Sequestration of Drugs from Biomolecular and Biomimicking Environments: Spectroscopic and Calorimetric Studies

: The binding of drugs to nucleic acids, proteins, lipids, amino acids, and other biological receptors is necessary for the transportation of drugs. However, various side effects may also originate if the bound drug molecules are not dissociated from the carrier, especially with the aid of non-toxic agents. The sequestration of small drug molecules bound to biomolecules is thus central to counter issues related to drug overdose and drug detoxiﬁcation. In this article, we aim to present several methods used for the dissociation of small drug molecules bound to different biological and biomimicking assemblies under in vitro experimental conditions. To this effect, the application of various molecular assemblies, like micelles, mixed micelles, molecular containers, like β -cyclodextrin, cucurbit[7]uril hydrate, etc., has been discussed. Herein, we also try to shed light on the driving forces underlying such sequestration processes through spectroscopic and calorimetric techniques.


Introduction
The study of the interaction between small molecules and biomolecules, like proteins, DNA, RNA, lipids membranes, etc. forms a key area of research in which a comprehensive understanding of such interactions lays the foundation for the requisite understanding toward the development of new drugs to counter various lethal diseases [1][2][3]. Various research groups have, over the years, investigated the interactions of small molecules with proteins, DNA, RNA, etc. by using experimental techniques based on spectroscopy, calorimetry, polarimetry, and so on [4][5][6][7][8][9][10]. In recent times, a significant body of computational or theoretical research endeavors has also been dedicated toward a better understanding of the interaction phenomena [11][12][13][14][15][16][17]. The increasing growth of attention, within the scientific community, to this field of research is not surprising because the site-specific targeted delivery of drug molecules and their excretion from the human body via a benign mechanism pose very crucial research questions toward the successful implementation of the therapeutic actions of a drug. The issue related to the excretion of drugs becomes even more pertinent with the view of the possibility of overdose, which may in turn lead to detrimental effects on various biomolecular assemblies within the human body [18][19][20]. In this context, it may be noted that in the last few years, the evolution in the field of nanomedicine has promised developments of smart systems aiming at in vivo functionality. However, it is important to realize that a large part of the promise developed in this area still relies on rational reasoning rather than concrete experimental evidence, thereby inviting further research to this effect [21,22]. Under real in vivo conditions, nanocarriers or nano-assemblies should first arrive at the site of interest, such as target organs/tissues, and then have the possibility of sequestering small drug molecules bound to biomolecules. However, nanocarriers or nano-assemblies themselves will always encounter issues related to nano-bio interactions. Therefore, the targeting ability becomes critical for these kinds

Sequestration of Small Molecules from DNA Using Micelles
In this section, we will discuss the mechanism of the sequestration of Harmane (HM) bound to herring sperm DNA, using the cationic surfactant CTAB [29]. Herein, the detergent molecules conventionally act as the hydrophobic sink for the dissociated drugs. Figure 1 depicts that in the presence of the cationic surfactant CTAB, the fluorescence spectral characteristics of DNA-bound HM are qualitatively reversed in comparison to those observed during the binding interaction of the drug with DNA, along with an increase in an additional fluorescence band at~375 nm [29] attributed to the neutral species of HM [49][50][51][52][53][54][55][56]. The modulation of the fluorescence intensity of HM (the cationic species) with added CTAB concentrations is shown in the inset of Figure 1. These results reflect the deintercalation of the bound drug from the HM-DNA complex in the presence of the surfactant. As a control experiment, the interaction of HM with the surfactant alone has also been performed with the result that the inception of saturation of the interaction of CTAB with the HM-DNA complex is observed at [CTAB]~0.6 mM, a considerably small concentration of the surfactant at which there is essentially no interaction of HM with the surfactant alone [29].
The CTAB-induced modulation of the fluorescence profile of the HM-DNA complex is thus reasonably assigned to the dissociation of the bound drug, while the development of the fluorescence band corresponding to the neutral species of HM may be due to enhanced hydrophobicity of the medium due to the presence of the surfactant. In this context, it is pertinent to state that similar observations were not found with an anionic surfactant, namely sodium dodecyl sulfate (SDS) [29]. This is probably due to the lack of an interaction of SDS with DNA owing to the electrostatic repulsion between the negatively charged polyphosphate DNA backbone and the anionic surfactant [26,27,[57][58][59][60][61][62][63]. Similarly, with a neutral surfactant (Triton X-100), no noticeable sequestration of the DNA-bound drug molecules could be observed [29]. These observations collectively lead to the conclusion that a stabilizing electrostatic interaction between the cationic surfactant CTAB and the negatively charged polyphosphate backbone of DNA probably plays an important role underlying the observed interactions leading to the sequestration of the bound drug molecules from the DNA duplex. It is relevant to state in this context that in the range of CTAB concentrations employed in the present study, the native conformation (B-form) of DNA remains practically unperturbed, as is characterized by the circular dichroic (CD) profile of DNA with added CTAB, which resembles the CD profile of the B-DNA [29]. modulation of the fluorescence intensity of HM (the cationic species) with added CTAB concentrations is shown in the inset of Figure 1. These results reflect the deintercalation of the bound drug from the HM-DNA complex in the presence of the surfactant. As a control experiment, the interaction of HM with the surfactant alone has also been performed with the result that the inception of saturation of the interaction of CTAB with the HM-DNA complex is observed at [CTAB] ~0.6 mM, a considerably small concentration of the surfactant at which there is essentially no interaction of HM with the surfactant alone [29].  In the study of the interaction of drug molecules with biological receptors, the kinetics of the association and dissociation of the drug is considered to have crucial diagnostic significance [26,27,[58][59][60]. A slow rate of deintercalation from the DNA-bound state has been widely argued in the literature as an important property toward efficient therapeutic functionality [26,27]. The fluorescence decay profile of HM in the presence of DNA (at λ~435 nm) represents a characteristic rate constant for the association process, as k a (±5%) = 0.0375 s −1 (Figure 2a), while the dissociation rate constant with added CTAB is found to be k d (±5%) = 0.0098 s −1 (Figure 2b) [29]. The CTAB-induced modulation of the fluorescence profile of the HM-DNA complex is thus reasonably assigned to the dissociation of the bound drug, while the development of the fluorescence band corresponding to the neutral species of HM may be due to enhanced hydrophobicity of the medium due to the presence of the surfactant. In this context, it is pertinent to state that similar observations were not found with an anionic surfactant, namely sodium dodecyl sulfate (SDS) [29]. This is probably due to the lack of an interaction of SDS with DNA owing to the electrostatic repulsion between the negatively charged polyphosphate DNA backbone and the anionic surfactant [26,27,[57][58][59][60][61][62][63]. Similarly, with a neutral surfactant (Triton X-100), no noticeable sequestration of the DNAbound drug molecules could be observed [29]. These observations collectively lead to the conclusion that a stabilizing electrostatic interaction between the cationic surfactant CTAB and the negatively charged polyphosphate backbone of DNA probably plays an important role underlying the observed interactions leading to the sequestration of the bound drug molecules from the DNA duplex. It is relevant to state in this context that in the range of CTAB concentrations employed in the present study, the native conformation (B-form) of DNA remains practically unperturbed, as is characterized by the circular dichroic (CD) profile of DNA with added CTAB, which resembles the CD profile of the B-DNA [29].
In the study of the interaction of drug molecules with biological receptors, the kinetics of the association and dissociation of the drug is considered to have crucial diagnostic significance [26,27,[58][59][60]. A slow rate of deintercalation from the DNA-bound state has been widely argued in the literature as an important property toward efficient therapeutic functionality [26,27]. The fluorescence decay profile of HM in the presence of DNA (at λ ~435 nm) represents a characteristic rate constant for the association process, as k 5% 0.0375 s (Figure 2a), while the dissociation rate constant with added CTAB is found to be k 5% 0.0098 s (Figure 2b) [29]. However, given that the dissociation kinetic data observed in the presence of CTAB should not be directly comparable with the association kinetics data found in the absence of the surfactant, the following expression has been used to calculate a value for the dissociation rate constant: However, given that the dissociation kinetic data observed in the presence of CTAB should not be directly comparable with the association kinetics data found in the absence of the surfactant, the following expression has been used to calculate a value for the dissociation rate constant: where, K = 3.34 × 10 3 represents the binding association constant of HM with DNA. Using this relationship, we have k −1 = 1.12 × 10 −5 M −1 s −1 [29], that is the prerequisite for a faster association, and the relatively much slower dissociation of the drug from the DNA duplex is realized under the presently employed experimental conditions. In another report by Paul et al. [35], the detergent-sequestered release of norharmane (NHM, Scheme 1) from the DNA double helix was addressed by using a series of cationic surfactants, namely cetyltrimethyl ammonium bromide (CTAB), tetradecyl trimethyl ammonium bromide (TTAB), and dodecyl trimethyl ammonium bromide (DTAB), with varying hydrophobic tail lengths (n = 16 for CTAB, n = 14 for TTAB and n = 12 for DTAB; n = number of carbon atoms in the surfactant tail length). where, K 3.34 10 represents the binding association constant of HM with DNA. Using this relationship, we have k 1.12 10 M s [29], that is the prerequisite for a faster association, and the relatively much slower dissociation of the drug from the DNA duplex is realized under the presently employed experimental conditions. In another report by Paul et al. [35], the detergent-sequestered release of norharmane (NHM, Scheme 1) from the DNA double helix was addressed by using a series of cationic surfactants, namely cetyltrimethyl ammonium bromide (CTAB), tetradecyl trimethyl ammonium bromide (TTAB), and dodecyl trimethyl ammonium bromide (DTAB), with varying hydrophobic tail lengths (n = 16 for CTAB, n = 14 for TTAB and n = 12 for DTAB; n = number of carbon atoms in the surfactant tail length). The observations ( Figure 3) of cationic surfactant-induced modulations in the fluorescence profile of DNA-bound NHM in a qualitatively reverse pattern in comparison to those observed for the binding of the drug to the DNA duplex is ascribed to the deintercalation of the drug from the DNA scaffolds, whereas the genesis of the additional fluorescence band at ~380 nm is attributed to the neutral species of the drug [29,35,[64][65][66]. The negligible interaction of NHM with the surfactants alone within the range of concentrations of the surfactants employed for deintercalation of the drug was also confirmed in suitably designed control experiments [35]. The important criterion of a slow rate of deintercalation of the drug compared to the rate of association is also achieved under the asemployed experimental conditions. The fluorescence decay traces describing the deintercalation of the DNA-bound drug in the presence of the surfactants are presented in the insets of Figure 3, and the relevant data are compiled in Table 2. In this context, it is important to note that simply through the rational choice of the surfactants, the rate of dissociation of the drug from the DNA scaffolds can be tuned effectively over an order of magnitude (Table 2). This in turn provides considerable flexibility in the wider perspective of research on drug delivery toward optimization of the ADME (Administration-Distribution-Metabolism-Elimination) profile of the drug, and our results appear to present a tenable initiative for this effect in an in vitro study.
Thermodynamics of dissociation. The thermodynamic parameters corresponding to the dissociation of the NHM-DNA complex with an added surfactant, as derived from isothermal titration calorimetry (ITC) measurements, are presented in Table 3 (and the results of ITC measurements for the interaction of CTAB with the NHM-DNA complex are displayed in Figure 4). That the native conformation of the DNA double helix is not The observations ( Figure 3) of cationic surfactant-induced modulations in the fluorescence profile of DNA-bound NHM in a qualitatively reverse pattern in comparison to those observed for the binding of the drug to the DNA duplex is ascribed to the deintercalation of the drug from the DNA scaffolds, whereas the genesis of the additional fluorescence band at~380 nm is attributed to the neutral species of the drug [29,35,[64][65][66]. The negligible interaction of NHM with the surfactants alone within the range of concentrations of the surfactants employed for deintercalation of the drug was also confirmed in suitably designed control experiments [35]. The important criterion of a slow rate of deintercalation of the drug compared to the rate of association is also achieved under the as-employed experimental conditions. The fluorescence decay traces describing the deintercalation of the DNA-bound drug in the presence of the surfactants are presented in the insets of Figure 3, and the relevant data are compiled in Table 2. In this context, it is important to note that simply through the rational choice of the surfactants, the rate of dissociation of the drug from the DNA scaffolds can be tuned effectively over an order of magnitude (Table 2). This in turn provides considerable flexibility in the wider perspective of research on drug delivery toward optimization of the ADME (Administration-Distribution-Metabolism-Elimination) profile of the drug, and our results appear to present a tenable initiative for this effect in an in vitro study.
Thermodynamics of dissociation. The thermodynamic parameters corresponding to the dissociation of the NHM-DNA complex with an added surfactant, as derived from isothermal titration calorimetry (ITC) measurements, are presented in Table 3 (and the results of ITC measurements for the interaction of CTAB with the NHM-DNA complex are displayed in Figure 4). That the native conformation of the DNA double helix is not discernibly perturbed in the presence of the surfactants within the range of concentrations of the latter implies the possibility of no direct interaction of DNA with the surfactants.
Consequently, it is argued that the enhanced hydrophobicity of the medium in the presence of the surfactants stimulates a transit opening of the compact double-helical DNA structure. This in turn provides a hydrophobic sink for deintercalation of the bound drug molecules from the drug-DNA complex by decreasing the energy of activation of the process [26,27]. The deintercalated drug molecules are subsequently associated with the surfactant molecules. The first step is typically governed by pivotal contributions from hydrophobic interactions and is usually recognized as the rate-limiting step. However, it is important to realize that the thermodynamic data derived from ITC measurements provide an equilibrium description of the inherently complex phenomenon. The observed increase in entropy (T∆S > 0) is in consensus with the typical thermodynamic signature for hydrophobic hydration in which a positive change of entropy is described based on the release of water molecules and the counterions from the interface of the interacting species [67][68][69][70][71][72].
Colloids Interfaces 2023, 7, x FOR PEER REVIEW 6 of 17 discernibly perturbed in the presence of the surfactants within the range of concentrations of the latter implies the possibility of no direct interaction of DNA with the surfactants. Consequently, it is argued that the enhanced hydrophobicity of the medium in the presence of the surfactants stimulates a transit opening of the compact double-helical DNA structure. This in turn provides a hydrophobic sink for deintercalation of the bound drug molecules from the drug-DNA complex by decreasing the energy of activation of the process [26,27]. The deintercalated drug molecules are subsequently associated with the surfactant molecules. The first step is typically governed by pivotal contributions from hydrophobic interactions and is usually recognized as the rate-limiting step. However, it is important to realize that the thermodynamic data derived from ITC measurements provide an equilibrium description of the inherently complex phenomenon. The observed increase in entropy T∆S > 0 is in consensus with the typical thermodynamic signature for hydrophobic hydration in which a positive change of entropy is described based on the release of water molecules and the counterions from the interface of the interacting species [67][68][69][70][71][72].

Sequestration of Small Drug Molecules from DNA Using Mixed Micelles
Nath and co-workers showed that anionic micelles (SDS micelles) have certain limitations for drug sequestration; for example, the sequestered cationic drug substantially inhabits the surface of micelles and is not encapsulated into the nucleus of micelles due to the high surface charge density, and cationic drug molecules are also unable to gain significant hydrophobic stabilization, etc. [36]. To overcome this challenge, they employed pluronic copolymers in addition to SDS micelles. Such polymer-micelle assemblies form mixed micelles and offer more hydrophobic and electrostatic environments to the bound drugs. These unique features of mixed micelles make them potent candidates for drug sequestration. It has been established that P123-SDS mixed micellar assemblies can successfully deintercalate DNA-bound ethidium bromide; this sequestration may be controlled by the varying chain length of the copolymer used [36,37]. Additionally, cationic surfactant-copolymer mixed micelles, such as P123-CTAB mixed micelles, can be utilized to diminish the denaturing capacity of surfactants on the native structures of biomolecules in the course of their functioning as sequestrating agents for protein-bound drugs [39]. Herein, we discuss the sequestration of an anticancer drug, epirubicin hydrochloride (EPR), from its intercalated complex with well-matched (WM) DNA and a series of mis-

Sequestration of Small Drug Molecules from DNA Using Mixed Micelles
Nath and co-workers showed that anionic micelles (SDS micelles) have certain limitations for drug sequestration; for example, the sequestered cationic drug substantially inhabits the surface of micelles and is not encapsulated into the nucleus of micelles due to the high surface charge density, and cationic drug molecules are also unable to gain significant hydrophobic stabilization, etc. [36]. To overcome this challenge, they employed pluronic copolymers in addition to SDS micelles. Such polymer-micelle assemblies form mixed micelles and offer more hydrophobic and electrostatic environments to the bound drugs. These unique features of mixed micelles make them potent candidates for drug sequestration. It has been established that P123-SDS mixed micellar assemblies can successfully deintercalate DNA-bound ethidium bromide; this sequestration may be controlled by the varying chain length of the copolymer used [36,37]. Additionally, cationic surfactant-copolymer mixed micelles, such as P123-CTAB mixed micelles, can be utilized to diminish the denaturing capacity of surfactants on the native structures of biomolecules in the course of their functioning as sequestrating agents for protein-bound drugs [39]. Herein, we discuss the sequestration of an anticancer drug, epirubicin hydrochloride (EPR), from its intercalated complex with well-matched (WM) DNA and a series of  The deintercalation of the drug from the DNA duplex in the presence of mixed micelles is further affirmed by fluorescence lifetime data, which show that the excited-state fluorescence lifetime of EPR in the EPR-DNA-mixed micelle complex (~1.44 ns) is strikingly close to that of the drug in the EPR-mixed micelle complex (in the absence of DNA) [38]. This result implies that the deintercalated drug molecules are entrapped within the mixed-micellar systems. The release of the drug from DNA with the mixed micelle is also established from other biophysical methods, like fluorescence anisotropy, UV-melting studies, and CD spectral studies. This investigation not only provides detailed insights into the interaction of anticancer drugs with DNA, but also highlights that a mixed-micellar system consisting of an anionic surfactant and neutral triblock copolymer can be employed for detoxification in the case of drug overdose [38].

Sequestration of Sanguinarine (SG) from the Liposome Membrane Using Cyclodextrin
Liposome membranes are self-assembled nanostructures that mimic the lipid bilayer of the plasma membrane. They roll up into a spherical shell having a hydrophilic interior and hydrophobic bilayer region capable of accommodating small amounts of water molecules inside the core and small hydrophobic molecules that can be non-covalently bind the lipid bilayer. These vesicles may comprise phospholipids, non-ionic or ionic surfactants, etc. The size and charge of phospholipid vesicles, known as liposomes, can be tuned The deintercalation of the drug from the DNA duplex in the presence of mixed micelles is further affirmed by fluorescence lifetime data, which show that the excitedstate fluorescence lifetime of EPR in the EPR-DNA-mixed micelle complex (~1.44 ns) is strikingly close to that of the drug in the EPR-mixed micelle complex (in the absence of DNA) [38]. This result implies that the deintercalated drug molecules are entrapped within the mixed-micellar systems. The release of the drug from DNA with the mixed micelle is also established from other biophysical methods, like fluorescence anisotropy, UV-melting studies, and CD spectral studies. This investigation not only provides detailed insights into the interaction of anticancer drugs with DNA, but also highlights that a mixed-micellar system consisting of an anionic surfactant and neutral triblock copolymer can be employed for detoxification in the case of drug overdose [38].

Sequestration of Sanguinarine (SG) from the Liposome Membrane Using Cyclodextrin
Liposome membranes are self-assembled nanostructures that mimic the lipid bilayer of the plasma membrane. They roll up into a spherical shell having a hydrophilic interior and hydrophobic bilayer region capable of accommodating small amounts of water molecules inside the core and small hydrophobic molecules that can be non-covalently bind the lipid bilayer. These vesicles may comprise phospholipids, non-ionic or ionic surfactants, etc. The size and charge of phospholipid vesicles, known as liposomes, can be tuned by varying the concentration and composition of the corresponding lipid molecules [73,74]. Over the years, liposomes have captured significant attention in drug delivery research due to their properties, like solubilization, drug loading, and the targeted delivery of drugs [44][45][46]75,76]. Besides this, Chakraborty and co-workers proposed that liposomes can deintercalate DNA-bound doxorubicin through lipoplex formation [41][42][43].
In this section, we discuss the sequestration of small drug molecules bound to liposome membranes by cyclodextrin [47]. Cyclodextrins (CDs) are non-toxic, cyclic sugar polymers, soluble in water and in polar solvents, comprised of a structurally well-defined hydrophobic cavity that can accommodate small "guest" molecules mainly via non-covalent interactions. Hence, CDs have been widely applied as "molecular hosts" in a variety of aspects [77][78][79][80][81]. Sanguinarine (SG) is an alkaloid belonging to the quaternary benzo[c]phenanthridine family, having a wide spectrum of therapeutic activities, including antibacterial, antimicrobial, and anti-inflammatory properties [82][83][84][85].
The binding interaction of SG with dimyristoyl-L-α-phosphatidylglycerol (DMPG) lipid is found to be reflected by a sharp enhancement of the fluorescence intensity of the band distinctive of the iminium form of SG (λ em~5 80 nm) together with the diminution of the fluorescence intensity of the band of the alkanolamine form (λ em~4 18 nm) with increasing DMPG concentrations ( Figure 6a); a schematic representation of the iminium and alkanolamine forms of SG is given in Scheme 2. This observation suggests that a preferential binding interaction of the cationic (iminium) form of the drug with the anionic surface charge of the lipid (DMPG) is assisted by a stabilizing electrostatic interaction [47]. Figure 6b reveals that with added β-cyclodextrin (βCD), the fluorescence intensity of the iminium form (λ em~5 80 nm) of DMPG-bound SG decreases coupled with an increase in the intensity of the alkanolamine form (λ em~4 18 nm). These observations are indicative of the expulsion of the drug molecules bound to the DMPG-lipid with added βCD [34,86,87]. Such release of the bound drug molecules is also accompanied by a significant reduction in the steady-state fluorescence anisotropy of the DMPG-bound drug in the presence of βCD, indicating the release of motional restrictions of the bound drug molecules [47]. It is pertinent to note that the complex interaction situation here can lead to several possible equilibria, such as the (i) interaction of DMPG lipid with βCD leading to disruption of the compact liposome structure, and (ii) inclusion of the drug molecules within βCD. With a view of the considerably weak interaction of SG with βCD (association constant in the order of~10 −3 M −1 [82]), the second possibility is rationally discarded [47].
Colloids Interfaces 2023, 7, x FOR PEER REVIEW 9 of 17 by varying the concentration and composition of the corresponding lipid molecules [73,74]. Over the years, liposomes have captured significant attention in drug delivery research due to their properties, like solubilization, drug loading, and the targeted delivery of drugs [44][45][46]75,76]. Besides this, Chakraborty and co-workers proposed that liposomes can deintercalate DNA-bound doxorubicin through lipoplex formation [41][42][43].
In this section, we discuss the sequestration of small drug molecules bound to liposome membranes by cyclodextrin [47]. Cyclodextrins (CDs) are non-toxic, cyclic sugar polymers, soluble in water and in polar solvents, comprised of a structurally well-defined hydrophobic cavity that can accommodate small "guest" molecules mainly via non-covalent interactions. Hence, CDs have been widely applied as "molecular hosts" in a variety of aspects [77][78][79][80][81]. Sanguinarine (SG) is an alkaloid belonging to the quaternary benzo[c]phenanthridine family, having a wide spectrum of therapeutic activities, including antibacterial, antimicrobial, and anti-inflammatory properties [82][83][84][85].
The binding interaction of SG with dimyristoyl-L-α-phosphatidylglycerol (DMPG) lipid is found to be reflected by a sharp enhancement of the fluorescence intensity of the band distinctive of the iminium form of SG (λem~580 nm) together with the diminution of the fluorescence intensity of the band of the alkanolamine form (λem~418 nm) with increasing DMPG concentrations ( Figure 6a); a schematic representation of the iminium and alkanolamine forms of SG is given in Scheme 2. This observation suggests that a preferential binding interaction of the cationic (iminium) form of the drug with the anionic surface charge of the lipid (DMPG) is assisted by a stabilizing electrostatic interaction [47]. Figure  6b reveals that with added β-cyclodextrin (βCD), the fluorescence intensity of the iminium form (λem~580 nm) of DMPG-bound SG decreases coupled with an increase in the intensity of the alkanolamine form (λem~418 nm). These observations are indicative of the expulsion of the drug molecules bound to the DMPG-lipid with added βCD [34,86,87]. Such release of the bound drug molecules is also accompanied by a significant reduction in the steadystate fluorescence anisotropy of the DMPG-bound drug in the presence of βCD, indicating the release of motional restrictions of the bound drug molecules [47]. It is pertinent to note that the complex interaction situation here can lead to several possible equilibria, such as the (i) interaction of DMPG lipid with βCD leading to disruption of the compact liposome structure, and (ii) inclusion of the drug molecules within βCD. With a view of the considerably weak interaction of SG with βCD (association constant in the order of ~10 −3 M −1 [82]), the second possibility is rationally discarded [47].  Association and dissociation kinetics. The increase in the fluorescence intensity of SG at λem = 580 nm (Figure 6a) upon interaction with the DMPG lipid has been monitored to study the association kinetics of the SG-DMPG interaction. Figure 7a shows the fluorescence trace describing the kinetics of the association of the SG-DMPG interaction, and the data are fitted to a nonlinear regression as follows: where, I(t) is the variation in fluorescence intensity (at the wavelength under investigation) with time t, with an apparent association rate constant being denoted by ka (β denotes the corresponding amplitude, and η is a constant). The interaction of SG with DMPG is found to reveal an apparent association rate constant of ka = (1.7 ± 0.08) × 10 −2 s −1 at T = 303 K [47]. Similarly, the βCD-induced sequestration of SG from the DMPG-bound state (studied by observing the diminution of fluorescence intensity at λem = 580 nm, Figure 7b) shows an apparent rate constant of dissociation as kd = (4.5 ± 0.18) × 10 −2 s −1 at T = 303 K [47].

Thermodynamics of the DMPG-βCD Interaction.
The thermodynamic parameters for the interaction of the DMPG lipid with βCD are compiled in Table 4, and the primary heat-burst curves are depicted in Figure 8a. An overall thermodynamically favorable interaction of the DMPG lipid with βCD is indicated by ΔG < 0, while the process is seen to be increasingly thermodynamically favorable (increasingly negative ΔG) with a rise in temperature. With an increasing temperature, the interaction is found to be increasingly Association and dissociation kinetics. The increase in the fluorescence intensity of SG at λ em = 580 nm (Figure 6a) upon interaction with the DMPG lipid has been monitored to study the association kinetics of the SG-DMPG interaction. Figure 7a shows the fluorescence trace describing the kinetics of the association of the SG-DMPG interaction, and the data are fitted to a nonlinear regression as follows: where, I(t) is the variation in fluorescence intensity (at the wavelength under investigation) with time t, with an apparent association rate constant being denoted by k a (β denotes the corresponding amplitude, and η is a constant). The interaction of SG with DMPG is found to reveal an apparent association rate constant of k a = (1.7 ± 0.08) × 10 −2 s −1 at T = 303 K [47]. Similarly, the βCD-induced sequestration of SG from the DMPG-bound state (studied by observing the diminution of fluorescence intensity at λ em = 580 nm, Figure 7b) shows an apparent rate constant of dissociation as k d = (4.5 ± 0.18) × 10 −2 s −1 at T = 303 K [47]. Association and dissociation kinetics. The increase in the fluorescence intensity of SG at λem = 580 nm (Figure 6a) upon interaction with the DMPG lipid has been monitored to study the association kinetics of the SG-DMPG interaction. Figure 7a shows the fluorescence trace describing the kinetics of the association of the SG-DMPG interaction, and the data are fitted to a nonlinear regression as follows: where, I(t) is the variation in fluorescence intensity (at the wavelength under investigation) with time t, with an apparent association rate constant being denoted by ka (β denotes the corresponding amplitude, and η is a constant). The interaction of SG with DMPG is found to reveal an apparent association rate constant of ka = (1.7 ± 0.08) × 10 −2 s −1 at T = 303 K [47]. Similarly, the βCD-induced sequestration of SG from the DMPG-bound state (studied by observing the diminution of fluorescence intensity at λem = 580 nm, Figure 7b) shows an apparent rate constant of dissociation as kd = (4.5 ± 0.18) × 10 −2 s −1 at T = 303 K [47]. Thermodynamics of the DMPG-βCD Interaction. The thermodynamic parameters for the interaction of the DMPG lipid with βCD are compiled in Table 4, and the primary heat-burst curves are depicted in Figure 8a. An overall thermodynamically favorable interaction of the DMPG lipid with βCD is indicated by ΔG < 0, while the process is seen to be increasingly thermodynamically favorable (increasingly negative ΔG) with a rise in temperature. With an increasing temperature, the interaction is found to be increasingly Thermodynamics of the DMPG-βCD Interaction. The thermodynamic parameters for the interaction of the DMPG lipid with βCD are compiled in Table 4, and the primary heat-burst curves are depicted in Figure 8a. An overall thermodynamically favorable interaction of the DMPG lipid with βCD is indicated by ∆G < 0, while the process is seen to be increasingly thermodynamically favorable (increasingly negative ∆G) with a rise in temperature. With an increasing temperature, the interaction is found to be increasingly less exothermic (∆H < 0), whereas the unfavorable entropic contribution (T∆S < 0) is found to be progressively less negative (Table 4). The variation in ∆H with temperature leads to a positive change in the heat capacity, ∆C p = (514 ± 20) J mol −1 K −1 , which in turn signifies an instrumental role of hydrophobic hydration in the interaction and is usually connected to the burial of polar surfaces of the interacting moieties (DMPG lipid and βCD) following the interaction [67,69,[86][87][88][89][90][91][92]. Table 4. Thermodynamic parameters for the DMPG-βCD interaction obtained from isothermal titration calorimetry (ITC).

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( less exothermic (ΔH < 0), whereas the unfavorable entropic contribution (TΔS < 0) is found to be progressively less negative (Table 4). The variation in ΔH with temperature leads to a positive change in the heat capacity, ΔCp = (514 ± 20) J mol −1 K −1 , which in turn signifies an instrumental role of hydrophobic hydration in the interaction and is usually connected to the burial of polar surfaces of the interacting moieties (DMPG lipid and βCD) following the interaction [67,69,[86][87][88][89][90][91][92].  Diffusion of the Drug. The translational diffusion coefficients (Dt) of SG in various experimental conditions employed in the study, as obtained from fluorescence correlation spectroscopy (FCS,) are summarized in Table 5. The variation in the Dt of SG in differen conditions clearly supports the conclusion drawn from spectroscopic and thermodynamic experiments. Hence, an increase in the Dt of the lipid-bound drug with added βCD can be interpreted from the release of the bound drug molecules and thus a release of the mo tional constraints on the SG molecules with added βCD [86,[93][94][95].  Diffusion of the Drug. The translational diffusion coefficients (D t ) of SG in various experimental conditions employed in the study, as obtained from fluorescence correlation spectroscopy (FCS,) are summarized in Table 5. The variation in the D t of SG in different conditions clearly supports the conclusion drawn from spectroscopic and thermodynamic experiments. Hence, an increase in the D t of the lipid-bound drug with added βCD can be interpreted from the release of the bound drug molecules and thus a release of the motional constraints on the SG molecules with added βCD [86,[93][94][95].

Sequestration of Cryptolepine Hydrate (CRYP) from RNA Using Cucurbit[7]uril
In this section, we discuss the sequestration of an antimalarial drug, cryptolepine hydrate (CRYP) bound to RNA, with the application of host:guest chemistry employing cucurbit [7]uril (CB7) as the host structure [48]. CB7 is well-known to form selective and compact host:guest inclusion complexes with guest molecules [96][97][98][99][100][101] and thus can be employed as sequestrating agents for small drug molecules bound to various biological receptors, such as RNA [102,103]. The occurrence of the interaction of CRYP with CB7 is evident from the decrease in absorbance (Figure 9a) and the remarkable increase in the fluorescence intensity (Figure 9b) of the drug with added CB7 [48]. The marked increase in fluorescence intensity may be due to the motional restrictions on the CRYP molecules upon encapsulation in CB7 that results in the lowering of the radiation-less decay channels of CRYP [104]. Figure 9c shows that the absorbance of the drug bound to RNA increases with added CB7, and the absorption spectrum finally resembles that of the interaction of CRYP with CB7 alone. With added CB7, the fluorescence intensity of the RNA-bound drug is found to increase and finally resemble that of the interaction of CRYP with CB7 alone (Figure 9d). Such observations that are qualitatively in a reverse pattern in comparison to those found during the binding of the drug with RNA can be interpreted on the basis of the CB7-induced deintercalation of the bound CRYP molecules from the RNA duplex [104].  [7]uril In this section, we discuss the sequestration of an antimalarial drug, cryptolepine hydrate (CRYP) bound to RNA, with the application of host:guest chemistry employing cucurbit [7]uril (CB7) as the host structure [48]. CB7 is well-known to form selective and compact host:guest inclusion complexes with guest molecules [96][97][98][99][100][101] and thus can be employed as sequestrating agents for small drug molecules bound to various biological receptors, such as RNA [102,103]. The occurrence of the interaction of CRYP with CB7 is evident from the decrease in absorbance (Figure 9a) and the remarkable increase in the fluorescence intensity (Figure 9b) of the drug with added CB7 [48]. The marked increase in fluorescence intensity may be due to the motional restrictions on the CRYP molecules upon encapsulation in CB7 that results in the lowering of the radiation-less decay channels of CRYP [104]. Figure 9c shows that the absorbance of the drug bound to RNA increases with added CB7, and the absorption spectrum finally resembles that of the interaction of CRYP with CB7 alone. With added CB7, the fluorescence intensity of the RNA-bound drug is found to increase and finally resemble that of the interaction of CRYP with CB7 alone (Figure 9d). Such observations that are qualitatively in a reverse pattern in comparison to those found during the binding of the drug with RNA can be interpreted on the basis of the CB7-induced deintercalation of the bound CRYP molecules from the RNA duplex [104].

Conclusions
In summary, this review predominantly outlines the in vitro studies on the release of several drugs from biological and bio-replicating systems, with the help of biophysical techniques, such as absorption spectroscopy, fluorescence spectroscopy, and isothermal titration calorimetry. In summary, the results discussed in this article are briefly presented below.
(i) The biological photosensitizer harmane (HM) strongly binds to the DNA duplex principally via the intercalation mode and can subsequently be sequestered using the cationic surfactant CTAB where the cationic surfactant acts as a hydrophobic sink for the drug molecules. (ii) Another biological photosensitizer, norharmane (NHM), which binds to the DNA duplex, has also been shown to have been sequestered with the aid of cationic surfactants, namely DTAB, TTAB, and CTAB, for which the rate of sequestration of the bound drug is found to be tunable with variations in the chain length of the surfactants. (iii) The deintercalation of the anticancer drug epirubicin hydrochloride bound to wellmatched and mismatched DNA has been shown using mixed micellar assemblies consisting of the anionic surfactant SDS and non-ionic surfactant P123. It was also noticed that the mixed micelles did not alter the native structure of DNA. (iv) The dissociation of the alkaloid sanguinarine cation bound to negatively charged DMPG liposomes using molecular guest β-cyclodextrin shows the application of host-guest chemistry in this context. (v) The application of host-guest chemistry in the sequestration of bound drug molecules is further demonstrated in the context of the deintercalation of the antimalarial drug cryptolepine hydrate (CRYP) from the RNA duplex using cucurbit [7]uril hydrate.
Thus, we are optimistic that these results could be helpful for studies on the excretion of drugs from biological environments and for understanding the accompanying mechanisms. Data Availability Statement: The data as represented in this review article has been collected from published papers related to the content of the review, duly acknowledging the respective publication.