Unveiling the Aggregation Behavior of Doxorubicin Hydrochloride in

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Unveiling the Aggregation Behavior of Doxorubicin Hydrochloride in Aqueous Solution of 1-octyl-3-methylimidazolium chloride and the Effect of Bile Salt on these Aggregates: A Microscopic Study Arghajit Pyne, Sangita Kundu, Pavel Banerjee, and Nilmoni Sarkar Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b00029 • Publication Date (Web): 23 Feb 2018 Downloaded from http://pubs.acs.org on February 25, 2018

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Unveiling the Aggregation Behavior of Doxorubicin Hydrochloride in Aqueous Solution of 1-octyl-3-methylimidazolium chloride and the Effect of Bile Salt on these Aggregates: A Microscopic Study Arghajit Pyne, Sangita Kundu, Pavel Banerjee and Nilmoni Sarkar* Department of Chemistry, Indian Institute of Technology, Kharagpur 721302, WB, India *Corresponding Author: Nilmoni Sarkar E-mail: [email protected] Fax: 91-3222-255303

Abstract In this article, we have unveiled the aggregation behavior of a potent chemotherapeutic drug, doxorubicin hydrochloride (Dox) in a well known imidazolium based surface active ionic liquid (SAIL), 1-octyl-3-methylimidazolium chloride (C8mimCl). The aggregates formed by Dox in C8mimCl have been characterized using dynamic light scattering (DLS), fluorescence lifetime imaging microscopy (FLIM), high resolution transmission electron microscopy (HR-TEM), analytical transmission electron microscopy (analytical TEM), field emission scanning electron microscopy (FESEM), atomic force microscopy (AFM) and fourier-transform infrared spectroscopy (FTIR) measurements. It is found that Dox forms large spherical aggregates in presence of C8mimCl SAIL. We have also explored the driving force behind this aggregation behavior of Dox in C8mimCl. Furthermore, it is observed that in presence of a common bile salt, sodium cholate (NaCh), Dox/ C8mimCl spherical aggregates disrupt to form rod like fibrillar aggregates. Therefore, formation of spherical aggregates and also its disruption into rod like fibrillar aggregates have been performed and this is expected to open a new scope for the design of a new generation smart drug delivery system where drug itself aggregates to form the delivery system. Key Words: chemotherapeutic drug, surface active ionic liquid, fluorescence lifetime imaging microscopy, spherical aggregates, rod like fibrillar aggregates. 1 ACS Paragon Plus Environment

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1. Introduction. In recent times, molecular self assembly and self organization bring a very different taste in the field of supramolecular chemistry1, physical chemistry2,3 and biology4 as they find extensive uses as molecular reaction center5-7, drug delivery system8-11, molecular catalysts12-14 etc. These molecular assemblies have a wide range of morphological diversities ranging from vesicles to nanofibers, tubes, rods, sheets etc. Among all these self assembled molecular frameworks, vesicles particularly draw significant amount of interests as they can mimic the biological cell membranes15,16 and also their organized structures composed of bilayers have the ability to encapsulate both hydrophobic and hydrophilic molecules.8,17 Besides, they can efficiently serve as molecular microreactors, where different nanoaggregates can be successfully synthesized.6 Commonly, the compounds having long hydrophobic tails are prone to form self assembled structures18,19 but nowadays, several other molecules like drug molecules9,10 and different biologically important molecules20,21 are also known to form self assembled molecular frameworks. In this respect, it is most important to mention here that electrostatic force of attraction, π-π, cation-π, hydrophobic and hydrogen bonding interactions are the main driving forces behind the formation of these self assembled supramolecular structures.22-24 Doxorubicin hydrochloride (Dox), also known as Aldrimycin or Rubex is an anthracycline drug, which is routinely prescribed as a potent chemotherapeutic agent. The amphiphilic nature of the drug molecule due to the presence of both hydrophilic daunosamine sugar moiety and hydrophobic anthracycline backbone with different polar functional groups25 leads to undergo aggregation in different environments similar to other amphiphilic molecules19-21,26 and several other drug molecules.9,10 Several research groups have studied the aggregation behavior of Dox in different media. Loftsson et al.27 have used the permeation method to quantify the degree of aggregation of Dox in water and they found that ~ 40 Dox molecules are self-aggregated together in 1 mM aqueous solution. Again, in the biological and acidic environments, this drug molecule exists in cationic form and thus is observed to undergo anion mediated aggregation in presence of phosphate28, citrate29, sulphate30 etc ions. This self aggregation behavior is likely to be the reason behind its cytotoxicity, multidrug resistance and reducing antitumor activity.28 Moreover, the unique structural formulation of this drug molecule results in its encapsulation in different organized assemblies to modulate their self assembled behavior31, as well as, formation of 2 ACS Paragon Plus Environment

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several interesting aggregated nanostructures with different amphiphilic molecules28. The self assemblies of Dox as described earlier are thought to be “harmful” due to lowering the biofunctionality and cellular internalization; while such intermolecular assemblies with foreign amphiphilic molecules reduce the toxicities of the drug self assemblies and thus revive their biofunctionalities.28 All these above facts suggest that the chemotherapeutic drug, Dox can be successfully employed as a model molecule to study its aggregation behavior. Nowadays, ionic liquids (ILs) have drawn a significant amount of interests due to their environment friendly nature and consequently applications in different chemical and biological arena.32 Actually, the organic salts comprising of sterically mismatched ions with melting point up to 100°C are ILs32,33 whereas, the long alkyl chains containing ILs with inherent amphiphilc character are known as surface active ionic liquids (SAILs). Now, imidazolium based ILs can assist to form a series of supramolecular aggregates with significant structural and morphological alterations34-36 primarily due to the presence of electrostatic, hydrophobic, hydrogen bonding, cation-π and π-π interactions.24,37,38 Apart from these, these imidazolium based ILs can also act as an important media to induce the aggregation behavior of several molecules39 and consequently several interesting molecular assemblies like vesicles40, fibrils21, nanoparticles41, ionogels42 etc are reported to form in presence of ILs. These supramolecular aggregates also show sound applications in different fields like drug delivery, gene delivery, nano carrier, bioimaging, photodynamic therapy etc.32,43 As a result, imidazolium based SAILs can be thought as very important systems to study aggregation behavior of different amphiphilic molecules. Again, the naturally occurring amphiphile, bile salts consist of both hydrophilic carboxylate ion and hydroxyl groups along with hydrophobic planar steroid backbone and thereby play a crucial role in fat digestion and also solubilising a large number of fat soluble compounds.44 The facial amphiphile bile salts are very much different from conventional “head-and tail” amphiphiles and induce morphological transitions of vesicles to several other nanostructures (mainly vesicles to micelles)45. Moreover, the bile salts are reportedly known to interact strongly with cationic drug molecules46 and also with imidazolium ionic liquids.47,48 The bile salts are known to induce morphological transitions of ionic liquid micelles47 and also improve the solubility of several hydrophobic drug molecules49 which increase their applicability in different research fields.

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Therefore, the above discussed scopes of the presence of different types of molecular interactions drive us to study the aggregation behavior of Dox in C8mimCl SAIL and further the effect of a well known bile salt, sodium cholate (NaCh) on the Dox/ C8mimCl aggregates. Different microscopic techniques like fluorescence lifetime imaging microscopy (FLIM), high resolution transmission electron microscopy (HR-TEM), analytical transmission electron microscopy (analytical TEM), field emission scanning electron microscopy (FESEM) and atomic force microscopy (AFM) are used to systematically characterize these different kinds of aggregates formed by the chemotherapeutic drug Dox in SAIL and bile salt. Although, there are some works regarding the aggregation behavior of Dox in different systems27-31, still the present study offers a topic of discussion for better understanding the aggregation mechanism of Dox and to the best of our knowledge, the aggregation of Dox in IL has not been explored yet. The effect of bile salt on Dox/ C8mimCl system further flavoured the work and thus the present work will expectedly open a new scope for the design of a new generation smart drug delivery vehicle where drug itself aggregates to form the delivery system and therefore can be potentially applicable in biological and pharmaceutical research fields. 2. Experimental section 2.1. Materials and Sample Preparation. We purchased the surface active ionic liquids (SAILs), 1-octyl-3-methylimidazolium chloride (C8mimCl),

1-dodecyl-3-methylimidazolium

chloride

(C12mimCl)

and

1-hexadecyl-3-

methylimidazolium chloride (C16mimCl) from Kanto Chemicals (purity is 0.98 mass fraction). Doxorubicin hydrochloride (Dox) and n-octyltrimethylammonium bromide (OTAB) were obtained from TCI Chemicals (India) Pvt. Ltd. The bile salt, sodium cholate (NaCh; purity is ~0.98 mass fraction) was obtained from Sigma Aldrich. All these materials were used as received without any further purification. We used milli-Q water (conductivity is 5.5 µS/m at 25°C) for the preparation of all the solutions. In our present study, firstly we have prepared 0.1 mM Dox in 10 mM C8mimCl solution. For this, methanol of Dox stock (Dox in methanol) was evaporated and subsequently 10 mM C8mimCl solution (neat C8mimCl SAIL in water to prepare 10 mM C8mimCl solution) was added to maintain the final Dox concentration 0.1 mM. Moreover, to check the effect of concentration variation of the two species on the aggregation behavior of Dox in C8mimCl, we have used 0.05, 0.025 mM Dox and 0.5, 1, 5, 20 and 60 mM C8mimCl solutions. These solutions were also prepared considering the same protocol as 4 ACS Paragon Plus Environment

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mentioned above. In the second part, we have observed the effect of three bile salt concentrations- 1mM, 10 mM and 20 mM to Dox/C8mimCl solution in 1:1 (v/v) ratio. These bile salt (NaCh) solutions were prepared by weighing appropriate amount of bile salts and dissolving in milii-Q water. Besides, for mechanistic studies, we have used 10 mM OTAB, 10 mM BmimOs, C12mimCl (0.5, 1, 5 and 10 mM) and C16mimCl (0.5 and 1 mM) solutions and these solutions were also prepared in the same manner. Finally all the solutions were kept at room temperature (25°C) prior to the experiments. The chemical structures of the materials are presented in Scheme 1. Scheme 1. The chemical structures of Dox, C8mimCl, NaCh bile salt and OTAB.

2.2. Instrumentation. The characterization of the aggregates formed by Dox in SAIL and also bile salt induced morphological transition were performed using dynamic light scattering (DLS), fluorescence lifetime imaging microscopy (FLIM), high resolution transmission electron microscopy (HRTEM), analytical transmission electron microscopy (analytical TEM), field emission scanning 5 ACS Paragon Plus Environment

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electron microscopy (FESEM), atomic force microscopy (AFM) and fourier transformed infrared spectroscopy (FTIR) measurements. We used DLS measurement for qualitative measurement of the size of the aggregates formed by Dox in C8mimCl SAIL. The measurement was carried out using Malvern Nano ZS instrument employing a 4 mW He−Ne laser (λ = 632.8 nm) equipped with a thermostated sample chamber. It is noteworthy to mention here that DLS measurement gives accurate results for spherical and near spherical aggregates whereas for nonspherical aggregates, it may produce erroneous results. On the other hand, FLIM is a very powerful tool for molecular level characterization of different large sized molecular aggregates. For this measurement, we used DCS-120 (complete laser scanning confocal FLIM microscope by Becker & Hickl GmbH) system equipped with an inverted optical microscope of Zeiss which is controlled by a galvo-drive unit (Becker &Hickl GDA- 120). For the acquisition of lifetime images, DCS 120 is equipped with a polarizing beam splitter and two avalanche photodiode detectors (ID-Quantique ID100). We did not use any external fluorescent probe because in our case, Dox itself acts as the fluorescent dye to probe the system. The FLIM measurements were carried out using a 20× objective (air immersion objective, NA 1.2). This instrument involves a 488 nm diode laser working at pulse mode (50 mW) to excite the sample solutions and long pass filters (498 nm) to separate the fluorescence signal from the excitation source. All the images shown in the paper were originated with the help of polarized fluorescence transient and this was further obtained by time correlated single photon counting detection electronics (Becker &Hickl SPC-152, PHD-400-N reference diode).50 For capturing the FLIM images, ~10 µL of the sample solution is placed on a clean glass slide and dried properly. High resolution transmission electron microscopy (HR-TEM) measurements were carried out using a JEOL Model JEM-2100 instrument operating at 200 kV. The sample preparation for the HR-TEM measurement involves blotting a drop of the sample solutions on a carbon-coated (50 nm carbon film) Cu grid (300 mesh, Electron Microscopy Science) and allowing to dry overnight prior to the experiments. We have performed analytical transmission electron microscopy (analytical TEM) measurements using FEI. TECNAIG220S-TWIN instrument operating at 120 kV acceleration voltage. The sample preparation for analytical TEM is exactly same as that for HR-TEM measurements.


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For field emission scanning electron microscopy (FESEM) measurements, we drop-casted the sample solution on a glass slide and air-dried overnight at room temperature, so that the solvent is completely evaporated prior to the measurement. Then the sample was gold coated carefully and finally mounted into the Supra 40 (Carl Zeiss Pvt. Ltd.) instrument for recording FESEM images. For recording the atomic force microscopy (AFM) images, we used Agilent 5500 instrument in the tapping mode. Briefly, the images were taken by drop-casting the sample solution on a newly cleaved mica surface and air-dried overnight before the experiment. The fourier transform infrared (FTIR) spectra of the samples presented in the manuscript were recorded in the range of 650–4000 cm-1 with a Nexus 870 FT-IR spectrometer. Each sample was recorded with 32 scans at a spectral resolution of 1.928 cm-1. We performed all the measurements at room temperature (250C). For spectral analysis, we used OMNIC software (version-6.0) of the Thermo Nicolet Corporation. Again, we used the same bandwidth and enhancement for each spectral deconvolution. 3. Result and Discussion: 3.1. Aggregation behavior of Dox in C8mimCl : To study the aggregation behavior of drug molecules in any microheterogeneous system is very much important because it provides an indication about the effectiveness of the drug inside the body system. Generally, the self aggregation of drug molecule leads to a decrease in the internalization and biofunctionality of the drug.28 Dox, the drug of our particular interest, is reported to form fibrillar aggregates in different systems like phosphate buffer28, citrate containing liposomal solutions etc.29 Moreover, in presence of amphiphilic poly (amino acid) derivative, Dox aggregates into some nano-spindles structures.28 These observations influence us to study the aggregation behavior of Dox in C8mimCl SAIL. Here it is important to mention that all the studies are performed using 0.1 mM Dox and 10 mM C8mimCl. From the DLS study, as shown in Figure 1A, it is evident that Dox forms some large aggregates in presence of 10 mM C8mimCl solution. The DLS study also gives information that the size of Dox/ C8mimCl aggregates are in the range of few µm. Since, DLS study gives only very qualitative information about the system, we have used FLIM technique because recently this technique is extensively used to get molecular level information of the system. The FLIM images of 0.1mM Dox in 10 7 ACS Paragon Plus Environment

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mM C8mimCl are shown in Figure 1B (i-ii). The images show that different sized large vesicular aggregates are present in the system. In the FLIM images, we have observed that spherical vesicular aggregates with very broad distribution in size are obtained (Figure 1C). These giant spherical aggregates are formed by Dox only in presence of C8mimCl since no such large spherical aggregates are obtained from the self-aggregation of Dox in water. Again, the concentration of C8mimCl used in the study is well below its critical micelle concentration (CMC) (CMC of C8mimCl is ~100 mM)51 and thus the SAIL is also not forming any such aggregated structure.

Figure 1. (A) DLS intensity profile of 0.1 mM Dox in 10 mM C8mimCl SAIL; (B) FLIM images of the aggregates formed by (i, ii) 0.1 mM Dox in 10 mM C8mimCl SAIL (all the scale bars are 5 µm), the left panel shows the lifetime images and the right panel shows the intensity images; (C) 3D surface view of figure (B,ii) shows the formation of giant spherical aggregates with some


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small sized aggregates also; (D) Lifetime distribution histogram of Dox aggregates in 10 mM C8mimCl obtained from the FLIM image (B, ii). Now, using the lifetime distribution histogram obtained from the FLIM image, we can easily comment on the lifetime of the aggregates formed by Dox in C8mimCl. The lifetime distribution histogram is provided in Figure 1D, where, we have observed that in C8mimCl SAIL, where Dox forms spherical vesicular aggregates, Dox shows a lifetime distribution ranging from ~700-1300 ps with a maximum at ~930 ps. Now, from our previous studies52, we have observed that the lifetime distribution corresponding to aggregated nanostructure lies in the higher lifetime side compared to that with less aggregation or no aggregation. Therefore, we can conclude that, our system i.e. 0.1 mM Dox in 10 mM C8mimCl which lies in significantly high lifetime region in the lifetime distribution histogram, afford a sufficiently rigid and confined environment. Moreover, we have also varied the concentrations of C8mimCl keeping the Dox concentration fixed and vice versa to explore the effect of concentration or molar ratio of each component to the Dox/C8mimCl aggregates. In each case (Table S1 of the supporting information), we have observed that similar spherical aggregates are formed. Therefore, we can qualitatively make a conclusion that, the presence of Dox and C8mimCl SAIL are sufficient for the formation of giant spherical aggregates. We have expressed this argument in terms of well known “AND” logic gate (Scheme 2) and pointed out that Dox/ C8mimCl supramolecular aggregates form only if both Dox and C8mimCl are present in the system.

(A)

(B)

Dox

Input C8mimCl

0 0 1 1

0 1 0 1

Output Dox/ C8mimCl spherical aggregates 0 0 0 1

Scheme 2. (A) The circuit symbol and (B) truth table for AND gate which shows the possibility of Dox/ C8mimCl spherical aggregate formation. The binary digits “0” and “1” in the “Input” 9 ACS Paragon Plus Environment

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column represent the absence and presence of the components in the system respectively, whereas, in the “Output” column “0” represents Dox/ C8mimCl aggregates do not form while “1” means the aggregates form in the system.

To further characterize these aggregates formed by 0.1 mM Dox in 10 mM C8mimCl, we have performed HR-TEM measurement. Figure 2 (a,b) shows the HR-TEM images of the large spherical Dox/C8mimCl aggregates which match well with the vesicular aggregates obtained in the FLIM measurement. The vesicular aggregates obtained from the HR-TEM measurement have an average diameter of ~2 µm and this is also in good agreement with the size obtained from the DLS and FLIM measurements. Although the HR-TEM images are reproduced several times, the aggregated microstructures may be sometimes destroyed partially during measurements. To avoid this problem, in addition to the HR-TEM measurements, we have also performed analytical TEM measurement operating at comparatively very less operating voltage (120 kV). The analytical TEM images of 0.1 mM Dox/10 mM C8mimCl aggregates are shown in the Figure S1 of the supporting information which clearly show that similar spherical aggregates as produced from HR-TEM measurement are obtained. These aggregates are also characterized by using FESEM technique, where, we have again reproduced the result as obtained in TEM and FLIM measurements. In Figure 2c, we have shown the FESEM image of the spherical aggregate as formed by Dox in presence of the SAIL, C8mimCl. Furthermore, we have also employed AFM technique which gives the presence of similar types of vesicular aggregates as obtained from other imaging measurements (i.e. FLIM, TEM and FESEM). The AFM image of Dox/ C8mimCl aggregate is shown in the Figure 2 (d,e) and from the AFM images again, we can tell that larger sized vesicular aggregate are formed which actually supports firmly the results obtained from other instrumental techniques.


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Figure 2. (a,b) HR-TEM images of the aggregates formed by 0.1 mM Dox in 10 mM C8mimCl (scale bars are 5 µm and 1 µm respectively); (c) FESEM image (scale bar is 1 µm) and (d,e) AFM images of the spherical aggregates formed by 0.1 mM Dox in 10 mM C8mimCl. Therefore, all the above mentioned imaging techniques along with DLS confirm that the aggregation of Dox in presence of SAIL, C8mimCl, results large spherical vesicular aggregates.

3.2. Proposed mechanism for the formation of Dox/ C8mimCl aggregates: The different types of well established supramolecular aggregates are formed mostly due to electrostatic, hydrophobic, hydrogen bonding, π-π and cation-π interactions.22-24,53 Molecular dissection of Dox and C8mimCl (Scheme 1) reveals that they can form aggregates due to the presence of all these above mentioned molecular interactions. Our experimental results provide us information about the existence of large spherical aggregates for Dox in C8mimCl SAIL. The aggregation of Dox in anionic buffers like phosphate28, citrate29, sulphate30 etc are well documented and establish the presence of anion mediated aggregation of the cationic drug


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molecules. Briefly, the anionic components of the buffer solutions act as a bridge to interconnect the neighbouring Dox molecules, where, they can easily stack through π-π interaction.28 Again, Li et al.29 have reported that in citrate containing liposomes, Dox molecules are stacked to form fibril like self aggregates which can be explained using hexagonal lattice model. Based on these explanations, we are interested to unlock the molecular mechanism for the formation of Dox/ C8mimCl spherical aggregates. In the later sections, we have elaborately discussed the role of different molecular interactions and try to make a logistic argument about the main driving force behind such aggregation. 3.2.1. Electrostatic force of attraction: Now, it is well known that the amphiphilic Dox molecule has two distinct pKa values. The first pKa value where the –NH3+ of the cationic Dox molecule is deprotonated to form neutral Dox molecule is ~8.2 whereas, the second pKa in which one –OH group of the anthracycline moiety of neutral Dox molecule gets deprotonated is ~9.5.27

Scheme 3. Dissociation equilibrium of Dox in aqueous solution. The two pKa s- pKa(1) and pKa(2) correspond to the dissociation of –NH3+ and –OH groups of Dox respectively. The dissociation equation of Dox in water and the respective pKa values are clearly depicted in the scheme 3. Now, we have measured the pH of the aqueous solution of 0.1 mM Dox, which is found to be ~6.10 and it decreases rapidly to ~3.98 upon the addition of 10 mM C8mimCl solution. The pH values indicate that in both bulk water and 10 mM C8mimCl solution, Dox 12 ACS Paragon Plus Environment

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present in cationic state (i.e. Dox-NH3+) and this restricts the electrostatic force of attraction between the cationic Dox and cationic C8mimCl species. This qualitative argument can be justified by providing a microscopic confirmation, where, we have replaced the SAIL, C8mimCl by another anionic SAIL, BmimOs (1-butyl-3-methylimidazolium octyl sulfate, CMC= 31−37 mM in water) so that, the concentrations of both Dox and BmimOs remain the same (i.e. 0.1 mM Dox and 10 mM BmimOs). From the FLIM image (in Figure S2), we have observed that Dox does not form similar kind of large vesicular aggregates as formed in presence of C8mimCl. On the structural aspect, C8mimCl has cationic methylimidazolium unit with eight carbon containing hydrophobic alkyl chain whereas, in BmimOs, the anion counterpart contains similar eight carbon containing alkyl chain with anionic sulphate head group. In spite of having the same alkyl branching in both the SAILs, Dox forms well defined vesicular aggregates only in presence of C8mimCl which conclude that electrostatic force of attraction is not playing the driving role in controlling the aggregation behavior of Dox in presence of SAIL. 3.2.2. Hydrophobic interaction: The hydrophobic interaction is very much important in forming supramolecular aggregates.23 To study the role of hydrophobic interaction in the formation of Dox/ C8mimCl vesicular aggregates; we have taken two other SAILs with different alkyl chain lengths- C12mimCl and C16mimCl. These SAILs have the same methylimidazolium head group with varying length of hydrophobic alkyl chains (number of carbon atoms in the alkyl chain is twelve in case of C12mimCl and sixteen for C16mimCl). The CMC of C12mimCl and C16mimCl are ~12.5 mM and ~1 mM respectively. Thus, for Dox/ C12mimCl aggregation study, we have used 0.1 mM Dox and varied the concentration of C12mimCl as 0.5, 1, 5 and 10 mM. Similarly, in Dox/ C16mimCl pair, we have taken 0.5 and 1 mM C16mimCl with 0.1 mM Dox solution. In all the cases (Figure S3 a-f), we have found no vesicular aggregate as obtained in case of Dox/ C8mimCl pair. Moreover, in all cases, we get some random morphologies which do not have any ordered structure. Again, in BmimOs, the cationic counterpart is similar to other SAILs (i.e. C8mimCl, C12mimCl and C16mimCl) only with difference in the length of the alkyl chain (i.e. in BmimOs, alkyl chain contains four carbons). But, we have mentioned previously (Figure S2) that in presence of BmimOs, Dox only forms some random small aggregates. Actually, for very short hydrophobic chain (considering the alkyl chain attaching with the methylimidazolium ring) 13 ACS Paragon Plus Environment

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containing BmimOs as well as very long hydrophobic chain containing C12mimCl and C16mimCl SAILs, there is a mismatch in hydrophobic interactions with Dox while such hydrophobic interactions match exactly between Dox and C8mimCl. As a consequence, effective molecular packing occurs which leads to large spherical aggregates formation and thus in our present study, the impact of hydrophobic alkyl chain length of the SAIL is very much important. 3.2.3. π-π and cation-π interactions: The π-π and cation-π interactions play a key role in the formation of different supramolecular aggregates. In our system, both the participating components i.e. Dox and C8mimCl have such structural features that they are quite expected to stack under π-π and cation-π interactions. Recently, imidazolium based ILs are found to be a good candidate for π-π and cation-π interactions24 and thus for our system also, we are very much keen to know about the role of such π-π and cation-π interactions in spherical aggregate formation. Therefore, we have taken another surfactant, n-octyltrimethylammonium bromide (OTAB), where, the imidazolium head group of C8mimCl is replaced by trimethylammonium (–NMe3+) group. The FLIM image of the aggregation of 0.1 mM Dox in 10 mM OTAB is shown in Figure S4, where, it is observed that some very small aggregates are formed. But these aggregates differ significantly from the Dox/ C8mimCl large spherical vesicular aggregates. This observation can be explained as, the presence of same hydrophobic C8 alkyl chain in OTAB as present in C8mimCl, exerts similar hydrophobic interaction which plays some role in the formation of such small aggregates; whereas, the presence of imidazolium head group in C8mimCl helps to form large vesicular aggregates. Actually, it is reported that, due to the lower electron density and effective π-π stacking ability of the imidazolium head groups; imidazolium based ILs can easily and favourably form aggregates compared to conventional alkyltrialkylammonium halide surfactants.35 Therefore, the presence of imidazolium head group of C8mimCl is very much important with respect to its ability to undergo π-π and cation-π interactions with Dox which results large spherical vesicular aggregates. Again, to get better insights into the role of this π-π and cation-π interactions, we have also varied the pH of 0.1 mM Dox in 10 mM C8mimCl solution. Since, Dox molecule has two pKa values-one is ~8.2 (above which –NH3+ of Dox gets deprotonated to form Dox-NH2) and another is ~9.5 (above this pKa, one -OH group of Dox gets deprotonated)27, Dox/ C8mimCl vesicular 14 ACS Paragon Plus Environment

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system is very much expected to get effected by changing the pH of the solution. In our present Dox/ C8mimCl vesicular system, the measured pH of the solution is ~3.98. We have varied the pH of the solution to attain two pH values- 7.1 and 8.9 and shown the results as obtained in Figure S5 of the supporting information. Interestingly, at pH value 7.1, the vesicular morphology of Dox/ C8mimCl system does not get affected anymore; whereas, at pH 8.9, we have observed that the Dox/ C8mimCl vesicular aggregates are getting disrupted. This interesting phenomena can be interpreted according to the general dissociation equilibrium reaction of Dox (as shown in scheme 3) in aqueous solution.27 At pH 8.9, which is above the first pKa of Dox molecule, the – NH3+ of Dox molecule gets dissociated to generate neutral –NH2 functionality. Now, as at this pH value, Dox/ C8mimCl vesicular aggregates get disrupted, we can conclude that the primary interaction between –NH3+ of Dox molecule and imidazolium based SAIL is diminished due to the dissociation of –NH3+ of Dox molecule to –NH2 which gives a very strong confirmation in favour of the cation-π interaction between the participating components. 3.2.4. Hydrogen bonding interaction: Hydrogen bonding interaction is also another important driving force behind the formation of stable supramolecular aggregates.23 The protons of imidazolium ring are highly acidic and thus imidazolium based ILs are very much prone to hydrogen bond formation.54,55This is also manifested from the hydrogen bonding interaction of histidin residues in the active site of serine hydrolases.56 In the previous section, we have discussed that replacement of imidazolium based SAIL, C8mimCl by conventional surfactant, OTAB diminishes the π-π and cation-π interactions which leads to inefficient packing between Dox and OTAB and thus very small aggregates are formed. Now, apart from these π-π and cation-π interactions, such replacement of C8mimCl by OTAB also loss the presence of highly acidic imidazolium protons and this fact also plays a crucial role in weakening the interaction between Dox and OTAB. On the other hand, strong and effective hydrogen bonding interactions are expected between Dox and C8mimCl, since both of them have large number of hydrogen bond donor and acceptor groups.25,55 Recently, Singh et al.57 have reported that hydrogen bonding interactions present among the carboxyl or amino groups of different amino acids of bovine serum albumin (BSA) and imidazolium protons result significant structural changes to the protein. Again, to get better insight into the role of hydrogen bonding interactions in Dox/ C8mimCl vesicular aggregates formation, we have employed FTIR 15 ACS Paragon Plus Environment

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spectroscopy as a key tool. The FTIR spectra of 0.1 mM Dox in water, 10 mM C8mimCl and 0.1 mM Dox in 10 mM C8mimCl solutions are shown in the Figure S6 of the supporting information. The stretching vibrational band appeared at ~ 3447 cm-1 corresponds to

O-H

-1

groups of Dox, whereas, for C8mimCl, the C=N stretching band appears at ~ 1569 cm . Now, both of these bands, as observed in the Figure S6, are shifted to ~3457 and 1572 cm-1 respectively upon Dox/ C8mimCl aggregates formation which indicates that hydrogen bonding interactions may be present among the O-H groups of Dox and C=N groups of C8mimCl. Again, the N-H stretching vibrational frequency, which appears at ~1634 cm-1 for Dox, slightly blue shifted to ~1630 cm-1 in Dox/ C8mimCl aggregates and thus supports the existence of hydrogen bonding interactions. Besides, several theoretical studies54 on the hydrogen bonding ability of imidazolium ILs reveal that for chloride ion containing imidazolium ILs, strong hydrogen bonding between imidazolium protons and chloride ion may also operate which can assist the aggregation behavior of several molecules in presence of such ILs. In our system also, such hydrogen bonding interactions among O-H groups of Dox and chloride ion of C8mimCl may be operative. However, for C8mimCl, the CH2 symmetric (νsym) and antisymmetric (νasym) stretching vibrations appear at ~2856 and 2926 cm-1 respectively which informs that the alkyl chains of C8mimCl are in the gauche conformation.58,59 But after the formation of Dox/ C8mimCl vesicular aggregates, these stretching frequencies do not get affected (Figure S6) which indicate that after the aggregate formation also, CH2 units do not face any conformational change and such observation is also supported from our previous study.59 Therefore, based on the above discussions, we can conclude that in spite of the presence of hydrophobic, π-π and cation-π interactions; hydrogen bonding interactions also play an important role in the formation of Dox/ C8mimCl vesicular aggregates. 3.3. Effect of bile salt on the Dox/ C8mimCl spherical vesicular aggregates. Bile salts are biologically very important facial amphiphilic molecules and potentially useful in disruption and solubilisation of vesicular aggregates.60 These molecules are also reported to bind effectively with several important biomolecules.61,62 Recently, Le Devédec et al.11 have shown that PEGylated bile salts can be effectively used for loading and solubilising hydrophobic drug, itraconazole. Again, Jiang and co-workers45 have established that sodium cholate (NaCh) and sodium

deoxycholate

(NaDC)

bile

salts

induce

vesicle


to

micelle

transition

in

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dodecyltriethylammonium bromide/sodium dodecyl sulfate vesicle system. Owing to these facts, we have added the bile salt sodium cholate (NaCh) to the Dox/C8mimCl solution maintaining 1:1 (v/v) ratio. To explore the effects of 1 mM, 10 mM and 20 mM NaCh on the Dox/ C8mimCl vesicular system, we have performed a time dependent study using FLIM technique. For 1 mM NaCh (Figure S7 (a)), there is no change in the morphology of Dox/ C8mimCl vesicular system even after 30 hour of incubation. A mixture of fibril and vesicle like aggregates are observed in presence of 10 mM NaCh, which is completely transformed into fibrillar morphology after ~12 hour as shown in Figure S7 (b-f) of the supporting information. Now, when we have added 20 mM bile salt, we have observed a complete transformation of vesicular to fibrillar structure at ~4 hour (Figure 3 a-d). Moreover, we have monitored this effect of 20 mM bile salt addition up to ~30 hour, where, we have clearly observed that the compactness of the fibrillar aggregates increase with the progress in the incubation time. The vesicular to fibrillar aggregate transition of Dox/ C8mimCl system in presence of highest bile salt concentration (i.e. 20 mM) is also supported by the HR-TEM and FESEM images which are shown in Figure 3 (e),(f) respectively.


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Figure 3. FLIM images of 0.1 mM Dox/ 10 mM C8mimCl/ 20 mM NaCh aggregates at (a) t=0 hr (b) t=2 hr, (c) t=4 hr and (d) t=10 hr (all the scale bars are in 5 µm); (e) HR-TEM image of 0.1 mM Dox/ 10 mM C8mimCl/ 20 mM NaCh aggregate (scale bar is in 5 µm); (f) FESEM image of 0.1 mM Dox/ 10 mM C8mimCl/ 20 mM NaCh aggregate (scale bar is in 10 µm) and Lifetime distribution histograms of (g) 0.1 mM Dox in 10 mM C8mimCl vesicular and (h) fibrillar aggregates formed by 0.1 mM Dox in 10 mM C8mimCl and 20 mM NaCh at t=10 hr. The fibrillar morphology, we obtained in these electron microscopic techniques matches well with that obtained in the FLIM technique. Similar as done in case of Dox/C8mimCl aggregates, we have also performed analytical TEM measurement for this Dox/C8mimCl/NaCh aggregates and shown the result as obtained in Figure S8 of the supporting information which also reproduce the HR-TEM result and thus support the presence of rod like fibrillar morphology as obtained in other microscopic techniques. To further corroborate, we have also taken the AFM images of the aggregated network obtained from the addition of bile salt into Dox/ C8mimCl spherical vesicular assembly. The AFM images, as shown in the Figure S9 of the supporting information, show similar types of fibrillar signature as shown in the other mentioned imaging techniques. Actually due to the facial amphiphilic architecture, bile salts impart strong steric interactions to force the headgroups of conventional amphiphiles apart from each other and as a result, the average headgroup area of the amphiphile increases which ultimately leads vesicle to other microstructure transition (the most common being vesicle to micelle transition).45 The lifetime distribution histograms (Figure 3g and 3h) of Dox/C8mimCl vesicular and Dox/C8mimCl/NaCh fibrillar systems as obtained from the FLIM images are discussed latter. Now, in our particular system, to find out the origin of this bile salt induced vesicular to fibrillar aggregate transition, we have performed some control experiments. For that, we have added 20 mM NaCh bile salt separately to 10 mM C8mimCl SAIL and 0.1 mM Dox at 1:1 (v/v) ratio. The FLIM (Figure 4 (a-c)) as well as HR-TEM (Figure 4 (d-f)) images clearly show that similar types of long thread like fibrillar morphologies, as obtained in case of Dox/ C8mimCl/ NaCh are observed.


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Figure 4. FLIM images of the aggregates of 10 mM C8mimCl in presence of 20 mM NaCh at (a) t = 2 hr, (b) t = 4 hr, (c) t = 10 hr (all the scale bars are 5 µm) (here we have used the external dye, 4-(Dicyanomethylene)-2-methyl-6-(4-dimethylaminostyryl)-4H-pyran (DCM) to capture the FLIM images), HR-TEM images of (d,e) 10 mM C8mimCl/ 20 mM NaCh (scale bars are 10 µm and 5 µm respectively) and (f) 0.1 mM Dox/ 20 mM NaCh fibrillar aggregates (scale bar is 5 µm). The small aggregated nanostructures as obtained in Dox/NaCh system are characterized using HR-TEM measurement (Figure 4 (f)), where, we have observed small spindle like nanofibrillar Dox/ NaCh aggregates are formed. Similar types of spindle like morphologies are also reported for Dox in presence of cholate grafted poly-L-lysine.28 Actually, due to the electrostatic force of attraction, the aggregation between oppositely charged Dox and NaCh are quite expected. Several research groups revealed that effective complexation between bile salts and cationic drug molecules take place which are primarily due to the electrostatic interactions among the participating components.46 Nevertheless, some significant contributions of hydrophobic and hydrogen bonding interactions are also thought to be another reason behind this aggregation.28 Again, the interactions between bile salts and ionic liquids are also very well documented.47,48 Bile salts are facial amphiphiles having hydrophilic carboxylate ion and hydroxyl groups and hydrophobic planar steroid moieties, whereas, the ILs are mainly imidazolium ring containing cationic species. Thus bile salts aggregate very strongly with ILs via electrostatic and 19 ACS Paragon Plus Environment

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hydrophobic interactions47,48 which may alter the micellar behavior of the IL. In our present system, as we have discussed earlier, NaCh bile salt forms rod like fibrillar microstructures with C8mimCl SAIL and this crucial step mainly modulate the bile salt induced transition of Dox/ C8mimCl vesicular to fibrillar aggregates. Very recently, Pillai et al.47 have used 1H NMR technique to establish the direct interaction between NaCh bile salt and imidazolium head group of C10mimCl SAIL, while, small upfield shifts in alkyl chain protons of SAIL propose some additional interactions between NaCh and SAIL alkyl chain are also present. Thus, in summary, C8mimCl-NaCh as well as, Dox-NaCh aggregation collectively pump up the transition from spherical vesicular to rod like fibrillar morphology and thus we have successfully performed the disruption of Dox/ C8mimCl spherical aggregates in presence of NaCh bile salt. Finally, from the FLIM images, we can get the lifetime distribution histograms which can be used to support the structural transition from Dox/ C8mimCl vesicular aggregates to Dox/ C8mimCl/ NaCh fibrillar aggregates. Actually the fluorescence lifetime of a fluorophore is highly sensitive towards the surrounding microenvironment of the fluorophore and therefore, any change in the rigidity or confinedness of the environment results in significant change in the lifetime distribution histogram of the fluorophore. The vesicular aggregates of 0.1 mM Dox in 10 mM C8mimCl, as observed in Figure 3 g, shows a lifetime distribution from ~ 700 ps to 1300 ps with the peak maximum at ~ 930 ps. On the other hand, in presence of NaCh bile salt, a transition from spherical vesicular to rod like fibrillar morphology occurs, where, the lifetime distribution histogram (Figure 3h) slightly shifts towards the higher lifetime side, ranging between ~600 ps and 1800 ps having the peak maximum at ~ 1150 ps. Now, we have discussed earlier that the lifetime distribution histogram in the higher lifetime side indicates the presence of more rigid and compact microenvironment surrounding the fluorophore compared to that present in the lower lifetime region. Although, the distribution histograms of Dox containing vesicular and fibrillar aggregates are not much separated, we can comment that the fibrillar aggregates provide slightly more rigidity and compactness around Dox since the lifetime histogram of Dox/C8mimCl/NaCh fibrillar aggregates shift at little bit higher lifetime side compared to the same corresponding to the Dox/C8mimCl vesicular aggregates. This may be due to the higher packing efficiency among the Dox containing fibrillar aggregates forming components than spherical vesicular aggregates forming components. Therefore, based on this lifetime distribution


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analysis also, we can successfully demonstrate the bile salt induced spherical vesicular aggregate to rod like fibrillar aggregate transition. 4. Conclusion. In conclusion, we have summarized the aggregation behavior of a well known anticancer drug, Dox in a common SAIL, C8mimCl. We have systematically characterized the aggregates using DLS and various imaging techniques (FLIM, HR-TEM, analytical TEM, FESEM and AFM) and found that large vesicular aggregates are formed by Dox in C8mimCl. To explore the mechanism behind the Dox/ C8mimCl spherical vesicular aggregates formation, we have carried out some control experiments and found that cation-π and π-π interactions along with hydrophobic and hydrogen bonding interactions play the key role behind the aggregates formation. Moreover, we have added an important bile salt, NaCh to Dox/ C8mimCl vesicular system and observed that the bile salt induces morphological transition from spherical vesicular aggregates to rod like fibrillar aggregates. We have also explored that this bile salt forms similar fibrillar aggregates with the SAIL, C8mimCl and small spindle like aggregates with Dox. Therefore, this Dox containing spherical vesicular aggregate formation and its disruption into fibrillar aggregate gives us choice to formulate a new generation drug delivery system based on the aggregation behavior of the drug itself and thus may be very much helpful in pharmaceutical and medicinal research field. Acknowledgment: N.S. gratefully acknowledges SERB and Department of Science and Technology (DST), Government of India for providing generous research grants. A.P, S.K and P.B are thankful to CSIR for their research fellowships. Supporting Information: Supporting FLIM images, TEM and AFM images and FTIR spectra are shown in the supporting information. References. 1. Stupp, S.I.; Palmer, L.C. Supramolecular Chemistry and Self-Assembly in Organic Materials Design. Chem. Mater. 2014, 26, 507−518.


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