Drug Partitioning in Micellar Media and its Implications in Rational

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Drug Partitioning in Micellar Media and its Implications in Rational Drug Design: Insights with Streptomycin Eva Judy, Darshna Pagariya, and Nand Kishore Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b04346 • Publication Date (Web): 26 Feb 2018 Downloaded from http://pubs.acs.org on February 27, 2018

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Drug Partitioning in Micellar Media and its Implications in Rational Drug Design: Insights with Streptomycin Eva Judy, Darshna Pagariya, Nand Kishore* Department of Chemistry, Indian Institute of Technology Bombay, Powai, Mumbai – 400 076, India. *Corresponding author (Email: [email protected])

_________________________________________________________ ABSTRACT: Oral bioavailability of a drug molecule requires its effective delivery to the target site. In general, majority of synthetically developed molecular entities have high hydrophobic nature as well as low bioavailability, therefore the need for suitable delivery vehicles arises. Self-assembled structures such as micelles, niosomes, and liposomes have been used as effective delivery vehicles and studied

extensively.

However,

the

information available in literature is mostly qualitative in nature. We have quantitatively investigated the partitioning of antibiotic drug streptomycin into cationic, non-ionic and mixture of cationic and non-ionic surfactant micelles and its interaction with the transport protein serum albumin upon subsequent delivery. A combination of calorimetry and spectroscopy has been used to obtain the thermodynamic signatures associated with partitioning and interaction with the protein, and the resulting conformational changes in the latter. The results have been correlated with other class of drugs of different nature to understand the role of molecular features in the partitioning process. These studies are oriented towards understanding the physical chemistry of partitioning of a variety of drug molecules into suitable delivery vehicles and hence establishing structure-propertyenergetics relationships. Such studies provide general guidelines towards a broader goal of rational drug design.

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INTRODUCTION Surfactants are widely used in extraction and purification of proteins from biological membranes,1 solubilisation of hydrophobic drugs,2 for pharmaceutical applications and as additives in creams, microemulsions and suspensions.3 Surfactants are amphiphilic molecules containing hydrophilic head group and lipophilic tail. They have a distinct property of associating as colloidal moieties, commonly known as micelles which are formed above a critical micelle concentration (cmc).4 Beyond cmc, the surfactant monomers structure themselves to form spherical aggregates by which hydrophobic tails are sequestered away from the aqueous polar environment and the hydrophilic head groups form an outer shell of polar ionic head groups.5 This process of micellization involves a series of steps with subsequent equilibria associated with respective equilibrium constants.5 Since they are used in solubilisation of drugs their effect on target oriented drug delivery has also been probed.6 Successful target oriented drug delivery requires enhanced bioavailability and minimal drug degradation and also their long sustenance in the circulatory system for prolonged supply and gradual accumulation in specified areas. 7- 10 Therefore, an understanding of interaction of proteins with surfactants and their subsequent structuring for drug delivery is important.11, 12 Efficient drug delivery by using surfactant micellar media is a combination of predominantly two types of interactions: partitioning of drugs into surfactants (loading of drugs) and their subsequent delivery to target site. Drug solubilisation in surfactants occurs as a result of partitioning of drugs.6 The drugs partition or arrange themselves into the different areas of the micelle (surface, intermediate palisade layer or into the core) depending on the hydrophobic nature of the drug.13 The partitioning of drugs depends vividly on their solubility, dissolution, area of partitioning and the proportion of sink condition.4, 14, 15 Moreover for surfactants, the hydrophilic-lipophilic balance (HLB) and the aggregation number also play a major role in partitioning,16 whereas the latter part involves the binding phenomenon of surfactant and drugs to the delivery site. A variety of factors such as steric nature of drugs, pH, temperature and ionic strength determine this interaction.1 In the present work, we have chosen an antibiotic drug, which are an important class of ribosomal ligands. Streptomycin belongs to amino glycosides category of drugs which promotes misreading of messenger RNA. Streptomycin is a bactericidal antibiotic obtained from

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Streptomyces griseus which is used in the treatment of tuberculosis.17 It acts by inhibiting bacterial protein synthesis by binding to the S12 protein of the 30S ribosomal unit.18 It also inhibits polypeptide chain initiation and increases the errors in polypeptide chain elongation.19 Cationic

surfactants

such

as

cetyltrimethylammonium

bromide

(CTAB)

and

tetradecyltrimethylammonium bromide (TTAB) have been extensively used in drug delivery since they can form a variety of interesting self-assembled structures frequently presenting low toxicity and good biocompatibility.7 The surfactant used in the study TTAB also possess antibacterial properties and alteration of cell permeability.5 They are considered as biocides, antispetics and preservatives.20 Nonionic surfactants such as Triton X-100 (TX-100) also possess wide biological applications. It solubilizes phospholipid membranes and is also known to be an actiator of lipolytic enzymes. The TX-100 is used to purify and isolate transmembrane proteins.21, 22 In addition to individual, a mixture of TTAB and TX-100 can also offer interesting drug partitioning abilities and subsequent delivery. Serum albumin is major constituent of blood comprising almost 60% of the total protein concentration (42 g L-1). It contributes to about 80% of the osmotic pressure of the blood and in addition acts as endogenous and exogenous carrier of substances throughout the body.23, 24 The present study involves bovine serum albumin (BSA) as a model target site for surfactant oriented drug delivery. It has an 80% sequence analogy to human serum albumin (HSA).25, 26 The primary structure is comprised of 583 amino acid residues and is characterised by high cysteine content as compared to low tryptophan content (two tryptophan residues at Trp 134 and Trp 212), stabilizing a series of 9 loops. Its secondary structure has 67% of helix of six turns and 17 disulphide bridges.27-29 The tertiary structure of the protein is composed of three domains I, II and III and each domain is further subdivided into two domains A and B.30, 31 In this work, we have used isothermal titration calorimetry in determining binding afﬁnity, enthalpy, entropy and stoichiometry of partitioning of streptomycin in TTAB, TX-100 and mixed micelles in the temperature range of 298.15 K to 313.15 K. Further, by using a combination of calorimetry and spectroscopy, experiments were also performed with streptomycin encapsulated in surfactant micelles and injected in BSA solution to understand the binding of the drug with BSA upon release. Understanding the strength and nature of interactions 3 ACS Paragon Plus Environment

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of drugs with micellar media in terms of partitioning constant, enthalpy and entropy provides guidelines for the modiﬁcation of drugs and choice of transport vehicles for effective drugdelivery. Isothermal titration calorimetric measurements enable an understanding of the nature of interactions of the drug with the micelles and the protein quantitatively in terms of energetics of interactions. This further allows identification of functional groups on the drugs responsible for interactions, and hence drawing structure-property-energetics relationships. The development of effective drug delivery strategies for bioavailability of poorly soluble compound in drug discovery process is very important. The drug delivery system based on surfactants offers significant advantage due to property of such molecule to form micelles in aqueous solution. A quantitative understanding of partitioning of drugs in system which form self-assembly such as, micelles is required for getting insight in to the release and interaction with target plasma protein. Such studies are essential to obtain general guidelines for improvement in the drugs and delivery vehicles for effective drug delivery.

MATERIALS AND METHODS Materials. Tetradecyltrimethylammonium bromide, TX-100, bovine serum albumin and streptomycin sulphate of the purest grade available were purchased from Sigma Chemical Co. USA or TCI Japan. The stock solution of the protein was prepared in 20 mM phosphate buffer at pH 7.4 and dialysed overnight at 277 K with a minimum of three buffer changes. The pH of the buffer was determined on a Standard Control Dynamics pH meter at ambient temperature which was standardized against reference solutions of pH 7 and pH 4. The protein concentration was determined on a JASCO V-550 UV-VIS spectrophotometer at 280 nm by using an extinction 32 % coefficient corresponding to A11cm = 6.8 . The measurements of mass were done on a Sartorius

211D Digital balance which has a readability of 0.01 mg. Isothermal Titration Calorimetry. The enthalpies of interaction were determined on a VP-ITC procured from Microcal, USA. The data were analysed using Microcal Origin software. Prior to filling the sample cell and syringe of the ITC, all the solutions were thoroughly degassed. This was done in order to avoid formation of any bubbles during the titration. The 4 ACS Paragon Plus Environment

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volume of the syringe and the cell of the ITC are 250 µL and 1.4206 mL, respectively. The experiments were programmed for a total of 25 injections, each injection devolving a volume of 10 µL in duration of 20 s with an interval of 240 s between successive injections. The concentration of streptomycin in the syringe was kept at 20 mM or 15 mM for partitioning studies whereas the concentration of the surfactant in the cell was kept relatively higher than respective CMC’s (5mM for TTAB, 1mM for TX-100) to maintain the micellar form of the surfactant during the complete titration. The concentration of streptomycin in the syringe of ITC was optimized to a value which was sufficient to achieve saturation in partitioning of the drug in the surfactant micelles. The mixed micelles were prepared by homogenously mixing both the surfactants to achieve final solution as (5 mM TTAB + 1 mM TX-100). The ITC experiments on drug partitioning were done in the temperature range of 298.15 K to 313.15 K with an interval of 10 K. The heats of dilution were determined by titrating drug into degassed water and water into the respective micellar or monomeric surfactant solutions at respective temperatures. For experiments on interaction of streptomycin with BSA when delivered from the micelles, the syringe contained optimised concentrations of streptomycin partitioned into micelles of individual or mixed surfactants whereas the cell contained 0.06 mM BSA. Here also, the respective dilution experiments were performed. After dilution corrections, the ITC profiles were analysed by suitable models as discussed in the Results and Discussion section. The effect of drug on micellization properties of the surfactants were further studied by using ITC. Here, the syringe contained 50 mM TTAB or 6 mM TX-100 or a mixture of the solutions for mixed micelles which was titrated into water and aqueous solutions of streptomycin at a concentration equal to that attained at the end of 25 injections of the drug into the ITC cell. Differential Scanning Calorimetry. The differential scanning calorimetric measurements were performed on a Nano DSC procured from TA Instruments with fixed cells having volume of 300 µL. The experiments were done at a heating scan rate of 1 K min-1. The amount of BSA was kept at 4 mg mL-1 for all the experiments and the thermograms were obtained in the temperature range of 298 K to 363 K. The baseline was obtained by filling both the sample and reference cells with buffer scanned at 1 K min-1. The reversibility of the thermal unfolding was checked by heating the sample to a temperature corresponding to 90% completion of the transition, cooling and then re-heating under the same experimental conditions. The 5 ACS Paragon Plus Environment

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baseline corrected DSC thermograms were analysed by using Nano Analyser software provided by TA Instruments to obtain the values of transition temperature (Tm) and calorimetric enthalpy (∆calH). Fluorescence spectroscopy. The fluorescence experiments were carried out on a Cary Eclipse fluorescence spectrophotometer procured from Varian, USA. The experiments were done in quartz cuvettes having a capacity of 3 ml and a path length of 1cm. The streptomycin partitioning in the surfactants was analysed by using pyrene as a probe with excitation and emission slit widths of the instrument kept at 5 nm and 2.5 nm, respectively. The excitation was performed at λex = 335 nm whereas the emission was observed in the range of

λem = 345 nm to 600 nm . The baseline corrections were done by subtracting the emission spectra of buffer containing the same amount of additive from those of the main experiments. Circular dichorism spectroscopy. The circular dichorism (CD) experiments were performed on a JASCO CD-180 spectropolarimeter to monitor the conformational changes in the protein at 298.15 K. The protein concentration and path length were kept at 5 µM and 0.2 cm for far UV-CD and 20 µM and 1 cm for near UV-CD measurements, respectively. The instrument was purged with nitrogen gas thoroughly before the experiments and the values of molar ellipticity were measured by using the following relation:

 θ  [θ ] =100×   c×l 

(1)

where c is the concentration of protein, θ is ellipticity and l is the path length of the traversed light.33 The corresponding baseline line corrections were done for all the experiments. The CD spectra were the average of three assessed plots scanned at 100 nm min-1. Dynamic light scattering. A Zetasizer Nano S from Malvern Instruments was used to perform the dynamic light scattering (DLS) measurements at 298.15 K to determine the particle size of the surfactants and the alterations caused by the partitioning of drugs in the surfactant micelles. The samples were irradiated with a monochromatic light source. Depending on the Brownian motion of the particles in the medium, the change in the wavelength of the light source


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is related to their spherical distribution. The translational diffusion coefficient of the particle can be measured using the following Stoke’s-Einstein equation d (H ) =

kT 3πηD

.

(2)

Here d(H) is the hydrodynamic radius, D is the translational diffusion constant, k is Boltzmann’s constant, T is absolute temperature and η is the viscosity of the medium. Transmission electron microscopy. The Transmission electron microscopy (TEM) images of the micellar structures of the surfactants were recorded using a JEM 2100 JEOL HRTEM, operating under an accelerating voltage of 200 kV. The samples were prepared in aqueous media consisting of 5 mM TTAB, 1 mM TX-100 and mixture of 5 mM TTAB and 1 mM TX-100 used in the partitioning studies. The samples were observed on a scale of 10 nm. The samples were loaded onto Formvar-coated 300 mesh copper grids and dried before being analysed.

RESULTS AND DISCUSSION Isothermal titration calorimetry. Since surfactants have been used in target oriented drug delivery as drug delivery vehicles, it is important to understand the interaction of a variety of drugs with the micellar and monomeric forms of the surfactants quantitatively. In view of this, we have attempted to understand energetics of interaction of antibiotic drug streptomycin quantitatively with the post and pre micellar forms of cationic, non-ionic and mixed surfactants. Interaction of streptomycin with micellar TTAB. The isothermal titration calorimetric studies on the interaction of streptomycin with TTAB were conducted by injecting the aqueous drug solution into the micellar and monomeric forms of TTAB in the temperature range of 293.15 K to 313.15 K. When the ITC experiments were done with the micellar form of TTAB, sufficiently high concentration of the latter over and above its cmc was taken in the cell to ensure that at the end of all the 25 injections, the surfactant in the cell of ITC remained in the micellar form. Further, the concentration of streptomycin in the syringe was optimised to ensure 7 ACS Paragon Plus Environment

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saturation of micelles with the drug molecules within added 25 injections. The sample cell of ITC was filled with 5 mM TTAB under micellar condition or with 0.4 mM TTAB under monomeric condition. A representative ITC profile for the interaction of streptomycin with micellar TTAB at 298.15 K is shown in Figure 1. The upper panel of the figure

_________________________________________________________

Figure 1. ITC profiles for the titration of 20 mM streptomycin with 5 mM TTAB at T = 298.15 K. The upper panel of the figure represents the raw data obtained on each titration, and the lower panel is the integrated heat profile.

_________________________________________________________ shows the raw data in terms of power versus time upon addition of 25 sequential injections each of 10µL volume. The lower panel in the figure represents the integrated heat profile as a function of the molar ratio of streptomycin to TTAB in the micellar form. The binding profile in this figure suggests interaction of streptomycin with the TTAB micelles in a partitioning manner


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leading to saturation level heat effects. The ITC profiles representing interactions at other studied temperatures are shown in the Supporting Information. A single set of binding site model fitted well to the experimental data points at each studied temperature, thereby providing the values of

binding / partitioning

constant (K),

standard molar enthalpy ( ∆H m0 ) and stoichiometry of binding/ partitioning (n). The values of these thermodynamic parameters thus obtained are given in Table 1.

_________________________________________________________ Table 1. The values of partitioning constant (K), change in standard enthalpy ( ∆H mo ), change in entropy ( ∆S mo ), change in standard Gibbs free energy ( ∆Gmo ), and stoichiometry of interaction (n) accompanying the partitioning of streptomycin in micelles of TTAB at different temperatures. T/K

K/M-1

∆H mo /

∆S mo /

∆Gmo /

(kJ mol-1)

(J K1 mol-1)

(kJ mol-1)

N

293.15

(2.70±0. 41)x103

20.50±0.20

136±2

-(19.26±0.37)

0.13±0.01

298.15

(2.33±0.14)x103

9.45±0.35

96±2

-(19.22±0.15)

0.17±0.01

303.15

(3.28±0.64)x103

4.29±0.31

81±4

-(20.41±0.50)

0.28±0.01

308.15

(2.46±0.40)x104

-(10.40±0.40)

50±2

-(25.90±0.41)

0.06±0.01

313.15

(1.01±0.27)x104

-(9.75±0.73)

46±3

-(24.01±0.69)

0.12±0.01

_________________________________________________________ The partitioning constant (K ) characterizes the equilibrium constant of the following reaction. nD + S x → Dn S x

(3)

Here n represents number of moles of the drug streptomycin interacting with surfactant micelles S x which has x moles of monomers per mole of micelles.34


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The value of partitioning constant (K), standard molar enthalpy

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( ∆H m0 ) and

stoichiometry (n) for partitioning of streptomycin in TTAB micelles are ( 2.33 ± 0.14) × 10 3 ,

(9.45 ± 0.35) kJ mol-1 and (0.17± 0.01), respectively at 298.15 K. As seen in Table 1, the value of partitioning constant increases slowly with rise in temperature from a value of (2.33 ± 0.14) × 10 3 at 298.15 K to (1.01 ± 0.27) × 104 at 313.15 K. The partitioning is associated with endothermic heat effects which decrease with rise in temperature becoming exothermic beyond 303.15 K. The endothermic enthalpy of partitioning suggests involvement of hydrophobic association in the partitioning process since hydrophobic interactions are accompanied with endothermic heat effects.35, 36 The endothermic nature of interactions can also include significant contribution from desolvation of both the drug and the surface of the micelle.37 This suggests that the major driving force for the partitioning arises from positive contribution of entropy, the extent of which also decreases with rise in temperature. This increase in entropy can be assigned to the desolvation of streptomycin and micelles as a result of partitioning. Even though, there is an enthalpic penalty; it is largely overcome by the release of water molecules during the association process, thereby driving the partitioning towards spontaneity leading to negative values of change in standard molar Gibbs free energy ( ∆G m0 ) [see Table 1]. The shape of TTAB micelles under the employed conditions is considered to be spherical as the concentration at which sphere to rod like transition occurs is reported to be as high as 26 mM.38 Therefore, the partitioning of streptomycin occurs in spherical micelles of TTAB. Beyond 303.15 K, the partitioning of streptomycin into TTAB micelles changes from endothermic to exothermic. For example, at 313.15 K , ∆H m0 = -(9.75± 0.73) kJ mol-1 (see Table 1). As seen in this Table, the value of partitioning constant is highest at 313.15 K. This strengthened partitioning associated with the exothermic heat of interaction suggests that the loss in structural rigidity of micelles at higher temperature allows streptomycin to be partitioned relatively more effectively, however, in a conformation which establishes polar interactions with the charged head groups of the micelles.


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The values of change in standard molar entropy upon partitioning ( ∆S m0 ) reported in Table 1, suggests decrease in dehydration upon partitioning with rise in temperature. Based on the knowledge of the stoichiometry of partitioning obtained from ITC measurements, the concentration of surfactant solution and the aggregation number of surfactant, the number of streptomycin molecules partitioned per micelle (N) can be calculated by using the following equation 34

N = x×n

(4)

Here x is the number of monomers per micelle of TTAB, and n is stoichiometry obtained from ITC measurements. The value of aggregation number ( x ) for TTAB micelles is reported to be nearly 55 at 298.15 K.39 By using this number in equation (2), it is calculated that about 10 streptomycin molecules are able to partition in one TTAB micelle. The concentration of micelles calculated by using 55 monomers per micelle is 0.090 mM in the cell of the ITC. With 10 streptomycin molecules partitioning per micelle, the maximum concentration of partitioned drug is 0.90 mM. Since we have taken 20 mM streptomycin in the syringe and added a total volume of 250 µL into the cell having a volume of 1.4206 ml, the final concentration of the drug in the solution comes out to be 2.99 mM. The ITC profile seen in Figure 1 is typical for a weak binding/partitioning which is supported by the calculations that out of total amount of streptomycin in the cell of ITC, only 30.1% of the available drug is partitioned. The interaction of streptomycin with the monomers of TTAB was also studied on ITC. For this the concentration of streptomycin in the syringe was maintained at 20 mM but the concentration of TTAB in the cell was lowered to 0.4 mM. Figure 2 represents heat change when streptomycin is injected in the TTAB monomers at different molar ratios. Interestingly, the monomers of TTAB also interact sufficiently with streptomycin, but with exothermic heat effects. However, the value of partitioning constant in this case was not determined since the monomers do not provide a self-assembled environment for effective drug partitioning. The exothermicity observed in the interaction of streptomycin with TTAB monomers can be attributed to polar interactions between the drug and surfactant molecules.


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_________________________________________________________

Figure 2. Heat changes observed upon titration of 20 mM streptomycin with micelllar (5 mM) or monomeric (0.4 mM) forms of TTAB at 298.15 K.

_________________________________________________________ Interaction of streptomycin with micellar TX-100. The interaction of streptomycin was also analysed with micellar and monomeric forms of TX-100 in the temperature range of 298.15 K to 313.15 K with a temperature interval of 10 K. Here also it was ensured that the final concentration of the surfactant remained in its micellar form at the end of 25 injections. The concentration of TX-100 in the ITC cell was 1 mM when the experiments were done with the micellar form of the surfactant, whereas for the interaction with monomers, the concentration of TX-100 was 0.1 mM.

Relevant control experiments were done at each temperature. A

representative ITC profile for the interaction of streptomycin with TX-100 at 298.15 K is shown in the Figure 3A. The profile clearly indicates that partitioning of streptomycin in TX-100 micelles leads to saturation levels of heat effects. The obtained experimental data could be best fitted by a model allowing sequential three binding sites, thus providing three values of partitioning constants ( K1 , K 2 and K 3 ), and accompanying values of standard molar enthalpies ( ∆H m0 ) , standard molar entropies ( ∆S m0 ) , and standard Gibbs free energies ( ∆G m0 ) (see Table


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2). We also tried fitting higher order sequential binding models to the experimental data points, however, the errors associated with various thermodynamic quantities were unacceptably large. The partitioning at these three sequential sites have two exothermic and one endothermic events. It is well established that exothermic association involves polar interactions and hydrophobic association is accompanied with endothermic heat effects.40 At 298.15 K, the values of ( ∆H m0 ) at these three sites are –(2.07 ± 0.12) kJ mol-1, (3.04 ± 0.26) kJ mol-1, and –(2.77 ± 0.22) kJ mol-1, respectively. The partitioning at all three sites are entropically favoured suggesting that the association process has a significant contribution from desolvation. The ITC profiles representing interactions at other studied temperatures are shown in the Supporting Information.

_________________________________________________________

Figure 3. (A) ITC profiles for the titration of 15 mM streptomycin in 1 mM TX-100 at 298.15 K. The upper panel of the figure represents the raw data, and the lower panel is the integrated heat profile. (B) Scheme for partitioning of streptomycin in TX-100 micelles in a sequential manner. The different colours of streptomycin represent sequential partitioning. As shown in the figure, green coloured streptomycin partitions initially, followed by red coloured and then by blue coloured drug molecules.

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It has been reported that TX-100 micelles have an effective hydrodynamic radius of 4.18 nm and 5.57 nm at 293.15 K and 303.15 K, respectively.41 The authors further conclude that the micelles of TX-100 have 2.50 nm to 2.70 nm of the hydrophobic region, and about 2.50 nm of hydrophilic region accounting for an overall radius of 5.10 nm. Assuming spherical nature of TX-100 micelles and based on these thermodynamic quantities, partitioning of streptomycin into TX-100 is expected to follow the scheme as shown in Figure 3B.

_________________________________________________________ Table 2. The values of partitioning constant (K), change in standard enthalpy ( ∆H mo ), change in entropy ( ∆Smo ), change in standard Gibbs free energy ( ∆Gmo ), and stoichiometry of interaction (n) accompanying the sequential partitioning of streptomycin in TX-100 micelles at different temperatures.

T/K 298.15

303.15

308.15

313.15

K/M-1 (9.22±0. 82) x 102 (2.36±0.28) x 103 (2.65±0.26) x 103 (11.20±0.41) x 102 (2.24±0.12) x 103 (1.92±0.19) x 103 (8.96±0.05) x 102 (2.63±1.4) x 103 (2.52±1.6) x 103 (6.77±0.02 ) x 102 (7.63±2.9) x 103 (5.52±2.6) x 103

∆H mo / (kJ.mol-1) -(2.07±0.12) 3.04±0.26 -(2.77±0.22) -(2.42±0.39) (5.47±0.24) -(3.49±0.19) -(2.68±1.02) (3.78±1.31) -(2.35±0.83) -(3.39±0.85) (4.03±0.85) -(1.28±0.29)

∆Smo / (J.K-1mol-1) 50±2 75±3 56±2 50±2 82±3 51±2 48±2 78±3 58±4 43±2 87±3 68±3

∆Gmo / (kJ.mol-1) -(16.92±0.22) -(19.25±0.29) -(19.54±0.24) -(17.70±0.09) -(19.44±0.14) -(19.06±0.25) -(17.41±0.01) -(20.18±0.41) -(20.07±0.61) -(16.97±0.01) -(23.27±0.31) -(22.43±0.28)

_________________________________________________________ As per this scheme, the interaction of streptomycin at the first set of sites takes place on the surface of the micelles through polar interactions. This is accompanied with a slight expansion of the TX-100 micelles allowing some of the streptomycin molecules to enter the palisade layer and undergo endothermic interactions. This is followed by the next set of streptomycin molecules further interacting at the surface of the micelles giving rise to exothermic heat effects. As


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mentioned earlier, the higher order sequential binding process is also possible, but the extraction of thermodynamic data led to large errors. In any case, the results suggest that the interaction of streptomycin with TX-100 modifies the structure of micelles in a concentration dependent manner. The value of aggregation number in the micelles of TX-100 is reported to be in the range from 79-121 which is higher than 55 reported for TTAB micelles.39, 42, 43, 44 Thus the TX100 micelles can offer flexibility to a greater extent than that by TTAB micelles. It is further seen from the data presented in Table 2 that the partitioning energetics of streptomycin in TX-100 micelles follow nearly the similar trend in the temperature range of 298.15 K to 313.15 K. Since the partitioning of streptomycin in the micelles of TX-100 occurs in a sequential manner, the stoichiometry of interaction could not be established, hence the number of molecules of the drug partitioning per TX-100 micelle could not be calculated.

_________________________________________________________

Figure 4. Heat released upon titration of 15 mM streptomycin with micelllar (1 mM) or monomeric (0.1 mM) forms of TX-100 at 298.15 K.

_________________________________________________________


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Figure 4 compares the heat effects associated with the interaction of streptomycin with the micelles and monomers of TX-100 at 298.15 K. The heat effects clearly demonstrate partitioning of the drug into the micelles of the surfactant, however, an interaction behavior with the monomers is also observed, but to a much smaller extent. Here also the interaction of streptomycin with the monomers of TX-100 is exothermic suggesting polar interactions. However, due to the absence of micelles, drug partitioning is not possible. The value of ∆H m0 for partitioning in TTAB varies from (20.50 ± 0.20) kJ mol-1 at 293.15 K to – (10.40±0.40) kJ mol-1 in the studied temperature range. However, for partitioning of the drug in TX-100 micelles, the value of ∆H m0 is in the range of - (1.28 ± 0.29) kJ mol-1 to

(5.47 ± 0.24) kJ mol-1. In general, the enthalpy of interaction of streptomycin with TTAB micelles is higher than that of the TX-100 micelles. The difference can be attributed to the molecular properties of the cationic and non-ionic micelles which not only alters the surface interactions but also leads to different extent of desolvation in the partitioning process.

Interaction of streptomycin with mixed micelles. The isothermal titration calorimetric studies were further performed to understand the partitioning of streptomycin in the mixed micellar system of cationic TTAB and non-ionic TX-100 surfactants. This should enable a comparative assessment of the partitioning behaviour of the drug in individual and mixed micelles of the surfactants. The experiments were designed such that 15 mM streptomycin was titrated from the syringe of ITC into mixed (5 mM TTAB + 1 mM TX-100) micelles in the temperature range of 298.15 K to 313.15 K. Figure 5 shows representative ITC profile


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_________________________________________________________

Figure 5. ITC profiles for the titration of 15 mM streptomycin in (5 mM TTAB + 1 mM TX100) mixed micelles at 298.15 K.

_________________________________________________________ accompanying the titration of streptomycin in mixed micelles at 298.15 K. The ITC profiles representing interactions at the other studied temperatures are shown in the Supporting Information. The basis of choosing these concentrations of the surfactant has been discussed earlier. The analysis of the respective dilution corrected ITC profiles which could be best fitted by three sequential binding sites model are presented in Table 3. The size and shape of mixed micelles of (TTAB + TX-100) are not available in literature. However, an analysis of the data presented in Table 3 suggests that the mixed micelles have predominant characteristics of TX100. Here the partitioning constant at each of the three sequential sites is of the order of 102. The major difference compared to that of interaction with pure TX-100 micelles is that the interaction of the drug is endothermic at two sites and exothermic at the third site. These results further suggest that the drug is able to partition more effectively in the palisade layers/hydrophobic regions of the mixed micelles. Thus the mode of interaction is similar to that described in Figure 3B with two relatively higher hydrophobic interaction sites and one polar site.


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_________________________________________________________ Table 3. The values of partitioning constant (K), change in standard enthalpy ( ∆H mo ), change in entropy ( ∆Smo ), change in standard Gibbs free energy ( ∆Gmo ), and stoichiometry of interaction (n) accompanying the sequential partitioning of streptomycin in (5 mM TTAB + 1 mM TX-100) mixed micelles at different temperatures.

T/K

298.15

303.15

K/M-1

∆H mo /

∆Smo /

∆Gmo /

(kJ.mol-1)

(J.K-1mol-1)

(kJ.mol-1)

(3.19 ±0.08)x102

1.07±0.01

52±2

-(14.29±0.06)

(3.28 ±0.08)x102

-(3.15±0.07)

38±2

-(14.36±0.06)

(3.18 ±0.08)x102

4.70±0.02

64±2

-(14.28±0.06)

(1.67 ±0.09)x102

1.45±0.04

(8.04 ±0.37)x102

-(3.14±0.08)

45±2

-(16.86±0.12)

3.43±0.17

60±2

-(14.86±0.17)

(2.17 ± 0.01)x102

-(9.36±0.53)

14±3

-(13.78±0.12)

(4.20±0.42)x103

1.38±0.10

74±2

-(21.38±0.26)

(4.50 ±0.04)x102

-(3.35±0.35)

(1.56 ±0.13 )x102

-(5.11±0.29)

26±2

-(13.15±0.15)

(9.43 ±0.91)x102

-(9.53±0.60)

27±4

-(17.83±0.25)

2

-(1.25±0.16)

45±2

-(15.30±0.23)

(3.64 ±0.24)x10 308.15

313.15

(3.57 ±0.31)x10

2

47±2

40±2

-(12.90±0.14)

-(15.65±0.23)

_______________________________________________________ Does Streptomycin alter the CMC of surfactants? It is known that additives can alter the physicochemical properties of surfactants,39 therefore, it is possible that streptomycin may also affect the CMC of the surfactant. In order to ensure that the surfactant in the cell of the ITC remains in the micellar form during the entire course of titration, the effect of streptomycin on CMC of the surfactants was studied by using ITC.


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________________________________________________________

Figure 6. ITC profiles accompanying titration of (A): 50 mM TTAB in water () 50 mM TTAB in 3 mM streptomycin (), and (B): 6 mM TX-100 in water () 6 mM TX-100 in 2.24 mM streptomycin () at 298.15 K. The injection volume in each case was 10 µL.

________________________________________________________ Here 50 mM TTAB taken in the syringe of ITC was titrated into to water taken in cell in the form of 25 sequential injections. Figure 6A represents the heat absorbed during demicellization of 50 mM TTAB when added from the syringe and formation of micelles in the cell. The value of CMC obtained from the inflection point of this figure is 3.6 mM which is in excellent agreement with those reported in literature.39, 43. The corresponding enthalpy of micellization is (6.14 ± 0.19) kJ mol-1which is in close agreement with the literature value of - (7.90 ± 0.40) kJ mol-1.45 Next, water in the cell of ITC was replaced by 3 mM Streptomycin solution and the syringe was filled with 50 mM TTAB. Here the integrated ITC profile showed an initial increase in endothermicity, followed by a decrease (Figure 6A). The nature of ITC profile is different 19 ACS Paragon Plus Environment

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than that observed when pure TTAB micelles are formed as a result of streptomycin – TTAB interactions. Even if the point where the heat starts decreasing is taken as CMC, its value is slightly lower than the value observed in water. Therefore, it is confirmed that during the entire course of titration, the surfactant in the cell remains in the micellar form. On performing similar procedure to check the alteration in the CMC of TX-100, 6 mM of the surfactant was taken in the syringe and titrated into water. The resulting ITC profile is shown in the Figure 6B. The value of cmc thus obtained is 0.28 mM which is also in excellent agreement with the literature.46, 47 The standard molar enthalpy of micellization of TX-100 thus obtained is ( ∆ mic H m0 ) = + (6.90 ± 0.12) kJ mol-1which is lower than the value [(11.4 ± 0.6) kJ mol-1] reported by Ruiz and co-workers.47 Similarly to check the alteration provided by the presence of streptomycin, 2.24 mM of the drug was taken in the cell and 6 mM TX-100 was titrated from the syringe of the ITC. It is clearly seen in Figure 6B that both the values of cmc and ( ∆ mic H m0 ) are not altered appreciably. Hence incorporation streptomycin into TX-100 micelles does not alter its CMC. The alteration in the CMC of mixed micelles was checked by titrating 50 mM TTAB into 6 mM TX-100 and 6 mM TX-100 into 50 mM TTAB ( Supporting Information.). On observation of the inflection points relating to the CMC, it is seen that the values of CMC of TTAB and TX-100 are in accordance with those reported in literature. Size of the micelles with and without streptomycin. The DLS studies were performed to determine the hydrodynamic radii of the surfactant micelles in with and without streptomycin. The size of micelles in terms of hydrodynamic radius in 5 mM TTAB was in the range of 5.2 nm to 5.5 nm and that for the micelles in 1 mM TX-100 was observed to be in the range of 8.7 nm to 9.4 nm. These values are in agreement with those reported in literature.41 The hydrodynamic radius of the mixed micelles in a mixture of 5 mM TTAB and 1 mM TX-100 was in the range of 5.9 nm to 6.2 nm. To the best of oue knowledge, the size of mixed micelles of TTAB and TX100 are not available in literature. The size of the mixed micelles is intermediate to that of the individual samples of TTAB and TX-100 micelles. This is in agreement with the ITC observations that the TX-100 micelles are not formed individually when taken along with the TTAB micelles. The size of the micelles was also determined in the presence of streptomycin 20 ACS Paragon Plus Environment

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which is observed to slightly increase upon interaction with the drug (see Table 4). As discussed earlier, streptomycin does not partition inside the micellar core but instead is mostly present on the surface of the micelles, hence a large variation in the particle size is not expected. The DLS results provide further support to the ITC and fluorescence observations as the change in the value of d ( H ) is not very significant. The d ( H ) of mixed micelles also support the mechanism

________________________________________________________ Table 4. Values of hydrodynamic radius d ( H ) of surfactant micelles in the presence and absence of streptomycin. d ( H ) / nm

System 5 mM TTAB micelles 5 mM TTAB micelles in presence of 3 mM streptomycin 1 mM TX-100 micelles 1 mM TX-100 micelles in the presence of 2.24 mM streptomycin Mixed micelles (5 mM TTAB+1 mM TX-100) Mixed micelles (5 mM TTAB+1 mM TX-100) in the presence of 2.24 mM streptomycin

5.2-5.5 6.0-6.2 8.7-9.4 9.9-10.6 5.9-6.2 7.0-7.3

________________________________________________________ of sequential partitioning as interpreted using ITC data since here also, the change in the size of the micelles is small. The shape and size of surfactant micelles were analysed using TEM. It was observed that the micelles of TTAB, TX-100 and mixed surfactants were spherical in size (see Figure 7). The scale range of these particles were less than 10 nm which is in accordance to the observed DLS results.


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________________________________________________________

A

B

C

Figure 7: TEM images of (A) 5 mM TTAB (B) 1 mM TX-100 and (C) Mixed micelles (5 mM TTAB + 1 mM TX-100)

________________________________________________________ Effect of composition of binary micellar pseudophases on synergistic interactions between TX-100 and TTAB in their mixed micelles. In order to check the extent of synergism in the mixed micelles of TTAB and TX-100, ITC experiments were designed to cover a broad range of the composition of the binary micellar pseudophases. For this, in the first set of experiments, the concentration of TX-100 was kept at 6 mM and the concentration of TTAB was varied such that the ratio r = [TTAB]/[TX-100] varies from a value of 8.33 to 16.67. In the second set of experiments, the concentration of TX-100 was doubled to 12 mM and the range of r was covered from to 4.17 to 8.33. Figure 8 represents the integrated ITC profiles accompanying the titration of mixed micelles of TTAB and TX-100 into water at 298.15 K at varying r values. As discussed earlier, upon injection of these solutions into the cell of ITC, intitally demicellization takes place followed by micellization when the concentration of the surfactant reaches its cmc. The extent of synergism in TTAB and TX-100 in the mixed micelles will not only be reflected in the resulting value of the cmc, but also in the value of standard molar enthalpy change.


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________________________________________________________

Figure 8: ITC profiles accompanying the injections of mixtures of TTAB and TX-100 at varying r values and 298.15 K reflecting the effect of composition of binary micellar pseudophases on the value of cmc.

________________________________________________________ The ITC profiles shown in Fig. 8 indicate that individual micellization of Triton X-100 is not seen in the presence of TTAB monomers. An initial endothermic hump followed by decrease in endothermicity until complete micellization suggests modification in micellization behavior of the mixed surfactant as compared to that observed in individual surfactants [see Fig. 6 and Fig. 8]. The values of the mid-point of the endothermic hump are 2.4 mM, 2.6 mM and 2.8 mM at r = 8.33, 12.50, and 16.67, respectively when the concentration of TX-100 is 6 mM in the initial surfactant mixture taken in the syringe of ITC. When the concentration of TX-100 in the surfactant mixture is increased to 12 mM, the values of mid-point of the hump are 1.9 mM and 2.1 mM at r = 4.17 and 8.33, respectively. Similarly the mid-point of the second half of the ITC profile is at 3.72 mM, 3.85 mM, and 3.95 mM at r = 8.33, 12.50, and 16.67, respectively when the concentration of TX-100 is 6 mM in the initial surfactant mixture taken in the syringe of ITC. When the concentration of TX-100 in the surfactant mixture is increased to 12 mM, these values 23 ACS Paragon Plus Environment

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are 3.56 mM and 3.63 mM at r = 4.17 and 8.33, respectively. It is clear from these values that the cmc of the mixed surfactant is observed to increase slightly when the concentration of TX-100 in the binary micellar pseudophase increases. The cmc of TTAB in water at 298.15 K is observed to be 3.6 mM which is close to the values obtained at the mid-point of the second half of the ITC profiles in the case of mixed surfactants. The initial endothermic hump observed in the ITC profiles accompanying the titration of mixed micelles into water is due to association of the TX100 molecules with the monomers of TTAB which is further followed up by the micellization of the mixed surfactant. Effect of composition of the binary micellar mixed phase on its interaction with streptomycin. The interaction of streptomycin and mixed micelles was studies for the different composition of the binary micellar mixed phase.

________________________________________________________

Figure 9. ∆Go accompanying the interaction of streptomycin with mixed micelles of TTAB and TX-100 at different r values at 298.15 K. Here r = [TTAB]/[TX-100]

______________________________________________________ 24 ACS Paragon Plus Environment

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Here ITC experiments were done by taking 15 mM streptomycin in the syringe of the ITC and mixture of TTAB and TX-100 at different r values in the sample cell (Supporting Information). In the entire range of the values of r from 2.50 to 16.67, the analysis of the respective dilution corrected ITC profiles were best fitted by three sequential binding sites model providing values of partitioning constant of the order of 102. The values of change in standard Gibbs free energy of interaction of streptomycin with the mixed micellar phases are shown in Figure 9. It is seen that the interaction behavior of streptomycin with the mixed micelles of TTAB and TX-100 remains similar both qualitatively and quantitatively.

Intrinsic Fluorescence. Due to its unique property, pyrene has frequently been used as a fluorescence probe especially in efficient formation of excimer.48, 49 The intensities of pyrene monomers due to vibronic band fluorescence are strongly influenced by the solvent environment. The perturbation of the vibronic band intensities in pyrene can be an effective tool to monitor the environment where it is present. Figure 10 shows the ratio of intensities of the vibronic bands at 373 nm (I1) and 384 nm (I3) at different concentrations of the drug streptomycin and also by the intensity ratio of the excimer (IE) at 475 nm and monomer band (IM) at 373 nm. The hydrophobic environment of pyrene molecules is measured in terms of I1/I3 and IE/IM intensities ratio. Therefore any change in the I1/I3 upon addition of streptomycin should indicate difference in micropolarity and microscopic viscosity induced as a result of drug partitioning. As seen in the Figure 10, minimum change is observed in the ratio of I1/I3 when the concentration of streptomycin is increased in the surfactant micelles. Since I1/I3 ratio in the surfactant micelles is almost unaffected even after streptomycin to surfactant molar ratio reaches up to saturation, it is inferred that encapsulation of pyrene molecules in the hydrophobic core of the micelles is not affected appreciably by the presence of the drug.


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________________________________________________________

Figure 10. Fluorescence intensity changes upon titration of Streptomycin drug into 5 mM TTAB containing pyrene and the plot of I1/I3 and IE/IM against the concentration of streptomycin.

________________________________________________________ These results compliment the ITC observations which also suggest that streptomycin molecules do not partition deep in the hydrophobic core of the micelles, rather they remain closer to the charged surface of the TTAB, TX-100 or mixed micelles of these surfactants. ITC of interaction of encapsulated streptomycin with BSA. The binding of streptomycin with BSA was studied at pH 7.4 when delivered under post micellar environment of the surfactant employing ITC (Figure 11). The sample cell of the ITC was filled with 0.06 mM BSA and the syringe contained 3 mM streptomycin dissolved in micellar form of the surfactant. With this arrangement, when the drug encapsulated in TTAB micelles is titrated into BSA, the micelles break down due to extensive dilution and the drug thus released binds to BSA. The effect of such a release of the drug on binding efficacy of streptomycin with BSA was examined based on the thermodynamic parameters of interaction.


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________________________________________________________

Figure 11. ITC profiles accompanying titration of (A) 3 mM streptomycin encapsulated in 5 mM TTAB, (B) 2.24 mM streptomycin in 1 mM TX-100, and (C) 2.24 mM streptomycin in (5 mM TTAB + 1 mM Troton X-100) at 298.15 K.

________________________________________________________ The binding of streptomycin with BSA in the absence of TTAB is reported to be accompanied by an association constant of (2.23 ± 0.07) × 103 at 298.15 K accompanied with standard molar enthalpy and standard molar entropy of binding as − ( 26 .4 ± 1 .6 ) kJ mol-1and 24.81 J K-1 mol-1, respectively.50 The data suggests that the association of streptomycin with BSA is largely driven by electrostatic interactions. Figure 11A represents the titration of 3 mM streptomycin encapsulated in 5 mM TTAB in 25 sequential injections into the cell containing 0.06 mM BSA. The integrated heat profile shown in the panel II of Figure 11A represents the binding of released streptomycin with the protein leading to saturation levels. The experimental data could not be fitted by a single site binding model. The best model which could satisfactorily fit the data points was a sequential binding site model with more than two sequential binding sites. The values of binding constants K1 and K2 are (1.59 ± 0.63) × 103 and (7.29 ± 0.20) × 103 , respectively at 298.15 K. The standard molar enthalpies of binding at these sites are -1 -1 − (37 .6 ± 1 .5) kJ mol and (52 .5 ± 0 .9 ) kJ mol , respectively. The thermodynamic parameters


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associated with subsequent sequential binding sites had large errors therefore, were not considered. As discussed above, streptomycin binds to BSA in 1:1 binding manner in the absence of surfactant. However in the presence of surfactant, satisfactory fitting of the data to sequential binding site may not necessarily mean opening up of new binding sites for the incoming drug. Therefore further experiments were designed to understand whether the additional binding data thus obtained is due to the association of TTAB with BSA. Figure 11A represents the titration of 3 mM streptomycin incorporated into 5 mM TTAB with 0.06 mM BSA at 298.15 K. A nonsigmoidal integrated binding profile shown in the lower panel of Figure 11A clearly suggests presence of more than one binding site for the surfactant on the protein. Such data generally can be fitted by higher order sequential binding sites model. Therefore, when the drug is delivered from the micellar TTAB (Figure 11A), the value of K1 remains of the order of 103 suggesting that the binding of streptomycin with BSA is not significantly altered whether it is delivered in the absence or presence of micelles. Interaction of encapsulated streptomycin in TX-100 with BSA. The binding studies of streptomycin encapsulated in TX-100 with BSA was performed at 298.15 K and pH 7.4. The post micellar concentration of TX-100 was kept at 1 mM whereas the pre-micellar concentration was 0.1 mM in these experiments. The amount of drug added to the surfactant solution was 2.24 mM which was its final concentration obtained during the ITC experiments. The drug encapsulated micelles were taken in the syringe whereas the cell contained 0.06 mM protein. Figure 11B represents interaction of 2.24 mM streptomycin encapsulated in 1 mM TX-100 with 0.06 mM BSA in 25 sequential injections which results in endothermic heat profiles. The data points could be fit satisfactorily by a sequential four binding sites model (Supporting Information). Here also, the binding of TX-100 with BSA was examined by performing additional ITC experiments. It is observed that TX-100 binds with BSA in a sequential manner accompanied with the thermodynamic parameters reported in table reported in Supporting Information. The interaction of TX-100 with BSA occurs at three binding sites which thus possibly explains the fitting of streptomycin binding data to four sequential binding sites under these conditions. Hence it can also be inferred that streptomycin released from the TX-100 micelle binds to the BSA along with the monomers of TX-100. The binding of the surfactant 28 ACS Paragon Plus Environment

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molecules and the drug encapsulated surfactant molecules to BSA was found to be of the order of 104. Interaction of encapsulated streptomycin in (TTAB+TX-100) mixed micelles with BSA. Here, the cell of the ITC was filled with 0.06 mM BSA and the syringe was filled with (5 mM TTAB + 1 mM TX-100) mixed micellar solution containing 2.24 mM streptomycin. Figure 11C represents the interaction of drug with BSA when released from the mixed micelles. Sequential mode of interaction of streptomycin is maintained in the mixed micelles (see Table in Supporting Information). The ITC data on the interaction of streptomycin with BSA when released from the micelles of TTAB, TX-100 or mixed surfactants suggest that the binding affinity of the drug with the target protein is unaffected in the presence of surfactant micelles under the employed experimental conditions. However, the data also suggest that the molecules of the delivery vehicle (surfactant in this case) do bind to the target protein, which may or not may affect the protein conformation. This was further investigated by using circular dichroism spectroscopy as discussed ahead. Self aggregation of streptomycin and its effect on partitioning. Amphiphilic pharmacologically active compounds have a tendency to self aggregate. It is possible that self aggregation may modulate the physicochemical properties of the drug and hence its interaction with the target. Self association of surface active drugs and its physicochemical and biological aspects has recently been reviewed.5 Streptomycin can also undergo self aggregation in aqueous solution mostly via formation of intermolecular hydrogen bonds via –OH or –NH2 groups. We have carefully analyzed the dilution profiles of streptomycin in aqueous solution by using ITC (Figure 12, Table 5). Figure 12A is a representative ITC profile accompanying the dilution of 15 mM streptomycin in water at 25oC. Various points in the initial part of the ITC profile correspond to de-aggregation of streptomycin. An analysis of the data as shown in the Figures 12A provides a critical aggregation concentration (cac) of 0.62 mM accompanied with an enthalpy of aggregation of (0.78 ± 0.03) kJ mol-1.


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________________________________________________________

Figure 12. ITC profiles accompanying dilution of 15 mM strepromycin in water at (A) 25oC and (B) at different temperatures. Analysis of the ITC profile to determine critical aggregation concentration is shown in (A). Enthalpies of aggregation at these temperature are shown in (C).

______________________________________________________ It must be noted here that the heats shown in this figure indicate demicellization process, hence enthalpy of micellization will be negative of the enthalpy of demicellization. Experiments at different temperatures (298.15 K to 313.15 K) suggest that cmc (cac) is not affected appreciably and remains almost the same at 0.62mM (Figure 12). However, the enthalpy of micellization of streptomycin varies from (0.78 ± 0.03) kJ mol-1 at 298.15 K to (2.12 ± 0.06) kJ mol-1 at 313.15 K. The information available in literature on the value of cmc of streptomycin in aqueous solution is scarce.5 These authors report the cmc of streptomycin as 90 µM which is lowest amongst the cmc of other reported antibiotics: actinomycin (0.1 mM), penicillin G (0.25 mM) and sodium fusidate (3.6mM). It is evident that the cmc is largely dependent on the molecular structure of the antibiotic. The value of cmc of streptomycin is lowest inspite of a large number of polar groups in the molecule which are capable of hydrogen bonding.


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______________________________________________________ Table 5. Enthalpy of aggregation (∆aggHo) of streptomycin in aqueous solution at different temperatures. T/K

∆aggHo/(kJ mol-1)

298.15

0.78±0.03

303.15

1.62±0.05

308.15

1.86±0.06

313.15

2.12±0.06

______________________________________________________ The value of cmc of streptomycin obtained in our work using ITC is 0.62 mM which is about 7.7 times higher than the earlier reports. We believe that the measurements based on heat changes provide a better estimate of the change in aggregation states of the solutes in aqueous solution. The enthalpy of micellization of streptomycin at each temperature is endothermic indicating significant role of the desolvation effect of the polar groups which are mainly responsible for the aggregation. Thus the micellization of streptomycin is entropically driven. The favourable entropic contribution to the micellization of streptomycin is stronger at higher temperature. Even though hydrophobic association is known to be endothermic in nature,40 streptomycin aggregation is unlikely to occur by this process due to several polar groups able to form hydrogen bonds. Though some drugs are known to self associate resulting into closed micelle like structure, continuous stacking mode of association has also been reported.51 In the case of streptomycin, since the critical aggregation number is temperature independent, stacking type of aggregation appears to be more likely. The aggregation of the drugs will be affected by the presence of another substance in solution, hence the value of cmc will either be lowered or increased. In the current studies it is assumed that the streptomycin interacts/partitions with the surfactants in the monomeric form and hence the results are likely to have slight errors arising out of such an assumption. In any case, the dilution corrections should largely reduce the errors though not completely.


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Circular Dichorism Spectroscopy: Effect of streptomycin binding on the conformation of BSA. Native BSA is known to predominantly exhibit α-helical structure with characteristic peaks at 208 nm and 222 nm.52 The secondary and tertiary structures of BSA were monitored by far and near UV-CD spectroscopy, respectively in the absence and presence streptomycin. Streptomycin induces secondary structure in BSA as indicated by enhanced molecular ellipticity at 210 nm and 222 nm (Figure 13). As seen from the near UV-CD spectra (Figure 13), the tertiary structure of the protein is not altered appreciably upon drug binding, although slightly strengthened, if monitored closely. These observations suggest that the binding of streptomycin to BSA imparts conformational stability to the protein. This slight strengthening of the conformation of BSA by streptomycin is in accordance with its small association constant and small enhancement in the thermal stability of the protein and gain in enthalpy of unfolding observed in DSC measurements as discussed ahead. Effect of surfactants on protein conformation. In order to establish that the surfactants under the employed experimental conditions do not disturb the integrity of the binding sites on the protein, effect of the former on secondary and tertiary structure of the latter was examined based on circular dichroism measurements. As discussed earlier, the final concentration of the surfactant in the protein in ITC experiments was 0.74 mM for TTAB, 0.14 mM for TX-100, and (0.74 mM TTAB + 0.14 mM TX-100) for the mixture. It is seen in Figure 13 that 0.74 mM TTAB or 0.14 mM TX-100 do not affect the tertiary structure of BSA appreciably. However, a slight strengthening of secondary structure of the protein is seen in the presence of 0.14 mM TX100 (Figure 13). TX-100, being non-ionic in nature, cannot establish strong hydrophobic interactions with the hydrophobic groups of the protein, hence rather shifts the N(Native) → D(Denatured) equilibrium slightly to the left. Mixture of (0.74 mM TTAB + 0.14 mM TX-100) leads to slight disruption of α-helical content of the protein (Figure 13).


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________________________________________________________

Figure 13. (a) and (b) Far UV-CD and Near UV-CD spectra of BSA with 0.74 mM TTAB, 0.4 mM streptomycin and mixture of 0.74 mM TTAB and 0.4 mM streptomycin. (c) and (d) Far UV-CD and Near UV-CD spectra of BSA with 0.14 mM TX-100, 0.3 mM streptomycin and mixture of 0.14 mM TX-100 and 0.3 mM streptomycin. (e) and (f) Far UV-CD and Near UVCD spectra of BSA with (0.74 mM TTAB+0.14 mM TX-100), 0.3 mM streptomycin and mixture of (0.74 mM TTAB+0.14 mM TX-100) and 0.3 mM streptomycin

_______________________________________________________ 33 ACS Paragon Plus Environment

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This modification in the action of the surfactants when they are in the mixture suggests reduction in the extent of non-ionic character of TX-100 when it forms micelles with a cationic surfactant. These results confirm that the TTAB and TX-100 individually do not affect the integrity of the binding sites for the drug on the protein under the employed conditions. However, in mixture form of the surfactants, a slight effect on the secondary structure of the protein is observed without affecting its tertiary structure. Effect of streptomycin on CD spectra of BSA in the presence of surfactants. Experiments were further done to understand how the binding of streptomycin with BSA may affect the conformation of the protein when the drug is delivered from the micellar media. The far and near UV-CD spectra of BSA in the presence of (streptomycin + 0.74 mM TTAB), (streptomycin + 0.14 mM TX-100) and mixture of (0.74 mM TTAB + 0.14 mM TX-100) are shown in Figure 13 It is observed that streptomycin induces secondary structure in the protein upon binding even in the presence of 0.74 mM TTAB, though the helical character is altered without affecting the tertiary structure. Similar effect is seen when the drug is deleivered from the TX-100 micelles (Figure 13). The secondary structural stabilization effect of streptomycin on the protein is seen even in the presence of mixture of (0.74 mM TTAB + 0.14 mM TX-100) with insignificant changes in the tertiary structure. The circular dichroism spectroscopic measurements confirm that the delivery of streptomycin to serum albumin when released from micellar environment under the employed conditions maintains conformational integrity of the protein with slight strengthening of its secondary structure as a result of drug-protein interactions. Surfactants, at higher concentration are known to disrupt the cellular membrane bilayers leading to formation of lipid-protein-surfactant complexes.5 Thus it is very important to choose optimum concentrations of the components of the drug delivery system to ensure conformational integrity of the protein. Differential scanning calorimetry (DSC). The assessment of thermal and conformational stability of protein during drug binding and effect of drug delivery vehicles on proteins is crucial. To assess the effects, DSC studies of BSA in the presence and absence of drugs and surfactants used as model micellar delivery vehicles were performed. 34 ACS Paragon Plus Environment

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Effect of streptomycin on the thermal stability of BSA. The thermal denaturation of BSA was studied by observing changes in the heat capacity as a function of temperature, in the presence and absence of streptomycin at pH 7.4 which is shown in the Figure 14. The thermodynamic parameters accompanying the thermal denaturation of BSA in the presence and absence of streptomycin are given in the Table 6.

_______________________________________________________

Figure 14. Thermal unfolding of 0.09 mM BSA in the absence and presence of streptomycin and (A) TTAB, (B) TX-100, and (C) mixture of TTAB and TX-100. The scan rate in these experiments was 1 K min-1.

________________________________________________________ It was observed that the thermal denaturation of BSA is irreversible both in the presence and absence of additives. In the absence of the drug, BSA unfolds at Tm = (334.15 ± 0.6) K and


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∆ cal H = (319 ± 23) kJ mol-1 which are in close agreement with those reported in literature.53 In the presence of 0.4 mM streptomycin, BSA exhibits slight stabilisation with Tm = (335.45 ± 1.3) K with marginal gain in calorimetric enthalpy at ∆ cal H = (340 ± 20) kJ mol-1. The final concentration of streptomycin in the cell of ITC after addition of 25 injections is 0.4 mM, which is the basis of choosing this concentration of drug in the DSC experiments. Since the changes in the transition temperature and enthalpy of transition are small, it is inferred that binding of streptomycin with native state of BSA is weak. This result is consistent with the ITC observations that streptomycin binds weakly with BSA with an affinity constant of the order of 103. Thermal denaturation of BSA in the presence of surfactants. The effect of TTAB, TX-100 and their mixture at specific concentrations on the thermal denaturation of BSA was studied. The concentrations of surfactants chosen were 0.74 mM TTAB, 0.14 mM TX-100 and mixture at these respective concentrations.

_________________________________________________________

Figure 15. Deconvolution of DSC profile accompanying thermal unfolding of BSA in presence of TTAB.

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The DSC profiles depicted in Figure 14 lead to the thermodynamic parameters accompanying the transition of BSA under these conditions reported in Table 6. The DSC endotherms for BSA in the presence of TTAB were best fitted by a model deconvoluting the overall transitions into two cooperative transitions (see Figure 15).

__________________________________________________________ Table 6. Transition temperature (Tm) and enthalpy change (∆calH) accompanying the thermal unfolding of 0.06 mM BSA in the absence and presence of streptomycin, TTAB, TX-100, mixture of these surfactants, and sterptomycin in the presence of these surfactants at pH 7.4. The DSC experiments were done at a fixed pressure of 3.0⋅105 Pa to avoid bubble formation. In this table (1) represents only one transition and (2) represents the second transition accompanying thermal unfolding of BSA. System

Tm(1)/K

Tm(2)/K

∆calH(1)/

∆calH(2)/

(kJ mol-1)

(kJ mol-1)

BSA

334.2±0.6

-

319±23

-

BSA + 0.40 mM STM

335.5±1.3

-

340±20

-

BSA + 0.74 mM TTAB

337.1±0.4

345.4±0.6

254±12

909±26

BSA + 0.14 mM TX-100

346.1±0.3

-

345±10

-

BSA + (0.74 mM TTAB + 0.14 mM TX-100)

334.7±0.4

342.1±0.5

269±8

541±14

BSA + 0.74 mM TTAB + 0.40 mM STM

336.7±0.5

344.9±0.6

245±12

658±21

BSA + 0.14 mM TX-100 + 0.40 mM STM

336.6±0.6

-

378±11

-

BSA + (0.74 mM TTAB + 0.14 mM TX-100) + 0.40 mM STM

335.2±0.5

342.5±0.4

255±14

775±24

STM: Streptomycin; TTAB: Tetradecyltrimethylammonium bromide; TX-100: TX-100

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The values of the two transition temperatures corresponding to the transitions in the presence of TTAB are (337 .1 ± 0 .4 ) K and (345 .4 ± 0.6 ) K which are associated with calorimetric enthalpies of ( 254 ± 12 ) kJ mol-1 and (909 ± 26 ) kJ mol-1 respectively. The increase in the first transition temperature by 2.9 K shows slight stabilization by TTAB in addition to the second transition at (345 .4 ± 0.6 ) K compared to that of the free protein. This increase can be attributed to structural

modifications in BSA upon binding with TTAB. Stabilization of proteins by surfactants at lower concentration of latter has also been reported earlier.54 TTAB being a cationic surfactant, binds to the surface of the negatively charged protein leading to deepening of the crevice in the segment containing Trp 212 in the subdomain II A, and hence division into two independent cooperative units giving rise to two transitions in the DSC profile. This relative weakening of structure of BSA has not been observed in the fluorescence and CD experiments. Hence in concordance with above mentioned reason that temperature plays a significant role in these binding interactions. TX-100 and mixture of TX-100 and TTAB under these conditions provide relative thermal stabilization to BSA. In the presence of TX-100 only one thermal transition was observed, whereas the mixture of TTAB and TX-100 yielded overall transitions as a sum of two individual transitions (Table 6). Enthalpic gain is observed in these profiles compared to that of free protein under the employed conditions. These results suggest that TTAB, TX-100 or the mixture of these surfactants do not destabilize the protein, hence rendering the integrity of binding sites for streptomycin on the protein. The calorimetric profiles for BSA in the presence of TX-100 yield one endotherm (Figure 14), whereas mixed micelles exhibit predominantly the characteristics of TTAB micelles, which is in agreement with ITC observations. Thermal denaturation of BSA in the presence of surfactants and streptomycin. After establishing the integrity of binding sites in the presence of surfactants, the effect of binding of streptomycin to BSA when released from micellar media was studied. The experimental conditions were 0.4 mM streptomycin with 0.74 mM TTAB or 0.14 mM TX-100 or in the mixture of these surfactants added to 4 mg ml-1 BSA. The thermodynamic parameters accompanying the transitions (see Figure 14) are reported in the Table 6. The data in the table suggest increase in thermal stability of BSA upon binding with streptomycin by 2.5 K in TTAB, 2.4 K in TX X-100 and 1 K in the presence of mixture of surfactants at the above mentioned 38 ACS Paragon Plus Environment

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concentrations. The binding of streptomycin with BSA when delivered from TTAB leads to DSC profiles with two independent thermodynamically cooperative units. In the presence of 0.74 mM TTAB and 0.4 mM streptomycin, the values of transition temperatures are (336 .7 ± 0.5 ) K and (344 .9 ± 0 .6 ) K respectively, which are in accordance with those values observed in presence of

TTAB. The calorimetric enthalpy values correspond to ( 245 ± 12 ) kJ mol-1 and (658 ± 21) kJ mol1

respectively. The reduction in the calorimetric enthalpy of the second transition in the presence

of streptomycin-TTAB system compared to that of only in the presence of TTAB arises due to alteration in the mode of interaction of TTAB with BSA in the presence of the drug. The DSC profile for BSA in the presence of streptomycin and TX-100 showed one endotherm similar to that observed in the presence of surfactant without the drug. A slight stabilization of about 2.4 K with small gain in enthalpy are observed (Table 6). Even the binding of streptomycin with BSA when released from mixed micelles led to slight thermal stabilization with DSC profiles similar to that in the presence of TTAB (Table 6). Overall, the DSC results of BSA with or without surfactant demonstrate that the drug binding is not affected at the experimental concentration levels of the surfactants. Further, small increase in thermal stability supports the ITC observations that the binding of streptomycin with BSA is weak. The DSC results have enabled a quantitative understanding of the effect of streptomycin on the protein with or without the employed delivery vehicle. Structure-property-energetics relationships. In order to gain insights into structure-propertyenergetics relationships, the data on partitioning of streptomycin into TTAB micelles has been correlated (Table 7) with those with naproxen,6, 34 diclofenac,6, 34 neomycin,6 and lincomycin.6 The partitioning of streptomycin, naproxen, and diclofenac sodium in TTAB micelles is of the order of 103 to 104.6, 34 Large differences in the enthalpies of partitioning are observed, though in each case partitioning is entropically supported. For example, the value of K for streptomycin is (2.33 ± 0.14) × 103 and that for naproxen is (6.10 ± 0.38) × 103 at 298.15 K.6, 34 Though the order of partitioning constant is same, the enthalpy of partitioning is endothermic [∆H o = (9.5 ± 0.4) kJ mol−1 ] for streptomycin and exothermic [∆H o = −(9.4 ± 0.9) kJ mol −1 ] for naproxen.6, 34 Even if the partitioning in the micelles of HTAB is considered, the value of K for neomycin is (0.862 ± 0.032) × 103 at 298.15 K (ref) which is associated with exothermic 39 ACS Paragon Plus Environment

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interactions [∆H o = −(11.86 ± 0.23) kJ mol −1 ] supported by increase in entropy by about 16 J K-1 mol-1.6 Streptomycin has a total of seven hydroxyl groups, four amino groups and one imine group which are capable of establishing polar interactions. The streptomycin molecule also has a bulky structure and significant hydrophobic component (Table 7). The partitioning of diclofenac into TTAB micelles is associated with a K value of (2.26 ± 0.13) × 10 4 at 298.15 K.6 The partitioning is exothermic [∆H o = −(22.80 ± 1.42) kJ mol −1 ] with increased entropy by (6.9 ± 4.8) J K-1 mol-1.6 Lincomycin with four hydroxyl groups, but significant hydrophobic component (hydropyran and pyrolidine rings) and four aromatic rings does not exhibit any partitioning into the surfactant micelles.6 These results suggest that even though the TTAB micellar surface is ionic/polar in nature, the interaction/partitioning is not directly proportional to the number of hydroxyl groups. Naproxen and diclofenac which are the smallest molecules amongst the drugs being compared establish stronger polar interactions with the TTAB micelles even though these molecules have only one hydroxyl group each, which is a part of carboxyl functional group. Steric factor appears to play a dominant deciding role in permitting the entry of the incoming drugs into the cationic surfactant micelles. In the case of lincomycin, the additional hydropyran and pyrolidine rings further prevent even polar association of the hydroxyl groups of this molecule with the micellar surface. In each case, the partitioning of the drug is accompanied with favourable entropy change suggesting desolvation predominance. One factor which could also contribute to relatively small endothermic enthalpy of partitioning of streptomycin into TTAB micelles is desolvation penalty due to seven hydroxyl groups which are strongly solvated. The desolvation enthalpic cost will be lesser in naproxen and diclofenac sodium due to only one carboxyl present in each of them. Exothermic partitioning of neomycin into TTAB micelles suggest compensation of desolvation enthalpic cost by establishing polar interactions, even though the value of partitioning constant remains nearly the same. These correlations suggest that a combination of steric factor, desolvation cost and enthalpy of packing of the drug into the micelles play a significant role in drug partitioning. The partitioning of drugs in non-ionic surfactants is not available in literature, hence we could not compare the data with that on streptomycin. However, the sequential partitioning mode of the drug into the micelles of TX-100 or mixed micelles of (TX-100 + TTAB) suggests better partitioning if the 40 ACS Paragon Plus Environment

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_________________________________________________________ Table 7. Structure-property-energetics relationships based on the thermodynamics of interaction of streprtomycin with serum albumin under different conditions Drug

Functionality

K

∆Ho/

∆So/

(kJ mol-1)

(kJ mol-1)

Seven –OH groups

In TTAB:

Four –NH2 groups

(2.33±0.14) (9.5±0.4) ⋅103

One –NH group

(96±2)

Three (Hydrofuran , cyclohexane and pyran) rings Seven –OH groups

In TTAB:

Six –NH2 groups

(0.86±0.03) -(11.9±0.2) (16±1) ⋅103

Six –O- groups Two Hydropyran, hydrofuran and cyclohexane rings

One –COOH group

In TTAB:

One –O- group

(6.10±0.38) -(9.4±0.9) ⋅103

(41±3)

Naphthalene moiety In HTAB: (0.93±0.05) ⋅103


-(54.6±0.7) -(126±2)

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One –COOH group

In TTAB:

One –NH group

(2.26±0.13) -(22.8±1.4) (7±5) ⋅104

Two –Cl groups Two benzene rings

In HTAB:

-(44.2±0.2) -(78±2)

(4.80±0.71) ⋅103 Four –OH groups

In HTAB :

One –NH group

No partitionin g

No partitioning

Two –R groups Hydropyran and pyrolidine ring

__________________________________________________________ charge on the surface of the micelles is reduced. These observations provide useful guidelines for engineering partitioning in a given drug molecule by exploring variations in its functional groups or the nature of the micellar system.

CONCLUSIONS AND FUTURE PERSPECTIVES Surfactant micelles, liposomes, nanoemulsions and various other structural formulations have been used in the past for efficient drug delivery at the target site. However, the qualitative information available on most of these systems is insufficient to provide guidelines for improving new molecular entities or drug delivery vehicles in terms of functional groups on each. High sensitivity isothermal titration calorimetry has provided the values of partitioning constant, the standard molar enthalpy and standard molar entropy of partitioning of streptomycin in cationic TTAB, non-ionic TX-100 and (TTAB + TX-100) mixed micelles. Further, the binding of the drug to bovine serum albumin when released from the micelles has also been studied in order to assess the effect of the molecules of the drug delivery vehicle on the integrity 42 ACS Paragon Plus Environment

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of the binding sites on the target protein. Streptomycin is observed to partition/interact weakly with cationic TTAB micelles, whereas, the partitioning is found to improve in the non-ionic or mixed non-ionic-cationic surfactant micelles. The results also suggest that initial interaction of streptomycin modifies the non-ionic/mixed cationic-non-ionic micellar shape allowing further partitioning of the drug molecule in a sequential manner. The binding of streptomycin with bovine serum albumin is not affected appreciably due to the presence of the molecules of drug delivery vehicle (surfactants) though the binding of surfactant molecules with BSA does take place. Conformational and thermal stability of the protein are also not appreciably altered as confirmed by differential scanning calorimetry. Comparison of the data on streptomycin, neomycin, lincomycin, naproxen and diclofenac partitioning in surfactant micelles suggest significance of desolvation process associated with the partitioning of the drugs in TTAB micelles. Structure-property-energetics relationships on partitioning of different drugs in TTAB and HTAB micelles suggest that in addition to desolvation of both the drug and surfactant micelles, steric factors and enthalpy of packing also play a significant role. Fluorescence properties of pyrene have further provided support in establishing the mode of partitioning. There is a serious need on understanding physical chemistry underlying the partitioning of a variety of drugs in different drug delivery vehicles. The future of such studies lies in obtaining enormous amount of thermodynamic data on partitioning of drugs with different structures and functions so that guidelines for suitable structure-property-energetics relationships can be developed. Such studies also establish the effect of the molecules of the delivery vehicles on the conformation of the target protein and hence improvements in the strategies for rational drug design.

ACKNOWLEDGMENTS The authors acknowledge financial support from the Board of Research in Nuclear Sciences, India.


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REFERENCES (1) Paul, B. K.; Sett, R.; Guchhait, N. Stepwise Unfolding of Ribonuclease A by a Biosurfactant. J. Colloid Interface Sci. 2017, 505, 673-681. (2) Alkan-Onyuksel, H.; Ramakrishnan, S.; Chai H-B.; Pezzuto, J. M. A Mixed Micellar Formulation Suitable for the Parenteral Administration of Taxol, Pharmaceutical Res. 1994, 11, 206-212. (3) Zhang, X. C.; Jackson, J. K.; Burt, H. M. Determination of Surfactant Critical Micelle Concentration by a Novel Fluorescence Depolarisation Technique. J. Biochem. Biophys Methods 1996, 31,145-150. (4) Chakraborty, S.; Shuklta, D.; Jain, A.; Mishra, B.; Singh, S. Assessment of Solubilisation Characteristics of Different Surfactants for Carvedilol Phosphate as a Function of pH. J. Colloid Interface Sci. 2009, 335, 242-249. (5) Schreier, S.; Malheiros, S. V. P.; de Paula, E. Surface Active Drugs: Self-Association and Interaction with Membranes and Surfactants. Physiochemical and Biological Aspects. Biochim. Biophys. Acta 2000, 1508, 210-234. (6) Choudhary, S.; Talele, P.; Kishore, N. Thermodynamic Insights into Drug–Surfactant Interactions: Study of the Interactions of Naproxen, Diclofenac Sodium, Neomycin, and Lincomycin with Hexadecytrimethylammonium Bromide by using Isothermal Titration Calorimetry. Colloids and Surfaces B: Biointerfaces, 2015, 132, 313-321. (7) Malmsten, M. (Ed.).Surfactants and Polymers in Drug Delivery, Marcel Deckker Inc., New York, USA, 2002. (8) Chein Y. W. (Ed.)., Novel drug delivery systems. Drugs and the Pharmaceutical Sciences, Marcel Deckker, New York, USA, 1992, p.50. (9) Torchilin, V.P. Structure and Design of Polymeric Surfactant Base Drug Delivery Systems, J. Control. Rel. 2001, 73, 137-172.


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(10) Zhang, H.; Annunziata, O. Diffusion of an Ionic Drug in Micellar Aqueous Solutions. Langmuir, 2009, 25(6), 3425-3434. (11) Basak, R.; Bandyopadhyay, R. Encapsulation of Hydrophobic Drugs in Pluronic F127 Micelles: Effects of Drug Hydrophobicity, Solution Temperature, and pH. Langmuir, 2013, 29(13), 4350-4356. (12) Valero, M.; Castiglione, F.; Mele, A.; da Silva, M. A.; Grillo, I.; González-Gaitano, G.; Dreiss, C. A. Competitive and Synergistic Interactions between Polymer Micelles, Drugs, and Cyclodextrins: The Importance of Drug Solubilization Locus. Langmuir, 2016, 32(49), 1317413186. (13) Rangel-Yagui, C. O.; Pessoa, A. Jr.; Tavares, L. C. Micellar solubilisation of Drugs. J. Pharm. Pharm. Sci. 2005, 8(2), 147-165. (14) Mooney, K. G.; Mintun, M. A.; Himmelstein, K. J.; Stella, V. J. Dissolution Kinetics of Carboxylic Acids II: Effect of Buffers. J. Pharm. Sci. 1981, 70(1), 22-32. (15) Aunins, J. G.; Southard, M. Z.; Myers, R. A.; Himmelstein, K. J.; Stella, V. J. Dissolution of Carboxylic Acids III: The Effect of Polyionizable Buffers. J. Pharm. Sci. 1985, 74(12), 1305-1316. (16) Frezzatti, W. A.; Toselli, W. R.; Schreier, S. Spin Label Study of Local AnestheticLipid Membrane Interactions. Phase Separation of the Uncharged from and Bilayer Micellization by the Charged Form of Tetracaine. Biochim. Biophys. Acta. 1986, 860(3), 531-538. (17) Eugene, T. M.; Henry,C. S.; Turner, G. C. Pulmonary Tuberculosis in Children Treated with Streptomycin. Pediatrics 1949, 3, 323-330. (18) Chang, F. N.; Flaks, J. G. Binding of Dihydrostreptomycin to Escherichia Coli Ribosomes: Characteristics and Equilibrium of the Reaction. Antimicrob. Agents Chemother. 1972, 2, 294-307. (19) Tai, P. C.; Wallace, B. J.; Davis, B. D. Streptomycin Causes Misreading of Natural Messenger by Interacting with Ribosomes after Initiation. Proc. Natl. Acad. Sci. USA 1978, 75, 275-279. 45 ACS Paragon Plus Environment

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Drug Partitioning in Micellar Media and its Implications in Rational

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