Columbia, MO, United States.
c
Department of Chemistry, University of Missouri, Columbia, MO, United States.
* Corresponding Author: Jing Wang, Ph. D. Phone: +86 17615695097 E-mail: [email protected] Address: 266 Xincun Road Shandong University of Technology Zibo, Shandong 255000 China
E-mail addresses: Jing Xue: [email protected] Xiaoqing Dong: [email protected] Qingsong Yu: [email protected] Sheila N. Baker: [email protected] Ming Wang: [email protected] Haofei Huang: [email protected]
1
Abstract Benzalkonium chloride (BC) is a quaternary ammonium antimicrobial agent used in a variety of applications. In this work, BC was prepared into deep eutectic solvent (DES) with acrylic acid (AA) or methacrylic acid (MA). Within the newly prepared DES, BC is responsible for antimicrobial properties, while AA and MA are responsible for polymerization. Three types of microorganisms, E. coli (gram-negative bacilli), S. aureus (gram-positive cocci) and C. albicans (fungi), were assessed for antimicrobial properties through agar diffusion test. DES viscosity measurements and polymerizations were also conducted to assist the antimicrobial performance analysis. From this study, stronger antimicrobial effectiveness of BC-AA DES towards S. aureus and C. albicans was observed, while smaller inhibition zone widths were obtained for BC-AA DES polymer compared to BC-AA DES monomer which may due to the limited active component transportation after polymerization. When changing AA to MA, increased structural complexity and decreased linearity may limit the molecule movement thus reduce the inhibition zone width, which could be proved by the calculated activation energy results. Accurately determined eutectic ratio of DES is recommended to get optimized drug release control. This work offers a new sight for preparation of antimicrobial materials with stronger effectiveness and limited release.
Keywords Antimicrobial property; benzalkonium chloride; deep eutectic solvent; drug release
2
1. Introduction Benzalkonium chloride (BC) is one of the most commonly used pharmaceuticals for antimicrobial purposes, which is frequently found in clinical, food line and domestic household cleaning products (Kampf and Kramer, 2004; Mangalappalli-Illathu and Korber, 2006). BC is an effective quaternary ammonium compound in inhibiting a variety of microbial such as bacteria, fungi and yeasts (Fazlara and Ekhtelat, 2012). When added into bulk materials, BC could ascribe the materials with antimicrobial properties, which is an easy approach to develop antimicrobial bulk materials. Instead of adding BC directly, we previously prepared BC into the form of deep eutectic solvent (DES) before incorporating it into an active dental composite (Wang et al., 2017), and an limited release of BC was observed with longer lasting antimicrobial effectiveness of the newly developed dental composite. In another study by Sánchez-Leija et al. (Sánchez-Leija et al., 2014), a controlled release of lidocaine hydrochloride was also reported when transformed into DES. Therefore, it is of great interest to target the BC derived DES to further study its antimicrobial performance before and after polymerization, which is beneficial to the development of antimicrobial materials with controlled drug release. DESs are regarded as an alternative to ionic liquids (ILs), and they share common properties like high viscosity, low vapor pressure and thermal stabilities (Tang and Row, 2013). ILs are salts at liquid state (Rogers and Voth, 2007), while DESs are mixtures of two or more components at a certain ratio leading to eutectic point commonly lower than the melting point of the single component (Abbott et al., 2003). The Type III DES is the most widely studied category, which is commonly formed of quaternary ammonium salt with halide anion and a hydrogen bond donor (HBD) (Zhang et al., 2012). DES has been utilized as solvent to increase the solubility of pharmaceuticals (Shekaari et al., 2017) and for antimicrobial applications (Wikene et al., 2015).
3
Rare study were found to prepare a specific pharmaceutical into DES for antimicrobial purposes. Hayyan et al. (Hayyan et al., 2013) investigated the antimicrobial properties of some common choline chloride based DESs with urea, glycerine, triethylene glycol, and ethylene glycol as HBDs. Although they didn’t find inhibition effect for the tested DESs, they suggested that the inhibition behavior of DESs depends on different component structures. Hayyan et al. (Hayyan et al., 2013) further studied the toxicity of some phosphonium-based DESs using two Gram positive bacteria, Bacillus subtilis (B. subtilis) and Staphylococcus aureus (S. aureus), and two Gram negative bacteria, Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa), and stronger antimicrobial effects were observed for DES compared to their individual components. The succedent studies on ammonium-based DESs (Hayyan et al., 2015) and cholinium-based DESs (Juneidi et al., 2015; Wen et al., 2015) reached a similar conclusion that the microbial inhibition effects of DESs strongly depend on the compositions of DESs. Similarly to ILs, DESs were also introduced into the polymer sciences, but not a lot of successfully remarkable work could be found for synthesis of polymers by using DESs as monomers (Carriazo et al., 2012). Monta-Morales et al. (Mota-Morales et al., 2013) mixed ammonium salts and HBDs of acrylic acid (AA) and methacrylic acid (MA) to obtain DESs for frontal polymerizations, and these DESs played an all-in-one role as monomer, filler and reaction medium. In our work, two designed BC derived polymerizable DES were prepared with BC&AA and BC&MA, which possess antimicrobial properties because of the incorporation of BC, and can perform polymerization as monomers due to the existence of AA and MA. These two DESs form through strong hydrogen bonding interactions between the ammonium and carboxyl structures. The eutectic point of the prepared eutectic mixtures was determined with Fourier transform infrared spectroscopy (FTIR) method. Viscosity measurement and degree of conversion (DC%)
4
tests were conducted for DES characterizations. Antimicrobial performances of DES before and after polymerization were evaluated through agar diffusion tests using three representative organisms, E. coli (gram-negative bacilli), S. aureus (gram-positive cocci) and C. albicans (fungi). The effect of DES components, polymerization and eutectic ratios on antimicrobial performance are thoroughly studied and discussed. This work is providing insightful information on development of antimicrobial materials with strengthened effectiveness and controlled drug release.
2. Materials and Methods
2.1 Reagents and Materials Benzalkonium chloride (BC), acrylic acid (AA), methacrylic acid (MA), camphorquinone (CQ) and ethyl 4-dimethylaminobenzoate (4E) were purchased from Sigma-Aldrich (St. Louis, USA). Potassium bromide (KBr) was purchased from Acros Organics (New Jersey, USA). Chloriform-d was purchased from Aladdin (Shanghai, China). All chemicals were used as received without further purification. Sterile filter paper (Qualitative P5) used for agar diffusion test was purchased from Fisher Scientific. The test organisms used in this study were Escherichia coli (E. coli, ATCC 5922, gramnegative bacilli), Staphylococcus aureus (S. aureus, NRS234, gram-positive cocci) and Candida albicans (C. albicans, ATCC 18804, fungi). The bacterial strains were purchased from ATCC (E. coli and C. albicans) and NARSA (S. aureus). The strains of E. coli and S. aureus were grown in Todd Hewitt broth containing 0.2% yeast extract and tested on Todd Hewitt Agar. The C. albicans was cultured in Sabouraud dextrose broth and tested on Sabouraud dextrose agar. All bacterial
5
cultures were incubated at 37 °C.
2.2 DES Preparation The eutectic mixtures were prepared by stirring BC & AA or BC & MA (Figure 1) with certain molar ratios at 60 °C until homogenous clear and colorless liquid formed. A Cary 660 FTIR spectrometer (Agilent Technologies) was used to determine the eutectic ratio of the newly prepared DES, where DES was pressed into a KBr pellet, and absorbance spectrum between 400 cm-1 and 4000 cm-1 were collected. A Bruker Avance III HD 400 MHz NMR was also used to further confirm the formation of hydrogen bond and the eutectic ratio of DES, where chloroform-d was used as solvent.
2.3 Viscosity Measurement Viscosities were measured using a Brookfield DV-III Ultra Programmable Rheometer (Spindle CPE-42, 0.2 – 6000 cP), and different temperatures were maintained through a Fischer Scientific Isotemp® temperature bath. Each temperature was maintained for at least 15 minutes before the readings, and the torque were controlled between 20-80% by changing the RPM for each reading. 5-10 viscosity readings were collected and averaged for each temperature, and modeling fittings were conducted using Sigma Plot 12.1.
2.4 DES Polymerization A light initiated polymerization system of DES/CQ/4E were constructed with camphorquinone (CQ) as the initiator and ethyl 4-dimethylaminobenzoate (4E) as the co-initiator. Since only small amount of initiators were needed for polymerization, CQ and 4E were dissolved in an acetone stock solution and was added upon requirements. KBr pellets containing DES/CQ/4E were dried 6
and measured using a Cary 660 FTIR spectrometer (Agilent Technologies) before and after light cure (Densply Spectrum® 800 curing light system). Absorbance spectrum between 400 cm-1 and 4000 cm-1 were collected at the sensitivity of 1.5, resolution of 4, background and sample scan numbers of 32. FTIR data were processed with Agilent Resolution Pro Software. DC% was able to be calculated by equation (1) based on the peak heights of absorbance. 𝑃𝑜𝑙𝑦𝑚𝑒𝑟
𝐴
𝑃𝑜𝑙𝑦𝑚𝑒𝑟
/𝐴
C=C C=O DC% = (1 − 𝐴𝑀𝑜𝑛𝑜𝑚𝑒𝑟 ) × 100% /𝐴𝑀𝑜𝑛𝑜𝑚𝑒𝑟 C=C
C=O
(1)
2.5 Agar Diffusion Test For the monomer samples used for agar diffusion test, filter paper disks (7mm in diameter) were soaked with DES monomers without CQ or 4E, and were dried with compressed air to eliminate excess liquid. For the polymer samples used for agar diffusion test, filter paper disks with the same size were soaked with DES/CQ/4E mixtures. After a similar drying step, the disks were light cured with a Densply Triad® light cure system (bulb #70353) for 2 minutes on each side. The tested microorganisms were grown in their respective media to reach the optical density of 1.0. Suspensions of 0.1 mL of the microorganisms were uniformly spread on agar plates by using a sterile cotton bud. Inhibition zone width is calculated by equation (2): W=(d2-d1)/2
(2)
where W is the inhibition zone width, d1 is the diameter of the filter paper disk, and d2 is the diameter of the inhibition zone diameter. Each sample was tested in six replicates.
3. Results and Discussion
3.1 Eutectic Point Determination
7
The most widely accepted method to determine the eutectic point is naked eye observation of freezing points of mixtures at different molar ratios (Abbott et al., 2004; Abbott et al., 2003). In this study, however, the mixtures of BC and HBDs are liquids at room temperature, and they have relatively lower freezing points. Thus the FTIR method and NMR method were used in this case for the determination of eutectic point. The eutectic phenomenon is mainly caused by relatively strong molecule interactions, hydrogen bond, in this case. The formation of hydrogen bond mainly affects the –COOH in AA and MA, leading to changes in the position and intensity of absorbance peak of the C=O stretch at 1700-1725 cm-1 (Figure 2(A)) in the FTIR spectra. Single component of AA showed two separated peaks at 1700 cm-1 and 1725 cm-1, but the peak at 1700 cm-1 diminished at the shoulder of the peak at 1725 cm-1 for AA derived eutectic mixtures. Single component of MA showed a sharp peak at 1700 cm-1, but this peak shifted to 1710 cm-1 for MA derived eutectic mixtures. The formation of hydrogen bond can also be proved by the NMR spectrum of BC-AA mixtures (Figure 3) and BCMA mixtures (Figure 4). The chemical shift of –COOH proton was at δ=11.38 for AA (Figure 3(A)) and at δ=11.31 (Figure 4 (A)) for MA. After mixing with BC, the chemical shift moved to a lower value for both BC-AA (δ=10.58, Figure 3(C)) and BC-MA (δ=9.84, Figure 4(C)). It was also observed that with the increase of BC molar fraction, the chemical shift of –COOH proton changed larger (Figure 3(C) - (F), Figure 4(C) - (F),), and it diminished at high BC molar ratios (Figure 3(G), Figure 4(G)). From this point, both of the FTIR spectrum and NMR spectrum evidenced the formation of hydrogen bond for BC-AA and BC-MA. In the FTIR spectra of the eutectic mixtures (Figure S1 and Figure S2), it was observed that the peak intensity of C=O stretch become smaller with the increase of BC molar ratio. Therefore, taking C=C stretch within AA or MA at 1633 cm-1 as basis, the peak intensity ratios of
8
1725 cm-1/1633 cm-1 for BC-AA mixtures and 1710 cm-1/1633 cm-1 for BC-MA mixtures were tracked (Figure 2(B)). With the increase of BC molar fraction, for both BC-AA and BC-MA mixtures, the C=O/C=C peak intensity ratios showed a decreasing trend. It could be observed that there was a downward sharp in the overall decreasing trend, and the sharp was at BC:AA=1:2 and BC:MA=1:2.5. Similarly, when we track the peak area of δ=7.60 over δ (-COOH) in the NMR spectra, a transition could be obtained in the increasing trend with the increase of BC molar fraction, and the transition also happened at BC:AA=1:2 (Figure 3(H)) and BC:MA=1:2.5 (Figure 4(H)). Therefore, the eutectic ratio (molar ratio) was determined as 1:2 for BC:AA and 1:2.5 for BC:MA, respectively. Both DESs have freezing/melting point below room temperature.
3.2 DES Viscosities The dynamic viscosities (η) of DESs were measured in the range of 25-85 °C (Table S1). The viscosity data were firstly fitted to the logarithmic form of Arrhenius model: ln(𝜂) = ln(𝜂0 ) +
𝐸𝜂 𝑅𝑇
(3)
where η is the dynamic viscosity, η0 is a coefficient, Eη is the activation energy for viscous flow, R is the universal gas constant, and T is the temperature in K. The ln(η) is linear to 1/T from this model, and The fitting parameters of the data and associated r2 are listed in Table 1. To better describe the curvature of the data, another Vogel-Fulcher-Tamman (V-F-T) model was introduced (Figure 5). V-F-T model (equation (4)) usually fits better for ILs or DESs with small or symmetric chemical structures (Jin et al., 2008). B
ln(𝜂) = A + 𝑇−𝑇
0
(4)
where A and B are constant parameters, and T0 is the divergent temperature of the configurational entropy of the system vanishing. Based on the fitting parameter of T0, activation energy of Eη has 9
been able to be calculated by equation (5). The results are summarized in Table 2. T 2 ) 𝑇−𝑇0
𝐸𝜂 = RB(
(5)
The activation energy at a certain temperature is the minimum energy barrier that the molecules need to overcome in order to move in the current system. Larger activation energy indicates higher difficulty for the molecule movement. The recovered activation energy from Arrhenius model (Table 1) is slightly lower than that from the V-F-T model (Table 2), but they closely follow the same trend. The activation energy of BC-MA DES is larger than that of the BCAA DES, so the molecule movement of BC-MA DES is expected to be more difficult than that of the BC-AA DES.
3.3 DES Polymerization The FT-IR spectra of DESs before and after polymerization are shown in Figure 6(A). It is clearly observed that the peak at 1633 cm-1 (C=C stretch) decreased for both BC-AA DES and BC-MA DES. DC% has been calculated by equation (1) based on the peak intensity before and after polymerization. From Figure 6(B), when 0.5% CQ and 0.5% 4E were added for initiation, DC% increased with the increasing of curing time. At the curing time of 20s, DC% have approached the maximum, but curing time of 60s was chosen for further experiment to ensure reaching the maximum DC%. The highest DC% is about 74% for BC-AA DES, and is about 80% for BC-MA DES. These are relatively high DC% for a CQ/4E initiated system (Guo et al., 2008) and solidified materials have been obtained for further experiments, so the initiator addition level was not further optimized. At the same curing condition and initiator addition level, DC% of BC-AA DES is slightly smaller than that of BC-MA DES. As for the polymerized DES, BC-AA DES polymer exhibited high flexibility, while BC-MA DES polymer exhibited high rigidity (Figure 6(C)). After
10
storing in water for 3 days at room temperature, both of BC-AA DES polymer and BC-MA polymer were dissolved.
3.4 Antimicrobial Performance 3.4.1 Antimicrobial performance of BC-AA DES The inhibition zone width for the individual components, BC-AA DES monomer and BC-AA DES polymer are shown in Figure 7(A). For S. aureus and C. albicans, extremely large inhibition zone widths were observed with AA. The antimicrobial activity of AA might be attributed to its acid nature, low molecular weight, and α, β-unsaturation (Stack, 1957). The BC-AA DES monomer and polymer inhibition zone widths are slightly larger but close to that of BC. This indicated that the antimicrobial effect of BC-AA DES monomer and polymer mainly come from BC rather than AA. The formation of BC-AA DES leads to changes on carboxylic functional group of AA, which has been proved by the FTIR spectrum used for eutectic point determination in this study. From this point, the DES formation is considered as the main reason for the ineffectiveness of antimicrobial activity of AA within DES. On the other hand, the antimicrobial activity of BC mainly comes from its cationic amphiphilic structure, which has a hydrophobic hydrocarbon region and a hydrophilic region (McDonnell and Russell, 1999). The hydrophilic cationic region could destabilize bacterial surface by forming electrostatic interactions with negative charged components on cell membrane walls. Once close contact is accomplished by the hydrophilic region, the BC’s hydrophobic region proceeds to penetrate through the hydrophobic bilayer to cause cell leakage and lysis (Fazlara and Ekhtelat, 2012). The formation of DES only affects the halide part of BC rather than the cationic part. In other words, the effectiveness of antimicrobial activity of BC has been able to be maintained even after the formation of DES. For E. coli (gram11
negative), there was no significant difference observed on the inhibition zone width of BC-AA DES monomer, polymer and BC only. For S. aureus (gram-positive) and C. albicans (fungi), in comparison with BC only, there were slightly increases for the samples of BC-AA DES monomers, which indicated the increased antimicrobial effectiveness. In general, gram-positive bacteria and fungi are more susceptible to BC than gram-negative ones because of their distinct membrane structures. Gram-positive bacteria and fungi generally have a single membrane surrounded by a thick mesh-like peptidoglycan that allows for the penetration of small molecules. In contrast, gram-negative bacteria have thin polysaccharide walls overlaid by a thin layer of lipopolysaccharides that modulates the accessibility of a cell to antiseptics and other small molecules (Coughlin et al., 1983). This indicates that the BC-AA DES monomer has better antimicrobial effect towards the gram-positive bacteria and fungi than gram-negative bacteria. From previous studies, hydrogen bond formation within DES may limit the movement of BC, slower the mass transport of antimicrobial agent, which may lead to smaller inhibition zone widths (Hayyan et al., 2015). In this study, however, larger inhibition zone widths were observed for S. aureus and C. albicans, which indicated stronger antimicrobial effectiveness of DES despite of lower mass transport. The inhibition zone widths of BC-AA DES polymers decreased compared with BC-AA DES monomers, and remained at almost the same level as BC only. This may due to the formation of polymer chains and networks after DES polymerization. The larger molecular weights and complicated tangled polymer chains limit the movement of BC component leading to smaller inhibition zone width. Compared to the molecule interaction, hydrogen bond in this case, the covalent bond formation during polymerization has larger influence on mass transport of BC.
12
3.4.2 Antimicrobial performance of BC-MA DES The inhibition zone widths of individual components, BC-MA DES monomers, and BCMA DES polymers are shown in Figure 7(B). Similar to AA, MA also exhibited strong antimicrobial effectiveness, especially to C. albicans. However, the inhibition zone widths are much smaller than those of AA. Two main reasons might lead to this difference, weaker antimicrobial effectiveness or limited mass transport. Neither reason is important because the antimicrobial effective structure within MA is altered after formation of DES. Also similar to the discussion based on BC-AA DES, this study focused on the inhibition zone width of BC only, BCMA DES monomer and BC-MA DES polymer. The main difference in chemical structures of AA and MA is the methyl branch, which increased the molecular weight and disordered the chain linearity. Compared to BC only, BC-MA DES monomer didn’t produce larger inhibition zone width, which is slightly different from the BC-AA DES monomer result. This change may due to the increased molecular weight and the disordered linearity which is unfavorable for the molecule movement. This result also matches the higher activation energy of BC-MA DES than BC-AA DES from the viscosity measurements. The BC-MA DES polymer also produced similar inhibition zone width to BC only for all of the tested organisms, which is similar to the BC-AA DES polymer result. Although DC% of BC-MA DES was slightly higher than that of BC-AA DES at the same polymerization condition, the inhibition zone widths were not significantly affected. Weaker antimicrobial effectiveness of MA compared to AA and limited molecule movement or mass transport of BC-MA DES compared to BC-AA DES contributed to the smaller inhibition zone widths, and polymerization played similar roles during the antimicrobial behavior.
13
3.4.3 Effect of polymerization on antimicrobial performance of DES Since increased antimicrobial effectiveness were observed for BC-AA DES monomers than BC only toward S. aureus and C. albicans, and after polymerization BC-AA DES polymers exhibited similar inhibition zone widths to BC only, the effect of polymerization on antimicrobial properties of BC-AA DES was further investigated. A challenging level of CQ up to 28% without 4E was added to BC-AA DES, and DC% was able to be increased to as high as 99% (Table 3). At the same monomer level and light-curing time of 60s, addition of initiators lead to higher DC% but smaller inhibition zone widths for both S. aureus and C. albicans. The antimicrobial effectiveness was not supposed to alter for the same DES, so the decrease of the inhibition zone widths was mainly due to the limited transport of the active components. The addition of initiator level was typically not as high as 28% (typically <1%), so there is always residue amount of uncured AA. The toxicity of uncured AA should be cautioned in future applications related to human consumption. This result further demonstrated the statement that the polymerization of BC-AA DES limited the movement of the antimicrobial components. 3.4.4 Effect of eutectic ratios to antimicrobial performance In order to test the sensitivity of antimicrobial performance to eutectic ratio of DES mixtures, six sets of agar diffusion tests were further conducted by varying the eutectic ratios and microbial organism types, and the results are shown in Figure 8. From the former sections, the eutectic point of BC-AA has been determined as 1:2 (BC molar fraction = 0.33), and the eutectic point of BCMA has been determined as 1:2.5 (BC molar fraction = 0.28). For BC-AA, the inhibition zone widths of polymers (Figure 8(A), 8(C) and 8(E)) were smaller than those of monomers, which followed the same trend in Figure 8(A). For BC-MA, the inhibition zone widths of polymers were not significantly different from those of monomers (Figure 8(B), 8(D) and 8(F)), which also 14
matches the results in Figure 8(B). Through Figure 8, slightly deviation of the eutectic ratios (BC molar fractions of the abscissa) lead to slightly increases in inhibition zone widths for both DES monomers and polymers. As was indicated in FTIR characterizations of Figure 2 - Figure 4, the strongest molecule interactions occur at the eutectic ratios, which means that the largest proportion of active components are trapped because of hydrogen bond formation. Therefore, deviations of the eutectic ratios may lead to increased portion of free-moving components, which favors the transportation of antimicrobial components producing larger inhibition zone widths. From this point, accurately determined eutectic ratios are suggested to get optimized drug release control in similar cases.
4. Conclusions Antimicrobial properties of DES monomers, polymers and their single components were evaluated through agar diffusion test in this work. Two main factors may contribute to the differences in inhibition zone widths, which are antimicrobial strength and active component transport. The formation of BC-AA DES may increase the antimicrobial strength compared to BC only, especially to the Gram positive bacteria (e.g. S. aureus) and fungi (e.g. C. albicans). The introduction of branch structure (e.g. methyl group within MA) may decrease the molecule movement of the prepared DES, which could be proved by the activation energy results calculated through viscosity measurements, thus limit the transportation of active components. Polymerization of DES monomers may further decrease the transportation of antimicrobial components, because smaller inhibition zone widths were observed with increased DC%. In addition, accurately determined eutectic ratio is recommended in order to get best release control of active antimicrobial component. This work provided some new hints for quaternary ammonium pharmaceutical applications that require strong effectiveness with lower release rate. 15
Acknowledgments Dr. Jing Wang would like to acknowledge the start-up fund from Shandong University of Technology that were used in partial support of this work.
References Abbott, A.P., Boothby, D., Capper, G., Davies, D.L., Rasheed, R.K., 2004. Deep eutectic solvents formed between choline chloride and carboxylic acids: versatile alternatives to ionic lquids. Journal of the American Chemical Society 126, 9142-9147. Abbott, A.P., Capper, G., Davies, D.L., Rasheed, R.K., Tambyrajah, V., 2003. Novel solvent properties of choline chloride/urea mixtures. Chemical Communications, 70-71. Carriazo, D., Serrano, M.C., Gutiérrez, M.C., Ferrera, M.L., Monte, F.d., 2012. Deep-eutectic solvents playing multiple roles in the synthesis of polymers and related materials. Chemical Society Reviews 41, 4996-5014. Coughlin, R.T., Tonsager, S., McGroarty, E.J., 1983. Quantitation of metal cations bound to membranes and extracted lipopolysaccharide of Escherichia coli. Biochemistry 22, 20022007. Fazlara, A., Ekhtelat, M., 2012. The disinfectant effects of benzalkonium chloride on some important foodborne pathogens. American-Eurasian J. Agric. & Environ. Sci. 12, 23-29. Guo, X., Wang, Y., Spencer, P., Ye, Q., Yao, X., 2008. Effects of water content and initiator composition on photopolymerization of a model BisGMA/HEMA resin. Dental Materials 24, 824-831. Hayyan, M., Hashim, M.A., Hayyan, A., Al-Saadi, M.A., AlNashef, I.M., Mirghani, M.E.S., Saheed, O.K., 2013. Are deep eutectic solvents benign or toxic? Chemosphere 90, 21932195. Hayyan, M., Looi, C.Y., Hayyan, A., Wong, W.F., Hashim, M.A., 2015. In vitro and in vivo 16
toxicity profiling of ammonium-based deep eutectic solvents. PloS one 10, e0117934. Jin, H., O’Hare, B., Dong, J., Arzhantsev, S., Baker, G.A., Wishart, J.F., Benesi, A.J., Maroncelli, M., 2008. Physical properties of ionic liquids consisting of the 1-butyl-3-methylimidazolium cation with various anions and the bis(trifluoromethylsulfonyl)imide anion with various cations. Journal of Physical Chemistry B 112, 81-92. Juneidi, I., Hayyan, M., Hashim, M.A., 2015. Evaluation of toxicity and biodegradability for cholinium-based deep eutectic solvents. RSC Advances 5, 83636-83647. Kampf, G., Kramer, A., 2004. Epidemiologic cackground of hand hygiene and evaluation of the most important agents for scrubs and rubs. Clinical Microbiology Reviews 17, 863-893. Mangalappalli-Illathu, A.K., Korber, D.R., 2006. Adaptive resistance and differential protein expression of salmonella enterica serovar enteritidis biofilms exposed to benzalkonium chloride. Antimicrobial Agents and Chemotherapy 50, 3588-3596. McDonnell, G., Russell, A.D., 1999. Antiseptics and disinfectants: activity, action, and resistance. Clinical Microbiology Reviews 12. Mota-Morales, J.D., Gutiérrez, M.C., Ferrer, M.L., Sanchez, I.C., Elizalde-Peña, E.A., Pojman, J.A., Monte, F.D., Luna-Bárcenas, G., 2013. Deep eutectic solvents as both active fillers and monomers for frontal polymerization. Journal of Polymer Science Part A 51, 1767-1773. Rogers, R.D., Voth, G.A., 2007. Ionic liquids. Accounts of Chemical Research 40, 1077-1078. Sánchez-Leija, R.J., Pojman, J.A., Luna-Bárcenas, G., Mota-Morales, J.D., 2014. Controlled release of lidocaine hydrochloride from polymerized drug-based deep-eutectic solvents. Journal of Matrials Chemistry B 2, 7495-7501. Shekaari, H., Zafarani-Moattar, M.T., Mokhtarpour, M., 2017. Solubility, volumetric and compressibility properties of acetaminophen in some aqueous solutions of choline based deep eutectic solvents at T= (288.15 to 318.15) K. European Journal of Pharmaceutical Sciences 109, 121-130.
17
Stack, V.T., 1957. Toxicity of α,β-unsaturated carbonyl compounds to microorganisms. Ind. Eng. Chem. 49, 913-917. Tang, B., Row, K.H., 2013. Recent developments in deep eutectic solvents in chemical sciences. Monatshefte für Chemie-Chemical Monthly 144, 1427-1454. Wang, J., Dong, X., Yu, Q., Baker, S.N., Li, H., Larm, N.E., Baker, G.A., Chen, L., Tan, J., Chen, M., 2017. Incorporation of antibacterial agent derived deep eutectic solvent into an active dental composite. Dental Materials 33, 1445-1455. Wen, Q., Chen, J., Tang, Y., Wang, J., Yang, Z., 2015. Assessing the toxicity and biodegradability of deep eutectic solvents. Chemosphere 132, 63-69. Wikene, K.O., Bruzell, E., Tønnesen, H.H., 2015. Characterization and antimicrobial phototoxicity of curcumin dissolved in natural deep eutectic solvents. European Journal of Pharmaceutical Sciences 80, 26-32. Zhang, Q., Vigier, K.d.O., Royera, S., Jérôme, F., 2012. Deep eutectic solvents: syntheses, properties and applications. Chemical Society Reviews 41, 7108-7146.
18
Figure and Table Legends Figure 1. Chemical structures of the quaternary salt, benzalkonium chloride (BC), and hydrogen bond donors, methacrylic acid (MA) and acrylic acid (AA), used for eutectic mixture preparations. Figure 2. Eutectic point determination with FTIR spectrum of BC-AA and BC-MA mixture. (A) FTIR spectrum of BC-MA and BC-AA mixtures and their single components; (B) C=O and C=C peak intensity ratios with different BC molar fractions. Figure 3. Eutectic point determination with NMR spectrum of BC-AA mixtures and single components. (A) AA only, (B) BC only, (C) BC:AA=1:4, (D) BC:AA=1:2.5, (E) BC:AA=1:2, (F) BC:AA=1:1.5, (G) BC:AA=3:2, (H) peak area ratios of δ (-COOH) and δ =7.60 with different BC molar fractions. Figure 4. Eutectic point determination with NMR spectrum of BC-MA mixtures and single components. (A) MA only, (B) BC only, (C) BC:MA=1:5, (D) BC:MA=1:4, (E) BC:MA=1:2.5, (F) BC:MA=1:1.5, (G) BC:MA=3:2, (H) peak area ratios of δ (-COOH) and δ =7.60 with different BC molar fractions. Figure 5. Viscosity in logarithm form versus temperature plots of BC-AA and BC-MA DES. Solid lines are fittings of the data to Arrhenius model, and dashed lines are fittings of the data to V-F-T model. Figure 6. DES polymerization. (A) FTIR spectrum of DES before (solid lines) and after (dashed lines) polymerization, (B) DC% of DES at different curing time, (C) mechanical properties of DES polymers.
19
Figure 7. Inhibition zone width of individual components, DES monomer and DES polymer towards different organisms, (A) BC-AA DES, (B) BC-MA DES. Figure 8. Inhibition zone width of eutectic mixtures at different ratios before and after polymerization for various organisms. Table 1. Fitting parameters of the viscosity data to Arrhenius Model. Table 2. Fitting parameters of the viscosity data to V-F-T Model. Table 3. Effect of degree of conversion (DC%) on inhibition zone width for S. aureus and C. albicans. Mean ± Standard Deviation, n=6.
20
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
GA
21
Author Contributions Jing Wang: conceptualization, methodology, investigation, writing – original draft, Jing Xue: Investigation, Writing- Original draft. Xiaoqing Dong: Investigation, writing – original draft; Qingsong Yu: Project administration; Sheila N. Baker: Project administration; Ming Wang: writing- reviewing and editing, Haofei Huang: validation.
22
Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
23
S0378-5173(19)31066-X https://doi.org/10.1016/j.ijpharm.2019.119005 IJP 119005
To appear in:
International Journal of Pharmaceutics
Received Date: Revised Date: Accepted Date:
16 July 2019 24 November 2019 26 December 2019
Please cite this article as: J. Wang, J. Xue, X. Dong, Q. Yu, S.N. Baker, M. Wang, H. Huang, Antimicrobial properties of benzalkonium chloride derived polymerizable deep eutectic solvent, International Journal of Pharmaceutics (2019), doi: https://doi.org/10.1016/j.ijpharm.2019.119005
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier B.V.
Antimicrobial properties of benzalkonium chloride derived polymerizable deep eutectic solvent Jing Wanga,b,*, Jing Xuea, Xiaoqing Dongb, Qingsong Yub, Sheila N. Bakerc, Ming Wanga, Haofei Huanga
a
School of Chemistry and Chemical Engineering, Shandong University of Technology, Zibo, Shandong, China
b
Department of Mechanical & Aerospace Engineering, University of Missouri, Columbia, MO, United States.
c
Department of Chemistry, University of Missouri, Columbia, MO, United States.
* Corresponding Author: Jing Wang, Ph. D. Phone: +86 17615695097 E-mail: [email protected] Address: 266 Xincun Road Shandong University of Technology Zibo, Shandong 255000 China
E-mail addresses: Jing Xue: [email protected] Xiaoqing Dong: [email protected] Qingsong Yu: [email protected] Sheila N. Baker: [email protected] Ming Wang: [email protected] Haofei Huang: [email protected]
1
Abstract Benzalkonium chloride (BC) is a quaternary ammonium antimicrobial agent used in a variety of applications. In this work, BC was prepared into deep eutectic solvent (DES) with acrylic acid (AA) or methacrylic acid (MA). Within the newly prepared DES, BC is responsible for antimicrobial properties, while AA and MA are responsible for polymerization. Three types of microorganisms, E. coli (gram-negative bacilli), S. aureus (gram-positive cocci) and C. albicans (fungi), were assessed for antimicrobial properties through agar diffusion test. DES viscosity measurements and polymerizations were also conducted to assist the antimicrobial performance analysis. From this study, stronger antimicrobial effectiveness of BC-AA DES towards S. aureus and C. albicans was observed, while smaller inhibition zone widths were obtained for BC-AA DES polymer compared to BC-AA DES monomer which may due to the limited active component transportation after polymerization. When changing AA to MA, increased structural complexity and decreased linearity may limit the molecule movement thus reduce the inhibition zone width, which could be proved by the calculated activation energy results. Accurately determined eutectic ratio of DES is recommended to get optimized drug release control. This work offers a new sight for preparation of antimicrobial materials with stronger effectiveness and limited release.
Keywords Antimicrobial property; benzalkonium chloride; deep eutectic solvent; drug release
2
1. Introduction Benzalkonium chloride (BC) is one of the most commonly used pharmaceuticals for antimicrobial purposes, which is frequently found in clinical, food line and domestic household cleaning products (Kampf and Kramer, 2004; Mangalappalli-Illathu and Korber, 2006). BC is an effective quaternary ammonium compound in inhibiting a variety of microbial such as bacteria, fungi and yeasts (Fazlara and Ekhtelat, 2012). When added into bulk materials, BC could ascribe the materials with antimicrobial properties, which is an easy approach to develop antimicrobial bulk materials. Instead of adding BC directly, we previously prepared BC into the form of deep eutectic solvent (DES) before incorporating it into an active dental composite (Wang et al., 2017), and an limited release of BC was observed with longer lasting antimicrobial effectiveness of the newly developed dental composite. In another study by Sánchez-Leija et al. (Sánchez-Leija et al., 2014), a controlled release of lidocaine hydrochloride was also reported when transformed into DES. Therefore, it is of great interest to target the BC derived DES to further study its antimicrobial performance before and after polymerization, which is beneficial to the development of antimicrobial materials with controlled drug release. DESs are regarded as an alternative to ionic liquids (ILs), and they share common properties like high viscosity, low vapor pressure and thermal stabilities (Tang and Row, 2013). ILs are salts at liquid state (Rogers and Voth, 2007), while DESs are mixtures of two or more components at a certain ratio leading to eutectic point commonly lower than the melting point of the single component (Abbott et al., 2003). The Type III DES is the most widely studied category, which is commonly formed of quaternary ammonium salt with halide anion and a hydrogen bond donor (HBD) (Zhang et al., 2012). DES has been utilized as solvent to increase the solubility of pharmaceuticals (Shekaari et al., 2017) and for antimicrobial applications (Wikene et al., 2015).
3
Rare study were found to prepare a specific pharmaceutical into DES for antimicrobial purposes. Hayyan et al. (Hayyan et al., 2013) investigated the antimicrobial properties of some common choline chloride based DESs with urea, glycerine, triethylene glycol, and ethylene glycol as HBDs. Although they didn’t find inhibition effect for the tested DESs, they suggested that the inhibition behavior of DESs depends on different component structures. Hayyan et al. (Hayyan et al., 2013) further studied the toxicity of some phosphonium-based DESs using two Gram positive bacteria, Bacillus subtilis (B. subtilis) and Staphylococcus aureus (S. aureus), and two Gram negative bacteria, Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa), and stronger antimicrobial effects were observed for DES compared to their individual components. The succedent studies on ammonium-based DESs (Hayyan et al., 2015) and cholinium-based DESs (Juneidi et al., 2015; Wen et al., 2015) reached a similar conclusion that the microbial inhibition effects of DESs strongly depend on the compositions of DESs. Similarly to ILs, DESs were also introduced into the polymer sciences, but not a lot of successfully remarkable work could be found for synthesis of polymers by using DESs as monomers (Carriazo et al., 2012). Monta-Morales et al. (Mota-Morales et al., 2013) mixed ammonium salts and HBDs of acrylic acid (AA) and methacrylic acid (MA) to obtain DESs for frontal polymerizations, and these DESs played an all-in-one role as monomer, filler and reaction medium. In our work, two designed BC derived polymerizable DES were prepared with BC&AA and BC&MA, which possess antimicrobial properties because of the incorporation of BC, and can perform polymerization as monomers due to the existence of AA and MA. These two DESs form through strong hydrogen bonding interactions between the ammonium and carboxyl structures. The eutectic point of the prepared eutectic mixtures was determined with Fourier transform infrared spectroscopy (FTIR) method. Viscosity measurement and degree of conversion (DC%)
4
tests were conducted for DES characterizations. Antimicrobial performances of DES before and after polymerization were evaluated through agar diffusion tests using three representative organisms, E. coli (gram-negative bacilli), S. aureus (gram-positive cocci) and C. albicans (fungi). The effect of DES components, polymerization and eutectic ratios on antimicrobial performance are thoroughly studied and discussed. This work is providing insightful information on development of antimicrobial materials with strengthened effectiveness and controlled drug release.
2. Materials and Methods
2.1 Reagents and Materials Benzalkonium chloride (BC), acrylic acid (AA), methacrylic acid (MA), camphorquinone (CQ) and ethyl 4-dimethylaminobenzoate (4E) were purchased from Sigma-Aldrich (St. Louis, USA). Potassium bromide (KBr) was purchased from Acros Organics (New Jersey, USA). Chloriform-d was purchased from Aladdin (Shanghai, China). All chemicals were used as received without further purification. Sterile filter paper (Qualitative P5) used for agar diffusion test was purchased from Fisher Scientific. The test organisms used in this study were Escherichia coli (E. coli, ATCC 5922, gramnegative bacilli), Staphylococcus aureus (S. aureus, NRS234, gram-positive cocci) and Candida albicans (C. albicans, ATCC 18804, fungi). The bacterial strains were purchased from ATCC (E. coli and C. albicans) and NARSA (S. aureus). The strains of E. coli and S. aureus were grown in Todd Hewitt broth containing 0.2% yeast extract and tested on Todd Hewitt Agar. The C. albicans was cultured in Sabouraud dextrose broth and tested on Sabouraud dextrose agar. All bacterial
5
cultures were incubated at 37 °C.
2.2 DES Preparation The eutectic mixtures were prepared by stirring BC & AA or BC & MA (Figure 1) with certain molar ratios at 60 °C until homogenous clear and colorless liquid formed. A Cary 660 FTIR spectrometer (Agilent Technologies) was used to determine the eutectic ratio of the newly prepared DES, where DES was pressed into a KBr pellet, and absorbance spectrum between 400 cm-1 and 4000 cm-1 were collected. A Bruker Avance III HD 400 MHz NMR was also used to further confirm the formation of hydrogen bond and the eutectic ratio of DES, where chloroform-d was used as solvent.
2.3 Viscosity Measurement Viscosities were measured using a Brookfield DV-III Ultra Programmable Rheometer (Spindle CPE-42, 0.2 – 6000 cP), and different temperatures were maintained through a Fischer Scientific Isotemp® temperature bath. Each temperature was maintained for at least 15 minutes before the readings, and the torque were controlled between 20-80% by changing the RPM for each reading. 5-10 viscosity readings were collected and averaged for each temperature, and modeling fittings were conducted using Sigma Plot 12.1.
2.4 DES Polymerization A light initiated polymerization system of DES/CQ/4E were constructed with camphorquinone (CQ) as the initiator and ethyl 4-dimethylaminobenzoate (4E) as the co-initiator. Since only small amount of initiators were needed for polymerization, CQ and 4E were dissolved in an acetone stock solution and was added upon requirements. KBr pellets containing DES/CQ/4E were dried 6
and measured using a Cary 660 FTIR spectrometer (Agilent Technologies) before and after light cure (Densply Spectrum® 800 curing light system). Absorbance spectrum between 400 cm-1 and 4000 cm-1 were collected at the sensitivity of 1.5, resolution of 4, background and sample scan numbers of 32. FTIR data were processed with Agilent Resolution Pro Software. DC% was able to be calculated by equation (1) based on the peak heights of absorbance. 𝑃𝑜𝑙𝑦𝑚𝑒𝑟
𝐴
𝑃𝑜𝑙𝑦𝑚𝑒𝑟
/𝐴
C=C C=O DC% = (1 − 𝐴𝑀𝑜𝑛𝑜𝑚𝑒𝑟 ) × 100% /𝐴𝑀𝑜𝑛𝑜𝑚𝑒𝑟 C=C
C=O
(1)
2.5 Agar Diffusion Test For the monomer samples used for agar diffusion test, filter paper disks (7mm in diameter) were soaked with DES monomers without CQ or 4E, and were dried with compressed air to eliminate excess liquid. For the polymer samples used for agar diffusion test, filter paper disks with the same size were soaked with DES/CQ/4E mixtures. After a similar drying step, the disks were light cured with a Densply Triad® light cure system (bulb #70353) for 2 minutes on each side. The tested microorganisms were grown in their respective media to reach the optical density of 1.0. Suspensions of 0.1 mL of the microorganisms were uniformly spread on agar plates by using a sterile cotton bud. Inhibition zone width is calculated by equation (2): W=(d2-d1)/2
(2)
where W is the inhibition zone width, d1 is the diameter of the filter paper disk, and d2 is the diameter of the inhibition zone diameter. Each sample was tested in six replicates.
3. Results and Discussion
3.1 Eutectic Point Determination
7
The most widely accepted method to determine the eutectic point is naked eye observation of freezing points of mixtures at different molar ratios (Abbott et al., 2004; Abbott et al., 2003). In this study, however, the mixtures of BC and HBDs are liquids at room temperature, and they have relatively lower freezing points. Thus the FTIR method and NMR method were used in this case for the determination of eutectic point. The eutectic phenomenon is mainly caused by relatively strong molecule interactions, hydrogen bond, in this case. The formation of hydrogen bond mainly affects the –COOH in AA and MA, leading to changes in the position and intensity of absorbance peak of the C=O stretch at 1700-1725 cm-1 (Figure 2(A)) in the FTIR spectra. Single component of AA showed two separated peaks at 1700 cm-1 and 1725 cm-1, but the peak at 1700 cm-1 diminished at the shoulder of the peak at 1725 cm-1 for AA derived eutectic mixtures. Single component of MA showed a sharp peak at 1700 cm-1, but this peak shifted to 1710 cm-1 for MA derived eutectic mixtures. The formation of hydrogen bond can also be proved by the NMR spectrum of BC-AA mixtures (Figure 3) and BCMA mixtures (Figure 4). The chemical shift of –COOH proton was at δ=11.38 for AA (Figure 3(A)) and at δ=11.31 (Figure 4 (A)) for MA. After mixing with BC, the chemical shift moved to a lower value for both BC-AA (δ=10.58, Figure 3(C)) and BC-MA (δ=9.84, Figure 4(C)). It was also observed that with the increase of BC molar fraction, the chemical shift of –COOH proton changed larger (Figure 3(C) - (F), Figure 4(C) - (F),), and it diminished at high BC molar ratios (Figure 3(G), Figure 4(G)). From this point, both of the FTIR spectrum and NMR spectrum evidenced the formation of hydrogen bond for BC-AA and BC-MA. In the FTIR spectra of the eutectic mixtures (Figure S1 and Figure S2), it was observed that the peak intensity of C=O stretch become smaller with the increase of BC molar ratio. Therefore, taking C=C stretch within AA or MA at 1633 cm-1 as basis, the peak intensity ratios of
8
1725 cm-1/1633 cm-1 for BC-AA mixtures and 1710 cm-1/1633 cm-1 for BC-MA mixtures were tracked (Figure 2(B)). With the increase of BC molar fraction, for both BC-AA and BC-MA mixtures, the C=O/C=C peak intensity ratios showed a decreasing trend. It could be observed that there was a downward sharp in the overall decreasing trend, and the sharp was at BC:AA=1:2 and BC:MA=1:2.5. Similarly, when we track the peak area of δ=7.60 over δ (-COOH) in the NMR spectra, a transition could be obtained in the increasing trend with the increase of BC molar fraction, and the transition also happened at BC:AA=1:2 (Figure 3(H)) and BC:MA=1:2.5 (Figure 4(H)). Therefore, the eutectic ratio (molar ratio) was determined as 1:2 for BC:AA and 1:2.5 for BC:MA, respectively. Both DESs have freezing/melting point below room temperature.
3.2 DES Viscosities The dynamic viscosities (η) of DESs were measured in the range of 25-85 °C (Table S1). The viscosity data were firstly fitted to the logarithmic form of Arrhenius model: ln(𝜂) = ln(𝜂0 ) +
𝐸𝜂 𝑅𝑇
(3)
where η is the dynamic viscosity, η0 is a coefficient, Eη is the activation energy for viscous flow, R is the universal gas constant, and T is the temperature in K. The ln(η) is linear to 1/T from this model, and The fitting parameters of the data and associated r2 are listed in Table 1. To better describe the curvature of the data, another Vogel-Fulcher-Tamman (V-F-T) model was introduced (Figure 5). V-F-T model (equation (4)) usually fits better for ILs or DESs with small or symmetric chemical structures (Jin et al., 2008). B
ln(𝜂) = A + 𝑇−𝑇
0
(4)
where A and B are constant parameters, and T0 is the divergent temperature of the configurational entropy of the system vanishing. Based on the fitting parameter of T0, activation energy of Eη has 9
been able to be calculated by equation (5). The results are summarized in Table 2. T 2 ) 𝑇−𝑇0
𝐸𝜂 = RB(
(5)
The activation energy at a certain temperature is the minimum energy barrier that the molecules need to overcome in order to move in the current system. Larger activation energy indicates higher difficulty for the molecule movement. The recovered activation energy from Arrhenius model (Table 1) is slightly lower than that from the V-F-T model (Table 2), but they closely follow the same trend. The activation energy of BC-MA DES is larger than that of the BCAA DES, so the molecule movement of BC-MA DES is expected to be more difficult than that of the BC-AA DES.
3.3 DES Polymerization The FT-IR spectra of DESs before and after polymerization are shown in Figure 6(A). It is clearly observed that the peak at 1633 cm-1 (C=C stretch) decreased for both BC-AA DES and BC-MA DES. DC% has been calculated by equation (1) based on the peak intensity before and after polymerization. From Figure 6(B), when 0.5% CQ and 0.5% 4E were added for initiation, DC% increased with the increasing of curing time. At the curing time of 20s, DC% have approached the maximum, but curing time of 60s was chosen for further experiment to ensure reaching the maximum DC%. The highest DC% is about 74% for BC-AA DES, and is about 80% for BC-MA DES. These are relatively high DC% for a CQ/4E initiated system (Guo et al., 2008) and solidified materials have been obtained for further experiments, so the initiator addition level was not further optimized. At the same curing condition and initiator addition level, DC% of BC-AA DES is slightly smaller than that of BC-MA DES. As for the polymerized DES, BC-AA DES polymer exhibited high flexibility, while BC-MA DES polymer exhibited high rigidity (Figure 6(C)). After
10
storing in water for 3 days at room temperature, both of BC-AA DES polymer and BC-MA polymer were dissolved.
3.4 Antimicrobial Performance 3.4.1 Antimicrobial performance of BC-AA DES The inhibition zone width for the individual components, BC-AA DES monomer and BC-AA DES polymer are shown in Figure 7(A). For S. aureus and C. albicans, extremely large inhibition zone widths were observed with AA. The antimicrobial activity of AA might be attributed to its acid nature, low molecular weight, and α, β-unsaturation (Stack, 1957). The BC-AA DES monomer and polymer inhibition zone widths are slightly larger but close to that of BC. This indicated that the antimicrobial effect of BC-AA DES monomer and polymer mainly come from BC rather than AA. The formation of BC-AA DES leads to changes on carboxylic functional group of AA, which has been proved by the FTIR spectrum used for eutectic point determination in this study. From this point, the DES formation is considered as the main reason for the ineffectiveness of antimicrobial activity of AA within DES. On the other hand, the antimicrobial activity of BC mainly comes from its cationic amphiphilic structure, which has a hydrophobic hydrocarbon region and a hydrophilic region (McDonnell and Russell, 1999). The hydrophilic cationic region could destabilize bacterial surface by forming electrostatic interactions with negative charged components on cell membrane walls. Once close contact is accomplished by the hydrophilic region, the BC’s hydrophobic region proceeds to penetrate through the hydrophobic bilayer to cause cell leakage and lysis (Fazlara and Ekhtelat, 2012). The formation of DES only affects the halide part of BC rather than the cationic part. In other words, the effectiveness of antimicrobial activity of BC has been able to be maintained even after the formation of DES. For E. coli (gram11
negative), there was no significant difference observed on the inhibition zone width of BC-AA DES monomer, polymer and BC only. For S. aureus (gram-positive) and C. albicans (fungi), in comparison with BC only, there were slightly increases for the samples of BC-AA DES monomers, which indicated the increased antimicrobial effectiveness. In general, gram-positive bacteria and fungi are more susceptible to BC than gram-negative ones because of their distinct membrane structures. Gram-positive bacteria and fungi generally have a single membrane surrounded by a thick mesh-like peptidoglycan that allows for the penetration of small molecules. In contrast, gram-negative bacteria have thin polysaccharide walls overlaid by a thin layer of lipopolysaccharides that modulates the accessibility of a cell to antiseptics and other small molecules (Coughlin et al., 1983). This indicates that the BC-AA DES monomer has better antimicrobial effect towards the gram-positive bacteria and fungi than gram-negative bacteria. From previous studies, hydrogen bond formation within DES may limit the movement of BC, slower the mass transport of antimicrobial agent, which may lead to smaller inhibition zone widths (Hayyan et al., 2015). In this study, however, larger inhibition zone widths were observed for S. aureus and C. albicans, which indicated stronger antimicrobial effectiveness of DES despite of lower mass transport. The inhibition zone widths of BC-AA DES polymers decreased compared with BC-AA DES monomers, and remained at almost the same level as BC only. This may due to the formation of polymer chains and networks after DES polymerization. The larger molecular weights and complicated tangled polymer chains limit the movement of BC component leading to smaller inhibition zone width. Compared to the molecule interaction, hydrogen bond in this case, the covalent bond formation during polymerization has larger influence on mass transport of BC.
12
3.4.2 Antimicrobial performance of BC-MA DES The inhibition zone widths of individual components, BC-MA DES monomers, and BCMA DES polymers are shown in Figure 7(B). Similar to AA, MA also exhibited strong antimicrobial effectiveness, especially to C. albicans. However, the inhibition zone widths are much smaller than those of AA. Two main reasons might lead to this difference, weaker antimicrobial effectiveness or limited mass transport. Neither reason is important because the antimicrobial effective structure within MA is altered after formation of DES. Also similar to the discussion based on BC-AA DES, this study focused on the inhibition zone width of BC only, BCMA DES monomer and BC-MA DES polymer. The main difference in chemical structures of AA and MA is the methyl branch, which increased the molecular weight and disordered the chain linearity. Compared to BC only, BC-MA DES monomer didn’t produce larger inhibition zone width, which is slightly different from the BC-AA DES monomer result. This change may due to the increased molecular weight and the disordered linearity which is unfavorable for the molecule movement. This result also matches the higher activation energy of BC-MA DES than BC-AA DES from the viscosity measurements. The BC-MA DES polymer also produced similar inhibition zone width to BC only for all of the tested organisms, which is similar to the BC-AA DES polymer result. Although DC% of BC-MA DES was slightly higher than that of BC-AA DES at the same polymerization condition, the inhibition zone widths were not significantly affected. Weaker antimicrobial effectiveness of MA compared to AA and limited molecule movement or mass transport of BC-MA DES compared to BC-AA DES contributed to the smaller inhibition zone widths, and polymerization played similar roles during the antimicrobial behavior.
13
3.4.3 Effect of polymerization on antimicrobial performance of DES Since increased antimicrobial effectiveness were observed for BC-AA DES monomers than BC only toward S. aureus and C. albicans, and after polymerization BC-AA DES polymers exhibited similar inhibition zone widths to BC only, the effect of polymerization on antimicrobial properties of BC-AA DES was further investigated. A challenging level of CQ up to 28% without 4E was added to BC-AA DES, and DC% was able to be increased to as high as 99% (Table 3). At the same monomer level and light-curing time of 60s, addition of initiators lead to higher DC% but smaller inhibition zone widths for both S. aureus and C. albicans. The antimicrobial effectiveness was not supposed to alter for the same DES, so the decrease of the inhibition zone widths was mainly due to the limited transport of the active components. The addition of initiator level was typically not as high as 28% (typically <1%), so there is always residue amount of uncured AA. The toxicity of uncured AA should be cautioned in future applications related to human consumption. This result further demonstrated the statement that the polymerization of BC-AA DES limited the movement of the antimicrobial components. 3.4.4 Effect of eutectic ratios to antimicrobial performance In order to test the sensitivity of antimicrobial performance to eutectic ratio of DES mixtures, six sets of agar diffusion tests were further conducted by varying the eutectic ratios and microbial organism types, and the results are shown in Figure 8. From the former sections, the eutectic point of BC-AA has been determined as 1:2 (BC molar fraction = 0.33), and the eutectic point of BCMA has been determined as 1:2.5 (BC molar fraction = 0.28). For BC-AA, the inhibition zone widths of polymers (Figure 8(A), 8(C) and 8(E)) were smaller than those of monomers, which followed the same trend in Figure 8(A). For BC-MA, the inhibition zone widths of polymers were not significantly different from those of monomers (Figure 8(B), 8(D) and 8(F)), which also 14
matches the results in Figure 8(B). Through Figure 8, slightly deviation of the eutectic ratios (BC molar fractions of the abscissa) lead to slightly increases in inhibition zone widths for both DES monomers and polymers. As was indicated in FTIR characterizations of Figure 2 - Figure 4, the strongest molecule interactions occur at the eutectic ratios, which means that the largest proportion of active components are trapped because of hydrogen bond formation. Therefore, deviations of the eutectic ratios may lead to increased portion of free-moving components, which favors the transportation of antimicrobial components producing larger inhibition zone widths. From this point, accurately determined eutectic ratios are suggested to get optimized drug release control in similar cases.
4. Conclusions Antimicrobial properties of DES monomers, polymers and their single components were evaluated through agar diffusion test in this work. Two main factors may contribute to the differences in inhibition zone widths, which are antimicrobial strength and active component transport. The formation of BC-AA DES may increase the antimicrobial strength compared to BC only, especially to the Gram positive bacteria (e.g. S. aureus) and fungi (e.g. C. albicans). The introduction of branch structure (e.g. methyl group within MA) may decrease the molecule movement of the prepared DES, which could be proved by the activation energy results calculated through viscosity measurements, thus limit the transportation of active components. Polymerization of DES monomers may further decrease the transportation of antimicrobial components, because smaller inhibition zone widths were observed with increased DC%. In addition, accurately determined eutectic ratio is recommended in order to get best release control of active antimicrobial component. This work provided some new hints for quaternary ammonium pharmaceutical applications that require strong effectiveness with lower release rate. 15
Acknowledgments Dr. Jing Wang would like to acknowledge the start-up fund from Shandong University of Technology that were used in partial support of this work.
References Abbott, A.P., Boothby, D., Capper, G., Davies, D.L., Rasheed, R.K., 2004. Deep eutectic solvents formed between choline chloride and carboxylic acids: versatile alternatives to ionic lquids. Journal of the American Chemical Society 126, 9142-9147. Abbott, A.P., Capper, G., Davies, D.L., Rasheed, R.K., Tambyrajah, V., 2003. Novel solvent properties of choline chloride/urea mixtures. Chemical Communications, 70-71. Carriazo, D., Serrano, M.C., Gutiérrez, M.C., Ferrera, M.L., Monte, F.d., 2012. Deep-eutectic solvents playing multiple roles in the synthesis of polymers and related materials. Chemical Society Reviews 41, 4996-5014. Coughlin, R.T., Tonsager, S., McGroarty, E.J., 1983. Quantitation of metal cations bound to membranes and extracted lipopolysaccharide of Escherichia coli. Biochemistry 22, 20022007. Fazlara, A., Ekhtelat, M., 2012. The disinfectant effects of benzalkonium chloride on some important foodborne pathogens. American-Eurasian J. Agric. & Environ. Sci. 12, 23-29. Guo, X., Wang, Y., Spencer, P., Ye, Q., Yao, X., 2008. Effects of water content and initiator composition on photopolymerization of a model BisGMA/HEMA resin. Dental Materials 24, 824-831. Hayyan, M., Hashim, M.A., Hayyan, A., Al-Saadi, M.A., AlNashef, I.M., Mirghani, M.E.S., Saheed, O.K., 2013. Are deep eutectic solvents benign or toxic? Chemosphere 90, 21932195. Hayyan, M., Looi, C.Y., Hayyan, A., Wong, W.F., Hashim, M.A., 2015. In vitro and in vivo 16
toxicity profiling of ammonium-based deep eutectic solvents. PloS one 10, e0117934. Jin, H., O’Hare, B., Dong, J., Arzhantsev, S., Baker, G.A., Wishart, J.F., Benesi, A.J., Maroncelli, M., 2008. Physical properties of ionic liquids consisting of the 1-butyl-3-methylimidazolium cation with various anions and the bis(trifluoromethylsulfonyl)imide anion with various cations. Journal of Physical Chemistry B 112, 81-92. Juneidi, I., Hayyan, M., Hashim, M.A., 2015. Evaluation of toxicity and biodegradability for cholinium-based deep eutectic solvents. RSC Advances 5, 83636-83647. Kampf, G., Kramer, A., 2004. Epidemiologic cackground of hand hygiene and evaluation of the most important agents for scrubs and rubs. Clinical Microbiology Reviews 17, 863-893. Mangalappalli-Illathu, A.K., Korber, D.R., 2006. Adaptive resistance and differential protein expression of salmonella enterica serovar enteritidis biofilms exposed to benzalkonium chloride. Antimicrobial Agents and Chemotherapy 50, 3588-3596. McDonnell, G., Russell, A.D., 1999. Antiseptics and disinfectants: activity, action, and resistance. Clinical Microbiology Reviews 12. Mota-Morales, J.D., Gutiérrez, M.C., Ferrer, M.L., Sanchez, I.C., Elizalde-Peña, E.A., Pojman, J.A., Monte, F.D., Luna-Bárcenas, G., 2013. Deep eutectic solvents as both active fillers and monomers for frontal polymerization. Journal of Polymer Science Part A 51, 1767-1773. Rogers, R.D., Voth, G.A., 2007. Ionic liquids. Accounts of Chemical Research 40, 1077-1078. Sánchez-Leija, R.J., Pojman, J.A., Luna-Bárcenas, G., Mota-Morales, J.D., 2014. Controlled release of lidocaine hydrochloride from polymerized drug-based deep-eutectic solvents. Journal of Matrials Chemistry B 2, 7495-7501. Shekaari, H., Zafarani-Moattar, M.T., Mokhtarpour, M., 2017. Solubility, volumetric and compressibility properties of acetaminophen in some aqueous solutions of choline based deep eutectic solvents at T= (288.15 to 318.15) K. European Journal of Pharmaceutical Sciences 109, 121-130.
17
Stack, V.T., 1957. Toxicity of α,β-unsaturated carbonyl compounds to microorganisms. Ind. Eng. Chem. 49, 913-917. Tang, B., Row, K.H., 2013. Recent developments in deep eutectic solvents in chemical sciences. Monatshefte für Chemie-Chemical Monthly 144, 1427-1454. Wang, J., Dong, X., Yu, Q., Baker, S.N., Li, H., Larm, N.E., Baker, G.A., Chen, L., Tan, J., Chen, M., 2017. Incorporation of antibacterial agent derived deep eutectic solvent into an active dental composite. Dental Materials 33, 1445-1455. Wen, Q., Chen, J., Tang, Y., Wang, J., Yang, Z., 2015. Assessing the toxicity and biodegradability of deep eutectic solvents. Chemosphere 132, 63-69. Wikene, K.O., Bruzell, E., Tønnesen, H.H., 2015. Characterization and antimicrobial phototoxicity of curcumin dissolved in natural deep eutectic solvents. European Journal of Pharmaceutical Sciences 80, 26-32. Zhang, Q., Vigier, K.d.O., Royera, S., Jérôme, F., 2012. Deep eutectic solvents: syntheses, properties and applications. Chemical Society Reviews 41, 7108-7146.
18
Figure and Table Legends Figure 1. Chemical structures of the quaternary salt, benzalkonium chloride (BC), and hydrogen bond donors, methacrylic acid (MA) and acrylic acid (AA), used for eutectic mixture preparations. Figure 2. Eutectic point determination with FTIR spectrum of BC-AA and BC-MA mixture. (A) FTIR spectrum of BC-MA and BC-AA mixtures and their single components; (B) C=O and C=C peak intensity ratios with different BC molar fractions. Figure 3. Eutectic point determination with NMR spectrum of BC-AA mixtures and single components. (A) AA only, (B) BC only, (C) BC:AA=1:4, (D) BC:AA=1:2.5, (E) BC:AA=1:2, (F) BC:AA=1:1.5, (G) BC:AA=3:2, (H) peak area ratios of δ (-COOH) and δ =7.60 with different BC molar fractions. Figure 4. Eutectic point determination with NMR spectrum of BC-MA mixtures and single components. (A) MA only, (B) BC only, (C) BC:MA=1:5, (D) BC:MA=1:4, (E) BC:MA=1:2.5, (F) BC:MA=1:1.5, (G) BC:MA=3:2, (H) peak area ratios of δ (-COOH) and δ =7.60 with different BC molar fractions. Figure 5. Viscosity in logarithm form versus temperature plots of BC-AA and BC-MA DES. Solid lines are fittings of the data to Arrhenius model, and dashed lines are fittings of the data to V-F-T model. Figure 6. DES polymerization. (A) FTIR spectrum of DES before (solid lines) and after (dashed lines) polymerization, (B) DC% of DES at different curing time, (C) mechanical properties of DES polymers.
19
Figure 7. Inhibition zone width of individual components, DES monomer and DES polymer towards different organisms, (A) BC-AA DES, (B) BC-MA DES. Figure 8. Inhibition zone width of eutectic mixtures at different ratios before and after polymerization for various organisms. Table 1. Fitting parameters of the viscosity data to Arrhenius Model. Table 2. Fitting parameters of the viscosity data to V-F-T Model. Table 3. Effect of degree of conversion (DC%) on inhibition zone width for S. aureus and C. albicans. Mean ± Standard Deviation, n=6.
20
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
GA
21
Author Contributions Jing Wang: conceptualization, methodology, investigation, writing – original draft, Jing Xue: Investigation, Writing- Original draft. Xiaoqing Dong: Investigation, writing – original draft; Qingsong Yu: Project administration; Sheila N. Baker: Project administration; Ming Wang: writing- reviewing and editing, Haofei Huang: validation.
22
Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
23