Deciphering the Aggregation Behaviour of CHAPS, a Steroidal Zwitterionic Surfactant, and its Interaction with Biologically Important Molecules and Macromolecules: A Spectroscopic Approach

Abstract

Steroidal biosurfactants, particularly bile salts have been the centre of attraction among the researchers due to their peculiar structure, aggregation pattern and a wide range of applications. The unconventional amphiphilic nature of bile salts is responsible for their surface-active property and thus responsible for the solubilization and absorption of cholesterol, lipids, dietary fats, and hydrophobic drugs. Bile salts have also been largely investigated as building blocks for the construction of supramolecular aggregates for drug delivery purposes. Moreover, the applications of biosurfactants in the field of proteomics have increased exponentially in recent years. Though bile salts provide alternatives to conventional surfactants by avoiding undesirable aggregations in proteins, yet their anionic nature can alter the native charge properties of proteins. In this regard, looking at the advantages of zwitterionic surfactants over the charged ones due to their mildness and neutral character, CHAPS (3-[(3-cholamidopropyl)-dimethylamino]-1-propanesulfonate), the zwitterionic derivative of cholic acid was conceptualized. CHAPS was originally synthesized in the 1980s, for the purification of membrane proteins considering its nondenaturing and disaggregating properties. Owing to its unique structure having a rigid steroid and a long zwitterionic tail, the aggregation behaviour of CHAPS cannot be explained through conventional micellization models. Hence, the micellization process of CHAPS has garnered ample curiosity; however, compared to bile salts, surface chemical research on CHAPS is only limited and it still remains an open question to be addressed. The extensive use of CHAPS in biochemical research necessitates a proper understanding of its aggregation behaviour at a molecular level. CHAPS being an electrically neutral surfactant over a large pH range can offer a better alternative to the bile salt-based delivery systems. Further, the successful applications of CHAPS as a drug carrier requires a comprehensive understanding on its interaction with the carrier proteins like serum albumins. With this motivation, the objective of the present thesis is to investigate the aggregation behaviour of CHAPS and to understand its interaction with certain biologically important small molecules and macromolecules. Chapter 1 introduces different types of conventional surfactants, their micellization pattern and applications. It highlights the importance of biosurfactants particularly the bile salts, the unique class of steroidal surfactants; their structure, amphiphilicity, aggregation patterns and applications have also been discussed. Owing to the widespread applications of bile salts, various derivatives of the bile salt variants with enhanced functionality have been synthesized, which have also been discussed. The present chapter also introduces CHAPS, the zwitterionic derivative of cholic acid. The surge in the application of zwitterionic moieties in the field of proteomics and pharmaceutics necessitates the understanding of the aggregation behaviour of CHAPS. Due to its biodegradable, biocompatible nature, and mild character, it can be a better alternative to the bile salts in the field of drug delivery. Hence an in-depth knowledge is required regarding the aggregation behaviour of this unique surfactant and its interaction with drugs and biological macromolecules like proteins. The present chapter also provides a summary of the literature regarding the aggregation behaviour and applications of CHAPS. Keeping the knowledge gap in mind the objectives of the present thesis have been articulated. Chapter 2 offers the information on the materials used and the methodologies employed for the studies. Chapter 3 deals with the understanding of the aggregation of CHAPS in aqueous solution using the modulation in the photophysical properties of Coumarin 1 (C1) and Coumarin 466 (C466). These two 7-aminocoumarins offer a large scope for research in both biological and chemical sciences due to the efficient intra-molecular charge transfer (ICT) process upon photoexcitation, which makes them highly sensitive to the local environment in terms of the polarity, viscosity, pH, and hydrogen bonding. They offer various fluorescence parameters such as emission intensity (quantum yield), energy, anisotropy, and lifetime to get insight into the aggregation behaviour and properties of surfactants. The present work contributes significantly to the general understanding of the microenvironment provided by the surfactant aggregates to the guest molecules. This chapter comprises of two parts: Part I focuses on the investigation of the aggregation behaviour of CHAPS under different physiological conditions by employing C1 as a fluorescent molecular reporter. Modulation in the photophysics of C1 has been utilized for making structural comparisons and in determining local structure variation of this unconventional surfactant and the cholates, its structural bile salt analogues. CHAPS exhibits a relatively speedier self-assembly and faster growth with slightly critical and cooperative micellization unlike the cholates, which is known to aggregate in a non-critical self-association manner i.e., the intermolecular interactions are less cooperative and occur in a progressive fashion over a
wide concentration range. The effect of salt concentration and temperature on the self-assembly process and micellar properties of CHAPS has been investigated. The effect of sodium cholate (NaC) and sodium taurocholate (NaTC) as co-surfactants has also been monitored to understand the formation of mixed micelles. The various photophysical parameters reveal that CHAPS micelles offer a relatively more hydrophobic, compact, and non-polar microenvironment to C1 as compared to the cholates, indicating efficient packing of CHAPS molecules in the micelles. Part II deals with the studies on the effect of CHAPS and different bile salts namely sodium deoxycholate (NaDC), sodium taurodeoxycholate (NaTDC), NaC, and NaTC, on the photophysical behaviour of C466 to understand the role of surfactants’ structure on the aggregation behaviour and micellar properties. This part also involves a comparative study on the aggregation behaviour and properties of these structurally different steroidal surfactants using C466 as a fluorescent molecular reporter. The photophysical properties of C466 have been found to be very sensitive towards the aggregation of CHAPS and the bile salts variants and their self-association has been reflected in the different fluorescence parameters. Though CHAPS shares maximum homology with the cholates, yet its aggregation behaviour is more inclined towards that of the deoxycholates and follows Small’s model of aggregation with three distinct stages: (i) pre-micellar range, (ii) micellization range, and (iii) stable primary micelles. The micellar properties such as hydrophobicity and rigidity of CHAPS aggregates are almost comparable to that of the deoxycholates and are significantly higher than the cholates. CHAPS has also been observed to form relatively larger micelles as compared to the bile salts. This chapter suggests a superior nature of CHAPS aggregates and better shielding of guest molecules inside the aggregates due to which, CHAPS based colloids could be promising candidates as potential drug delivery systems. Chapter 4 evaluates and compares CHAPS and the four bile salts namely NaDC, NaTDC, NaC, and NaTC, as potential drug carriers for curcumin under different physiological conditions to address the two major challenges that limit the practical applications of curcumin i.e., its poor aqueous solubility and lack of stability leading to low bioavailability. Various fluorescence parameters such as emission intensity, emission energy, fluorescence anisotropy, quantum yield, and fluorescence lifetime of curcumin are compared in the presence of these steroidal surfactants to get a comprehensive idea about the microenvironment of curcumin in these aggregates. The extent of increase in the fluorescence parameters of curcumin has been observed to be considerably higher in the presence of CHAPS and the two conjugated bile salts NaTDC and NaTC, in comparison to that of the unconjugated bile salts i.e., NaDC and NaC. The presence of the basic carboxylate end group in the side chain of NaDC and NaC introduces an additional non-radiative decay pathway due to the possibility of excited-state proton transfer (ESPT) between curcumin and the carboxylate end group. In this chapter, the concentration of maximum solubilized curcumin inside the aggregates of CHAPS and the bile salts has also been estimated, and CHAPS is found to solubilize the maximum amount of curcumin as compared to the bile salts. The effect of physiological conditions such as pH, ionic strength, and temperature on the stability of curcumin in these biosurfactants has been studied and among these steroidal surfactants, CHAPS has been found to offer better stability to curcumin under all conditions. The biological activity of curcumin loaded micelles has also been evaluated in the presence of these surfactants. The present study reveals that CHAPS based colloids are promising candidates as potential delivery systems for biomedical applications. Chapter 5 deals with the effect of CHAPS on the structure and function of the two widely studied serum albumins BSA (Bovine serum albumin) and HSA (Human serum albumin). The two proteins, BSA and HSA with two and one tryptophan residues respectively, provide an opportunity for an interesting comparison of tryptophan fluorescence behaviour on interaction with CHAPS. To understand the effect of CHAPS on protein conformation, different spectroscopic techniques have been utilized along with molecular docking. From the fluorescence studies, a significant difference has been observed in the fluorescence parameters of the two homologous proteins in the presence of CHAPS. The different fluorescence parameters of BSA exhibit considerably larger changes than those of HSA, which can probably be due to the involvement of the additional tryptophan in the subdomain IB i.e., tryptophan 134 of BSA in interaction process. From the binding analysis, a sequential interaction between CHAPS and BSA occurring in three different stages depending on the concentrations of CHAPS has been identified. Stage I: up to 1 mM concentration, CHAPS binds at the highly specific and energetic sites of the serum protein; Stage II: In the concentration range of 18 mM, CHAPS interacts cooperatively with BSA; Stage III: in the concentration range of 832 mM of CHAPS, a plateau in tryptophan fluorescence is observed, which indicates that the protein is either almost saturated with CHAPS and further binding may not be consequential or the binding of CHAPS happens at the low-affinity sites in such a manner that the microenvironment of tryptophan residues remains unaffected. Moreover, in this concentration range, self-aggregation of CHAPS has already commenced and there is possible adsorption of CHAPS micelles on the surface of BSA. Unlike BSA-CHAPS system, a bimodal variation is observed in different fluorescence parameters of HSA in the studied concentration range of CHAPS and the changes are significantly smaller in comparison to that of BSA. Studies are also carried out in the presence of two well-known site markers, Warfarin, and Ibuprofen, to perceive the effect of CHAPS on the two principal drug binding sites. The effect of CHAPS on the secondary structure of the proteins has also been studied from the far-UV CD profiles of the two serum albumins. A comparison has been carried out between CHAPS and its bile salt analogues NaC and NaTC in terms of their interaction with the two serum albumins in order to understand the role of the surfactants’ structure on the interaction process. The present study reveals the mildness of CHAPS towards these drug-binding proteins in comparison to its bile salt analogues, which makes CHAPS a better alternative to the bile salts as a drug delivery. Chapter 6 provides a summary of the important findings in the present work and also discusses the future scope.