CHANGES IN THE ELECTRICAL PROPERTIES OF AGARGEL SUPPORTED BILAYER LIPID MEMBRANE BROUGHT ABOUT BY MIDAZOLAM

Accepted Manuscript Title: CHANGES IN THE ELECTRICAL PROPERTIES OF AGARGEL SUPPORTED BILAYER LIPID MEMBRANE BROUGHT ABOUT BY MIDAZOLAM Authors: S. Rameshkumar, Mallaiya Kumaravel PII: DOI: Reference:

S0013-4686(17)31212-4 http://dx.doi.org/doi:10.1016/j.electacta.2017.05.180 EA 29615

To appear in:

Electrochimica Acta

Received date: Revised date: Accepted date:

8-1-2017 11-4-2017 27-5-2017

Please cite this article as: Rameshkumar S., Kumaravel Mallaiya, CHANGES IN THE ELECTRICAL PROPERTIES OF AGARGEL SUPPORTED BILAYER LIPID MEMBRANE BROUGHT ABOUT BY MIDAZOLAM, Electrochimica Actahttp://dx.doi.org/10.1016/j.electacta.2017.05.180 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

CHANGES IN THE ELECTRICAL PROPERTIES OF AGARGEL SUPPORTED BILAYER LIPID MEMBRANE BROUGHT ABOUT BY MIDAZOLAM S.Rameshkumara, Mallaiya Kumaravelb* a

.Department of Chemistry, Sri Vasavi College, Erode,Tamilnadu,India-638 316.

b

.Department of Chemistry, PSG College of Technology, Coimbatore,Tamilnadu,India-

641 004. *corresponding Author Mail ID: [email protected] Graphical Abstract

Various regions of sb-BLM , at different applied AC frequency of Bode Plots in EIS. Highlights    

Non specific interaction of Midazolam with bilayer lipid membrane is confirmed. Long time interaction of MDZ with artificial bilayer membrane system was studied. Effect of bath concentration on the electrical properties of bare and drug doped membrane was explained. The various regions of sb-BLM are electrically characterized using EIS.



Cylic voltammetric and polarization studies supported the EIS studies.

Abstract This work describes the studies on the interaction of Midazolam (MDZ) with agar gel salt bridge supported bilayer lipid membrane ( sb-BLM) using electrochemical impedance spectroscopy, cyclic voltammetry and potentiodynamic polarization methods. The non-specific interaction of MDZ with sb-BLM was studied by following the dose-dependent changes in its electrical properties. MDZ was found to interact with the membrane surface at lower concentrations and impart a tightening effect which is indicated by the increase in capacitance of the membrane. At higher concentrations, MDZ molecules get partitioned into the BLM phase leading to fluidization of the membrane and in turn an increase in ionic conductance across BLM phase. Ionization of midazolam hydrochloride (MDZH+Cl-) depends on the ionic strength of the medium supporting the membranes. The ionic form of the drug interacts preferentially with the polar region of the membrane while the unionized forms are found to interact strongly with its hydrocarbon core regions. Thus, the overall electrical properties of the sb-BLM, observed with MDZ doses, are a consequence of the competitive effects brought about by these drug species on both the regions of the sb-BLMs. Keywords: sb-BLM; Impedance spectroscopy; Drug-membrane interaction; Cyclic voltammetry; Self assembly. Introduction

The bilayer lipid membranes form a protective boundary for the living cell, wherein two amphiphilic phospholipid mono layers are oriented with their hydrocarbon tails directed toward each other and the polar heads turned towards the aqueous electrolytic solutions [1]. The lipid bilayers are impermeable to most of the ions and molecules and represent selective barrier between inside and outside of the living cells. Moreover, proteins embedded in these membranes carry out a wide range of physiological and biochemical processes such as capture and transformation of energy, materials transport across BLM and exchange of nutrients [2, 3]. Methods of forming model membranes have evolved over the years, ranging from planar BLMs to those formed on metal surfaces (s-BLMs) and on gel electrolyte surfaces (sb-BLMs). Analytical applications of BLMs started with the incorporation of ion channels, ion carriers, enzymes and receptors into the lipid bilayers [4]. However, the successful applications of BLMs for drug membrane interaction studies and BLM based biosensors depend mainly on their stability and reproducibility. Supported bilayer lipid membranes overcame many of the drawbacks of black lipid membranes, namely the fragility and sensitivity to electrical and mechanical disturbances [5-7] and represent a useful model system to study the basic interactions in biological cell membranes and are of great interest for technological applications such as biosensors and molecular electronic devices [8-18]. Among the supported BLMs, though s-BLM, formed on the metallic surface is a stable and long- lasting, its resistance is smaller and specific capacitance is 20-50 times higher than that of conventional bilayer lipid membranes [19, 20]. Hence, formation of sb-

BLMs proves to be a promising approach, wherein BLMs are formed on the surface of agarose salt bridge, electrode [21-25] for studying properties and functions cell membranes. Midazolam (MDZ) is a tricyclic benzodiazepine used in anesthetic practice. Its pharmacological activity is due to binding to the specific sites in membrane bound proteins [25]. Being lipophilic, MDZ also interacts with the biomembrane architecture and its concentration in the biomembranes becomes one order higher than that in surrounding medium [26-28] and this type of non specific interaction changes the properties of membrane [29-31]. The drug affects the physical state of membrane [32]. MDZ differs from most of the “traditional benzodiazepines” in having a nitrogen atom in its additional ring structure [25]. This nitrogen is not sufficiently basic to be protonated at physiological pH, but is basic enough to give water soluble salts when treated with strong acids [25]. Its hydrochloride salt (MDZH+Cl-) displays an important pH dependent ring opening reaction [33-36]. It forms soluble salt when the pH of the medium is below 4. At physiological pH, the ring closes and becomes highly lipid soluble. Only few studies have been made on the interaction of benzodiazepines with model membranes [26, 30, 37] and little work has been done on MDZ-BLM interaction. In our previous work, we studied the interaction between MDZ and BLM using planar lipid membrane model, at lower concentrations. The long term interaction and changes in the properties of BLMs at the higher MDZ concentrations were not studied using conventional lipid bilayer systems due to their short life time.

Changes in the electrical properties of BLM with addition of MDZ in 0.01 M NaCl bath was not well established using planar BLM model. The BLM formed over the agar gel surface (sb-BLM) was found to have relatively longer life time than planar BLM. Studies with agar gel supported BLMs on non specific interaction of drug molecules are almost scanty. The purpose of this work is to study the interaction between midazolam and agar gel supported bilayer lipid

membranes

using

electrochemical

impedance

spectroscopy,

cyclic

voltammetry and potentiodynamic polarization studies in NaCl bath solutions. An attempt is also made to explain effect bath concentration on the electrical properties of sb-BLM and drug membrane interaction. Electrochemical impedance spectroscopy monitors the integrity of bilayer lipid membranes in real time and is shown to be very sensitive to the changes brought about by drug molecules in the properties of BLMs [38]. Electrochemical impedance spectroscopy also enables us to evaluate the capacitance and resistance of the bilayer membrane phase in addition to those of the electrical adjacent ionic layer [26]. Cyclic voltammetry and potentiodynamic polarization methods provide the informations on the electrical properties of BLM surface – electrolyte interface. 2. Materials and methods The hydrochloride salt of midazolam (MDZH+Cl-), obtained from Neon Laboratories Ltd, India, was used for the studies. A stock solution of egg lecithin containing greater than or equal to 99% L-α-Phosphatidylcholine (procured from Sigma Aldrich) was prepared by dissolving (5mg/mL) in chloroform. The BLM forming dispersion was

prepared by evaporating 100 µL of the stock solution in a 2 mL screw-cap tube under nitrogen atmosphere and dissolving the resulting lipid film in 200 µL of n-decane (Merck,Germany). The agar gel was filled in 4cm long Teflon tubes with 0.8 mm inner diameter using standard procedure [21]. 0.3 g of agarose ( Sigma Aldrich) was dissolved in 15 mL of boiling saturated KCl solution and the teflon tubes were immersed so that the agar gel filled the tubes completely. A silver/silver chloride electrode, prepared by anodizing a silver wire of diameter 0.35 mm in 0.1 M KCl solution was introduced into the gel in the teflon column to make the electrical contact and allowed to cool to room temperature. The end of the Teflon tube was cut by a very sharp knife and 3 µL of lipid solution in ndecane was applied on the fresh surface of the agarose gel and allowed to dry under nitrogen atmosphere. Again 3 µL of the lipid solution was applied and immersed in 3.5 mL NaCl bath solution for 30 minutes, where the L-α-Phosphatidylcholine molecules underwent self assembly to form the lipid bilayer and the electrical impedance | z | and phase angle remained almost stable for 40 hours in most of the experiments. All the aqueous solutions used in our studies were prepared using Miili-Q water (R>18MΩ cm-1). Stirring device, vibration isolated platform, faraday cage and Ag/AgCl electrodes were fabricated following standard procedures [39-43]. The electrochemical studies were carried out using PARSTAT 2273, Advanced Electrochemical System with a three electrodes set up, where in a Pt foil served as the counter electrode, a Ag wire coated with AgCl served as the reference electrode, while the BLM formed on the agarose gel surface served as the working electrode. After every drug

dose, a stabilization period of 30 minutes was allowed for the drug to equilibrate between the two phases. The electrochemical impedance spectra of the bare and drug doped sbBLMs were recorded in the frequency range 1 MHz to 10 mHz at the open circuit potential by superimposing a sinusoidal AC signal of small amplitude 25 mV. Analysis of the impedance data was done using ZSimpWin 3.21 software. The cyclic voltammograms of bare and drug doped sb-BLMs were recorded in the potential range 0.5 V to -0.5 V at a scan rate of 100 mV/s. The potentiodynamic polarization studies were carried out in the potential range -0.5 V to 0.5 V at the scan rate of 0.166mV/s. 3. Results and discussion 3.1 Electrochemical Impedance spectroscopy The spontaneous self assembly of phospholipid molecules into a bilayer on the agar surface and its interaction with midazolam at various concentrations were followed by monitoring the electrical impedance | z | and the phase angle shift (). The Nyquist plots obtained for bare and drug doped sb-BLMs formed in 1.0M, 0.1M and 0.01M NaCl bath solutions are shown in Fig.1a, 1b and 1c. The typical impedance spectra obtained are semicircles with mass transfer phenomenon at lower frequencies and the nature of the Nyquist plot in these frequencies end depends on the gel concentrations [44] and the spur observed characterizes the agarose electrode [44]. The semicircle nature of the impedance spectra indicates that the lipid bilayers formed are dielectric in nature with leakage [45, 46]. Considering the bilayer - solution interface and

the lipid bilayer phase, the capacitance(C) and conductance (G) of the bilayer system respectively are given by the expressions [47].

𝐶=

𝐺=

2 2 𝐶𝑀 𝐺𝑃 +𝐶𝑃 𝐺𝑀 +(𝜔2 𝐶𝑀 𝐶𝑃 )(𝐶𝑃 +𝐶𝑀 )

(1)

(𝐺𝑃 +𝐶𝑀 )2 +𝜔2 (𝐶𝑃 +𝐶𝑀 )2 2 𝐺𝑀 𝐺𝑃 (𝐺𝑀 +𝐺𝑃 )+𝜔2 (𝐶𝑀 𝐺𝑃 +𝐶𝑃2 𝐺𝑀 )

(2)

(𝐺𝑃 +𝐶𝑀 )2 +𝜔2 (𝐶𝑃 +𝐶𝑀 )2

Thus, both capacitance (C) and conductance (G) (or membrane resistance R) are dispersed with the frequency of the AC perturbation. In general the impedance spectra obtained are not straight forward and the corresponding impedance parameters and the interpretation of data can be done using equivalent circuits [25, 48]. The observed impedance spectra showed the best fit with the circuit shown in Fig.2 [20]. In this circuit, Rs corresponds to the resistance of the electrolyte, connectors, wires etc and its value is proportional to length of the gel column in the working electrode [20]. For long term investigation, the length of the agar column is very important and as recommended agar columns of length 3.0 to 4.0 cm were used for the studies. CPE is constant phase element which is used in place of a capacitor for bilayer lipid membrane -electrolytic solution interface and its impedance function is represented by the expression [49, 50];

𝑍𝐶𝑃𝐸 =

1 𝑌0 (𝑖𝜔𝑧)𝑛

(3)

Where 𝑌0 represents the admittance of the system, ‘𝜔’ is angular frequency and n is a

constant (-1≤ n ≤1). The CPE represents pure resistor when n=0 and pure capacitor when n=+1. If n=-1 then the CPE represents an inductor [49, 51]. The presence of CPE in the proposed equivalent circuit can be easily detected from the depressed nature of the semicircle in the impedance spectra, which indicates a non-ideal capacitive behavior of the interface [49, 51]. In general the depression of the semicircles in the impedance spectra is attributed to the chemical inhomogeneities and anion adsorption [49,50]. Rp represents charge transfer resistance of the membrane-solution interface. CM and RM represent the capacitance and resistance of the phospholipid bilayer phase. Ca, Ra and W correspond to the capacitance, resistance and Warburg impedance of the agar gel- bilayer interface. Qualitative treatment of the observed impedance spectra of sb-BLMs can be made by considering slabs of different dielectric properties present in them [ 25, 52] as in the case of black lipid membrane. The flow of ions in each slab gives rise to an ionic current while accumulation of ions at the boundary between contiguous dielectric slabs under AC conditions gives rise to a capacitive current [52]. Hence, each dielectric slab in the proposed equivalent circuit, shown in Fig.2, is simulated by a parallel combination of a resistor and a capacitor, namely by a RC mesh [25, 52]. However, when the net impedance is also affected by mass transfer in a slab, Warburg impedance (W) is also used in the RC mesh. The contribution from each dielectric slab to the net impedance at different frequency ranges can be determined as follows[25,52]: When two circuit elements have appreciably different impedances, their overall impedance is controlled by the circuit element of higher impedance, if they are in series and by the circuit element of lower

impedance if they are in parallel. The impedance of the capacitor under AC conditions is given as

𝑋𝐶 =

1 𝜔𝐶

=

1

(4)

2𝜋𝑓𝐶

At the highest frequencies the overall impedance |Z| is determined by the resistance Rs, since at these frequencies, 𝑅𝑠 ≫

1 𝜔𝐶𝑃

and same is true for the impedances of Rm Cm and Ra

Ca meshes, whose impedances are determined by the lowest of the impedances of these two elements in parallel. It can be seen from bode impedance plot (Fig.3) that at these frequencies the phase angle 1 𝜔𝐶𝑃

is nearly equal to zero and with decreasing frequency

becomes greater than both Rs and

1 𝜔𝐶𝑎

but still lower than that of RM. Hence the plot

|Z| vs log(f) shows a straight line with a slope of -1 in these frequencies. With further decrease in frequency

1 𝜔𝐶𝑀

increases and its contribution to the total impedance is greater

than the other RC meshes. Hence, again a straight line with a slope -1 continues (Fig.3) and becomes comparable with RM. When

1 𝜔𝐶𝑀

becomes comparable and lesser than RM, at these frequencies, the overall

impedance is independent of frequency and is completely controlled by RM and, the Bode phase at these frequencies decreases towards zero. But before reaching the phase angle zero, with a further decrease in frequency,

1 𝜔𝐶𝑎

becomes greater than RM and once again a

new straight line with slope -1 is observed and hence phase angle in the Bode plot moves towards 900.

The electrochemical parameters obtained using the proposed equivalent circuit for the MDZ doped sb-BLMs in 1M, 0.1M and 0.01M NaCl bath solutions are given in Table 1. The specific membrane capacitance observed is consistent with those reported for classical BLM [20]. The thickness of the membranes formed on the agar surface is calculated from the specific membrane capacitance following the equation [25].

𝐶𝑀 =

𝜀0 ε 𝑑

(5)

Where 𝜀0 is the permittivity of free space (𝜀0 =8.854*10-12 FM-1], 𝜀 is dielectric constant of the lipid bilayer phase 𝜀=2.05 [25 53]. The calculated thickness of the BLMs in 1.0M , 0.1M and 0.01M NaCl bath solutions respectively are 4.67, 5.1 and 6.1nm. These values are very close to twice the thickness of the lecithin monolayer (2 – 5 nm) and consistent with those values reported in the literature [25, 54-56] providing evidence for the formation of bilayer lipid membranes on the agar gel surface. From the electrochemical parameters given in Table 1, it is seen that capacitance of the polar region (CP) i.e. the capacitance membrane surface - bath solution, decreases and reaches a constant value, while that of the lipid bilayer phase increases with drug dose. The initial increase in capacitance of polar region can be attributed to the higher relative permittivity of ( D = 60 ) of the polar region of the membrane than the core of the lipid bilayer, where the ionized form of midazolam interacts[37].

Midazolam in solution exists in ionized, neutral and ion pair forms [25]. The extent of these forms in solution is strongly influenced by the concentration of Cl- ions in the bath solution. A complex equilibrium existing among these species in solution is represented as follows [25,57].

(6)

Evidently due to common ion effect of the Cl- ions, the proportion of neutral and ion pair forms of midazolam in 1.0M NaCl bath solution is larger than that in 0.1 M NaCl, which in turn is greater than that in 0.01M NaCl solution. At neutral pH, the positive charges on the surface of lipid bilayer due to nitrogenous base of phospholipid molecules are covered by Cl- ions from bath solution and hence the surface of BLM is negatively charged due to uncovered negatively charged phosphate groups [59]. However, this negative charge is partially neutralized by the adsorption of Na+ ions from bath solution [58]. The surface negative charge of BLM decreases with increase in NaCl concentration, due to the increased adsorption of Na+ ions at the BLM surface [ 58 ] or in other words the excess surface negative charge of BLM increases as the bath concentration decreases. Hence, the interaction between ionized form of midazolam at the and bilayer - solution interface is larger in 0.01 M NaCl bath solution when compared to that in 0.1M and 0.01M solutions.

From Table 1 it can also be seen that the capacitance of bilayer lipid membrane phase increases with drug dose, which is biphasic as shown in Fig. 4. The specific membrane capacitance of the lipid bilayer system can be expressed interms of capacitance of polar region (CP) and that of bilayer membrane phase (CM) by the following expression. 1 𝐶

=

1 𝐶𝑃

+

1

(7)

𝐶𝑀

Therefore,

𝐶=

𝐶𝑃 𝐶𝑀

(8)

𝐶𝑃 +𝐶𝑀

The capacitance of lipid bilayer phase is related to its thickness and the dielectric constant of the lipid core by the expression.

𝐶𝑀 =

𝜀0 𝜀𝑀 𝑑

(9)

Where 𝜀𝑀 and d are the relative permittivity and thickness of the lipid bilayer phase respectively. The initial increase in the capacitance of the lipid bilayer phase is due to the interaction of the ionized form of midazolam at the polar region of membrane surface which tightens the lipid bilayer phase and thereby the membrane thickness decreases. At the saturation concentration, where CP attains a constant value, the penetration of neutral and ion pair forms into the core of bilayer lipid phase is larger and this can be attributed to

the second phase increase in capacitance of lipid bilayer systems. When the MDZ molecules are inserted into the lipid bilayer phase, the capacitance of the lipid bilayer system is given by the following expression,

𝐶 𝑀𝐷𝑍 =

𝐶𝑃 (𝐶𝑀 +𝐶𝑀𝐷𝑍 )

(10)

𝐶𝑃 +(𝐶𝑀 +𝐶𝑀𝐷𝑍 )

𝐶 𝑀𝐷𝑍 can also be expressed as a function that depends on the capacitance of the membrane in the absence of MDZ, as follows [25] 𝐶 𝑀𝐷𝑍 = (𝐶 +

𝐶𝑃 𝐶𝑀𝐷𝑍 𝐶𝑃 +𝐶𝑀

)(

𝐶𝑃 +𝐶𝑀 𝐶𝑃 +𝐶𝑀 +𝐶𝑀𝐷𝑍

)

(11)

Where,

𝐶𝑀𝐷𝑍 =

𝜀0 𝜀𝑀𝐷𝑍 𝑁𝑀𝐷𝑍 𝑉𝑀𝐷𝑍

(12)

𝑁𝑀𝐷𝑍 = average number of MDZ molecules per unit area of the bilayer 𝑉𝑀𝐷𝑍 = the molecular volume of MDZ 𝜀𝑀𝐷𝑍 = the dielectric coefficient of MDZ The 𝐶𝑀𝐷𝑍 will be smaller than C and 𝐶𝑃 even at very high concentrations of MDZ, because the cross sectional area of phospholipid occupied domain is much higher than the cross sectional area of the MDZ occupied domain. Hence, this condition makes, 𝐶𝑃 +𝐶𝑀 𝐶𝑃 +𝐶𝑀 +𝐶𝑀𝐷𝑍

≃1

(13)

and

𝐶 𝑀𝐷𝑍 = 𝐶 +

𝐶𝑃 𝐶𝑀𝐷𝑍 𝐶𝑃 +𝐶𝑀

(14)

and hence, the capacitance of bilayer lipid system increases with drug dose. 3.2 Effect of NaCl concentration in the bath solution on the capacitance, thickness and conductance of lipid bilayer phase The increase in adsorption of Cl- ions on the lipid bilayer surface with NaCl concentration as discussed earlier, brings a tightening effect on the membrane and in turn decrease in thickness of lipid bilayer phase [25]. The capacitance of lipid bilayer phase (CM), which is inversely proportional to thickness of the membrane, increases with NaCl concentration as seen in Table 1 is a result of the arrangement of large number of Na+ and Cl- ions parallel to BLM surface. From the binding constants of Na+ and Cl- ions on the BLM surface, it is known that the adsorption of Cl- ions on BLM surface takes place to a larger extent when compared to Na+ ions [58] and also the surface negative charge buildup on BLM surface increases with decrease in NaCl concentration [25,58]. Though the BLM system is rigid, it is associated with definite ionic conductance due to its leaky nature to smaller cations such as Na+ and K+ ions [60].When the NaCl concentration is high in the bath solution the adsorption of Cl- ion will be larger and will cover the maximum BLM surface and block the minute pores or holes on the BLM surface and thus provides resistance for the flow of smaller ions like Na+, K+ across the BLM phase. Hence, the membrane conductance (G) decreases or membrane resistance(R) increases with increase in NaCl concentration in the

bath solution. The partition of lipophilic drugs such as MDZ into the BLM causes fluidization of the BLM phase which tends to increase its leaky nature to smaller cations [25,59].The ionic pressure on the BLM surface also influences the ionic resistance or conductance across membrane - agar gel interface, the ionic pressure exerted on the BLM surface from bath solution makes it to stick firmly on the agar surface and block the nano holes on the BLM surface, pointing agar surface and hence resistance for the flow of ions across BLM - agargel interface increases with increase in NaCl concentration in the bath solution, which can be seen from the Nyquist plots shown in Figure 1a,1b and 1c. The ionic pressure on the BLM surface in the different bath solutions are in the order 1.0 M > 0.1 M > 0.01 M and the thickness of the BLM phase followed reverse order. The ionic pressure is always there even the drug is partitioned into the membrane and initially it increases slightly with increase in MDZ concentration, where the ionized form (MDZH+) of midazolam gets attached to the BLM surface on the negative charges on BLM surface and decreases the leaky nature of BLM phase. From table 1 it is also clear that initially the capacitance (CP) of the BLM – electrolyte solution interface decreases and reaches almost a constant value while charger transfer resistance (RP) increases and then decreases with drug concentration. The decrease in capacitance (CP) is due to the adsorption of ionized form of midazolam molecules on the negative charges of BLM surface and at the saturation concentration CP attains a constant value. The adsorption of ionized form of MDZ molecules decrease the negative charge on the BLM surface and offers resistance for movement of smaller ions like Na+ and K+ across BLM – electrolyte solution interface, while at higher drug concentration the BLM phase is fluidized due to partition of drug

molecules into the membrane phase and allows the flow ions across it, this increase in ionic conductance across BLM phase is only possible when the ions move across the BLM – electrolyte solution interface, which is actually observed and this the reason for increase in ionic conductance across the BLM – electrolyte solution interface . 3.3 Cyclic voltammogram The cyclic voltammograms recorded for the bare and drug doped sb- BLMs in 1.0 M, 0.1M and 0.01 M NaCl bath solutions, are shown in Fig.5a,5b and 5c. The cyclic voltammograms obtained have triangular wave shapes indicating the absence of redox reactions in the recorded potential range. The capacitance values obtained from these voltammograms, presented in Table 2, correspond to the capacitance of membrane-solution interface, which has the same order as CP determined from EIS with slightly larger value. From table 2

it can be seen that the capacitance increases with decrease in NaCl

concentration in the bath solution, this is due to a fact that when NaCl concentration in the bath solution decreases the adsorption of Cl- ions on the membrane surface decreases, hence net negative charge on the membrane surface increases, because the Na+ ions have lower adsorption than Cl- ions [58]. On running cyclic voltammograms, when the potential is scanned in the positive direction, the Cl- ions and water dipole arrange parallel to the membrane surface and while scanning the potential in the negative direction the positively charged forms of MDZ molecules, Na+ ions and water dipole arrange parallel to the membrane surface. The arrangement of ions parallel to membrane surface increases with decrease in NaCl concentration, since the concentration of unneutralized positive and

negative charges increases with decrease in NaCl concentration. Hence, the capacitance at the membrane – solution interface increases while scanning between positive and negative potentials with decrease in NaCl concentration in bath solution. The positively charged midazolam molecules may also attach to the membrane surface through Cl- bridge, as explained for the adsorption of organic compounds through sulphate and chloride bridges [46]. 3.4 Polarization studies The Tafel plots recorded for bare and drug doped BLMs in 1.0 M, 0.1 M and 0.01 M NaCl bath solutions are shown in Fig.6a, 6b and 6c. From these plots it is clear that current density in the cathodic region increases with increase in MDZ concentration while it decreases in the anodic region. This is due to the fact that in the cathodic region membrane surface is negatively charged which favors the adsorption of Na+ ions and ionized form of midazolam (MDZH+). The ionized form of midazolam can directly attached to the negative charges (phosphate groups) on the membrane surface and also through Cl- ion bridges. The concentration of ionized form of midazolam increases with increase in drug dose and due to this attachment of ionized form of MDZ through Cl- ion bridges on the BLM surface also increased. This ionized form of MDZ attached to the BLM surface through Clion bridges can get partitioned into the BLM phase. The partition of MDZHCl into the BLM can cause fluidization of bilayer lipid membrane phase and increases the flow of smaller cations such as Na+ and K+ ions across the BLM phase. In the anodic region the

membrane surface is positively charged and will repel the ionized form (MDZH+) of midazolam and Na+ ions, but attracts the Cl- ions. The Cl- ions get attached to BLM surface largely due to its high binding constant on BLM surface and provide resistance for movement of smaller ions such as Na+ and K+ across BLM phase. Both in 1.0 M and 0.1M NaCl bath solutions there is shift in the equilibrium membrane potential (EEMP) in the positive direction with drug dose and the shift is greater than 78 mV indicating that the addition of drug to the bath solution greatly affects the anodic process. In contrary, the current density increases both in anodic and cathodic regions of Tafel plots of BLM in 0.01 M NaCl bath solution without almost any change in equilibrium membrane potential, which indicates that in 0.01M NaCl bath solution both anodic and cathodic regions are equally affected with drug dose and the increase in current density observed in the anodic region is due to lesser ionic pressure of bath solution as discussed earlier. In all the cases equilibrium current density also increases with drug concentration in the bath solutions indicating increase in conductance across BLM phase due to fluidization effect of drug molecules partitioned into the BLM phase. Conclusions In solution, various forms of midazolam exist in a complex equilibrium and the extent of these forms depends on NaCl concentration of the medium. While the ionized forms of midazolam interact with the BLM surface, neutral forms interact with the hydrocarbon core of BLM.. The net charge of BLM surface also depends on NaCl concentration in bath solution. The behavior of BLM-agar surface interface in 0.01 M NaCl bath solution is

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Figure.1a,1b and 1c AC Impedance Spectroscopy of bare and drug-doped sb-BLMs NaCl bath solutions



Figure.2 An equivalent circuit for sb- BLM Model

Figure.3

Bode Plots of lZl (black squares) and phase angle (green squares) against log(frequency) for a bare membrane.



Figure.4

Effect of MDZ concentration on the capacitance of BLM phase







Figure. 5a,5b and 5c Cyclic Voltammograms recorded for interaction of MDZ with sb-BLM in NaCl bath solutions.





Figure.6a,6b and 6c Potentiodynamic polarization curves recorded for the interaction of MDZ with sb-BLM in NaCl bath solutions.

Table.1 Electrochemical Impedance parameters of bare and drug doped membrane in NaCl bath solutions S.N o

Concentrati on of MDZ ( µM)

1.0 M NaCl RP CP CM Ω (pF) (nF X10

1

0

92.61

2

10

92.58

3

20

92.49

4

40

101.5

5

60

113.4

6

80

119.0

7

100

121.6

8

200

128.0

9

400

133.1

10

600

138.0

11

800

142.2

12

1000

145.2

RM 9 (10 Ω

7

)

)

7.0 1 7.1 1 7.2 1 6.9 1 6.5 2 6.3 5 6.0 2 5.3 7 5.1 1 3.6 1 2.9 0 2.4 3

1.9 5 1.9 8 2.0 9 2.8 2 2.9 3 2.9 6 2.9 5 2.9 4 2.9 7 2.9 4 2.9 8 2.9 7

1.39 1.47 1.56 1.33 1.27 1.06 0.92 1 0.73 3 0.41 5 0.26 1 0.10 5 0.09 1

CP (pF) 49.0 9 48.8 8 48.0 3 47.8 6 47.3 7 46.9 4 48.6 1 49.2 2 51.2 5 57.2 7 62.2 6 69.2 8

0.1 M NaCl RP CM Ω (nF

RM 8 (10 Ω

X107

)

)

8.14

1.7 9 1.8 3 1.8 7 1.8 9 1.9 0 2.6 3 2.6 6 2.6 5 2.6 9 2.6 7 2.6 6 2.6 9

3.89

8.16 8.31 8.40 8.52 8.55 8.61 8.63 8.65 1.44 0.98 5 0.59 2

4.13 5.41 7.64 8.71 2.16 1.98 0.83 7 0.64 0 0.31 2 0.18 2 0.06 8

CP (pF ) 49. 1 48. 9 48. 0 47. 8 47. 3 46. 9 46. 6 46. 2 46. 2 68. 4 79. 5 81. 2

0.01 M NaCl RP CM RM Ω (nF (107Ω X107

)

)

9.32

1.5 2 1.5 7 1.6 2 1.6 7 1.7 1 1.7 6 1.7 9 1.8 2 2.1 2 2.4 9 2.5 1 2.5 2

9.65

9.35 9.82 9.89 9.92 8.45 6.25 3.84 1.27 0.98 1 0.77 5 0.51 1

9.26 8.35 8.11 7.56 6.92 6.12 0.86 1 0.57 4 0.43 2 0.27 3 0.19 3

Table.2 Changes in the Capacitance of sb-BLM – Electrolytic Solution Interface with MDZ Concentration derived from Cyclic Voltammograms Midazolam Concentration (µM) 0 10 100 1000

1.0 M NaCl 452 507 591 713

Capacitance (pF) 0.10 M NaCl 466 582 657 797

0.01 M NaCl 2257 2522 2741 6382