GHENT UNIVERISTY

FACULTY OF PHARMACEUTICAL SCIENCES

Vakgroep Geneesmiddelenleer

Laboratory of Pharmaceutical Technology

Academic year 2008-2009

DEVELOPMENT OF A THERMOSENSITIVE AND

MUCOADHESIVE VEHICLE, BASED ON

POLOXAMER AND CHITOSAN, FOR VAGINAL

ADMINISTRATION OF DRUGS

Evelien MORLION

Eerste Master in de Farmaceutische Zorg

Promotor

Prof. Dr. J.P. Remon

Commissarissen

Dr. E. Mehuys

Prof. Dr. C. Vervaet

AUTEURSRECHT

“De auteur en de promotor geven de toelating deze masterproef voor consultatie beschikbaar te

stellen en delen ervan te kopiëren voor persoonlijk gebruik. Elk ander gebruik valt onder de

beperkingen van het auteursrecht, in het bijzonder met betrekking tot de verplichting

uitdrukkelijk de bron te vermelden bij het aanhalen van de resultaten uit deze masterproef.”

2 juni 2009

Promotor Auteur

Prof. Dr. J.P. Remon Evelien Morlion

WORDS OF THANKS

By the completion of this master thesis, I would like to thank everyone who

provided a contribution to the finishing point of this work.

Prof. Dr. J.P. Remon, for the general control about these scription and the

possibility to participate to this Erasmus-Socrates project.

Furthermore, I am very grateful to Professoressa Silvia Rossi of the

University of Pavia (Italy), for her work, assistance, patience and lovely

words. It was a very great fortune to have the possibility to work for her.

Grazie mille!

People that I surely may not forget to mention, are all the wonderfull

people of the “Laboratorio di Preformulazione e Sviluppo galenico”! Angela,

for her patience to explain everything and all the efforts to study English;

Giulia and Stefania, Monica and Krupal, Ivan (B.F.G.) and all the others, for

all the nice times, the words of courage and teaching Italian words! Grazie

tutti!!

My parents, who gave me the opportunity and possibility to have this

unique experience abroad. Additionally their never ending support and

encouraging words, are extremely valuable and precious for me!

My family and many friends, (which I unfortunately cannot mention all by

name in order not to forget someone by emotion) both the current loveable

ones in Belgium as the new fine international friendships, for their interest

and support during the making of this trip and carrying out the research.

In particular a warm thanks as well to a special person in my life, who was

so patiently to listen to me in my inferior moments, to support and

encourage me and give me so much love... Pieter.

Also a great appreciation to Lies Dunscombe, for her valuable time and

grammatical help in the English language. To Peter-Jan Pertry , for his

informatical help at the lay-out of the work and to Maurits Timperman, for

his reading support.

LIST OF ABBREVIATIONS

AUC Area under the curve

Da Dalton

DD Degree of deacetylation

LMW Low Molecular Weight

Pa Pascal

PEO Poly(ethylene oxide)

PF-127 Pluronic® F-127

PPO Poly(propylene oxide)

SVF Simulated Vaginal Fluid

USP United States Pharmacopeia

1. INTRODUCTION.......................................................... Fout! Bladwijzer niet gedefinieerd.

1.1. ANATOMY AND PHYSIOLOGY OF THE VAGINA RELEVANT TO DRUG DELIVERY... Fout!

Bladwijzer niet gedefinieerd.

1.2. VAGINAL CAVITY AS A ROUTE FOR DRUG ADMINISTRATIONFout! Bladwijzer niet

gedefinieerd.

1.3. CONVENTIONAL VAGINAL DRUG DELIVERY STYSTEMSFout! Bladwijzer niet

gedefinieerd.

1.3.1. Solutions and foams .................................... Fout! Bladwijzer niet gedefinieerd.

1.3.2. Tablets ......................................................... Fout! Bladwijzer niet gedefinieerd.

1.3.3. Suppositories (Pessaries) ............................. Fout! Bladwijzer niet gedefinieerd.

1.3.4. Creams and gels ........................................... Fout! Bladwijzer niet gedefinieerd.

1.4. MODIFIED RELEASE FORMS ................................ Fout! Bladwijzer niet gedefinieerd.

1.4.1. Rings ............................................................ Fout! Bladwijzer niet gedefinieerd.

1.5. BIOADHESIVE DRUG DELIVERY SYSTEMS ............ Fout! Bladwijzer niet gedefinieerd.

1.5.1. Bioadhesion: definition and mechanism ..... Fout! Bladwijzer niet gedefinieerd.

1.5.1.1. Bioadhesion theories .......................................Fout! Bladwijzer niet gedefinieerd.

1.5.2. Bioadhesive polymers .................................. Fout! Bladwijzer niet gedefinieerd.

1.5.2.1. Chitosan as bioadhesive polymer ....................Fout! Bladwijzer niet gedefinieerd.

1.6. THERMOGELLING SYSTEMS ................................ Fout! Bladwijzer niet gedefinieerd.

2. AIM OF THE WORK..................................................... Fout! Bladwijzer niet gedefinieerd.

3. EXPERIMENTAL PART ................................................. Fout! Bladwijzer niet gedefinieerd.

3.1. MATERIALS ......................................................... Fout! Bladwijzer niet gedefinieerd.

3.2. METHODS ........................................................... Fout! Bladwijzer niet gedefinieerd.

3.2.1. Sample preparation ..................................... Fout! Bladwijzer niet gedefinieerd.

3.2.2. Characterization of poloxamer solutions and poloxamer/chitosan lactate mixtures

..................................................................... Fout! Bladwijzer niet gedefinieerd.

3.2.2.1. pH measurements ...........................................Fout! Bladwijzer niet gedefinieerd.

3.2.2.2. Rheological measurements .............................Fout! Bladwijzer niet gedefinieerd.

3.2.2.3. Mucoadhesion measurements ........................Fout! Bladwijzer niet gedefinieerd.

3.2.3. Characterization of 15% (w/v) poloxamer/0,8% (w/v) chitosan mixture diluted

with simulated vaginal fuid (SVD)................ Fout! Bladwijzer niet gedefinieerd.

3.2.3.1. Sample preparation .........................................Fout! Bladwijzer niet gedefinieerd.

3.2.3.2. Rheological measurements .............................Fout! Bladwijzer niet gedefinieerd.

3.2.3.3. Mucoadhesion measurements ........................Fout! Bladwijzer niet gedefinieerd.

4. RESULTS AND DISCUSSION ........................................ Fout! Bladwijzer niet gedefinieerd.

4.1. CHARACTERIZATION OF POLOXAMER SOLUTIONS AND 15% (W/V) POLOXAMER/

CHITOSAN LACTATE MIXTURES ........................... Fout! Bladwijzer niet gedefinieerd.

4.1.1. pH measurements ....................................... Fout! Bladwijzer niet gedefinieerd.

4.1.2. Rheological measurements.......................... Fout! Bladwijzer niet gedefinieerd.

4.1.2.1. Poloxamer solutions ........................................Fout! Bladwijzer niet gedefinieerd.

4.1.2.2. Poloxamer/chitosan lactate mixtures ..............Fout! Bladwijzer niet gedefinieerd.

4.1.3 Mucoadhesion measurements .................... Fout! Bladwijzer niet gedefinieerd.

4.2. CHARACTERIZATION OF 15% (W/V) POLOXAMER/0.8% CHITOSAN LACTATE DILUTED

WITH SIMULATED VAGINAL FLUID (SVF) ............. Fout! Bladwijzer niet gedefinieerd.

4.2.1. Rheological analysis ..................................... Fout! Bladwijzer niet gedefinieerd.

4.2.2. Mucoadhesion measurements .................... Fout! Bladwijzer niet gedefinieerd.

5. CONCLUSIONS ............................................................ Fout! Bladwijzer niet gedefinieerd.

6. BIBLIOGRAPHY ........................................................... Fout! Bladwijzer niet gedefinieerd.

1

1. INTRODUCTION

1.1 ANATOMY AND PHYSIOLOGY OF THE VAGINA RELEVANT TO DRUG DELIVERY

In the pharmaceutical literature, the human vagina is described as a highly expandable,

slightly S-shaped, fibromuscular, collapsible tubular tract, with a length between 6 to 10 cm. It

extends from the lower part of the uterine cervix to the external part of the vulva, known as the

labia minor, and is situated between the urinary bladder and the rectum, like illustrated in Figure

1.1. (Bernkop-Schnürch et al., 2003).

FIGURE 1.1.: REPRESENTATION OF THE FEMALE REPRODUCTIVE SYSTEM

(http://training.seer.cancer.gov/module_anatomy/unit12_3_repdt_female.html)

The vaginal wall consists of three layers: the epithelial layer, the muscular coat and the

tunica adventia. The surface of the vagina is composed of numerous folds, called rugae, which

increase the surface area of the vaginal wall. The vagina is characterized by a non-keratinized,

stratified squamous epithelium, with varying thickness influenced by the estrogen level changes

throughout the menstrual cycle. Even the different life phases, among which newborn, child,

adult and menopause, effect the thickness of this epithelium. Until puberty, the vaginal

epithelium remains relatively thin, it leads to an improved drug absorption via vaginal route.

2

From menarche to menopause, the vaginal epithelium thickness increases, so that drug

absorption becomes lower. When no more estrogenic secretions appear, like in the menopause

or after ovariectomy, the vaginal epithelium becomes thin and atrophic, like in the prepuberty

state (Sandri G. et al., 2006 ; Valenta C., 2005).

Besides the altering thickness of the vaginal epithelium, also the pH is an important

parameter to consider for the development of vaginal drug delivery. In prepuberty the pH of the

vaginal fluids remains nearly neutral. In the phase before menopause a decrease in pH until 4

and 5 occurs, while pH increases up to 7 after the menopause (Justin-Temu M. et al., 2004). The

vaginal pH is maintained by the buffering action of the bacteria Lactobacillus acidophilus, which

converts glycogen from exfoliated epithelial cells into lactic acid. An increasing pH in the vagina is

observed during menstruation and coitus, because both vaginal transudate and ejaculate are

alkaline. Like the thickness of the vaginal epithelium and the pH, the amount and composition of

the hydrophilic vaginal fluid also changes throughout the menstrual cycle, which can affect the

drug release profile from intravaginal dosage forms (Hussain A. et al.,2005).

The presence of a thick network of blood vessels makes the vagina an excellent route for

drug delivery, both for systemic and local effects. The main blood supply to the vagina is through

the vaginal branch of the uterine artery. Blood leaving the vagina enters the peripheral

circulation via a rich venous plexus, which empties primarily into the internal iliac veins and

secondarily into the inferior hemorrhoidal veins. For this reason drugs absorbed through the

vagina don’t undergo first-pass metabolism, which is an important advantage of administering

drug via vaginal route (Alexander N.J. et al. ,2004).

3

1.2 VAGINAL CAVITY AS A ROUTE FOR DRUG ADMINISTRATION

The vaginal cavity and its effectiveness as a site of drug administration have been well

established in the last decades. The pharmaceutical dosage forms used for vaginal application

are traditionally employed for drug delivery in the local treatment of specific gynaecological

pathologies, such as candidiasis, genital Herpes or vaginitis caused by bacteria, fungi, protozoa or

virus (Pavelic Z. et al., 2001). In the last years, major advancements have been reported to

microbicides in formulations to prevent the transmission of sexually transmitted diseases.

Additionally the vaginal route can also be used for labour-inducing drugs, spermicidal agents and

steroids. Nowadays, vaginal delivery systems are used as an alternative to parenteral

formulations for drugs that can’t be given advantageously per os because of presystemic

metabolism (Sandri G. et al., 2006).

Conventional drug administration trough the vagina has following main advantages: the

ability to by-pass first uterine pass effect, ease of administration and high permeability for low

molecular weight drugs. Besides these facts, it can also reduce the appearance and severity of

gastro-intestinal diseases and of hepatic side effects subsequent to hormone administration.

However, gender aspect, personal hygiene and menstrual cycle variations, seem to be the main

limitations of the vaginal administration of drugs (Valenta C.,2005).

Besides locally acting drugs, that work by drug penetration into the deep cell layers after

administration, drug permeation across the epithelium can occur and reach the systemic

circulation. These local or systemic effects can be achieved by drug dissolution in vaginal lumen

and the formation of a hydrodynamic layer on the vaginal walls, pursued by penetration of the

epithelium corresponding to a concentration gradient. Therefore, it is necessary for the

penetrating or permeating substance to possess adequate lipophilicity to diffuse through the

lipidic components of the membrane and have a certain degree of aqueous solubility as well to

make sure that dissolution in the vaginal fluid is possible (Sandri G. et al., 2006).

Estrogens and sexual arousal increase the volume and composition of the vaginal fluid,

which has an expanding effect on the absorption of poorly water soluble drugs. Besides this,

excessively fluid may remove the drug from the application site or cause an unpredictable drug

4

distribution. Furthermore pH variation, correlated with the menstrual cycle, has an influence on

drug ionization (by varying the degree of ionization of weak electrolytes) and the consequent

drug release profiles (Hussain A. et al., 2005; Sandri G. et al., 2006).

The vagina may be able to absorb molecules with a higher molecular weight than other

mucosal regions, which creates the possibility to modify the bioavailability of a drug applied on

the vaginal mucosa, by the use of chemical penetration enhancers and by the suitable dosage

form, despite the fact that the general permeability of the vagina is already greater than that of

the rectum, skin and buccal mucosa. This characteristic is particularly important when

considering the vaginal absorption of high molecular weight molecules, such as peptides and

proteins (Richardson J.L. et al., 1992).

1.3 CONVENTIONAL VAGINAL DRUG DELIVERY STYSTEMS

Several kinds of pharmaceutical dosage forms have been developed for the release of

drug in the vaginal environment. These traditional formulations include solutions, foams, tablets

and suppositories, in particular pessaries, as well as rings, creams and gels. The choice of the

most appropriate delivery system will depend on different considerations: required effect (local

or systemic), drug release (immediate or controlled) and patient acceptability (Malcolm R.K. et

al., 2006).

1.3.1. Solutions and foams

Both solutions (lavages) and foams are liquid formulations. They differ in the fact that in

foams a suitable propellant is present. They are administered by means of a pressurized delivery

device (G. Sandri, et al., 2006).

5

1.3.2. Tablets

This vaginal dosage form has the advantages of ease of manufacturing, ease of

application and low cost. They contain binders, disintegrants and other excipients that are used

to prepare conventional oral tablets. Absorption of a drug across the vaginal mucoa can be

impaired in presence of hydrophobic and release retarding materials in the formulation (Hussain

A. et al., 2005).

1.3.3. Suppositories (Pessaries)

Suppositories are very common vaginal delivery systems, comparable to those intended

for rectal administration. These formulations are created to melt or dissolve in the vaginal cavity;

depending on the choice of excipients they can ensure a sustained drug release. Suppository

systems are now most commonly used to administer drugs for cervical ripening prior to child

birth and local delivery of drugs in the treatment of vaginal infections (Sandri G. et al., 2006).

1.3.4. Creams and gels

Creams and gels represent the greatest number of vaginal formulations, frequently used

for topical delivery of contraceptives and anti-microbial drugs. Vaginal gels are also used to

administer drugs used in the cervical ripening and labor induction. Hydrophilic formulations,

particularly hydrogels, swell when placed in the aqueous vaginal environment and release the

drug mainly by diffusion. Swelling augments the spreading of the formulation on the mucosal

surface and consequently heightens the contact between the drug and the vaginal epithelium.

The main drawback of such formulations on the other hand is the impossibility to administer an

exact drug dose (Sandri G. et al., 2006).

6

1.4 MODIFIED RELEASE FORMS

These formulations are characterized by excipients and/or technology capable of

controlling and prolonging drug release.

1.4.1. Rings

Vaginal rings are described as circular devices designed to release a drug in a controlled

manner after insertion in the vagina. They measure about 5.5 cm in diameter with a circular

cross section of 4-9 mm. Depending on drug release kinetic, vaginal rings can be subdivided in

simple and sandwich or reservoir types. The latter devices are designed to create a constant drug

release, instead of a fast release at the surface and a slower release of the drug in the inner layer

as is used for the simple devices. Besides drug release, properties are affected by the type of

employed polymer. Among these, poly(dimethylsiloxane), ethylene vinyl acetate and styrene

butadiene block copolymers are counted.

Drugs commonly administered in vaginal rings are hormones in hormonal replacement

therapy and in contraception. Advantages of this form are that it is easy to use, does not

interfere with coition, does not require daily intake of pills and allows a continuous delivery of

low dose steroids (Hussain A. et al., 2005).

1.5 BIOADHESIVE DRUG DELIVERY SYSTEMS

An important drawback related to the administration of conventional vaginal drug

delivery systems is their short permanence in the vaginal cavity. This implies repeated

administrations which result in poor patient compliance.

To overcome this kind of problem, bioadhesive drug delivery systems have been

developed. The insertion of bioadhesive polymers into the formulation should allow an increase

of the contact time of the drug with the vaginal mucosa in order to reduce drug dosages or

applications and accordingly improve patient compliance (Robinson J.R. et al., 1994).

7

1.5.1. Bioadhesion: definition and mechanism

The term bioadhesion refers to any bond formed between two biological surfaces or a

bond between a biological and a synthetic surface. In the case of bioadhesive drug delivery

systems, the term bioadhesion is typically used to describe the adhesion between polymers,

either synthetic or natural, and soft tissues (i.e. vaginal mucosa). The target of these systems

may be either the mucus layer lining the mucosa, or the epithelial cells, or a combination of the

two (Chikering D.E., 1999).

The so-called ‘mucoadhesion’ is actually another version of ‘bioadhesion’, although both

are often used synonymously. In general, bioadhesion is the term used to describe adhesive

interactions with any biological or biological derived substance, while mucoadhesion is used

specifically to describe interactions involving mucus covering a mucosal surface, like the vagina.

Even though the vaginal epithelium is usually considered to be a mucosal surface, it has no

goblet cells nor glands and lacks the direct release or production of mucin (Valenta C. et al.,

2005; Chickering D.E. et al., 1999).

In the formation of such bioadhesive bonds, three steps are involved: (1) wetting and

swelling of polymer to permit an intimate contact with biological tissue, (2) interpenetration of

bioadhesive polymer chains and mucin chains, which forms the main component of mucus, and

(3) formation of physical or chemical bonds between the biological substrate and the

mucoadhesive polymer (Chickering D.E. et al., 1999).

1.5.1.1. Bioadhesion theories

The design of bioadhesive drug delivery systems should take the mechanisms on which

the bioadhesion phenomenon is founded in consideration. Hereafter the different theories that

have been proposed to clarify the bioadhesive systems, are summarized. In general, five theories

have been adapted: the electronic, absorption, wetting, diffusion and fracture theories.

8

The electronic theory

The proposition of the electronic theory relies on the hypothesis that both the

bioadhesive material and the target biological substrate have different electronic structures.

When these layers come in contact with each other, an electronic transfer occurs, which evolves

in the formation of a double layer of electrical charges at the bioadhesive interface. The

bioadhesive force is supposed to be due to attractive forces across this electrical double layer.

This theory evoked some debates regarding whether these electrostatic forces are the cause or

rather the result of the contact between the bioadhesive and the biological component

(Chickering D.E. et al., 1999).

The adsorption theory

This theory assumes that a mucoadhesive polymer adheres to mucus because of van der

Waals interactions, hydrogen bonds, electrostatic interactions or hydrophobic interactions. The

complete number of this individually weak interactions can as a whole produce a strong

adhesive interaction (Chickering D.E. et al., 1999).

The wetting theory

The wetting theory emphasizes the concern of the ability of liquid adhesives to spread

and develop intimate contact. This theory thus uses interfacial tensions to predict spreading and

subsequently adhesion (Chickering D.E. et al., 1999).

The diffusion theory

The concept that interpenetration to a sufficient depth (in a range of 0.2-0.5 µm) of

bioadhesive polymer chains and mucin glycoprotein chains produce semipermanent adhesive

bonds through entanglement, is supported by this theory. It is believed that bond strength

increases with the degree of penetration of the polymer chains into the mucous layer (Chickering

D.E. et al., 1999).

9

The fracture theory

The fracture theory analyses the force that is required for separation of two surfaces after

adhesion. It is considered to be appropriate for the calculation of fracture strengths of adhesive

bonds concerning rigid mucoadhesive materials (Chickering D.E. et al., 1999). The work of

adhesion of a bioadhesive system fixed on a biological tissue, e.g. excised pig vagina, can be seen

as a good indicator of its bioadhesive power (Lejoyeux F. et al, 1989).

1.5.2. Bioadhesive polymers

The permanence of the formulation at the application site for an adequate period of time

can be achieved by including bioadhesive polymers in the formulation. To obtain adhesion at

least one of the following polymer characteristics should be required: (a) sufficient quantities of

hydrogen-bonding chemical groups (-OH and –COOH), (b) anionic charges on the surface, (c) high

molecular weight, (d) high chain flexibility, and (e) surface tensions that will induce spreading

into the mucous layer. Each of these characteristics support the formation of bonds that can

have a chemical or a mechanical origin (Chickering D. E. et al., 1999).

Chemical bonds can be divided in strong primary bonds (i.e. covalent bonds) and weaker

secondary bonds, for instance ionic bonds, van der Waals interactions and hydrogen bonds.

Although permanent bonding may be very useful, some considerations have to be made.

Permanent bonds with the epithelium may not produce permanently retained delivery devices,

due to epithelium turn-over (of about 4-5 days). Moreover, biocompatibility of such binding has

not been investigated in detail and could cause significant problems. For these reasons the

formation of weak secondary bonds is preferred. It is the case of some hydrogels that are able to

interact with mucus glycoproteins by means of ionic, van der Waals and hydrogen bonds

(Chickering D. E. et al., 1999).

Mechanical bonds can be seen as physical connections between surfaces. Microscopically,

they involve physical entanglement of mucin strands with flexible polymer chains and/or

interpenetration of mucin strands in a porous polymer substrate. The adhesive bond strength

can be influenced by the presence of water, the contact time between the materials, and the

10

length, flexibility and depth of penetration of the polymer chains. The rate of this penetration on

the other hand, is dependent on chain flexibility and diffusion coefficients of the polymer strand

and the mucin layers (Chickering D.E. et al., 1999).

Some of the polymers reported to possess such bioadhesive properties are Polycarbophil,

Carbopol, Hydroxypropylmethylcellulose (HPMC), and chitosan (Vermani K. et al., 2002).

1.5.2.1. Chitosan as bioadhesive polymer

Chitosan is an aminopolysaccharide industrially derived from the alkaline deacetylation of

chitin, a natural polymer, which is present in the exoskeleton of shrimps and crabs. The structural

formula and synthesis of chitosan is shown in Figure 1.2. This natural biopolymer chitosan,

consisting of glucosamine and N-acetylglucosamine units, is a biocompatible, biodegradable,

non-toxic, cationic polymer, insoluble in water, but soluble in dilute aqueous acids up to pH 6.2

(Ruel-Gariépy E. et al., 2004; Lehr C-M., 1992). Furthermore it has wound healing and

antimicrobial properties which has made chitosan a very attractive biopolymer for use in

biomedical and pharmaceutical applications for a long time (Ahmadi R. et al., 2007).

This polymer possesses OH and NH2 groups that can give rise to hydrogen bonding,

considered essential for mucoadhesion (Valenta C., 2005).

11

FIGURE 1.2.: REPRESENTATION OF THE CHEMICAL STRUCTURE AND SYNTHESIS OF

CHITOSAN

(http://commons.wikimedia.org/wiki/File:Chitosan_Synthese.svg)

In the last few years, many authors have studied the mucoadhesive properties of

chitosan, which are markedly affected by environmental pH (Lehr C.M. et al., 1992; Sandri G et

al., 2004; Şenel S. et al., 2000).

The effectiveness of chitosan as mucoadhesive agent is damaged by its insolubility at pH

above 6.2. To solve this difficulty of chitosan’s solubility, chitosan derivatives like methyl

pyrrolidinone chitosan (MPC) and N-trimethyl chitosan (TMC) were synthesized (Sandri G. et al.,

2004/2005).

12

1.6 THERMOGELLING SYSTEMS

An increasing number of in-situ forming systems have been described in the past few

years in literature, because of their valuable biomedical applications in drug delivery, cell

encapsulation and tissue repairing. Different mechanisms are known to express the formation of

the gel in-situ: solvent exchange, UV-irradiation, ionic cross-linking, pH change and temperature

modulation. Among these, temperature modulation can be seen as the most advantageous for

particular applications, in view of the fact that a thermogelling system doesn’t require organic

solvents, co-polymerization agents, nor an externally applied trigger for gelation (Ruel-Gariépy E.

et al., 2004).

This thermoreversible gel can be prepared by the use of polysaccharides like

Hydroxypropyl methylcellulose (HPMC), Methylcellulose (MC) or Ethyl(hydroxyethyl)cellulose

(EHEC), but also by N-isopropylacrylamide copolymers and poloxamer and its copolymers (Ruel-

Gariépy E. et al., 2004).

Poloxamer is a thermosensitive non-ionic amphiphilic block copolymer, from the ABA-

type composed of PEO (A) an PPO units (B) (Ruel-Gariépy E. et al., 2004). Figure 1.3. shows the

chemical structure of poloxamer 407, with a central lipophilic poly propyleenoxide, bordered by

two hydrophilic poly ethyleenoxide chains.

FIGURE 1.3.: CHEMICAL STRUCTURE OF PLURONIC F-127 (a: ETHYLENE OXIDE

PORTION; b: PROPYLENE OXIDE PORTION)

(http://www.ualberta.ca/~csps/JPPS9_3/Article_349/art349.html)

13

Poloxamer 127 is frequently used and commercially known as Pluronic® F127, where the

“F” refers to the flake form of the product. This polymer is widely common in medical,

pharmaceutical and cosmetic field due to its ability to interact very well with other chemicals, its

high solubilizing capacity for different drugs, good drug release characteristics, low toxicity and

the capability to undergo phase reverse thermal gelation. For all these reasons, PF-127 is

considered as a good medium for drug delivery systems (Mayol L. et al.,2008).

Since it has an amphiphilic character, pluronic F-127 is a surfactant soluble in water,

where concentrated aqueous solutions of poloxamer form thermoreversible gels.

Gels were phenomenological defined in 1993 by Almdal et al, which states that a gel is a

soft, solid or solid-like material which consists of at least two components, one of which is a

liquid present in a large quantity. The solid-like characteristics of a gel can be defined in terms of

two dynamic mechanical properties: an elastic (or storage) modulus, G’ (Pa) which, when plotted

against frequency, presents a pronounced plateau, and a viscous (or loss) modulus, G” (Pa),

which is considerably smaller than the elastic modulus in the plateau region (Figure 1.4.).

a) b)

G’ G’

G”

G”

Log Frequency (Hz) Log Frequency (Hz)

FIGURE 1.4.: COMPARISON OF DYNAMIC MECHANICAL SPECTRUMS FOR (a) A

TYPICALLY CROSS-LINKED POLYMER GEL AND (b) AN ENTANGLED

POLYMER SOLUTION (WHITOUT CROSS-LINKS)

Log

G’ ;

G”

(P

a)

Log

G’ ;

G”

(P

a)

14

PF-127 is classified as an inorganic hydrogel in spite of the fact that the gelation

mechanisms of poloxamer solutions, including packing of micelles and micelle entanglements,

are still debated. Micelle formation occurs at the critical micellization temperature and

concentration. In an aqueous solution, micelles are formed by the aggregation of multiple

molecules with their polar, hydrophilic polyoxyehtylene oxide chains forming the outer shell,

covering the hydrophobic central core. One of the gelation hypotheses is that when the

concentration reach values higher than a critical gel concentration, the micelles order into a

lattice, which can be seen as the driving force for gel formation ( Escobar-Chàvez J.J. et al., 2006).

A fixed statement on the other hand, is that PF-127 aqueous solutions have the interesting

attributes of reverse thermal gelation. More precisely it means that they are liquid at

refrigerated temperatures (4-5°C) and gel while warming to room temperature (25°C). This

gelation can be made undone, by cooling in ice (Escobar-Chávez J.J. et al., 2006). When the

mobile viscous liquid at room temperature is applied at body temperature (37°C), it transforms

to a transparent gel (Ruel-Gariépy E. et al., 2004).

In previous research an exponential relationship was found between viscosity and

temperature, with a rise dependent on the concentration of PF-127. From this phenomenon, we

can suppose that Pluronic F-127 micelles in aqueous solution indeed undergo a thermally

induced swelling and desolvation (Escobar-Chávez J.J., 2006).

Poloxamer formulations are usually found to increase the drug residence time at the

application site, which results in an enhanced bioavailability and efficacy (Chang J.Y. et al., 2002).

15

2. AIM OF THE WORK

In the last decades a great deal of work has been devoted in the pharmaceutical field to

the development of drug delivery systems intended for drug administration through the so called

transmucosal/mucosal routes. Among these, the vaginal route is gaining an increasing interest.

Across vaginal route, a transmucosal administration which implies drug appearance in the

systemic circulation, and a local administration which implies a site specific action of the drug on

the vaginal mucosa, can be achieved.

In the first case the advantages with respect to an oral administration are: to avoid first-

pass effect, to avoid drug degradation by gastrointestinal fluid and to reduce the drug absorption

variability that is very high after oral administration, due to food presence and gastrointestinal

mobility.

As for mucosal administration, vaginal cavity can be site of different microbial and viral

infections that can be successfully treated with a local administration of anti-infective or of anti-

inflammatory agents.

For both systemic and local treatments, the achievement of a prolonged contact of the

drug with vaginal mucosa allows an improvement of drug efficacy and patient compliance by

reducing the number of administrations. With this objective, different mucoadhesive

formulations intended for vaginal administration have been developed. Another approach that

can be used to prolong drug permanence onto mucosa is the employment of in situ gelling

systems such as thermal sensitive solutions. Such solutions present the advantage to be easily

administered and spreaded onto mucosa, due to its low consistency at room temperature.

Moreover they should be able to withstand the physiological removal mechanism because

characterized at 37°C by high consistency due to gel formation.

Given these properties, the aim of the present work was the development of a

pharmaceutical vehicle having both thermal sensitive and mucoadhesive properties. In

particular, poloxamer, a block copolymer made of polyoxyethylene and polyoxypropylene, was

used as thermal sensitive agent. Chitosan, a natural polymer derived from chitin, well known for

16

its mucoadhesive properties, was used as mucoadhesive agent. The research work was divided in

three different phases.

In a first phase the rheological properties of two poloxamer solutions prepared at 15%

and 16% (w/v) were investigated in order to find the optimal poloxamer concentration. These

concentrations were chosen on the basis of literature data. Some authors found that poloxamer,

when hydrated at a concentration close to 16%, was able to gelify at the physiological

temperature. Different rheological tests were employed. In particular viscosity tests at 25° and

37°C were carried out to evaluate the increase in sample viscosity at the physiological

temperature due to gel formation. Viscoelastic tests were also performed to assess: 1) the

sample gelation temperature by evaluating the variation of elastic properties on increasing

temperature; 2) the gelation rate by investigating the influence of the time on elastic properties;

3) the gel elastic properties at 37°C by estimating the variation of the elasticity on increasing

frequency.

The subsequent phase of the research was devoted to the preparation and

characterization of mucoadhesive poloxamer/chitosan lactate mixtures. In particular a chitosan

with a low molecular weight was chosen in order to obtain mixtures characterized by low

viscosity at room temperature, necessary to permit an easy application. The optimal chitosan

lactate concentration was chosen on the basis of rheological and mucoadhesion tests. The

mucoadhesive properties of the mixtures were assessed by using two different biological

substrates: mucin suspension and pig vaginal mucosa.

In the last part of the research the most promising poloxamer/chitosan lactate mixture,

developed in the previous phases, was diluted with simulated vaginal fluid in order to mimic the

in vivo administration environment and was then characterized for rheological and

mucoadhesive properties.

17

3. EXPERIMENTAL PART

3.1 MATERIALS

Pluronic® F127 (Prill, BASF, USA)

Chitosan LMW 1504 (MW 250000 Da; DD 98%) (Giusto Faravelli, Milan, Italy)

Lactic acid 80% E 270 (A.C.E.F., Fiorenzuola d’Arda (PC), Italy)

Mucin from porcine stomach (Type 2) (Sigma Chimica, Milan, Italy)

Sodium acetate (Carlo Erba Reagenti, Rodano (MI), Italy)

Acetic Acid 96% (Carlo Erba Reagenti, Rodano (MI), Italy)

Hydrochloric Acid 37% (Riedel- de Haën, Seelze, Germany)

Bovine serum albumine (Sigma-Aldrich, Steinheim, Germany)

Sodium Chloride (Carlo Erba Reagenti, Rodano (MI), Italy)

Calcium hydroxide (Sigma-Aldrich, Milan, Italy)

Acetic Acid 96%, (Carlo Erla Reagenti, Rodano (MI), Italy)

Urea (Sigma-Aldrich, Steinheim, Germany)

Potasium hyroxide (Carlo Erba Reagenti, Rodano (MI), Italy)

D (+) Glucose (Sigma-Aldrich, Steinheim, Germany)

30° Bé Glycerol (Carlo Erba Reagenti, Rodano (MI), Italy)

3.2 METHODS

3.2.1 Sample preparation

Poloxamer solutions were prepared according to the cold method (Choi et al., 1998). The

following concentrations were considered: 15 and 16% (w/v). The solutions were prepared under

stirring in a beaker put in an ice bath to keep the solution fluid.

Mixtures based on poloxamer and chitosan lactate were also prepared. For this aim,

chitosan lactate solutions were prepared by dissolving chitosan in distilled water containing an

exact amount of lactic acid (to obtain a 1:1 molar ratio between acid and chitosan deacetylated

amine groups).

18

The mixtures contained a fixed poloxamer concentration equal to 15% (w/v) and differing

chitosan lactate concentrations (0.4%, 0.8%, 1.2%, 1.6% w/v). The samples were stored at 4°C

before testing.

3.2.2 Characterization of poloxamer solutions and poloxamer/chitosan lactate mixtures

3.2.2.1 pH measurements

The pH value of the poloxamer/chitosan lactate mixtures and of the poloxamer solutions

was measured by means of pH 210 Microprocessor pH Meter (Hanna Instruments, Italy).

3.2.2.2 Rheological measurements

The rheological analyses were performed by the use of a rotational rheometer

(Rheostress RS600, Haake, Karlsruke, Germany), equipped with a cone plate combination (C35/1:

Ø = 35 mm; angle = 1°) as measuring system (see Figure 3.1.).

All the poloxamer/chitosan lactate mixtures and poloxamer solutions were subjected to

viscosity and dynamic viscoelastic measurements.

19

FIGURE 3.1.: REPRESENTATION OF RHEOSTRESS RS600, HAAKE, KARLSRUKE,

GERMANY

(http://www.rheologysolutions.com/rs600_sensors.html)

Viscosity measurements

Viscosity measurements were performed at 25°C and 37°C to reproduce room and

physiological temperature, respectively. The apparent viscosity was measured on increasing

shear rate values ranging into the interval 20- 300 s-1.

A waiting time equal to 900s was used in order to thermostate the sample at the desired

temperature and to simulate the possible gelation in the vagina. Shear stress and viscosity

values were plotted against shear rate values to obtain, respectively, flow and viscosity curves.

Dynamic viscoelastic measurements

20

Stress sweep test

In the stress sweep test increasing shear stress values are applied to the sample at a

constant value of frequency and the elastic response of the sample is measured in terms of

storage modulus (G’). Such a test is performed to individuate for each sample the so called

“linear viscoelastic region” that is the range of shear stress for which the elastic response of the

sample does not change (G’ is constant). The shear stress at which G’ deviates from a constant

(plateau) value indicates deviation from linear viscoelastic behaviour. This is due, in the case of

polymeric gels, by a disruption of the inner structure. In Figure 3.2. a typical G’ profile obtained

from stress sweep test is reported.

FIGURE 3.3.: TYPICAL G’ PROFILE OBTAINED FROM STRESS SWEEP TEST

In order to individuate for each sample the linear viscoelastic region, stress sweep tests

were carried out at 37°C applying shear stress increasing from 0.1 to 200 Pa at a constant

frequency of 0.1 Hz, while a waiting time of 300 s was used.

Oscillation measurements

0

30

60

90

0 20 40 60 80 100

shear stress (Pa)

G' (P

a)

Linear viscoelastic

region

21

Polymer solutions and polymer/chitosan lactate mixtures were subjected to oscillation

measurements, which provide the application of a constant shear stress value (chosen in the

linear viscoelastic region, previously determined) and the measurement of the elastic response

of the sample expressed by the following parameters: storage modulus G’ (Pa), loss modulus

G”(Pa) and loss tangent tg. The application of a shear stress chosen in the linear viscoelastic

region ensures that during the test the inner structure of the sample is not destroyed. Three

independent variables were considered: temperature, frequency of the applied stress and time.

In particular at first oscillation tests were performed at a constant value of frequency

(0.1Hz) and at temperature values ranging between 8 and 50°C, to evaluate the gelation

temperature of the samples. In order to standardize the sample condition, before each

measurement a fixed shear stress equal to 20 Pa was applied for 180 s (waiting time).

Then the gelation rate of the sample was assessed by performing oscillation

measurements at constant values of temperature (37°C) and frequency (0,1Hz) and at increasing

time, which meant that no waiting time was applied

To assess the elastic properties of the gel formed at physiological temperature, oscillation

tests were also performed at a constant value of temperature (37°C) and at increasing frequency

values ranging between 0.1 and 10 Hz. The stress applied was chosen within the linear

viscoelastic region. Before each measurement, a shear stress of 20 Pa was applied for 300s.

3.2.2.3 Mucoadhesion measurements

The mucoadhesive potential of each sample (poloxamer solutions and

poloxamer/chitosan lactate mixtures) was determined by means of a tensile stress tester (TA.XT.

plus Texture Analyser, Stable Micro Systems, UK) (see Figure 3.3.). The mucoadhesion test

provides the measurement of the force required to detach the sample from a biological

substrate as a function of the displacement occurring at the bioadhesive interface (Sandri G. et

al., 2002). The parameter considered was the Work of Adhesion, calculated by using the

22

trapezoidal method as the area under the detachment force versus displacement curve (AUC)

(Ferrari F., 1995).

The apparatus employed was equipped with 1 kg ‘charge cell’ and an A/MUC measuring

system. Such system consists of a support (see (A) in Figure 3.3.) and a probe (see (B) in Figure

3.3.). The support consists of two concentric cylinders hold together by four screws. The upper

cylinder has a circular hole in the center (see (C) in Figure 3.3.) with a diameter of 14 mm to put

the sample or the biological substrate into. The probe is cylindrical and has a diameter of 10 mm.

Furthermore, the support is placed in a water bath thermostated at 37°C.

Probe (B)

Circular hole (C)

Support (A) [Upper view]

FIGURE 3.2.: REPRESENTATION OF TA.XT. plus TEXTURE ANALYSER, STABLE MICRO SYSTEMS, UK

(http://www.stablemicrosystems.com/)

Mucoadhesion measurements in presence of mucin suspension

23

A first series of tests were carried out using a mucin suspension as biological substrate. At

this aim 8% mucin dispersion was prepared in pH 4,5 acetate buffer (USP). A cylindrical filter

paper disc with 10 mm diameter was stuck on the probe (B) by means of adhesive tape. Then 30

l of the mucin dispersion was layered on the paper disc. Another paper filter disc was fixed

between the two cylinders of the support (A), on which 30 l of the sample (poloxamer/chitosan

lactate mixtures or poloxamer solution) was placed. After a waiting time of 600 s (necessary to

allow the sol/gel transition of the sample) the probe was lowered to put the mucin dispersion in

contact with the sample. A preload of 600 mN was applied in order to allow the formation of the

mucoadhesive joints. After a 60 s rest, the preload was removed and the probe was moved at a

constant speed (2,5 mm/min) up to the complete separation of the two surfaces. Both

displacement of the probe and force of detachment data were recorded and simultaneously

collected on a personal computer.

Tests were also performed using 30 l of pH 4,5 acetate buffer instead of mucin

dispersion (blank measurements).

In order to compare the mucoadhesive performance of the different samples better, the

differential mucoadhesive parameter AUC was calculated according to the following equation:

AUC= AUCbs – AUCblank

where:

AUCbs = work of adhesion obtained in presence of the biological substrate (mucin

suspension)

AUCblank = work of adhesion obtained from blank measurements

Mucoadhesion measurements in presence of porcine vaginal mucosa

24

The poloxamer/chitosan lactate mixture, which has demonstrated the best mucoadhesive

properties (i.e. the highest capability to interact with mucin), was subjected to tensile tests,

using pig vaginal mucosa as biological substrate.

The vaginal mucosa was obtained from a local slaughterhouse immediately after the

sacrifice of the animal. Vaginal tissue was then cleaned and separated from the supporting

muscular and connective tissues with surgical scissors paying attention to maintain the integrity

of the vaginal structure. The isolated tissue was incised longitudinally and then stored at -20°C

before testing.

A circular portion of the mucosa (with a diameter of about 10 mm) was fixed on the

probe (B) using a cyanoacrilate glue, while 100 µl of the sample was layered on the filter paper

disc fixed on the support (A).

The conditions employed (preload, rate of probe movement, waiting time) were the same

as described above for the measurements performed in presence of mucin.

The same test was also carried out on 15% (w/v) poloxamer solution and was used as

reference.

3.2.3 Characterization of 15% (w/v) poloxamer/0,8% (w/v) chitosan lactate mixture diluted

with simulated vaginal fuid (SVF)

25

3.2.3.1. Sample preparation

Simulated vaginal fluid (SVF) was prepared according to Owen and Katz (1999). The SVF

composition was: 0.040 g urea, 0.351 g NaCl, 0.140 g KOH, 0.250 g Lactic Acid 80%, 0.104 g

Acetic Acid 96%, 0.500 g glucose, 0.022 g Ca(OH)2, 0.016 g glycerol, 0.002 g Bovine Serum

Albumin, distillated water up to 100 ml. After stirring the solution, the pH was measured and

adjusted to 4.2 with 4 N HCl.

15% (w/v) poloxamer solution/0,8 % (w/v) chitosan lactate mixture was diluted with SVF

according to a volume ratio equal to 1 : 4.

3.2.3.2. Rheological measurements

15% (w/v) poloxamer/0,8% (w/v) chitosan lactate mixture diluted with SVF was subjected

to viscosity measurements at 37°C as described in the paragraph 3.2.2.1.

Dynamic viscoelastic tests were also performed. In particular, after the determination of

the linear viscoelastic region by means of the stress sweep test (see paragraph 3.2.2.1), the

sample was subjected to oscillation measurements which provide time and frequency as

independent variables. All the measurements were carried out at 37°C. The conditions employed

were the same described in paragraph 3.2.2.1

3.2.3.3. Mucoadhesion measurements

The mucoadhesive potential of 15% (w/v) poloxamer/0,8% (w/v) chitosan lactate mixture

upon dilution with SVF was assessed in presence of mucin dispersion as described in the

paragraph 3.2.2.3.

26

4. RESULTS AND DISCUSSION

4.1. CHARACTERIZATION OF POLOXAMER SOLUTIONS AND 15% (W/V) POLOXAMER/

CHITOSAN LACTATE MIXTURES

4.1.1. pH measurements

Table 4.1. reports pH values obtained for the poloxamer solutions and for the considered

poloxamer/chitosan lactate mixtures. It can be observed that the addition of chitosan lactate to

15% (w/v) poloxamer solutions produces a decrease in pH down to values close to 5. This

decrease is favorable for a vaginal administration, because like mentioned in the introduction

section the pH of the vaginal cavity during the fertile life of the woman ranges from 4 to 5. An

increase in the pH due, for example, to the administration of a neutral or alkaline formulation

could produce modifications of the vaginal microbial flora with a consequent onset of infections.

TABLE 4.1.: pH VALUES OF THE POLOXAMER SOLUTIONS AND OF THE

POLOXAMER/ CHITOSAN LACTATE MIXTURES

Poloxamer

% (w/v) Chitosan lactate % (w/v) pH

16% - 7,4

15% - 7,4

15% 0,4% 5

15% 0,8% 5

15% 1,2% 4,7

15% 1,6% 4,7

4.1.2. Rheological measurements

27

4.1.2.1. Poloxamer solutions

Figure 4.1. (a) and (b) reports the viscosity and flow curves obtained at 25°C for

poloxamer solutions prepared at 15% and 16% (w/v) concentrations. The poloxamer

concentrations were chosen on the basis of literature data. In fact, some authors found that

poloxamer solutions prepared at concentrations close to 16% were able to gelify at 37°C (Koffi A.

A. et al., 2008).

FIGURE 4.1.: VISCOSITY (a) AND FLOW (b) CURVES OBSERVED AT 25°C FOR THE POLOXAMER

SOLUTIONS (15% (W/V) AND 16% (W/V)) (mean values ± s.e.; n=3)

0,0

0,5

1,0

1,5

2,0

2,5

0 50 100 150 200 250 300 350

Shear rate (1/s)

Vis

cost

ity

(Pa.

s)

Poloxamer 15% (w/v)

Poloxamer 16% (w/v)

a)

b)

0

30

60

90

120

150

0 50 100 150 200 250 300 350

Shear rate (1/s)

Sh

ear

stre

ss (

Pa)

28

Both solutions show pseudoplastic behaviour; they are in fact characterized by viscosity

values that decrease on increasing shear rate. As expected an increase in polymer concentration

corresponds to an increase in viscosity values. The viscosity observed for both the two solutions

are compatible with an easy vaginal administration and with a good spreading onto mucosa.

In Figure 4.2. G’ (storage elastic modulus) vs temperature profiles of the two poloxamer

solutions are mentioned. The beginning of sample gelation is proven by a steep increase in G’

values with increasing temperature. The two solutions used show a different gelation

temperature: while 15% (w/v) poloxamer solution starts to gelify at 30°C, the gelation

temperature of 16% (w/v) poloxamer solution is close to 25°C. Therefore, only the behaviour of

15% (w/v) solution is compatible with an in situ gelation after vaginal administration.

FIGURE 4.2.: G’ vs TEMPERATURE PROFILES OBSERVED FOR THE POLOXAMER SOLUTIONS

(15% (W/V) AND 16% (W/V)) (mean values ± s.e.; n=3)

Figure 4.3. shows G’ vs time profiles obtained at 37°C for the two poloxamer solutions. It

can be observed that while 16% (w/v) poloxamer solution gelifies immediately at 37°C, 15% (w/v)

poloxamer solution shows a slower gelation, since it needs more than 200 s to start the gelation.

0

5000

10000

15000

20000

0 10 20 30 40 50 60

Temperature (°C)

G' (P

a)

Poloxamer 15% (w/v)

Poloxamer 16% (w/v)

29

FIGURE 4.3.: G’ vs TIME PROFILE OBSERVED AT 37°C FOR THE POLOXAMER SOLUTIONS (15%

(W/V) AND 16% (W/W)) (mean values ± s.e.; n=3)

The viscosity and flow curves obtained at 37°C for poloxamer solutions prepared at 15%

and 16% (w/v) concentrations are reported in Figure 4.4. (a) and (b). No significant differences

are observed in the viscosity of the two solutions which show comparable viscosity and flow

curves. Both solutions are characterized by pseudoplastic behaviour and by viscosity values

higher than those observed at 25°C (see Figure 4.1.). This means that the gelation of the two

samples occurs at 37°C. It is necessary to point out that for such viscosity measurements a

waiting/thermostatation time of 900 s was used. Even if 15% poloxamer solution is characterized

by a slow gelation rate, a time of 900 s appears sufficient to obtain a gel that has the same

consistency of that formed by 16% poloxamer solution.

0

2000

4000

6000

8000

10000

12000

0 5 10 15

Frequency (Hz)

G' (

Pa

)

Poloxamer 15% (w/v)

Poloxamer 16% (w/v)

30

FIGURE 4.4.: VISCOSITY (a) AND FLOW (b) CURVES OBSERVED AT 37°C FOR THE POLOXAMER

SOLUTIONS 15% (W/V) AND 16% (W/V) (mean values ± s.e.; n=3)

0

2

4

6

8

10

12

14

0 50 100 150 200 250 300 350

Shear rate (1/s)

Vis

cosi

ty (

Pa.

s)

Poloxamer 15% (w/v)

Poloxamer 16% (w/v)

0

50

100

150

200

250

300

0 50 100 150 200 250 300 350

Shear rate (1/s)

She

ar s

tres

s (P

a)

a)

b)

31

Figure 4.5. shows the values of the storage elastic modulus G’ obtained at 37°C for the

two poloxamer solutions as a function of frequency. The 16% poloxamer solution is characterized

by higher values of G’ in comparison to the 15% poloxamer solution. This means that at 37 °C

after 300 s (this is the waiting time used in these measurements) 16% poloxamer solution forms

a more elastic gel.

FIGURE 4.5.: G’ vs FREQUENCY PROFILE OBSERVED AT 37°C FOR THE POLOXAMER SOLUTIONS

((15% (W/V) AND 16% (W/V)) (mean values ± s.e.; n=3)

In Figure 4.6. loss tangent (tg δ) vs frequency profiles obtained at 37°C for the two

poloxamer solutions are reported. Since loss tangent is the ratio between the loss (G”) and the

storage (G’) modulus, it indicates which of the two components (elastic or viscous) prevails in

sample viscoelastic behaviour. Both solutions are characterized by loss tangent values lower than

1 for frequency values higher than 0,3 Hz; this means that for such frequency values both

samples show a prevalence of the elastic component to the viscous one.

0

2000

4000

6000

8000

10000

12000

0 5 10 15

Frequency (Hz)

G' (

Pa

)

Poloxamer 15% (w/v)

Poloxamer 16% (w/v)

32

FIGURE 4.6.: LOSS TANGENT vs FREQUENCY PROFILE OBSERVED AT 37°C FOR THE POLOXAMER

SOLUTIONS (15% (W/V) AND 16% (W/V)) (mean values ± s.e.; n=3)

On the basis of the results so far obtained, 15% (w/v) poloxamer solution was chosen for

the subsequent phase of the work which provides the preparation and characterization of

poloxamer/chitosan lactate mixtures.

4.1.2.2. Poloxamer/chitosan lactate mixtures

In Figure 4.7. (a) and (b) the viscosity and flow curves obtained at 25°C for

poloxamer/chitosan lactate mixtures can be seen. The addition of chitosan lactate to poloxamer

solution produces a slight increase in sample viscosity. However the viscosity values observed are

compatible with an easy vaginal administration and with a good spreadibility onto mucosa.

0,1

1,0

10,0

0 2 4 6 8 10 12

Frequency (Hz)

Lo

ss

ta

ng

en

t (G

"/G

')

Poloxamer 15% (w/v)

Poloxamer 16% (w/v)

33

FIGURE 4.7.: VISCOSITY (a) AND FLOW (b) CURVES OBSERVED AT 25°C FOR THE

POLOXAMER/CHITOSAN LACTATE MIXTURES (mean values ± s.e.; n=3)

Figure 4.8. represents G’ (storage modulus) vs temperature profiles of the

poloxamer/chitosan lactate mixtures. The addition of chitosan lactate to poloxamer solution

produces a slight increase in sample gelation temperature. Such an increase is evident for the

mixtures containing 0.4; 1.2 and 1.6% chitosan lactate. The mixture based on 0,8% (w/v) chitosan

0,0

0,5

1,0

1,5

2,0

2,5

0 50 100 150 200 250 300 350

Shear rate (1/s)

Vis

co

sti

ty (

Pa.s

)

Poloxamer 15%

Polox 15% - ChitLact 0,4%

Polox 15% - ChitLact 0,8%

Polox 15% - ChitLact 1,2%

Polox 15% - ChitLact 1,6%

0

20

40

60

80

100

120

140

160

0 50 100 150 200 250 300 350

Shearrate (1/s)

She

arst

ress

(Pa)

a)

b)

34

lactate seems to gelify at the same temperature of 15% poloxamer solution. All the mixtures are

still showing gelation temperatures in the physiological range.

FIGURE 4.8.: G’ vs TEMPERATURE PROFILES OBSERVED FOR THE POLOXAMER/CHITOSAN

LACTATE MIXTURES (mean values ± s.e.; n=3)

In Figure 4.9. G’ vs time profiles obtained at 37°C for the poloxamer/chitosan lactate

mixtures are reported. It can be observed that among the considered mixtures, the one based on

0.8% chitosan lactate is characterized at 0 time by a high value of G’. This indicates an immediate

gelation of the sample placed in contact with the plate of the rheometer thermostated at 37°C.

In comparison, the other mixtures are characterized by G’ profiles comparable to that observed

for the 15% poloxamer solution.

0

1000

2000

3000

4000

0 10 20 30 40 50 60

Temperature (°C)

G' (

Pa

)

Poloxamer 15%

Polox 15% - ChitLact 0,4%

Polox 15% - ChitLact 0,8%

Polox 15% - ChitLact 1,2%

Polox 1,6% - ChitLact 1,6%

35

FIGURE 4.9.: G’ vs TIME PROFILE OBSERVED AT 37°C FOR THE POLOXAMER/CHITOSAN LACTATE

MIXTURES (mean values ± s.e.; n=3)

In Figure 4.10. (a) and (b) viscosity and flow curves obtained at 37°C for

poloxamer/chitosan lactate mixtures are reported. All the mixtures show, at 37 C°, viscosity

values higher than those observed at 25°C, which is the result of gel formation. The presence of

chitosan lactate at 0.4% (w/v) produces a decrease in mixture viscosity in comparison with

poloxamer solution. For higher chitosan lactate concentration such effect is less evident:

viscosity profiles rise up to become comparable (for 1.2% chitosan lactate) to that of poloxamer

solution.

0

1000

2000

3000

4000

5000

6000

0 200 400 600 800

Time (s)

G' (

Pa

)

Poloxamer 15%

Polox 15% - ChitLact 0,4%

Polox 15% - ChitLact 0.8%

Polox 15% - ChitLact 1.2%

Polox 15% - ChitLact 1.6%

36

FIGURE 4.10.: VISCOSITY (a) AND FLOW (b) CURVES OBSERVED AT 37°C FOR THE

POLOXAMER/CHITOSAN LACTATE MIXTURES (mean values ± s.e.; n=3)

Figure 4.11. shows the values of the storage modulus G’ obtained at 37°C for

poloxamer/chitosan mixtures as a function of frequency. The addition of chitosan lactate at 0.4%

(w/v) concentration produces a reduction of G’ profile, which indicates a decrease in gel elastic

0

2

4

6

8

10

12

0 50 100 150 200 250 300 350

Vis

co

sit

y (

Pa.s

)

Shear rate (1/s)

Poloxamer 15%

Polox 15% - 0,4% ChitLact

Polox 15% - 0,8% ChitLact

Polox 15% - 1,2% ChitLact

Polox 15% - 1,6% Chitlact

0

50

100

150

200

250

300

0 50 100 150 200 250 300 350

Shear rate (1/s)

Shea

r stre

ss (P

a)a)

b)

37

properties. An increase in chitosan lactate concentration up to 0.8% causes a steep increase in G’

values. The 15% poloxamer/0.8% chitosan lactate mixture is in fact characterized by the highest

G’ profile, which means that its elastic properties are more pronounced than those of the

poloxamer solution. A further increase in chitosan lactate concentration is responsible for a

decrease in gel elastic properties as evidenced by the lower G’ values observed for 15%

poloxamer/1,2% chitosan lactate and by 15% poloxamer/1.6% chitosan lactate mixtures.

FIGURE 4.11.: G’ vs FREQUENCY PROFILE OBSERVED AT 37°C FOR POLOX AMER/CHITOSAN

LACTATE MIXTURES (mean values ± s.e.; n=3)

In Figure 4.12. loss tangent vs frequency profiles obtained at 37°C for poloxamer/chitosan

lactate mixtures are reported. For frequency values higher than 0.6 Hz all the mixtures are

characterized by a prevalence of the elastic component on the viscous one (as evidenced by loss

tangent values lower than 1). The loss tangent profiles of the mixtures are lower or comparable

with respect to 15% poloxamer solution with the exception of that observed for 15%

poloxamer/0.4% chitosan lactate mixture.

0

1000

2000

3000

4000

0 5 10 15

Frequency (Hz)

G' (

Pa)

Poloxamer 15%

Polox 15% - ChitLact 0,4%

Polox 15% - ChitLact 0,8%

Polox 15% - ChitLact 1.2%

Polox 15% - ChitLact 1.6%

38

FIGURE 4.12.: LOSS TANGENT vs FREQUENCY PROFILE OBSERVED AT 37°C FOR

POLOXAMER/CHITOSAN LACTATE MIXTURES (mean values ± s.e.; n=3)

4.1.3 Mucoadhesion measurements

Figure 4.13. reports the values of the mucoadhesion parameter Work of adhesion (AUC)

observed in presence of mucin suspension for 15% (w/v) poloxamer solution and its mixtures

with chitosan lactate. Such values are compared in the same graph to those obtained for the

same samples using pH 4.5 acetate buffer instead of mucin suspension (blank measurements).

AUC values obtained from blank measurements are indications of the contribution of sample

consistency to the mucoadhesion phenomenon. The achievement of AUC values in presence of

mucin higher than those observed in blank measurement indicates that an interaction between

polymer chains and mucin glycoproteins occurs.

All the considered samples, except the 15% poloxamer solution, are characterized by a

statistically significant (Anova one way, post hoc test) increase in AUC values in presence of

0,1

1

10

100

0 2 4 6 8 10 12

Frequency (Hz)

Lo

ss

ta

ng

en

t (G

"/G

')

Poloxamer 15%

Polox 15% - ChitLact 0,4%

Polox 15% - ChitLact 0,8%

Polox 15% - ChitLact 1.2%

Polox 15% - ChitLact 1.6%

39

mucin in comparison to blank measurements. It indicates that while 15% poloxamer solution

does not possess mucoadhesion properties, all the mixtures are capable to interact with mucin.

Moreover, in presence of mucin, the mixtures with chitosan lactate are characterized by

higher values of AUC with respect to 15% poloxamer solution. This indicates that the addition of

chitosan lactate causes an increase in sample mucoadhesive properties. Among the mixtures, the

highest AUC value is shown by the mixture containing 0.8% (w/v) chitosan lactate.

In Table 4.2. results obtained from statistical analysis are reported.

FIGURE 4.13.: VALUES OF WORK OF ADHESION (AUC) OBTAINED FOR 15% POLOXAMER

SOLUTION AND ITS MIXTURES WITH CHITOSAN LACTATE (mean values ± s.e.; n=7)

0

200

400

600

800

1000

1200

1400

1600

Polox 15% Polox 15% -

0,4%ChitLact

Polox 15 % -

0,8% ChitLact

Polox 15 % -

1,2% ChitLact

Polox 15% - 1,6

% ChitLact

AU

C (

mN

*mm

)

pH 4.5 acetate buffer

Mucin suspension

40

TABLE 4.2.: RESULTS OF THE STATISTICAL ANALYSIS (ANOVA ONE WAY, POST HOC TEST)

EMPLOYED TO COMPARE AUC VALUES OBTAINED IN PRESENCE AND IN ABSENCE

(BLANK MEASUREMENTS) OF MUCIN

BLANK MEASUREMENTS

IN P

RES

ENC

E O

F M

UC

IN

15%

poloxamer solution

15% poloxamer/

0,4% chitosan lactate

15% poloxamer/

0,8% chitosan lactate

15% poloxamer/

1,2% chitosan lactate

15% poloxamer/

1,6% chitosan lactate

15% poloxamer

solution

Not significant

15% poloxamer/

0,4% chitosan lactate

p< 0,001

15% poloxamer/

0,8% chitosan lactate

p< 0,001

15% poloxamer/

1,2% chitosan lactate

p< 0,01

15% poloxamer/

1,6% chitosan lactate

p< 0,001

To compare the mucoadhesive potential of the different samples better, the differential

parameter ΔAUC was calculated by subtracting the values of work of adhesion obtained in blank

41

measurements from those obtained in presence of mucin. Higher ΔAUC values correspond to

better mucoadhesive properties.

In Table 4.3. the ΔAUC values calculated for 15% (w/v) poloxamer solution and its

mixtures with chitosan lactate are described. The highest value is shown in the mixture

containing 0,8% (w/v) chitosan lactate. This mixture, being characterized by the highest values of

AUC in presence of mucin and of ΔAUC, demonstrates the best mucoadhesive properties.

TABLE 4.3.: REPRESENTATION OF ΔAUC VALUES CALCULATED FOR 15% (W/V) POLOXAMER

SOLUTION AND ITS MIXTURE WITH CHITOSAN LACTATE

ΔAUC

(mN.mm)

15% poloxamer

solution

257

15% poloxamer/

0,4% chitosan lactate

701

15% poloxamer/

0,8% chitosan lactate

933

15% poloxamer/

1,2% chitosan lactate

581

15% poloxamer/

1,6% chitosan lactate

763

In order to confirm the mucoadhesive potential of 15% poloxamer/0.8% chitosan lactate

mixture, tensile tests were also performed using porcine vaginal mucosa as biological substrate,

while 15% poloxamer solution was used as reference. The obtained values are, respectively, 561

± 169 (s.e., n=7) mN*mm for poloxamer solution and for 1329 ± 107 (s.e., n=7) mN*mm for the

mixture with chitosan lactate.

42

The obtained results confirm that, also in presence of vaginal mucosa, the mixture with

chitosan lactate is characterized by higher mucoadhesive properties in comparison to the 15%

poloxamer solution.

4.2. CHARACTERIZATION OF 15% (W/V) POLOXAMER/0.8% CHITOSAN LACTATE DILUTED WITH

SIMULATED VAGINAL FLUID (SVF)

4.2.1. Rheological analysis

In Figure 4.14. the G’ vs temperature profile obtained for diluted 15% (w/v)

poloxamer/0.8% (w/v) chitosan lactate mixture is reported. It can be observed that the dilution

with simulated vaginal fluid causes a lag time of about 450 s before gelation occurs. Such lag time

can be considered compatible with a vaginal administration: the patient should be at rest for 7-8

minutes after administration.

FIGURE 4.14.: G’ vs TEMPERATURE PROFILE OBSERVED FOR THE 15% (W/V) POLOXAMER/

0.8% (W/V) CHITOSAN LACTATE MIXTURE AFTER DILUTION WITH SVF (mean

values ± s.e.; n=3)

0

200

400

600

800

1000

0 200 400 600 800

Time (s)

G' (P

a)

43

In Figure 4.15. the viscosity curve obtained at 37°C for 15% (w/v) poloxamer/ 0.8% (w/v)

mixture diluted with SVG is showed. As expected the dilution with SVF causes a decrease in

viscosity. In spite of the dilution with SVF, such a mixture shows at 37°C viscosity values higher

than those observed at 25°C for the not diluted mixture (see Figure 4.7.).

Figure 4.16. reports the values of the storage modulus G’ obtained at 37°C for 15% (w/v)

poloxamer/ 0.8% (w/v) mixture diluted with SVF as a function of frequency. Analogously to

viscosity, elastic properties of the mixture decrease upon dilution. The diluted mixture is

characterized by high G’ values ranging between the interval 790 – 1500 Pa. This proves that,

even if diluted, the mixture forms a strong gel. This is also confirmed by loss tangent values that

are, for all the frequency values considered, lower than 1 indicating that elastic component

prevails on the viscous one (Figure 4.17.).

FIGURE 4.15.: VISCOSITY CURVES OBSERVED AT 37°C FOR THE 15% POLOXAMER/0.8%

CHITOSAN LACTATE MIXTURE UPON DILUTION WITH SVF (mean values ± s.e.; n=3)

0

1

2

3

4

5

6

0 50 100 150 200 250 300 350

Shear rate (1/s)

Vis

cosi

ty (P

a.s)

Polox 15% - 0,8% ChitLact

Polox 15%- 0,8% ChitLact + SVF

44

FIGURE 4.16.: G’ vs FREQUENCY PROFILE OBSERVED AT 37°C FOR THE 15% POLOXAMER/ 0.8%

CHITOSAN LACTATE MIXTURE UPON DILUTION WITH SVF (mean values ± s.e.; n=3)

FIGURE 4.17.: LOSS TANGENT vs FREQUENCY PROFILE OBSERVED AT 37°C FOR THE 15%

POLOXAMER/ 0.8% CHITOSAN LACTATE MIXTURE UPON DILUTION WITH SVF

(mean values ± s.e.; n=3)

0

1000

2000

3000

4000

0 5 10 15

Frequency (Hz)

G' (

Pa

)

Polox 15% - ChitLact 0,8%

Polox 15% - ChitLact 0,8% + SVF

0,01

0,1

1

10

100

0 2 4 6 8 10 12

Frequency (Hz)

Lo

ss

ta

ng

en

t (G

"/G

')

Polox 15% - ChitLact 0,8%

Polox 15% - ChitLact 0,8% + SVF

45

4.2.2 Mucoadhesion measurements

In Figure 4.18. the values of the mucoadhesion parameter Work of adhesion (AUC)

observed in presence and in absence of mucin suspension for 15% (w/v) poloxamer/0.8% (w/v)

chitosan lactate mixture diluted with SVF solution are reported. Upon dilution, the mixture

maintains the capability to interact with mucin; in presence of mucin it is in fact characterized by

an AUC value statistically higher (p < 0.01) than that observed in presence of acetate buffer

(blank measurements).

FIGURE 4.18.: VALUES OF WORK OF ADHESION (AUC) OBTAINED FOR 15% (W/V) POLOXAMER/

0.8% (W/V) CHITOSAN LACTATE MIXTURE DILUTED WITH SVF (mean values ±

s.e.; n=7)

0

50

100

150

200

250

Polox 15% - 0.8% ChitLact + SVF

AU

C (

mN

*mm

)

pH 4,5 acetate buffer Mucin suspension

46

5. CONCLUSIONS

Both the two poloxamer solutions (15% and 16% (w/v)) are characterized at room

temperature by a viscosity which permits an easy vaginal application and a good spreading onto

mucosa. As expected, both the two poloxamer solutions are able to gelify on increasing

temperature but only 15% poloxamer solution shows a gelation temperature compatible with an

in situ gelation into vaginal cavity. For this reason such solution was chosen for the preparation

of the mixtures with chitosan lactate.

The addition of chitosan lactate to poloxamer solution produces a decrease in pH value

up to 5. Such a decrease is favorable for a vaginal administration since the pH of the vaginal

environment of fertile women is in a range of 4- 5.

The viscosity of all the mixtures at 25°C is still compatible with a vaginal application.

Moreover the addition of chitosan does not produce marked changes in gelation temperature

that remains in the physiological range. At 37°C all the mixtures are characterized by a marked

prevalence of the elastic modulus on the viscous one, behaviour typical of a gel. Another

parameter to be considered in view of a vaginal application is the time of gelation which has to

be as short as possible. All the mixtures considered show short gelation times, more in particular

the one based on 0.8% chitosan lactate seems to be characterized by an immediate gelation at

37°C.

The presence of chitosan lactate in the mixtures is responsible for the acquisition of

mucoadhesive properties. In fact the solution of poloxamer alone is not able to interact with the

biological substrate (mucin dispersion). In presence of mucin, no significant differences are

observed among AUC values of the mixtures based on chitosan lactate concentration 0.8%

(w/v). Among these mixtures, the one with 0.8% chitosan lactate concentration is characterized

by the highest value of AUC (i.e. by the best mucoadhesive potential).

The same mixture upon dilution with simulated vaginal fluid maintains the mucoadhesive

properties and is able to form at 37° C a viscoelastic gel. The diluted mixture shows a lag time of

gelation of about 450 s thus, after the patient had applied the formulation, a 7-8 minute rest

should be left.

47

Even if further studies are necessary to confirm the results so far obtained, it can

generally be concluded that 15% poloxamer/chitosan lactate mixtures, particularly the mixture

based on 0.8% (w/v) chitosan lactate, seem promising vehicles for vaginal administration of

drugs. For this reason, the 15% poloxamer solution combined with 0.8% chitosan lactate solution

deserves the most interests for further research in the use of thermoreversible mucoadhesive

gels as vaginal drug delivery systems.

Later on this vehicle will be used for the vaginal delivery of antibiotic drugs in the

treatment of vaginal mucositis. In fact the presence of chitosan lactate into the formulation

should provide, besides mucoadhesive properties, also intrinsic antimicrobial and tissue repairing

ones.

The work is in progress to investigate the capability of the poloxamer-chitosan lactate

mixtures to interact with microbial cells and to favour wound-healing.

48

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