Enhanced Transport of Nandrolone Decanoate Drug by Human Serum Albumin in Presence of [ BMIM ] PF 6 and [ BMIM ] BF 4

The interaction of nandrolone decanoate drug dissolved in (2%) [BMIM]BF4 or [BMIM]PF6 with human serum albumin (HSA) at different temperatures in the range of 285–310 K was examined by fluorescence quenching. Stern-Volmer equation and its modified form were used to determine the interaction parameters K and n. The results revealed that binding affinities of HSA for nandrolone decanoate drug in 2% [BMIM]BF4 or [BMIM]PF6 are in the order of 105 M–1 and the number of bound drug molecules per HSA macromolecule are approximated to 1 at all temperatures studied. The thermodynamic parameters: free energy change (∆Go), enthalpy change (∆Ho) and entropy change (∆So) for HSA– nandrolone decanoate/ionic liquid were calculated according to van’t Hoff equation. Data analysis indicated that both electrostatic interactions and hydrophobic ineractions played important roles in the interaction of nandrolone decanoate drug with HSA.


Introduction
The nature and types of interaction between small molecules and biomacromolecules characterize an active area of research.2][3][4][5] These interactions can also regulate the drug stability as well as toxicity for the duration of the chemotherapeutic course of action. 1 Ionic liquids are remarkable for their tunable nature and exceptional properties.They have attracted considerable attention in various areas of chemistry, synthesis and catalysis, 1 electrochemistry, [5][6][7] biomass conversion, 8,9 biotechnology 10,11 and many other fields.The studies on ionic liquids cover a wide range of topics that include structure and properties as well as advanced chemical transformations. 12,13Improvements of separation, 14 electrochemical behavior, 15 analytics, extraction, biocatalytic properties 16 are some established areas.The ecological impact of ionic liquids has been recently recognized. 17,18ere are three generations of ionic liquids depending on their chemical structure and properties. 19Limited water solubility and low dissolution rate are among the major problems of modern drug development.7][28][29] Ionic liquids can enhance water solubility of hydrophobic compounds, possibly due to formation of associates between ionic liquids and biomolecules. 30androlone decanoate (Fig. 1) is a parenteral anabolic steroid.It is primarily used to treat anemia, chronic Considering limited solubility of the drug in water, it is of interest to study the effect of some ionic liquids, namely 1-butyl-3-methyl-1-imidazolium tetrafluoroborate [BMIM][BF 4 ] (Fig. 2.a) and 1-butyl-3-methyl-1-imidazolium hexafluorophosphate [BMIM][PF 6 ] (Fig. 2.b) to increase its solubility in water and to enhance its ability to transfer the drug through the body.Their behaviors were examined by following and analyzing the fluorescence spectra of human serum albumin-nandrolone decanoate mixture in the absence and presence of the selected ionic liquids as solvents.Since the changes in the fluorescence of HSA are indeed related to changes in the fluorescence of tryptophan residue caused by interactions with a variety of quenchers.
In this work, the quenching of HSA fluorescence intensities by the studied drug/2% ionic liquid systems was explored for the first time at different temperatures in the range of 285-310 K.
Binding parameters (K and n) were determined and thermodynamic parameters of HSA-nandrolone decanoate /ionic liquid interaction were calculated.Data analysis indicated that electrostatic interactions and hydrophobic associations played different roles for the two ionic liquids.
effect.All HSA solutions were prepared in 50 mM potassium phosphate buffer solutions of pH 7.0 containing 25 mM NaCl to maintain constant ionic strength.Considering large molar absorptivity of HSA, the solutions were properly diluted to give absorbance in the range 0.2 to 0.7 in the linear response covered by the instrument.The HSA stock solution was kept in the dark at 281 K and the pH was checked with a suitably standardized pH meter.During the protein-drug binding experiments, the HSA concentrations were fixed at 2 μM and the drug concentrations were varied from 0 up to 6.5 µM.

Apparatus
Lambda 20, a Perkin Elmer UV/Vis spectrophotometer, was used for determination of the concentration of HSA using ε = 35219 M -1 cm -1 at 278 nm as the molar extinction coefficient of HSA. 31 Fluorescence spectra were obtained using Perkin Elmer luminescence (series no.70412) spectrometer equipped with a water-jacketed cuvette holder that was maintained at each chosen temperature by means of a circulatory water bath.

3. Fluorescence Measurements
Fluorescence spectra (in the range of 280-450 nm upon excitation at 280 nm) were recorded at various temperatures; 285, 293, 303 and 310 K.The reactants were kept at the same temperature before mixing and making fluorescence measurements.The fluorescence intensities were automatically recorded by injecting 20 µL samples of 80 µM drug solution containing 2% ionic liquids into the HSA solution which makes the final concentration of the test solution 0.148% considering the dilution effect.Each titration was made three times.
The inner-filter effect corrections 32 were done according to the formula: (1)   where E corr is the corrected fluorescence value, Fobs the measured fluorescence value, A exc and A em the measured absorbance values at the excitation and emission wavelengths, respectively.The interval between every two connective sample additions was 2 minutes.

Results and Discussion
Fluorescence data were corrected for the inner filter effect caused by attenuation of the excitation beam and emission signal because of absorption by quencher and fluorophore to obtain the proper fluorescence intensity values.The value of the correction factor was 1.05 that incurs a negligible effect to the results.
Solutions of HSA containing amounts up to 6.5 micromolar were obtained by dilution with buffer containing the ionic liquid and used further with the drug to study its Yasseen et al.: Enhanced Transport of Nandrolone Decanoate Drug ... Nandrolone decanoate drug was selected as a model drug due to its limited solubility in water.In addition, organic solvent may cause denaturation to HSA.Therefore, utilization of selected ionic liquids is justified.
This study is on the effect of ionic liquids on drug-protein interaction.The interaction between nandrolone decanoate and HSA in absence of ionic liquids showed very little quenching in fluorescence intensity which is an indication of marginal interaction.On addition of ionic liquid to the nandrolone decanoate-HSA mixture, a notable drop in fluorescence intensity indicating remarkable effect of ionic liquid on the drug-protein interaction.

1. Interaction of Nandrolone Decanoate Drug with HSA in Presence of Ionic Liquids
Emission spectra of the protein's intrinsic fluorescence was performed to investigate the interaction between the drug (nandrolone decanoate) in 2% ionic liquid ([BMIM]BF 4 or [BMIM]PF 6 ) and HSA at four different temperatures: 285, 293, 303 and 310 K.
The fluorescence spectra of HSA (2 μM), obtained in the emission range of 250-450 nm in the absence and the presence of increasing nandrolone decanoate concentrations (0-6.5 μM) containing 2% ionic liquids ([BMIM] BF 4 or [BMIM] PF 6 ) were studied in phosphate buffer at pH 7.0 and are shown in Figs. 3 and 4, respectively.
The fluorescence spectrum of HSA produced an emission maximum at 343 nm due to the presence of sole tryptophan (Trp-214) residue which is located in subdomain IIA. 33ddition of nandrolone decanoate to HSA resulted in a gradual decrease in the fluorescence intensity.However, no shift in the emission maximum was noticed upon nandrolone decanoate addition to HSA.][36][37] Fluorescence quenching of the single tryptophan residue in HSA was used to measure drug-binding affinity.Tryptophan fluorescence is the most frequently examined A very small shift (about 3 nm in the wave length of the fluorescence intensity) was observed on addition of increasing amounts of the ionic liquids to the test solution.It is inferred that the protein retains its native three-dimensional structure under the conditions studied in this ex-periment.In this system, quenching in fluorescence was due to interaction of nandrolone decanoate and HSA but effects of ionic liquids were negligible and the main effect of which was a solubilizing role.
The quenching data were analyzed using the Stern-Volmer equation, Where F 0 and F are the relative fluorescence intensities in the absence and presence of quencher drug, [Q] is the concentration of quencher, K sv is the Stern-Volmer quenching constant and K is the binding constant for the accessible fluorophores. 32

2. Stern-Volmer Analysis of HSA Interaction with Nandrolone Decanoate Drug
The fluorescence quenching data for nandrolone decanoate -HSA in presence of the two selected ionic liquids were analyzed by Stern-Volmer equation (equation 2).
The values of K SV constants at different temperatures are listed in Table 1.The patterns of the F 0 /F versus [Q] (Stern-Volmer) plots for HSA were shown in Figs, 5 and 6.These plots present good linear relationships between F 0 /F versus drug concentration.
The fluorescence intensities were quenched by the interaction of HSA with nandrolone decanoate in the presence of the ionic liquids ([BMIM]BF 4 and [BMIM]PF 6 ).
This behavior is attributed to nandrolone decanoate binding to HAS, which may be facilitated by both ionic liquids due to their hydrophobicity and enhanced transport of the drug to the binding site.
From table 1, it is noticed that the binding constants for the two systems: nandrolone decanoate -HSA in presence of the studied ionic liquids, [BMIM]BF 4 and [BMIM]PF 6 ,were of the same order of magnitude, being slightly higher with [BMIM]PF 6 as compared to that with the former.This result is in accordance with expectations based on the similarity in structure of the two compounds.The hydrophobicity of hexaflurophosphate is more than that of tetrafluoroborate anion.Therefore, the ionic liquid containing hexaflurophosphate can penetrate into the hydrophobic region of HSA making stronger bonding with higher binding constants and explains the observation that this ionic liquid is more effective on the protein.

Modified Stern-Volmer Analysis of Binding Equilibria
Quenching data were also analyzed according to the modified Stern-Volmer equation, equation 3. It is shown in Figs.7 and 8 that the dependence of log [(F 0 -F)/F] versus log[quencher] is linear with a slope equal to the value of binding sites (n) and y-intercept corresponding to the log of the effective quenching constant (K).The corresponding results of the linear regression equations at four different temperatures are shown in Table 1.The decreas-   ing trend of K with increasing temperature is in accordance with the K SV dependence on temperature resulting in a reduction of the stability of the nandrolone decanoate -HSA complex at higher temperatures.

4. Types of interaction force between HSA and the nandrolone drug
The changes of free energy, enthalpy and entropy were estimated using the Van't Hoff 's equation: (4)   and the relationship, (5)   Using van't Hoff equation that can br rewritten as: (6)   Integration produces the equation: (7)   Since: (8)   Equating the right sides of the equations produces: (9)   This equation is used for calculation of the equilibrium values obtained for the interaction of drug and HSA.
According to the effective binding constants of nandrolone decanoate drug to HSA obtained at the studied temperatures, the thermodynamic parameters were determined from linear Van't Hoff plot (Fig. 9) and the values are presented in Table 2.The formation of drug -HSA complexes in presence of the selected ionic liquids were carried out spontaneously through exothermic reaction accompanied by positive ∆S o values.Ross and Subramanian 38 have characterized the sign and magnitude of the thermodynamic parameter associated with various kinds of interaction that may take place in protein association processes.
Ionic liquid facilitated binding process of nandrolone decanoate to HSA in aqueous solution is governed by hydrophobic as well as specific interactions (electrostatic and/or hydrogen bond).Binding was characterized by positive entropy and negative enthalpy changes and is more effective in the presence of hexafluorphosphate anions although the size of the anionic part in the [BMIM]BF 4 is smaller and can more easily approaches the hydrophobic core of HSA.

Conclusion
The interaction between nandrolone decanoate drug and HSA in presence of ionic liquids [BMIM]BF 4 and [BMIM]PF 6 was investigated through fluorescence quenching methods.
The results indicate that the studied ionic liquids affect the binding of nandrolone decanoate drug to HSA at various temperatures in the range of 285-310 K.It was observed that the binding constants between nandrolone and HSA were decreased with temperature.
In addition, the binding constants in presence of ionic liquid [BMIM]PF 6 is slightly greater than that in presence of [BMIM]BF 4 in the drug solution.
The study opened the door to new avenues in the screening and design of appropriate safe drugs containing ionic liquids that will likely find important applications in modern medical research.

Table 1 :
Results of the binding in the nandrolone decanoate/ionic liquid/HSA systems..

Table 2 :
Thermodynamic parameters of the nandrolone decanoate/2% ionic liquid-HSA interaction at different temperatures and pH 7.0.