Molecular Interactions and Aggregation Behavior of Cloxacillin Sodium in Water and Aqueous NaCl Solutions through Volumetric and Ultrasonic Sound Velocity Studies at 298.15 K

Introduction

In pharmacokinetics, drug is a very essential chemical material that used for the treatment of various disease such as therapy, prevention /diagnosis, develop mental well-being ¹ etc. To recognize the drug action in human body at the molecular level, the physicochemical properties of drugs are very important².  By studying the physicochemical properties the binding possibility of biologically active molecules in presence of several solvents or co-solutes can be well examine³.

Cloxacillin sodium (CloNa) is a sodium salt of cloxaclliin and semi synthetic beta-lactamase penicillin antibiotic having remarkable antibacterial actions. Cloxacillin Sodium (CloNa) shows^4–6 noticeable vulnerability to acid and base reagents, several nucleophiles, oxidizing agents,  metal ions, and many solvents and is broadly used in the treatment of blood poisoning, parenchyma of skin infection, pneumonia, etc.⁷.

Volumetric^2,8–12 and ultrasonic sound velocity^13,14 technique is very convenient and reliable tool to well investigation in case of drugs,  amino acids, polymers etc analysis. Thermodynamic properties such as, ,  and aggregation concentration of any solution are very crucial parameter to recognize the solute–solvent and solute–solute interactions¹⁵.

For cloxacillin Sodium (CloNa), there are some reports on the stability of solid CloNa, degradation¹⁶, interactions with protein¹⁷, Taboada¹⁸ examined aggregation behavior of CloNa in aqueous and aqueous NaCl with light-scattering, NMR techniques. However, there is still a lack of knowledge about the interactions among solute-solvent-co-solute and aggregation behavior of CloNa in water or electrolyte solvents. In this background, this work presents a study on the solute-solvent, solute-solute interactions and aggregation behavior or properties of cloxacillin sodium (CloNa) in water and aqueous NaCl solutions through volumetric and ultrasonic velocity method. In this study, NaCl (co-solute) is taken because it is the chief electrolyte in the extracellular fluid¹⁹ in human body rather than other ions, such as Ca⁺ , K⁺ , Mg⁺ , HPO4⁻, and HCO³⁻. The range of Na+ in human body is ∼92 % in case of over-all positive ions and Cl− is ∼68 % for negative ions.

Figure 1: Structure of Cloxacillin Sodium (CloNa) Click here to View figure

To understand the complete interactions of CloNa with electrolytes, density, ρ, and ultrasonic sound velocity, u, of CloNa in water and NaCl aqueous solutions was measured at 298.15 K and atmospheric pressure. From density and ultrasonic sound velocity data, apparent molar volumes,, adiabatic compressibility, β_S, apparent molar adiabatic compressibility, , their parallel limiting parameters, and aggregation concentrations (CMC) were calculated. The tremendous results are represented in our paper.

Experimental Details

Materials

CloNa and sodium chloride were used in this study and collected from Beximco Pharmaceuticals Limited (Bangladesh), and Loba Chemie Pvt. Ltd. (India) respectively. The chemicals specifications are given below in Table 1 that used in this investigation.

Table 1: Chemicals Specifications

Chemical title	Molar mass/(kg·mol−1)	Clarity declared by provider	Origin
Cloxacillin sodium(CloNa)	0.457864	mass fraction, ≥ 0.990	Beximco Pharmaceuticals Limited (Bangladesh)
NaCl	0.058442	mass fraction, ≥ 0.995	Loba Chemie Pvt. Ltd(India)

Density and Ultrasonic Sound Velocity Measurement

Each solution prepared instantly with highly purified water. Because water was redistilled and degassed with specific conductance of < 10⁻⁶ S (cm⁻¹). By weighing, solution was prepared in molality using a balance (B204-S, Mettler Toledo, Switzerland) having a precision of ± 0.0001 g. Molality uncertainty for solutions is up to ± 2·10⁻⁵(mol kg⁻¹) . An analyzer (DSA 5000, Anton Paar, Austria) was used to examine density, ρ, and ultrasonic sound velocity, u, of the solutions. A density check carried out at 20 °C with a) distilled and degassed water, b) dry air. For each measurement, the density meter calibrated with redistilled and gas free water in the investigational temperature area. Temperature sensitivity was controlled by a built-in Peltier method to ± 1·10⁻³ K. The instrument sensitivity for density with respect to an accuracy is 1·10⁻³(kg m⁻³)  and for ultrasonic sound velocity 1·10⁻²(m s⁻¹). The density and ultrasonic sound velocity uncertainty was obtained ± 5·10⁻³ kg m⁻³ & ± 5·10⁻² m s⁻¹, respectively.

Results and Discussion

The measured density ρ & ultrasonic sound velocity u of CloNa (as a function of molality) in water and aqueous sodium chloride solution at different concentration are taken in Table 2. The apparent molar volume,, adiabatic compressibility, β_S and apparent molar adiabatic compressibility, calculated from measured density ρ and ultrasonic sound velocity u using equation (1), (2) & (3) are shown in Table  2.

where,

m = molality, ρ = density of solution,= adiabatic compressibility, ρ₀ = density of solvent,= adiabatic compressibility and M₂ = molar mass

The reported standard combined uncertainties in m, , , ,  and CMC were calculated based on first order Taylor series approximation with the help of their equivalent error equations of the following type,

where,

Δx_1, Δx₂…Δx_n= standard errors,

x_1, x₂… x_n =independent variables

The changes of apparent molar volume,  and apparent molar adiabatic compressibilities, of CloNa with respect to molality   at 298.15K are shown in Figure 2(a) and 2(b). The values of, are increased with respect to CloNa concentration, indicate the strong solute-solute interaction. At a certain point the increasing rate is changed and shows the break point. To investigate the properties i.e. solute-solvent, solute-solute interaction and aggregation behavior,, and CMC are very useful and these parameters can be obtained by regression of and  vs molality data to piece-wise linear model of the following type :

where,

y =or,

m = molality,

X = CMC,

y⁰ = limiting parameter at infinite dilution or,

b= slopes S_v and S_k^,

Subscripts 1 = for pre-CMC region,

Subscripts 2= for post-CMC region,

Fitting parameters with their standard uncertainties are presented in Table 3.

Figure 2: The variation of (a)and (b) values of CloNa in m = (■- 0.000, ■- 0.10049,■- 0.0586; ■-0.30012,■-0.40623,■-0.50572, ■-0.60480) M aqueous NaCl solutions at 298.15 K. Click here to View figure

Solute-Solvent Interactions

The calculated, &indicates the interactions between solute and solvent molecules (solute-solvent). The changes of of CloNa with respect to NaCl molality at various temperatures are shown in Figure 3(c). The values of&are goes to minima with the concentration of sodium chloride that is sign for the presence of strong interactions among solute, solvent and co-solute in the mixtures.

According to the configuration of CloNa (Figure. 1), the probable interactions of CloNa with NaCl are(a) two types ion−ion interactions such as  i) COO⁻(CloNa) with Na⁺(NaCl) ii) Na⁺ (CloNa) with Cl− (NaCl), (b) Ion−hydrophilic interactions of  Na⁺(NaCl) with the hydrophilic groups (O, F, N, S and Cl) of CloNa, and(c) Ion−hydrophobic interactions of Na⁺(NaCl) with the hydrophobic groups (alkyl groups, benzene ring) of CloNa. The co-sphere overlap model²⁰ recommends that (a) and (b) type interactions gives positive contribution to  , however type (c) gives an opposite contribution to i.e. negative. The decrease ofvalues with respect to increase in NaCl concentration suggests that at the beginning the ion− hydrophobic interaction exceeds over the ion− hydrophilic and ion−ion. After that ion− hydrophilic, ion−ion interaction exceeds owing to the increasing interactions with excess co-solute (NaCl) molecules. The variations of of CloNa against the concentration of NaCl are shown in Figure 3(d). The  values are more negative at the minima with the rising of NaCl concentration, indicates water clustering occurrences by the increasing of hydrogen bond. Allowing the Frank and Wen²¹ model, around the ions (low-charge density) water molecules are randomly distributed and bulk water molecules arranged orderly. The water molecules are more compressible as a monomeric form. The O, F, N, S and Cl (hydrophilic groups) of FluNa, have weak partial charges and (b) type interaction gives monomeric water molecules to bulk due to relaxation, where they form cluster water molecules and gives a negative role to. This is also supported well-accommodated aromatic rings²² in electrolyte solution instead of pure water. After minima, the magnitude of increase i.e, compressibility increases due to dominate (a) type interactions.

Figure 3: The variation of  (c) and  (d) of CloNa with respect to molality of NaCl at 298.15K Click here to View figure

Solute-Solute Interactions

The interactions of solute with solute (solute-solute) in terms of slope S_v and S_k are displayed in Figure 4 with respect to NaCl molality at 298.15 K. The slopes S_v1, S_k1 (pre- CMC) and S_v2, S_k2 (post-CMC) goes to the maxima. It is clearly declaring that S_v1, S_k1 provide information about the interactions between CloNa molecules, while S_v2, S_k2 deliver about the micelle CloNa interactions. The S_v1, S_k1, S_v2 and S_k2 values show strong interactions among the CloNa-CloNa molecules and micelle- micelle. And increasing trend with the NaCl concentration may be due to decreasing electrostatic repulsion among their head group charge. But for decreasing trend, the exact interpretation is difficult. However, in case of pre-CMC slopes there may be increase electrostatic repulsion among their head group charge due to excess co-solute (NaCl) molecule interaction and for post-CMC slopes transition happen from spherical micelles to other geometrical shapes^23-25.

Figure 4: The variation of slopes of CloNa with respect to NaCl molality at 298.15K. The figure (e), (f) represent pre-CMC and (g), (h) represent post-CMC slopes. The presented slopes are achieved from and Click here to View figure

Aggregation Behaviors

The piecewise linear regression model is used for determining CMC point as shown in equation 5.  X is the critical micelle concentration. The variation of CMC values of CloNa are presented in Figure 5 with respect to NaCl molality. Previously CMC values reported by light scattering^18,26 and NMR¹⁸ techniques in water and aqueous NaCl solutions exhibits a near agreement with these CMC values. It is observed from Figure 5(i) and 5(j) that the CMC values attained from – m, – m data show similar action to NaCl concentration. The gradually decreasing CMC values acquire a minimum value and then increasing with the increasing NaCl concentration. The decreasing trend is owing to the decreasing of electrostatic repulsion between the polar head groups (O, F, N, S and Cl) in presence of NaCl. And after that at the higher concentration of NaCl, the transition of spherical micelle²⁵ to cylindrical micelle is responsible for increasing CMC values. Because at higher salt concentrations, solute molecules are packed compactly and they successfully adopt a more cylindrical shape and cylindrical shape contain additional solute molecules rather than spherical that is form at upper solute concentrations.

Figure 5: The CMC of CloNa attained from (i)  and (j) with respect to NaCl concentration at T = 298.15K. Click here to View figure

Table 2: The Density, ρ, and Ultrasonic Sound Velocity, u, Apparent Molar Volume, ϕ_v , Adiabatic compressibility, β_s , Apparent molar Adiabatic compressibility, ϕ_k of CloNa in Water and Aqueous NaCl Solutions at 298.15 K and Atmospheric Pressure^a.

m/mol·kg-1	ρ /kg m−3	u /m s−1	ϕv /10−6 m3 mol−1	βs/10-10. Pa-1	ϕk /10−14 m3 mol−1 Pa−1
	Cloxa+water
0.00000	994.069	1519.98	–	4.3542±0.0006	–
0.03709	999.780	1525.15	303.10±0.20	4.3000±0.0006	-1.70±0.20
0.07391	1005.293	1529.84	303.49±0.08	4.2503±0.0006	-1.20±0.10
0.12465	1012.670	1535.36	303.90±0.06	4.1890±0.0005	-0.60±0.06
0.16916	1018.936	1539.36	304.22±0.04	4.1416±0.0005	-0.04±0.05
0.20779	1024.205	1542.20	304.60±0.04	4.1052±0.0005	0.45±0.04
0.22741	1026.844	1543.90	304.70±0.03	4.0856±0.0005	0.57±0.04
0.27564	1033.193	1547.89	304.96±0.00	0.0000±0.0000	0.84±0.00
0.33466	1040.711	1552.10	305.23±0.02	3.9887±0.0005	1.19±0.02
0.36240	1044.138	1553.97	305.40±0.02	3.9660±0.0005	1.34±0.02
	Cloxa+water+NaCl(0.10049m)
0.00000	998.077	1525.87	–	4.3033±0.0006	–
0.04265	1004.609	1531.81	303.00±0.20	4.2422±0.0006	-1.50±0.20
0.09145	1011.840	1537.39	303.50±0.10	4.1814±0.0005	-0.67±0.09
0.12129	1016.131	1540.28	303.83±0.07	4.1481±0.0005	-0.22±0.07
0.16344	1022.045	1543.73	304.23±0.05	4.1057±0.0005	0.38±0.05
0.20903	1028.260	1547.37	304.59±0.04	4.0617±0.0005	0.79±0.04
0.25362	1034.172	1550.95	304.85±0.03	4.0199±0.0005	1.06±0.03
0.27238	1036.599	1552.44	305.00±0.03	4.0028±0.0005	1.15±0.03
0.31669	1042.225	1555.38	305.30±0.03	3.9661±0.0005	1.44±0.03
	Cloxa+water+NaCl(0.20058m)
0.00000	1002.042	1531.63	–	4.2541±0.0006	–
0.01466	1004.329	1533.79	300.90±0.40	4.2325±0.0006	-2.00±0.60
0.07899	1013.985	1541.70	302.74±0.10	4.1492±0.0005	-0.70±0.10
0.11657	1019.421	1545.22	303.20±0.06	4.1083±0.0005	-0.03±0.07
0.15175	1024.219	1548.05	304.64±0.05	4.0742±0.0005	0.58±0.05
0.19914	1030.754	1551.77	304.61±0.04	4.0289±0.0005	0.99±0.04
0.23176	1035.045	1554.16	305.06±0.03	3.9999±0.0005	1.26±0.03
0.28156	1041.314	1557.65	306.02±0.03	3.9580±0.0005	1.62±0.03
	Cloxa+water+NaCl(0.30012m)
0.01027	1005.835	1537.12	–	4.2078±0.0005	–
0.03577	1007.442	1538.76	300.00±0.80	4.1922±0.0005	-2.50±0.70
0.08030	1011.349	1542.23	301.20±0.00	0.0000±0.0000	-1.50±0.00
0.12389	1017.875	1547.00	303.40±0.10	4.1051±0.0005	-0.26±0.09
0.16407	1024.089	1550.85	304.06±0.07	4.0600±0.0005	0.48±0.06
0.19320	1029.646	1554.01	304.55±0.05	4.0217±0.0005	0.97±0.04
0.23903	1033.459	1556.09	305.49±0.04	3.9961±0.0005	1.31±0.04
0.26144	1039.426	1559.37	306.08±0.03	3.9565±0.0005	1.66±0.03
0.01027	1042.369	1561.01	305.97±0.03	3.9370±0.0005	1.75±0.03
	Cloxa+water+NaCl(0.40623m)
0.00000	1009.984	1543.16	–	4.1578±0.0005	–
0.01265	1011.970	1545.25	298.80±0.60	4.1384±0.0005	-2.80±0.60
0.04111	1016.243	1549.00	302.20±0.20	4.1011±0.0005	-1.30±0.20
0.10324	1025.172	1554.15	304.54±0.07	4.0385±0.0005	0.86±0.07
0.11808	1027.298	1555.49	304.37±0.06	4.0232±0.0005	0.96±0.06
0.17294	1034.861	1560.17	304.82±0.04	3.9699±0.0005	1.34±0.04
0.18248	1036.192	1560.43	304.64±0.04	3.9634±0.0005	1.53±0.04
0.24694	1044.550	1564.40	305.65±0.03	3.9118±0.0005	2.09±0.03
0.29255	1050.206	1566.47	306.35±0.03	3.8804±0.0005	2.50±0.03
	Cloxa+water+NaCl(0.50572m)
0.00000	1013.645	1548.47	–	4.1144±0.0005	–
0.02042	1016.819	1551.41	299.50±0.40	4.0860±0.0005	-1.50±0.30
0.03887	1019.579	1553.68	301.30±0.20	4.0631±0.0005	-0.80±0.20
0.04994	1021.205	1555.04	302.10±0.10	4.0495±0.0005	-0.60±0.10
0.07263	1024.417	1557.18	304.10±0.10	4.0257±0.0005	0.20±0.10
0.09956	1028.180	1559.41	305.24±0.07	3.9995±0.0005	0.82±0.07
0.13802	1033.397	1562.24	306.45±0.05	3.9649±0.0005	1.46±0.05
0.21541	1043.699	1567.77	306.81±0.03	3.8982±0.0005	2.06±0.03
0.25704	1049.040	1570.61	306.96±0.03	3.8643±0.0005	2.26±0.03
	Cloxa+water+NaCl(0.60480m)
0.00000	1017.540	1554.11	–	4.0690±0.0005	–
0.03186	1022.407	1558.55	301.00±0.50	4.0266±0.0005	-1.00±0.20
0.06342	1026.985	1561.87	303.30±0.20	3.9916±0.0005	0.10±0.10
0.10141	1032.314	1564.92	304.84±0.10	3.9555±0.0005	1.06±0.07
0.14013	1037.516	1567.70	306.28±0.07	3.9217±0.0005	1.68±0.05
0.19528	1044.759	1571.46	307.14±0.05	3.8759±0.0005	2.19±0.04
0.23221	1049.570	1574.03	307.09±0.04	3.8456±0.0005	2.35±0.03
0.25649	1052.689	1575.68	307.01±0.03	3.8262±0.0005	2.44±0.03
0.29076	1056.876	1577.87	307.42±0.02	3.8004±0.0005	2.60±0.03
0.35519	1064.600	1582.11	307.77±0.02	3.7527±0.0005	2.80±0.02

 

Table 3: Fitting values or parameters of eq 5 for  and with respect to NaCl molality at 298.15 K and Atmospheric Pressure^a.

m/mol.kg-1 (NaCl)	ϕv0	Sv1	Sv2	CMC ϕv	ϕk0	Sk1	Sk2	CMC ϕk
0.00000	302.82±0.03	8.52±0.24	5.08±0.32	0.212±0.012	-2.15±0.01	12.49±0.10	5.74±0.14	0.209±0.003
0.10049	302.57±0.02	10.24±0.21	6.64±0.24	0.173±0.008	-2.13±0.04	15.56±0.38	6.00±0.43	0.174±0.006
0.20058	300.57±0.39	25.00±4.80	11.80±3.70	0.147±0.040	-2.27±0.04	19.43±0.46	8.02±0.35	0.145±0.004
0.30012	299.48±0.25	48.60±4.90	15.40±2.20	0.082±0.011	-2.75±0.11	31.50±2.20	9.30±1.00	0.097±0.008
0.40623	297.29±0.42	119.00±14.00	10.00±1.70	0.054±0.005	-3.47±0.08	52.70±2.60	8.76±0.32	0.077±0.003
0.50572	297.78±0.42	87.30±8.50	9.60±2.60	0.089±0.006	-2.02±0.09	29.30±1.50	6.90±1.10	0.114±0.006
0.60480	299.46±0.40	54.70±5.60	6.10±1.70	0.127±0.009	-1.88±0.16	29.50±2.30	5.01±0.67	0.122±0.007

 

Conclusions

This work reports experimental information about the molecular interactions and aggregation behavior of cloxacillin sodium in water and in sodium chloride solution at 298.15K. From the measurement apparent molar volume, , apparent molar adiabatic compressibility, , limiting apparent molar volume,  , limiting apparent molar adiabatic compressibility,  , experimental slopes S_v , S_k and critical micelle concentration (CMC) of cloxacillin sodium were calculated and explain in terms of solute-solute, solute-solvent interactions and aggregation behavior. Thedecreases with the increase in NaCl concentration due to the disruption of hydrophobic hydration layer of cloxacillin sodium due to ion-hydrophobic interactions and increases for the ion-ion, ion-hydrophilic interactions. The shows same trend with respect to increasing NaCl concentration which supports. At first critical micelle concentration were found decreasing with respect to NaCl concentration due to minimization of electrostatic repulsion among the charged head groups, and increase in CMC at higher NaCl concentration is due to shape changes of micelles from spherical to different new, i.e cylindrical rod. So, apparent molar volume, limiting apparent molar volume and CMC gives us appreciated information about solute-solvent, solute-solute interaction and aggregation actions, which is very important for our body plasma co-solute interactions. Various ultrasound parameters support the existence of strong solute-solute, solute – solvent interactions and micelle phenomena. The results obtained from these studies can thus be helpful for pharmacological application of drugs.

Acknowledgments

The researchers show gratitude to Department of Chemistry, University of Rajshahi, Bangladesh for given that top laboratory accommodations to convey this research work.

Conflict of Interest

The researchers declare that they have no conflict of interest.

Reference

1. Sarkar, A.; Pandit, B. K.; Sinha, B. J. Chem. Thermodyn.2016, 96, 161–168.
   CrossRef
2. Devunuri, N.; Kancherla, S.; Chennuri, B. K.; Gardas, R. L. J. Mol. Liq.2016, 216, 347–353.
   CrossRef
3. Scott, M. J.; Jones, M. N. Biochim. Biophys. Acta – Biomembr.2000, 1508 (1–2), 235–251.
   CrossRef
4. Mincy, J. W.; Hem, S. L. Journal of Pharmaceutical Science.1975,65(8), 1165-1170.
   CrossRef
5. Hartman, B.; Tomasz, A. Antimicrob. Agents Chemother.1981, 19 (5), 726–735.
   CrossRef
6. Mays, D. L. Anal. Profiles Drug Subst. Excipients.1975, 4 (C), 113–136.
   CrossRef
7. Lei, W.; Li, Z. J. Chem. Technol. Biotechnol.2004, 79 (3), 281–285.
   CrossRef
8. Roy, M. N.; Dewan, R.; Roy, P. K.; Biswas, D. J. Chem. Eng. Data2010, 55 (9), 3617–3624.
   CrossRef
9. Zhuo, K.; Zhang, Q.; Xuan, X.; Zhang, H.; Wang, J. Front. Chem. China2007, 2 (2), 193–198.
   CrossRef
10. Chauhan, S.; Kumar, K.; Singh, K.; Jyoti, J. J. Surfactants Deterg.2013, 17 (1), 169–175.
    CrossRef
11. Bahadur, I.; Deenadayalu, N. J. Solution Chem.2011, 40 (9), 1528–1543.
    CrossRef
12. Khatun, M. R.; Islam, M. M.; Rima, F. R.; Islam, M. N. J. Chem. Eng. Data. 2016, 61 (1), 102–113.
    CrossRef
13. Khatun, M. R.; Islam, M. M.; Islam, M. D. N.; Rhaman, M. D. M.; Nath, R. K. Asian J. Chem.2019, 31 (5), 1113–1127.
    CrossRef
14. Chauhan, S.; Rana D. S.; Akash.; Rana K.; Chauhan, M. S.; Ahmad Umar. Advanced Science Letters. 2012, 5, 1-4.
    CrossRef
15. Naseem, B.; Jamal, A.; Jamal, A. J. Mol. Liq.2013, 181, 68–76.
    CrossRef
16. Elmolla, E. S.; Chaudhuri, M. Desalination.2010, 252 (1–3), 46–52.
    CrossRef
17. Paula V. Messina.; Jose Miguel Besada-Porto.; Juan M. Ruso. Current Topics in Medicinal Chemistry. 2014, 14, 555-571.
    CrossRef
18. Taboada, P.; Attwood, D.; Ruso, J. M.; Sarmiento, F.; Mosquera, V. Langmuir.1999, 15 (6), 2022–2028.
    CrossRef
19. Talwar, G. P.; Srivastava, L. M. Textbook of Biochemistry and Human Biology; Prentice-Hall of India, 2003.
20. Franks. Water Science Reviews. Hydration Phenomena in Colloidal Systems. 1989, 172.
    CrossRef
21. Frank, H. S.; Wen, W.Y. Discuss. Faraday Soc.1957, 24, 133.
    CrossRef
22. Masterton, W. L.; Volume, F.; Molal, P. J. Chem. Phys.1954, 22 (11), 1830.
    CrossRef
23. Noriyuki, Y,;Kensuke L,; Susumu O.  J. Chem. Phys.2006, 124, 184901.
24. Alargova, R. G.; Danov, K. D.; Petkov, J. T.; Kralchevsky, P. A.; Broze, G. Langmuir,1997, 7463 (14), 5544–5551.
    CrossRef
25. Sammalkorpi, M.; Karttunen, M.; Haataja, M. J. Phys. Chem.2009, 5863–5870.
    CrossRef
26. Attwood, D. Adv. Colloid Interface Sci.1995, 55, 271–303.
    CrossRef

---

This work is licensed under a Creative Commons Attribution 4.0 International License.