β-CD conjugated dendrimer

II

Preface

Dendrimers are a new class of synthetic polymers based on a well-defined

cascade motif. These macromolecules may be synthesized to reach the size of

nanoobjects having dimensions similar to proteins. Dendrimers are regarded as

ideal candidates for use in biomedical applications. Dendrimers containing

cyclodextrin exhibited that the -CD-grafted biodegradable dendrimers is a

potential candidate as an efficient drug carrier systems due to its relative

stability in aqueous solution and its drug encapsulation and release properties.

Cyclodextrins and their complexes constitute a fascinating subject that, due to

its specific chemical structure. This great attention makes today’s CD science

and technology extremely involved in a large number of important areas of

research covering both basic and applied science in chemistry and biology.

Their ability to modify the behavior of trapped guests is being used for example,

in chemistry, biology, food, pharmacy/medicine and agriculture.

This book reflects the height of the latest knowledge of these sweet nanocavities

to host different kinds of guests on the drug delivery systems.

I would like to thank the University of Tabriz and Tabriz University of medical

science, RCPN that enabled my research at faculty of chemistry and leading to

my deeper understanding of supramolecular complexes. Finally, I hope this

volume will be beneficial to readers of this book who like dendrimer and

cyclodextrin chemistry to enter the fascinating field of supramolecular

chemistry.

Prof. Hassan Namazi

Yousef Toomari

University of Tabriz, Iran, July 2012

III

Table of Contents

Preface ................................................................................................................. II

Table of Contents ............................................................................................. III

List of Figures .....................................................................................................V

List of Tables ................................................................................................. VIII

List of Symbols and Abbreviations ................................................................ IX

Chapter 1: Background and Literature Review .............................................. 1

1.1. Dendrimers ................................................................................................. 11.1.1. Introduction .......................................................................................... 11.1.2. History and Progress of Dendrimer Research ...................................... 21.1.3. Molecular Structure of Dendrimers ..................................................... 31.1.4. Synthesis Methods ............................................................................... 31.1.5. Properties of Dendrimers ..................................................................... 51.1.6. Application of Dendrimer in Drug Delivery Systems ....................... 141.1.7. Mechanism of Drug Delivery Using Dendrimers .............................. 151.1.8. Release of Guest Molecules ............................................................... 221.1.9. Modification of Periphery Groups of Dendrimer .............................. 231.1.10. Aim of Work .................................................................................... 25

Chapter 2: Experimental Section .................................................................... 27

2.1. Materials and Methods ............................................................................. 272. 1.1. Materials ............................................................................................ 272.1.2. Instrumental Measurement ................................................................. 28

2.2. Methods .................................................................................................... 292.2.1. Preparation of , -di(chlorocarbonylmethylene)poly(ethyleneglycol) (ClOC-PEG-COCl) .................................................... 292.2.2. Preparation of G1 Using Thionylchloride ......................................... 292.2.3. Preparation of G2 Using DCC ........................................................... 302.2.4. Preparation of G3 Using DCC ........................................................... 312.2.5. Synthesis of G1- -CD Using DCC .................................................... 322.2.6. Synthesis of G2- -CD Using DCC .................................................... 332.2.7. Synthesis of G3- -CD Using DCC .................................................... 34

IV

2.2.8. Synthesis of G1-NH- -CD Using DCC ............................................. 352.2.9. Synthesis of G2-NH- -CD Using DCC ............................................. 372.2.10. Synthesis of G3-NH- -CD Using DCC ........................................... 382.2.11. Preparation of Gn- -CD (n=1–2) and Gn-NH- -CD (n=1–2)/NLXComplexes .................................................................................................... 402.2.12. In Vitro Release Studies ................................................................... 402.2.13. Particle Size Analysis ....................................................................... 41

Chapter 3: Results and Discussion .................................................................. 42

3.1. Investigation of Dendrimer Synthesized Based on Citric acid ................ 423.1.1. Synthesis of G1 Compound ............................................................... 423.1.2. Synthesis of G2 Compound ............................................................... 453.1.3. Synthesis of G3 Compound ............................................................... 473.1.4. Synthesis of G1- -CD Compound ..................................................... 513.1.5. Synthesis of G2- -CD Compound ..................................................... 553.1.6. Synthesis of G3- -CD Compound ..................................................... 593.1.7. Synthesis of G1-NH- -CD Compound .............................................. 623.1.8. Synthesis of G2-NH- -CD Compound .............................................. 693.1.9. Synthesis of G3-NH- -CD Compound .............................................. 73

3.2. Investigation of Nanocarriers Size ........................................................... 773.3. In Vitro Release Studies ........................................................................... 80

3.3.1. Loading of NLX Drug Molecule into the Dendrimers ...................... 803.3.2. Calculating the Amount of the Trapped and Released Drug Molecule ...................................................................................................................... 803.3.3. Studying the Controlled Release of NLX from the Nanocarriers...... 82

Chapter 4: Conclusions .................................................................................... 87

Chapter 5: References ...................................................................................... 88

V

List of Figures

Figure 1. Typical architecture of dendrimer. ........................................................ 3

Figure 2. Schematic drawing showing the divergent method for synthesis of

dendrimers. ............................................................................................................ 4

Figure 3. Schematic drawing showing the convergent method for synthesis of

dendrimers. ............................................................................................................ 5

Figure 4. The close dimensional size (nm) of selected proteins to respective

generations of PAMAM dendrimers [12]. ............................................................ 8

Figure 5. Schematic illustration of the result of back-folding in an increased

molecular density in the interior of a dendrimer...................................................9

Figure 6. The effect of the solvent on the conformational state of a dendrimer. 12

Figure 7. Drawing of a dendrimer carrier encapsulating hydrophobic drug

molecules in the dendrimer’s voids to increase their aqueous solubility and

control their release rate. ..................................................................................... 17

Figure 8. Schematic drawing showing a dendrimer-drug conjugate where the

drug molecules (red ovals) are either directly coupled (solid lines) to

dendrimer’s surface groups or via a pH-sensitive linkage (blue rectangle). ...... 22

Figure 9. Chemical structure of naltrexone. ....................................................... 27

Figure 10. 1H NMR spectrum of G1 in DMSO-d6. ............................................ 44

Figure 11. FT-IR spectrum of G1. ...................................................................... 44




Figure 15. 1H NMR spectrum of G3 in acetone deuterium. ............................... 50


Figure 17. 1H NMR spectrum of G1- -CD in DMSO-d6. .................................. 54

Figure 18. FT-IR spectrum of G1- -CD. ............................................................ 54

Figure 19. 1H NMR spectrum of G2- -CD in DMSO-d6. .................................. 58

VI



Figure 22. 1H NMR spectrum of -CD-Tos in DMSO-d6. ................................. 65

Figure 23. 1H NMR spectrum of G1-NH- -CD in DMSO-d6. ........................... 68

Figure 24. FT-IR spectrum of G1-NH- -CD. ..................................................... 68

Figure 25. 13C NMR spectrum of G1-NH- -CD in DMSO-d6. .......................... 69







Figure 32. The size distribution profiles of G1- -CD and G2- -CD dendrimers

estimated by DLS. ............................................................................................... 78

Figure 33. Particle Diameter of G1-NH- -CD by using LDPSA. ...................... 79



Figure 36. Release of NLX from Gn (n=1-3)/NLX complexes as a function of time

and pH (pH 7.4, 37 ˚C). ...................................................................................... 84

Figure 37. Release of NLX from Gn (n=1-3)-NH- -CD/complexes as a function of

time and pH (pH 7.4, 37 ˚C). .............................................................................. 84

Figure 38. Release of NLX from Gn (n=1-2)- -CD/complexes as a function of time

and pH (pH 7.4, 37 ˚C). ...................................................................................... 85

Figure 39. Release of NLX from G3, G3- -CD and G3-NH- -CD/complexes as a

function of time and pH (pH 1, 37 ˚C). .............................................................. 85

Figure 40. Release of NLX from G3-NH- -CD/complexes as a function of time

and pH (pH 1, 7.4 and 10 and 37 ˚C). ................................................................. 86

Figure 41. Release of NLX from G2- -CD/complexes as a function of time and

pH (pH 1, 7.4 and 10 and 37 ˚C)......................................................................... 86

VII

List of Schemes

Scheme 1. Synthesis of G1. ................................................................................ 43



Scheme 4. Synthesis of G1- -CD. ...................................................................... 53

Scheme 5. . Synthesis of G2- -CD. .................................................................... 57

Scheme 6. Synthesis of G3- -CD. ...................................................................... 61

Scheme 7. Hierarchical synthesis of -CD-NH2. ............................................... 64

Scheme 8. Synthesis of G1-NH- -CD. ............................................................... 67

Scheme 9. Synthesis of G2-NH- -CD. ............................................................... 71

Scheme 10. Synthesis of G3-NH- -CD. ............................................................. 75

VIII

List of Tables

Table 1. Physical characteristics of PAMAM dendrimers. .................................. 7

Table 2. Diameter of nanocarriers obtained using laser diffraction particle size

analyzer. .............................................................................................................. 78

Table 3. Drug loading efficiency of Gn(n=1-3), Gn(n=1-3)- -CD and Gn(n=1-3)-NH- -

CD/NLX complexes. ........................................................................................... 81

IX

List of Symbols and Abbreviations

13C NMR carbon-13 nuclear magnetic resonance spectroscopy 1H NMR proton nuclear magnetic resonance spectroscopy

% percentage(s)

% w/v percentage weight per volume

vibration modes

chemical shift

extinction coefficient

celsius temperature or centigrade scale

aq aqueous

br broad

-CD -cyclodextrin

-CD-NH2 6-Deoxy-6-amino- -cyclodextrin

-CDTos 6-Deoxy-6-(p-toluenesulfonyl)- -cyclodextrin

-CD-N3 6-Deoxy-6-azido- -cyclodextrin

CD cyclodextrin

CDs cyclodextrins

cm-1 wavenumbers

Co company

D2O deuterium Oxide

DDS drug delivery systems

DCC dicyclohexylcarbodiimide

DLS dynamic light scattering

DMF N, N-dimethylformamide

DMSO dimethyl sulfoxide

DMSO-d6: perdeuterated dimethyl sulfoxide

e.g. for example

FT-IR fourier transform infrared spectroscopy

X

Fig. figure

G1- -CD Gn(n=1)- -cyclodextrin



G1-NH- -CD Gn(n=1)-NH- -cyclodextrin



G generation

g gram

m medium/multiplet

MHz mega Hertz

mol mole

MS mass spectrometry

nm nanometer(s)

NLX naltrexone

PAMAM poly(amidoamine)

PD pharmacodynamics

pH -log of proton concentration

PK pharmacokinetic

PPI poly(propylene imine)

ppm parts per million

PEG polyethylene glycol

PEO poly(ethylene oxide)

PEG–COCl , -di(chlorocarbonylmethylene) poly(ethyleneglycol)

Rf retention Factor

s strong/singlet

SEC size-exclusion chromatography

t triplet

TEM transmission electron microscopy

XI

THF tetrahydrofuran

TLC thin layer chromatography

TSA -toluenesulfonic acid

UV-vis ultraviolet-visible

v:v volume/volume

1

Chapter 1: Background and Literature Review

1.1. Dendrimers

1.1.1. Introduction

Dendrimers are a family of nanosized and perfect monodisperse polymers with

a regular and highly branched three-dimensional macromolecules and compact

spherical geometry in solution. Their name comes from the Greek word

“dendron”, meaning “tree” and meros meaning “part”. In comparison to the

traditional polymers, dendrimers are relative newcomers. Dendrimers differ

from many traditional polymers, such as linear and branched polymers, because

they are well defined size, with a high degree of molecular uniformity and

monodispersity, in addition to a highly functional surface [1-6]. Generally, low

generation dendrimers have an open structure, but as the generation increases

the structure becomes more spherical and dense. Dendrimers offer a plenty of

advantages compared to other architectural forms of polymers that have been

used in drug-delivery systems (DDS). Compared with classical linear polymers,

dendrimers offer several featured advantages as drug carrier applicants. These

advantages consist of: (1) high density and reactivity of functional groups on the

outside of dendrimers [7, 8]; (2) well-defined globular structure, predictable

molecule weight and monodispersity [9]; (3) controllable size [10-12]; (4) high

penetration abilities of dendrimers through the cell membrane [7, 13]; (5)

enhanced penetration and retention effect of dendrimers offers preferential

uptake of the materials by cancer tissues [8]; (6) programmed release of drugs

from the matrixes leads to reduced toxicity, increased bioavailability and

simplified dosing schedule [14, 15]. Commonly, the size, shape, and surface

properties of the polymeric carriers greatly influence the pharmacodynamic

(PD) and pharmacokinetic (PK) behaviors of drugs encapsulated in/complexed

2

to/conjugated to the carrier [16]. In the case of dendrimers, the loading ability of

drug molecules and other bioactive agents can be altered by varying dendrimer

generations, the water solubility, biodistribution, circulation time in blood and

therapeutic efficiency of drugs in dendrimer-based formulations can be tuned by

varying dendrimer surface components, the release of drugs from dendrimer

scaffolds can be controlled by using different degradable linkers between

dendrimers and drugs, and the specific accumulation of the dendrimer-based

therapeutics can be achieved by further modifying the dendrimers with targeting

moieties [16]. These properties together prove dendrimer perfect candidates in

the design of new DDS. This thesis will give an overview of the specific use of

modified dendrimers with -CD in drug delivery and how it relates to the

specific properties of these materials.

1.1.2. History and Progress of Dendrimer Research

The successful laboratory synthesis of dendrimers initially reported by Vögtle

and his group at the end of the 1970’s [17] with the so-called “cascade

molecules”. The concept of repetitive growth with branching was first reported

in 1978 by Vögtle. The result was a new core-shell macromolecular

architecture, now recognized as dendrimers. This was followed closely by the

Tomalia group [2], the first paper showing in great detail the preparation of

poly(amidoamine) (PAMAM) dendrimers appeared in 1985, the same year a

communication reported the synthesis of arborols by Newkome et al. [18], and

others to give rise to the larger dendritic structures [19, 20]. These

hyperbranched molecules were called “dendrimers” or “arborols” (from the

Latin “arbor” also meaning tree).

3

1.1.3. Molecular Structure of Dendrimers

In contrast to traditional polymers, dendrimers are unique core–shell structures

possessing three basic structural components (Fig. 1): a core (central point

known as the initiator) (I), branched units (II), and end groups (the outer shell or

periphery) (III).

Figure 1. Typical architecture of dendrimer.

1.1.4. Synthesis Methods

1.1.4.1. Divergent Approach

In the divergent synthesis strategy, the construction of the dendrimer takes place

in a stepwise manner starting from the core and building up the molecule

towards the outside using two basic procedures (1) coupling of the monomer

and (2) deprotection or conversion of the monomer end-group to create a new

reactive surface functionality and then coupling of a new monomer. The process

4

is repeated for several generations and a dendrimer is built layer after layer. The

divergent synthesis was initially applied extensively in the synthesis of

poly(propylene imine) (PPI) and PAMAM dendrimers, but has also found wide

use in the synthesis of dendrimers having other structural designs [20, 21]. The

divergent technique is successful for the construction of large quantities of

dendrimers. Problems happen from side reactions and imperfect reactions of the

ending groups that lead to structure defects. To avoid side reactions and to force

reactions to achievement large excess of reagents is required. It causes some

difficulties in the purification of the ultimate product [22-24] (Fig. 2).

Figure 2. Schematic drawing showing the divergent method for synthesis of

dendrimers.

1.1.4.2. Convergent Approach

Fréchet and Hawker first proposed an alternative approach to dendrimer

syntheses, known as the convergent method. The convergent synthesis strategy

was developed as a comeback to the limitations of the divergent synthesis [24,

25]. The convergent method, proceeds in the opposite direction, from the

periphery to the core that is from the outside inwards. When the growing

branched polymeric arms, called dendrons, are large enough, dendrons are

attached to a multifunctional core molecule. The convergent growth technique

5

has several benefits. In this method the number of reactive sites during the

creation process remains minimal leading to faster reaction rates and yields.

Another benefit of this approach is the large “molecular difference” between the

reactant molecule and the product, facilitating the separation of the reactants

from the product during the purification process (Fig. 3).

Figure 3. Schematic drawing showing the convergent method for synthesis of

dendrimers.

1.1.5. Properties of Dendrimers

1.1.5.1. Nanoscale Monodispersity

Dendrimers can be constructed with a well-defined molecular structure

(monodisperse), dissimilar to linear polymers. The monodispersity of

dendrimers has been studied broadly by size-exclusion chromatography (SEC),

mass spectrometry (MS), gel electrophoresis and transmission electron

microscopy (TEM) [26]. The level of monodispersity is controlled by the ability

of the synthetic chemist, as well as the isolation and purification techniques

utilized. Generally, convergent technique create the most closely monodisperse

dendrimers as determined by mass spectrometry. This is because the convergent

method allows purification at each step of the synthesis and removes cumulative

effects because of unsuccessful couplings [27]. However, mass spectrometry

6

has shown that PAMAM dendrimers produced by the divergent technique are

remarkably monodisperse for earlier generation (G= 0–5) [28].

1.1.5.2. Functionalization of Dendrimer Surface Groups

The periphery groups of dendrimers within a generational series can be

estimated at least three different styles, specifically as a flexible, semiflexible,

or inflexible functionalized scaffolding. Based on mathematically defined

dendritic branching rules, the several exterior exhibitions become more crowded

and inflexible as a function of growing generation level. Additionally,

dendrimers may be viewed as adaptable and can be surface-functionalized with

a great range of chemical and application features. The capacity to engineer and

control these factors provides a limitless list of possibilities for utilizing

dendrimers as modules for nanodevice design [28-31].

1.1.5.3. Nanoscale Dimensions and Shapes that Mimic Proteins

Considering the extraordinary structure control and nanoscale dimensions

observed for dendrimers, it is not amazing to find extensive interest in their use

as globular protein mimics (Fig. 4) [32]. Based on their systematic, size-scaling

properties, electrophoretic and hydrodynamic [33] behavior, dendrimers are

referred to as synthetic proteins [34, 35]. Substantial effort has been focused

recently on the use of dendrimers for site-isolation mimicry of proteins [1], drug

delivery [35], surface engineering [36], and light harvesting [37]. These

fundamental properties have in fact led to their commercial use as globular

protein replacements for gene therapy, immunodiagnostics [38], and a variety of

other biological applications. Dendrimers are generally produced in an

interactive sequence of reaction steps, in that each additional repetition leads to

higher generation dendrimer. Each of the new layer creates a novel ‘generation’

7

along with doubling the number of terminal groups (or active sites) and almost

double the molecular weight of the previous generation, too (Table 1) [1].

Generation Number of surface groups Molecular weight a Diameter (nm)b

0 4 517 1.5

1 8 1430 2.2

2 16 3256 2.9

3 32 6909 3.6

4 64 14215 4.5

5 128 28826 5.4

6 256 58048 6.7

7 512 116493 8.1

8 1024 233383 9.7

9 2048 467162 11.4

10 4096 934720 13.5

a Molecular weight is based on defect-free, ideal-structure, amine terminated

dendrimers. b Molecular dimensions determined by size-exclusion chromatography.

Table 1. Physical characteristics of PAMAM dendrimers.

8

Figure 4. The close dimensional size (nm) of selected proteins to respective

generations of PAMAM dendrimers [12].

1.1.5.4. Physicochemical Properties of Dendrimers

As the dendrimer grows, the various parts of the dendritic architecture (the

dendrimer structure may be divided into three parts: a: The surface of dendrimer

b: the outer shell c: the core) begin to show different features which are

improved with increasing generation. In the subsequent section

physicochemical properties of dendrimers will therefore be presented in more

general terms.

1.1.5.4.1. Dendrimers and the Effect of Molecular Growth

The conformational behaviors of a dendrimer upon growing to upper

generations are defined by (1) the molecular dimensions of the monomers (2)

the flexibility of the dendrons and (3) the capability of the interactions of end-

groups with each other (by hydrogen bonding creating a dense outer shell).

In 1983 de Gennes and Hervet presented limits to the growing of branched

molecules, paying due attention to the effect of steric hindrance [39]. Their

studies determined that upon growth, the outside of the dendrimer becomes

9

gradually crowded while the molecular density of the core section remains low

during the molecular growth. As no back-folding (dendrons folding into the

inside of the dendrimer) is taken into account, the increasing molecular

crowding in the outside shell will give a limitation on the generation number

that a starburst dendrimer can grow to.

Dendrimers with amine surface groups have a relatively high mobility due to

the absence of binding interactions between both the dendrimer arms and the

functional groups at the superficial. This larger mobility enables the dendrons to

back-folding the dendrimer interior as a significance of entropy [40]. Thus, the

structural behavior of the dendrimer upon growing to higher generations is

estimated by the ability of the surface functionalities to form a network with

each other through, e.g. hydrogen bonding or ion pairing thus consolidating a

dense outer shell [41]. Also, study based on molecular dynamics indicate that

low generation dendrimers have conformations with low degree of back-folding

(“density overlap”) compared to higher generations [42] (Fig. 5).

Figure 5. Schematic illustration of the result of back-folding in an increased

molecular density in the interior of a dendrimer.

10

1.1.5.4.2. Dendrimers and the Effect of pH

PAMAM and PPI dendrimers with amino terminal groups have basic exterior

groups as well as a basic inner. For these types of dendrimers with interiors

containing tertiary amines, the low pH area commonly leads to extended

conformations because of electrostatic repulsion between the positively charged

ammonium groups. At this pH (pH 4), the interior is getting increasingly

“hollow” as the generation number rises because of repulsion between the

positively charged amines both at the dendrimer exterior and the tertiary amines

in the internal.

At neutral pH, back-folding occurs which may be a consequence of hydrogen

bonding between the uncharged tertiary amines in the internal and the positively

charged exterior amines. At higher pH (pH 10) the dendrimer contract as the

charge of the molecule becomes neutral, obtaining a more globular structure

based on a loose dense network, where the repulsive forces between the

dendrimer arms and between the surface groups reaches a minimum. At this pH,

the conformation has a higher degree of back-folding as a significance of the

weak “inter-dendron” repulsive forces.

PPI dendrimers having carboxylic acid end-groups at pH 2 has the most

extended conformation because of the electrostatic repulsion between the

positively charged protonated tertiary amines, leading to a large radius of the

core, while at pH 6 radius of the dendrimer because of equals of positively

charged amines with amount of negatively charged carboxylic groups is

decreased. At pH 11 the electrostatic repulsion between the negative charged

forces the surface groups apart to give a more extended conformation with a

highly expanded exterior area [43].

11

1.1.5.4.3. Dendrimers and the Effect of Salt

In general molecular simulations in the PPI dendrimers display that high

concentration of salts has a strong influence on charged of PPI dendrimers and

lead to compact conformation of dendrimers, with a high degree of back-folding

[44, 45]. At low concentration of salts, the repulsive forces between the charged

dendrimer fragments resulted in an extended conformation to minimize charge

repulsion in the construction.

1.1.5.4.4. Dendrimers and the Effect of Concentration

The conformation of dendrimers with flexible constructions is not only affected

by small molecules like solvents, salts or protons, but may also be sensitive to

other dendrimers or surfaces which can have a great effect on the molecular

density and conformation of the dendrimer. Conformation of PPI dendrimers

(G4, G5) in a polar solvent like methanol achieved with small angle X-ray

scattering (SAXS) experiments show that upon increasing concentration

becomes increasingly contracted. This molecular contraction may diminish the

repulsive forces between the dendrimer molecules and growth the ability of the

dendrimers to display a more tight intermolecular packing [46].

1.1.5.4.5. Dendrimers and the Effect of Solvent

The role of the solvent to solvate the dendrimer structure is a very significant

factor in the conformational state of a dendrimer. Investigations with molecular

dynamics show that the dendrimer conformation depends on dendrimer

generation in different solvents [42]. Generally all generations of dendrimers

show extend of back-folding with decreasing solvation. However, the low

generation dendrimers show the highest affinity towards back-folding because

of poor solvation compared to the higher generation dendrimers.

12

NMR studies performed on PPI dendrimers indicate that an apolar solvent,

poorly solvates the dendrons favoring intramolecular interactions between the

dendrimer sections and back-folding. However, a weakly acidic solvent like

chloroform can act as a hydrogen donor for the interior amines in a basic

dendrimer like PPI, leading to an extended conformation of the dendrimer due

to extensive hydrogen bonding between the solvent and the dendrimer amines

[47]. Both experimental as well as theoretical studies on amino-terminated PPI

and PAMAM dendrimers display the trend that apolar aprotic (“poor”) solvents

induce higher molecular densities in the core area as a result of back-folding,

however polar (“good”) solvents solvate the dendrimer arms and induce a

higher molecular density on the dendrimer surface (Fig. 6) [48].

Figure 6. The effect of the solvent on the conformational state of a dendrimer.

1.1.5.5. Biocompatibility of Dendrimers

In order to incorporate dendrimers as biological agents, they have to accomplish

definite biological requests. The dendrimer should be:

13

non-toxic;

non-immunogenic (except for vaccines);

able to cross biobarriers (biopermeable) for example, the intestines, the

blood–tissue barriers, cell membranes, etc.;

able to stay in circulation in the biological system for the time needed to

have the desired clinical effect;

able to target to particular structures

Biological properties like the toxicity or the immunogenicity profile of a

dendrimer is to a large extent governed by the size of the dendrimer and by the

exterior groups existing on the particular dendrimer. Dendrimers have to show

low toxicity and be non-immunogenic in order to be broadly used in biomedical

applications. Till now, the cytotoxicity of dendrimers has been chiefly

investigated in vitro, though; a few in vivo studies have been reported. Duncan

and co-workers have studied the relationship between structure and

biocompatibility of PAMAM, PPI, poly(ethylene oxide) (PEO) grafted

carbosilane dendrimers with cationic (NH2-terminated) and anionic (COONa-

terminated) dendrimers in vitro. They have reported that, regardless of structure,

cationic dendrimers were usually more hemolytic and cytotoxic effect at even

relatively low concentration in relationship to anionic dendrimers. Furthermore,

the cytotoxicity was found to be generation-dependent, with higher generation

dendrimers being the most toxic [49]. One more study reported that anionic

PAMAM dendrimers have displayed a significantly lower cytotoxicity than

cationic dendrimer using Caco-2 cells [50]. One way to decrease the

cytotoxicity of cationic dendrimers may reside in partial surface derivatization

with chemically inert functionalities such as PEG or fatty acids. Hydroxy- or

methoxy-terminated polyester dendrimers have been reported to have low

toxicity in both in vitro and in vivo studies. During in vitro study, at high

concentration, these dendrimers have induced some inhibition of cell growth but

14

no increase in cell death was observed. The results make these new dendritic

motifs promising candidates for drug-delivery devices [51].

Immunogenicity is one of the vital biological properties of the dendrimers. In

brief, dendrimers have varying degrees of immunostimulatory properties

depending on their surface functionalities. Generally, the immunogenicity of

unmodified amino dendrimers is low, but seems to increase with increasing

generation of the dendrimer. Initial reports on the immunogenicity of

dendrimers showed low or modest immunogenicity of unmodified amino

terminated PAMAM dendrimers (G3–G7). However, later investigations

showed some immunogenicity of these dendrimers and found that modification

of amino-terminated PAMAM dendrimers with polyethylene glycol (PEG)

chains decreases immunogenicity and gives longer life-time in the blood stream

in comparison to unmodified dendrimers [52].

1.1.6. Application of Dendrimer in Drug Delivery Systems

Drug delivery is a significant feature in the design of a drug because the suitable

choice of delivery system can control the bioavailability, concentration profile

and unwanted side effects (by targeted delivery). Dendrimers and polymers

have some properties in common, when considering drug delivery. The chief

problem related with using polymers for drug delivery is the wide molecular

weight dispersal often found in polymers, which in terms can lead to

irreproducible pharmacokinetic behavior. Dendrimers are in general useful as a

part of the molecular toolbox for drug delivery. Toxicity and biodistribution of

dendrimers are significant in medical use of any drug, and for dendrimers this is

highly dependent on the actual structure of the dendrimer [49, 53].

General principles are being established for designing dendrimer structures as

delivery systems which contain: 1) negatively-charged and neutral dendrimers

are commonly biocompatible, whereas positively-charged species display

15

varying degrees of toxicity; 2) PEGylation growths water solubility and

dendrimer size, and can lead to progressed retention and biodistribution

characteristics; 3) therapeutic agents can be internalized into the void space

between the edge and core, or covalently attached to functionalized exterior

groups; 4) targeting moieties bound to the dendrimer surface can be used to

specially treat cancer cells with definite over-expressed receptor targets. As

dendrimer structures have become more specialized, improved efficiency in in

vitro and in vivo models is being realized. The applications of such systems

have been several-fold, including the use of dendrimers to increase drug

solubility and bioavailability, and to act as release modifiers and platforms for

drug targeting.

1.1.7. Mechanism of Drug Delivery Using Dendrimers

Drug delivery with dendrimers can basically take place by two different types of

mechanisms: (1) by in vivo degradation of a drug–dendrimer conjugate, where

the drug is covalently bound to the dendrimer, or (2) by utilizing host–guest

chemistry where the drug is existing as a guest in the dendrimer, and is released

because of changes in the physical environment such as pH, temperature or

simply released by diffusion out of the dendrimer. Two other significant issues

in drug delivery are targeted delivery to a specific type of cell or controlled

release from a depot, which may be present in circulation or imbedded in some

suitable tissue. Host–guest based systems are treated first in the following

sections, since they are the most studied dendrimer-based drug delivery

systems.

The ideal dendrimer carrier should show high aqueous solubility and drug-

loading ability, biodegradability, low toxicity, favorable retention and

biodistribution characteristics, specificity, and suitable bioavailability. In

dendrimer-based drug delivery, a drug is either non-covalently encapsulated in

16

the inside of the dendrimer or covalently conjugated to form macromolecular

prodrugs.

An area that has attracted great interest is the interaction between drugs and

dendrimers. Numerous types of interactions have been discovered, which can be

generally subdivided into the entrapment of drugs within the dendritic

architecture (involving electrostatic, hydrophobic and hydrogen bond

interactions) and the interaction between a drug and the surface of a dendrimer

(electrostatic and covalent interactions). In this overview a range of drug/model

compound–dendrimer interactions will be considered together.

1.1.7.1. Noncovalent Encapsulation of Drugs

The nature of drug encapsulation inside a dendrimer may be simple physical

entrapment, or can involve non-bonding interactions with particular structures

within the dendrimer. Much of the work in this field has been based on the

hypothesis that dendrimers have a hollow core and a dense shell [7], and in fact,

much of the text based on drug encapsulation supports this theory (Fig. 7).

Primary studies of dendrimers as potential delivery systems focused on their use

as unimolecular micelles and ‘dendritic boxes’ for the noncovalent

encapsulation of drug molecules. For example, hydrophobic drugs and dye

compounds were incorporated into several dendrimer cores [54-57]. An

advantage of using dendritic unimolecular micelles rather than conventional

polymeric micelles is that the micelles structure is maintained at all

concentrations because the hydrophobic pieces are covalently attached.

Although the introduction of stabilizing PEO chains on the dendrimer outside

has expanded the scope of dendritic unimolecular micelles to incorporate

anticancer drugs such as 5-fluorouracil [58], methotrexate [15] and doxorubicin

[15] and can slow the drug release rates in these systems to some extent, this

method has yet to be demonstrated as a general strategy.

17

The encapsulation efficiency of drug molecules is influenced by dendrimer size,

exterior structure, solvent pH and functionality of the drug molecule. Generally,

higher generation dendrimers explain improved capability for drug

solubilization compared to lower generation dendrimers because of the larger

void volume available for drug encapsulation [59], and enhanced electrostatic

interactions with increasing dendrimer generation [60-63].

The encapsulation of guest molecules which is based on non-covalent

interactions (hydrophobic interactions, physical entrapment, hydrogen bonding,

electrostatic bonding, or a combination of these methods) will be discussed in

more detail below with the help of examples.

Figure 7. Drawing of a dendrimer carrier encapsulating hydrophobic drug

molecules in the dendrimer’s voids to increase their aqueous solubility and

control their release rate.

18

1.1.7.1.1. Hydrophobic Interactions

The first method, which is most broadly applied, is based on the nature of the

micelles core: Hydrophobic cores interact favorably with hydrophobic guest

molecules [57, 64, 65]. Dendrimers consisting of an apolar core and polar shell

have been referred to as “unimolecular micelles”. Unlike usual micelles, the

dendritic structure is independent of dendrimer concentration [56, 65-68], i.e.

the dendrimers did not have a critical micelle concentration. The unimolecular

micelles structure of dendrimers was reported by Newkome et al. [56, 69]. The

ability of dendritic micelles to solubilize hydrophobic guest molecules in

aqueous media through hydrophobic interactions was first proved by Newkome

et al [70]. The location of the guest is not controlled nor is it known. The design

of dendrimers with well-defined and localized cavities or binding sites can be

realized by the specific incorporation of a hydrophobic core, hydrogen-bonding

moiety, or a metal ion coordination site.

PEG has been used to alter dendrimers in the design of solubilizing and drug

delivery systems [15, 58, 59, 71-75]. PEG is naturally conjugated to the outward

of a dendrimer to supply a hydrophilic shell around a hydrophobic dendritic

core to form a unimolecular micelle. PEG is of specific attention in the design

of dendrimer systems for pharmaceutical applications due to its high water

solubility, biocompatibility and ability to modify the biodistribution of carriers

[76]. Water-soluble dendritic unimolecular micelles of G3 dendrimer with

surface shell of PEG chains was synthesized by Liu et al. [69, 71] and the drug

loading of indomethacin was examined. The drug-loaded dendrimer supplied

sustained release of indomethacin over a period of approximately 30 h.

19

1.1.7.1.2. Physical Entrapment

Meijer et al. [54, 55] developed the famous ‘dendritic box’ idea, whereby

hydrophobic guest molecules are physically entrapped inside the internal

cavities of the dendrimers. During the synthetic process guest molecules could

be entrapped inside the cavities of the dendritic boxes, with a dense exterior

shell preventing dispersion from the structures, even after solvent extraction,

prolonged heating or sonication. The guest molecule becomes physically

trapped inside the core and can be released only when the ‘dense shell’ is

disrupted. The synthesis of dendritic boxes based on PPI dendrimers with

primary amine end groups were synthesized by Jansen et al [54, 77]. The

inflexible shell end group was obtained through modification with a bulky

amino acid derivative to yield a dense and rigid chiral shell with solid-phase

properties and a flexible core capable of entrapping molecules. A number of dye

molecules were encapsulated in the dendritic box, for example, up to 4

molecules of Bengal Rose could be encapsulated per dendrimer.

1.1.7.1.3. Hydrogen Bonding Interactions

Hydrogen Bonding interactions are possible between guest molecules and the

core or the exterior of dendritic micelles if appropriate functional groups are

existing on both components [78].

1.1.7.1.4. Electrostatic Interactions

The presence of large numbers of ionizable groups on the periphery and interior

of dendrimers supplies an interesting prospect for electrostatic attachment of

many ionizable drugs, providing the resulting complex retains adequate water

solubility [79-81]. For example, full generation PAMAM dendrimers have

primary amine ending groups (–NH2) on the periphery and tertiary amine

20

groups ( N–) positioned at the branching positions in the core both are

titratable having pKa values of 10.7 and 6.5, respectively [82].

The non-steroidal anti-inflammatory drug such as ibuprofen drug has been most

widely complexed to full generation of PAMAM dendrimers. Electrostatic

interaction can occur between the carboxyl groups of this poorly acidic drug and

the amine groups of the dendrimers. It has been estimated that approximately 40

ibuprofen molecules interact with G4 PAMAM dendrimer at pH 10.5 causing a

significant improvement of drug solubility [83].

The solubility improvement of the poorly water-soluble drug indomethacin in

the presence of a series of G4 and G4.5 PAMAM dendrimers and also G4

dendrimers with exterior hydroxyl groups was examined by Chauhan et al. [84].

The increasing solubility of indomethacin with G4 dendrimer was explained on

the basis of electrostatic interaction between the carboxyl group of

indomethacin and the amino groups of the dendrimer. As no electrostatic

interaction can be expected between the G4.5 and drug, the increased solubility

was thought to be a result of molecular encapsulation resulting from non-

specific, non-covalent interactions. Possible association of weak hydrogen

bonding was also thought to be effective in causing an increase of solubility in

the attendance of the hydroxyl-terminated G4 dendrimer. All three types of

dendrimer were shown to be efficient in increasing the change of indomethacin

crossways skin in both in vitro and in vivo experiments.

1.1.7.1.2. Covalent Dendrimer–drug Conjugates

The covalent binding of drugs to the exterior groups of dendrimers through

hydrolysable or biodegradable connections offers the opportunity for a greater

control over drug release than can be succeed by electrostatic complexation of

drugs to the dendrimers (Fig. 8). An alternative methodology to the

development of dendrimers as anticancer drug carriers is to exploit their well-

21

defined multivalency for the covalent attachment of drug molecules to the

dendrimer periphery. The drug loading can be altered by varying the generation

number of the dendrimer, and release of the drug can be controlled by

incorporating degradable linkages between the drug and dendrimer. For

example, Duncan and co-workers [85] have prepared conjugates of PAMAM

dendrimers with cisplatin, a potent anticancer drug with nonspecific toxicity and

poor water solubility. The conjugates display increased solubility, decreased

systemic toxicity and selective accumulation in solid tumors.

The preparation of multivalent folic acid conjugates of dendrimers [86] has

important suggestions for targeting to tumor cells, and the multivalent character

of dendrimers facilitates the attachment of several payloads, including targeting,

diagnostic and therapeutic molecules, as well as combinations of these agents.

Because expression of the folate receptor is improved in several human cancers

and restricted in most normal tissues [87], folic acid is an interesting candidate

for the active targeting of dendrimer–drug conjugates to tumors. Inspired by the

concepts of Esfand and Tomalia [35], and the multivalent dendrimer-folate and

dendrimer-methotrexate conjugates of Fréchet and co-workers [86], Quintana et

al. [88] prepared analogous PAMAM dendrimers with methotrexate conjugated

to their outside via either a stable amide or an ester linkage that could be

hydrolyzed under biological states. As expected, the introduction of folic acid

into these conjugates was found to increase their cellular uptake, resulting in an

increase in cytotoxicity of the methotrexate ester conjugate relative to that of the

free drug in vitro.

22

Figure 8. Schematic drawing showing a dendrimer-drug conjugate where the

drug molecules (red ovals) are either directly coupled (solid lines) to

dendrimer’s surface groups or via a pH-sensitive linkage (blue rectangle).

1.1.8. Release of Guest Molecules

Dendrimers have been used as devices for the release of hydrophobic

compounds [89]. Investigations have shown that these compounds structures are

able to release hydrophobic drugs in a sustained mode, as release has been

found at times to continue for several hours [89]. Release of hydrophobic

molecules from dendritic structures is dependent on both the construction of the

host and the size and shape of the guest molecules. The release of hydrophobic

compounds from amphiphilic dendritic polymers can also be affected by the

type of host-guest interactions involved. While weaker host-guest interactions

like hydrophobic interactions allow the almost complete release of the

encapsulated molecules from the micelle, partial release of the compounds has

23

been found to occur when stronger interactions (hydrogen and ionic bonding)

are involved. Release studies of lidocaine from a dendritic micelle have been

achieved in an aqueous medium [90]. At pH 7 lidocaine is hydrophobic and the

interactions between the core of the micelle and the drug are hydrophobic. The

release profile displays sustained character and it is detected that almost all the

drug molecules encapsulated are released after about 30 hours.

Encapsulation studies of ibuprofen in PAMAM dendrimers end-functionalized

with -NH2 groups showed two types of host-guest interactions: hydrophobic

interactions between the drug molecules and the hydrophobic core, and ionic

interactions between the carboxylic group of the drug and the amino termination

groups of the PAMAM dendrimer [91]. It was observed that the release of the

drug from the dendrimer was considerably slower, with 70% and 80% of the

drug being released from G3 and G4 dendrimers, respectively, after 8-9 hours. It

was demonstrated that because of the small size of the G3 dendrimer, ibuprofen

molecules were not solubilized in the hydrophobic internal of the micelle but

rather formed complexes through ionic interactions with the peripheral amino

groups of the dendrimer. In the G4 micelle, however, a small fraction of

ibuprofen (~ 18%) was also encapsulated within the hydrophobic voids of the

dendritic core, leading to the higher release of the drug from the G4 dendritic

micelles. In methanol and deionized water, less than 15% of the drug was

released from the G4 dendritic micelle even after 9 hours. This suggests that the

stronger the drug-dendrimer complex, the lower the fraction of drug released.

1.1.9. Modification of Periphery Groups of Dendrimer

Because of their perfect structure and the large number of chain ends present, it

is convenient to functionalize dendrimers at their periphery. Dendrimers with

hydrophobic cores derived from hexatricontaalchohol and poly(aryl ether)s,

were functionalized by Newkome et al. [56] and Hawker et al. [57] with polar

24

carboxylic acid groups, respectively. In this work, we conjugated -CD groups

on the surface of the dendrimers and investigated how the guest molecules get

trapped in the dendritic compounds in detail.

Cyclodextrins (CDs) are cyclic oligosaccharides created with 6, 7 or 8

glucopyranose units, respectively named as -, - or -cyclodextrin. Their

hydrophobic apolar interior (cavity) allows to form inclusion complexes with

organic compounds through formation of non-covalent inclusion complexes

[92]. The cavity size of -CD is insufficient for many drugs and -CD is

expensive. In general, -CD has weaker complex forming ability than

conventional CDs. -CD has been widely used in the early stages of

pharmaceutical applications because of its ready availability and cavity size

suitable for the widest range of drugs. But the low aqueous solubility and

nephrotoxicity limited the use of -CD especially in parenteral drug delivery.

Chemically modified CD derivatives have been prepared with a view to extend

the physicochemical properties and inclusion capacity of parent CDs. Several

amorphous, non-crystallizable CD derivatives with enhanced aqueous

solubility, physical and microbiological stability, and reduced parenteral

toxicity have been developed by chemical modification of parenteral CDs [93,

94].

-CD have a polar exterior and an apolar interior cavity [95] and is well known

for its capability to form host–guest inclusion complexes with hydrophobic

drugs [96]. Encapsulation ability of CDs has found numerous applications in

fields such as drug delivery systems. -CD is the most positive to be solubilized

of molecules but the solubilization capability of -CD is restricted by its poor

solubility in water (1.85 g/100 ml at 20 °C) [97] and almost unsolvable

complexes are often achieved with greatly a polar molecules [92]. Diverse

chemical alterations have been so far projected for improvement of solubility of

-CD and its complexes in aqueous medium, such as preparation of -CD

conjugates with biocompatible polar artificial polymers [98, 99]. In recent

25

times, it has been publicized that the merging of CDs into polymeric

nanoparticles can enhance the drug loading and modifies drug release [100-

102].

1.1.10. Aim of Work

Literature shows that due to their multivalency and globular shape dendrimers

are regarded as ideal candidates for use in biomedical applications. A number of

challenging targets remain in the design and synthesis of dendrimers for this

purpose. In this thesis, the supramolecular encapsulation of guest molecules in

the interior of a dendrimer is evaluated by using specific interactions

(electrostatic, hydrophobic and hydrogen bonding).

In this work, the synthesis of a new class of dendrimers is described that can be

applied in drug delivery systems (DDS). For this purpose, were described the

synthesis and characterization of multivalent citric acid based dendrimers and

modified citric acid dendrimers. Modified citric acid dendrimers are synthesized

via ester and amide linkage with -cyclodextrin ( -CD) and 6-Deoxy-6-amino-

-cyclodextrin ( -CD-NH2) for non-toxicity and enhanced water-solubility.

Cyclodextrins (CDs), including hydrophobic apolar interior (cavity) allows to

form inclusion complexes with organic compounds through formation of non-

covalent inclusion complexes

First, second and third generation (G) of citric acid based dendrimers are

prepared and the modification of synthesized dendrimers has been investigated

in detail. This thesis reports on the preparation of a citric acid-based dendrimers

bearing -CD as grafted side-chains by using dicyclohexyl carbodiimide (DCC)

as a coupling agent and an evaluation of its ability for the encapsulation of a

Naltrexone (NLX) and a biocompatible surface. The use of these dendrimers as

pH sensitive carriers for NLX has also been studied. Dendrimers were loaded

with NLX and the release pattern of the drug was investigated. Influences of the

26

dendrimer generation and number of grafted -CD with dendrimers on their

encapsulation ability have been described.

27

Chapter 2: Experimental Section

2.1. Materials and Methods

2. 1.1. Materials

Poly(ethylene glycol) 600 diacid (acid number 175, 96–98%, from Fluka) was

dried over Na2SO4 (Merck Chemical Co., Germany). -cyclodextrin (from

Fluka) dried in oven at 90 ˚C for 8 h. Citric acid and pyridine (purified with

refluxing over NaOH for 2 h and subsequent distillation) were obtained from

Merck Chemical Co. (Germany). -Toluenesulfonic acid (TSA) were purified

by conventional methods before use. Thionyl chloride (Merck Chemical Co.,

Germany) was refluxed on linseed oil for 2 h. Sodium azide was purchased

from Fluka. Dicyclohexyl carbodiimide (DCC) was purchased from Merck

Chemical Co. (Germany). N, N-dimethylformamide (DMF) (from Fluka) was

dried and distilled under reduced pressure. Dialysis membrane D7884 was

purchased from Sigma-Aldrich Co. (Steinhein, Germany) (retains molecular

weights greater than 2000 and releases smaller than 1200). Naltrexone (NLX)

hydrochloride was obtained from Sigma (St. Louis, MO, USA) and neutralized

with NaOH 0.25M. Fig. 9 shows the structure of NLX. Other reagents and

solvents purchased from Merck Chemical Co. (Germany).

Figure 9. Chemical structure of naltrexone.

28

2.1.2. Instrumental Measurement

FT–IR spectra were recorded on a Bruker Model Tensor-27 spectrometer.

Under appropriate conditions in a magnetic field, a sample can absorb

electromagnetic radiation in the radio frequency region at frequencies governed

by the characteristics of the sample. A plot of frequencies of the absorption

peaks versus peaks intensities constitutes a nuclear magnetic resonance

spectroscopy (NMR) spectrum. Thus, NMR is a very useful technique in

obtaining detailed information about molecular structure. In this dissertation,

NMR is used to identify the formation of specific products of reactions.1H and13C NMR spectra were recorded with a Bruker Advance 400 spectrometer

operating at 400.132 MHz (1H) and 100.623 (13C) using DMSO-d6, acetone

deuterium and D2O as a solvent. Analytical ultraviolet-Visible (UV-vis)

absorption spectroscopy is described by Beer’s Law (equation 2.1), where the

absorbance (A) of a molecule is a function of the molar absorptivity ( ), the path

length (l), and the concentration (c).

A = lc (2.1)

The UV absorption spectra were recorded using 1700 Shimadzu

spectrophotometer. Particle sizes were determined with dynamic light scattering

by commercially available equipment Zetasizer Nano ZS from Malvern using a

4-mW He–Ne laser (633 nm wavelength) with a fixed detector angle of 173°

and Laser Diffraction Particle Size Analyzer with model of SALD-2101 from

Shimadzu.

29

2.2. Methods

2.2.1. Preparation of , -di(chlorocarbonylmethylene) poly(ethyleneglycol)

(ClOC-PEG-COCl)

The diacyl halide poly(ethyleneglycol) was prepared by literature method [103].

In this procedure, dry poly-(ethyleneglycol) 600 diacid (PEG-A) was

chlorinated with refluxing in thionylchloride and ClOC–PEG–COCl obtained as

the light yellow oil, yield 100%.

2.2.2. Preparation of G1 Using Thionylchloride

A solution of citric acid (10.27 g, 5.83 10-2 mol) in 25 ml dry (THF) was

placed in a round-bottom flask equipped with a reflux condenser, dropping

funnel, argon inlet and magnetic stirrer. Dry pyridine (0.2 ml, 2.48 10-3 mol)

was added to this solution at 15 min through dropping funnel and mixture was

stirred for 20 min. A solution of ClOC–PEG–COCl (15.52 g, 2.43 10-2 mol) in

15 ml dry THF was added at for 30 min. The mixture was stirred at 0 for 1

h then at room temperature for 3 h and finally at 50 for additional 6 h (all

steps of reaction was carried out under argon) then was cooled and filtered off

and was precipitated in diethylether. The product was washed using

dichloromethane, cooled acetone, toluene and dissolved in 10 ml THF and

reprecipitated in diethylether and n-hexane for several times. The mixture was

poured in 5 ml of water at 25 . The mixture was conducted into cellophane

membrane dialysis bag. The bag was closed and transferred into a flask

containing 100 ml of water maintained at 25 . The external water was

continuously stirred for two days. The external water was removed after 24 h

and added 100 ml fresh water, then the product was removed from dialysis bag

and dried under vacuum at 50 as the reddish oil, yield 73% [20, 104].

30

1H NMR (DMSO-d6) [400 MHz,δ,ppm]: 2.67-2.77 (citric acid CH2), 3.6-3.8 (-

OCH2CH2O) and 4.1-4.2 (-COCH2O-). IR (KBr,cm-1): 3439-2610 (COOH),

1738 (C=O), 1180 and 1106 (C-O).

2.2.3. Preparation of G2 Using DCC

A solution of G1 (12.8 g, 1.35 10-2 mol) in 20 ml dry THF was added to a

round-bottom flask equipped with reflux condenser, argon inlet, dropping

funnel and magnetic stirrer. Dry pyridine (0.5 ml) was added to this solution by

dropping funnel (15 min). The mixture was stirred vigorously for 10 min. A

solution of DCC (18.38 g, 9.72 10-2 mol) in 15 ml dry THF was added to

mixture at 0 by dropping funnel. The mixture was stirred for 30 min. Then a

solution of citric acid (17.12 g, 9.72 10-2 mol) in 15 ml THF was added

dropwise to this solution. The mixture was stirred at 0 for 1.5 h then at room

temperature for 24 h under argon. The solution was filtered off and was placed

at 5 for 24 h and again the solution was filtered off. The product was

precipitated in diethylether and was washed by acetone dichloromethane,

toluene then dissolved in THF and reprecipitated in diethylether for several

times. The product was dissolved in 5 ml water and stirred for 24 h at room

temperature. The solution was filtered off and poured in 5 ml of water at 25 .

The mixture was conducted into cellophane membrane dialysis bag. The bag

was closed and transferred into a flask containing 100 ml of water maintained at

25 . The external water was continuously stirred for two days. The external

water was removed after 24 h and added 100 ml fresh water. The product was

removed from dialysis bag and dried under vacuum at 50 as the amorphous

compound, yield 60% [20, 104].

31

1H NMR (DMSO-d6) [400 MHz,δ,ppm]: 2.6-2.8 (citric acid CH2), 3.57-3.8 (-

OCH2CH2O) and 4.1-4.2 (-COCH2O-). IR (KBr,cm-1): 3581-2620 (COOH),

1732 (C=O), 123180 and 1096 (C-O).

2.2.4. Preparation of G3 Using DCC

A solution of G2 (8.3 g, 4.16 10-3 mol) in 35 ml dry THF was added to a



dropping funnel at 20 min. The mixture was stirred vigorously for 20 min. A

solution of DCC (23.17 g, 1.12 10-1 mol) in 25 ml of dry THF was added to

mixture at 0 by dropping funnel. The mixture was stirred for 30 min. Then a

solution of citric acid (21.58 g, 1.12 10-1 mol) in 20 ml of THF was added

dropwise to this solution. The mixture was stirred at 0 for 2 h then for

additional 72 h under argon at room temperature. The solution was filtered off

and placed at 5 for 24 h and again the solution was filtered off. The product

was precipitated in diethylether and washed by acetone, dichloromethane (for

removing the excess of citric acid), and toluene then dissolved in THF and

reprecipitated in diethylether for several times. The product was dissolved in 5

ml water and stirred for 24 h at room temperature. The solution was filtered off

and poured in 5 ml of water at 25 . The mixture was conducted into

cellophane membrane dialysis bag. The bag was closed and transferred into a

flask containing 100 ml of water maintained at 25 . The external water was


and added 100 ml fresh water. The product was removed from dialysis bag and

dried under vacuum at 50 as the solid, yield 18% [20, 104]. 1H NMR (DMSO-d6) [400 MHz,δ,ppm]: 2.62-2.76 (citric acid CH2), 3.57-3.77

(-OCH2CH2O) and 4.1-4.2 (-COCH2O-). IR (KBr,cm-1): 3439-2610 (COOH),

1730 (C=O), 1260 and 1096 (C-O).

32

2.2.5. Synthesis of G1- -CD Using DCC

G1 was prepared as previously reported [20]. A solution of G1 (0.25 g, 2.6 × 10-3

mol) in 20 ml dry DMF was added to a round-bottom flask equipped with reflux

condenser, argon inlet, dropping funnel and magnetic stirrer. Dry pyridine (0.2

ml) was added to this solution by dropping funnel (15 min). The mixture was

stirred vigorously for 15 min. To this solution, DCC (0.385 g, 1.87 × 10-3 mol)

in 15 ml dry DMF was added as a coupling agent at 0 °C by dropping funnel.

The mixture was stirred for 30 min. Then a solution of -CD (1.795 g, 1.58 ×

10-3 mol) in 15 ml DMF was added dropwise to this solution. The mixture was

stirred at 0 °C for 3 h then at room temperature for 54 h and finally at 65-75 °C

for 5 h under argon. The solution was filtered off and was placed at 4 °C for 24

h and again the solution was filtered off and the solvent was removed under

vacuum. The product was precipitated in diethylether and was washed by

acetone. Then the product was dissolved in the methanol, and filtered off then

were precipitated in diethylether several times for removal of unreacted -CD.

The solution was filtered and the solvent was removed under vacuum. The

product was dissolved in 5 ml water and stirred for 24 h at room temperature.

The solution was filtered off and poured in 5 ml of water at 25 °C. The mixture

was conducted into cellophane membrane dialysis bag. The bag was closed and

transferred into a flask containing 100 ml of water maintained at 25 °C. The

external water was continuously stirred for two days. The external water was

removed after 24 h and added 100 ml fresh water. The product was removed

from dialysis bag and dried under vacuum at 50 °C and purified compound was

obtained as a light brown solid, yield 51%. 1H NMR (400 MHz, DMSO-d6): =2.8-2.9 (q, citric acid CH2), 3.26-3.8 (br, -

CD H2, H4, H5, H3, H6a, H6b and polyethylene glycol (PEG) -OCH2CH2O-), 3.92-

4.2 (br, PEG -COCH2O-), 4.2-4.3 (br, β-CD -CH2OCO-dendrimer), 4.57 (br, -

CD OH6), 4.81 (br, -CD H1), 5.62 (br, -CD OH3) and 5.79 (br, -CD OH2). IR

33

(KBr, cm-1): 3400 ( , OH β-CD and COOH dendrimer), 2928 and 2860 ( , C–

H), 1739 and 1704 ( , ester and acid C=O), 1151 ( , C-O-C), 1106 and 1033 ( ,

C-O).


G2 was prepared according to literature [20]. A solution of G2 (0.25 g, 1.25 ×

10-4 mol) in 20 ml dry DMF was added to a round-bottom flask equipped with

reflux condenser, argon inlet, dropping funnel and magnetic stirrer. Dry

pyridine (0.2 ml) was added to this solution by dropping funnel within 15 min.

The mixture was stirred vigorously for 20 min. To this solution, DCC (0.56 g,

2.7 × 10-3 mol) in 15 ml dry DMF was added as a coupling agent at 0 °C by

dropping funnel. The mixture was stirred for 30 min. Then a solution of -CD

(2.56 g, 1.29 × 10-3 mol) in 15 ml DMF was added dropwise to this solution.

The mixture was stirred at 0 °C for 3 h then at room temperature for 72 h and

finally at 65-75 °C for 6 h under argon. The solution was filtered off and was

placed at 4 °C for 24 h and again the solution was filtered off and the solvent

was removed under vacuum. The product was precipitated in diethylether and

was washed by acetone. Then the product was dissolved in the methanol, and

filtered off then were precipitated in diethylether several times for removal of

unreacted -CD. The solution was filtered and the solvent was removed under

vacuum. The product was dissolved in 5 ml water and stirred for 24 h at room

temperature. The solution was filtered off and poured in 5 ml of water at 25 °C.



25 °C. The external water was continuously stirred for two days. The external


removed from dialysis bag and dried under vacuum at 50 °C and purified

compound was obtained as a brown solid, yield 32%.

34

1H NMR (400 MHz, DMSO-d6): =2.72-2.88 (q, citric acid CH2), 3.16-3.83 (br,

-CD H2, H4, H5, H3, H6a, H6b and PEG -OCH2CH2O-), 4-4.1 (br, PEG -

COCH2O-), 4.2-4.4 (br, β-CD -CH2OCO-dendrimer), 4.6 (br, -CD OH6), 4.83

(br, -CD H1), 5.6 (br, -CD OH3) and 5.73 (br, -CD OH2). IR (KBr, cm-1):

3423 ( , OH β-CD and COOH dendrimer), 2931 and 2859 ( , C–H), 1741 and

1705 ( , ester and acid C=O), 1151 ( , C-O-C), 1100 and 1031 ( , C-O).


G3 was prepared as previously reported [20]. A solution of G3 (0.25 g, 4.85 ×

10-5 mol) in 20 ml dry DMF was added to a round-bottom flask equipped with

reflux condenser, argon inlet, dropping funnel and magnetic stirrer. Dry

pyridine (0.2 ml) was added to this solution by dropping funnel within 15 min.

The mixture was stirred vigorously for 20 min. To this solution, DCC (0.65 g,

3.15 × 10-3 mol) in 15 ml dry DMF was added as a coupling agent at 0 °C by

dropping funnel. The mixture was stirred for 30 min. Then a solution of -CD

(2.99 g, 5.8 × 10-4 mol) in 15 ml DMF was added dropwise to this solution. The

mixture was stirred at 0 °C for 3 h then at room temperature for 96 h and finally

at 65-75 °C for 8 h under argon. The solution was filtered off and was placed at

4 °C for 24 h and again the solution was filtered off and the solvent was

removed under vacuum. The product was precipitated in diethylether and was

washed by acetone. Then the product was dissolved in the methanol, and

filtered off then were precipitated in diethylether several times for removal of

unreacted -CD. The solution was filtered and the solvent was removed under

vacuum. The product was dissolved in 5 ml water and stirred for 24 h at room

temperature. The solution was filtered off and poured in 5 ml of water at 25 °C.



35

25 °C. The external water was continuously stirred for two days. The external


removed from dialysis bag and dried under vacuum at 50 °C and purified

compound was obtained as a brown solid, yield 25%.

1H NMR (400 MHz, DMSO-d6): =2.72-3.06 (q, citric acid CH2), 3.16 (m, -

CD H2), 3.3-3.9 (br, -CD H4, H5, H3, H6a, H6b and PEG -OCH2CH2O-), 4-4.3

(br, β-CD -CH2OCO-dendrimer), 4.57 (br, -CD OH6), 4.83 (br, -CD H1), 5.63

(br, -CD OH3) and 5.73 (br, -CD OH2). IR (KBr, cm-1): 3416 ( , OH β-CD

and COOH dendrimer), 2930 and 2858 ( , C–H), 1743 and 1705 ( , ester and

acid C=O), 1154 ( , C-O-C), 1186 and 1031( , C-O).

2.2.8. Synthesis of G1-NH- -CD Using DCC

In order to obtain a G1-NH- -CD, 6-Deoxy-6-amino- -cyclodextrin ( -CD-

NH2) was first synthesized. -CD-NH2 was prepared as previously reported

[98]. Briefly, -CD-NH2 was obtained through a three-step synthetic pathway.

In the first step 6-Deoxy-6-(p-toluenesulfonyl)- -cyclodextrin ( -CDTos) was

prepared. In this method, dry -CD dissolved in pyridine. The resulting solution

was cooled in an ice bath and reacted with a solution of p-toluenesulfonyl

chloride in pyridine. The mixture was stirred at room temperature under

nitrogen for 12 h. Then pyridine was evaporated under a reduced pressure at 40

°C to obtain a viscose material, then diethyl ether was added. The white

precipitate was recrystallized in distilled water three times [99]. Then -CD

converts to the 6-Deoxy-6-azido- -cyclodextrin ( -CD-N3). For this purpose -

CD-N3 was prepared by reacting at 85 °C an aqueous suspension of -CDTos

with sodium azide. The reaction was monitored by TLC on silica plates with the

mixture n-BuOH/EtOH/H2O 5:4:3 (v/v) as mobile phase until the complete

disappearance of the spot of -CD-Tos (Rf=0.45). The azide product was finally

36

recovered by precipitation in acetone as a white powder. Then -CD-N3 and

triphenylphosphine were dissolved in DMF, and to the solution was added

concentrated NH3 (aq). The mixture was stirred at room temperature for about 4

h, and the solution was poured into acetone, giving a crude product as a white

precipitate. The resulting product was purified and -CD-NH2 was obtained

[98]. The spectroscopic data show the complete agreement with the proposed

structures.

A solution of G1 (0.25 g, 2.6 × 10-3 mol) in 20 ml dry DMF was added to a



dropping funnel (15 min). The mixture was stirred vigorously for 20 min. To

this solution, DCC (0.385 g, 1.87 × 10-3 mol) in 15 ml dry DMF was added as a

coupling agent at 0 °C by dropping funnel. The mixture was stirred for 30 min.

Then a solution of -CD-NH2 (1.79 g, 1.89 × 10-3 mol) in 15 ml DMF was

added dropwise to this solution. The mixture was stirred at 0 °C for 3 h then at

room temperature for 54 h and finally at 45-55 °C for 5 h under argon. The

solution was filtered off and was placed at 4 °C for 24 h and again the solution

was filtered off and the solvent was removed under vacuum. The product was

precipitated in diethylether and was washed by acetone. Then the product was

dissolved in the methanol, and filtered off then were precipitated in diethylether

several times for removal of unreacted -CD-NH2. The solution was filtered and

the solvent was removed under vacuum. The product was dissolved in 5 ml

water and stirred for 24 h at room temperature. The solution was filtered off and

poured in 5 ml of water at 25 °C. The mixture was conducted into cellophane


containing 100 ml of water maintained at 25 °C. The external water was



37

dried under vacuum at 50 °C and purified compound was obtained as a brown

solid, yield 43%.

1H NMR (400 MHz, DMSO-d6): = 2.06 (s, dendrimer -CH2CONH-), 2.7-2.92

(q, citric acid CH2), 3.11 (m, β-CD -CONHCH2-), 3.3-3.8 (br, -CD H2, H4, H5,

H3, H6a, H6b and PEG -OCH2CH2O-), 4.1-4.2 (br, PEG -COCH2O-), 4.57 (br, -

CD OH6), 4.81 (br, -CD H1), 5.62-5.64 (br, -CD OH3), 5.74-5.81 (br, -CD

OH2) and 7.97-8 (br, NH-C=O). 13C NMR (100 MHz, DMSO-d6): =31 and 70

(citric acid CH2), 50.22 ( -CD CH2-NH-CO), 60.49 ( -CD C6), 72.3-73.4 (β-

CD C2, C3 and C5) 70 and 72.3 (PEG carbons), 81 (β-CD C4), 101 (β-CD C1),

and 157, 162 and 167 (C=O). IR (KBr, cm-1): 3359 ( , OH β-CD and COOH

dendrimer), 2928 and 2862 ( , C–H), 1739 and 1705 ( , ester and acid C=O),

1653 ( , amide C=O), 1522 ( , CO–N–H), 1149 ( , C-O-C), 1109 and 1035 ( ,

C-O).





dropping funnel within 15 min. The mixture was stirred vigorously for 20 min.

To this solution, DCC (0.58 g, 2.79 × 10-3 mol) in 15 ml dry DMF was added as

a coupling agent at 0 °C by dropping funnel. The mixture was stirred for 30

min. Then a solution of -CD-NH2 (2.65 g, 1.35 × 10-3 mol) in 15 ml DMF was

added dropwise to this solution. The mixture was stirred at 0 °C for 3 h then at

room temperature for 72 h and finally at 45-55 °C for 6 h under argon. The

solution was filtered off and was placed at 4 °C for 24 h and again the solution

was filtered off and the solvent was removed under vacuum. The product was


38











solid, yield 30%.

1H NMR (400 MHz, DMSO-d6): = 2.08 (s, dendrimer -CH2CONH-), 2.7-2.92

(q, citric acid CH2), 3.06 (m, -CD -CONHCH2-), 3.32-3.83 (br, -CD H2, H4,

H5, H3, H6a, H6b and PEG -OCH2CH2O-), 4.1-4.2 (br, PEG -COCH2O-), 4.6 (br,

-CD OH6), 4.82 (br, -CD H1), 5.62-5.64 (br, -CD OH3), 5.74-6 (br, -CD

OH2) and 8.1-8.2 (br, NH-C=O). 13C NMR (100 MHz, DMSO-d6): =31 and

69.8 (citric acid CH2), 49.53 ( -CD CH2-NH-CO), 60.2 ( -CD C6), 68-70 (β-

CD C2, C3 and C5) 72.3and 72.6 (PEG carbons), 81 (β-CD C4), 101 (β-CD C1),

and 156, 167 and 170 (C=O). IR (KBr, cm-1): 3388 ( , OH β-CD and COOH

dendrimer), 2930 and 2858 ( , C–H), 1743 and 1706( , ester and acid C=O),

1649 ( , amide C=O), 1561 ( , CO–N–H), 1223 ( , C-O-C), 1115 and 1032 ( ,

C-O).





39

dropping funnel (15 min). The mixture was stirred vigorously for 20 min. To

this solution, DCC (0.65 g, 3.15 × 10-3 mol) in 15 ml dry DMF was added as a

coupling agent at 0 °C by dropping funnel. The mixture was stirred for 30 min.

Then a solution of -CD-NH2 (3.98 g, 5.8 × 10-4 mol) in 15 ml DMF was added

dropwise to this solution. The mixture was stirred at 0 °C for 3 h then at room

temperature for 96 h and finally at 45-55 °C for 8 h under argon. The solution

was filtered off and was placed at 4 °C for 24 h and again the solution was

filtered off and the solvent was removed under vacuum. The product was












solid, yield 24%.

1H NMR (400 MHz, DMSO-d6): =1.84 (s, dendrimer -CH2CONH-), 2.72-2.88

(q, citric acid CH2), 3.09 (m, -CD -CONHCH2-), 3.29-3.8 (br, -CD H2, H4,

H5, H3, H6a, H6b and (PEG) -OCH2CH2O-), 4.1-4.2 (br, PEG -COCH2O-), 4.6

(br, -CD OH6), 4.83 (br, -CD H1), 5.62-6 (br, -CD OH3 and OH2) 8.1-8.2 (br,

NH-C=O). 13C NMR (100 MHz, DMSO-d6): =30.8 and 69.8 (citric acid CH2),

50 ( -CD CH2-NH-CO), 64 ( -CD C6), 70-72.6 (β-CD C2, C3, C5 and PEG

carbons), 81 (β-CD C4), 106 (β-CD C1), and 156, 162 and 167 (C=O). IR (KBr,

cm-1): 3416 ( , OH β-CD and COOH dendrimer), 2930 and 2856 ( , C–H),

40

1740 and 1706( , ester and acid C=O), 1657 ( , amide C=O), 1564 ( , CO–N–

H), 1228 ( , C-O-C), 1156 and 1035 ( , C-O).

2.2.11. Preparation of Gn- -CD (n=1–2) and Gn-NH- -CD (n=1–2)/NLX

Complexes

For the preparation of Gn- -CD (n=1–2) and Gn-NH- -CD (n=1–2)/NLX complexes,

first the dendrimers were dissolved in 20 ml DMF and these solutions were

added to a round-bottom flask equipped with reflux condenser and magnetic

stirrer containing a solution of drug (excess of NLX) in 20 ml DMF. The

solutions were stirred for 2h at 35-45 ˚C. Complexes were precipitated in n-

hexane and then dissolved in water, filtered and precipitated in diethylether. The

sample obtained was dried at 35 ˚C for 3 h in a vacuum oven.

2.2.12. In Vitro Release Studies

In vitro release of drug–dendrimer complexes was carried out by the dialysis

method. A quantity of dried dendrimer/drug complexes (66 mg) were added to a

5 ml of aqueous buffered solution (pH 10, 7.4, and 1) at 37 °C. The resulting

solution was loaded into a cellophane membrane dialysis bag. The cellophane

bag was closed and transferred into glass beakers containing 25 ml of the equal

buffer solution maintained at 37 °C. The outer phase was stirred continuously

with a magnetic stirrer at 800 rev/min and a sampling (3 ml) was withdrawn at

regular selected intervals and replaced with 3 ml fresh buffer. The NLX released

in to the medium was analyzed through a UV spectrophotometer to characterize

the release of NLX and determined from the calibration curve achieved

previously under the same conditions. The absorbance of the outer phase was

monitored at 282 nm (pH=1 and 7.4) and 291 nm (pH=10) by a 1700 Shimadzu

UV spectrophotometer to characterize the concentration of NLX using a 1 cm

41

quartz cell. The results were presented in the terms of cumulative release as a

function of time.

2.2.13. Particle Size Analysis

Particle size of complexes were measured by dynamic light scattering (DLS)

using a Malvern Zeta-Sizer Nano ZS equipped with a 4-mW He–Ne laser (633

nm wavelength) with a fixed detector angle of 173°. Samples were dissolved in

distilled water, and Light scattering experiments were carried out at 25 °C and

were started 10 min after the cuvette was placed in the DLS apparatus to allow

the temperature to equilibrate. About 1 ml of the sample was transferred to a

special dust free light-scattering cell. The temperature was controlled to within

±0.02 °C. All samples were quietly inverted for homogenization before

measurement. The particle sizes were measured based on their average

diameters. Also, Particle sizes of resulted dendrimers were measured by Laser

Diffraction Particle Size Analyzer (SA-2101).

42

Chapter 3: Results and Discussion

3.1. Investigation of Dendrimer Synthesized Based on Citric acid

3.1.1. Synthesis of G1 Compound

The compound G1 was synthesized from the reaction of ClOC–PEG–COCl

with anhydrous citric acid and ClOC–PEG–COCl was prepared through

chlorination of diacid poly(ethyleneglycol) using thionylchloride in yield 100%

[20]. The general synthesis pathway is presented in Scheme 1. The condition of

reactions and the characterization of products are described in the

“Experimental section”. The structure of G1 was verified by 1H NMR and FT-

IR spectroscopy. Fig. 10 shows the 1H NMR spectrum of G1 compound. In Fig.

10 the 1H NMR of G1which shows a quartet at 2.67–2.77 ppm as a AB system

for the CH2 protons of citric acid, the protons of PEG at 3.6–3.8 ppm (–

OCH2CH2O–) and 4.1–4.2 ppm (–COCH2O–) can be recognized also the

chemical shifts at 2.5 ppm is related to the DMSO as the solvent. The integral

ratio of aliphatic protons of PEG to the citric acid part of the molecule is 6 (in

comparison to 6 as a theoretical calculation). This is evidence, which shows the

reaction has been accomplished completely and both of the acyl halide functions

of chlorinated PEG have been reacted with citric acid.

FT-IR spectrum of G1 (Fig. 11) shows a band around 1106 cm-1 can be

assigned as characteristic stretching vibration of C–O of G1. In the FTIR

spectrum the absorption band at 1738 cm-1 is attributed to the carbonyl groups

of ester. A strong and wide band at 3439 cm-1 is the absorption of -COOH

groups from dendrimer component. The FT-IR spectrum of G1 indicates a C–H

asymmetrical stretching vibration of the dendrimer segment at 2858-2860 cm-1.

The peak at 1180 cm-1 (C–O stretch) confirmed the presence of ether group of

PEG. Both these data confirmed the formation of ester bond between the acyl

group of ClOC–PEG–COCl and the hydroxyl group of citric acid.

43

Scheme 1. Synthesis of G1.

44

Figure 10. 1H NMR spectrum of G1 in DMSO-d6.

Figure 11. FT-IR spectrum of G1.

45


The compound G2 was prepared via the reaction of G1 and dry citric acid in the

presence of DCC as a coupling agent. The general synthesis pathway is

presented in Scheme 2. The condition of reactions and the characterization of

products are described in the “Experimental section”. The structure of G2 was

verified by 1H NMR and FT-IR spectroscopy. Fig. 12 display 1H NMR of G2,

the chemical shifts at 2.6–2.8 ppm (CH2) of protons of citric acid as a quartet

(AB system), protons of PEG at 3.57–3.8 ppm (–OCH2CH2O–) and 4.1–4.2

ppm (–COCH2O–) can be recognized. In 1H NMR spectroscopy the comparison

of the proton numbers of CH2 of G2 shows that the number of protons of citric

acid versus number of protons of PEG is grown related to the G1, which

indicates the formation of dendrimer (G2). Also integral ratio of aliphatic

protons of PEG to citric acid is shows that the reaction was completed and the

growth of dendrimer is confirmed (G2). This is an evidence, which shows the

reaction has been accomplished completely and both of the between the

carboxylic group of G1 have been reacted with citric acid.

FT-IR spectrum of G2 (Fig. 13) shows a band around 1096 cm-1 can be

assigned as characteristic stretching vibration of C–O of G2. In the FTIR

spectrum the absorption band at 1734 cm-1 is attributed to the carbonyl groups

of ester. A strong and wide band at 3400 cm-1 is the absorption of -COOH

groups from dendrimer component. The peak at 1180 cm-1 (C–O stretch)

confirmed the presence of ether group of PEG. Both these data confirmed the

formation of ester bond between the between the carboxylic groups of G1 and

the hydroxyl group of citric acid.

46



47



The preparation of G3 was carried out via the reaction of G2 with dry citric acid

in the presence of DCC as a coupling agent and DMF as solvent. The general

synthesis pathway is presented in Scheme 3. The condition of reactions and the

characterization of products are described in the “Experimental section”. The

structure of G3 was verified by 1H NMR and FT-IR spectroscopy. Fig. 14 and

15 display 1H NMR of G3, the chemical shifts at 2.62–2.76 ppm (CH2) of

protons of citric acid as a quartet (AB system), protons of PEG at 3.57–3.77

ppm (–OCH2CH2O–) and 4.1–4.2 ppm (–COCH2O–) can be recognized. In 1H

NMR spectroscopy the comparison of the proton numbers of CH2 of G3 shows

that the number of protons of citric acid versus number of protons of PEG is

grown related to the G2, which indicates the formation of dendrimer (G3). The

number of protons of G3 in the same chemical shifts displays the growth of

citric acid part in comparison with the protons of PEG as a core and also in G2.

48

Also integral ratio of aliphatic protons of PEG to citric acid is 0.47 (in

comparison to 0.5 as a theoretical calculation) shows that the reaction was

completed and the growth of dendrimer is confirmed (G3). This is an evidence,

which shows the reaction has been accomplished completely and both of the

between the carboxylic group of G2 have been reacted with citric acid.

FT-IR spectrum of G3 (Fig. 16) shows a band around 1096 cm-1 can be assigned

as characteristic stretching vibration of C–O of G2. In the FTIR spectrum the

absorption band at 1730 cm-1 is attributed to the carbonyl groups of ester. A

strong and wide band at 3300 cm-1 is the absorption of -COOH groups from

dendrimer component. The FT-IR spectrum of G2 indicates a C–H

asymmetrical stretching vibration of the dendrimer segment at 2858-2860 cm-1.

The peak at 1260 cm-1 (C–O stretch) confirmed the presence of ether group of

PEG.

49


50


Figure 15. 1H NMR spectrum of G3 in acetone deuterium.

51


3.1.4. Synthesis of G1- -CD Compound

Compound G1- -CD was synthesized from the conjugation of G1 with -CD by

‘‘one-step synthesis reaction’’, through the formation of an ester linkage in the

presence of pyridine after activation of acidic groups with DCC as a coupling

agent. The general synthesis pathway is presented in Scheme 4. The condition

of reactions and the characterization of products are described in the

“Experimental section”. The 1H NMR spectrum (Fig. 17) of the G1- -CD

conjugate shows signals originating from both G1 and -CD. 1H NMR data of

these compounds shows a quartet at 2.8–2.92 ppm as AB system for the CH2

protons of citric acid, the anomeric protons of -CD at 4.82 and CH2OCO-

dendrimer of β-CD at 4.2-4.4 can be recognized. All these chemical shifts were

in agreement with the projected structure of these compounds. The average

number of -CD in the G1- -CD dendrimers prepared was evaluated from their1H NMR spectrum. 1H-NMR spectrum of the resulting G1- -CD was measured

and the molar ratio of dendrimer and -CD was calculated from the peak areas

52

of anomeric protons of -CD and citric acid protons of the dendrimer. From the

integration ratio of the signal at 4.82ppm (corresponding to the anomeric

protons of -CD) to the signal at 2.8-2.92 ppm corresponding to the four

protons of citric acid, approximately 2 molecule of -CD were attached to each

molecule of G1.

FT-IR spectrum (Fig. 18) of this compound showed a band around 1033 cm-1

that could be assigned as characteristic stretching vibration of C–OH of -CD.

A strong and wide band at 3361cm-1 was the absorption of hydroxyl groups

from both -CD and dendrimer components. In the FT-IR spectrum of G1- -

CD, the strong peak at 1739 cm-1 could be assigned to the stretching vibration of

carbonyl group in which was not observed in -CD spectrum. The results

indicated that the carboxymethylation occurs at C6 position. The FT-IR

spectrum of G1- -CD indicated a C–H asymmetrical stretching vibration of the

dendrimer segment at 2926-2860 cm-1, which is not appeared in -CD. The

absorption at 1106 cm-1 (C–O stretch) confirmed the presence of ether group of

PEG. The results of FT-IR absorption and 1H NMR spectra confirmed the

successful synthesis of dendrimers.

53

Scheme 4. Synthesis of G1- -CD.

54

Figure 17. 1H NMR spectrum of G1- -CD in DMSO-d6.

Figure 18. FT-IR spectrum of G1- -CD.

55







“Experimental section”. The 1H NMR spectrum (Fig. 19) of the G2- -CD

conjugate shows signals originating from both G2and -CD. 1H NMR data of

these compounds shows a quartet at 2.72–3.04 ppm as AB system for the CH2

protons of citric acid, the anomeric protons of -CD at 4.83 and CH2OCO-

dendrimer of β-CD at 4-4.2 can be recognized. All these chemical shifts were in

agreement with the projected structure of these compounds. The average

number of -CD in the G2- -CD dendrimers prepared was evaluated from their1H NMR spectrum. 1H-NMR spectrum of the resulting G2- -CD was measured

and the molar ratio of dendrimer and -CD was calculated from the peak areas

of anomeric protons of -CD and citric acid protons of the dendrimer. From the

integration ratio of the signal at 4.83 ppm (corresponding to the anomeric



molecule of G2.



A strong and wide band at 3423 cm-1 was the absorption of hydroxyl groups






56





57

Scheme 5. . Synthesis of G2- -CD.

58

Figure 19. 1H NMR spectrum of G2- -CD in DMSO-d6.


59







“Experimental section”. The 1H NMR spectrum of the G3- -CD conjugate

shows signals originating from both G3 and -CD. 1H NMR data of these

compounds shows a quartet at 2.72–3.06 ppm as AB system for the CH2 protons

of citric acid, the anomeric protons of -CD at 4.83 and CH2OCO-dendrimer of

β-CD at 4.2-4.4 can be recognized. All these chemical shifts were in agreement

with the projected structure of these compounds. The average number of -CD

in the G3- -CD dendrimers prepared was evaluated from their 1H NMR

spectrum. 1H-NMR spectrum of the resulting G3- -CD was measured and the

molar ratio of dendrimer and -CD was calculated from the peak areas of

anomeric protons of -CD and citric acid protons of the dendrimer. From the

integration ratio of the signal at 4.83 ppm (corresponding to the anomeric



molecule of G3.



A strong and wide band at 3416 cm-1 was the absorption of hydroxyl groups






60





61

Scheme 6. Synthesis of G3- -CD.

62


3.1.7. Synthesis of G1-NH- -CD Compound

Also in this work, we describe the functionalization of -CD and its coupling to

synthesized dendrimers. Selective and mono-functionalization of -CD is a

most important challenge due to the existence of seven equivalent 6-primary

and 14 2, 3-secondary hydroxyl functions of the glucose units. In order to, -CD

was monofunctionalized into its amino derivative and then conjugated to the

carboxylic-side of dendrimers. For this purpose, we functionalized the -CD to

6-Deoxy-6-(p-toluenesulfonyl)- -cyclodextrin ( -CDTos) and 6-Deoxy-6-

amino- -cyclodextrin ( -CD-NH2) so as to selectivity and reactivity of mono

substitutions of -CD-NH2 that was prepared as previously reported. Then the

dendrimer Gn (n=1-3) covalently conjugated to the -CD-NH2, through the

formation of an amide band by Using DCC as a coupling agent.

63

3.1.7.1. Preparation of 6-Deoxy-6-amino- -cyclodextrin ( -CD-NH2)

-CD-NH2 was obtained through a three-step synthetic pathway. The first step

was the mono-tosylation of -CD [102]. This reaction was carried out in water,

in the presence of p-toluenesulfonyl chloride and sodium hydroxide, at 0 °C.

The 6- deoxy-6-(p-toluenesulfonyl)- -cyclodextrin ( -CD-Tos) was insoluble in

the reaction mixture and precipitated as a white solid during the process, thus

preventing further functionalization. The product was recovered by filtration

and washed with acetonitrile [105]. 6-Deoxy-6-azido- -cyclodextrin ( -CD-N3)

was prepared by reacting at 85 °C an aqueous suspension of -CD-Tos with

sodium azide. The reaction was monitored by TLC on silica plates with the

mixture isopropanol/ethyl acetate/water/NH4OH 5:2:3:1 as mobile phase until

the complete disappearance of the spot of -CD-Tos (Rf=0.52) and the presence,

besides a trace of unsubstituted -CD (Rf=0.28), of a single spot attributed to -

CD-N3 (Rf=0.40) [105]. -CD-N3 was finally recovered by precipitation in

acetone. Then crude -CD-NH2 was prepared by reacting mono-6-azido-6-

deoxy-CD with triphenyl phosphine in DMF at ambient conditions for 2 h.

After precipitation in acetone, filtering and washing with acetone, pure amine

was obtained as white solid [106]. All products were fully characterized by

NMR and FTIR. The spectroscopic data were shown in complete agreement

with the proposed structures. The general synthesis pathway is presented in

Scheme 7.

In the 1H NMR spectrum of -CD-Tos (Fig. 22) diagnostic peaks characteristic

of tosyl moiety were present, in particular the peaks at 7.4 and 7.7 ppm,

corresponding to the aromatic hydrogen’s of tosyl group, the peak at 4.27 ppm,

corresponding to the methylene adjacent to the sulfonyl moiety, and the

resonance peak at 2.41 ppm, corresponding to the methyl group of the tosyl

moiety. The anomeric protons of -CD appeared at 4.83 ppm. In the 1HNMR

spectrum of -CD-N3 the diagnostic peak of methylene group adjacent to the

64

azide moiety overlapped with those of the -CD hydrogen’s in position 3, 5 and

6 giving rise to a broad peak between 3.67 and 3.56. In the spectrum of -CD-

N3, all peaks relative to the tosyl moiety disappeared after reaction of with

sodium azide. The 1H NMR spectrum of -CD-NH2 lacked diagnostic peaks.

However, the completion of the reduction was as certained by the disappearance

in its FTIR spectrum of the diagnostic band at 2041 cm−1.

Scheme 7. Hierarchical synthesis of -CD-NH2.

65

Figure 22. 1H NMR spectrum of -CD-Tos in DMSO-d6.

3.1.7.2. Synthesis of G1-NH- -CD Compound

Compound G1-NH- -CD was synthesized from the conjugation of G1 with -

CD-NH2 through the formation of an amide linkage in the presence of pyridine

after activation of acidic groups with DCC as a coupling agent. The general



obtained products were characterized using 1H NMR, 13C NMR and FTIR

spectroscopy. The 1H NMR spectrum of the G1-NH- -CD (Fig. 23) conjugate

shows signals originating from both G1 and -CD-NH2. 1H NMR data of these

compounds shows a quartet at 2.7–2.92 ppm as a AB system for the CH2

protons of citric acid, the anomeric protons of -CD at 4.81, the -CH2CONH-

dendrimer protons at 1.84-2.08 ppm and NH-C=O of β-CD at 7.97-8 ppm can

be recognized. All these chemical shifts were in accordance with the proposed

structure of these compounds. The average number of -CD in the G1-NH- -CD

dendrimers prepared was estimated from their 1H NMR spectra. 1H-NMR

66

spectrum of the resulting G1-NH- -CD was measured and the molar ratio of

dendrimer and -CD was calculated from the peak areas of anomeric protons of

-CD and citric acid protons of the dendrimer. From the integration ratio of the

signal at 4.81 ppm, which corresponds to the anomeric protons of -CD, to the

signal between 2.7 and 2.92 ppm corresponding to the four protons of citric

acid, approximately 2 molecule of -CD were attached to each molecule of G1-

NH- -CD.

FTIR spectrum of these compounds shows a strong and wide band at 3359 cm-1

is the absorption of hydroxyl groups from both -CD and dendrimer

components while peak at 1739 cm-1 confirmed the ester linkage. The peaks at

1653 and 1522 cm-1 were assigned to C=O stretching vibration of amide

carbonyl group and N–H bending vibration of a secondary amine, respectively.

The FTIR spectrum of these compounds indicates a C–H asymmetrical

stretching vibration of the dendrimer segment at 2928-2862 cm-1, which is not

appeared in -CD. Stretch vibration for ether band of PEG appeared at 1109-

1251 cm-1 (C–O stretch) (Fig. 24). The attachment of -CD to the chain end of

the dendrimer was further confirmed by 13C NMR spectrum for these

dendrimers. 13C NMR data of these compounds, shows signals at approximately

31, 70 and 72, 73 ppm are related to the carbon atoms of CH2 citric acid and

PEG respectively, also signals of C4 and C1 of β-CD carbons are appeared at 81

and 102 ppm, respectively. The signals of carbonyl groups are appeared at 157,

162 and 167 ppm for the carbon atoms of amide, ester and acid, respectively.

Thus, the 13C NMR resonances at 157 ppm confirmed the presence of the amide

band (Fig. 25). The results of FT-IR absorption, 1H NMR and 13C NMR spectra

confirmed the successful synthesis of these compounds.

67

Scheme 8. Synthesis of G1-NH- -CD.

68

Figure 23. 1H NMR spectrum of G1-NH- -CD in DMSO-d6.

Figure 24. FT-IR spectrum of G1-NH- -CD.

69

Figure 25. 13C NMR spectrum of G1-NH- -CD in DMSO-d6.








spectroscopy. The 1H NMR spectrum of the G2-NH- -CD conjugate shows

signals originating from both G2 and -CD-NH2. 1H NMR data of these

compounds (Fig. 26) shows a quartet at 2.7–3 ppm as a AB system for the CH2


dendrimer protons at 2.08 ppm and NH-C=O of β-CD at 8.1 ppm can be

recognized. All these chemical shifts were in accordance with the proposed


70


spectrum of the resulting G2-NH- -CD was measured and the molar ratio of

dendrimer and -CD was calculated from the peak areas of anomeric protons of

-CD and citric acid protons of the dendrimer. From the integration ratio of the

signal at 4.82 ppm, this corresponds to the anomeric protons of -CD, to the

signal between 2.7 and 3 ppm corresponding to the four protons of citric acid,

approximately 4 molecule of -CD was attached to each molecule of G2-NH- -

CD.

FTIR spectrum of these compounds shows a strong and wide band at 3359–

3416 cm-1 is the absorption of hydroxyl groups from both -CD and dendrimer


1649 and 1551-1649 cm-1 were assigned to C=O stretching vibration of amide







dendrimers. 13C NMR data of these compounds, shows signals at approximately

31.7, 69.8 and 70.5, 70 ppm are related to the carbon atoms of CH2 citric acid

and PEG respectively, also signals of C4 and C1 of β-CD carbons are appeared

at 81 and 101 ppm, respectively. The signals of carbonyl groups are appeared at

156, 167 and 170 ppm for the carbon atoms of amide, ester and acid,

respectively. Thus, the 13C NMR resonances at 156 ppm confirmed the presence

of the amide band (Fig. 28). The results of FT-IR absorption, 1H NMR and 13C

NMR spectra confirmed the successful synthesis of these compounds.

71


72



73









spectroscopy. The 1H NMR spectrum of the G3-NH- -CD conjugate shows

signals originating from both G3 and -CD-NH2. 1H NMR data of these

compounds shows a quartet at 2.72–2.88 ppm as a AB system for the CH2


dendrimer protons at 1.84 ppm and NH-C=O of β-CD at 7.97-8 ppm can be

recognized. All these chemical shifts were in accordance with the proposed

74



spectrum of the resulting G3-NH- -CD (Fig. 29) was measured and the molar

ratio of dendrimer and -CD was calculated from the peak areas of anomeric

protons of -CD and citric acid protons of the dendrimer. From the integration

ratio of the signal at 4.83 ppm, which corresponds to the anomeric protons of -

CD, to the signal between 2.72 and 2.88 ppm corresponding to the four protons

of citric acid, approximately 6 molecule of -CD were attached to each

molecule of G3-NH- -CD.

FTIR spectrum of these compounds shows a strong and wide band at 3416 cm-1

is the absorption of hydroxyl groups from both -CD and dendrimer


1657 and 1564 cm-1 were assigned to C=O stretching vibration of amide







dendrimers. 13C NMR data of these compounds (Fig. 31), shows signals at

approximately 31, 69 and 72, 72.4 ppm are related to the carbon atoms of CH2

citric acid and PEG respectively, also signals of C4 and C1 of β-CD carbons are

appeared at 81 and 106 ppm, respectively. The signals of carbonyl groups are

appeared at 157, 162 and 167 ppm for the carbon atoms of amide, ester and

acid, respectively. Thus, the 13C NMR resonances at 157 ppm confirmed the

presence of the amide band. The results of FT-IR absorption, 1H NMR and 13C

NMR spectra confirmed the successful synthesis of these compounds.

75


76



77


3.2. Investigation of Nanocarriers Size

Particle sizes were determined with DLS experiments in distilled water and

laser diffraction particle size analyzer. DLS results present that the average

diameters of G1- -CD and G2- -CD/NLX complexes were 50 and 70 nm,

respectively (Fig. 32). Typical examples of size distributions by laser diffraction

particle size analyzer (LDPSA) for -CD-modified dendrimers (Gn(n=1-3)-NH-

-CD) are presented in Fig. 33-35. These dendrimers exhibited a narrow size

distribution. Median diameter of G1-NH- -CD, G2-NH- -CD and G3-NH- -CD

were 63, 69 and 82 nm, respectively. It shows that have been increased with

increasing of generation of dendrimers. Size distribution profiles of these

complexes by laser diffraction particle size analyzer are also presented in Table

2.

78

Compound Diameter (nm)

G1-NH- -CD 63

G2-NH- -CD 69

G3-NH- -CD 82

Table 2. Diameter of nanocarriers obtained using laser diffraction particle size

analyzer.

Figure 32. The size distribution profiles of G1- -CD and G2- -CD dendrimers

estimated by DLS.

79

Figure 33. Particle Diameter of G1-NH- -CD by using LDPSA.


80


3.3. In Vitro Release Studies

3.3.1. Loading of NLX Drug Molecule into the Dendrimers

The resultant dendrimer-cyclodextrin conjugates revealed enormously high

water solubility. Generally, dendrimers serve as carriers of biologically active

agents by encapsulating them in the interior or, joining them on the exterior

edging of the dendrimers. When the guest molecules are drugs, the resulted

drug/dendrimer complexes could be used as ideal candidates for carriers and in

drug release. Herein, Gn(n=1-3), Gn(n=1-3)- -CD and Gn(n=1-3)-NH- -CD as

biocompatible compounds have been synthesized, and the encapsulation of

NLX drug, using these structures has been attempted. The measurements of UV

from complexes confirmed the presence of drug in the resulted complexes.

3.3.2. Calculating the Amount of the Trapped and Released Drug Molecule

For determining the amount of drug encapsulated in the complex, 66 mg of

dried drug/dendrimer complexes was dissolved in 100 ml of water. After

complete dissolution of the drug/dendrimer complexes, the amounts of drug in

81

drug/dendrimer complexes was measured by UV spectrometer and the amounts

of the trapped drug for each generation before and after the functionalization

with -CD are displayed in Table 3. The UV detection for to determine the

quantity of drug inside the complexes was measured at 282 nm.

NLX loading efficiency of the complexes was determined as fallowing:

Amount of trapped drug in the complexes (g) = (A/B)

Loading efficiency (%) = (C/A) × 100

where A, B and C are the primary feeding amount of drug (g), determined free

drug content (g) and amount of trapped drug in the complexes (g) respectively.

Compound Naltrexone

Loading (%)

G1/NLX 8

G2/NLX 22

G3/NLX 27

G1-β-CD/NLX 17

G2-β-CD/NLX 32

G3-β-CD/NLX 37

G1-NH-β-CD/NLX 22

G2- NH-β-CD/NLX 35

G3- NH-β-CD/NLX 39

Table 3. Drug loading efficiency of Gn(n=1-3), Gn(n=1-3)- -CD and Gn(n=1-3)-NH- -

CD/NLX complexes.

As shown in Table 3 the loading efficiency% of carried drug by dendrimers was

enhanced with the rising of dendrimer generations. The drug content and

percent encapsulation efficiency was varying between 8 and 39%. The

82

maximum encapsulation of NLX was observed with G3-NH-β-CD in distilled

water of about 39%. The increased entrapment was possibly due to increase the

number of acidic groups of dendrimers (for electroestatic interactions), cavities

(for trap of drugs) in higher generations and the number of grafted -CD cavity.

Coating with -CD further increased drug entrapment because of the

hydrophobic cavity of CDs is capable of including a variety of hydrophobic

compounds using host-guest complexation. Whenever, it seems the ionic

interaction between dendrimers and guest molecule and host–guest interactions

including hydrophobic interactions, hydrogen-bonding and Vander Waals

interactions influence the ability of dendrimers as drug carriers.

3.3.3. Studying the Controlled Release of NLX from the Nanocarriers

The controlled release of NLX from the nanocarriers was investigated in pH 1,

7.4, and 10.

In vitro release behavior of NLX from the NLX/dendrimer complexes were

examined in buffered solution at different pH values (pH=1 and pH=7.4

max=282, pH=10 max=291 and 37 °C). Hydrolysis was carried out in a

cellophane membrane bags permeable to low molecular weight of compounds

and the amount of the drug release was determined in different pH values (pH 1,

7.4, and 10 all at 37 °C). UV absorbance measurements were carried out for the

characterization of NLX concentration in the complex solution. In vitro release

rate of NLX from dendrimers having -CD and without -CD in end groups of

dendrimer is compared and subsequent results were obtained. The results

displayed that both the rate and the amount of the release of the drug from

dendrimer/drug complex is influenced by some factors such as the generation of

the host molecule, biodegradability of the dendrimers, functional groups on the

surface of the dendrimer, solubility of the drug, the interactions between the

drug and the dendrimer in the complex and pH and the effects of each factor is

83

considered as the following. The comparison of the data showed that in

different pH’s the NLX released in different rates. In pH 10 the rate of release

of NLX was slower than pH 1 and 7.4. Interestingly, in all cases after

approximately 600 min, the release of NLX from the complexes is

approximately completed and is changed to a very slow rate. Figs. 36, Fig. 37

and Fig. 38 displays the controlled release of NLX from Gn (n=1-3), Gn (n=1-2)- -CD

and Gn (n=1-3)-NH- -CD at pH 7.4, respectively. As shown, the rate of release and

the amount of released drugs in complexes (pH 7.4) were increased with the

increase of generations but the rate of release in Gn (n=1-3)-NH- -CD was found

to be less compared to their corresponding dendrimers (Gn (n=1-3)) and these

results is related the hydrophobic interactions between -CD cavity and NLX

that leading to stable complexes and steric hindrance preventing the drug release

from open structure. The comparison of the data was confirm that the rate of

release in Gn (n=1-3)-NH- -CD dendrimers slightly slow than Gn (n=1-3)- -CD.

Because amide linkage in Gn (n=1-3)-NH- -CD were comparatively stable against

hydrolysis, whereas the ester band showed pH dependent release and the degree

of release varied with pH. Also, Fig. 39 shows the release profiles of NLX from

G3, G3- -CD and G3-NH- -CD at 37 °C at pH 1. As shown, the rate of release

for G3 faster than to G3- -CD and G3-NH- -CD, respectivly. Control released in

this case is related to the stable complexes between drug and -CD cavity. The

Fig. 40 and Fig. 41 shows the rate of release of NLX from G3-NH- -CD and G2-

-CD at pH 1, 7.4 and 10. As shown, the rate of release in pH 1 is faster,

because the complexes in this pH are not stable and the esteric bonds are

hydrolyzed. Also, the results showed that the drug release rate from all kinds of

the complexes was faster in the initial period of time. This rapid release was due

to the free drug remaining at the surface that was not entrapped competently

inside the dendrimer matrix. After this initial burst, slow and constant release

rate is observed. The slow release after the initial burst release was as a result of

the low dispersion of NLX from complexes. Again as shown the rate of released

84

drug at pH 7.4 is higher than pH 10 and lower than the pH 1. This result could

be related to the relative stability of the complexes in the above different pH’s.

Figure 36. Release of NLX from Gn (n=1-3)/NLX complexes as a function of time

and pH (pH 7.4, 37 ˚C).

Figure 37. Release of NLX from Gn (n=1-3)-NH- -CD/complexes as a function of

time and pH (pH 7.4, 37 ˚C).

pH=7.4

0102030405060708090100

0 100 200 300 400 500 600 700

Time (min)

Cum

ulativerelease%

G1

G2G3

pH=7.4

0

20

40

60

80

100

0 100 200 300 400 500 600 700

Time (min)

Cum

ulativerelease%

G1-NH- -CD

G2-NH- -CD

G3-NH- -CD

85

Figure 38. Release of NLX from Gn (n=1-2)- -CD/complexes as a function of time

and pH (pH 7.4, 37 ˚C).

Figure 39. Release of NLX from G3, G3- -CD and G3-NH- -CD/complexes as a

function of time and pH (pH 1, 37 ˚C).

0

10

20

30

40

50

60

70

0 100 200 300 400 500 600 700

Cum

ulat

ive

rele

ase

%

Time (min)

pH=7.4

G1- -CD

G2- -CD

pH=1

0102030405060708090100

0 100 200 300 400 500 600 700

Time (min)

Cum

ulativerelease%

G3

G3- -CD

G3-NH- -CD

86

Figure 40. Release of NLX from G3-NH- -CD/complexes as a function of time

and pH (pH 1, 7.4 and 10 and 37 ˚C).

Figure 41. Release of NLX from G2- -CD/complexes as a function of time and

pH (pH 1, 7.4 and 10 and 37 ˚C).

G3-NH- -CD

01020

3040506070

8090100

0 100 200 300 400 500 600 700

Time (min)

Cum

ulativerelease%

PH=1

PH=7.4

PH=10

87

Chapter 4: Conclusions

In this study dendrimers of citric acid and poly (ethylene glycol) as a core

compound was synthesized through divergent method. Then the synthesized

dendrimers modified with -CD and or with -CD-NH2 groups through ester

and amide linkage using DCC as a coupling agent, as a novel drug carrier

having an interior for encapsulation of drugs and a biocompatible surface. The

results were shown that the numbers of conjugated -CD units to dendrimers

increased with modification of -CD into -CD-NH2. Also -CD-modified

dendrimers having a shell of amphiphilic residues in the peripheral moiety of

the dendrimer in order to increase their encapsulation ability designed.

Complexes of the synthesized dendrimers with NLX have been developed. Rate

of release of guest molecules shows that the release depends on interaction

between host and guest molecule, generation of linear–dendritic

macromolecules, biodegradability of the dendrimers, functional groups on the

surface of the dendrimer and pH. Also, findings show that the -CD-coated

dendrimers can be utilized for sustained release delivery of NLX. Thus, results

exhibited that the -CD-grafted biodegradable citric acid dendrimers is a

potential candidate as an efficient drug carrier systems due to its relative

stability in aqueous solution and its drug encapsulation and release properties.

-CD groups were conjugated onto the surface of the dendrimers and the size of

the nanocarriers in the distilled water was investigated using DLS and LDPSA

technique which was 50, 70 nm (Gn(n=1-2)- -CD) and 63, 69 and 82 nm for G1-

NH- -CD, G2-NH- -CD and G3-NH- -CD, respectively.

88

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