Encapsulation of Biologically Potent Neurotransmitters into HP-β-CD Probed by Physicochemical Approach simultaneously Optimized by Computational Studies

The inclusion complexes of the neurotransmitters, namely (±) epinephrine hydrochloride, dopamine hydrochloride, and tyramine hydrochloride in an aqueous medium, were studied physicochemically and spectroscopically. These rendered systems of drug delivery with the potential to ensure delivery to desired targets. The stoichiometry of the 1:1 ratio of host-guest binding was established using Job plots. Conductimetric studies gained an understanding of inclusion as a process. Furthermore, 1 H NMR was used to characterize the inclusions, and the Benesi Hildebrand equation was applied to obtain the association constants. Thermodynamics, which are important in predicting the feasibility of any process, were in favor of inclusion as obtained by the evaluation of various thermodynamic parameters from Van᾿t Hoff's equation.


Introduction
The encapsulation of neurotransmitters of utmost biological importance is a key to interpreting and developing an understanding of formed molecular assemblies as an extension to find applications in potential drug delivery to the targeted sites of their action [1][2][3].HP-β-CD owes its application to enabling pharmaceutical excipients by enhancing the solubility of a spectrum of drugs in the aqueous phase [4,5].This is attributed to the toroidal configuration with innumerable hydroxyl groups at each of its ends, imparting it the ability to bind drug molecules with an even higher degree of hydrophobicity [6][7][8], thereby releasing it into biological systems with absolute ease while keeping its solubility in water intact.The polar exterior with hydroxyl groups is the key to aqueous solubility [2,3].The factors stabilizing the binding inside the hydrophobic cavity are non-covalent Vander Waals forces, electrostatic forces, H-bonding, removal of energetically high water bound to the cavity, and also the reduction in conformational strain post to the binding [6,7,9].The formation of such supramolecular assemblies based on such forces was understood by interpreting results obtained through physicochemical studies like conductance measurement [6].Controlled delivery of drugs based on HP-β CD as binders or carriers of drug molecules is a key to therapeutics [1].It surpasses the lipophilic membranes of appreciable hydrophobicity [4,5] under the head of the inclusion complex to exert therapeutic action as desired by its target.This approach has potency in enhancing stability, bio-availability, and solubility in drug formulations [10][11][12] The three chosen amines in their hydrochloride combinations are our molecular guests.Each of them belongs to the class of neurotransmitters occurring in biological systems that make significant contributions in conducting nervous impulses from one axon to the next dendritic tip of the neurons [6].Dopamine is a neurotransmitter occurring in mammals that is necessary to ensure homeostasis [6,13,14].Parkinsonism, a diseased condition, is again due to the deficiency in the occurrence of this biogenic amine and leads to improper neural and neuromotor functions [15].Tyramine is yet another NT of interest and a Catecholamine releaser [16,17].Epinephrine, the third amine taken, is also an NT and a hormone that chemically mediates impulses to the required organs [18,19].It is an active medication for treating patients with cardiac arrest and other such disorders.[20][21][22] Here, the formulations of drugs studied were dopamine, tyramine, and epinephrine in their hydrochloride combinations with HP-β-CD, respectively.The understanding of such supramolecular assemblies was gained using Job plots, conductance measurements, UV-VIS Spectroscopy, and 1 H NMR. The addition of a thermodynamic standpoint drew the parameters, the key to quantifying the complexation of each such amine with HP-β-CD.Thereby, the overall thermodynamics involved in the process are established [1].Scheme 1.Molecular structure of the neurotransmitters.

Materials
The procurement of dopamine hydrochloride (DH), tyramine hydrochloride (TH), and epinephrine hydrochloride (EH) were done from the German Company Sigma-Aldrich.These were used as obtained in hands.The associated purity of HP-β-CD, DH, TH, and EH were 99 %, 98 %, and 99 %, respectively.

Apparatus and procedure
Solubilities of the HP-β-CD and the biogenic amines (NT) selected were thoroughly checked with the triply distilled and degassed water before the experimental run.The water used had a specific conductance value of 1×10 -6 S Cm -1 .In observation, all such NTs were soluble in every quantity of the aqueous solutions of the HP-β-CD.Stock solutions were prepared according to mass for each of the NT᾿S (weighing was done with the Mettler Toledo AG -285, and the uncertainty associated was 0.0003 g).At a temperature of 298.15K, a mass dilution was done to the prepared solutions.A handful of precautionary measures were followed during mixing to cut losses due to evaporation.
The spectrophotometer JASCO V-530 UV/VIS was used to record the UV-visible spectra.The uncertainty associated with its wavelength resolution was ± 2 nm.Constancy in measuring temperature was kept intact using a digital thermostat.Systronics 308 Conductivity Bridge and immersion conductivity cell CD-1 dip-type were used in the conductance studies.The conductivity cell was reported to have a cell constant value of 0.1 ± 0.001 cm -1 .HPLC grade water of Specific conductance 6.0 Sm -1 was used.Fresh 0.001 M aqueous KCl solutions were used to calibrate the conductivity cell before the experiment.An uncertainty of 3 % was found for the specific conductance values obtained.
Bruker ADVANCE 400 MHz was used to record the NMR spectra in D2O.The δ values in ppm are the signal quotations.Other residuals were treated as due to internal standards, i.e.D2O: 4.79 ppm.The chemical shifts are our data in hand.

Stoichiometry of host-guest binding through job plots
The UV-visible spectroscopy-based continuous variation method of Job was a key to understanding the stoichiometry of host-guest binding [6,23].The plot of ΔA × R VS R generated the Job plots, here, ΔA denotes the absorbance difference values for the NT᾿s while complexes and free of HP -β-CD denoted as R = [NT] / ([NT] + [CD]).Then, variation in mole fraction was undertaken for the NTs in the 0-1 range (Supporting information is held up with tables S1, S2, S3) [6,24].The values for the absorbances were recorded at their respective λmax for the experimental solutions, all recorded at a temperature of 298.15 K.At the maximum deviation, the value of R was evident in the stoichiometry of the inclusion complex (IC) (e.g., 1:1 complexes, 1:2 complexes, and 2:1 complexes, respectively, gave R values 0.5, 0.33 and 0.66.Our maxima were all found at R = 0.5 for all of the three plots.This obtained value of R established a stoichiometry of 1:1 binding for the host-guest.

Conductance illustration of the inclusion and the stoichiometry involved
The conductance measurements for the NTs' aqueous solutions were performed to determine if the ICs were produced with the addition of the host HP-β-CD to these solutions [25,26].An appreciable value for the ̔ к᾿ was an illustration of the NTs as charged structures.Furthermore, the values for ̔ к᾿ were seen to exhibit a decreasing trend upon the addition of the host HP-β-CD, indicative of the binding of the guest NTs to the host.A break in each of these curves for conductance for a specific concentration of HP-β-CD indicated successful inclusion.The HP-β-CD concentration and the к value corresponding to the breaks in each of these curves are tabulated in Table (S4).This established that the breakpoint ratio of the [NT] / [HP-β-CD] is approximately 1:1 and thus follows to establish the host-guest ratio binding to be 1:1.
The breakpoint corresponds to the concentration with the maximum possible inclusion.The process upholds an equilibrium of the dynamic type involved, but the keynote is that the encapsulation or formation of assemblies is more ready for most molecules only at this breaking point (Host (conc) / Guest (conc)) = 1:1.The concentration of HP-β-CD exceeds that of NT beyond this breakpoint and is indicative of the equilibrium shifting towards the inclusion formation.

1 H NMR studies
The NMR study is key to interpreting the extent of inclusion of the guest molecule in the host [27,28].For the taken NTs, the values of the H3 and H5 post-to inclusion explain the ease with which each inclusion of the guests occurs in the hydrophobic cavity of the HP-β-CD [29,30].The values of chemical shifts for H3 and H5 were recorded respectively as (0.00, -0.01) for tyramine, (-0.02, -0.02) for dopamine, and (-0.04, -0.04 ) for epinephrine.This indicates that the extent of inclusion is maximum for epinephrine, with the most negative values of the chemical shift for H3 and H5, followed by tyramine with intermediate negative values of the chemical shifts, further followed by the least negative value of the chemical shifts for the same in case of dopamine.Alternatively, the ease of inclusion with HP-β-CD decreases from epinephrine to dopamine to tyramine.

Calculation of the association constants and thermodynamic parameters
The UV-VIS Spectroscopy was used to evaluate the association constants (Ka) for the ICs of NTs with the HP-β-CD.This was obtained due to the change in values of the Δε, the molar extinction coefficient, of the NT+HP-β-CD complexes.This observation is attributed to the variation in polarity of the immediate vicinity of the Chromophores present in the neurotransmitters on the transition to the hydrophobic non-polar toroidal cavity from an entirely polar aqueous surrounding [31,32].The determination of Ka was done by studying the change in ΔA, the absorption intensity of the DH (S5), TH (S6), and EH (S7) as HP-β-CD᾿s concentration function.The Benesi-Hildebrand method was applied further to obtain the double reciprocal plots, as is the case for 1:1 host-guest ICs. 1 The Intercept obtained for the double reciprocal plot was divided to obtain the values of Ka for the three inclusions [33].
A probe to the thermodynamic standpoint was delved to evaluate parameters of thermodynamic importance and extended to the inclusion as a process with the well-known Van᾿t Hoff equation.It correlates thermodynamic parameters with Ka.Thus, known Ka parameters like ΔH°, ΔS°, and ΔG° were derivable from the equation stated below: The equation shows linearity between ln Ka/2.303 and 1/T.The thermodynamic parameters stated above, along with ΔG°, are all derivable for the inclusions performed [34].
The study undertaken for the ΔH 0 and ΔS 0 are constant for the short range of temperature taken here.However, a Non-Linear approach is to be used while studying the same parameters for a wider range of temperatures.Direct calorimetric methods are associated with more accuracy concerning the ΔH 0 , as these are dependent on equilibrium constants for the IC as a function of temperature [35].The Δε of the NTs was the determinant for the obtained Ka, which was the basis for the obtained thermodynamic parameters.This followed as a direct consequence of the changes near the chromophores in the NTs.Owing to the transition to an apolar cavity from a polar region.The solvent interactions are noncontributing to the obtained ΔH° and ΔS° and are only defined for the formed molecular complexes of interest.
A feasible process that is an inclusion formation was laid from the negative values of the ΔG°.The negative enthalpy (ΔH°) and negative entropy (ΔS°) indicate that the process of inclusion is an exothermic.It also establishes that the process is not entropy-driven.The above-mentioned observations are in favor of the association developed in the course of inclusion between the host-guest pair, thereby cutting down randomization and a drop in entropy,favoring inclusion on the whole.The mentioned entropy effect is more than overweighed by the larger negative values for the ΔH°, establishing overall favorable thermodynamics for inclusion formation.
The Table S8 for the order of -ΔG° and Ka is EH> DH> TH.The ease of getting included inside the non-polar cavity is in the same order as that stated above.This is an immediate result following the structures of the guest NTs with comparable hydrophobic moieties.However, a point of difference is the variable numbers of the polar -OH groups in each of them.With 2 -OH groups of phenolic nature and 1-OH group of alcoholic nature in EH, it forms a strongly associated inclusion by H-bond formation.Following EH with the strongest such inclusion among the three, DH and TH form less and least stronger associations on account of the possession of the two and only one -OH group in their structures, respectively.

Preparation of 3D-structures of T.Y., D.O.P., EPI and HP-β-CD
The three-dimensional structures of DH, TH, and EH were obtained from the PubChem website (http://pubchem.ncbi.nlm.nih.gov).HP-β-CD was constructed from β-CD by adding a hydroxypropyl group, and β-CD was taken from Cambridge Crystal Data Centre (CCDC) with a deposit number (CCDC ID: 762697) [36].Before conducting the computational investigations, the missing hydrogen atoms and the charges of the atoms were added to HP-β-CD as well as DH, TH, and EH structures, and the optimization was done with the DFT-B3LYP method using Gaussian 09 software [37].

Molecular docking evaluation
Molecular docking is a computer-based analysis that assists in determining the preferred binding mode of a guest with a host [38,39].A well-established docking procedure for the PyRx host-guest system was employed.[40].Before conducting docking, PDB files of DH, TH, EH, and HP-β-CD were transformed into PDBQT format, and the atomic coordinates were generated using PyRx software.For all three DH-HP-β-CD, TH-HP-β-CD, and EH-HP-β-CD complex, the center values of the grid box were set as center x= 8.4381, center_y= 24.3525 and center_z= 1.6111, and the values of dimension as size_x= 25, size_y = 25, and size_z = 25 for all three coordinates.The grid box size was taken around the binding cavity of HP-β-CD (receptor) to sufficiently allow the ligand to move without any constraint in the search space.Based on the docking score, the conformer with the lowest docked energy was selected [41], and for visualizations, Discovery Studio Visualizer, v21.1.0.20298,BIOVIA, was used [42].

Molecular docking studies
Molecular docking has been widely utilized to anticipate the binding orientation of a guest into the host pocket [43,44].With the help of PyRx software, the docking has been done between NTs and HP-β-CD.The most favorable docked conformation of the Guest-HP-β-CD complex is shown in Fig 3 .The negative binding energy (ΔG) with the lowest value associated with the docked conformation, which has maximum stability of TH-HP-β-CD, DH-HP-β-CD, and EH-HP-β-CD-complex was found to be -5.20 kcal/mol, -5.60 kcal/mol and -5.70 kcal/mol which is very close to the value collected from other studies, implicating that the binding energy obtained from docking is almost similar to the value obtained from other analysis.Docking results suggested that all three complexes and the guest molecules were completely incorporated in the hydrophobic cavity of HP-β-CD through the wider end.These interactions of guest molecules with HP-β-CD gathered from the docking study are aligned with the observations of 1 H NMR analysis also.

Conclusion
The exploration of ICs with NTs attempted to develop potential drug deliveries for applications in medicinal chemistry.The Jobs plot and conductance study established 1:1 inclusion.Furthermore, the association constants also indicated that the extent of inclusion is maximum for epinephrine, followed by dopamine, which tyramine again follows.
The thermodynamic parameters ΔH°, ΔS°, and ΔG° also indicated the same as those established before.The NMR study also provides evidence of the same fact.Additionally, the ΔG° obtained either from the molecular docking or association constant assumes values very close to each other.This also indicated that the extent of inclusion is maximum for the epinephrine and minimum for the tyramine.The result obtained is the same for all approaches.

Fig. 2 .
Fig. 2. Change in the conductivity of aqueous (a) DH solution, (b) TH solution, and (c) EH solution with the addition of HP-β-cyclodextrin at 298.15 K.

Table 1 .
Shifting of 1 H NMR values of NTs.

Table 2 .
Shifting of 1 H NMR values of HP-β-CD.
a Standard ambiguities in temperature (T) = 0.01 K