Establishing Structure Property Relationship in Drug Partitioning into

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Establishing Structure Property Relationship in Drug Partitioning into and Release from Niosomes: Physical Chemistry Insights with Anti-Inflammatory Drugs Moumita Dasgupta, and Nand Kishore J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b06141 • Publication Date (Web): 31 Aug 2017 Downloaded from http://pubs.acs.org on September 1, 2017

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The Journal of Physical Chemistry

Establishing Structure Property Relationship in Drug Partitioning into and Release from Niosomes: Physical Chemistry Insights with Anti-inflammatory Drugs

Moumita Dasgupta, Nand Kishore*

Department of Chemistry, Indian Institute of Technology Bombay, Powai, Mumbai 400 076, India

*Corresponding author. Email: [email protected]

1 ACS Paragon Plus Environment


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ABSTRACT: Understanding physical chemistry underlying interactions of drugs with delivery formulations is extremely important in devising effective drug delivery systems. The partitioning and release kinetics of diclofenac sodium and naproxen from Brij 30 and Triton X-100 niosomal formulations have been addressed based on structural characterization, partitioning energetics and release kinetics, thus establishing relationship between structures and observed properties. Both the drugs partition in nonpolar regions of TX-100 niosomes via stacking of aromatic rings. The combined effects of interactions of the drugs with polar head groups and the rigidity of the niosome vesicles determine entry and partitioning of drugs into niosomes. The observed slower rate of release of the drugs from the drug encapsulated niosomes of TX-100 than those of Brij 30, suggest stable complexation of drugs in the nonpolar interior of the former. No release of drugs from the niosomes was observed till 24 h even upon varying pH conditions without SDS. However SDS in drug loaded niosomes led to release of drugs in as early as 6 h. The sustained pattern of in vitro release kinetics of the drugs thus observed from our niosomal preparations suggest these vesicular systems to be promising for pharamaceutical applications as potential drug delivery vehicles.


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1. INTRODUCTION Encapsulation of drugs into carriers or delivery vehicles such as surfactant micelles, nanoemulsions, liposomes, ethosomes and various other engineered formulations

not only

increase the stability and bioavailability of the drugs but also provide an opportunity of controlled and sustained release of the drugs over longer duration in the body.1,2 Niosomes are vesicular systems with an aqueous interior core consisting of non-ionic surfactants, which constitute the bilayer of these vesicles.3-5 Presence of hydrophobic bilayer with an aqueous interior, provides the advantage of packaging both hydrophobic and hydrophilic drug molecules in the niosomes.3 Niosomal system provides advantages over liposomes with respect to the ease and low cost of production, chemical stability and storage.6,7 Other than drugs, niosome vesicles have also been studied for the encapsulation and controlled release of various other functional molecules such as β-carotene,8 antioxidants (for example, gallic acid, ascorbic acid, curcumin, quercetin, α-tocopherol),1,9 human insulin10 and peptides.11 Niosomes find their potential applications not only in pharmaceutical but also in the food and cosmetic industries.1,12 Drug loaded niosome vesicles can be administered in the body via different routes viz. ocular, oral, transdermal, nasal and intravenous among others.13-17 Not only passive targeting but also active targeting of ligand conjugated niosomes formed from pluronic surfactant, and carrying anticancer therapeutics to tumour tissue has been reported.18 The transition of micelles formed from pluronic triblock copolymer F127 to nisomes in presence of cholesterol using fluorescence resonance energy transfer has also been reported.19 An earlier study has explored the heterogeneity of the inside architecture of niosomes employing steady state and time-resolved fluorescence spectroscopy.20 Formation of hybrid niosomes from nonionic Tween 20 and anionic



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dicethylphosphate and their subsequent interaction with linear polycations for the purpose of development of composite colloidal multidrug delivery systems have also been explored earlier.5 In the current study we prepared two different kinds of niosomes, one made of the non-ionic surfactant Brij 30 (polyoxyethylene (4) lauryl ether) while the other made of TX-100 (Triton X100) with their HLB (hydrophilic-lipophilic balance) values being 9.721 and 13.5,22 respectively, which are in coherence with the larger polar head group of TX-100 compared to Brij 30 surfactant (Figure 1). Entrapment and in vitro release of tretinoin, effective in dermatological disorders, from Brij 30 niosomes, have been reported earlier.23 Characterization of empty niosomes of TX-100 using fluorescence correlation spectroscopy has also been done previously.24,25 The non-ionic surfactants thus used for niosome preparation are generally less toxic compared to their ionic counterparts,26 which also makes the non-ionic ones more preferable for use in development of drug delivery vehicles. We have studied the partitioning behaviour and release kinetics of each of the drugs, diclofenac sodium and naproxen, from the individual niosomes of Brij 30 and TX-100. In order to gain physicochemical insights of the above processes, we employed a combination of calorimetric and spectroscopic techniques. The characterization of the structure and morphology of the niosomes, was done from light scattering and electron microscopic techniques. The main aim of the study is to look into the energetics of interaction of the drug molecules with that of the two types of niosomal preparations of differing chemical structures using isothermal titration calorimetry. The results obtained from isothermal titration calorimetry experiments would provide insights into the nature of the stabilizing interactions between the drug molecules and that of the niosomes thereby indicating the possible loci of partitioning of each of the drugs, diclofenac sodium and naproxen into the niosomes of Brij 30 and TX-100. Therefore, the


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purpose of the study is to understand the role of the functional groups on the drugs and the chemical structure of the niosomes that would guide the stable complexation of the drugs with these vesicular drug delivery vehicles based on favourable interactions. Again, the rate of diffusion controlled release of the drugs from the niosomal formulation is expected to be determined by the extent of stabilizing interactions between them as well as structural rigidity of the vesicles. Hence understanding the role of the functional groups on the drugs, the nature of the niosomes and energetics of their interactions are extremely important in establishing their structure property relationship. A recent literature report employed isothermal titration calorimetry to obtain the thermodynamic parameters of interaction of norharmane and βcyclodextrin with the niosomes of Triton X-100.27 Other than partitioning, the current study also explores the kinetics of release of the drugs from the niosome vesicles and that would provide an idea of how effective these drug loaded delivery vehicles are, with respect to their roles in controlled and sustained release of the encapsulated drug molecule. Also to the best of our knowledge understanding the energetics of interaction of the drugs diclofenac sodium and naproxen with the niosomes of Brij 30 and TX-100 and its correlation with the observed release kinetics of these drugs from the niosomal preparations, have not been addressed. The information and insights gained from this study would thus be immensely beneficial for guiding the pharmaceutical industries to design and develop appropriate niosomal formulations for the pupose of effective and controlled drug delivery thereby increasing the bioavailability of the drugs in vivo. Both naproxen and diclofenac sodium belong to the group of nonsteroidal anti-inflammatory drugs (NSAIDs) which carry out their anti-inflammatory and analgesic effect by inhibiting the COX enzymes in the body.28,29 Among the NSAIDs, diclofenac sodium is known for having the



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highest therapeutic index.30 While naproxen relieves moderate fever and inflammatory responses,31 diclofenac treats pain due to dysmenorrhea, trauma and post surgery.32 The chemical structures of naproxen and diclofenac sodium are shown in Figure 1.

Figure 1. Chemical structures of (A) Brij 30, (B) TX-100, (C) Diclofenac sodium and (D) Naproxen

2. MATERIALS AND METHODS Materials. The surfactants, Brij 30 and Triton X- 100 (TX-100), used for niosome preparation as well as the drugs diclofenac sodium and naproxen of the best available purity grade were purchased from Sigma –Aldrich Chemical Company, USA. Bovine serum albumin and sodium dodecyl sulfate was also procured from Sigma–Aldrich Chemical Company, USA. Choloroform and Cholesterol were purchased from Sisco Research Lab (SRL) PVT. LTD. India. The stock solutions of the drugs, sodium dodecyl sulfate and bovine serum albumin were prepared in 20 mM phosphate buffer of pH 7.4. Extensive overnight dialysis of the stock protein solutions, at 4 °C against the phosphate buffer with at least three changes, was done before using the protein solutions for experiments on Isothermal Titration Calorimetry. A Jasco V-550 double 6 ACS Paragon Plus Environment

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beam spectrophotometer was used for determination of concentration of the dialysed protein solution using the extinction coefficient corresponding to A2801% = 6.8.33 For weighing of mass, Sartorious BP 211D digital balance, that has a readability of 0.01 mg, was used. Preparation of Niosomes. Two different types of niosome vesicles were made from the two neutral surfactants namely, Brij 30 and Triton X-100 (TX-100). The niosomes were prepared by the method of lipidic film hydration.1,24,25,34,35 Cholesterol was added to the surfactants individually, in the molar ratio of Brij 30: cholesterol = 4:1 and TX-100: cholesterol = 2:1 for the Brij 30 and TX-100 niosomes, respectively. Due to higher HLB value of TX-100 than that of Brij 30 as mentioned in the introduction, the formation of TX-100 niosomes required a higher molar ratio of cholesterol: surfactant compared to that of Brij 30. The concentrations of the surfactants in the niosomal preparation were kept at 0.4 mM and 1.25 mM, for Brij 30 (cmc = 7 to 14.52 mgl-1)36 and TX-100 (cmc= ≈ 0.22 mM)37 respectively, which were much higher than their respective cmc values. The required quantities of the surfactant and cholesterol were dissolved in chloroform followed by drying of the organic solvent in a rotary evaporator to form a thin lipid film in the round bottomed flask. The lipid film was subsequently hydrated with aqueous stock solutions of the drugs (10 mM of diclofenac or 10 mM of naproxen) or only buffer to form drug loaded or empty niosomes of Brij 30 and TX-100 respectively, under continuous stirring condition for 30 minutes at 40 °C. This hydrated lipid film was then kept at 25 °C for a period of 24 h to achieve stable annealing, followed by removal of the unbound, excess drug molecules by dialysis at 4 °C, against phosphate buffer of pH 7.4 with 4 changes of buffer at 1 h interval each. Prior to monitoring the release of these drugs from the niosomes of Bij 30 as well as those of TX-100, these drug loaded noisome preparations of Brij 30 and TX-100 were



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sonicated for 15 minutes and 30 minutes respectively, using a bath sonicator. For all ITC experiments non-sonicated niosomal preparations were used. Methods Isothermal Titration Calorimetry (ITC). Isothermal Titration Calorimetry was employed to study both the direct partitioning/interaction of the drugs with the empty niosomes of Brij 30 and TX-100 individually as well as the release of these drugs from the drug loaded niosomes of Brij 30 only, over a period of 24 h, at pH 7.4 and 298.15 K. For direct interaction studies, 10 mM of the drug was loaded fully in the 250 µl syringe while the cell contained the solution of empty niosomes of Brij 30 or TX-100. In case of studying the release of the drugs from the corresponding drug loaded niosomes of Brij 30, the diclofenac sodium encapsulated Brij 30 niosomes (DB) or naproxen encapsulated Brij 30 niosomes (NB) were loaded into the syringe and titrated into the solution of 0.060 mM of BSA contained in the cell. All the ITC experiments were done on VP-ITC (Microcal, Northampton, MA, USA) at 298.15 K. The reference cell in all the above experiments contained buffer. Aliquots of 10 µl of the solution in the syringe were injected into the sample cell at an interval of 4 minutes. A total of 25 injections, each lasting for 20 s, were made from the computer controlled syringe. Stirring of the solution in the sample cell was done at a speed of 250 rpm. For each experiment, corresponding control experiments (dilutions of the material in the syringe and cell) were carried out and then subtracted from the main experiment. This was followed by data analysis for each of the experiments using the Origin 7.0 software, provided by Microcal, to obtain the thermodynamic parameters- binding constant ( K ), stoichiometry of binding ( n ), standard molar enthalpy change ( ∆H ), and o

standard molar entropy change ( ∆S o ) associated with the binding interactions. The uncertainties reported for each of these thermodynamic parameters have been derived from replicate


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experiments and include errors arising due to repeatability and sample impurity. The heat content

Q of the solution contained in the active cell volume V 0 (determined relative to zero for the unliganded species) at fractional saturation Θ , after the i th injection is determined from eq (1),

Q = n Θ M t ∆ HV 0

(1)

where, ∆H is the molar heat of partitioning of the drug into a particular niosomal media, M t is the total concentration of the surfactant, and n is the number of partitioning sites in the receptor niosomes. The heat released ∆Q (i ) from the i th injection for an injection volume dV i is given by the following equation.

∆Q(i ) = Q(i ) +

dVi  Q(i) + Q(i − 1)   − Q(i − 1) V0  2

(2)

Fluorescence Spectroscopy. Fluoromax-4 spectrofluorometer (Horiba Scientific) was used for measuring the steady state fluorescence emission of the samples, in a quartz cuvette of 1 cm path length. Two types of experiments were designed on fluorescence spectroscopy to monitor the time dependent release profile of the drugs from the niosomes. In one type, the intrinsic fluorescence emission of 0.1 mg/ml of BSA was monitored upon addition of 5 µl of the drug encapsulated and dialysed niosomal preparation at pH 7.4, 5.5 and 9.0, at different time intervals over a period of 24 h and at 298.15 K. For this experimental set up, the excitation wavelength was fixed at 295 nm in order to selectively excite the tryptophan residues of BSA and the background emission due to buffer and drug loaded niosomal preparations were duly subtracted from the main experiment. In the other set of experiments, the drug encapsulated and dialysed niosomal preparations at pH 7.4 were incubated at 310.15 K in absence of any protein over 9 ACS Paragon Plus Environment


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several days while monitoring the changes in the fluorescence emission spectra of the drugs over different time (t) intervals at 298.15 K. For these latter experiments, the excitation wavelengths were fixed at 230 nm and 287 nm for naproxen38 and diclofenac sodium39 respectively. The background emissions due to the buffer, devoid of any niosomes, were duly subtracted from the main experiments. The excitation and emission slit widths for all the above experiments were fixed at 5 nm each. Absorbance Spectroscopy. The absorbance measurements were done on Jasco V-550 double beam spectrophotometer. 5 µl of naproxen loaded Brij 30 (NB) or diclofenac sodium loaded Brij 30 (DB) niosome solution of pH 7.4 was added to 2.995 ml of buffer of same pH and their absorbance spectra were monitored at a scan speed of 100 nm/min over a wavelength range of (190-450) nm and at different time intervals over a period of 24 h at 298.15 K. The baseline correction in presence of buffer was done before noting each of the spectra. For study on SDS mediated rupture of niosomes of Brij 30 and TX-100 to release the encapsulated drug molecules of naproxen or diclofenac sodium into the buffer, 5 µl of each of these drug loaded niosomal solutions of pH 7.4 were added to 2.995 ml of 30 mM of SDS solution (pH 7.4) and kept at 298.15 K. The absorbance spectra of the above mixtures of SDS and aliquots of each type of niosomes, were noted at a speed of 100 nm/min over a wavelength range of (190-450) nm, at several time intervals over a period of 24 h at 298.15 K. Prior to each reading, the above solutions were briefly vortexed. Baseline corrections in presence of 30 mM of SDS under similar experimental set up and conditions were done before noting each of these spectra. Dynamic Light Scattering. A 90 Plus Particle Size Analyzer from Brookhaven Instruments Corp., New York, USA, was used for determination of size distribution of the drug (naproxen or


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diclofenac sodium) encapsulated niosomes of Brij 30 and TX-100, at 298.15 K and pH 7.4. The instrument laser operates at 658 nm. Small particles in suspension undergo random thermal motion known as Brownian motion. This random motion is modeled by the Stokes-Einstein equation, which is given below and used for particle size analysis

D = k BT / 3πηd

(3)

where, D is translational diffusion coefficient, k B is Boltzman constant, T is temperature in Kelvin, η is viscosity of the solvent and d is hydrodynamic diameter of the drug encapsulated niosomes.40 Transmission Electron Microscopy (TEM). A Philips CM 200 Transmission Electron Microscope, operating under an accelerating voltage of 200 kV, was used for imaging the drug (naproxen or diclofenac) encapsulated niosomes of Brij 30 and TX-100. For each sample preparation, equal volumes of the niosomal solution and 1% phosphotungstic acid solution were mixed and then a drop of the mixture was loaded onto Formvar-coated 300 mesh copper grids and air dried before collecting the images. Scanning Electron Microscopy (SEM). The SEM imaging was done on an FEI Quanta 200 Environmental Scanning Electron Microscope and JEOL JSM-7600F FEG-SEM operating under an accelerating voltage of 0.2 kV to 30 kV and 0.1 to 30 kV, respectively.

3. RESULTS AND DISCUSSION Structural Characterization of the Niosomal Vesicles. The results on size distribution of the drug (diclofenac sodium or naproen) loaded niosomes of Brij 30 and TX-100 done using a



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combination of dynamic light scattering (DLS) and transmission electron microscopy (TEM) are presented below in the respective subsections. Scanning electron microscopy (SEM) was used to characterize the morphologies of the empty niosomes of Brij 30 and TX-100 and Dcf encapsulated TX-100 niosomes (DX). We did not obtain homogenous size distribution after sonication. Thus with an aim of complete structural and morphological characterization of niosomes, we employed a combination of experimental techniques such as DLS, TEM and SEM. Dynamic Light Scattering (DLS). The results of dynamic light scattering for solutions of diclofenac sodium (D) or naproxen (N) encapsulated niosomes of Brij 30 (B) and TX-100 (X) namely- DB, DX, NB and NX- are shown in Figure 2. The results are represented in the number versus diameter format (Figure 2) to understand the exact number of a particular species/size of the vesicles. The size distribution of DB shows two peaks, one at 89 nm and another at 511 nm, the former being higher in abundance (Figure 2A). For NB, the size distribution shows the peaks at 96 nm and 905 nm with relatively higher abundance of the former (Figure 2C). Similar multiple size distribution implying heterogeneity in sizes of niosomal vesicles has been reported earlier.41 In case of DX and NX, only one peak was obtained, at 4.5 µm

and 1.5 µm,

respectively (Figures 2B and 2D). The drug encapsulated niosomes formed from TX-100 are larger in size than those of Brij 30 niosomes.


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Figure 2. Size distribution of diclofenac sodium (D) or naproxen (N) encapsulated niosomes of Brij 30 (B) and TX-100 (X). (A) DB, (B) DX, (C) NB and (D) NX.

Transmission Electron Microscopy (TEM). The TEM images of diclofenac (D) or naproxen (N) loaded niosomes of Brij 30 (B) and TX-100 (X) - DB, DX, NB and NX- are shown in Figure 3. The black dots represent the encapsulated drug molecules within DB and NX niosomes (Figures 3A and 3D). The dark interior of niosomes of DX and NB represent the possible location of the drugs (Figures 3B and 3C). The TEM images of drug encapsulated



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niosomes of Brij 30 show more heterogeneity in size compared to those of TX-100, as per our observation from DLS results as well.

Figure 3. Transmission electron microscopy images of (A) DB, (B) DX, (C) NB and (D) NX. Scale bar = 200 nm for (A) and (B), 1 µm for (C) and 2 µm for (D).

Scanning Electron Microscopy (SEM). The SEM was done to achieve imaging of the niosomal vesicles of Brij 30 and TX-100 in the absence of any drug molecule, at a lower magnification compared to TEM, in order to view the micron sized vesicles. In order to confirm and visualize the presence of the larger niosome vesicles specifically, Scanning Electron 14 ACS Paragon Plus Environment

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Microscopy (SEM) was done with the empty niosomes of the non-ionic surfactants which cannot be imaged with Transmission Electron Microscope (TEM) owing to the small field of view and higher magnification of the latter. The large vesicles are probably the Multivesicular vesicles (MVV) and Multilamellar large vesicles (MLV).42-44 The population of these larger vesicles is much lower than that shown in the number percent versus Diameter plots obtained from DLS. Observing the possible heterogeneity caused due to the presence of larger sized vesicles of dissimilar sizes was the main purpose of SEM imaging, which are otherwise not obtained from TEM or DLS results. The results are shown in Figures 4A and 4B. To correlate the size distribution of diclofenac sodium encapsulated TX-100 niosome (DX) vesicles, obtained from DLS with that of electron microscopic imaging, SEM imaging was done for DX vesicles which show the presence of micron sized vesicle (Figure 4C).



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Figure 4. Scanning electron microscopy of empty niosomes of (A) Brij 30 and (B) TX-100 and (C) diclofenac sodium encapsulated TX-100 niosome (DX). Partitioning of Diclofenac and Naproxen into the Empty Niosomes of Brij 30 and TX100 Isothermal Titration Calorimetry (ITC). The representative ITC thermograms, for titration of separate solutions of 10 mM of diclofenac sodium and naproxen into separate solutions of Brij 30 and TX-100 empty niosomes at temperature 298.15 K, are shown in Figure 5. The upper panels in these figures show the raw data for 25 sequential injections of 10 mM of diclofenac sodium or naproxen into empty niosomes of Brij 30 or TX-100, (labeled in the figure legend) while the lower panels show the integrated heat values in kcal mol-1 as a function of molar ratio of drug to niosomes. The results show binding profiles for titration of both the drugs individually, into the empty niosomes of TX-100 while no binding/partitioning of these drugs was observed when titrated into the empty niosomes of Brij 30. The line of best fit for the data obtained for titration of these drugs into the empty niosomes of TX-100, was obtained by fitting the data to the sequential binding model for two sites. This provided the thermodynamic parameters- binding constant ( K ), standard molar enthalpy change ( ∆H ), and standard molar o

entropy change ( ∆S o ) associated with the sequential binding of the drugs with the empty niosomes of TX-100. These thermodynamic parameters are given in Table 1. The objective behind the design of such experiments on direct interaction of the drugs with the empty niosomes is similar to those of partitioning of drugs into micellar media for micelle mediated drug delivery systems.



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Figure 5. ITC profiles for titration of (A) 10 mM diclofenac sodium into empty niosomes of TX100, (B) 10 mM naproxen into empty niosomes of TX-100, (C) 10 mM diclofenac sodium into empty niosomes of Brij 30 and (D) 10 mM naproxen into empty niosomes of Brij 30. Upper


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panels show the raw heat of the sequential injections of the drug into the empty niosomes while lower panels show the integrated heat values in terms of kcal mol-1 as a function of molar ratio.

Table 1. The Values of Partitioning Constant ( K ), Stoichiometry ( n ), Standard Molar Enthalpy Change ( ∆H o ), Standard Molar Gibbs Free Energy Change ( ∆G° ) and Standard Molar Entropy Change ( ∆S o ) Associated with the Partitioning of Diclofenac sodium and Naproxen into the Empty Niosomes of TX-100 at 298.15 K Solution

∆H ° /

K/ -1

(kcal mol )

∆G° / (kcal mol-1)

-1

(M )

∆S ° / (cal K-1 mol-1)

Diclofenac sodium Site 1 Site 2

(1.53 ± 0.33)×103 (9.81 ± 1.80)×102

4.86 ± 0.51 -(4.10 ± 0.44)

-(4.35 ± 0.13) -(4.08 ± 0.11)

30.9 ± 1.8 -0.1 ± 1.5

Naproxen Site 1 Site 2

(2.11 ± 0.46)×103 (1.12 ± 0.21)×103

-(0.20 ± 0.01) 0.20 ± 0.02

-(4.55 ± 0.13) -(4.16 ± 0.11)

14.6 ± 0.4 14.6 ± 0.4

The best fitting to the data was obtained by the sequential binding model which suggests that partitioning at site 1 helps and drives the subsequent association of the drug molecule at the site 2 of the TX-100 niosomes. The values of ∆H ° {(4.86 ± 0.51) kcal mol-1} and ∆S ° {(30.9 ± 1.8) cal K-1 mol-1 } (Table 1) associated with site 1 of interaction suggests nonpolar mode of partitioning while the values of ∆H ° [{-(4.10 ± 0.44)} kcal mol-1] and ∆S ° [{-(0.1 ± 1.5)} cal K-1 mol-1] (Table 1) associated with binding of diclofenac sodium at site 2 of TX-100 niosomes suggests the occurrence of polar interactions. The small change in standard molar entropy [ ∆S ° ={-(0.1 ± 1.5)} cal K-1 mol-1] associated with the partitioning of diclofenac sodium at site 2 of TX-100 niosomes suggests negligible changes in the solvent structure upon such complexation. However, the change in standard molar entropy { ∆S ° = (30.9 ± 1.8) cal K-1 mol-1 } associated with the partitioning of diclofenac sodium at site 1 of TX-100 niosomes is much higher than that at site 2. This is because of release of the water molecules that were hydrating the diclofenac 19 ACS Paragon Plus Environment


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molecules, into the bulk solvent upon entry of the diclofenac molecules into the nonpolar lipid bilayer of the TX-100 niosomes as well as the additional disorder introduced into the system by the molecular motions within the niosome vesicles to accommodate the drug molecules. The chemical structures (Figure 1) of diclofenac and that of the ethylene oxide units of TX-100 with the lone pair of electrons on oxygen atoms show that both are electron rich systems. Therefore the injection of diclofenac solution into TX-100 niosomal solution prohibits immediate partitioning at the head group and the diclofenac molecules penetrate deep into the nonpolar lipid bilayer where the aromatic rings of the drug and the TX-100 molecules can stack together. The structural loosening between the packed monomers within TX-100 niosome allows the partitioning of diclofenac at site 2 which is the polar head region. The affinity of binding ( K of the order 103) of diclofenac at site 1 (nonpolar lipid bilayer) of TX-100 niosomes is also higher than that at site 2 (polar head region) ( K of the order 102). The site 2 of interaction on TX-100 niosomes is expected to be at the first or second oxygen atom of the ethylene oxide units from the outer aqueous periphery so that there is not much of entropic changes [ ∆S ° ={-(0.1 ± 1.5)} cal K-1 mol-1] accompanying the process of diclofenac binding at this site of TX-100 niosomes as discussed above. At this site, the hydrogen atom of the —NH moiety from diclofenac molecule (Figure 1C) can form hydrogen bond with the lone pair of electrons on oxygen atoms of ethylene oxide units of the polar head region yielding the observed exothermic enthalpic changes [ ∆H ° ={-(4.10 ± 0.44)} kcal mol-1]. The association of diclofenac at the polar head region of the TX100 niosomes is comparatively less stable [ ∆G° = {-(4.08 ± 0.11)} kcal mol-1for site 2] than that for partitioning at the nonpolar lipid bilayer of TX-100 niosomes [ ∆G° = {-(4.35 ± 0.13)} kcal mol-1 for site 1]. This lower stability at site 2 of interaction (polar head of TX-100 niosomes) could be due to the competing water molecules in the surrounding while such competing


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interactions are absent at the site 1 (nonpolar lipid region) which is away from the solvent molecules. Therefore, the values of these thermodynamic parameters (Table 1) associated with partitioning of diclofenac sodium into TX-100 niosomes suggests the binding to be entropically and enthalpically driven at site 1 (nonpolar lipid bilayer) and site 2 (polar head groups) of TX100 niosomes, respectively. Based on the above discussion, the probable sites and modes of partitioning of diclofenac molecules into the niosomes of TX-100 are represented pictorially in Figure 6A.

Figure 6. Schematic representation of sites of partitioning of (A) diclofenac and (B) naproxen into the niosomes of TX-100.



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On the other hand in case of partitioning of naproxen into TX-100 niosomes, the values of -1 -1 ∆H ° are {-(0.20 ± 0.01)} kcal mol and (0.20 ± 0.02) kcal mol for site 1 and site 2 of

interaction, respectively and those of ∆S ° are (14.6 ± 0.4) cal K-1 mol-1 and (14.6 ± 0.4) cal K-1 mol-1 for site 1 and site 2 of interaction, respectively. Also the affinities of binding of naproxen at both sites on TX-100 niosomes are same ( K of the order 103 for both sites). These similar values of ∆H ° and ∆S ° (Table 1) associated with partitioning of naproxen into TX-100 niosomes indicate similar endothermic mode of binding of the drug with the TX-100 niosomes thereby suggesting both the loci to be in the hydrophobic lipid bilayer and in the vicinity of one another. The small negative value of ∆H ° [{-(0.20 ± 0.01)} kcal mol-1] could be due to weak interactions of naproxen molecules with the polar heads of the TX-100 niosomes, thereby suggesting the site 1of naproxen - TX-100 interaction to be at the juncture between the outer head group and the lipid bilayer. At this site, one of the aromatic rings of naproxen and that of TX-100 can stack together while the other aromatic ring of naproxen can interact with the nearest H-atom of >CH2 moieties of ethylene oxide units of TX-100 head group via CH-π interaction where this latter type of interaction imparts slight exothermicity to the value of ∆H ° . The other site of interaction (site 2) of TX-100 niosomes with naproxen molecules is slightly away from the polar head groups and relatively interior in the hydrophobic region of the lipid bilayer, compared to site 1, evident from the similar but small positive value of ∆H ° {(0.20 ± 0.02) kcal mol-1} value for site 2 of interaction. The binding of naproxen at site 1 [ ∆G° = {-(4.55 ± 0.13)} kcal mol-1] of TX100 niosomes was observed to be slightly more stable as compared to that of site 2 [ ∆G° = {(4.16 ± 0.11)} kcal mol-1]. The values of ∆S ° , being (14.6 ± 0.4) cal K-1 mol-1 for each of site 1 and site 2 of partitioning of naproxen into TX-100 niosomes suggests that binding of naproxen at both the sites on TX-100 niosomes, is entropically driven. The above discussed loci of


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partitioning of naproxen into TX-100 niosomes suggest that these observed large positive values of standard molar entropy changes associated with complexation of the naproxen molecules with TX-100 niosomes are expected to be caused due to the release of the water of hydration around the naproxen molecules as they penetrate into the lipid bilayer of the TX-100 niosomes. However, these ∆S ° values are half as much as ∆S ° {(30.9 ± 1.8) cal K-1 mol-1} associated with the partitioning of diclofenac sodium at site 1 of TX-100 niosomes. This observation suggests the absence of huge molecular motions induced within the niosome structures upon partitioning of naproxen into TX-100 niosomes as compared to that of partitioning of diclofenac into these niosomes. Based on the above discussion, the probable sites and modes of partitioning of naproxen molecules into the niosomes of TX-100 are represented pictorially in Figure 6B. The absence of partitioning of these drugs into the niosomes of Brij 30 (Figures 5C and 5D) highlights the combined effect of rigidity of the niosome vesicles as well as the attractive/repulsive role of the polar head groups in the stable binding of the drug molecules. The polar head groups of both TX-100 and Brij 30 contain ethylene oxide units which repel the approaching molecules of the drugs diclofenac sodium and naproxen which are also electron rich systems as evident from their chemical structures (Figure 1). The observed results suggest higher fluidity in the structure of the TX-100 niosomes over Brij 30 niosomes, such that the approaching drug molecules may penetrate into the nonpolar interior of the former where favorable stacking interactions can take place between the drug and TX-100 molecules. The difference in rigidity of the niosome vesicles formed from these two types of surfactants is inherent in their chemical structures (Figure 1) with hydrophilic-lipophilic balance (HLB) values of 9.721 and 13.522 for Brij 30 and TX-100 respectively as mentioned earlier. The surfactants with HLB values between 14-17 cannot form niosome vesicles unless cholesterol is added45



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which also imparts rigidity to the vesicles thus formed. The niosomal preparations of TX-100, in the current study, contained cholesterol in the molar ratio of TX-100 : cholesterol = 2:1 which still maintained certain extent of fluidity in the TX-100 niosomes to allow the partitioning of the said drugs. Supporting Information Figure S1 gives a pictorial representation of the absence of partitioning of diclofenac and naproxen into the empty niosomes of Brij 30. Diffusion Controlled Release of the Drugs Diclofenac and Naproxen from the Drug Loaded Niosomes of Brij 30 and TX-100 – Naproxen/Brij 30 (NB), Diclofenac/Brij 30 (DB), Naproxen/TX-100 (NX) and Diclofenac /TX-100 (DX) The kinetics of the process was monitored for different time intervals and various physical conditions (pH, temp) etc. The time (t) point t = 0 hour (h) refers to the time of completion of dialysis of the drug loaded niosomal preparation to remove the excess, unbound drug molecules. Isothermal Titration Calorimetry (ITC) of Interaction of Drug Released from Drug Loaded Niosomes with Bovine Serum Albumin as a Function of Time. The drug (diclofenac sodium or naproxen) loaded niosomes of Brij 30 (contained in the syringe) were allowed to interact with 0.060 mM of BSA (contained in the cell) at several time (t) points from t = 0 h to 24 h to obtain the values of the thermodynamic parameters for the above interaction with the progress of time upto 24 h, from the ITC experiments. The rationale behind the designing of such experiments is that the time dependent diffusion controlled process of release of the drugs from the niosomes of Brij 30 would lead to increased binding of the drugs with the protein BSA in the cell, as time progresses, leading to corresponding changes in the thermodynamic parameters associated with the interaction. The binding of the drugs diclofenac sodium46 and naproxen47 with BSA has already been characterized earlier and therefore BSA is known to contain the


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binding sites for these drugs. The results obtained for separate titrations of diclofenac sodium loaded Brij 30 niosomes (DB) and naproxen loaded Brij 30 niosomes (NB), into the solution of BSA at several time intervals upto t = 24 h under physiological pH condition at 298.15 K, are shown in Table 2. The t = 0 h time point has already been defined above at the beginning of the section “Diffusion Controlled Release of the Drugs Diclofenac and Naproxen from the Drug Loaded Niosomes of Brij 30 and TX-100 – Naproxen/Brij 30 (NB), Diclofenac/Brij 30 (DB), Naproxen/TX-100 (NX) and Diclofenac /TX-100 (DX)”. The results show no change in the values of the associated thermodynamic parameters signifying no release of the drugs from the Brij 30 niosomes at pH 7.4 and 298.15 K temperature, upto a period of 24 h. This provides evidence for stability of the drug encapsulated niosomal structures of Brij 30 upto a period of 24 h at 298.15 K and physiological pH. However, this diffusion controlled release of the drugs from both types of niosomes were further studied using spectroscopy. Table 2. Values of the Thermodynamic parameters - Binding Constant ( K ), Standard Molar Enthalpy Change ( ∆H ° ), Standard Molar Gibbs Free Energy Change ( ∆G° ), Standard Molar Entropy Change ( ∆S ° ) and Stoichiometry of Binding ( n ) - Associated with Titration of Diclofenac sodium and Naproxen Encapsulated Niosomes of Brij 30 (DB and NB) into 0.060 mM BSA at Several Time (t) Intervals from t = 0 to 24 h, at pH 7.4 and 298.15 K n Solution ∆G° / ∆S ° / K/ ∆H ° / (M-1) (kcal mol-1) (kcal mol-1) (cal K-1 mol-1) BSA-DB 0h (3.75 ± 0.30)×103 -3.37 ± 0.02 -4.87 ± 0.05 4h (3.90 ± 0.31)×103 -3.36 ± 0.16 -4.90 ± 0.05 8h (3.96 ± 0.31)×103 -3.26 ± 0.14 -4.91 ± 0.05 24 h (3.81 ± 0.30)×103 -3.10 ± 0.13 -4.88 ± 0.05 BSA-NB 0h (1.14 ± 0.09)×104 -1.32 ± 0.03 -5.53 ± 0.05 4h (1.23 ± 0.10)×104 -1.33 ± 0.03 -5.58 ± 0.05 8h (1.27 ± 0.11)×104 -1.34 ± 0.03 -5.60 ± 0.05 24 h (1.88 ± 0.30)×104 -1.21±0.04 -5.83 ± 0.09


5.0 ± 0.2 5.2 ± 0.6 5.5 ± 0.5 6.0 ± 0.5

9.3 ± 0.3 9.4 ± 0.3 10.1 ± 0.3 10.5 ± 0.3

14.1 ± 0.2 14.2 ± 0.2 14.3 ± 0.2 15.5 ± 0.3

11.0 ± 0.2 10.6 ± 0.2 10.4 ± 0.2 10.2 ± 0.3


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Absorbance Spectroscopy. These experiments were done to correlate with the results of ITC (Table 2) on release of diclofenac sodium (D) and naproxen (N) from the Brij 30 (B) niosomes over a period of 24 h at pH 7.4 and 298.15 K. Figure 7 shows the absorbance spectra of the same niosomal solutions of these drugs (pH 7.4) monitored at same time intervals as those of ITC experiments, upto 24 h at 298.15 K. The results show the characteristic absorption peaks at 200 nm and 276 nm for diclofenac sodium48 and at 230 nm for naproxen.49,

50

Absence of any

significant changes in the peak intensities for both the niosomal preparations (DB and NB) suggest no release of these drugs from the Brij 30 niosomes over a period of 24 h at 298.15 K and physiological pH. This observation is in coherence with the results obtained from the corresponding ITC experiments discussed above.

Figure 7. Absorbance spectra of (A) diclofenac sodium and (B) naproxen encapsulated niosomes of Brij 30, monitored over a period of 24 h at pH 7.4 and 298.15 K.

Fluorescence Spectroscopy.


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pH Dependent in Vitro Release of Diclofenac Sodium and Naproxen from the Niosomes of Brij 30 and TX-100. Experiments were designed to monitor the release kinetics of diclofenac sodium (D) and naproxen (N) from the niosomes of Brij 30 (B) and TX-100 (X) at pH 5.5, 7.4 and 9.0, over a period of 24 h at 298.15 K. For this purpose, the intrinsic fluorescence emission of BSA was noted in presence of DB, DX, NB and NX niosomes at different time intervals t = 0 h, 4 h, 8 h and 24 h after dialysis of each of the niosomes. The representative results of emission spectra of BSA in presence of DB, DX, NB and NX niosomes at different time intervals at pH 7.4 are shown in Figure 8 while those at pH 5.5 and 9.0 are shown in Supporting Information Figures S2 and S3. Gradual release from the vesicles with subsequent build up of concentration of the drug molecule in the solution with time would lead to enhanced binding of the drug with BSA, leading to corresponding changes in the fluorescence emission spectra of the latter. The aim was to note such changes in the intrinsic fluorescence emission of BSA with time, in presence of the drug encapsulated niosomes. The pH conditions (pH 5.5, 7.4 and 9.0) were so chosen in order avoid adverse effects of extreme acidic or alkaline conditions on the protein conformation. For each experiment the protein concentration was fixed at 1.5 µM. The maximum emission wavelength ( λmax ) of BSA obtained in absence of any niosomes at pH 5.5, 7.4 and 9.0 are 337 nm, 339 nm and 334 nm, respectively. The results show that in presence of DB and DX niosomes, there is a slight decrease and a small red shift in the intensity and λmax of intrinsic fluorescence emission of BSA, respectively, signifying the occurrence of some interaction between the protein and diclofenac sodium loaded niosomes of Brij 30 and TX-100. The extent of this initial interaction does not change over a period of 24 h as evident from the absence of any significant difference or specific trend in the emission spectra of BSA in presence of DB or DX niosomes, from t = 0 to 24 h at 298.15 K under various pH conditions. However



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there is some difference in the peak intensity of emission at t = 4 h (Figure 8B and Supporting Information Figure S3A) in presence of DX at pH 7.4 and 5.5. Similar absence of specific trend or significant difference in the intrinsic fluorescence emission of BSA from t = 0 to 24 h in presence of naproxen loaded niosomes of Brij 30 and TX-100 (namely, NB and NX respectively) was also observed. In presence of NX niosomes, there is a slight increase and red shift in the intensity and λmax of intrinsic fluorescence emission of BSA, respectively, signifying interaction between BSA and NX niosomes, which does not differ significantly over a period of 24 h under various pH conditions. This effect of small increase in the intensity of λmax of BSA was observed in presence of NB at pH 7.4 and 9.0 with small red shifts at pH 7.4 and 5.5 respectively. The effects on intensity and wavelength of intrinsic fluorescence of BSA in presence NB or NX are however much less compared to that in presence of DB or DX. Therefore, these results suggest that upto a period of 24 h (+ 4 h of dialysis for removal of unbound drugs) under conditions of pH 5.5, 7.4 and 9.0, at 298.15 K, the niosomal complexes of Brij 30 or TX-100 are stable enough to prevent any diffusion of the drug molecules (diclofenac sodium or naproxen) encapsulated within these vesicles.


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Figure 8. Representative intrinsic fluorescence emission spectra of BSA monitored over time interval, t = 0-24 h at 298.15 K and pH 7.4, in presence of diclofenac sodium encapsulated niosomes of (A) Brij 30 (DB), (B) TX-100 (DX) and in presence of naproxen encapsulated niosomes of (C) Brij 30 (NB) and TX-100 (NX).

In Vitro Release of Diclofenac Sodium and Naproxen from the Niosomes of Brij 30 and TX100 under Conditions of Physiological pH and Temperature. Experiments were designed to monitor the in vitro release kinetics of diclofenac sodium and naproxen from the niosomes of Brij 30 and TX-100 under physiological conditions and in absence of any protein. To mimick the physiological condition of the body, the drug encapsulated niosomes (DB, DX, NB and NX) prepared at pH 7.4, were incubated at 310.15 K over several days. The niosomal solutions (DB, 29 ACS Paragon Plus Environment


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DX, NB or NX) were selectively excited for the drug molecule (diclofenac sodium or naproxen) contained within the vesicles and the changes in their fluorescence emission spectra were observed at different time intervals over several days, starting from t = 0 h. The results are shown in (Figures 9B, 9C, 10B and 10C). As reference experiments, the concentration dependent changes in the fluorescence emission spectra of only the drug molecule (diclofenac sodium or naproxen) in buffer (pH 7.4) were also monitored (Figures 9A and 10A). The results obtained from the reference experiments show the typical emission maxima at 362 nm for diclofenac sodium50 and 352 nm for naproxen51. The trends obtained from these reference experiments (Figures 9A and 10A) show that there is an initial increase in the fluorescence intensity upto the build up of a certain concentration of diclofenac sodium followed by continuous decline with further increasing concentration of diclofenac sodium in buffer (pH 7.4) while that for naproxen there is a continuous decrease in the fluorescence emission intensity upto a constant value, with increasing concentration of naproxen in buffer (pH 7.4). The quenching of the fluorescence emission intensity of either of the drugs (diclofenac sodium or naproxen) with their increasing concentration could be attributed to the possible intermolecular interactions. The changes obtained in fluorescence emission spectra of the drug encapsulated niosomes, over several days, were then compared to the above mentioned results of the reference experiments. The release kinetics of diclofenac sodium from the Brij 30 niosomes were monitored from the changes in the fluorescence emission spectrum of diclofenac sodium over a period of t = 0 h to 1190 h at different time intervals (Figure 9B and Supporting Information Figure S4B). The results show a typical fluorescence emission spectra with emission maximum at 362 nm for the solution of DB niosomes and the emission intensity remains constant upto t = 352 h followed by its initial increase at t = 380 h that again remains constant upto t = 638 h. Therefore the first round of


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release of diclofenac sodium from Brij 30 niosomes takes place after 380 h which is after 15-16 days. Then there is a further increase of the emission intensity at t = 706 h which remains constant upto 754 h followed by decrease at t = 802 h and a further decrease at t = 850 h (Figure 9B and Supporting Information Figure S4B). This increase followed by a decrease in the emission intensity of the diclofenac sodium encapsulated Brij 30 niosomes is in coherence with the trend obtained for diclofenac sodium in buffer (pH 7.4) with gradually increasing concentration of the drug in buffer (Figure 9A and Supporting Information Figure S4A). The fluorescence emission intensity further decreases at t = 970 h and at t = 1190 h (Figure 9B and Supporting Information Figure S4B). Hence the release of diclofenac sodium from niosomes of Brij 30 continues over a period of about 49-50 days thereby pertaining to the principle of controlled and sustained release of drugs from vesicular formulation. In case of release of diclofenac sodium from TX-100 niosomes the release kinetics of the drug was monitored from its changes in fluorescence emission over a period of t = 418 h only (Figure 9C and Supporting Information Figure S4C), owing to observation of comparatively smaller changes in the fluorescence spectrum than that of DB. The typical fluorescence emission spectra with emission maxima at 362 nm was obtained for the solution of DX as well. However Figure 9C and Supporting Information Figure S4C show that the first significant increase in fluorescence emission intensity of DX solution was observed at t = 49 h which remained constant upto a period of t = 196 h. This was followed by a slight decrease at t = 244 h and that remained steady upto t = 418 h. Therefore, the first round of release of diclofenac sodium from TX-100 occurs after t = 49 h (in 2 days) and again after t = 244 h (10 days). Absence of pronounced/significant changes in the emission intensities of DX solution denote the absence of significant quantity of release of diclofenac sodium from TX-100 niosomes at the observed time intervals. Figures 10B,



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10C and Supporting Information Figures S5B and S5C show the respective release kinetics of naproxen from the niosomes of Brij 30 and TX-100 obtained from the changes in fluorescence emission spectra of naproxen over a time period of t = 963 h. The fluorescence emission spectra of both the solutions of NB and NX show the typical emission maxima at 352 nm with a decrease in the emission intensity with time. This decrease in emission intensity is again in coherence with the decreasing trend observed for increasing concentration of only naproxen in buffer (pH7.4) (Figure 10A). This observation therefore denotes gradual release of naproxen with time from the niosomes of Brij 30 and TX-100. Significant changes in fluorescence emission intensity, denoting the release of naproxen from the niosomes, is observed to begin from t = 50 h and t = 124 h in cases of NB and NX niosomes, respectively (Figures 10B, 10C, Supporting Information Figures S5B and S5C). This denotes a faster release kinetics of naproxen from Brij 30 niosomes compared to those of TX-100 signifying higher stability of naproxen-TX100 complex. Also the release of naproxen from niosomes of Brij 30 seem to reach a saturation by t = 291 h (about 12 days) (Figure 10B and Supporting Information Figure S5B). Therefore, for prolonged retention and release of naproxen in the blood circulation, TX-100 niosomes are desirable. The stability against diffusion controlled release process of these drugs, the niosomal vesicles of DB, DX and NX seem to be much higher than that of NB. This also suggests that the entrapped naproxen molecules are positioned at or closer to the polar head groups of the outer layer of the lipid bilayer of Brij 30 thereby making the diffusion process faster.


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Figure 9. Fluorescence emission spectra of (A) increasing concentration of only diclofenac sodium (Dcf) (16.67 µM to 228.01 µM) in buffer (pH 7.4) and of diclofenac sodium encapsulated niosomes of (B) Brij 30 and (C) TX-100, monitored over several days ( from time, t = 0 to 1190 h and t = 0 to 418 h, respectively) under physiological conditions of pH and



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temperature. Detailed figure mentioning the time intervals of noting each spectra is given in the Supporting Information Figure S4.

Figure 10. Fluorescence emission spectra of (A) increasing concentration of only naproxen (Nap) (16.64 µM to 228.01 µM) in buffer (pH 7.4) and of naproxen encapsulated niosomes of 34 ACS Paragon Plus Environment

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(B) Brij 30 and (C) TX-100, monitored over several days (from time, t = 0 to 963 h) under physiological conditions of pH and temperature. Detailed figure mentioning the time intervals of noting each spectra is given in the Supporting Information Figure S5. Sodium Dodecyl Sulfate Mediated Rupture of the Drug Loaded Niosomes (NB, DB, NX and DX) and Release of the drugs Absorbance Spectroscopy. The absorbance spectra of the solutions of diclofenac sodium or naproxen encapsulated niosomes of Brij 30 and TX-100 namely- DB, DX, NB and NX- in presence of 30 mM sodium dodecyl sulfate (SDS), were monitored over a time period of 24 h at 298.15 K and pH 7.4. The objective was to observe the kinetics of SDS mediated rupture of the niosome vesicles with subsequent release of the entrapped drug molecule over the given period of time and under given physical conditions. The results are shown in Figure 11. SDS mediated solubilization of Span 80 niosomes has been reported earlier.52 The diclofenac sodium encapsulated niosomes of Brij 30 and TX-100 (DB and DX) show the characteristic absorption peaks at 200 nm and 276 nm for diclofenac while the naproxen encapsulated niosomes of Brij 30 and TX-100 (NB and NX) show characteristic absorption peak at 230 nm for naproxen (Figure 11). The observed increase in absorption intensities of each of the drug loaded niosomal solutions with time, in presence of SDS, denotes the release of the corresponding drug molecules from the niosomal vesicles into the buffer (Figure 11). The observed absorbance values at the time intervals t = 0 h, 6 h and 24 h were used to calculate the concentration of the drug at each of these time points using the values of molar extinction coefficients corresponding to ε276 = (1.16 x 104) L mol–1 cm–1 and ε262 = (4.15 x 103) L mol–1 cm–1 for diclofenac sodium and naproxen respectively, as per our experimental results. The values of molar extinction coefficients reported in literature are ε276 = (1.04 ± 0.02) x 104 L mol–1 cm–1 and ε262 = (5.62 x 103) L mol–1 cm–1 for



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diclofenac sodium48 and naproxen53 respectively. Thus the amount of drug released (in µM) in presence of SDS, at time intervals, t = 6 h and t = 24 h relative to that at t = 0 h, is shown in the insets of Figure 11 for each of the four drug loaded niosomal preparations (Figure 11 insets). Further experiments beyond 24 h in presence of SDS were not carried out. In presence of SDS, the kinetics of drug release is found to be much faster than the simple diffusion controlled process in absence of any additive. The release of drug from the niosomal vesicles, took place within 24 h in presence of SDS unlike the simple diffusion controlled process in absence of any additive.


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Figure 11. Absorbance spectra of (A) diclofenac sodium encapsulated niosomes of Brij 30 (DB), (B) diclofenac sodium encapsulated niosomes of TX-100 (DX), (C) naproxen encapsulated niosomes of Brij 30 (NB) and (D) naproxen encapsulated niosomes of TX-100 (NX), monitored over a time period of t = 24 h in presence of 30 mM SDS at pH 7.4 and 298.15 K. The insets show the amount of drug released (in µM) from the respective niosomes, at time intervals, t = 6 h and t = 24 h, with respect to the concentration at t = 0 h.

Determination of Rate Constant for Diffusion Controlled Release of the Drugs under Physiological Conditions of pH and Temperature and SDS Mediated Release from the Niosomes. The maximum fluorescence intensity values versus time obtained from time dependent fluorescence emission spectra of NB and NX under physiological conditions of pH and temperature and the maximum absorption values versus time obtained from time dependent changes in absorbance spectra of all the niosomal solutions in presence of SDS were fitted by a first order rate equation to obtain the rate of release of the drugs from the respective niosome vesicles to the buffer medium. Eq 4 represents the first order rate equation where y is the observed fluorescence intensity or absorbance at time t , A is the initial fluorescence intensity or absorbance obtained at time t = 0 h and k denotes the rate constant for release of the drugs from the niosome vesicles.

y = Ae− kt

(4)

The rates of release of the drugs from the niosomes thus calculated, are given in Table 3. From the absorption spectra obtained in presence of SDS, the spectral changes at the 37 ACS Paragon Plus Environment


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characteristic wavelengths of 276 nm for diclofenac sodium and 230 nm for naproxen while from the fluorescence emission spectra, the intensity changes at the emission maximum of 352 nm for naproxen were used for the above calculation of rate constants. Calculation of rate of release of diclofenac sodium from the niosomes of Brij 30 and TX-100 could not be done owing to the abnormal/irregular trend of the fluorescence intensities shown by the drug upon its gradual release from the vesicles under the physiological conditions of pH and temperature (Figure 9 and Supporting Information Figure S4).

Table 3. Values of Rate Constant, k , for Release of Naproxen from Naproxen Encapsulated Niosomes Brij 30 (NB) and TX-100 (NX) at pH 7.4 and 310.15 K, in the Absence of SDS and for Release of Naproxen and Diclofenac sodium from their Respective Drug Encapsulated Niosomes of Brij 30 (NB and DB) and TX-100 (NX and DX) at pH 7.4 and 298.15 K, in the Presence of SDS 102 k /h-1

Niosomal Solution At pH 7.4, 310.15 K NB NX

(0.97 ± 0.08) (0.32 ± 0.04)

At pH 7.4, 298.15 K NB+SDS NX+SDS DB+SDS DX+SDS

(3.81 ± 0.21) (1.65 ± 0.50) (3.27 ± 0.66) (0.43 ± 0.18)

The results thus obtained (Table 3) show that the rate of diffusion controlled release of naproxen from the niosomes of Brij 30 [ k = {(0.97±0.08)·10-2} h-1] is faster than that from TX100 niosomes [ k = {(0.32 ± 0.04)·10-2} h-1]. The increased rate of release of these drugs from the niosomes of Brij 30 and TX-100 [ k = {(3.81 ± 0.21)·10-2} h-1 and (1.65 ± 0.50)·10-2} h-1 for release of naproxen from Brij 30 and TX-100 niosomes respectively, while k = {(3.27 ± 0.66)·10-2} h-1 and k = {(0.43 ± 0.18) ·10-2} h-1 for release of diclofenac sodium from Brij 30 and TX-100 niosomes respectively] in presence of SDS suggests the possible destabilizing effect 38 ACS Paragon Plus Environment

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of SDS on the niosome vesicles. This destabilization effect is expected to be brought about by the interaction between SDS and those of the drug loaded niosomes and affects the vesicular architecture of the latter thereby releasing the entrapped drug molecules into the external medium. The higher values of k obtained (Table 3) for release of both the drugs from Brij 30 niosomes than those of TX-100 in presence of SDS, once again suggest the higher stability of the drug loaded TX-100 niosomes which correlate with the diffusion controlled release kinetics observed in absence of any additive under physiological conditions. In presence of SDS, the smallest value of k [{(0.43 ± 0.18) ·10-2} h-1] was observed for the release of diclofenac sodium from TX-100 niosomes which denote the highest stability of diclofenac sodium encapsulated TX-100 niosomes (DX) among all the four drug loaded niosomal preparations (Table 3) which also correlates with the results and discussion under section “In vitro release of diclofenac sodium and naproxen from the niosomes of Brij 30 and TX-100 under conditions of physiological pH and temperature”. Correlation between Site of Partitioning of the Drugs into the Niosomes of Brij 30 and TX-100 and that of Their Release Kinetics. The results obtained from ITC experiments provided important insights on the probable loci of partitioning of the drugs diclofenac sodium and naproxen in the niosomes of Brij 30 or TX-100. The preferred locus for both the drugs on TX-100 niosomes is the nonpolar lipid bilayer where favourable stacking interactions between the aromatic rings of the drugs and that of TX-100 molecules led to stable complexation of each of these drugs with the empty niosomes of TX-100. On the other hand absence of favourable interactions with Brij 30 prohibited partitioning of these drugs into the empty niosomes of Brij 30. To study the diffusion controlled release kinetics of the same drugs under several physical conditions, the drug encapsulated niosomes of Brij 30 or TX-100 were formed by hydrating the



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dried lipid layer of the surfactant and cholesterol mixtures of certain molar ratios with the aqueous solution of the drug molecules (pH 7.4) such that the drug molecules get trapped within the niosome vesicles. The rate of release of these drugs (diclofenac or naproxen) from the drug loaded niosomes of Brij 30 or TX-100 was then studied using spectroscopic techniques. Both quantitative and qualitative analysis of rate of release of these drugs from the said drug loaded niosomes show slower kinetics of diffusing out of the TX-100 niosomes compared to that of Brij 30 niosomes, in absence and presence of SDS at pH 7.4. This observation when compared to the ITC results of partitioning of these drugs into the empty niosomes suggest that both diclofenac and naproxen molecules were preferentially trapped at nonpolar region of TX-100 containing the aromatic ring, when the individual drug encapsulated niosomes were prepared. The observed absence of partitioning of these drugs into empty niosomes of Brij 30 suggest that the drug molecules were localized at the nonpolar bilayer of Brij 30 niosomes to avoid the electron rich head group when the drug loaded Brij 30 niosomes were prepared lipidic film hydration. Therefore, due to the favourable stacking of the aromatic rings of the drug molecules and that of TX-100, which is absent in the system of Brij 30 niosomes, evident from the chemical structures and corresponding ITC results of drug partitioning, the rate release of the drugs is relatively faster from within the Brij 30 niosomes. Another factor that contributes to the slower rate of diffusion of the drugs from the TX-100 niosome compared to Brij 30 niosomes, is the presence of longer electron rich polar head group of the former surfactant. Therefore, the rate of release of the given drug molecules from the drug encapsulated niosomes of Brij 30 and TX-100 is determined and dictated mostly by the stabilizing interactions taking place between the respective drug molecule and the surfactant units of the niosomes rather than the fluidity of the vesicular architecture of these niosomes.


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4. CONCLUSIONS We have addressed the energetics of partitioning of the drugs diclofenac and naproxen into the empty niosomes of Brij 30 and TX-100. The thermodynamic parameters, thus obtained from isothermal titration calorimetry experiments, such as the standard molar enthalpy change ( ∆H ), o

and standard molar entropy change ( ∆S o ) associated with the binding process, provided fine details of molecular interactions with insights into possible loci of partitioning within the niosome vesicles. The other thermodynamic parameters such as the binding constant ( K ) and standard molar Gibbs free energy change ( ∆G° ) provided the affinity and stability of binding at the respective binding sites. These results and insights thus gained have been correlated with the observed release kinetics of diclofenac and naproxen from their corresponding drug encapsulated niosomes which gave guidelines in drawing the conclusion on structure property relationship. None of the drugs partitioned into the empty niosomes of Brij 30. Both the drugs however, partitioned preferably at the nonpolar lipid bilayer region of the empty TX-100 niosomes diffusing through the polar head group region into the interior, signifying the fluidity of the TX100 niosomal vesicles as compared to Brij 30 niosomes. Amount of cholesterol present, determines the vesicular rigidity and even helps in formation of stable niosome vesicles for surfactants with higher HLB values due to their longer polar head groups.54,55 The structural characterization using light scattering and electron microscopy, reveals heterogeneous size distribution of these niosomal vesicles. The kinetics of diffusion controlled release of these drugs, diclofenac and naproxen, from the drug encapsulated niosomes of Brij 30 and TX-100 is gradual and continues over several days, under physiological conditions of pH and temperature. Therefore prolonged or sustained release of these drugs into blood circulation, from the niosomes 41 ACS Paragon Plus Environment


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of Brij 30 and TX-100 can be achieved. The observed slower rate of release of the drugs from the drug encapsulated niosomes of TX-100 than those of Brij 30, not only suggest stable stacking of the aromatic rings of the drugs with those of TX-100 but also the effect of its longer electron rich polar head group containing multiple ethylene oxide units. No drug release was observed within 24 h after their preparation even upon changing the pH to acidic and alkaline conditions. This observation suggests that our drug encapsulated niosomal preparations are not going to be affected by the pH changes in the gastrointestinal tract of the body thereby signifying their stability and potential to be successfully used for oral administration of our niosomal formulations carrying the anti-inflammatory drugs, diclofenac sodium and naproxen. However, the denaturing effect of SDS mediates the release of these drugs within a span of 24 h. The nonionic surfactants being less toxic compared to the ionic ones give an advantage to these niosomal preparations for effective pharmaceutical application. Therefore these niosomal vesicles of Brij 30 and TX-100 seem to be promising enough for their application as delivery vehicles in controlled delivery of drugs as well as achieve sustained release of the drug molecules into the blood circulatory system. Our study therefore addressed both the qualitative as well as quantitative aspects of drug-niosome complexation and in vitro drug release kinetics, thereby establishing their structure property relationship.

SUPPORTING INFORMATION AVAILABLE: Schematic representation of absence of partitioning of diclofenac and naproxen into the niosomes of Brij 30, Figures 9 and 10 in details and intrinsic fluorescence emission spectra of BSA monitored over time interval, t = (0-24) h at 298.15 K, in presence of diclofenac and naproxen encapsulated niosomes of Brij 30 (DB) and TX-100, at pH (A) 5.5 and (B) 9.0.


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ACKNOWLEDGEMENT The authors are grateful to Board of Research in Nuclear Sciences, Mumbai for the financial support.



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Establishing Structure Property Relationship in Drug Partitioning into

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