1. INTRODUCTION

The idea of developing mucoadhesive polymers for drug delivery

has been introduced into the pharmaceutical product development for

more than 40 years ago and nowadays it has been subjected as a

promising strategy to improve the residence time and the specific

localization of drug delivery systems on various mucus membranes.

The term bioadhesion may be defined as a condition in which two

materials are held together for extended periods of time by interfacial

forces, out of which one material is in biological nature. In the

pharmaceutical literature, when the adhesive attachment is to mucus

or a mucous membrane, the phenomenon is classified as

mucoadhesion1.The mucoadhesive formulations have been

successfully used for the effective delivery of drugs for systemic

action/localized action and also to deliver the difficult molecules

(proteins and oligonucleotides) into the systemic circulation.

1.1. Mucoadhesion

For the last two decades the research on mucoadhesion has

become of interest because of its usefulness in improvising the

localized drug delivery, by retaining a formulation at the site of action

(e.g. within the gastrointestinal tract) or systemic delivery, by retaining

a formulation in intimate contact with the absorption site (e.g. the

nasal cavity). Moreover the mucoadhesive materials could also be

used as therapeutic agents by coating and protecting damaged tissues

(gastric ulcers or lesions of the oral mucosa) or by acting as

lubricating agents2 (in the oral cavity, eye and vagina).

1.1.1. Structure of mucus membrane

Mucus membranes (mucosa) are the moist surfaces lining the

walls of various body cavities such as the gastrointestinal and

respiratory tracts. The mucus consists of a connective tissue layer (the

lamina propria) above which is an epithelial layer and the mucus layer

makes the surface moist. The epithelia of mucosa may be either single

layered (e.g. the stomach, small and large intestine and bronchi) or

multilayered/stratified (e.g. in the esophagus, vagina and cornea). The

former contain goblet cells which secrete mucus directly onto the

epithelial surfaces, the latter contain, or are adjacent to tissues

containing, specialized glands such as salivary glands that secrete

mucus on to the epithelial surface. Mucus exists either as a gel layer

adherent to the mucosal surface or as a luminal soluble or suspended

form. The composition of mucus gels consists of mucin glycoproteins,

lipids, inorganic salts and water. The water portion accounts for more

than 95% of its weight and makes it a highly hydrated system3. The

mucin glycoproteins are the most important structure-forming

component of the mucus gel and are responsible for its characteristic

gel-like, cohesive and adhesive properties. The thickness of this

mucus layer varies on different mucosal surfaces, from 50 to 450 µm

in the stomach4-5 to less than 1 µm in the oral cavity6. The major

functions of mucus are that of protection and lubrication (they could

be said to act as anti-adherents).

1.1.2. Mechanisms of mucoadhesion

For the occurrence of mucoadhesion, the molecules must

involve in bond formation across the interface. The mechanism of

bond formation is varied and can be divided into following ways. (1)

Ionic bonds—In this the two oppositely charged ions comes together

through electrostatic interactions and forms a strong bond (e.g. in a

salt crystal). (2) Covalent bonds—this bond forms by sharing the

electrons, in pairs, between the bonded atoms in order to fill the

orbitals in both.

(3) Hydrogen bonds—here a hydrogen atom, when covalently bonded

to electronegative atoms such as oxygen, fluorine or nitrogen, carries

a slight positively charge and is therefore is attracted to other

electronegative atoms. The hydrogen bond considered to be weaker

than ionic or covalent bonds. (4) van der Waals bonds—this bond is

because of interaction that arise from dipole–dipole and dipole-

induced dipole attractions in polar molecules, and dispersion forces

with non-polar substances. 5) Hydrophobic bonds—this is a indirect

way of bond formation occurs when non-polar groups are present in

an aqueous solution by hydrophobic effect.

1.1.3. Theories of adhesion

There are six general theories of adhesion, which have been

adapted for the investigation of mucoadhesion7-8.

1) The electronic theory indicates the transfer of electrons between

the materials and adhering surface because of differences in the

electronic structure. This is proposed to result in the formation of an

electrical double layer at the interface, with subsequent adhesion due

to attractive forces.

2) The wetting theory is defined by surface and interfacial energies

between the adhesive material and mucus membrane and applied

mainly to liquid systems. It is the ability of a liquid to spread

spontaneously onto surface as a prerequisite for the development of

adhesion. The measure of contact angle defines the spread of liquid on

surface and as general rule the lower the contact angle, the greater

the affinity of the liquid to the solid. The spreading coefficient (SAB)

can be calculated from the surface energies of the solid and liquids

using the equation.

SAB = γB - γA - γAB

where γA is the surface tension (energy) of the liquid A, γB is the

surface energy of the solid B and γAB is the interfacial energy between

the solid and liquid. SAB should be positive for the liquid to spread

spontaneously over the solid.

The work of adhesion (WA) represents the energy required to separate

the two phases, and is given by,

WA = γA + γB - γAB.

The greater the individual surface energies of the solid and liquid,

relative to the interfacial energy, the greater the work of adhesion.

3) The adsorption theory explains the adhesion on the basis of

hydrogen bonding and van der –Waal‘s forces. It has been proposed

that these forces are the main contributors to the adhesive

interaction. A subsection of this, the chemi sorption theory, assumes

an interaction across the interface occurs as a result of strong

covalent bonding.

4) The diffusion theory laid down on the basis of the capability of

polymeric chains to diffuse across the adhesive interface. This process

is driven by concentration gradients and is dependent on the available

molecular chain lengths and their motilities. The depth of

interpenetration depends on the diffusion coefficient and the time of

contact. Sufficient depth of penetration creates a semi-permanent

adhesive bond.

5) The mechanical theory assumes that adhesion arises from an

interlocking of a liquid adhesive setting into irregularities on a rough

surface. However, rough surfaces also provide an increased surface

area available for interaction along with an enhanced visco elastic and

plastic dissipation of energy during joint failure, which are thought to

be more important in the adhesion process than a mechanical effect.

6) The fracture theory differs a little from the other defined

mechanisms in that it relates the adhesive strength to the forces

required for the detachment of the two involved surfaces after

adhesion. This assumes that the failure of the adhesive bond occurs

at the interface. However, failure normally occurs at the weakest

component, which is typically a cohesive failure within one of the

adhering surfaces.

1.1.4. Process of mucoadhesion

Because of its complexity, the process of mucoadhesion cannot

be defined by just one of the above defined mechanism. By

considering the mechanism of mucoadhesion, the possible ways of

mucoadhesive bond formation in in-vivo is described as below

(Fig.1.1)

These include:

(1) Dry or partially hydrated dosage forms contacting surfaces with

substantial mucus layers (typically particulates administered into the

nasal cavity).

(2) Fully hydrated dosage forms contacting surfaces with substantial

mucus layers (typically particulates of many First Generation

mucoadhesives that have hydrated in the luminal contents on delivery

to the lower G.I. tract).

(3) Dry or partially hydrated dosage forms contacting surfaces with

thin/discontinuous mucus layers (typically tablets or patches in the

oral cavity or vagina).

(4) Fully hydrated dosage forms contacting surfaces with

thin/discontinuous mucus layers (typically aqueous semisolids or

liquids administered into the esophagus or eye).

Fig. 1.1. Some scenarios where mucoadhesion can occur in in-

vivo

The mucoadhesive process between mucoadhesive material and a

mucus membrane takes place in two steps (Fig.1.2).

Step 1 —Contact stage: An intimate contact (wetting) occurs between

the mucoadhesive and mucous membrane.

Step 2 —Consolidation stage: Various physicochemical interactions

occur to consolidate and strengthen the adhesive joint, leading to

prolonged adhesion.

Fig 1.2: The two stages in mucoadhesion

1.1.4.1. The contact stage

The mucoadhesive and the mucous membrane have initially

come together to form an intimate contact. In some cases these two

surfaces can be mechanically brought together, e.g. placing and

holding a delivery system within the oral cavity, eye or vagina. In

others the deposition of a particle is encouraged via the aerodynamics

of the organ. For example within the nasal cavity or bronchi of the

respiratory tract deposition onto the sticky mucus coat is encouraged

by processes such as inertial impaction, in order to filter out particles

from the air stream9.

1.1.4.2. The consolidation stage

It has been proposed that if strong or prolonged adhesion is

required, a second consolidation stage is required. Mucoadhesive

materials reported to adhere most strongly to solid dry surfaces10 as

long as they are activated by the presence of moisture. Moisture will

effectively plasticize the system allowing mucoadhesive molecules to

become free, conform to the shape of the surface, and bond

predominantly by weaker van der-Waal and hydrogen bonding. In the

case of cationic materials such as chitosan, electrostatic interactions

with the negatively charged groups (such as carboxyl or sulphate) on

the mucin or cell surfaces are also possible. The mucoadhesive bond

formation is heterogeneous and is very difficult to identify, however

hydrogen bond formation is believed to be the most important. To

achieve strong adhesion, a change in the physical properties of the

mucus layer will be required otherwise it will readily fail on

application of a dislodging stress. Two theories explain by how a gel

strengthening/consolidation occurs. One is based on a

macromolecular interpenetration effect, which has been dealt with in

a theoretical basis (Fig.1.3). The second theory is the dehydration

theory. When a material capable of rapid gelation in an aqueous

environment is brought into contact with a second gel water

movement occurs between gels until equilibrium is achieved. The

dehydration theory is limited to explaining the adhesion arising when

a dry or partially hydrated formulation are brought into contact with a

substantial mucus gel, and will not apply to the occasions where

hydrated gels are involved.

Fig.1.3: The interpenetration theory; three stages in the interaction between a mucoadhesive polymer and mucin

glycoprotein

Fig. 1.4: The dehydration theory of mucoadhesion

1.1.5. Removal mechanisms

Adhesive failure will normally occur at the weakest component

of the joint. For weaker adhesives this would be the mucoadhesive–

mucus interface, for stronger adhesives this would initially be the

mucus layer, but later may be the hydrating mucoadhesive material.

Fig. 1.5: The possible regions for mucoadhesive joint failure

1.1.6. Factors affecting mucoadhesion

The process of mucoadhesion is complex and many factors

influence the mucoadhesion11. These most important factors that

influence the mucoadhesion process are

Optimum Molecular weight

Flexibility of polymer chains

Ionizable functional groups in the polymer

pH of suurounding medium

Presence of metal ions

1.1.7. Mucoadhesive materials

The types of materials used are very important in determining

the strength of mucoadhesion. The functional groups of the materials

are critical and the most widely explored group of mucoadhesives are

hydrophilic macromolecules containing numerous hydrogen bond

forming groups12-13, the so-called ―first generation‖ mucoadhesives.

The presence of hydroxyl, carboxyl or amine groups on the molecules

favours adhesion. They are called wet adhesives in that they are

activated by moistening and will adhere non-specifically to many

surfaces14. Typical examples are carbomers, chitosan, sodium alginate

and the cellulose derivatives (Fig.1.6). These materials have the

regulatory approval and considered as ―off-the-shelf‖, but in the last

few years, novel materials have been investigated. These novel

materials overcome the limitations of first generation off-the-shelf

mucoadhesive materials, and allow specificity, or prolong and

strengthen the mucoadhesion process. In some cases, existing

mucoadhesive polymers have been modified, while in others, new

materials are developed. One approach to produce improved

mucoadhesives has been to modify existing materials. For example

thiol groups (by coupling cysteine, thioglycolic acid, cysteamine) have

been placed into a range of mucoadhesive polymers such as the

carbomers, chitosans and alginates by Bernkop-Schnurch et al15-16.

The concept is that in-situ they will form disulphide links not only

between the polymers themselves thus inhibiting over hydration and

formation of the slippery mucilage, but also with the mucin

layer/mucosa itself, thus strengthening the adhesive joint and leading

to improved adhesive performance. This interesting approach appears

to be meeting with some success. The incorporation of ethyl hexyl

acrylate into a copolymer with acrylic acid in order to produce a more

hydrophobic and plasticized system was considered by Shojaei et al17.

The grafting of polyethylene glycol (PEG) onto poly(acrylic acid)

polymers and copolymers has also been investigated18-19. These

copolymers were shown to have favorable adhesion relative to poly

(acrylic acid) alone, in that the polyethylene glycol is proposed to

promote interpenetration with the mucus gel. Poly (acrylic acid)/PEG

complexes have also been developed as mucoadhesive materials20.

Poloxomer gels have been investigated as they are reported to show

phase transitions from liquids to mucoadhesive gels at body

temperature and will therefore allow in-situ gelation at the site of

interest. Pluronics have also been chemically combined with

poly(acrylic acid)s to produce systems with enhanced adhesion and

retention in the nasal cavity21. Dihydroxyphenylalanine (DOPA), an

amino acid found in muscle adhesive protein that is believed to lend

to the adhesive process, has also been combined with pluronics to

enhance their adhesion22. Lectins are proteins or glycoproteins that

have been considered second-generation bioadhesives, and differ

significantly from the polymeric systems described above. There is a

range of lectins available that interact with specific sugar residues via

relatively weak (secondary) interactions and have been considered for

use in targeted drug delivery23.

Fig.1.6. The structure of some common “first generation” mucoadhesive polymers

Out of all these attempts in the process of developing newer

mucoadhesive polymers the thiol bearing polymers the so called

‗Thiomers‘ have shown to be promising in the designing of new drug

delivery systems.

1.2. Thiomers

Right from the concept of mucoadhesion has arrived in the

mucoadhesive research, various attempts have been undertaken in

order to improve the adhesive properties of polymers. These

approaches include the use of linear poly(ethylene glycol) as adhesion

promoter for hydrogels, the neutralization of ionic polymers,

mucoadhesion by a sustained hydration process and the development

of polymer–adhesion conjugates providing a specific binding to

epithelia. However, all these systems are based on the formation of

non-covalent bonds such as hydrogen bonds, van der-Waal‘s forces,

and ionic interactions. Accordingly, they ended with a relatively weak

mucoadhesion, in many times failed in the localization of a drug

delivery system at a given target site for longer duration of time.

Mucoadhesive polymers have therefore in many cases not proven to be

effective as pharmaceutical glue. In the process of identifying novel

mucoadhesive excipients a presumptive new generation of

mucoadhesive polymers, thiolated polymers—designated thiomers,

were discovered24. In contrast to well-established mucoadhesive

polymers these novel polymers are capable of forming covalent bonds.

These thiomers creates a disulfide bond with biological systems

thereby been discovered for the covalent adhesion of polymers to the

mucus gel layer of the mucosa. Thiomers are mucoadhesive basis

polymers, which display thiol bearing side chains (Fig.1.7). Based on

thiol/disulfide exchange reactions and/or a simple oxidation process

as illustrated in, disulfide bonds are formed between such polymers

and cysteine-rich subdomains of mucus glycoproteins (Fig.1.8).

Hence, thiomers mimic the natural mechanism of secreted mucus

glycoproteins, which are also covalently anchored in the mucus layer

by the formation of disulfide bonds25.

Fig. 1.7: Synthesis of Thiolated polymers (Thiomers)

Fig. 1.8: Mechanism of disulfide bond formation between

Thiomers and mucus glycoproteins (mucins)

1.2.1. Types of Thiomers

Based on the surface functional groups generated on the

polymers thiomers are classified into cationic thiomers and anionic

thiomers.

1.2.1.1. Cationic thiomers

Cationic thiomers mainly exhibits primary amino group as

anionic substrate. Chiotsans are widely studied substance in the

preparation of cationic thiomers. The primary amino group at the 2-

position of the glucosamine subunits of this polymer is the main

target for the immobilization of thiol groups. Various carboxylic acid

group containing ligands such as cysteine and thioglycolic acid can

be covalently attached to this primary amino group via the formation

of amide or amidine bonds. These carboxylic groups react with amine

groups of chitosan mediated for instance by carbodiimides. An

unintended oxidation of thiol groups during synthesis can be avoided

by performing the reaction under inert conditions or at controlled pH

conditions. Furthermore, disulfide bonds can be reduced after the

synthesis process by the addition of reducing agents such as

dithiotreithol or borohydride. 2-Iminothiolane is also can be used as

coupling agent to attach by amidine bonds 2-iminothiolane. It offers

the advantage of a simple one step coupling reaction. In addition, the

thiol group of the reagent is protected towards oxidation due to its

chemical structure26.

1.2.1.2. Anionic thiomers

Anionic thiolated polymers represent the presence of anionic

substructures on the polymer surface such as carboxylic groups.

These carboxylic acid groups can be easily attached to polymers

through the formation of amide bonds. Sulfahydral bearing ligands

such as cysteine, cysteamine and homocysteine are used. The

formation of amide bonds can be mediated by carbodiimides. The

formation of oxidative products can be minimized by conducting the

reaction at controlled conditions. In addition, disulfide bonds formed

during synthesis can be cleaved thereafter by the addition of reducing

agents such as dithiothreitol or NaBH4.

1.2.2. Mechanisms for improved mucoadhesion by thiomers

The formation of disulfide bonds between the thiomers and the

mucus gel layer takes place either via thiol/disulfide exchange

reactions or via a simple oxidation process of free thiol groups.

Generally there are no mucosal surfaces in which mucins with

cysteine rich subdomains are not present. In contrast to noncovalent

bonds disulfide bonds are not influenced by factors such as ionic

strength and pH. Velocity and extent of disulfide bond formation

depends on the concentration of thiolate anions representing the

reactive form for thiol/disulfide exchange reactions and oxidation

processes. The concentration of thiolated anions in turn depends on:

a) The pKa value of the thiol group in dependence on the polymer

backbone and the chemical structure of the ligand, more or less

reactive thiomers can be designed. Thiol groups of the chitosan–

thiobutylamidine conjugate, for instance, exhibit a pKa value of 9.9,

whereas the pKa value of the thiol groups of poly(acrylate)–cysteine

conjugates is 8.35.

b) The pH of the thiomer. As only ionic thiomers are used, they all

display a high buffer capacity. The buffer capacity of a sodium

poly(acrylate) matrix tablet, for instance, can be compared with that of

an at least 25 M acetate buffer. As all charged groups remain

concentrated on the polymeric network a kind of ―microclimate‖ can

be established. The reactivity of thiol groups can consequently be

controlled by adjusting the pH of the polymer to a certain level. The

higher the pH is adjusted, the more reactive are the thiol groups and

vice versa

c) And the pH of the surrounding medium. The reactivity of thiol

groups inside the polymeric network is mainly controlled by the pH of

the thiomer, whereas the reactivity on the surface of the polymer is

more controlled by the pH of the surrounding medium. As the mucus

gel layer being close to the epithelium has a pH around 7, thiol groups

penetrating into the mucus are always sufficiently reactive. Leitner et

al27 could show by four different methods including rheological,

diffusion, gel permeation and certain mucoadhesion studies the

formation of disulfide bonds between thiolated polymers and mucus

glycoproteins.

1.2.2.1. In situ cross-linking process

Another likely mechanism being responsible for the improved

mucoadhesive properties of thiomers is based on their in situ cross-

linking properties28. The mechanism is illustrated in Fig.1.9. It is

similar to the mechanism on which the adhesive properties of most

adhesive are based on, i.e. a penetration of the adhesive into a certain

surface structure followed by a stabilization process of the adhesive.

In case of superglues, for instance, monomeric cyanoacrylates

penetrate into raw surfaces followed by a polymerization process.

Thiolated polymers display in situ gelling properties due to the

oxidation of thiol groups at physiological pH-values, which results in

the formation of inter- and intramolecular disulfide bonds. These in

situ gelling properties are in particular of interest for liquid or

semisolid vaginal, nasal and ocular formulations, which should

stabilize themselves once applied on the site of drug delivery.

Fig. 1.9: Schematic presentation of improved mucoadhesion by an in- situ cross-linking in comparison to chain links.

1.2.3. Features of thiomers

The thiomers because of its surface bearing sulfahydral groups

and its polymeric nature, they exhibit different properties and found

to be advantageous for variety of applications. These include

Mucoadhesive and cohesive properties29-30

Enzyme inhibitory properties30-31

Permeation enhancing properties32-33

Thiomers as matrices for controlled drug release34

1.2.4. Stability of thiomers

Because of the sensitivity of thiol groups towards oxidation, the

chemical stability of thiomers has already been investigated in detail.

PCP-Cys and chitosan-TGA were tested both as representative anionic

and cationic candidates, respectively. And found to be stable under all

storage conditions when compressed into matrix tablets35.

With the introductory note above described about the

advantages of mucoadhesive systems and applications of thiomers, it

is concluded that the potential of thiomeric mucoadhesive drug

delivery systems for the designing of formulations for small molecule

drugs, proteins and peptides for various routes of administration is

enormous, however so far synthesized thiomers have limited

sulfahydral groups and the resultant mucoadhesion is weak. For

producing the longer mucoadhesion with the same thiomers require

larger quantity of polymer which is an inhibiting factor. And some

polymers are of carbohydrate based and may be antigenic. The

polymers which are having multiple reactive functional groups to

conjugate with thiol bearing ligands, biocompatible, non antigenic will

be of advantage in preparing the thiomeric polymers. Dendrimers with

its multiple surface functional groups are suitable for synthesizing the

thiomers with high amount of thiol per mole of dendrimer.

1.3. Dendrimers

As polymer science has evolved over the past two centuries, the

number of compositions and architectures of macromolecules

synthetically accessible has also grown. The ability to easily tune the

size, chemistry, topology and ultimately the properties of polymers

through chemical synthesis inevitably has led to their widespread use

in variety of technological applications. The myriad properties and

functions that can be designed into polymeric systems are prompting

the medical community to use polymers in the drug delivery, tissue

engineering and biological imaging. The highly branched and

symmetrical molecules as dendrimers are the most recently

recognized members of the polymer family, with the first dendrimer

reports published in the late 1970s and early 1980s by the several

groups36-39. Dendritic polymers can differ significantly from linear

polymers in their properties. They have a number of beneficial

attributes for biomedical applications, including the following:

Bio distribution and pharmacokinetic properties that can be

tuned by controlling dendrimer size and conformation. This can

be achieved with precision by varying dendrimer generation

number or by creating dendrimer-polymer hybrids.

Huge structural and chemical homogeneity, dendrimer

biological properties can be attributable to a single molecular

entity and not a statistical distribution of polymeric or self

assembled materials facilitating the reproducibility of

pharmacokinetic data within and between different synthetic

loss.

Ability to be functionalized with multiple copies of drugs,

chromophores or ligands either at their peripheries and/or their

interiors. Dendrons also can be used to precisely increase the

drug loading capacity of carriers, such as antibodies and

biocompatibility polymers like poly(ethylene glycol).

High ligand density, unlike in linear polymers as dendrimer‘s

generation increases, the multivalent ligand density at the

surface increases, which can strength ligand receptor binding

and improve the targeting of attached components.

Controlled degradation: This can be achieved by judicious

choice of dendrimer chemistry, with unique modes of

decomposition accessible through use of self immolative

dendrimers.

1.3.1. Dendrimer chemistry and structure

A dendrimer is a polymeric molecule composed of multiple

perfectly branched monomers that emanate radially from a central

core reminiscent of a tree, hence dendrimers derive their name (Greek

Dendra). A dendron can be divided into three different regions: The

core, the interior (or branches) and the periphery (or end groups). The

number of branch points encountered upon moving outward from the

core of the dendron to its periphery defines its generation (G-1, G-2,

G-3), dendrimers of higher generations are larger, more branched and

have more end groups at their periphery than dendrimers of lower

generations (Fig.1.10). A dendrimer may be based on practically any

type of chemistry, the nature of which can determine its solubility,

degradability and biological activity. Some of the commonly

encountered types of dendrimers in biological applications are based

on polyamidoamines40, polyamines41, polyamides (polypeptides),

poly(aryl ethers), polyesters, carbohydrates and DNA42 (Fig 1.11). By

far the most common dendrimer scaffold is that of the

polyamidoamine (PAMAM) dendrimers, which are available

commercially with a wide variety of generation and peripheral

functionalities.

Perhaps the most exploited property of dendrimers is their

multivalency. Unlike in linear polymers, as dendrimer molecular

weight and generation increases, the terminal units become more

closely packed, a feature exploited by many investigators as a means

to achieve concentrated payloads of drugs or spectroscopic labels for

therapeutic and imaging applications. The many end groups can also

greatly modulate dendrimers solubility: hydrophilic end groups can

make water soluble a dendrimer with a hydrophobic core; where as

hydrophobic peripheral moieties can make a dendrimer with a

hydrophilic interior soluble in oil. Dendrimer multivalency is

particularly useful when multiple copies of ligands are affixed to the

periphery of the molecule. Dendrimer multivalency has lent itself to

applications ranging from the prevention of tumour cell adhesion and

metastasis by carbohydrate modified dendrimers43 (in vitro) to the

inhibition of HIV infection by sulphate modified dendrimers in primate

studies43.

Fig.1.10: A dendrimer and dendron are represented with solid

lines. (The colored, broken lines identify the various key regions of the dendrimer)

Fig.1.11: The variety of dendrimers used in biology. A few examples of the types of dendrimer chemistries used in

biological applications. (a) G-2 poly(glutamic acid) dendrimer. (b) G-2 polyamidoamine (PAMAM) dendrimer. (c) G-3

polypropyleneimine (PPI) dendrimer. (d) G-3 polymelamine

dendrimer. (e) G-2 polyester dendrimer

1.3.2. Biological Applications

1.3.2.1. Drug and gene delivery

By attaching a drug to a suitable carrier it is possible to

enhance its aqueous solubility, increases its circulation half life,

target the drug to certain tissues while minimizing drug exposure to

healthy tissues, results in therapeutic efficacy. Numerous reports on

the in vitro efficacy of purely dendrimer based drug carriers have been

published, but only a few in vivo therapeutic studies exist. One of the

earliest examples of anti tumour drug delivery with dendrimers was

achieved by complexing cisplatin (20-25% by weight) to the surface

groups of G-4 carboxylate terminated PAMAM dendrimer45.

Conjugation of cisplatin to the dendrimer led to a tenfold increase in

cisplatin solubility, but the drug also caused cross linking between

dendrimers, resulting in aggregates with diameters 3-40 nm. When

administered intravenously to mice, the aggregates targeted

subcutaneous tumours via a passive targeting mechanism known as

the enhanced permeation and retention effect. PAMAM dendrimers

have also been used as anti tumour targeted carriers of

methotrexate46. The peripheral amines of G-5 PAMAM dendrimers

were first partially modified with acetyl groups to reduce dendrimer

surface charge. The acetylated PAMAM was subsequently

functionalized with folate as a targeting ligand.

1.3.2.2. Imaging

In vivo imaging is an increasingly useful tool in biomedicine, as

it is non-invasive and provides a wealth of information regarding the

native states of a variety of tissue types. The earliest in vivo uses of

dendrimers were as carriers for magnetic resonance imaging contrast

reagents47. Another non invasive imaging application of dendrimers

involves photonic oxygen sensing. By encapsulating hydrophobic

metalloporphyrins in the cores of variously sized poly (glutamic acid),

poly (aryl ether) or poly (ether amide) dendrimers Vinogradov et al48

have prepared water soluble oxygen sensors whose phosphorescence

is quenched upon collision with dissolved oxygen.

1.3.2.3. Intrinsic drug properties

Whereas the majority of dendrimer designs have been used as

carriers for drugs and nucleic acids, some dendrimers act as drugs

themselves. Supattapone et al49 discovered that branched polyamines

including PAMAM dendrimers and hyper branched polymers stimulate

the removal of prion proteins present in infected cells. The branched

architecture appears essential to this application because linear

polyamines and small molecule amines are ineffective.

Multivalent display of ligands on the surface of a dendrimer has

also proven to be a viable method of inhibiting multivalent binding

between cells, viruses, bacteria, proteins and combinations thereof.

For example, a G-4 poly(L-Lysine) dendrimer bearing sulphate groups

at its periphery is being evaluated as an anti viral topical ointment50.

1.3.3. Biocompatibility of dendrimers

The success of dendrimers as carriers or biomaterials will

depend in large part on their biocompatibility-whether dendrimers

elicit an undesirable response from their biological host. Long term

accumulation of low molecular weight compounds is not often a

problem because they are excreted in the urine or in the faeces after

metabolism. However, injected polymers are not eliminated as easily,

especially if they are not readily degraded into smaller units51 or are

too large to be filtered via the kidneys. Thus for dendrimers which can

be classified as low molecular weight or polymeric depending on their

generation, acceptable biocompatibility must be accompanied by a

reasonably fast renal elimination rate or biodegradation rate.

1.3.3.1. In vitro toxicity

In most cases, the nature of a dendrimers numerous end

groups dictate whether or not it displays significant toxicity. For

example, cationic dendrimers with terminal primary amino groups

such as PAMAM and polypropyleneimine (PPI) dendrimers generally

display concentration dependent toxicity and haemolysis52, where as

dendrimers containing only neutral or anionic components have been

shown to be much less toxic and less haemolytic. Cytotoxicity of

amino terminated dendrimers can be lessened by partial or complete

modification of the dendrimer periphery with negatively charged or

neutral groups53. The toxicity of cationic PAMAM dendrimers with

each generation, but surprisingly cationic PPI dendrimers does not

follow this trend.

1.3.3.2. In vivo toxicity

In vivo toxicity correlates reasonably well with in vitro toxicity.

Mice tolerate low intraperitoneal doses of positively charged PAMAM

dendrimers (~ 10 mg/kg). Acute and sub chronic toxicity studies in

mice with melamine dendrimers bearing cationic surface charges

revealed that intraperitoneally administered doses above 10 mg/kg

produced liver toxicity, as demonstrated by increased levels of alanine

transaminase in serum and liver necrosis upon histopathological

analysis. When ~ 50 % of the cationic groups of a structurally similar

dendrimer were replaced with neutral polyethylene oxide chains, no

acute or sub chronic toxicity was observed after intraperotoneal of

intravenous injection of doses greater than 1 g/kg54, similarly, a

family of non charged polyester dendrimer showed very low toxicity.

1.3.4. Degradation behavior of dendrimers

Biodegradability of dendrimers is a valuable attribute that can

prevent bioaccumulation and the possible toxic effects associated with

its occurrence. The most widely studied dendrimers, PAMAMs, are

hydrolytically degradable only under harsh conditions because of their

amide backbones and hydrolysis proceeds slowly at physiological

temperatures. Photolytically labile dendrimers may allow external

initiator and spatially addressable dendrimer degradation. Dendrimers

in which the dendrons are released from the core in which the

dendrimer peripheral groups are cleaved55 or in which the dendrimer

peripheral groups are cleaved or in which the entire dendrimer

degrades into a identical small molecule fragments upon ultraviolet

irradiation have been prepared. Although the aromatic decompositions

products of some of the dendrimers are non-toxic. It will be interesting

to earn if less hydrophobic aliphatic molecules cab be used to increase

dendrimer solubility and ensure their biocompatibility.

1.3.5. Pharmacokinetics behavior of dendrimers

An understanding of dendrimer pharmacokinetics is essential

for their application in medicine because the bioavailability, toxicity

and ultimately efficacy of dendrimer based drugs and imaging agents

will depend on heir tissue accumulation profiles, drug release rates

and elimination rates56. The important feature is he that drugs that

are loaded into dendrimers using non covalent hydrophobic or

hydrogen bonding interactions are rapidly released when the

dendrimer drug combination is placed into the bio phase and thus

drug targeting is not optimal because the drug leaves the carrier

before the carrier arrives at its intended target.

In the view of above described advantages of dendrimers here in

the present project it is decided to prepare novel thiolated dendrimer

excipients for designing the mucoadhesive systems. Hence by virtue of

combining the advantages of thiomers in mucoadhesion and

dendrimers in mucosal penetration behavior the prepared thiolated

dendrimers will be tested for their efficacy by applying to different

mucosal organs such as ocular, oral and nasal.

1.4. Ocular delivery

Topical administration of therapeutic moieties to the eye is the

most popular and well recognized route of administration for the

treatment of ocular disorders. However, because of efficient protective

mechanisms of the eye, such as blinking, baseline and reflex

lachrymation and drainage remove rapidly foreign substances,

including drugs, from the surface of the eye, the ocular formulations

are rapidly drained from the ocular surface and limits the

bioavailability only to below 5% and also the residence time of the

formulation is very short57. Moreover, the anatomy, physiology and

barrier function of the cornea compromise the rapid absorption of

drugs58. In order to achieve the therapeutic levels of drugs, frequent

dosage of formulations is required but it may result in inducing toxic

side effects and cellular damage at the ocular surface59-60. To improve

the ocular bioavailability and penetration, the applied formulations

shall contact the cul-de-sac for prolonged time. Various strategies

have been developed and met some success in meeting the

therapeutic efficacy.

1.4.1. Anatomy and physiology of eye

The structure and construction of eye is very complex. The

protective wall of the eyeball has consists of three layers: the outer

coat or the sclera and cornea, a middle layer or uveal coat and the

inner coat or retina. The sclera is made of fibrous tissues shaped as

segments of two spheres, the sclera and cornea61. The external part of

the eye is covered by the mobile tarsal part of the eyelids. The eyelids

are under involuntary and voluntary control. They play role in

distributing the tear fluid over the eye, providing an optically smooth

surface over the cornea. The shear rate during blinking is estimated to

be about 20,000 s_1, and influences ocular bioavailability of applied

drugs by influencing the rheological properties62.

The cornea is a clear, transparent, avascular tissue to which

nutrients and oxygen are supplied by the lachrymal fluid and aqueous

humour. It is composed of five layers: epithelium, Bowman‘s layer,

stroma, Descemet‘s membrane and endothelium

The conjunctiva is a thin transparent membrane, which lines

the inner surface of the eyelids and is reflected onto the globe. At the

corneal margin, it is structurally continuous with the corneal

epithelium. The membrane is vascular and moistened by the tear film.

The conjunctiva is composed of an epithelium, a highly vascularised

substantia propria, and a submucosa or episclera. The bulbar

epithelium consists of 5 to 7 cell layers. At the surface, epithelial cells

are connected by tight junctions, which makes the conjunctiva

relatively impermeable. The conjunctival tissue is permeable to

molecules up to 20,000 Da, whereas the cornea is impermeable to

molecules larger than 5000 Da.

The mucus layer is very sensitive to hydration and forms a gel-

layer with viscoelastic rheological properties. It protects the epithelia

from damage and facilitates the movements of the eyelids. Mucins

improve the spreading of the tear film and enhance its stability and

cohesion. Mucus is wiped over the surface of the eye by the upper

eyelid during blinking. The mucus gel entraps bacteria, cell debris,

and foreign bodies, forming mucous threads consisting of thick fibers

arranged in bundles. These threads are transported during blinking to

the inner canthus and expelled onto the skin. The mucus layer can

form a diffusion barrier to macromolecules depending on the degree of

network entanglement. On the other hand, mucus can bind cationic

substances because of the negative charges of mucins. Mucus

consists of glycoproteins, proteins, lipids, electrolytes, enzymes, muco

polysacchrides and water. The primary component of mucus is mucin,

a high-molecular-mass glycoprotein with subunits containing a

protein core, approximately 800 amino acids long, of which about 200

are bearing polysaccharide side-chains. The protein core consists of

tandem repeat regions, which are repeated sequences of mainly

serine, threonine and proline. The polysaccharide side chains are

linked to the protein core by an O-glycosidic bond between N-

acetylgalactosamine on the sugar chain and the hydroxyl groups of

the serine and threonine residues on the protein backbone. As the

polysaccharide side chains usually terminate in either fucose or sialic

acid (N-acetylneuraminic acid, pKa=2.6), the glycoprotein is negatively

charged at physiological pH.

1.4.2. Ocular Pharmacokinetics

The main routes of drug administration and elimination from

the eye have been shown schematically in the below figure (Fig. 1.12).

They are

1) Transcorneal permeation from the lacrimal fluid into the

anterior chamber.

2) Non-corneal drug permeation across the conjunctiva and sclera

into the anterior uvea

3) Drug distribution from the blood stream via blood-aqueous

barrier into the anterior chamber

4) Elimination of drug from the anterior chamber by the aqueous

humor turnover to the trabecular meshwork and Sclemm's

canal

5) Drug elimination from the aqueous humor into the systemic

circulation across the blood-aqueous barrier

6) Drug distribution from the blood into the posterior eye across

the blood-retina barrier

7) Intravitreal drug administration

8) Drug elimination from the vitreous via posterior route across

the blood-retina barrier

9) Drug elimination from the vitreous via anterior route to the

posterior chamber

Fig. 1.12: Schematic presentation of the ocular structure with the

routes of drug kinetics

1.4.3. The barriers for corneal absorption of drugs

a) Drug loss from the ocular surface

After administration, the flow of lacrimal fluid removes the

therapeutic agents from the surface of the eye. After application the

dosage form, the excess volume is cleared from nasolacrimal duct63.

Another way of drug removal from ocular surface is by systemic

absorption through conjunctival sac or through nasal cavity64.

b) Lacrimal fluid-eye barriers

Another barrier which prevents the drug absorption from

corneal epithelium is the lacrimal fluid. The corneal barrier is formed

upon maturation of the epithelial cells. They migrate from the limbal

region towards the center of the cornea and to the apical surface. The

most apical corneal epithelial cells form tight junctions that limit the

paracellular drug permeation65. Hence, lipophilic drugs have

increased permeability than the hydrophilic drugs. Despite the

tightness of the corneal epithelial layer, transcorneal permeation is

the main route of drug entrance from the lacrimal fluid to the aqueous

humor. It is considered that, the conjunctiva is more leaky epithelium

than the cornea and its surface area is also nearly 20 times greater

than that of the cornea.

c) Blood-ocular barriers

The eye is protected from the forgein matter in the blood stream

by blood-ocular barriers. These barriers have two parts: blood-

aqueous barrier and blood-retina barrier. The anterior blood-eye

barrier is composed of the endothelial cells in the uvea. This barrier

prevents the access of plasma albumin into the aqueous humor, and

also limits the entry of hydrophilic drugs from plasma into the

aqueous humor. The posterior barrier between blood stream and eye

is comprised of retinal pigment epithelium (RPE) and the tight walls of

retinal capillaries. Unlike retinal capillaries the vasculature of the

choroid has extensive blood flow and leaky walls. Drugs easily gain

access to the choroidal extravascular space, but thereafter

distribution into the retina is limited by the RPE and retinal

endothelia. Despite its high blood flow the choroidal blood flow

constitutes only a minor fraction of the entire blood flow in the body.

Therefore, without specific targeting systems only a minute fraction of

the intravenous or oral drug dose gains access to the retina and

choroid. Unlike blood brain barrier, the blood-eye barriers have not

been characterised in terms of drug transporter and metabolic enzyme

expression.

1.4.5. Drug elimination from lacrimal fluid

Several mechanisms such as a relatively impermeable cornea1

barrier and rapid drainage of the instilled solution protect the eye.

Mechanistic studies by Lee and Robinson66 have shown that drugs are

mainly eliminated from the pre corneal lacrimal fluid by solution

drainage, lacrimation and nonproductive absorption to the

conjunctiva of the eye (Fig. 1.13). These factors and the cornea1

barrier limit the penetration of the topically administered drug into

the eye. Only a few percent of the applied dose is delivered into

intraocular tissues, while the major part (50-100%) of the dose is

absorbed systemically (Fig. 1.14).

Fig. 1.13: Elimination of topically applied drugs from cornea

Fig. 1.14: Fate of Topically applied drugs in cornea.

1.4.6. Novel drug delivery systems for increased ocular

bioavailability

Wide research has been conducted for last two decades for

improvising the delivery efficacy of the ocular dosage forms. Increasing

the corneal penetration and improving the corneal residence time are

the two focussed areas considered for effective delivery of ophthalmic

drugs. Improving the viscosity of the formulation allows residing the

formulation for longer time at the cornea and improving the ocular

bioavailability of drugs67-68. Apart from the increasing the viscosity,

the application of bioadhesive polymers for reducing the drainage loss

after instillation of ophthalmic formulations, hence improving drug

absorption or local action is reported69. In designing the bioadhesive

polymers various properties of polymers such as hydration or degree

of swelling, molecular weight, functional groups, molecular

conformation or chain flexibility and mobility, and concentration

influence the adhesion to the cornea70-71.

The polymers by its charge get interact with mucus layer of the

eyes and enhance the residence time of the formulation. Apart from

increasing the residence time some mucoadhesive polymers also act

as protective and healing agents, bioavailability enhancers. The

composition and properties of the tear film will influence the

mucoadhesion of ocular delivery systems. Various theories (electronic,

adsorption, wetting, diffusion or interpenetration) were proposed to

explain bioadhesion or mucoadhesion. In order to be a good

mucoadhesive adjuvant, the polymer of the drug delivery system must

make intimate contact with the mucus layer. The polymer chains

must be mobile and flexible enough to inter diffuse into the mucus

and penetrate to a sufficient depth in order to create a (strong

entangled) network.

1.4.7. Thiomer preparations in ocular drug delivery

The so far described mucoadhesive polymers forms non covalent

bond with tear film and may not be sufficient enough to increase the

residence time of the formulation at the cornea. In the process of

exploiting the new polymers, thiomers have been designed. Thiomers

are thiol bearing polymers and helps in the formation of covalent

linkage with cornea. The rationale of the strategy is based on

knowledge concerning the role of disulfide bridges in the three-

dimensional mucin network formation72. Thiolated polymers, or so

called thiomers, are capable of forming covalent bonds with cysteine-

rich subdomains of mucins, where as mucoadhesive polymers

discussed so far formed non-covalent bonds (hydrogen bonds or ionic

interactions) with mucus, or exhibited physical entanglements73. The

schematic representation of interactions of thiomers with mucin

present on mucosa was given in Fig. 1.15. The extensive cross-linking

process of the thiomers with mucins resulted in a tremendous

increase in viscosity and mucoadhesion independent of pH or ionic

strength of the medium. The mucoadhesive properties of a chitosan

thioglycolic acid conjugate and a poly(acrylic acid)–cysteine conjugate

improved 10-fold and even 100-fold, respectively, compare to the

native polymers74-75. The thiomer formulation improve the cohesive

properties by a simple oxidation process in aqueous media and results

in the formation of inter- and/or intrachain disulfide bonds within the

polymeric network76. And also some of the thiomers have permeation

capability and promote the paracellular uptake of drugs by opening

the tight junctions. An in vitro study on the cornea of rabbits with

polycarbophil–cysteine showed a 2.2-fold and 2.4-fold increase in the

transcorneal permeation of sodium fluorescein and dexamethasone

phosphate, respectively, when compared to the unmodified poly

carbophil. Thiomers could be useful additives in artificial tear

formulation due to anti oxidative and radical scavenging properties.

Moreover, thiomers are similar to ocular mucins displaying numerous

thiol groups. Thiomers also mimic the physiological process of the

mucus layer such as tear film stabilization. The formation of disulfide

bonds with mucins leads to strong mucoadhesion, prolonged

residence time and protective effect for the corneal/conjunctival

epithelium. Considering mucoadhesion of thiomers based on covalent

bond formation with mucins, inserts (diameter 2mm) consisting of

PAA 450–cysteine conjugate, a thiolated poly(acrylic acid) 450 kDa,

were prepared by direct compression and evaluated by

fluorophotometry. In humans, the inserts based on thiolated

poly(acrylic acid) or PAA 450–cysteine conjugate provided a sufficient

fluorescein concentration in the tear film for more than 8 h. Inserts

made of thiomers were not soluble, and had good cohesive properties,

due to the formation of inter- and/or intra chain disulfide bonds

within the polymeric network after hydration. The general irritation

score indicated that the inserts were well accepted and tolerated77.

Fig. 1.15: Schematic representation of the interactions between thiomers and mucins

1.5. Oral delivery

Oral delivery of drugs is the most promising approach and

convenient way of administering the drugs. However, various factors

influence the oral absorption and limits bioavailability thereby

restricting the oral delivery for drugs. Poor uptake of the drugs and

short residence time of formulations are limiting factors in successful

administration of drugs by oral route. Gastric emptying plays an

important role in the dynamics of drug absorption and can lead to

variable and unpredictable availability. It is common for conventional

oral dosage forms to transit rapidly through the stomach, both in the

fasted and fed conditions, although the latter prolongs transit time to

some extent. Numerous strategies have been proposed to modify the

GI transit of oral pharmaceutical formulations. Approaches include

formulations that swell or expand in the gastric content and are

retained in the stomach by floating on the gastric contents or else are

too large to pass through the pylorus78-79. Alternative potential

approach is to design a mucoadhesive formulation which can adhere

to the lining of the stomach or small intestine, thus retaining the drug

at the target absorption site for a prolonged period.

1.5.1. Mucoadhesion approach for oral drug delivery

In order to develop an ideal oral bioadhesive system it is

important to have a thorough understanding of mucosa, bioadhesive

polymers and mucin-polymer interactions in the physiological

environment. Mucoadhesive drug delivery systems exploit the

attraction between polymers used in drug formulations and the

mucus layer that covers epithelial surfaces throughout the body,

including the gastrointestinal tract80-81. Mucus occurs in vivo both as

a gel layer that adheres strongly to mucosal surfaces and as a luminal

soluble or suspended forms82. Mucus gels consist more than 95% of

water and thoroughly hydrate the other mucus components: lipids,

inorganic salts, and mucins. Mucins tend to have a blocky

architecture: most of the glycans are confined to certain densely

glycosylated regions of the chain, separated from each other by

‗‗naked‘‘ protein stretches bearing only few sugar residues. Mucins are

large macromolecules, with molecular weights ranging from 10 to 50 ·

106 g/mol. They tend to assemble into even larger assemblies, via

hydrophobic interactions between amino acid residues, hydrogen

bonding among sugar units, or disulfide linkages involving cysteines83.

This variability among mucus throughout the body suggests roads

towards site-specific delivery of mucoadhesive drug formulations

through control, on the molecular level, of the interactions between

mucins and mucoadhesive polymers84.

Various investigators have proposed different mucin-polymer

interactions, such as:

Wetting and swelling of the polymer to permit intimate contact

with the biological tissue;

Interpenetration of bioadhesive polymer chains and

entanglement of polymer and mucin chains;

Formation of weak chemical bonds

Sufficient polymer mobility to allow spreading

Water transport followed by mucosal dehydration.

As the mucus layer comes into contact with bioadhesive coated

system, various non specific (van der walls, hydrogen bonding and /or

hydrophobic interactions) or specific interactions occur between the

complimentary structures. In order to improve the targeting and

delivery of orally administered drugs to the stomach and the small

intestine, the interest in mucoadhesive polymers has greatly increased

in the last two decades. The use of these polymers as carriers is

supposed to enhance the bioavailability of orally given drugs because

of a lengthened contact time of the drug with the gastrointestinal (GI)

mucosa. When mucoadhesive polymers also exhibiting permeation

enhancing properties are used, the intensified contact with the

mucosa should provide the prerequisite for an increased epithelial

permeability for many drugs mediated by the polymeric carrier

system.

1.5.2. Bioadhesive polymers

Bioadhesive polymers can be defined as natural or synthetic

materials capable of adhering to the biological substrate for an

extended duration. The extended duration should be sufficient time to

allow for a reduced frequency of administration compared to

conventional non bioadhesive polymers. The key attributes of

polymers contributing to bioadhesion are:

Sufficient quality of hydrogen bonding functional groups (-OH-

and –COOH)

High molecular weight and chain flexibility

Anionic surface charges

Adequate surface tension to promote spreading into the mucus

layer

Surface anchored groups with affinity to form bridges between

polymer and mucin.

1.5.3. Desired attributes of bioadhesive polymers for target oral delivery

In order for a bioadhesive polymer to work effectively, it should

possess the following multifunctional physio chemical properties

appropriate for oral drug delivery system:

Rapid adherence to mucosa

Demonstrate strong interactions with the mucin epithelial

tissue

Regenerate new bioadhesive surface and thus maintain

adherence over longer duration

Remain unaffected by the hydrodynamic conditions, food and

pH changes

Maintain bioadhesiveness upon hydration

Available in different bioadhesive grades for different

applications

Able to work effectively with various classes of BCS drugs

Minimum impact on the drug release

No breakdown at the mucosa

Wide margin of safety both locally and systemically

Easy to incorporate into various dosage forms

Long shelf life

Low cost.

1.5.4. Thiomers oral drug delivery

Because of advantages with the mucoadhesive systems,

numerous attempts have been undertaken in order to improve the

mucoadhesive properties of polymeric excipients. The mucoadhesive

properties of the first studied polymers were based on the formation of

non-covalent bonds such as hydrogen bonds and ionic interactions

with the mucus layer. The most commonly bridging structure in

biological systems, the disulfide bond, is thereby utilized to improve

adhesion of polymeric carrier systems to mucosal membranes. This

new generation of mucoadhesive polymers is based on so-called

thiomers. Thiomers are polymers bearing thiol groups that lead to the

formation of disulfide bonds with cysteine-rich subdomains of mucus

glycoproteins, the main constituents of the mucus layer covering

gastro-intestinal epithelia85. Thiolated polymers, are believed to

interact with cysteine-rich subdomains of mucus glycoproteins

forming disulfide bonds between the mucoadhesive polymer and the

mucus layer. Owing to the immobilization of thiol groups on already

well-established mucoadhesive polymers, their mucoadhesive

properties are strongly enhanced. Examples for this improved

mucoadhesion are given in Table1.1. Covalent bonds are believed to

be formed not only between thiomer and mucus, but also within the

thiomer itself. This theory was confirmed by the decrease in free thiol

groups within thiomers resulting in an increase in Viscosity86. Inter-

and intramolecular disulfide bonds improve the cohesive properties of

the thiolated polymer compared to the unmodified polymer. In

addition, the cohesive properties within the mucus gel layer are also

enhanced. Because disulfide bonds are formed during and after

thiomer mucus interpenetration, the mucus layer on which the

delivery system is adhering becomes comparatively more stable so

that the adhesive bond does not fail within the mucus gel layer itself.

Although thiomers show strongly increased mucoadhesive properties,

the adhesion of delivery systems being based on such polymers is

nevertheless limited by the natural mucus turnover. The mucus

turnover in the human intestine, for instance, was determined to be in

the range of 12–24 h87. Time of adhesion is therefore limited by this

permanent renewal process to several hours. These efficacies of

thiomers can be explained by the fact that the thiomers combines two

different mechanisms of mucoadhesion: (a) improved ionic

interactions between polymer and membrane and (b) the formation of

disulfide bonds due to the introduction of thiol. Huge literature is

available showing the potential of thiomers in enhancing the contact

time, bioavailability and permeation of administered small molecule

drugs, proteins and peptides88-90.

Table 1.1: Thiolated polymers, which are interesting candidates

as auxiliary agents91

Polymer Additional information

Chitosan–iminothiolane

250-fold improved mucoadhesive

properties

Poly(acrylic acid)–cysteine

100-fold improved mucoadhesive

properties.

Poly(acrylic acid)–homocysteine

Approximately 20-fold improved

mucoadhesive properties.

Chitosan–thioglycolic acid

Ten fold improved mucoadhesive properties

Chitosan–thioethylamidine

Nine fold improved mucoadhesive properties

Alginate–cysteine

Four fold improved mucoadhesive properties

Poly(methacrylic acid)–cysteine

Improved cohesive and mucoadhesive properties

Sodium carboxymethylcellulose–cysteine

Improved mucoadhesive properties

1.6. Nasal delivery

Nasal drug delivery has been recognized as potential route from

ancient days and nowadays it becomes an important tool in the

treatment of various disorders. Even though nasal route is practised

primarily for the treatment of local disorders, in the modern days it‘s

been exploited for the systemic application of many drugs including

protein and peptide molecules. For the last two decades numerous

articles and reviews have been published emphasizing the potential of

nasal drug delivery92-94. Nasal route, because of its richly supplied

vascular nature of nasal mucosa, has received a great deal of focus as

a convenient and reliable method for the systemic administration of

drugs, especially those which are ineffective orally and must be

administered by injection. The therapeutic agents which are

susceptible for hepatic metabolism or poor permeability in the GI tract

can be administered by nasal route for effective delivery of such drugs.

The ideal properties of drugs to be considered for nasal drug

delivery:

Used chronically

Ineffective orally

Used in small doses

Rapid entry to the general circulation is desirable

Rapid entry to CNS

1.6.1. Advantages of nasal drug delivery

Rapid absorption, higher bioavailability, therefore lower

doses

Avoidance of liver first pass metabolism

Fast onset of therapeutic action

Avoidance of metabolism by the gastrointestinal tract

Avoidance of irritation of the gastrointestinal membrane

Reduced risk of overdose

Non-invasive, therefore, reduced risk of infection

Ease of convenience and self-medication

Improved patient compliance

Can be a beneficial adjunct product to an existing

product;

Reduced risk of infectious disease transmission.

1.6.2. Limitations of nasal drug delivery

Not all are drugs are nasally permeable

Amount of administered volume is low (25-200 µl)

Some drugs may cause nasal irritation.

Some drugs may undergo metabolic degradation

1.6.3. Nasal anatomy and physiology

The nasal cavity is divided into two halves by the nasal septum

and extends posteriorly to the nasopharynx, while the most anterior

part of the nasal cavity, the nasal vestibule, opens to the face through

the nostril (Fig. 1.16). The atrium is an intermediate region between

the vestibule and the respiratory region. The respiratory region, the

nasal conchae or turbinates, which occupies the major part of the

nasal cavity, possesses lateral walls dividing it into 3 sections: the

superior, middle and inferior nasal turbinates. These folds provide the

nasal cavity with a very high surface area compared to its small

volume. The epithelial cells in the nasal vestibule are stratified,

squamous and keratinized with sebaceous glands. Due to its nature,

the nasal vestibule is very resistant to dehydration and can withstand

noxious environmental substances and limits permeation of

substances. The atrium is a transitional epithelial region with

stratified, squamous cells anteriorly and pseudostratified columnar

cells with microvilli posteriorly. Pseudostratified columnar epithelial

cells (Fig. 1.17) interspersed with goblet cells, seromucus ducts, the

openings of subepithelial seromucus glands cover the respiratory

region (the turbinates). Furthermore, many of these cells possess

actively beating cilia with microvilli. Each ciliated cell contains about

100 cilia, while both ciliated and non ciliated cells possess about 300

microvilli each.

Fig.1.16: Schematic of a sagittal section of human nasal cavity showing the nasal vestibule (A), atrium (B), respiratory

region: inferior turbinate (C1), middle turbinate (C2) and the superior turbinate (C3), the olfactory region (D)

and nasopharynx (E).

Fig.1.17. Cell types of the nasal epithelium showing ciliated cell

(A), non-ciliated cell (B), goblet cells (C), gel mucus layer (D), sol layer (E), basal cell (F) and basement membrane (G)

Table 1.2: Structural features of different sections of nasal cavity and their relative impact on permeability95

Region Structural features Permeability

Nasal vestibule Nasal hairs(vibrissae)

Epithelial cells are stratified, squamous and keratinized

Sebaceous glands present

Least permeable because of the presence of keratinized cells

Atrium Transcpicthelial region

Stratified squamous

cells present interiorly and pseudo stratified cells with microvilli present posteriorly

Less permeable as it has small surface area

and stratified cells are present anteriorly

Respiratory region (inferior turbinate middle turbinate superior turbinate)

Pseudo stratified columnar cells with microvilli (300 per cell), large surface area

Receives maximum nasal secretions because of the presence of seromucus glands, nasolacrimal duct and goblet cells

Richly supplied with blood for heating and humidification of inspired air, presence of paranasal sinuses

Most permeable region because of large surface area and rich vasculature

Olfactory region Specialized ciliated olfactory nerve cells for smell perception

Receives ophthalmic and maxillary divisions of trigeminal nerve

Direct access to cerebrospinal fluid

Direct access to cerebrospinal fluid

Nasopharynx Upper part contains ciliated cells and lower part contains squamous epithelium

Receives nasal cavity drainage

With the initial success in the development of nasal delivery

products has led to intensive research, however the enthusiasm was

confronted with disappointing in vivo results showing poor

bioavailability. The reasons for the failure seem to be the low

residence time of the formulations in the nasal tract. Apart from this

low permeability and enzymatic degradation also considered as the

culprits for the failure. This failure led to the development of new

strategies, consequently the use of mucoadhesive polymers based

formulations demonstrated enhanced residence time and permeation

enhancing capabilities96-97. The encouraging results and the desire to

overcome some new challenges stimulated the development of new

generations of polymers based on pH or thermal responsiveness or

modified existing polymers having improved bioadhesive or

permeation-enhancing properties98-99.

1.6.4. The factors that influence nasal drug delivery (NDD) as

potential route

A large surface area available for drug deposition and

absorption. The effective absorptive surface area of the nasal

epithelium is even higher as a result of the presence of

microvilli.

The nasal epithelium is thin, porous (especially when compared

to other epithelial surfaces) and highly vascularized. This

ensures high degree of absorption and rapid transport of

absorbed substances into the systemic circulation for initiation

of therapeutic action.

A porous endothelial basement membrane that poses no

restriction to transporting the drug into general circulation.

Absorbed substances are transported directly into the systemic

circulation thereby avoiding the first pass metabolic effect

generally experienced following oral drug administration.

In some cases, drugs can be absorbed directly into the CNS

after nasal administration bypassing the tight blood–brain

barrier.

Generally speaking, the enzymatic activity of the nasal

epithelium is lower than that of the GIT or liver and higher

bioavailability of drugs especially proteins and peptides can be

achieved. In addition, enzyme inhibitors are more effective

following nasal than oral application because of a higher degree

of dilution in the latter than in the former.

Realization of pulsatile delivery of some drugs like human

growth hormone, insulin, etc., is higher with NDD.

The nose is amenable to self-medication that not only lowers the

cost of therapy but improves patient compliance as well. The

risk of over-dosage is low and nasal lavage can be used to

remove unabsorbed excess drug.

Reformulation of existing drugs as NDD products offers

companies the possibility to extend the life cycle of their

products

1.6.5. Mucoadhesion as a strategy to improve systemic drug delivery via the nasal route

As an alternative to the oral and parenteral route of

administration, nasal route has emerged as effective way for the

administration of small and large therapeutic moieties. A number of

approaches are used to counter the various limitations of nasal drug

administration. The three major approaches that have been attempted

are: the use of chemical enhancers to improve absorption;

incorporation of enzyme inhibitors; and increasing drug local

residence time using mucoadhesive polymers. An alternative approach

to the use of chemical enhancers to improve nasal drug absorption is

to increase the duration of formulation residence within the nasal

cavity. This is achieved by the use of bioadhesive polymers.

1.6.6. New generation polymers used on nasal drug delivery

Initial research on nasal mucoadhesion employed polymers

manufactured for other purposes in the pharmaceutical and food

industries several different types of polymers have been employed for

delivery of different types/classes of drugs. In a lot of other cases only

marginal or even no successes were obtained. Further examination of

the causes of failure pointed lack of 1) tissue specificity with respect to

adhesion, (2) reduced adhesion time, (3) lack of permeation

enhancement capability, (4) interaction between drug and polymer

leading to decreased release of the drug from the dosage form,

increased drug instability, etc., and (5) toxicity induced by the

polymer. Efforts to overcome such problems have lead researchers to

develop new polymers, the so-called second-generation

mucoadhesives, a lot of which have been developed and tested for

nasal drug delivery. Even though the mechanism of adhesion is the

same at nasal or gastrointestinal tract sites, the fictionalization of the

polymers leads to more tissue/organ specificity to issues like (1)

delivery from devices and deposition in the appropriate region of the

nasal cavity, (2) lack of tissue specificity with respect to adhesion, (3)

reduced adhesion time, (4) lack of permeation enhancement

capability, (5) interaction between drug and polymer leading to

decreased release of the drug from the dosage form, increased drug

instability, etc., and (6) toxicity induced by the polymer.

1.6.7. Thiomers in nasal drug delivery

Pharmaceutical technological attempts to overcome these

barriers include the use of enzyme inhibitors, permeation enhancers

and multifunctional polymers ideally guaranteeing both enzyme

inhibition permeation enhancement and mucoadhesion. Among this

group of multifunctional polymers exhibiting all these mentioned

properties, thiolated polymers-designated thiomers—are the most

promising for nasal delivery. Due to the immobilisation of thiol groups

on well-established multifunctional polymers their enzyme inhibitory,

permeation enhancing and mucoadhesive properties can be strongly

improved. Recently, the potential of a thiomer gel formulation could be

demonstrated by in vivo studies. Leitner et al100 developed a nasal gel

formulation for systemic delivery of hGH. The efficacy of a

mucoadhesive gel formulation being based on unmodified

polycarbophil and polycarbophil–cysteine was compared in rats.

Results demonstrated a significantly higher and prolonged nasal

bioavailability of hGH, which was incorporated in the thiomer gel

formulation. Utilizing the thiomer gel formulation an absolute nasal

bioavailability of 2.75±0.37% was achieved. As thiomers also exhibit a

strong permeation enhancing effect, however, it is difficult to attribute

this improved in vivo efficacy exclusively to the improved

mucoadhesive properties. In another study thiolated polyacrylate

microparticles were generated for the nasal delivery of hGH. The

intranasal administration of this microparticulate formulation to rats

resulted in a relative bioavailability of 8.11±2.15% that represents a 3-

fold improvement compared to microparticles comprising the

corresponding unmodified polymer.

1.6.8. Nose to brain delivery of drugs to the brain via the nasal

route

Recent research on the nasal delivery has highlighted the

possibility of exploiting this route for direct transport of drugs from

nose to brain. Absorption of drugs at the olfactory region of the nose

provides a potential for a therapeutic agent available in the brain.

Numerous studies in the animals provide the evidence for the

presence of a direct pathway from the olfactory region to the brain101-

103. Hence, nasally administered drugs might be able to reach a target

in the brain to a greater concentration that any other route. Reports in

the literature of studies in animal models and in man have shown this

to be a distinct possibility with results showing the uptake of drugs

into the cere-brospinal fluid and the brain tissue being dependent

upon molecular weight and the lipophilicity. The route by which

nasally delivered drugs can reach the cerebro-spinal fluid (CSF),

which surrounds the brain and the actual brain tissue, is depicted in

the Fig.1.18. The drug can also follow the systemic pathway to reach

the brain by crossing the blood brain barrier but the use of this

pathway is highly dependent on the properties of the drug104. Of

special interest is the transport of drug across the olfactory region in

the nasal cavity directly into the brain tissue (e.g. olfactory bulb) or

the CSF. The olfactory epithelium is situated between the nasal

septum and the lateral wall of each side of the two nasal cavities and

just below the cribriform plate of the ethmoid bone separating the

nasal cavity from the cranial cavity. The drug can cross the olfactory

pathway by one or a combination of pathways105. Firstly, the drug can

use a transcellular pathway, where the drug is transferred by receptor

pathway, where the drug is transferred by receptor passive diffusion.

This pathway is especially suited for small lipophilic molecules or

large molecules. Secondly, the drug can use the para cellular pathway

by passing through the tight junctions or through open clefts in the

membrane. This pathway is especially suited for smaller hydrophilic

molecules. Thirdly, the drug can be transported through the olfactory

neuron cells by intracellular axonal transport primarily to the

olfactory bulb.

Fig. 1.18: Possible routes of transport between the nasal cavity and the brain and CSF

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