1

ROMANIAN ACADEMY

„Ilie Murgulescu” Institute of Physical Chemistry

THESIS ABSTRACT

THE STUDY OF THERMODYNAMIC STABILITY OF LIGAND – CYCLODEXTRIN

MOLECULAR STRUCTURES. STRUCTURAL AND MORPHOLOGICAL ASPECTS

CORRELATED WITH THERMODYNAMIC DATA.

Scientific Advisor,

CS I Dr. Tănăsescu Speranţa Valeria

PhD Student,

Neacșu Dana-Andreea

BUCUREŞTI

2018

2

Summary

1

I. Current state of research in the field 6

1. Introduction 7

2. Theoretical aspects related to phase equilibria thermodynamics in "receptor-ligand"

systems

11

2.1 The phase rule 11

2.2 Thermodynamic functions for phase transformations in the guest – cyclodextrin

molecular systems

11

2.3 Equilibrium conditions between phases 12

2.4 Thermodynamic theory involving First-Order Phase Transitions 13

2.5 Phase equilibria thermodynamics in one-component systems 14

2.6 Phase equilibria thermodynamics in multicomponent systems 15

3. Current state of research in the thermodynamics field of cyclodextrine inclusion

compounds

18

3.1 Aspects involved in the cyclodextrine characterisation 18

3.1.1 General considerations, structure and properties of cyclodextrins 18

3.2 Correlations between the structure and thermodynamics of compound complexation 22

3.2.1 Basic interactions involved in the stability of complexes 23

3.2.2 Steric factors affecting the inclusion phenomena 24

3.2.2.1 Effect of ligand structure 24

3.2.2.2 Effect of cyclodextrin cavity 25

3.2.2.3 Effect of cyclodextrin cavity functionalization 25

3.2.3 Enthalpic and entropic contributions 26

3.2.3.1 Effect of enthalpy-entropy compensation 26

3.2.4 Influence of the reaction medium on the complexity thermodynamics 27

3

3.3 Methods of preparation widely used in the preparation of inclusion compounds 28

3.4 Applications. Advantages of cyclodextrins and cyclodextrin-ligand complexes 30

II. Experimental methodes 33

4. Techniques used for the characterisation of inclusion complexes 34

4.1 Differential scanning calorimetry (DSC) and thermogravimetry (TG) 34

4.1.1 The relative degree of crystallinity (RDC) 38

4.1.2 Determination of the purity of a substance 38

4.2 Isothermal titration calorimetry (ITC) 39

4.3 Spectral and morphological analysis 41

4.3.1 Ultraviolet–visible absorption spectroscopy (UV-VIS) 41

4.3.1.1 Stoichiometry determination 41

4.3.1.2 Binding constant determination 41

4.3.1.3 The obtaining of thermodynamic parameters from UV-VIS spectroscopy

data using van’t Hoff analysis

42

4.3.2 Fourier transform infrared spectroscopy (FT-IR) 43

4.3.3 Scanning electron microscopy (SEM) 44

4.4 Other methodes involved in sample characterisation 44

4.4.1 Quantum chemistry calculations (QC) 44

4.4.2 pH measurements 44

III. Original contributions to the characterization of ligand-cyclodextrin type systems 45

5. Thermodynamic characterization of cyclodextrins used in the synthesis of complexes 46

5.1 Thermodynamic study of αCD, βCD, HPαCD, HPβCD, γCD 46

5.2 Conclusions 58

6. Comparative study of the inclusion compounds of uracil and 5-fluorouracil with

cyclodextrins

60

4

6.1 General considerations. Study motivation 60

6.2 Experimental details and results from UV-Vis measurements 61

6.2.1 Materials and synthesis of inclusion compounds 61

6.2.2 Determination of stoichiometry of cyclodextrin-uracil and cyclodextrin-5-

fluorouracil inclusion compounds by UV-Vis spectroscopy measurements

62

6.3 The experimental study of inclusion compounds of the cyclodextrin-uracil and

cyclodextrin-5-fluorouracil, in solid state

64

6.3.1 The thermodynamic study of inclusion compounds between uracil/5-

fluorouracil and cyclodextrins

64

6.3.1.1 Experimental results obtained by TG/DTG 64

6.3.1.2 Experimental results obtained by DSC 68

6.3.2 Structural and morphological characterization of pure compounds and inclusion

complexes

73

6.3.2.1 Analysis of the results obtained by FTIR 73

6.3.2.2 Morphological characterization by SEM 76

6.4 Theoretical study of inclusion compounds of the cyclodextrin-uracil and

cyclodextrin-5-fluorouracil

78

6.4.1 Evaluation of inclusion compounds based on QC 78

6.5 Conclusions 88

7. Cyclodextrin inclusion compounds with L- and D- alpha-amino acids stereoisomers 90

7.1 General considerations. Study motivation 90

7.2 Materials and synthesis of inclusion compounds 92

7.3 The thermodynamic study of pure amino acids and inclusion compounds between the

optical isomers of arginine, histidine, tryptophan with cyclodextrins

94

7.3.1 TG/DTG analysis of pure amino acids used in the synthesis of complexes

formed with cyclodextrins

94

7.3.2 TG/DTG results for the inclusion compounds between the optical isomers of

arginine and cyclodextrins

96

5

7.3.3 TG/DTG results for the inclusion compounds between the optical isomers of

histidine and cyclodextrins

99

7.3.4 TG/DTG results for the inclusion compounds between the optical isomers of

tryptophan and cyclodextrins

102

7.3.5 DSC results for the inclusion compounds between the optical isomers of

arginine and cyclodextrins

104

7.3.6 DSC results for the inclusion compounds between the optical isomers of

histidine and cyclodextrins

108

7.3.7 DSC results for the inclusion compounds between the optical isomers of

tryptophan and cyclodextrins

111

7.4 Structural and morphological characterization of the inclusion compounds between

the optical isomers of arginine, histidine, tryptophan and cyclodextrins

114

7.4.1 FT-IR spectra of the inclusion compounds between the optical isomers of

arginine, histidine, tryptophan and cyclodextrins

114

7.4.2 SEM images of the inclusion compounds between the optical isomers of

arginine, histidine, tryptophan and cyclodextrins

119

7.5 Correlations between thermodynamics, structure and morphology of inclusion

compounds

122

7.6 Conclusions 124

8. The study of interaction between cichonine and cyclodextrins 125

8.1 General considerations. Study motivation. 125

8.2 Sample synthesis 127

8.3 Thermodynamic study of inclusion compounds between cinchonine and cyclodextrins 128

8.3.1 TG/DSC results for pure cinchonine 129

8.3.2 TG/DTG results for the inclusion compounds of cinchonine and beta-

cyclodextrin, at various molar ratios

130

8.3.3 TG/DTG results for the inclusion compounds of cinchonine and 2-

hydroxypropyl-beta-cyclodextrin, at various molar ratios

132

8.3.4 TG/DTG results for the inclusion compounds of cinchonine and gamma- 135

6

cyclodextrin, at various molar ratios

8.3.5 DSC results for the inclusion compounds of cinchonine and beta-cyclodextrin,

at various molar ratios

138

8.3.6 DSC results for the inclusion compounds of cinchonine and 2-hydroxypropyl-

beta-cyclodextrin, at various molar ratios

142

8.3.7 DSC for the inclusion compounds of cinchonine and gamma-cyclodextrin, at

various molar ratios

141

8.4 Structural and morphological characterization of inclusion compounds between

cinchonine and cyclodextrins

147

8.4.1 FT-IR spectra for the inclusion compounds of cinchonine and cyclodextrins, at

various molar ratios

147

8.4.2 SEM imagines for the inclusion compounds of cinchonine and 2-

hydroxypropyl-beta-cyclodextrin, at 1:1 and 1:2 molar ratios

151

8.5 Correlations between thermodynamics, structure and morphology of inclusion

compounds

152

8.6 Conclusions 156

9. Study of interaction between doxorubicin hydrochloride (Dox) and γ-cyclodextrin 158

9.1 General considerations. Study motivation. 158

9.2 Sample synthesis 159

9.3 Experimental study of the complexity between Dox and γ-CD, in liquid state 160

9.3.1 Dox/γCD analysis by UV-Vis spectroscopy 160

9.3.1.1 Experimental details about UV-Vis measurements 160

9.3.1.2 Analysis of UV-Vis spectra and determination of stoichiometry 160

9.3.1.3 Determination of association constants and thermodynamic parameters

for the Dox / γCD system

162

9.3.2 pH measurements and distribution of Dox microspecies 165

9.3.3 Dox / γCD system analysis by isothermal calorimetric titration (ITC) 167

9.3.3.1 Experimental details related to ITC measurements 167

7

9.3.3.2 Analysis of thermodynamic parameters obtained from ITC 168

9.3.4 Correlations between the thermodynamic parameters and the driving

forces involved in the structure of the complex

170

9.4 Experimental study of the complexity between Dox and γCD, in solid state 172

9.4. Thermodynamic study of inclusion compounds between Dox and γCD 172

9.4.1.1 TG/DTG results 172

9.4.1.2 DSC results 173

9.4.2 Structural and morphological characterization of the Dox / γCD system 175

9.4.2.1 FT-IR spectra 175

9.4.2.2 SEM images 177

9.5 Conclusions 178

10. General Conclusions. Original contributions 179

11. Perspectives of expanding and continuing research 182

Bibliography 183

List of scientific articles and communications 204

KEYWORDS: thermodynamics, stability, morphology, cyclodextrin, cinchonine, uracil

1. Introduction

“Cyclodextrins are universal molecular recipients for: organic, inorganic and organometallic

compounds, neutral, cationic, anionic or even organic radicals" [1]. The main property of

cyclodextrins is to form complexes with almost any type of guest molecule. Due to its remarkable

molecular architecture, cyclodextrins play an important role in medicine, biology, pharmaceuticals,

food and consumer goods, with particular importance being reflected in the number of papers and

patents dedicated to cyclodextrin and its inclusion compounds.

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In the thesis were made original contributions regarding the thermoanalytic study of some

complexes of organic molecules with cyclodextrins and their thermal behavior was correlated with

the structural and morphological aspects involved in their formation. In order to support the

information derived from the thermodynamic data, quantum chemistry calculations were used to

obtain the energy parameters of the complexation interaction and new structural and morphological

data were presented in relation to the studied complexes.

The motivation of the study is also related to other aspects, such as:

- the need of new information on the structure, morphology and quantum chemistry

calculations for ligand / cyclodextrin systems with biochemical importance;

- the role of energy parameters in highlighting the correlation between the ligand

structure and the properties of the complex obtained with cyclodextrin;

- the importance of the studied systems, which can be models within a series of

complexes obtained between cyclodextrins and ditopic molecules.

The study in the frame of this thesis is based on the analysis of the results obtained by

correlating several experimental methods, namely:

- Thermal analysis and calorimetry methods (Differential Scanning Calorimetry - DSC,

thermogravimetric analysis - TG, isothermal titration calorimetry - ITC).

- Structural and morphological characterization methods (Fourier transformed infrared

spectroscopy measurements - FT-IR, scanning electron microscopy - SEM, ultraviolet –

visible absorption spectroscopy - UV-Vis).

- Determination of pH.

- Quantum chemistry (QC) calculations.

In this study, a series of well-known amino acids and active biochemicals have been selected

as guest molecules for which inclusion complexes with different cyclodextrins [alpha-cyclodextrin

(αCD), beta-cyclodextrin (βCD), 2-hydroxypropyl-alpha-cyclodextrin (HPαCD), 2-

hydroxypropyl-beta-cyclodextrin (HPβCD), gamma-cyclodextrin (γCD)] have been synthesized.

In the table below are listed the studied systems and the methods of investigation used within this

thesis.

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Cyclodextrin type /

Ligand type

αCD βCD 2HPαCD 2HPβCD γCD Analysis methods

L-Arginine ● ● ● DSC, TG/DTG, FTIR, SEM,

D-Arginine ● ● ● DSC, TG/DTG, FTIR, SEM,

L-Histidine ● ● ● DSC, TG/DTG, FTIR, SEM,

D-Histidine ● ● ● DSC, TG/DTG, FTIR, SEM,

L-Tryptophan ● ● ● DSC, TG/DTG, FTIR, SEM,

D- Tryptophan ● ● ● DSC,TG/DTG, FTIR, SEM,

Uracil ● ● ● ● DSC, TG/DTG, FTIR, SEM, QC,

UV-Vis

5-Fluorouracil ● ● ● ● DSC, TG/DTG, FTIR, SEM, QC,

UV-Vis

Doxorubicin ● DSC, TG/DTG, ITC, FTIR, SEM,

UV-Vis, pH

Cinchonine ● ● ● DSC,TG/DTG, FTIR, SEM, UV-

Vis

Taking into consideration the approached field of study, this thesis aimed to achieve the

following specific objectives:

1) Synthesis of inclusion complexes starting from the ligand and the corresponding cyclodextrin,

using inexpensive and simple preparation methods.

2) Thermochemical characterization of pure substances from which the synthesis of complexes was

started and determination of the corresponding thermochemical parameters.

3) Thermodynamic and thermochemical characterization, stability and essential thermal

characteristics of the synthesized complexes using appropriate thermal analysis and calorimetry

methods.

4) Spectral analysis in order to check the formation of inclusion complexes.

5) Morphological characterization of the complexes

6) Use of UV-Vis, QC analysis methods for the obtaining of energetic parameters and

stoichiometry of complexes.

7) To highlight the influence of the reaction medium and of the component structure on the process

of formation of inclusion complexes.

The PhD thesis is structured in 11 Chapters, included in three main parts:

Ist part (chapters 1, 2 3) is devoted to general considerations of phase equilibria

thermodynamics in receptor-ligand systems as well as to the state of the art of cyclodextrin-

inclusion compounds thermodynamics.

IInd

part (chapter 4) is dedicated to the experimental methods and techniques involved in

sample characterization.

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IIIrd

part (chapters 5, 6, 7, 8, 9, 10) of the thesis is devoted to the original contributions to the

ligand-cyclodextrin systems.

At the end of the thesis are presented the general conclusions and perspectives related to this

study, followed by the bibliographic references and the list of scientific articles and communications

related to the thesis theme.

Chapter 1 presents a brief introduction that discloses information on the importance of cyclodextrins

and inclusion complexes formed with cyclodextrins. The goal/aim, the specific objectives and the

structure of the PhD thesis are presented.

Chapter 2 contains essential information related to the thermodynamics of first-order phase

transitions. In this chapter are presented fundamental aspects related to the thermodynamic functions

of the phase transformations involved in the guest-molecule – cyclodextrin type systems. It is also

described based on thermodynamic equations approach, the theory applied in determining the purity

of substances.

Chapter 3 contains generalities on the structure and properties of cyclodextrins, as well as the

description of the most commonly methods in preparing inclusion compounds. This chapter

provides the main information about driving forces and steric factors that influence the structure and

thermodynamics of the complexation process.

Chapter 4 presents the equipments and analysis methods involved in this thesis to study pure

components and synthesized complexes.

Chapter 5 contains the thermal analysis data obtained for pure cyclodextrins (αCD, βCD, HPαCD,

HPβCD, γCD). The characterization of pure cyclodextrins is imperiously needed in order to evaluate

the properties of CDs before preparing the complexes, as well as to analyze their formation.

Chapter 6 presents the original contributions resulted from the investigation of inclusion complexes

between the uracil or 5-fluorouracil guest molecules and cyclodextrines. There are 16 systems

synthesized that were thermodynamically investigated using DSC / TG. The complexation process

was validated by the FTIR and SEM methods. The stoichiometry of the complexes was determined

before obtaining the solid powders, and quantum chemistry calculations were performed to provide

the energetic parameters characteristic for the complexation interaction.

Chapter 7 includes experimental data on the solid state study of complexes formed between the

optical isomers of amino acids: arginine, histidine and tryptophan with cyclodextrins.

Thermodynamic parameters of pure amino acids and complexes were established by thermal

11

analysis. Comparative discussions on the complexes stability were performed. FTIR and

morphology data for all studied complexes and for the corresponding pure amino acids were

provided. Taking into account the obtained data, the influence of the various factors that determined

the thermodynamics of the complexation and the structure of the complexes was discussed.

Chapter 8 presents the study of inclusion complexes between cinchonine and cyclodextrins for

molar ratios 1: 1, 2: 1 and 1: 2 (ligand-cyclodextrin). Based on thermal analysis data the

thermograms and the parameters corresponding to the thermal effects were provided and the

stability of the complexes was discussed, the thermochemical data being supported by the structural

and morphological analysis. Influence of structural factors and of the reaction medium on the

complexity behavior were correlated.

Chapter 9 contains the original contributions resulting from the analysis of the complex formed

between doxorubicin and γ-cyclodextrin, in solid and liquid state. The stoichiometry of the solution

was investigated and the thermodynamic parameters were determined by isothermal calorimetric

titration and UV-Vis spectroscopy data. The solid powder of the complex was investigated by

thermal analysis and the corresponding thermograms and thermochemical parameters were

presented. Based on the FTIR spectroscopy data, the complexation porcess was evaluated. The

morphological analysis was also presented.

Chapter 10 and 11 outline the final conclusions and prospects for expanding and continuing

research.

Original contributions to the characterization of ligand-cyclodextrin type systems

2. Comparative study of the inclusion compounds of uracil and 5-fluorouracil with

cyclodextrins

The preparation of solid state inclusion compounds (U/αCD, 5FU/αCD, U/βCD, 5FU/βCD,

U/HPαCD, 5FU/HPαCD, U/HPβCD, 5FU/HPβCD) was done by “melting in solution” method,

using 1:1 molar ratios of ligand-CD. The system stoichiometry was determined by the continuous

variation method. Each system was equilibrated 24h, after that the absorbance variation was

observed at 258nm for U systems and at 265nm for 5FU formed systems respectively. In this

12

experiment, stoichiometry was determined for 10-5

M aqueous solution and for each of the studied

systems, the stoichiometric ratio was 1:1.

These determinations were necessary to verify if the stoichiometry of the diluted aqueous

solution correspond to the molar ratio of the systems

DSC thermograms of complexes formed between 5FU or U with cyclodextrins show no

presence of thermal process attributed to the melting of the ligand in presence of CD, thus for all the

studied complexes the existence of strong interactions between the guest molecule and the CD

cavity took places, as indicated in Fig. 2.1.

50 100 150 200 250 300 350 400 450

Flu

x c

alo

ric

(m

W)

En

do

Temperatura (C)

CD

5FU/CD

5FU

U/CD

U

50 100 150 200 250 300 350 400 450

Flu

x c

alo

ric

(m

W)

En

do

Temperatura (C)

HPaCD

5FU/HPCD

5FU

U/HPCD

U

50 100 150 200 250 300 350 400 450

Flu

x c

alo

ric

(m

W)

En

do

Temperatura (C)

CD

5FU/CD

5FU

U/CD

U

50 100 150 200 250 300 350 400 450

Flu

x c

alo

ric

(m

W)

En

do

Temperatura (C)

HPCD

5FU/HPCD

5FU

U/HPCD

U

Fig. 2.1 DSC thermograms for U, 5FU, αCD, βCD, HPαCD, HPβCD and synthesized inclusion

compounds.

Formation of complexes with functionalised cyclodextrins was more effective than

formation of complexes with native cyclodextrins, and this aspect could be highlighted by following

the decomposition temperature of the complexes, as shown in Table 2 - 1.

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Tabelul 2 - 1

The order of decreasing the decomposition temperature of the inclusion complexes formed between

U/5FU and cyclodextrins

Fig. 2.2 TG curves (continuous line) and DTG (dotted line) for U, 5FU and for complexes formed

between U, 5FU and cyclodextrins.

The formation of inclusion complexes for all studied systems took places and the analysis of

TG/DTG data (Figure 2.2) confirms the results obtained by DSC [2].

Fig. 2.3 shows FTIR-ATR spectra for U, 5FU, cyclodextrins and inclusion compounds. For

pure cyclodextrins, broadband present in the spectrum with the maximum absorption at 3288 cm-1

for αCD, 3314 cm-1

for βCD, 3302 cm-1

for HPαCD and 3330 cm-1

for HPβCD is characteristic.

Complex 5FU/HPβCD>5FU/HPαCD>U/HPβCD>U/HPαCD>5FU/βCD>U/βCD>5FU/αCD>UαCD

T (°C) 299.2 262.1 260.7 258.7 254.1 253.2 252.1 239.4

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This band is attributed to the O-H stretching vibration in symmetric and anti-symmetric modes and

is affected when the inclusion complex was formed [3]. For the 5FU/αCD, U/αCD, 5FU/HPαCD

and U/HPαCD systems there are no significant band changes at approximately 3300 cm-1

. This band

is shifted much more for the complexes 5FU/HPβCDCD, U/HPβCD, U/βCD and 5FU/βCD; it can

be said that ligand inclusion occurs with the involvement of a larger number of hydrogen bonds [4].

Fig. 2.3 FT-IR spectra for: U, 5FU, cyclodextrins and inclusion compounds formulated for 1: 1

molar ratio (ligand/CD).

15

Quantum chemistry (QC) calculations were performed using the GAMESS software package

facilities installed on an IBM cluster of 65 computing units. The studied systems geometries were

optimized by using enhanced wave function sets: STO-3G, STO-6G, n311-6G, and finally, zeta

triple valence (TZV) function [5, 6]. Quantum chemistry calculations have shown that the results

obtained by applying DFT (density functional theory) confirm that inclusion complexes have been

formed between the considered ligands (U/5FU) and the cyclodextrins CD, CD, HPCD,

HPCD.

MPE energies (MPE = reciprocal distortion energy) are represented for the position of the

guest molecule inside the CD cavity, for all HG structures (CD/ligand) - Fig. 2.4. In Fig. 2.5, it was

observed for the R-configurations (the ligand molecule placed in the center of the small opening of

the CD parallel to it), that the plane of the 5FU molecule remained perpendicular to the Oz axis, thus

the molecule 5FU was strongly bound to the host molecule by hydrogen bonds (Fig. 2.5).

Fig. 2.4 The variation of MPE corresponding to the position of the guest molecule inside the CD

cavity for the HG assembly formed between: (left) - the U molecule with CD, CD, HPCD,

HPCD and (right) - the 5FU molecule with CD, CD, HPCD, HPCD

In the case of H-5FU-R complexes, it can be seen (Figure 2.4) that MPE (Mutual

Disturbance Energy) energies are significantly higher for HPαCD and HPβCD. It can be argued that

5FU forms more stable complexes with modified cyclodextrins than with native ones.

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Fig. 2.5 The final configurations of the H-5FU-R inclusion complexes: a) CD-5FU-R, b) CD-

5FU-R, c) HPCD-5FU-R, d) HPCD-5FU-R

Based on the obtained results, it was found that:

- Both U and 5FU molecules interact weaker with αCD, and U molecule interacts weaker than 5FU

molecule with HPβCD and βCD cyclodextrins.

- FTIR and DSC/TG data suggest inclusion complex formation of all synthesized samples,

indicating the existence of interactions between the constituent molecules of the inclusion complex.

- Considering calculated energies, QC results indicate higher stability for complexes of 5FU with

modified cyclodextrins than for complexes of 5FU with native cyclodextrins. The QC calculations

agreed with the results indicated by DSC analysis.

3. Cyclodextrin inclusion compounds with L- and D- alpha-amino acids stereoisomers

The complexation process between the optical isomers of the amino acids Arg, His and Trp

with αCD, βCD, HPaCD in the solid state was evaluated. It was performed for the first time a

systematic analytical study of this type of complexes prepared in 1:1 molar ratio by the co-

precipitation method [7]. Thermal analysis data showed that LArg/βCD, DArg/βCD, LHis/βCD,

LTrp/βCD and DTrp/βCD are the most thermally stable. In the case of the complexes: LArg/αCD,

LArg/HPαCD, DArg/HPαCD, LHis/αCD, LHis/HPaCD, DHis/αCD, DHis/HPaCD, DHis/βCD,

LTrp/HPaCD have been made illustrations showing the variation of the starting (Ton), transition (Tm)

and enthalpy (ΔH) temperatures corresponding to the melting endotherm (Figures 3.1, 3.2 and 3.3).

Thus, comparisons could be made between these inclusion complexes. The complex with the highest

17

thermal stability could be determined, considering as criteria the lowest value of ΔH and the highest

transition temperature.

Fig. 3.1 Variation of onset temperature, transition temperature and enthalpy for the melting

endotherm of LArg/αCD, DArg/αCD, LArg/HPαCD

Fig. 3.2 Variation of onset temperature, transition temperature and enthalpy for the melting

endotherm of complexes formed between His isomers and αCD, HPαCD and for DHis/βCD

complex

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Fig. 3.3 Variation of onset temperature, transition temperature and enthalpy for the melting

endotherm of complexes formed between Trp isomers and αCD, HPαCD

Considering for the studied systems the correlation of the resulted data, the following were found:

1) in the case of His, a higher affinity of cyclodextrins for the levogir isomer is observed;

2) in the case of Arg, both isomers are preferred with higher affinity than native cyclodextrins;

3) the most stable inclusion compounds are obtained between the Trp and αCD isomers, βCD,

HPαCD, and the most energetically favored ones are those formed with native cyclodextrins.

4) it can be concluded that the amino acids Arg, His and Trp interact with αCD, βCD more strongly

than with HPαCD.

4. The study of interaction between cinchonine and cyclodextrins

Solid form complexes formed between Cinc and cyclodextrins: βCD, HPβCD, γCD for

molar ratios 1:1, 2:1 and 1:2 were synthesized by co-precipitation method using a mixed water

solution of alcohol 50% Vol.

The differences regarding the thermal behavior of systems formed between Cinc and native

βCD and γCD cyclodextrins, are shown in Fig. 4.1.

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Fig. 4.1 Variation of onset temperature, transition temperature and enthalpy for the melting

endotherm of complexes formed between Cinc and cyclodextrins considering the molar ratios of

1:1, 2:1 and 1:2 (guest:host)

DSC/TG analysis of the compounds formed between Cinc and HPβCD showed the formation

of the complexes by partial inclusion of Cinc molecule into HPβCD cavity, for the 1:1 and 1: 2

molar ratios (guest: host), (presented in Fig. 4.2 and 4.3).

220 240 260 280 300 320 340 360 380 400

HPCD

Cinc/HPCD(2:1)

Cinc/HPCD(1:2)

Cinc/HPCD(1:1)

Cinc

Flu

x c

alo

ric

(u

. a

.) E

nd

o

Temperatura (C)

Fig. 4.2 DSC curves for pure Cinc, HPβCD and

inclusion complexes formed from different molar

ratios.

Fig. 4.3 TG/DTG curves for inclusion

complexes between Cinc and HPβCD for

different molar ratios

20

Based on thermal analysis data, it was found that:

• There is a possibility of favorable energy interactions between the components of Cinc systems

and native cyclodextrins: βCD and γCD, for the 1:1 and 1:2 molar ratios (guest:host), and for

Cinc/HPβCD(2:1), by partially inclusion of the two Cinc molecules into the HPβCD cavity.

• The physical size of the CD cavity, the solvent and the CD rim functionalization influence the

formation of inclusion complexes.

5. Study of interaction between doxorubicin hydrochloride (Dox) and γ-cyclodextrin

The complexation process between Dox and γCD in the liquid and solid state was evaluated,

performing for the first time a systematic thermodynamic study of this interaction.

The non-covalent interaction between Dox and γCD was evaluated in the liquid state using

pH measurements, UV-Vis spectroscopy and ITC measurements.

The pH of each Dox/γCD molar ratio was monitored when the complex stoechiometry was

evaluated by the continuous variation method and the results are shown in Fig. 5.1.

Fig. 5.1 Stoechiometry determination by the

continuous variation method for the Dox/γCD

system at 25 ° C (λ = 473 nm).

Fig. 5.2 Variation of temperature equilibrium

constant for Dox/γCD system.

The results showed for Dox/γCD inclusion complex the 1:1 stoichiometry. The data on the

thermodynamic processes that took place in the liquid state confirmed that the interaction was

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thermodynamically favorable having a negative variation of Gibbs free energy. It was highlighted

that the complexation process is predominantly driven by entropy and moderately driven by

enthalpy, this was resulted from van't Hoff analysis based on UV-Vis data and ITC measurements

(Figures 5.2 and 5.3).

Fig. 5.3 ITC data obtained for the binding interaction

between Dox (40.4 mM) and γCD (1.01 mM) in aqueous

solution at 25 ° C (p = 0.1 MPa). The upper panel shows

gross ITC data. The lower panel shows the integrated and

normalized data for the representation of heat values

according to the molar ratio, and the continuous line

represents the least squares estimated curve.

The TG/DTG analysis of pure and Dox/γCD complexes was performed in the temperature range

from 25 °C to 450 °C, and the resulting curves are shown in Fig. 5.4 and 5.5.

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Fig. 5.4 Curbele DSC pentru substanțele pure și

pentru complexul de incluziune

Fig. 5.5 Curbele TG si DTG (inset) pentru

substanțele pure și pentru complexul Dox/γCD

The thermal behavior of the Dox/γCD complex is presented in the DSC/TG thermograms of

Fig. 5.4 and 5.5. In the temperature range from 25 °C to 130 °C, the dehydration process takes place

in three successive steps with a total mass loss of 2.6%. After the dehydration phenomenon,

Dox/γCD presents a sequential decomposition process, as evidenced by the DTG profile asymmetry

(inserted in Fig. 5.5) [8].

The morphology of pure compounds as well as the Dox/γCD complex were evaluated using

SEM, and the obtained images are shown in Fig. 5.6.

Fig. 5.6 SEM images obtained at 25 ± 1 ° C for: a) Dox, b) Dox/γCD inclusion complex and

c) γCD.

It can be observed that the morphology of the Dox/γCD complex presents a new solid phase

with no presence of the morphological characteristics of Dox or γCD.

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6. General Conclusions. Original contributions

The aim of the thesis is the thermoanalytical study of complexes of cyclodextrins and the

correlation of the thermodynamic behavior with the structural and morphological aspects involved

in their formation.

In the thesis, characterization of ligand-CD systems was performed by differential dynamic

calorimetry, thermogravimetry, isothermal calorimetry titration, and the results were supported by

spectral and morphological data.

In this work, the synthesis of inclusion complexes was accomplished by "melting and

solution" and "coprecipitation" methods, starting from the 1: 1 molar ratio.

Ligands were chosen to belong to different structural classes to investigate complexation

behavior under the influence of various factors such as: molar ratio, synthesis mode, solubility.

The objectives proposed in this paper led to the following conclusions and original contributions:

- In this thesis a systematic thermoanalytic study of the solid powders of the complexes

formed between the ligands: uracil (U), 5-fluorouracil (5FU) and cyclodextrins: αCD,

HPαCD, βCD și HPβCD, were done. The resulted values of the decomposition temperatures

of the complexes were used in order to evaluate the stability.

- Quantum chemistry calculations were done for systems formed between U, 5FU and

cyclodextrins: αCD, βCD, HPαCD, HPβCD. The values of the binding, deformation and

mutual perturbation energy values were established by DFT calculations.

- The investigation of systems formed between optical isomers of amino acids: arginine (Arg),

histidine (His), tryptophan (Trp) wih cyclodextrins: αCD, βCD, HPαCD was accomplished.

The experimental results related to thermal, spectral and morphological behavior solid

powders of the complexes formed between His, Arg and cyclodextrins, were reported.

Comparative issues of the isomers of amino acids considered with cyclodextrins have been

discussed considering the contribution of driving forces of the complexation process.

- It was shown that native cyclodextrins αCD and βCD interact strongly with amino acids than

HPαCD. Based on the obtained data, it was found that the size of the CD cavity and the

existence of the functionalization of CD cavity can acting as chiral selectors for the optical

isomers of amino acids.

24

- The influence of structural factors on the thermodynamic behavior was discussed for the first

time and the corresponding energetic parameters were specified for both the aqueous

solution and solid powder complex formed between doxorubicin (Dox) and γCD (Dox/γCD).

For a 10-5

M aqueous solution of Dox and γCD, the stoichiometry was found to be 1:1. In

solid state, FTIR and SEM thermal data confirm that the Dox/γCD complex was formed by

partial inclusion of the Dox molecule in the γCD cavity.

- For the first time, the complexes formed between cinconin (Cinc) and cyclodextrins were

investigated in solid state and it was shown that the presence of the cosolvent influences the

complexation process. Thus, the thermal analysis data and FTIR results showed the existence

of interactions between the components without inclusion of Cinc in the CD cavity for

systems formed between Cinc and native cyclodextrins, considering the molar ratios of 1:1

and 1:2. An important role is played by CD functionalization, justifying for Cinc/HPβCD the

posibility to form both the 1:1 molar ratio complex and also the 1: 2 molar ratio complex.

- In the case of ligand molecules (Cinc and Dox) made up of two structural units, it was

observed that for the 1:1 molar ratio (in the solid state), a partial inclusion of the ligand

molecule in the γCD cavity was happend.

- Based on the thermodynamic parameters obtained from the DSC, comparative diagrams

were done considering the variation of onset temperature, transition temperature and

enthalpy for the melting endotherm of the complexes formed without inclusions into CD

cavity of the guest molecule (systems formed between Cinc or optical isomers of the amino

acids with cyclodextrins). It was shown there is a tendency that the lowest enthalpy values

correspond to the highest values of the start and transition temperature of the melting

endotherm. This can be considered as a selection criterion for the determination of stability

within the same set of complexes formed without inclusion of guest.

- The importance of the driving forces in the complexation process, such as: hydration,

presence of cosolvent, the possibility of self-assembly, the size and functionalization of CD

cavity and the structural characteristics of the ligand molecule were discussed.

- It was emphasized that the diluted aqueous solutions used in synthesis of the complexes

determined a 1:1 stoichiometry of the complexes formed between cyclodextrins and the

ligands: Dox, Cinc, U and 5FU.

25

- In this thesis the SEM images corresponding to inclusion complexes formed between the

considered ligands and cyclodextrins were analyzed for the first time.

- In this work, the importance of characterization of pure substances before the synthesis steps

is underlined. Considering the thermal analysis of cyclodextrins, it is pointed out that the

presence of different thermal behavior can be induced by a series of factors such as:

commercial variety, storage time and conditions.

- The results obtained in this thesis emphasize the importance of methods of thermal analysis

in the study of complexes formed with cyclodextrins, together with other methods of

investigation.

7. Perspectives of expanding and continuing research

The properties of cyclodextrins have led to the development of many research branches, and

inclusion complexes formed with cyclodextrins constitute an enormous research area with new

perspectives on combinatorial chemistry.

Appreciation of complexity stability and reactivity are important in application development,

so data on energy factors are needed in areas such as molecular engineering and supramolecular

chemistry.

The scientific data resulting from this study may be starting points for new research such as

chiral separation and catalysis, as well as starting points for making realistic models of more

complicated processes such as macromolecular hydration, hydrophobic proteins, various types of

interaction etc.

The new research will allow experimental measurements to be expanded in terms of the

influence of physicochemical factors such as: the concentration of the constituent molecules of the

system, the concentration and type of the solvent, the pH, the temperature, complexes stoichiometry

control etc.

Bibliography (selection)

[1] Szejtli J, Osa T (eds). Comprehensive supramolecular chemistry, vol 3: Cyclodextrins.

Pergamon, New York, 1999.

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[2] Neacsu A, Pincu E, Munteanu G, Munteanu C, Meltzer V. Characterization and comparison of

the solid state inclusion compounds of α- , β- cyclodextrins and its 2-hydroxypropyl derivatives with

uracil and 5-fluorouracil, Rev. Chim. 2017;12:2756-2760.

[3] Rocha BA, Rodrigues MR, Bueno PCP, de Mello Costa-Machado AR, de Oliveira Lima Leite

Vaz MM, Nascimento AP, Barud HS, Berretta-Silva AA. Preparation and thermal characterization

of inclusion complex of Brazilian green propolis and hydroxypropyl-β-cyclodextrin. J Therm Anal

Calorim. 2012;108:87-94.

[4] Kavitha K, Srinivasa RA, Nalini CN. An investigation on enhancement of solubility of 5

fluorouracil by applying complexation technique - characterization, dissolution and molecular -

modeling studies. J App Pharm Sci. 2013;3:162-166.

[5] Schmidt KK, Baldridge, Boatz JA, Elbert ST, Gordon MS, Jensen JH, Koseki S, Matsunaga N,

Nguyen KA, Su SJ, Windus TL. General Atomic and Molecular Electronic Structure System. J

Comput Chem 1993;14:1347-1363.

[6] Binning RC, Curtiss LA. Compact contracted basis sets for third‐row atoms: Ga–Kr. J Comput

Chem 1990;11:1206-1216.

[7] Neacsu A, Neacsu A, Contineanu I, Munteanu G, Tanasescu S, Solid state study of the inclusion

compounds of alpha-, beta- cyclodextrins with D-, L-tryptophan isomers, Rev Rou Chim.

2013;58(11):863-870.

[8] Neacșu A. Physicochemical investigation of the complexation between γ-cyclodextrin and

doxorubicin in solution and in solid state. Thermochimica Acta. 2018;661:51-8.