Polymer-coated liposomes with potential of hydrating dry mucous membranes

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Polymer-coated liposomes with potential of hydrating dry mucous

membranes

Ljubica Mihailovic

Master Thesis in Pharmaceutics 45 study points

Section for Pharmaceutics and Social Pharmacy Department of Pharmacy

The Faculty of Mathematics and Natural Sciences

UNIVERSITY OF OSLO

January 2019

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Polymer-coated liposomes with potential of hydrating dry mucous

membranes

Ljubica Mihailovic

Supervisors:

Marianne Hiorth Gro Smistad Joseph Azumah

Section for Pharmaceutics and Social Pharmacy Department of Pharmacy

The Faculty of Mathematics and Natural Sciences

UNIVERSITY OF OSLO

January 2019

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Polymer-coated liposomes with potential of hydrating dry mucous membranes Ljubica Mihailovic

http://www.duo.uio.no/

Print: University Print Centre, University of Oslo

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Acknowledgements

The present work was carried out at the Section for Pharmaceutics and Social Pharmacy, Department of Pharmacy, University of Oslo, during the period may 2018 to january 2019.

I would like to express my sincere gratitude to my supervisors, Marianne Hiorth, Gro Smistad and Joseph Azumah for guidance, support and understanding through my work.

A special thanks to Marianne Hiorth for her insightful remarks, encouragement and fruitful discussions. Thank you for your patience, time, effectiveness, prompt and helpful answers on all my doubts and that your doors were always open for me.

My appreciation also goes to Tove Larsen for positivity and quick solutions for all obstacles I had during laboratory work.

I am very grateful for great colleges I met during my work in a lab, for sharing experiences and joy in the difficult moments.

Finally, I would like to express my deeply gratitude to my lovely family for understanding and great support and motivation during my work on the master thesis.

Oslo, January 2019 Ljubica Mihailovic

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Abstract

Xerostomia and hyposalivation can cause increased morbidity and decreased quality of life of an aging population. Formulations available for local alleviation of the symptoms have limited effect due to the harsh conditions in the oral cavity.

A new approach could be to use liposomal formulations with encapsulated glycerol. These particles can potentially be spread over the whole surface of oral cavity, adsorb onto it and perform prolonged lubrication and hydration.

In this thesis liposomes with different surface charge, with or without encapsulated glycerol, were produced. Different polymers (HM-HEC, alginate, LM-pectin and chitosan) were used to coat and modify the liposome characteristics in order to select those with best stability and mucoadhesive properties. The particle size and charge were characterized by dynamic light scattering and laser Doppler electrophoresis. Selected formulations were tested for interaction with mucin by rheology and the principle of rheological synergism after mixing with mucin solution. The stability of all the formulations were followed during storage in refrigerator by measuring the change in the particle size and the zeta potential. The results showed that all formulations were successfully prepared without markedly influence of glycerol on their size, charge and mucoadhesivness. The chitosan coated liposomes were pointed out as the most mucoadhesive formulation. Storage-life of the formulations was shown to be dependent on the glycerol content for negative SOYA-PC-EggPG liposomes that disintegrating after 14 weeks. This was prevented by coating these liposomes with chitosan. Encapsulation of glycerol influenced oppositely the neutral liposomes. The stable neutral liposomes with glycerol disintegrated during 18 weeks of storage when they were coated with HM-HEC.

Positive liposomes SOYA-PC-DOTAP were the most stable in the presence of glycerol.

Coating of those liposomes with two different negative polymers demonstrated oppositely results. LM-pectin coating showed stable particle size and charge while coating with alginate caused instability probably due to desorption of the alginate from the liposome surface.

Overall, the most promising formulation for prolonged effect on mouth dryness could be chitosan coated liposomes with encapsulated glycerol. These particles interacted strongly with mucin and were stable during the whole test period of 18 weeks. Further investigations on in vivo stability, toxicity and hydration effect would be interesting to conduct.

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1 Introduction

1.1 Background

Xerostomia or dry mouth syndrome and salivary gland hypofunction (SGH) have lately been more in focus due to an aging population with increased polypharmacy. Oral dryness can be a profoundly discomfortable symptom per se, especially during the night. Consequences of inadequate salivation, such as deterioration of the dental health, oral ulcers and infections and halitosis, have more influence on life impairment. Salivary production or composition can be decreased reversibly or irreversibly due to different effects, such as side-effects of different medications, radiation towards the head and the neck region and other diseases such as Sjogren’s syndrome.

Current treatment with systemic medicines to increase production of saliva have many adverse effects while local salivary stimulants or substitutes have limited effects due to harsh conditions in the oral cavity that prevent prolonged effect especially during the night.

Mucoadhesive tablets can also irritate the mucous membrane and cause low compliance.

Liposomes are biocompatible, biodegradable and non-toxic particles that have been

established at the marked as a drug carrier. Different active substances could be encapsulated in the aqueous core or phospholipid layer of liposomes and released after their degradation on the place of action. Biopolymers can be used to modify the properties of the liposomes by adsorbing in a layer around them. In this way steric and/or electrostatic repulsion prevents liposomes from aggregation and prolong their stability. Mucodhesive properties of the biopolymers prolong the contact time of the polymer-coated liposomes on the mucosal surfaces. Those advantages of the liposomes can be utilized for developing promising system for the treatment of xerostomia. Mucoadhesivness can assure longer residential time and better distribution of the formulation on the oral surface. This can be beneficial especially for use during night or in patients with low compliance due to discomfort of the presence of the product in the mouth. Also, the nanosized particles may mimic the layer that natural saliva creates on the oral surface constantly bathing the oral cavity. Polyalcohol glycerol is a broadly used humectant that keeps moisture. Encapsulation of glycerol in the polymer-coated

mucoadhesive liposomes could be advantageous strategy for developing a local product for treatment of mouth dryness.

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1.2 Aim of the study

The overall aim of this thesis was to develop a liposomal formulation with encapsulated glycerol and good mucoadhesive properties with the potential of giving more effective and prolonged treatment of dry mouth.

The investigation was divided into 3 sub-aims:

1. To produce and characterize different liposomal formulations with encapsulated glycerol

2. To develop a method for investigating the interaction of liposomal formulations with mucin by rheology and evaluate the mucoadhesive properties of selected formulations by this method

3. To examine the stability of the prepared liposomal formulations with encapsulated glycerol

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1.3 Abbreviations

DLS Dynamic light scattering DA Degree of acetylation DDA Degree of deacetylation DE Degree of esterification

DOTAP Dioleoyltrimethylammoniumpropane Egg PG Egg-phosphatidylglycerol

HM-HEC Hydrophobically modified hydroxyethylcellulose LM-pectin Low methoxyl pectin

Mw Molecular weight

PBS Phosphate-buffered saline PDI Polydispersity Index SPC Soya-phosphatidylcholine

Wt% Weight percentage

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2 Theory

2.1 Oral cavity

The oral cavity is located at the entrance of the digestive and respiratory systems. It is naturally exposed to different nutrients, xenobiotics and microorganisms that can be stopped by the oral epithelial barrier, absorbed through it, or transported further through the digestive or respiratory systems. Therefore, it represents an interesting area that is largely utilized for drug delivery. The oral cavity has very dynamic environment that changes with constant salivation, food and drink intake and myriad of microorganisms that inhabit its surfaces.

The anatomical parts of the oral cavity are engaged in producing speech, mastication and swallowing (Figure 2.1). There are four areas with distinct tissue types in the oral cavity:

palatal, gingival, sublingual and buccal mucosa (Pfister and Ghosh 2005).

Figure 2.1. Anatomical structure of oral cavity and types of mucous membrane, modified from the reference (Bergmeier 2018)

2.1.1 Oral mucosa

The oral cavity is covered by oral mucosa, that is, on one side, a barrier, and on the other side, a high degree of vascularized tissue with a large surface/volume ratio, with potential for drug

Masticatory mucosa Lining mucosa Specialized mucosa Upper lip

Alveolar mucosa Hard palate Soft palate Cheek

Tongue

Underside of tongue

Gingiva Floor of mouth

Lower lip

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5 absorption. Oral epithelium consists of several layers of stratified squamous epithelial layer (Figure 2.2), separated with tight cell-packed basement membrane from a thin layer of connective tissue (lamina propria). Superficial epithelial cells in the area of hard palate and some parts of gingiva are keratinized and termed masticatory mucosa, while other parts of oral cavity are covered with lining mucosa with non-keratinized epithelium (Figure 2.1).

Specialized mucosa is present on the dorsal side of the tongue in the taste buds (Bergmeier 2018). The morphology and thickness of the mucous membrane varies, for example buccal mucosa can be 500 – 600 μm and sublingual mucosa 100 – 200 μm thick (Tsibouklis et al.

2013).

Figure 2.2. Schematic structure of the oral mucosa (Tsibouklis et al. 2013)

Several hundred minor salivary glands are widespread through the oral mucosa. Mucus gel- like layer covers the oral epithelium, lubricates it and protects it as a barrier. It can be 40 – 300 μm tick and has turnover time 12-24 hours (Tsibouklis et al. 2013).

Mucous membrane of the mouth is normally colonized with fungi, viruses and many bacterial arts that coexist with other structures in formation of biological plaque that covers the

surfaces in the oral cavity.

2.1.2 Saliva

The oral cavity is constantly flushed with fluid, composed of salivary secretion, gingival crevicular fluid, and also includes food and cell debris and bacteria. This fluid is known as whole saliva (Humphrey and Williamson 2001).

Stratum corneum Stratum granulosum

Stratified squamous epithelial layer Stratum spinosum

Stratum basale

Basement membrane

Lamina propria

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Saliva has many important roles in the oral cavity. It has the function of digesting due to the presence of alpha-amylases, lipases and serine-protease. Salivary secretion dissolves the substances so that they can produce the taste sensation. It lubricate the surfaces in the mouth to make speaking, mastication and swallowing easier. Saliva is the water medium that buffers the oral cavity, while calcium and phosphate concentrations balance between demineralization and remineralization of the dentin of the teeth. Protection of the oral cavity from

microorganisms is achieved by secretion of enzymes (lysozyme, lactoferrin and peroxidase) and glycoproteins (Bhayani and Lai 2018; Humphrey and Williamson 2001).

Salivary glands produces about 1-1.5 L saliva daily, of which 90% is produced by the paired salivary glands (parotid, submandibular end sublingual) while 10% comes from numerous minor salivary glands (Humphrey and Williamson 2001). Basal saliva is produced

predominantly by submandibular glands (65%) and it is secreted 14 – 16 hours daily and responsible for oral comfort and protection of the mouth (Sreebny and Schwartz 1997). In stimulated salivation, parotid glands are dominated with 50% output (Humphrey and Williamson 2001). Stimulated saliva is produced about 2 hours daily and plays a role in swallowing and clearance of the substances from the oral cavity (Sreebny and Schwartz 1997). There are individual variations in salivary production, circadian variations (with lowest production during sleep) and seasonal variations (lower during the summer and peak in the winter) (Bhayani and Lai 2018).

Hyposalivation, decreased salivary production, is predominantly diagnosed by measuring the salivary flow rates (sialometry) as unstimulated or stimulated saliva flow rate (Ying Joanna and Thomson 2015).

2.1.3 Xerostomia

Xerostomia or dry mouth syndrome is the term defined as subjective sensation of dryness in the oral cavity and should be clearly distinguished from salivary gland hypofunction (SGH) that is objectively measured reduction in salivary flow (Bhayani and Lai 2018). Mouth dryness can be caused both by reduced salivary flow or change in salivary composition.

Decrease in salivary flow of 30 % can be already perceived as dryness in the mouth (Bhayani and Lai 2018).

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7 Prevalence of xerostomia is controversial and one systematic review study found it to be 8% - 42%, with variations due to the lack of agreement on definition and method used to diagnose it. Salivary gland hypofunction had a similar prevalence in the same population, 11-47%, while on the other hand, prevalence of both conditions present together was only 2-6%

(Hopcraft and Tan 2010). Xerostomia is more often related to women and elderly, while SGH is generally agreed not to be influenced by aging alone (Hopcraft and Tan 2010).

Xerostomia and SGH can be caused by body dehydration, cytotoxicity to the salivary glands or impairment in neural stimulation of the glands. Dehydration of different derivations can be linked to xerostomia, like diabetes insipidus, renal disease, diarrhea and vomiting or use of diuretics. Damage to the salivary glands is seen in systematic diseases like Sjogren’s syndrome, diabetes mellitus, rheumatoid arthritis or infections with viruses: human

immunodeficiency virus (HIV), hepatitis C virus and mumps. Radiotherapy of head and neck carcinoma can result in dry mouth in almost to thirds of the patients (Bhayani and Lai 2018).

Psychogenic origin of dry mouth can be seen in anxiety and depression. Mouth breathing can also worsen mouth dryness, especially because basal salivary flow rates are lowest during the night. In people over 50 years old predominant cause of xerostomia is use of more than one of xerogenic medications, where over 400 from 42 groups are found to cause dry mouth and reduced salivary flow (tricyclic antidepressants with anticholinergic side effects,

antihistamines, α-adrenergic antagonists, diuretics, antihypertensives) (Bhayani and Lai 2018). The most important population that is affected by dry mouth is the elderly due to frequent presence of medical conditions and polypharmacy and they are more susceptible to the adverse drug effects. Xerostomia causes burden on the morbidity and quality of life and is getting more focus nowadays with the aging population. Dry mouth syndrome may be

reversible, as in the case of use of xerogenic drugs, or irreversible, caused by Sjogren’s syndrome or radiotherapy.

Typical disturbances in quality of life that results from dry mouth are dysphagia, reduced sensitivity to taste perception, problems with speaking, oral fungal infections, dental caries, dental erosion, oral ulcers, dry cracked lips and corners of the mouth and dry and coarse tongue (Ying Joanna and Thomson 2015).

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Table 2.1. Some of the products used for treatment of dry mouth on the Norwegian market

Product Pharmaceutical form

Active ingredients Other ingredients XyliMelts Adhering

pastilles

500 mg xylitol slowly releasing

over 1 - 6 hours

Gummi arabicum

Cellulose gum (hydroxymethylcellulose)

Saliva Orthana Spray Mucin

Xylitol Biotène

Oralbalance

Gel Xylitol

Sorbitol Glycerol Lactoperoxidase

Hydrated silica, Silica, Hydroxyethylcellulose, Isoceteth-20, Cellulose gum, Menthol

Biotène Tooth paste Fluor, enzimes and proteins found naturally in

saliva, Sorbitol, Glycerol

Hydrated silicia, PEG-8, Cocamidopropyl betaine, Xanthan gum, Limonene

Salivin Lozenge Xylitol

Malic acid

Maltitol, Acesulfame K, Gummi arabicum Flux Dry

Mouth

Gel Fluor

Xylitol Glycerol Sorbitol

Hydroxyethylcellulose, PEG-40, Hydrogenated castor oil, Chamomilla Recutita

flower extract, Allantoin Flux Dry

Mouth

Mouth wash Fluor

Xylitol Glycerol

Sorbitol

PEG-40, Hydrogenated castor oil, Chamomilla Recutita flower extract, Hydroxyethylcellulose, Potassium sorbate,

Allantoin Flux Dry

Mouth

Lozenge Fluor

Xylitol

Isomalt, Maltitol, Tartaric acid, Caramelized sugar syrup

Proxident Spray Fluor

Xylitol Malic acid

Carbamid Nycodent

saliva

Lozenge Fluor

Xylitol Malic acid

Sorbitol

Isomalt,

Magnesiumsalts of fetty acids

Xerodent Lozenge Fluor

Xylitol Malic acid

Macrogol 6000, Povidone, Sodium stearyl fumarate, Anhydrous colloidal silica

Treatment of xerostomia is comprised of local symptomatic treatment with salivary stimulants (sialogogues) and substitutes, that both have limited time of action and systemic therapy with parasympathomimetic drugs. Unselective parasympathomimetics pilocarpine and M1, M3

receptor selective cevimeline, are used with documented effect (Hamad et al.), but both with typical adverse effects as sweating, urinary frequency, headache, dizziness, blurry vision and nausea. Electrostimulation of salivary glands, gene therapy and acupuncture are also tried with controversial results due to poor quality of evidence.

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9 Different products that stimulate or substitute saliva secretion are marketed for treatment of xerostomia and can be found in Table 2.1. Products are available in different pharmaceutical forms: spray, mouth wash, gel, lozenges, while prolonged residence time is assured with mucoadhesive slow releasing pastilles. Effect are based on ingredients that are established humectants (polyols, sugar alcohols), liposoluble ingredients (Hydrogenated castor oil), thickeners (Gummi arabicum, hydroxymethylcellulose, silica) and ingredients that prevents consequences of hyposalivation (fluor, lactoperoxydase, extract Camomille, allantoin).

In the management of xerostomia there is need for a product that is easy to use, with

acceptable taste and lubrication effect lasting longer than 4-6 hours, especially for symptoms manifesting during night.

2.2 Mucoadhesion

Mucoadhesion is defined as attachment of macromolecules to mucus and/or epithelial surface (Rathbone, Senel, and Pather 2015).

Figure 2.3. Structure of mucin glycoproteins and potential interaction domains (Yang et al. 2012)

Mucus

Mucus comprises mainly water (95%) and the rest are glycoproteins, mucopolysaccharides, proteins and lipids. The most important functional component in mucus is the glycoprotein mucin, secreted by mucosal goblet cells (Figure 2.3). Mucins are group of glycoproteins with

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molecular weight from 1 000 to 40 000 kDa (Rathbone, Senel, and Pather 2015). They have protein backbone rich in serine and threonine and attached oligosaccharide side chains, that compose 50-80% dry weight (Rathbone, Senel, and Pather 2015). Oligosaccharide chains consist of glucose, galactose, N-acetylglucosamine, N-acetylgalactosamine and fucose, which are further functionalized with sulphate esters and sialic acid. Mucins are negatively charged at physiological pH due to the sialic acid residues. There are two types of mucins, soluble and membrane-bound that comprises hydrophobic moiety that is attached to cell membrane (Rathbone, Senel, and Pather 2015).

Mechanisms of mucoadhesion

The interaction between the mucoadhesive polymer and mucous membrane is termed mucoadhesive joint and it is created in three steps:

1. Contact stage, where the mucoadhesive is placed in direct contact with the mucosa causing reduction in surface free energy. This stage depends on hydration of the materials and in the case of semisolid or liquid dosage forms, wetting and spreading are promoted by increasing the contact surface, while for dry materials are first wetting, hydrating and swelling necessary for contact to occur. Considering this, it might be concluded that liquid formulations are preferable over solid dosage forms when applied to patients with extent hyposalivation.

2. Interpenetration stage, where polymer chains diffuse into the mucus layer and entanglements occurs (mechanical bond) depending on polymer chain flexibility and length.

3. Consolidation stage is the next step where mechanical and/or chemical bonds create resulting in stronger mucoadhesive joint and prolonged adhesion. Chemical bonds (Yang et al. 2012) comprise strong interactions:

a. ionic bonds, for example in interaction of positively charged chitosan and mucin b. covalent bonds,

and weak secondary interactions:

c. hydrogen bonds, between slightly positively charged hydrogen atom and electronegative oxygen or nitrogen atom

d. van der Waals bonds

e. hydrophobic bonds between non-polar groups that associates together in aqueous medium (Rathbone, Senel, and Pather 2015).

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11 Several theories can explain mucoadhesion depending on the characteristics of the

mucoadhesive material: electronic, adsorption, wetting, diffusion, and fracture theories.

Factors influencing mucoadhesion are related to polymer properties but also to environment characteristics. The presence of the functional groups on the polymer able to form hydrogen or ionic interactions and higher charge density (amount of charged groups on the polymer chain) promotes mucoadhesion. Molecular weight of the polymer influences the process of its interpenetration and diffusion in mucus layer and should be preferably about 100 kDa for linear polymers, while chain flexibility, high molecular weight and conformation (for polymers with branched structure) are more important for chain entanglement. Polymer concentration affects mucoadhesion not only with larger amount of chains available for interaction. Namely, if polymer concentration is hypertonic, water is attracted by osmosis from biological surface to polymer, stimulating mucus cohesion and mucoadhesive joint (Rathbone, Senel, and Pather 2015).

Environmental factors, such as fast turnover of the mucus layer, saliva secretion, food and drink intake and their influence on pH and roughness and hydration of the mucosal

membrane, are also important factors for mucoadhesion and its duration (Rathbone, Senel, and Pather 2015).

2.2.1 Mucoadhesive polymers

Polymers are a very miscellaneous group with broad utilization in different industries

according to their beneficial characteristics. They can be natural, synthetic or semi-synthetic, water soluble or insoluble and charged or uncharged (Rathbone, Senel, and Pather 2015).

In this work semi-synthetic (HM-HEC) and natural polymers (chitosan, LM-pectin and sodium alginate) are used for coating of liposomes to promote their mucoadhesive properties and stability. These polymers are polysaccharides, composed of many monosaccharide units connected by glycosidic bonds. They are non-toxic, biocompatible and biodegradable and therefore favorable for use in pharmaceutical preparations.

Hydrophobically modified hydroxyethyl cellulose (HM-HEC)

HM-HEC is an amphiphilic, non-ionic polysaccharide derived from cellulose. Cellulose is comprise of 1,4-β-D-anhydroglucose repeating monomers with limited bound rotations and chain flexibility (Vadodaria and English 2016). Modifications of the hydroxyl groups in

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cellulose to hydroxyethyl ether results in hydroxyethyl cellulose, HEC with better water solubility, stability and non-toxicity (Vadodaria and English 2016). HEC is used in a wide range of pharmaceutical formulations as a thickener (Meland et al. 2014). With hydrophobic modifications by grafting alkyl chains on the HEC backbone, amphiphilic property of this non-ionic polymer is achieved in the form of HM-HEC (Figure 2.4).

Figure 2.4. Schematic structures of a) HEC and b) HM-HEC (Laschet et al. 2004)

Sodium alginate

Sodium alginate is a water soluble linear polysaccharide that naturally originates from many species of brown seaweed. It is composed of various ratios of (1-4) linked β-D-mannuronic acid (M) and α-L-guluronic acid (G) units (Nie et al. 2008) (Figure 2.5).

Figure 2.5. Schematic structures of sodium-alginate (Pistone et al. 2017)

Alginate has been widely utilized as a gelation agent. Negative charge of alginate comes from the carboxyl groups in the monomer units.

LM-pectin

Pectins are water soluble polysaccharides originated from the cell wall of many plants. They have well-established use in many industries as stabilizer, gelling and thickening agent.

Pectins consists of linear chains of (1–4)-linked α-D-galacturonic acid residues, with side- chains composed of neutral sugars like L-rhamnose. Carboxyl groups of galacturonic acids can be esterified to methoxyl esters or amidated to carboxamide groups, which determines the types of pectin and their characteristics. The degree of esterification (DE) of 50% is the

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13 differentiating border between low methoxy (LM) and high methoxy (HM) pectin and degree of amidation (DA) is used to determine amino pectins (AP) (Thirawong, Kennedy, and Sriamornsak 2008).

Figure 2.6. Schematic structures of pectins (Klemetsrud et al. 2018)

LM-pectin, used in this study is more negatively charged charged and therefore has a more extended chain formation.

Chitosan chloride

Chitosan ([1–4] 2-amino-2-desoxy-β-D-glucan) is a biopolymer, derived from the chitin of crabs and lobsters by N-deacetylation with alkali (Rossi et al. 2001).

Figure 2.6. Schematic structure of chitosan chloride (Pistone et al. 2017)

At the pH 6.8 chitosan is positively charged due to protonation of amino groups and thus proportionate to degree of deacetylation. Lower charge and lower degree of deacetylation favors the more contracted formation of random coils of chitosan in the solution.

2.3 Liposomes

Liposomes are spherical vesicles, formed spontaneously in contact of amphiphilic lipids with water. They are composed of one or more lipid bilayers which are enclosing a water

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compartment. Liposomes were first used to study the cell membrane in 1960s and during period of twenty years were successfully developed into a drug delivery system.

2.3.1 Liposomes in pharmaceutical application

Characteristics of liposomes (size, charge, flexibility) are related to different types of lipids and conditions and methods of the preparation. Lipid components in liposomes are usually neutral phosphatidylcholine of soya or egg origin and cholesterol and can be combined with positive or negative charged lipids to result in stable liposome composition.

There are several reasons liposomes are recognized as convenient carriers for drug delivery.

Liposomes are biocompatible and biodegradable drug carriers, easy to manufacture, versatile in size (30 nm to 10 µm), with possibility of carrying hydrophilic or/and lipophilic drugs in different compartments (Devarajan and Jain 2015). Encapsulation of the drug in the carriers improves stability of the drug in physiological environment (degradation by enzymes, pH, early clearance) and decreases its toxicity and side effects (Hillery, Lloyd, and Swarbrick 2001). In addition, liposomes can potentially carry drugs to the body compartments that are not easily accessible by conventional pharmaceutical forms, for example through crossing the blood-brain barrier. Cationic liposomes have been used as vectors for gen transfer with benefit of being non-infectious comparing to viral carriers (Hillery, Lloyd, and Swarbrick 2001). Targeted drug delivery systems can be achieved with physical, passive or active targeting. Liposomes, that have long circulation life and size 20-200 nm, can passively cross capillaries in tumor tissue with larger openings that is known as enhanced permeability and retention effect (EPR) (Cheng, Lee, and Mitra 2014). Low pH that is present in tissue with inflammation and tumor tissues, and in intracellular organelles, lysosomes, can be used for passive delivery of the drugs encapsulated in pH sensitive liposomes that will be released in acid environment. Active drug targeting is related to binding carriers to ligands, receptors or monoclonal antibodies with high affinity to the targeted cells or cell compartments. In addition to minimizing toxic effects of therapy achieved by encapsulation of cytotoxic drugs in liposomes, functionalization of liposomes with folate results in higher affinity to cancer cells with expressed folate receptor on their surfaces (Devarajan and Jain 2015). Glycoprotein asialofetuin have been used as ligand on the liposomes to reach the targeted receptors on hepatocytes (Hillery, Lloyd, and Swarbrick 2001).

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15 Disadvantages of liposomes as drug delivery systems are limited drug loading capacity, short shelf life of the formulation and their instability towards the immune system. Negatively charged liposomes, are being recognized rapidly in the circulation by reticuloendothelial system and removed by the process of phagocytosis. This can be prevailed with steric stabilization of liposomes by grafting with polyethylene glycol (well known as “stealth”

liposomes). This sterically prevents opsonisation and consequently phagocytosis of liposomes (Hillery, Lloyd, and Swarbrick 2001). On the other hand, phagocytosis of liposomes by the macrophages invaded by pathogen microorganisms inside them could be used to target Micobacterium tuberculosis.

2.3.2 Polymer-coated liposomes

Polymers are usually flexible molecules where bonds rotate into the most stable positions and result in different conformations in solutions. Conformations of the polymer in solution depend on the intramolecular interaction between the main and side chains, but also on the interaction with molecules of the dispersant, pH and ionic strength. Random coil

conformation of the polymer can therefore be stretched in different grade. For example, polymers without or with low charge are more likely to have more contracted, spherical conformation, like HM-HEC and chitosan. Highly charged polymers have extended, stretched, almost rod-like conformation (Bajpai 1997). Alginate and LM-pectin with many negative carboxyl groups along the chain will probably have more extended formation at pH 6.8.

Polymers can be adsorbed on the surface with the whole chain, taking “train” formation, or only with some parts, making “train”, “loops” and “tails” (Figure 2.7). Coating with a neutral polymer results usually in the train, loop and tail conformation. Coating with an electrically charged polymer conformation will depend on pH, ionic strength, charge of the surface etc.

When poor solvent is used or with increased ionic strength the amount of adsorbed polymer increases and it becomes dependent on the polymer molecular weight (Bajpai 1997).

Figure 2.7. Adsorption of the polymer chain in the formation of “trains”, “loops” and “tails”

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Liposomes can come close to each other and if attraction forces can overcome repulsion forces they will aggregate or fuse. Coating of liposomes is used to increase their stability toward aggregation by steric or/and electrostatic repulsions. A mucopolysaccharide layer attached to the liposome surface will also promote their adhesion to the mucosal membrane.

Coating increase liposome size in different grade according to liposome and polymer type and conditions (pH, type of solvent). This will influence the polymer conformation and amount of polymer adhered to liposomes and consequently size increase and compactness of the coating layer.

2.3.3 Liposomes with local effect on mucosal membranes

Formulation of the products with local effect in the oral cavity has many challenges, such as stability issues and systemic absorption. Dilution of a therapeutic agent by constantly

secretions and swallowing limits the time of its contact with the site of action and effectivity.

Therefore, the prolonged residence time is the desired characteristic of preparations with local effect in the oral cavity.

Liposomes could be platform for overcoming those obstacles. The therapeutic agent could be encapsulated and protected from degradation and rapid clearance while potential side-effects or irritation could be decreased. Coating liposomes with mucoadhesive biocompatible, biodegradable and non-toxic polymer can prolong contact time at the place of action. In addition, liposome surface can be grafted with ligands with high affinity to specific cells or tissues. Adhering mucoadhesive liposomes on the specific structures in the oral cavity, for example hydroxyapatite or mucin has been extensively studied in the last years (Nguyen, Hiorth, et al. 2011; Pistone et al. 2017; Klemetsrud et al. 2018). This also opens possibility for drug delivery systems that could selectively bound to the salivary glands to stimulate their secretion. This could decrease the side-effects of the medications used systemically.

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2.4 Glycerol

Glycerol is small trihydroxyl sugar alcohol. It is viscous liquid, with 60 % sweetness of sucrose. It is less polar than water and used as a solvent in water mixtures. It creates intramolecular hydrogen bonds in the water solutions.

Figure 2.8. Schematic structure of glycerol (Mario Pagliaro)

Glycerol is broadly utilized as a humectant and moisturizer in pharmaceutical and cosmetic products. It also provides lubrication, smoothness and texture when added to the

pharmaceutical preparations (Mario Pagliaro). In the commercial OTC product Orajel, dry mouth moisturizing gel for relieving discomfort on the irritated mucous membrane, the glycerol concentration is 18 %. Glycerol has antimicrobial properties in concentration over 20 %.

2.5 Particle size and the zeta potential

Particle size

Characterization of the liposomes and estimation of their physical stability are performed by measuring the hydrodynamic diameter. It corresponds to the radius of a sphere that would diffuse in the solution in the same way as measured particle. The hydrodynamic diameter is measured by dynamic light scattering (DLS). Particles are moving by Brownian motion and scatter the light from the laser. Larger particles scatter the light more and can mask the small.

The rate of the intensity fluctuations of the scattered light is measured so that Zetasizer software obtain correlation function that is fitted with a general purpose fitting model. The Stokes-Einstein equation is used for calculation and results expressed as zeta-average and polydispersity index.

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Stoks-Einstein equation:

dH = hydrodynamic diameter k =Boltzmann’s constant T = absolute temperature µ = viscosity

D = translational diffusion coefficient

The polydispersity index shows the broadness of the size distribution and it can be between 0 and 1, while values above 0.3 show that the system is probably poly-disperse.

The zeta potential

Measuring of the zeta potential is based on laser Doppler electrophoresis. The zeta potential is a potential on the slipping plane of the particle, ie. on the surface of the electrical double layer. Generally, particles with absolute value of the zeta potential higher than 30 mV are considered to have enough repulsive energy to remain stable.

The zeta potential is calculated from measured mobility during electrophoresis using Smoluchowski approximation for Henry equation:

where UE = electrophoretic mobility, z = zeta potential, ε = dielectric constant, µ = viscosity and f(ka) = Henry’s function

The Smoluchowski approximation is used for polar media and moderate electrolyte concentration in which case f(ka) is 1.5.

2.6 Rheological synergism

Rheology comprises the study of the flow and deformation of matter under applied forces using rheometer. In this study a cone and plate geometry is used for steady shear

measurements and viscosity is measured at different shear rates. When the viscosity is independent of the shear rate system it is a Newtonian system. Dilute polymer solutions in this study exhibited shear-thinning behavior where the viscosity decreases with increasing

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19 shear rate. This is explained by disrupting the intermolecular connections and entanglements between the chains or alignment of the chains in the direction of flow seen in diluted polymer solutions.

Figure 2.9. Schematic illustration of the type of interactions that can occur in the mixtures of protein and polymer solutions

Rheological synergism is the principle that reveals interactions between the mixed

components when the viscosity of the mixture deviates from expected additive viscosity of components with the same concentration.

Δƞ = ƞ^mix - (ƞ^m+ ƞ^x)

ƞ^mix – viscosity of the component (x) and mucin mixture ƞ^m– viscosity of the mucin solution

ƞ^x – viscosity of the component (x) solution Δƞ – deviation from additive viscosity

Possible interactions in the mixtures of protein and polysaccharide solutions are shown in Figure 2.9. Both increase and decrease in viscosity of the mixture can indicate interaction.

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20

3 Materials and instruments

3.1 Materials

3.1.1 Lipids and polysaccharides

Lipids Abbreviation Molecular weight (g/mol)

K - number Producer

L-α-phosphatidylcholine

(Soya) SPC 775.037 792044-01/907

Lipoid GmbH, Germany 1,2-dioleoyl-3-

trimethylammonium-propane (chloride salt)

DOTAP 698.542 890890P

Avanti Polar Lipids, USA L-α-phosphatidylglycerol

(Egg, Chicken) (sodium salt) Egg PG 782.284 EPG-202

Avanti Polar Lipids, USA

Polysaccharides Abbreviation Characteristics K - number Producer Hydrophobically

modified

hydroxyethylcellulose (Natrosol Plus 330 CS)

HM-HEC Mw=300 kDa NT1E9478 Ashland, Wilmington, USA

Sodium alginate (Protanal LF 10/60)

Alg Mw=147 kDaâ G=65-75%â M=25-35%â

GQ0907101 FMC BioPolymer, USA

Low methoxyl pectin (Genu pectin

LM-12 CG-Z)

LM-pec Mw=76 kDa^b DE=35%^b Degree of methyl

esterification

GR32494 CP Kelco

Chitosan chloride (Protasan

UP CL-213)

Chi Mw=307 kDa^c DDA=83%^a

Degree of deacetylation

BP-0805-04 Novamatrix, FMC Biopolymer, Norway

a (Pistone et al. 2015) b(Nguyen, Alund, et al. 2011)

c(Jonassen, Kjøniksen, and Hiorth 2012)

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21 3.1.2 Other chemicals

Other chemicals Molecular weight (g/mol

K - number Producer

Chloroform, CHCl3 119.38 K48213145636 Merck, Germany

Sodium dihydrogen phosphate monohydrate, NaH2PO4 x H2O

137.99 A942646 706 Merck, Germany Sodium dihydrogen phosphate

dihydrate, NaH2PO4 x 2H2O

156.01 04G230009 VWR International, Leuven, Belgium Disodium hydrogen phosphate

dihydrate,Na2HPO4 x 2H2O

177.99 K25001880 814 Merck, Germany

85% glycerol 16B136/3,

15B039/4

A-PRO, Den norske Eterfabrikk, Norway

Dextran from Leuconostoc mesenteroides

~150 000 SLBR0333V Sigma Life Science, USA Mucin from bovine submaxillary

glands (BSM), Type I-S

SLBS0651V Sigma-Aldrich, St. Louis, USA

3.1.3 Stock solutions of lipids

Soya-PC 10 mg/ml in chloroform

Desired amount of Soya-PC, equilibrated in room temperature for about 30 minutes, was weighted on the analytical balance in the glass weighing boat. The content was flushed with chloroform from the weighing boat to an appropriate volumetric flask until the desired volume was reached. The solution was mixed, transferred to injection vial, flushed with nitrogen gas, sealedand stored in the freezer (-20°C).

DOTAP 2 mg/ml in chloroform

The solution was already prepared in the lab by previous user.

Egg-PG 2 mg/ml in chloroform:methanol = 2 : 1

The whole content of an ampule with Egg-PG, equilibrated in room temperature for about 30 minutes, was transferred to an appropriate volumetric flask by flushing it with

chloroform:methanol=2:1 mixture until the desired volume was reached. The solution was

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22

mixed, transferred to injection vial, flushed with nitrogen gas, sealedand stored in the freezer (-20°C).

3.2 Solutions

Phosphate buffer, 5 mM pH 6,8 (PBS)

The Solution I and II were first made in concentration 5mM.

The Solution I was prepared by weighing 690 mg sodium dihydrogen phosphate monohydrate (NaH2PO4 x H2O) (alternatively 780 mg sodium dihydrogen phosphate dihydrate (NaH2PO4 x 2H2O)) in a plastic weighing boat on the analytical balance. It was transferred to a 1 L

volumetric flask and dissolved in MilliQ water (18,2 MΩcm) ad 1 L.

Similarly, the solution II was prepared by weighing 890 mg Na2HPO4 x 2H2O and dissolving it to 1 L. Solution I and II were mixed in a 2:1 proportion. The pH was checked on pH-meter and eventually adjusted to pH 6.80±0.03 with the parts of the solutions.

The solution was filtered through 200 nm polycarbonate membrane into the glass bottle with plastic cover and stored in the refrigerator.

Glycerol solutions in Phosphate buffer, 5 mM pH 6,8

Phosphate buffer 5 mM pH 6.8 was also used to make 8.5%, 17%, and 25.5% glycerol solutions by weighing calculated amount of 85% glycerol in the Erlenmeyer flask on the balance and diluting it to a determined weight.

All solutions were filtered through 200 nm polycarbonate membrane into the glass bottle with plastic cover and stored in the refrigerator.

Polysaccharide solutions 0.1 wt% and 0.16 wt%

A certain amount of polysaccharide was weighed in a beaker on the analytical balance and diluted with the correct amount of the corresponding hydration medium to obtain the correct concentration. The mixture was set on the magnetic stirring (level 4) covered with parafilm to dissolve overnight.

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23 The solution was filtrated through 5 μm filter the following day and used for coating of

liposomes (0.1 wt%) and 0.16 wt% for making samples for rheological measurements.

Mucin 0.5 wt% in 5 mM phosphate buffer, pH 6,8 / 17 wt% glycerol

A certain amount of mucin was weighed in a beaker on the analytical balance and diluted with the correct amount of the corresponding hydration medium to obtain the correct

concentration. The mixture was set on the magnetic stirring (level 4) covered with parafilm to dissolve overnight.

The solution was filtrated through 5 μm filter the following day and used for making samples for rheological measurements.

3.3 Instruments and equipment

3.3.1 Preparation of liposomes

Instrument/equipment Model Producer

Rotary evaporator Rotavapor Hei-VAP Advantage Heidolph, Germany Associated vacuum pump PC 511 Vacuubrand, Germany Freeze-dryer Christ-Alpha 2-4 LOC -1M Martin Christ, Germany

Associated vacuum pump RV 8 Edwards High Vacuum International, England Extruder Lipex Thermobarrel Extruder, 10 ml Lipex Biomembranes Inc,

Canada

3.3.2 Measuring the particle size and the zeta potential

Particle size and zeta potential Zetasizer Nano - ZS Malvern Instruments, England

Dip cell Dip Cell: MAL1140660

Disposable Folded Capillary Cell

Malvern Instruments, England

Standard Zeta potential transfer standard - 42mV+/-4.2mV

Malvern Instruments, England

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24

3.3.3 Measuring the viscosity

Rotational rheometer Paar-Physica MCR 301 Anton Paar, Austria

Spindel CP75-1-SN23951

Associated water bath F 12 Julabo, Germany

3.3.4 Miscellaneous

Analytical Balance AG 204 Delta Range Mettler Toledo, USA

Balance PB30002 Delta Range Mettler Toledo, USA

Finnpipette, 20-200 μm J59016 Thermo Labsystems Inc, USA Finnpipette, 200-1000 μm U27916, CH 08819 Thermo Electron Corporation, USA Finnpipette, 1-5 ml U31616 Thermo Electron Corporation, USA Dispensette, 1-10 ml Organic-Easy Calibration BRAND GMBH + CO KG,

Germany

pH-meter SevenCompact pH/Ion

meter S220 Mettler Toledo, USA/Switzerland Calibration solutions Buffer solution pH 4

Buffer solution pH 7

Prolabo, France

Magnetic stirrer RO 10power IKA-Werke GmbH & Co, Germany

Filter Millex SV 5 μm,

R2JA18358K Merck Millipore, USA

Polycarbonat membrane for filtration of hydration medium

Nuclepore Track-Etch Membrane,

Whatman, 0,2 μm, 47 mm, 7005367

GE Healthcare, England

Polycarbonat membrane for extrudation

Nuclepore Track-Etch Membrane,

Whatman, 0,2 μm, 7008689

Supportive strain disk Whatman Drain disc PE 25mm,7006171

Peristaltic pump 520S Watson Marlow, USA

Disposable cuvettes Polystyrene cuvettes Sarstedt, Germany Dialysis membrane

Spectra/Por 6, Pre-wetted RC tubing (MWCO 8 kD, diameter 32 mm), 132586

Spectrum Laboratories, Inc., CA, USA

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25

4 Methods

4.1 Preparation of liposomes

4.1.1 Preparation of the lipid film

Liposomes were made in series of 10 ml, in concentration 3 mM for stability investigations, and again later in 10 mM (further diluted to 3 mM for larger volume) for rheological

measurements.

Desired quantities of stock solutions of lipids (Chapter 3.1.3) and chloroform were pipetted into a 250 ml round-bottom flask and mixed. The flask was attached to a Rotavapor to remove the organic solvent while rotating at 90 rpm immersed in a water bath with a temperature of 40°C. The pressure was gently reduced from atmospheric pressure to 200 mbar while all the solvent was evaporated and thereafter reduced to about 60 - 65 mbar for additional 20 minutes. The obtained lipid film was left on the freeze-dryer overnight for removing of eventual remains of the solvent.

4.1.2 Hydration of the lipid film

The next day the lipid film was hydrated with 10 ml of the corresponding hydration medium (phosphate buffer 5mM pH 6.8 or 8.5%/17%/25.5% glycerol in phosphate buffer 5mM pH 6.8), flushed with nitrogen gas to protect the unsaturated fatty acids and placed on rotary stirring at 90 rpm for 10 minutes and then stored at room temperature protected from light for the next 2 hours while it was mixed gently every 30 minutes. Afterward, it was stored in the refrigerator.

4.1.3 Extrusion

The following day the room temperated dispersion was extruded in a Lipex extruder according to standard procedure, through to 200 nm polycarbonate membranes to achieve formation of unilamellar liposomes. Extrusion was conducted 10 times at room temperature

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26

into the particle free injection vial and finally flushed with nitrogen gas and stored in the refrigerator.

4.2 Coating of liposomes with polymer

Liposomes in concentration 3 mM, were coated with a 0.1wt% polymer solution (containing the same glycerol concentration and the same pH as the liposomes) filtered through the 5 μm filter. Liposome and polymer solutions were mixed in a 1:4 ratio.

20 ml vials and 12 mm magnets were rinsed with MilliQ water and the corresponding solution to remove eventual dust and filled with polymer solution (4 ml or 8 ml). The polymer solution was set on stirring with a magnet (level 7) causing swirl in the solution, while the dispersion of the liposomes (1 ml or 2 ml, respectively) from Eppendorf tube was added using a

peristaltic pump with 20 revolutions per minute. Stirring was continued for additional 5 minutes after the whole dispersion of liposomes was added. Subsequently, magnets were carefully removed from the vials, dispersion flushed with nitrogen gas, sealed and stored in refrigerator. Characterization of the liposomes was performed the day after extrusion and the day after coating.

4.3 Measuring the particle size and the zeta potential

The particle size and the zeta potential were measured on the Zetasizer Nano ZS. Polystyren cuvettes, packed without dust, were inspected and filled with 500 µl solution, then 500 µl coated liposome dispersion was added. The volume for the uncoated liposomes was 100 µl, added to 900 µl solution (Table 4.1).

Table 4.1. Preparation of the samples for measuring the particle size and the zeta potential Uncoated liposomes,

0.3 mM

Uncoated liposomes 3 mM Solution (0/8.5/17/25.5%

glycerol in 5 mM PBS pH 6.8)

100 µl 900 µl

Coated liposomes, 0.3 mM

Coated liposomes 0.6 mM Solution (0/8.5/17/25.5%

glycerol in 5 mM PBS pH 6.8)

500 µl 500 µl

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27 The samples were gently mixed and the sample controlled visually for presence of particles.

Dilution was made in such a manner that the final concentration of the liposomes was 0.3 mM. All the results for particle size represented the mean value of three measurements on the same sample. The settings used for measuring the particle size are shown in Table 4.2. The Z- average was chosen as the correct estimate of the size of the hydrodynamic diameter of the liposomes and the polymer-coated liposomes.

Table 4.2. Settings used for measuring the particle size Dispersant, viscosity,

refractive index Water, 0.8872 cP, 1.330

8.5% glycerol, 1.1213 cP, 1.340 17% glycerol, 1.4334 cP, 1.350 25.5% glycerol, 1.8720 cP, 1.360

Approximation Mark-Houwink parameter

Temperature 25˚C

Equilibration time 300 seconds

Cell type Disposable cuvettes

Measurement angle 173˚C

Measurement duration Automatic Number of measurements 3

Positioning method Seek for optimum position Attenuator Automatic attenuation selection

Analyse model General options

The zeta potential of a standard was measured first and considered satisfying if within 42 +/- 4.2 mV. After the particle size of the sample was measured, the same sample was used for measuring the zeta potential by carefully dipping the dip cell lit tilted into the solution to avoid contaminating the sample with particles or air bubbles. Alternatively, the sample was transferred by injection into the disposable folded capillary cell for measuring the zeta potential. All the results for the zeta potential represented mean value of five measurements on the same sample and the settings used are shown in Table 4.3.

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28

Table 4.3. Settings used for measuring the zeta potential

Material, refractive index Polystyrene latex, 1.590 Dispersant, dielectric constant Water, 78.5

8.5% glycerol, 78.5 17% glycerol, 78.5 25.5% glycerol, 78.5

Approximation Smoluchowski

Temperature 25˚C

Equilibration time 300 seconds

Dip cell Dip cell for zeta potential

Disposable folded capillary cell

Measurement duration Automatic, 10-100

Number of measurements 5

Attenuator Automatic

Voltage Automatic

Analyse model Auto mode

4.4 Investigating the interaction of the formulations with mucin

The interaction of the liposomes with mucin was measured based on the principle of

rheological synergism and the calculation of discrepancies between the additive viscosity of the single components compared to viscosity of the mixture. Dispersions of coated liposomes were up-concentrated to insure measurable interaction with mucin.

Up-concentration of liposome dispersions

The coated liposomes (45 ml or 30 ml) were dialyzed against a dextran solution 200 g/L in 5 mM phosphate buffer pH 6.8 with or without 17% glycerol. 12-13 cm of a dialysis membrane with Mw cut off 8 kDa was used.

There were tried with up-concentration 3 times (0.6 mM to 1.8 mM) and up-concentration 2 times (0.6 mM to 1.2 mM) where the final concentration was calculated from weigh of the dialyzed dispersion in the tube before and after the up-concentration. Table 4.4 shows time needed for up-concentration of the different samples.

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29 Table 4.4. Up-concentration of coated liposomes dispersions

Up-concentration of

liposomes From 0.6 mM to 1.8 mM From 0.6 mM to 1.2 mM

Time needed 24 – 36 h 12 - 24 h

Sample volume change 45 ml to 15 ml 30 ml to 15 ml Preparation of the samples for rheology measurements

Mucin solution 0.5 wt% and polymer solution 0.16 wt% were set on stirring the day before measuring while the coated liposome dispersion was up-concentrating. Polymers and mucin solution were filtrated through a 5 μm filter.

Mixing the polymers, uncoated or coated liposomes with the mucin solution was performed by adding the mucin solution into the vial with samples (1:1) in dropwise manner (3.5 ml/min) with magnet stirring making swirl in the solution (level 7), followed by additional 5 minutes stirring. Mixtures with mucin were stored at room temperature protected from light and rheological measurements were conducted the next day.

Concentrations of liposomes in all samples were the same 0.6 mM (coated and uncoated) and concentration of polymer solution in sample was assumed to be equal to concentration of the polymer on the coated liposomes 0.08 wt%.

Measurements of the viscosity

The viscosity was measured on the rotational rheometer at a controlled shear rate. A spindle with cone-and-plate geometry, cone angle of 1° and diameter 75 mm was used. Before measurements, air check and motor adjustment, were performed and the viscosity of distilled water at 37°C was measured. Samples were measured at 37°C with equilibration time of 5 minutes at shear rates from 0.1 – 100 1/s. Measuring position was 0.151 mm and sufficit of the samples were “trimmed” at trimming position 0.161 mm to assure equal quantity of all samples. Three samples were measured to obtain the mean value.

Afterwards, differences between measured viscosity of the mixtures with mucin and theoretical additive viscosity of the components were calculated (equation 1). This was expressed as the percentage deviation with respect to theoretical additive viscosity of mixture without interaction (equation 2), and analyzed.

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30

(Equation 1) Δƞ = ƞ^mix - (ƞ^m+ ƞ^x)

ƞ^mix – viscosity of the component (x) and mucin mixture ƞ^m– viscosity of the mucin solution

ƞ^x – viscosity of the component (x) solution Δƞ – deviation from additive viscosity

(Equation 2) % Δƞ = Δƞ / (ƞm+ ƞx) * 100

% Δƞ – percentage deviation from additive viscosity

4.5 Measurements of pH

The pH-meter was first calibrated with buffer solutions pH 4 and pH 7. The electrode were rinsed with distilled water and dried before soaked into the sample. pH was measured at room temperature.

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5 Experimental design

5.1 Preparation of liposomes with encapsulated glycerol

Liposomes were made in order to test their ability to encapsulate and be stable in a glycerol solution. Neutral, positive and negative liposomes were made and coated with polymer solutions of opposite charge. The HM-HEC coated liposomes and the alginate coated liposomes were made in several variants: with 0%, 8.5%, 17% and 25.5% glycerol used as hydration medium. Those concentrations actually represented solutions with 10, 20 and 30%

of the 85% glycerol solution, commonly used in the laboratory. Table 5.1. shows the list over the prepared formulations.

Table 5.1. Overview of the prepared formulations investigating the encapsulation of glycerol

Formulation Hydration medium Polymer

Main lipid

Charged Lipid

5mM phosphate buffer pH 6.8 (PBS)

8.5%

glycerol in PBS

17%

glycerol in PBS

25.5%

glycerol in PBS

Polymer 0.1%

dissolved in solution corresponding hydration medium

SPC x HM-HEC

SPC 10% DOTAP x Alginate

SPC 10% DOTAP x LM-pectin

SPC 10% Egg PG x Chitosan

The possibility of encapsulating was investigated by measuring particle size and zeta potential of both uncoated and coated liposomes, with and without glycerol.

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32

5.2 Investigating the interaction of the formulations with mucin

The residence time in the oral cavity of the formulations was estimated by measuring the mucoadhesive properties of the liposomes in a test system. A mucin solution from bovine submaxillary glands was used as a model for testing, and the interaction was tested based on the principle of rheological synergism i.e. viscosity measured before and after mixing the components.

Dispersions of coated liposomes were up-concentrated to 1.2 mM before mixing with mucin in order to detect measurable interaction with mucin.

The liposomes with 17% glycerol were chosen, and series without glycerol and series with 17% glycerol solution were included in the testing. The viscosity was measured before and after mixing with mucin for all polymer solutions, uncoated liposomes and coated liposomes.

For example, when HM-HEC coated liposomes in phosphate buffer were tested, 7 samples were prepared: 4 samples of the single components (mucin solution, HM-HEC solution, uncoated liposomes dispersion and HM-HEC coated liposomes dispersion) and 3 samples where corresponding components were mixed with mucin. An overview of the tested samples for HM-HEC coated liposomes in PBS is shown in Table 5.2. The same 7 combinations were also tested when 17% glycerol was used as the hydration medium for liposomes and the solvent.

Table 5.2. Overview of the samples tested for interaction with mucin (example for HM-HEC coated liposomes in PBS medium)

HM-HEC coated liposomes in PBS

Sample Concentration in PBS

Mucin 0.25 wt%

HM-HEC 0.08 wt%

Uncoated liposomes 0.6 mM

HM-HEC coated liposomes 0.6 mM

HM-HEC + Mucin

0.08 wt%

0.25 wt%

Uncoated liposomes + Mucin

0.6 mM 0.25 wt%

HM-HEC coated liposomes + Mucin

0.6 mM 0.25 wt%

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33 The same procedure was repeated with the alginate-, LM-pectin- and chitosan coated

liposomes, without and with 17% glycerol, totally 8 groups (Table 5.3) with 7 samples in each group.

Table 5.3. 8 groups tested for interaction with mucin

PBS medium 17% glycerol medium

HM-HEC coated liposomes HM-HEC coated liposomes with 17 % glycerol Alginate coated liposomes Alginate coated liposomes with 17 % glycerol LM-pectin coated liposomes LM-pectin coated liposomes with 17 % glycerol Chitosan coated liposomes Chitosan coated liposomes with 17 % glycerol

Alginate coated liposomes were tested in concentration 0.6 mM and 0.3 mM (not up- concentrated samples, since up-concentration resulted in aggregation)

The concentrations of all the liposomes were 0.6 mM and the concentration of the polymer solutions were 0.08 wt%, approximately the same as the concentration of the polymer on the coated liposomes.

5.3 Stability measurements

Testing of the in vitro stability was performed for all of the prepared formulations (Table 5.1) by measuring the particle size and zeta potential one day after preparation and at several time points during the storage in refrigerator.

Samples for measurements were prepared in such a manner that concentration of the

liposomes was 0.3 mM (Table 4.1). Solution used for dilution was the same as the one used as hydration medium.

Polymer-coated liposomes with potential of hydrating dry mucous membranes

Polymer-coated liposomes with potential of hydrating dry mucous

membranes

Ljubica Mihailovic

Master Thesis in Pharmaceutics 45 study points

Section for Pharmaceutics and Social Pharmacy Department of Pharmacy

The Faculty of Mathematics and Natural Sciences

UNIVERSITY OF OSLO

Polymer-coated liposomes with potential of hydrating dry mucous

membranes

Ljubica Mihailovic

Supervisors:

Marianne Hiorth Gro Smistad Joseph Azumah

Section for Pharmaceutics and Social Pharmacy Department of Pharmacy

The Faculty of Mathematics and Natural Sciences

UNIVERSITY OF OSLO

Acknowledgements

Abstract

Table of contents

1 Introduction

1.1 Background

1.2 Aim of the study

1.3 Abbreviations

2 Theory

2.1 Oral cavity

2.2 Mucoadhesion

2.3 Liposomes

2.4 Glycerol

2.5 Particle size and the zeta potential

2.6 Rheological synergism

3 Materials and instruments

3.1 Materials

3.2 Solutions

3.3 Instruments and equipment

4 Methods

4.1 Preparation of liposomes

4.2 Coating of liposomes with polymer

4.3 Measuring the particle size and the zeta potential

4.4 Investigating the interaction of the formulations with mucin

4.5 Measurements of pH

5 Experimental design

5.1 Preparation of liposomes with encapsulated glycerol

5.2 Investigating the interaction of the formulations with mucin

5.3 Stability measurements