Testing of adhesive properties of plasticized polyesters using rotational rheometer. Diploma Thesis

Transcription

1 Testing of adhesive properties of plasticized polyesters using rotational rheometer Diploma Thesis Hradec Králové 2017 Chiazor Ugo Ogadah 1

2 Statement of originality I declare that this diploma thesis is my own personal work and that I worked on it on my own. All literature and other resources that I used are listed in the references list and are properly cited. Date: Chiazor Ugo Ogadah 2

3 Acknowledgments Foremost, I would like to express my deepest appreciation to my supervisor PharmDr. Eva Šnejdrová, Ph.D. for her guidance throughout my thesis. Her patience and immense knowledge made this project a success. To my friends, thank you for your prayers and kind words. It gave me the strength to persevere. Finally, a special gratitude is extended to my family for the endless support and encouragement during this academic journey. I will forever be grateful for your love. 3

4 1 Table of contents 1 Table of contents Abstract Aim of the diploma thesis Introduction Theoretical section Polymers in Pharmaceutical technology Polymers from natural sources Semi synthetic polymers Synthetic biodegradable polymers Plasticizers in Pharmaceutical Technology Mechanism of plasticization Classification of plasticizers Role of plasticizers in Pharmaceutics Plasticized drug delivery systems Bioadhesion The bioadhesive materials Mechanism of bioadhesion Application of bioadhesives in pharmacy Methods for testing bioadhesion Experimental section

5 6.1 Materials Instrument Methods Plasticization of polyesters Starting up the rheometer Test for flow properties Test for adhesive properties Results Flow properties Adhesive properties Discussion Flow properties of plasticized polyesters Adhesive properties of plasticized polyesters Conclusions Literature

6 2 Abstract Title of thesis: Testing of adhesive properties of plasticized polyesters using rotational rheometer Author: Ogadah Chiazor Ugo Department: Pharmaceutical Technology Supervisor: PharmDr. Eva Šnejdrová, Ph.D. In this thesis, the rheological and adhesive properties of three polyesters intended for use as drug carriers in polymeric drug delivery systems were studied. The theoretical section summarizes the polymers used as drug carriers. Plasticizers, mechanism of action, types and uses and finally bioadhesion mechanism, application of the bioadhesive preparations, and methods of testing bioadhesion are reviewed. In the experimental section, ethyl salicylate, ethyl pyruvate and triethyl citrate were selected as the plasticizers to decrease the viscosity of the polyesters for easy processability, and setting of the optimal adhesive properties. Flow properties of the plasticized systems was measured with a rotational rheometer at temperature 37 o C and a shear rate range from 0.10 to 100 s -1. The analysis of the viscosity curves revealed that the resulting plasticized systems are mostly Newtonian. All tested plasticizers decrease the viscosity of the systems. The most effective is ethyl pyruvate. The adhesive properties were determined by the pull away test on the rotational rheometer, and evaluated as the peak in negative normal force, the area under the force-time curve and the time taken for the peak force to decay by 90 %. The peak in negative normal force is recommended as the best parameter for comparing the adhesive properties of the systems. The influence of the plasticizer type on the adhesive properties was evaluated. The highest adhesiveness was found using triethyl citrate, followed by ethyl salicylate, and ethyl pyruvate. Within the range of measured values, the viscosity values of plasticized polyesters correspond to their adhesive properties. Keywords: branched polyesters, plasticizer, flow properties, adhesive properties 6

7 3 Aim of the diploma thesis The overall aim of this thesis is the testing of the rheological and adhesive properties of plasticized star-like polyesters of glycolic and lactic acids, and selection of the plasticizer type and concentration suitable for formulation and application of polymeric drug delivery systems of various application forms. This study includes: 1. Plasticizing of star-like polyesters of glycolic and lactic acids with plasticizers type and concentration relevant in the field of pharmacy and medicine. 2. Evaluation of the method suitable for testing of adhesive properties using rotational rheometer. 3. Testing of the flow and adhesive properties of plasticized polyesters. 4. Evaluation of the tests with parameters suitable for characterization and comparison of the polymeric systems. 5. Studying the influence of the type and concentration of plasticizer on flow and adhesive properties of plasticized polyesters. 7

8 4 Introduction Methods of optimizing drug delivery are constantly evolving. The concept of bioadhesion started to be applied to drug delivery systems in the 1980s and the interest has grown over the past decades 1. A bioadhesive polymer maintains an intimate and prolonged contact with the biological surface 2. As a result, increase in bioavailability can be achieved 3. Several polymers which are available as drug delivery carriers have found their application in the formulation of bioadhesive drug delivery systems 1. The polyesters of D, L-lactic acid and glycolic acid are employed in the current study. They fulfil all the criteria for polymers employed in drug delivery, which is biodegradability, biocompatibility and low toxicity 4. However, the amorphous nature of these polyesters makes it difficult to manipulate with the materials. The viscosity must be decreased for better processability. A satisfactory approach for this is by plasticization. It has been demonstrated that the plasticized polyesters have better bioadhesive parameters than the commonly used bioadhesive polymers 5. Since different types and/or concentrations of plasticizers will influence the characteristic of the polyesters differently, it is therefore necessary to investigate the efficiency of different plasticizers in achieving the desirable properties of the candidate polyesters for possible application in various formulations. The work is a continuation of studies on characterization of star-like polyesters for drug delivery made previously 6,7. 8

9 5 Theoretical section 5.1 Polymers in Pharmaceutical technology Polymers have a long-standing significance in pharmaceutical technology. They have been employed for various purposes such as auxiliary substances for conventional dosage forms and for packaging materials. The peculiarities of polymers are being exploited in drug delivery technologies. They are employed as drug carriers for the design of various drug delivery systems which aim at controlling drug release characteristics, prolonging residence time of the drug, reducing drug toxicity, increasing absorption and bioavailability 4. Several natural, semi-synthetic and synthetic polymers have been used in formulating drug delivery systems. The most crucial criteria in choosing polymers for this purpose is biodegradability. This is because the material can be broken down into smaller non-toxic molecules which can easily be eliminated by the body after the drug release 8. In addition, the materials are required to be biocompatible i.e. they must not evoke any immunogenic responses in the organism 4. The subsequent paragraphs will give a brief overview of some of the commonly encountered polymers as well as some of their applications and prospects in modern drug delivery Polymers from natural sources The naturally occurring biodegradable polymers discussed in this review have been broadly classified as polysaccharide based and protein based polymers. Polysaccharide based polymers Alginate is a polysaccharide derived from seaweed. It is not enzymatically degradable in mammals 9. However, it is extensively investigated as drug delivery vehicles due to its biocompatibility and non-immunogenicity. The gel forming properties of alginates create the possibility to encapsulate various substances without altering their biological activity 4. There are on-going investigations pertaining the potential use of alginate for encapsulation of beta cell islet for the therapy of type 1 diabetes 10. 9

10 Chitosan is a cationic polysaccharide derived from chitin (a constituent of the shell of sea crustaceans) by deacetylation. Its wide use in drug delivery is due to its high biodegradability and biocompatibility. In addition, it is non-toxic and shows good bioadhesive properties 11. Chitosan and its derivatives have been employed successfully in the formulation of various mucoadhesive drug delivery systems. For instance, Sandri et al. reported a successful buccal delivery of hydrophilic macromolecule using trimethylated chitosan 12. Dextran is a polysaccharide derived from bacteria. It has been employed over the last five decades primarily as a plasma volume expander. However, owing to its favourable biodegradable properties among other qualities, it is actively being investigated for sustained delivery of drugs and proteins 13. Protein based polymers Collagen is a non-toxic, biodegradable natural polymer found in skin, bones, cartilages of vertebrates 4. Collagenous drug delivery systems have been investigated for local delivery of low molecular weight drugs. A delivery vehicle comprising of collagen and gentamicin (Sulmycin -Implant, Collatamp -G) is available worldwide for the prolonged local delivery of the antibiotic with very minimal systemic exposure. However, the major drawback for widespread application of collagen-based materials is due to immunogenic responses and high cost of purification 9. Gelatin is obtained by partial hydrolysis of collagen. Due to its high water solubility and poor mechanical properties, it is often cross-linked with other materials 4. It is extensively investigated as drug delivery carriers for various drug classes due to its versatility and long history of safe use in many medical and pharmaceutical applications (as a plasma expander) 14. Albumin is a protein found in the human plasma. Its biodegradability, biocompatibility and non-toxicity makes it attractive for use in drug delivery. Albumin microspheres has been reported to be a suitable drug carrier for targeted drug delivery in cancer therapy 15. This can be attributed to the fact that cancer cells utilize albumin as a source of nitrogen and energy. The albumin microspheres are preferentially taken up by the tumour cells, and the drugs are delivered to the specific site of action. Albumin-based drug targeting is a therapeutic approach in the treatment of breast cancer 16,17. 10

11 5.1.2 Semi synthetic polymers Cellulose derivatives are biocompatible polymers obtained by the chemical modification of the cellulose biopolymer 4. Several cellulose derivatives have been employed for various drug delivery systems. As an example, hydroxy propyl methylcellulose is herein discussed. Hydroxy propyl methylcellulose (HPMC) is a hydrophilic cellulose ether. It can be employed in the formulation of controlled release tablets. Recently, Avachat et al designed and studied a controlled release drug delivery system intended for coadministration of two drugs (diclofenac sodium and chondroitin sulphate) orally 18. A HPMC based matrix tablet was formulated using wet granulation technique and loaded with different concentration of each drug. The results of the in vitro drug release study indicated that the HPMC matrix system can offer a controlled release of both drugs from one single tablet. In another study, the influence of HPMC concentration of the rate of release of the anti-inflammatory drug, naproxen was evaluated. The dissolution results indicated a reduced rate of drug release following an increase in HPMC concentration Synthetic biodegradable polymers The synthetic polymers have the advantage of having their properties tailored to suit the desired biological application. Various synthetic biodegradable polymers such as poly lactic acid, poly lactic-co-glycolic acid, polyorthoesters, polyalkyl cyanoacrylates, polyanhydrides, polyamides, polycarbonates and polyphosphoesters are employed in drug delivery. In depth reviews on them can be seen in various literatures 4,20,21. The poly(α) esters are the earliest and most widely studied group of biodegradable polymers 9. They are easy to synthesize (by ring opening or condensation polymerization) and are readily available commercially

12 Poly (lactic) acid (PLA) Polylactic acid possesses chiral molecules. As a result, four forms can be obtained. Two optically active stereoisomers; poly L-lactic acid and poly D-lactic acid, a racemic mixture poly D,L-lactic acid and a meso-poly(lactic acid) 21. However, only poly L-lactic acid PLLA and poly D,L-lactic acid PDLLA have been studied extensively. PLLA has a very slow degradation time (up to 5 years) 9. PDLLA has a faster degradation rate compared to PLLA therefore, it is the preferred choice in developing drug delivery vehicles. Poly (lactic-co-glycolic acid) PLGA is derived by random copolymerization of polylactic acid (both D,L- and L- forms) and polyglycolic acid. It the most desirable biomaterial for drug delivery in terms of design and performance 22. It is the most investigated biodegradable polymer due to its vast commercial availability and ease of processing 21. Furthermore, it is USA FDA approved for use in humans 23. Several PLGA drug delivery vehicles have been developed, thanks to its excellent biodegradability, biocompatibility and low toxicity. An excellent review on the use of PLGA micro/nanoparticles in the controlled delivery of proteins, genes, growth factors, vaccines and antigens was compiled by Mundargi et al. 24. The rate of degradation can be tailored by varying the lactic acid isomer (D- or L-lactic acid) or the ratios in which PLA and PGA are combined 4. This is evident in the degradation times of PLGA 50:50 (refers to 50% lactic acid and 50% glycolic acid composition in the copolymer) PLGA75:25 and PLGA 80:15 being 1-2 months, 4-5 months and 5-6 months respectively 21. Polycaprolactone (PCL) is a non-toxic, semi-crystalline polyester with a very low glass transition temperature. It undergoes slow degradation (2-3 years) hence it is ideal in formulation of devices for prolonged release 25. The contraceptive device Capronor, for the long term release of the hormone levonorgestrel is composed of polycaprolactone 26. It is highly permeable to many drugs 4. 12

13 5.2 Plasticizers in Pharmaceutical Technology A plasticizer according to the International Union of Pure and Applied Chemistry (IUPAC) definition is a substance or material incorporated in a material (usually a plastic or an elastomer) to increase its flexibility, workability, or distensibility. Plasticizers are non-volatile, low molecular weight additives which improve flexibility and processability of the polymer by decreasing the glass transition temperature (Tg) 27. In addition, they should lower the melt viscosity, elastic modulus and tensile strength of the polymer and at the same time increase the toughness and elongation at break 28. Generally, the plasticizers are liquids of low volatility. They are straight or cyclic molecules with average molecular weight ranging from g/mol 29. The molecular weight, the functional group in the molecule, concentration, chemical structure, miscibility and compatibility of the plasticizer with the polymers influence the effectiveness of the plasticizer 30. Usually, small molecular weight molecules provide a higher plasticization efficiency. Efficiency of the plasticizer can be evaluated as the extent to which a plasticizer is able to lower the glass transition temperature 31. The application of plasticizers gained significance in the 19 th century. Camphor and castor oil served as the plasticizers in the manufacture of cellulose based materials. But the final results of the products were unsatisfactory. The search for better plasticizers led to the discovery of phthalic acid esters in the 20 th century. They are the largest class of plasticizers till date. Diethylhexylphthalate (DEHP) also known as dioctylphtalates (DOP) has been the most widely used 32. However, a lot of concerns have been raised regarding their toxic health effects due to leaching 33. Numerous application of plasticized products in various areas prompted the introduction of new plasticizers with improved quality. They include some fatty acid esters, benzoates, tartrates and chlorinated hydrocarbons, esters of adipic, azelaic and sebacic acid 34. Natural-based substances such as epoxidized triglyceride vegetable oils from soybean oil, linseed oil, castor-oil, sunflower oil, and fatty acid esters 35 could offer the advantage of low migration and low toxicity. 13

14 5.2.1 Mechanism of plasticization The plasticizers act by inserting their molecules between the molecules of the polymer. They increase the space between the polymer molecules thereby weakening the polymerpolymer interactions and resulting in a free movement of the polymer chains and a significant decrease in glass transition temperature 36. This explanation alone is not able to account for all the ways that the plasticizers act in modifying the polymer properties. The mechanism of action of plasticizers is therefore described using the plasticization theories according to a recent review by Daniels 37. The lubricity theory holds that the plasticizer acts as a lubricant enabling the polymer chains to move freely when a force is applied to the plasticized system. This theory assumes that the movement restrictions of the unplasticized polymer is due to surface irregularities and van der Waals interactions. Therefore, upon plasticization, the polymerpolymer interactions are weakened as the plasticizer diffuses into the polymer. The gel theory assumes that the polymer is rigid because of a three-dimensional structure or gel sites formed as a result of weak van der Waals forces or hydrogen bonds holding the polymer closely together along the chains. The plasticizer interacts with the polymer chains at these sites and breaks the polymer-polymer interactions. Thereby making the polymer less rigid and allowing the molecules to move more freely. The free volume theory measures the internal space available within the polymer. In the amorphous state, the molecules of the polymer are held closely together. They cannot move freely due to a low free volume. Increasing the free volume provides more space for the polymer molecules to move freely. This theory assumes that free volume can be increased by the addition of a plasticizer. The plasticizer separates the polymer molecules and lowers the Tg thus making the polymer more flexible. The mechanistic theory views the polymer-plasticizer and plasticizer-plasticizer interactions. It assumes that the interactions are temporary and constantly changing. Since these interactions are weak, the plasticizer can move from one site of the polymer to another and it can displace and replace another plasticizer at another site. The plasticizer-polymer interactions predominate at low plasticizer concentration. While the plasticizer-plasticizer interactions become more dominant at higher concentration of plasticizers. 14

15 In polymeric systems, even liquid drugs or liquid excipients can act as plasticizers. They are referred to as non-conventional or non-traditional plasticizers. The physichochemical, thermal and mechanical properties of polymeric systems loaded with different active ingredients; ibuprofen, metoprolol tartrate, chlorpheniramine maleate were studied. The results showed that the Tg of the polymeric system decreased significantly with increasing content of the drugs When the plasticizer is in low concentration, an opposite effect of plasticization occurs. This phenomenon is known as antiplasticization or effects of low plasticizer concentration. The polymer becomes more rigid. The tensile strength and the elastic modulus is increased and the elongation is decreased Classification of plasticizers Plasticizers are classified as internal or external. Internal plasticizers are integrated into the original structure of the polymer. Their molecules are mostly bulky in size, they can increase the side chain. Which makes it possible to create more space between the polymer molecules and allow them to move freely. Whereas, the external plasticizers are added to the polymer during processing 42. No chemical bonds are created during plasticization, it means the plasticizer molecules are not attached to the polymer matrix and can move freely. Therefore the external plasticizers can be lost through evaporation, extraction or migration 42,43. Nevertheless, the use of external plasticizers is preferred because it offers the possibility to select the suitable plasticizer type and concentration based on the desired properties of the product 31. Another classification of plasticizers is primary and secondary 44. This is according to the solvent power of the plasticizer. If at a high concentration, the polymer is soluble in the plasticizer, it is regarded as a primary plasticizer. The secondary plasticizers can be mixed with the primary plasticizers either to further improve the properties of the product or just minimizing cost. 15

16 Classification of plasticizers into hydrophobic and hydrophobic is based on their miscibility with water. Most of the plasticizers for pharmaceutical use belong in this category. The hydrophilic plasticizers include glycerine, polyethylene glycols, polyethylene glycol monomethyl ether, propylene glycol, sorbitol sorbitan solution. The hydrophobic plasticizers are usually biocompatible esters of di- or tricarboxylic acids or glycerol esters. Di and tri carboxylic esters include: Triethyl citrate (TEC) Tributyl citrate (TBC) Acetyl triethyl citrate (ATEC) Dibutyl sebacate (DBS) Diethyl phthalate (DEP) Dibutyl phthalate (DBP Di- and triesters of alcohol include: Triacetin (TA) Vegetable oils Fractionated coconut oil Acetylated monoglycerides Pharmaceutically used plasticizers are further classified based on their molecular weight as: low molecular weight, monomeric or polymeric plasticizers. Rationally, the plasticizers for biodegradable polymers should also be biodegradable 45. Research focused on finding biodegradable plasticizers to be used alongside the biodegradable polymers especially in parenteral controlled-release drug delivery systems has increased recently. For plasticization of the biodegradable polyesters polylactic acid (PLA) and poly(lactic-co-glycolic)acid (PLGA) it is possible to use oligoesters of similar aliphatic hydroxy acids 46. Other suitable plasticizers suggested for the biodegradable polymers include polyesteramides 47, polypropylene glycol 48 and polyethylene glycol (PEG). The higher the molecular mass of PEG, the better the plasticization Another useful effect of oligomeric or polymeric plasticizers is their ability to minimize or completely prevent plasticizer migration from materials

17 Water can act as an internal plasticizer in many hydrophilic biopolymers and some synthetic polymers. But it can exhibit antiplasticization effect when the content is very low 53. A decrease in Tg by up to 15 C was observed in a PLGA system in an environment of water vapours with water content up to 2.6 %. Theophylline pellets coated with Eudragit RS exhibited some changes in mechanical and dissolution properties upon storage in relative humidity Role of plasticizers in Pharmaceutics Generally, in choosing plasticizers, the most important factors are high compatibility with the polymer. However, in selecting plasticizers for pharmaceutical use, the pivotal criteria are biocompatibility and low toxicity 32. Other criteria for selection in this area are based on the influence of plasticizer on drug release, influence of plasticizer on mechanical properties and processing characteristics as well as price. Numerous plasticizers exist on the market especially for use in the chemical industry. But only a few of them meet the requirements for application in pharmacy. A good plasticizer for pharmaceutical use should be resistant to leaching or migration 55. In addition, it should comply with all health and safety regulations. The role of plasticizer in decreasing glass transition temperature, decreasing viscosity, etc can be exploited in many unit operations in the manufacturing of pharmaceutical dosage forms for better processability and application. Some of the most important pharmaceutical application of plasticizers include film coating for oral dosage form, and additives in formulation of drug delivery systems. A film coat is applied to the oral solid dosage forms such as tablets, capsules, pellets, powders and granules for various purposes like masking unpleasant taste and odour, enhancing colour, modifying drug release, etc. The coating comprises of a polymer and suitable additives, among them is the plasticizer. The major groups of polymers used for this purpose are the cellulose ethers and cellulose esters, and the plasticizers could be alkyl citrates, phthalates, trimellitates, sebacates, glycols, polyethers, adipates. 17

18 The role of plasticizer in film coating is to ensure optimal conditions for the film formation. The minimum temperature at which a coating will form a homogenous, continuous film without cracks is known as the minimum film forming temperature (MFFT). It is slightly above the Tg of the polymer. The MFFT should be lower than the coating temperature 56. The plasticizer lowers the Tg of the polymer, which means the temperature required for film coating will be lower 57. Another role of the plasticizer in film coating is for improving the flexibility of the coatings 58. A flexible coating has a good mechanical impact resistance thus it can withstand any mechanical impact force that it could be subjected to during the coating process as well as in the site where the coated dosage form will be applied. The plasticizer influences the mechanical properties of the polymer by decreasing the intermolecular forces along the polymer chains. In turn, the tensile stress and elastic modulus of the polymer is decreased and the strain elongation is increased 59. In addition, the plasticizer ensures good adhesion between the polymer and the surface of the material that is being coated. This is necessary for mechanical protection of the dosage form as well as modified drug release from the coated dosage form. The forces responsible for adhesion are the intermolecular forces between the polymer and the surface of the solid and the internal stresses within the film. When the plasticizer is added to the coating formulations, it decreases the elastic modulus and Tg, thus reducing the internal stresses 60. A decrease in the internal stresses in the film coating will increase the force of adhesion. This is further increased with increased plasticizer concentration. The hydrophilic plasticizers triethyl citrate and polyethylene glycol 6000 show a greater influence in lowering the Tg of the polymers than the lipophilic plasticizers tributyl citrate and dibutyl sebacate. In addition, the coatings plasticized with the hydrophilic plasticizers exhibit stronger adhesion 61. Influence of plasticizer on adhesion of acrylic polymer to a hydrophilic or hydrophobic compact tablet was studied. The result showed that the acrylic polymers adhesion on the hydrophobic tablets is not strong 62,63. 18

19 The influence of plasticizers on the film depends on the type of plasticizer type and concentration. The permeability of water vapours, humidity content and optical transparency of films prepared from protein isolated from pea seeds was investigated. The plasticizers employed were glycerol and sorbitol in 3-7% and 4-8% concentration respectively. Increasing concentration of glycerol only increased permeability of water vapours and humidity, with no effect on solubility. Sorbitol on the other hand increased the solubility, but water vapour permeability and humidity content were lower 64. Various drug delivery systems require plasticizers as additives to modify the properties of the polymers in terms of processability, drug incorporation and drug release, etc. examples of some plasticized drug delivery systems are discussed below. The role of plasticizers in each system is further highlighted Plasticized drug delivery systems Drug delivery systems are important for drug targeting and modified or controlled release. Various polymers of both natural and synthetic origin are used for formulating these systems. Some of the polymers properties need to be modified to suit the application. Choosing plasticizers for drug delivery systems and dosage forms require a very wide background of information. Because the formulation may contain other additives that behave differently in the presence of a plasticizer 65. Examples of some plasticized drug delivery systems include: free membranes, polymeric membranes for transdermal systems, matrix polymeric systems, in situ forming implants, plasticized polymeric microparticles and bioadhesive plasticized polymers 66. In situ forming implants: The in situ forming implants require low viscosity for ease of administration through a trocar needle. The role of the plasticizer as a viscosity decreasing agent can be employed here. Upon administration into a tissue, they change mechanical properties. They offer prolonged release of the active ingredient and are slowly dissolved or biodegraded in the body. 19

20 Bioadhesive plasticized polymers: in agreement with the wetting theory of bioadhesion 3, lower viscosity enhances better spreadability of the polymer on a surface which is required for sufficient adhesion of the bioadhesive formulations. The plasticizer can serve in achieving the sufficiently low viscosity. Plasticized microparticles: The aim of plasticization of polymers in microparticulate drug delivery systems is to modify drug release kinetics. The plasticizer can accelerate the drug release rate. PLGA microspheres for targeted drug delivery, loaded with different concentrations of the chemotherapeutic agent etoposide was plasticized with 25 and 50% tricaprin. The system showed a significant increase in the velocity of release of the active ingredients compared to the unplasticized microspheres Bioadhesion Adhesion is the ability of one thing to stick firmly to another. The adhesion between two materials where at least one of them is of biological origin is known as bioadhesion. As defined by various authors, bioadhesion is the ability of a material to stick to a biological surface for an extended period of time 68,69. A subgroup of bioadhesion where the biological surface interacting with the bioadhesive system is the mucus layer of a mucous membrane is known as mucoadhesion 70. However, the two terms are used interchangeably in practice 71. Generally, the aim of developing drug delivery systems with bioadhesive materials is to improve absorption and bioavailability of drugs. The bioadhesive drug delivery systems can be used to administer drugs to achieve both local and systemic effect. They can be designed in various dosage forms such as tablets, patches, films, beads, sprays, etc. for application to oral cavity, nasal cavity, eye, vagina, oral cavity and gastrointestinal tract. In most applications of bioadhesive polymers, the primary site for bioadhesion is the mucus layer of the mucous membrane

21 The mucous membrane (mucosa) is the moist surface which lines the walls of various organs and body cavities such as the eye, mouth, nasal cavity, respiratory and gastrointestinal tracts. The mucous membrane is made up of a layer of epithelial cells. Some of the epithelial cells (goblet cells) secrete a slimy fluid known as the mucus. The mucus exists as a thin viscoelastic gel layer sticking to the mucosal epithelial cell surface 68. Except for the high water content (95-99 %), which makes the mucus a highly hydrated system, the major component of the mucus is the glycoproteins (1-5 %) 72. The glycoproteins (mucin) impact the mucus with the gel-like characteristics and are believed to be responsible for the interaction with the bioadhesive polymers. It consists of a protein core and carbohydrate side chains linked by covalent bonds. Other components of the mucus include free lipids, inorganic salts and enzymes. The compositions vary depending on the body region, the epithelia secreting the mucus and the state of health of the organism. The mucus is responsible for protecting the epithelial cells from mechanical or chemical injury as well as maintaining moisture content of the membrane The bioadhesive materials The bioadhesive materials are mainly polymers of natural or synthetic origin. The bioadhesive properties depend on the character of the material used in the formulation 1. The most widely researched group of polymers suitable for the development of bioadhesives are high molecular weight, polar molecules with many hydrogen bond forming groups 73,74. The most important properties of the polymer necessary for sufficient adhesion include: 1. Hydrophilicity: the polymers which possess hydrophilic functional groups such as carboxyl or hydroxyl groups, are able to swell and therefore expose the maximum number of adhesive sites for hydrogen bonding or electrostatic interaction between he polymer and the mucus network High degree of swelling: higher cross-linked density of the polymer leads to a decrease in the diffusion of water into the polymer network, thereby resulting to insufficient swelling and decreased interpenetration between polymer and mucin Molecular weight: polymers of high molecular weight provide more bonding sites. Optimal molecular weight for bioadhesion often varies depending on the type of polymers. For linear polymers, mucoadhesion increases with increasing molecular weight, but the same relationship does not hold for non-linear counterparts 1. 21

22 4. Chain flexibility: polymer chains flexibility aid in the interpenetration and entanglement between the mucosal/epithelial surface and the polymer chains. Therefore adhesive properties increase with increasing flexibility 76. Polymer chain flexibility can be decreased by crosslinking Charge: anionic and cationic polymers are used. However, the anionic polymers are better than the cationic polymers in terms of adhesion and potential toxicity 77. Bioadhesive drug delivery systems can be formulated using biodegradable or nonbiodegradable, natural or synthetic polymers. The most used synthetic bioadhesive polymers are represented by the cellulose derivatives (methylcellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, sodium carboxy methylcellulose), derivatives of polyacrylic acid (carbomers, polycarbophil), polyvinylpyrrolidone, polyvinyl alcohol, polyethylene oxide. Meanwhile, the bioadhesive polymers from natural sources include tragacanth, sodium alginate, karaya gum, guar gum, xanthan gum, lectin, gelatin, pectin and chitosan 78. The bioadhesive polymers are classified as first and second generation according to their specificity 79. The first generation bioadhesive polymers such as carbopols and carbomers lack specificity and they are able to bind to both mucosal layer and to cell surfaces, while the second generation bioadhesive polymers e.g. lectins have specific ligands that they interact with in the biological molecule 76. This highly specific receptor-mediated type of bioadhesion is known as cytoadhesion. The cytoadhesive property of the lectin based polymers is being explored for the formulation of targeted drug delivery system Mechanism of bioadhesion Adhesion is a two-step process. It consists of the wetting/contact stage and the consolidation stage 3. 22

23 Stage 1: the wetting/contact stage. This involves intimate contact between the bioadhesive and the substrate. This can be from sufficient wetting or from swelling of the bioadhesive material. For the successful creation of a strong adhesive bond, an increase in the initial contact time is required. The initial contact time also determines the extent to which the polymer swells as well as the penetration of the polymer chains into the mucosal surface 81. Stage 2: the consolidation stage. Following the establishment of contact in the first phase, the bioadhesive then penetrates the crevices of the tissue surface (interdiffusion) or the chains of the bioadhesive molecule entangles with those of the mucus (interpenetration). Several chemical interactions occur to strengthen the adhesive joint 3. This stage is important when a strong adhesion is required, especially in targeting an area which is under constant stress 82. Figure 1: The two stages in mucoadhesion 3 Several theories have been postulated to account for the adhesion mechanism. One or more theories can contribute equally to the formation of bioadhesive bonds. Therefore, a combination of the theories can be used to describe the phenomenon

24 The wetting theory applies mainly to liquid bioadhesive systems. It considers the spreadability of a liquid drug delivery system over a biological surface as a requirement for the development of bioadhesion. The contact angle can be measured to determine the affinity of the liquid on the surface (Figure 2). The general rule holds that if the contact angle of the liquid on the substrate surface is lower, then there is a higher affinity of the liquid to the surface 3. Adequate spreadability is provided by contact angle close to or equal to zero. Figure 2: Scheme of contact angle 1 The electronic theory suggests that the opposing electrical charges of the mucus and the mucoadhesive system gives rise to adhesion by the means of electron transfer. Contact between adhesive polymer and the glycoprotein results in the formation of an electrical double layer on the interface. Attractive forces across the double layer results in adhesion 84. The adsorption theory postulates that secondary bonds which arise mainly due to Van der Waals forces and hydrogen bonding is responsible for surface interactions in mucoadhesion. After an initial contact between two surfaces, the materials will adhere due to forces acting between the chemical structures at the two surfaces, a semipermanent bond which require less energy to break is formed 1. 24

25 The mechanical theory explains the diffusion of liquid adhesives into the crevices and irregularities on the surface of the substrate. Adhesion then arises from the formation of an interlocked structure 3. Surface roughness results in more interfacial area available for interactions. The fracture theory assesses the difficulty to separate the two surfaces after adhesion 85. The force required to detach the polymer from the mucus represents its adhesive bond strength. The diffusion theory states that the polymer chains penetrate into the mucin chains through diffusion to a sufficient depth as to create a semi-permanent adhesive bond (Figure 3). The depth of penetration and intermingling depend on the diffusion coefficient. This in turn is dependent on the molecular weight of the polymer, chain length and chain mobility and contact time 86. Figure 3: Interdiffusion of polymer chains of bioadhesive devices and mucus 1 25

26 5.3.3 Application of bioadhesives in pharmacy The bioadhesive properties of the polymers can be employed in the development of drug delivery systems intended for targeted delivery or controlled release in the GIT, vagina, rectum, oral cavity, nasal cavity, eye. They are used for local drug therapy as well as systemic delivery of drugs. Several bioadhesive drug delivery systems have been designed using existing conventional dosage forms. The conventional dosage forms are redesigned to become bioadhesive by the incorporation of a bioadhesive material in the formulation 1. The ability to retain the polymer on the mucus layer and the sustained release of the drug are the basis of a successful mucoadhesive drug delivery system 87. The mucoadhesive delivery systems can be formulated as tablets, patches, microparticles. The drug can be dispersed in the adhesive polymer matrix or the adhesive polymer can form a barrier through which the drug can be released by diffusion. In the powdered form, the mucoadhesive can be incorporated into ointments and pastes. Gels, vaginal rods, pessaries and suppositories can also be formulated with mucoadhesive material 3. Examples of commercially available mucoadhesive preparations for different application routes can be seen in Table 1. Table 1: Commercially available mucoadhesive preparations 78,88,89 Application site Active substance Name and form Bioadhesive Polymer Eye Hypotears Poly (vinyl) alcohol Nasal cavity Triamcinolone Nasacort spray Microcrystalline cellulose Buccal cavity Nitroglycerine Suscard tablet Hypromellose Vagina Progesterone Crinone gel Carbomers Oral cavity Hydrocortisone Corlan tablet Arabic gum 26

27 Drug delivery to the gastrointestinal tract: despite the ease and acceptability of drug administration through the oral route, a short retention time due to GIT motility and gastric emptying and high mucus turnover can decrease the absorption and bioavailability of the dosage forms administered via this route. The use of mucoadhesive polymers however is aimed at slowing down the movement of the drug along the GIT to increase the residence time of the drug in the GIT, improve absorption and increase bioavailability 90. Drug delivery to the oral cavity: The oral cavity is a target site for bioadhesives because first pass metabolism and GIT degradation are bypassed 91. Delivery of drugs to the oral cavity can be via the buccal or sublingual routes. The sublingual mucosa exhibits a higher permeability than the buccal mucosa. As a result, sublingual mucoadhesives are designed in such a way that the active ingredients are released quickly and this is used when a rapid response is required. The buccal mucoadhesives are more favourable where a controlled release of the active ingredient is desired 76. For example, in the treatment of chronic diseases, the lower permeability of the buccal mucosa is exploited for gradual delivery and prolonged action 90. Ocular drug delivery: In the treatment of various eye disorders, topical application of ophthalmic preparations is the most acceptable way to administer drugs to the eye 92. The drugs may be absorbed systemically or act locally on the eye surface. However, the residence time is extremely short. Frequent administration is required to reach therapeutic levels and this can often lead to low patient compliance. The mucoadhesive preparations can be employed to increase the effect of ophthalmic preparations by prolonging the residence time of the active substance in the eye surface. This will in turn reduce the frequency of administration and improve patient compliance. Dosage forms for drug delivery to the eye include liquid drops, gels, and ointment. Some patches and ocular inserts have also been designed for drug delivery to the eye 84. The in-situ gelling polymers can also be used as ocular drug delivery platforms. These systems are liquid before administration and in response to stimuli such as temperature or PH within the eye fluid they are transformed into highly viscous rheologically structured networks

28 Nasal drug delivery: It has been demonstrated that administration of various drugs (gentamicin, ergotamine tartrate) to the nasal cavity leads to plasma concentrations comparable to the parenteral drug delivery 94. This is because the blood is drained directly from the nose into systemic circulation 95. However, drug delivery and absorption through this route is limited by the short residence time of drugs in the nasal mucosa due to mucociliary clearance. The use of the bioadhesive system in this route will prolong the residence time. Vaginal drug delivery: Administration of drugs via the vaginal route offers the possibility for both local and/or systemic effects. Drug delivery via this route minimizes hepatic first-pass metabolism. Despite the large surface area, high permeability and vascularity, the dosage form fails to be retained for a longer period of time due to the self-cleansing action of the vagina tract 96. The bioadhesive preparations are used to retain the drug longer in the vagina, thereby improving absorption and bioavailability Methods for testing bioadhesion In the formulation of mucoadhesive systems of a new kind, adhesive properties of polymers are tested to ensure the efficiency of the proposed polymer 68. However, results from different groups cannot be directly compared since there is no standard method to test for bioadhesion 2. Methods of testing bioadhesive formulations can be categorized as in vitro and in vivo methods. The in vitro methods are usually based on the measurement of adhesive strength that is, the force required to break the bond between the model substrate and the mucoadhesive. The force is categorized as shear, tensile or peeling forces depending on the direction in which the adhesive is separated from the substrate 1. Some of the common in vitro tests in literature are discussed in the subsequent paragraphs. 28

29 Measurement of tensile strength: in the tensile strength method, the force required to break the adhesive bond between the model membrane and the polymers is measured. the apparatus for this measurement is usually a modified balance or tensile testers. A representative example is demonstrated by Robinson and his group 97. The force required to separate the bioadhesive sample from a tissue freshly resected from the stomach of a Rabbit was determined. In this experiment (Figure 4), a part of the tissue which had the mucus side exposed was secured on a weighed glass vial and submerged in a beaker containing a USP simulated gastric fluid. Another portion of the same tissue, again with the mucus side exposed, was placed over a rubber stopper and secured with a vial cap. A small amount of polymer was placed between the surface of the two mucosal tissues. The amount of force which the polymer used to detach from the tissue was recorded. Figure 4: Illustrating the modified dual tensiometer used by Leung and Robinson 98 Measurement of shear strength: it measures the force required to separate two parallel glass slides covered with a polymer and a mucus film 99. This can be done using Wilhemy s model. A glass plate suspended from a microforce balance was immersed in a sample of mucus under regulated temperature. The force required to pull the plate out of the sample is measured under constant experimental conditions 2. 29

30 Figure 5: Apparatus to determine mucoadhesion in vitro, using Wilhemy s technique 1 Rheological method can be used to test semi-solids and liquid dosage forms. This method was first proposed by Hassan and Galo 100. In this test, the polymer solution is mixed with the mucin solution to simulate an interpenetration layer. For a polymer with strong mucoadhesive ability, this mixture is expected to have a higher viscosity in comparison to the sum of the polymer and the mucin viscosities separately. This rheological synergism is used to evaluate the adhesive properties. The viscosity or elasticity is determined for the mixture and the result is compared to the rheological properties of the polymer and mucin separately. The flow-through or wash away test This method of testing evaluates drug release in areas with a constant flow of biological fluid 101. It is beneficial when developing mucoadhesive formulations for targeting areas, such as the eye where constant lacrimation and blinking could displace the mucoadhesive tablet 86. The principle of the flow-through test is schematically represented in Figure 6. This method was first described by Rango Rao and Buri 102. In the experiment, a mucosal tissue is secured on a slide surface and covered with a product to be tested. A simulated body fluid is maintained at constant flow to wash off a formulation from the surface of the tissue. A spectrophotometric or chromatographic analysis can be performed to monitor the drug or mucoadhesive polymer washed off into the collected fluid. In addition, the dosage formed retention on the mucosal tissue can also be monitored. 30

31 Figure 6: Flow-through experimental set up for evaluating mucoadhesive properties 86 Other in vitro testing methods include: colloidal gold staining method proposed by Park in late 1980s 103, fluorescent probe technique a method by Park and Robinson 77, viscometric method by Hassan and Galo 100. A few in vivo experiments have also been implemented: gamma scintigraphy, transit studies with fluorescent-coupled or radio labelled dosage forms, isolated loop techniques The limitations in testing for bioadhesion using some in vivo techniques, lies in the cost and ethical concerns. Nevertheless, they are an important and more accurate representation of the true mucoadhesive potential of the drug delivery system

32 6 Experimental section 6.1 Materials Copolymer of Lactic and Glycolic acid (Faculty of Pharmacy Hradec Králové) (PLGA) Ethyl pyruvate (Sigma-Aldrich, USA) Ethyl salicylate (Sigma-Aldrich, USA) PLGA branched with 2% Polyacrylic Acid (Faculty of Pharmacy Hradec Králové) (A2) PLGA branched with 3% Tripentaerythritol (Faculty of Pharmacy Hradec Králové) (T3) Triethyl citrate (Sigma-Aldrich, USA) 6.2 Instrument Kinexus Pro+ Rheometer (Malvern instruments Ltd, UK) Figure 7: Kinexus absolute rotational rheometer

33 6.3 Methods Plasticization of polyesters A specified amount of the selected polyester (Table 2) was weighed into tarred glass beakers on analytical balance and heated in the oven at 90 o C for 30 min. The plasticizer ethyl salicylate (ES) was added to the samples and heated again. Covering with aluminium foil helped to prevent evaporation during heating. The samples were mixed at intervals using a metal lance until homogenous i.e. until plasticizer was fully incorporated into the polymer. Initially, the plasticizer was added in 10 %, 20 %, and 30 % concentrations. It means 9.0 g, 8.0 g and 7.0 g of polyester and 1.0 g, 2.0 g and 3.0 g of plasticizer respectively. Each sample had a final mass of 10.0 g. The 10 % and 20 % concentration of ethyl salicylate plasticizer did not bring about a satisfactory decrease in the viscosity. Therefore, the concentration was increased to 40 % and 50 % (calculated by mixed equations). In this manner, samples of PLGA and T3 were prepared using 30 % concentration of ethyl pyruvate (EP) and triethyl citrate (TEC) as plasticizers. List of samples tested shows Table 3. Table 2: Characteristics of branched polyesters 108 Polyester Monomer ratio (%) LA:GA:T/PAA Mn (g/mol) Mw (g/mol) PDI g Tg ( C) PLGA 50:50:50 1,700 2, T3 48.5:48.5:3 5,300 17, A2 49:49:2 8,600 14, LA lactic acid GA glycolic acid T tripentaerythritol PAA polyacrylic acid 2,200 g/mol Mn number average molecular weight (g/mol) Mw weight average molecular weight (g/mol) PDI polydispersity index calculated as Mw/Mn g` index of branching determined by intrinsic viscosity of linear and branched polymer of the identical molar mass ( br/ lin) glass transition temperature ( C) Tg 33

34 Table 3: Composition of plasticized systems to be tested Polymer Plasticizer type Plasticizer concentration TEC 30 % - - PLGA EP 30 % - - ES 30 % - - T3 TEC 30 % - - EP 30 % - - ES 30 % - - TEC 30 % - - A2 EP 30 % - - ES 30 % 40 % 50 % Starting up the rheometer First, it was necessary to confirm that the air supply was turned on. By checking if the green light of the compressed air indicator was glowing. Then the pressure on the device was checked. The left and right pressure gauge showed 4 bars and 3 bars respectively. The instrument was switched on and left to stabilize for 5 minutes. The computer was switched on and the r-space software for Kinexus was launched by double clicking on the desktop icon. The initialization of the device was completed by clicking Next. The selected upper geometry was then inserted into the device and was detected by the software. The final step according to the instructions from the software was setting the zero gap. The upper geometry moved down and met the lower geometry and then separated shortly afterwards. At this point, the instrument was ready. 34

35 6.3.3 Test for flow properties The list of sequences was opened by double clicking the rfinder toolbar in the software. The sequence was opened. The shear rate range was chosen. After clicking on the Load sample icon, the type of material used was selected from the list. The sample was applied in sufficient amount on the lower geometry using a plastic spatula (to avoid scratching the instrument), and the experiment was named as follows: the polymer type, concentration, and type of plasticizer (e.g. PLGA30%ES). Upon clicking Next, the upper geometry went down and pressed on the loaded sample and the excess was trimmed off using the plastic spatula. The solvent trap system was employed to prevent evaporation of the plasticizer from the sample during the measurements. The temperature was set and the samples per decade (frequency of measuring for every shear rate) was also set. At the end of each measurement, the sample was unloaded according to instructions on the software and the data was saved on the computer. Before loading the next sample, the lower geometry and the upper geometry were cleaned using acetone. The data obtained was evaluated using the Newtonian model fit and the Power law model fit for viscometry. All measurements were done three times, and average and standard deviation were calculated. Results of the rheological properties were represented graphically on the viscosity curves as the dependence of viscosity on shear rates. Parameters for measurement 1. Sequence: a. Toolkit_V001 Table of Shear Rates - Equilibrium Flow Curve b. Analyse_0021 Newtonian model fit for viscometry c. Analyse_0004 Power law model for viscometry 2. Shear rate range: s Upper geometry: cone plate 2 o /20mm 4. Temperature: 37 C 5. Samples per decade: 10 35

36 6.3.4 Test for adhesive properties A tack and pull away test on the Malvern Kinexus absolute rotational rheometer was used to determine tackiness and adhesion of the polyester materials. The sequence Tack original 1 was selected in the r-space software. The upper geometry was mounted on the device. The value for the temperature was set. The required working gap was set. The sample was loaded, the upper geometry went down to press on the sample and the excess was trimmed. Vertical gapping parameters (gapping speed and working gap) were entered. After starting the test, it was necessary to wait for stability. When the upper geometry detached from the lower geometry, the result was retrieved by clicking on peak/valley analysis or feedback. After each sample was unloaded, both geometries were cleaned with acetone. All measurements were done five times and the data were saved. The average and standard deviation were calculated. The result was reported back as: 1) the peak tension (negative normal force) that is, the maximum force required for detachment of the upper geometry from the lower geometry 2) area under force-time curve which represents the adhesive/cohesive strength and 3) time(action) which is a measure of the failure rate or time i.e. the time required for the force to decay by 90%. Parameters for measurement 1. Sequence: tack original 1 2. Upper geometry: cone plate PU20 3. Temperature: 37 C 4. Working gap: 0.5 mm 5. Gapping speed: 10 mm/s 6. Final gap: 100 µm 36

37 7 Results 7.1 Flow properties Figure 8: Viscosity curves of polyester PLGA plasticized by 30 % triethyl citrate Table 4: Newtonian model fit of polyester PLGA plasticized by 30% triethyl citrate Sample Description PLGA30%TEC Experiment Name Shear viscosity (Pa s) Viscometry_0009 Table of shear rates with Newtonian model fit Average ± SD ± 2.42 Correlation coefficient

38 Figure 9: Viscosity curves of polyester PLGA plasticized by 30 % ethyl pyruvate Table 5: Newtonian model fit of polyester PLGA plasticized by 30% ethyl pyruvate Sample Description PLGA30%EP Experiment Name Shear viscosity (Pa s) Viscometry_0009 Table of shear rates with Newtonian model fit Average ± SD ± 0.57 Correlation coefficient

39 Figure 10: Viscosity curves of polyester PLGA plasticized by 30 % ethyl salicylate Table 6: Newtonian model fit of polyester PLGA plasticized by 30% ethyl salicylate Sample Description PLGA30%ES Experiment Name Shear viscosity (Pa s) Viscometry_0009 Table of shear rates with Newtonian model fit Average ± SD ± Correlation coefficient

40 Figure 11: Viscosity curves of polyester T3 plasticized by 30 % triethyl citrate Table 7: Newtonian model fit of polyester T3 plasticized by 30% triethyl citrate Sample Description T30%TEC Experiment Name Shear viscosity (Pa s) Viscometry_0009 Table of shear rates with Newtonian model fit Average ± SD ± Correlation coefficient

41 Figure 12: Viscosity curves of polyester T3 plasticized by 30 % ethyl pyruvate Table 8: Newtonian model fit of polyester T3 plasticized by 30% ethyl pyruvate Sample Description T30%EP Experiment Name Shear viscosity (Pa s) Viscometry_0009 Table of shear rates with Newtonian model fit Average ± SD ± 9.20 Correlation coefficient

42 Figure 13: Viscosity curves of polyester T3 plasticized by 30% ethyl salicylate Table 9: Newtonian model fit of polyester T3 plasticized by 30% ethyl salicylate Sample Description T30%ES Experiment Name Shear viscosity (Pa s) Viscometry_0009 Table of shear rates with Newtonian model fit Average ± SD ± 1.01 Correlation coefficient

43 Figure 14: Viscosity curves of polyester A2 plasticized by 30% triethyl citrate Table 10: Newtonian model fit of polyester A2 plasticized by 30% triethyl citrate Sample Description A30%EP Experiment Name Shear viscosity (Pa s) Viscometry_0009 Table of shear rates with Newtonian model fit Average ± SD ± 4.4 Correlation coefficient

44 Figure 15: Viscosity curves of polyester A2 plasticized by 30% ethyl pyruvate Table 11: Newtonian model fit of polyester A2 plasticized by 30% ethyl pyruvate Sample Description A30%EP Experiment Name Shear viscosity (Pa s) Viscometry_0009 Table of shear rates with Newtonian model fit Average ± SD ± 2.34 Correlation coefficient

45 Figure 16: Viscosity curves of polyester A2 plasticized by 30% ethyl salicylate Table 12: Newtonian model fit of polyester A2 plasticized by 30% ethyl salicylate Sample Description A30%ES Experiment Name Shear viscosity (Pa s) Viscometry_0009 Table of shear rates with Newtonian model fit Average ± SD ± 6.67 Correlation coefficient

46 Figure 17: Viscosity curves of polyester A2 plasticized by 40% ethyl salicylate Table 13: Power law model fit of polyester A2 plasticized by 40% ethyl salicylate Sample Description A40%ES Experiment Name K n Correlation coefficient Viscometry_ Table of shear rates with Power law model fit Average ± SD ± ±

47 Figure 18: Viscosity curves of polyester A2 plasticized by 50% ethyl salicylate Table 14: Power law model fit of polyester A2 plasticized by 50% ethyl salicylate Sample Description A30%ES Experiment Name K n Correlation coefficient Viscometry_ Table of shear rates with Power law model fit Average ± SD ± ±

48 7.2 Adhesive properties Figure 19: Record of tack test of polyester PLGA plasticized by 30% triethyl citrate Table 15: Adhesive properties of polyester PLGA plasticized by 30% triethyl citrate Sample Average ± SD Fmax (N) ± 2.42 Area (N/s) ± 2.40 Time (s) ±

49 Figure 20: Record of tack test of polyester PLGA plasticized by 30% ethyl pyruvate Table 16: Adhesive properties of polyester PLGA plasticized by 30% ethyl pyruvate Sample Average ± SD Fmax (N) ± 1.02 Area (N/s) Time (s) ± ±

50 Figure 21: Record of tack test of polyester PLGA plasticized by 30% ethyl salicylate Table 17: Adhesive properties of polyester PLGA plasticized by 30% ethyl salicylate Sample Average ± SD Fmax (N) ± 0.88 Area (N/s) ± 0.93 Time (s) ±

51 Figure 22: Record of tack test of polyester T3 plasticized by 30% triethyl citrate Table 18: Adhesive properties of polyester T3 plasticized by 30% triethyl citrate Sample Average ± SD Fmax (N) ± 1.08 Area (N/s) ± 1.91 Time (s) ±

52 Figure 23: Record of tack test of polyester T3 plasticized by 30% ethyl pyruvate Table 19: Adhesive properties of polyester T3 plasticized by 30% ethyl pyruvate Sample Average ± SD Fmax (N) ± 2.13 Area (N/s) ± 0.75 Time (s) ±

53 Figure 24: Record of tack test of polyester T3 plasticized by 30% ethyl salicylate Table 20: Adhesive properties of polyester T3 plasticized by 30% ethyl salicylate Sample Average ± SD Fmax (N) ± 1.02 Area (N/s) ± 0.50 Time (s) ±

54 Figure 25: Record of tack test of polyester A2 plasticized by 30% triethyl citrate Table 21: Adhesive properties of polyester A2 plasticized by 30% triethyl citrate Sample Average ± SD Fmax (N) ± 0.65 Area (N/s) ± 1.64 Time (s) ±

55 Figure 26: Record of tack test of polyester A2 plasticized by 30% ethyl pyruvate Table 22: Adhesive properties of polyester A2 plasticized by 30 % ethyl pyruvate Sample Average ± SD Fmax (N) ± 0.16 Area (N/s) ± 0.04 Time (s) ±

56 Figure 27: Record of tack test of polyester A2 plasticized by 30% ethyl salicylate Table 23: Adhesive properties of polyester A2 plasticized by 30 % ethyl salicylate Sample Average ± SD Fmax (N) ± 0.47 Area (N/s) ± 0.42 Time (s) ±

57 Figure 28: Record of tack test of polyester A2 plasticized by 40% ethyl salicylate Table 24: Adhesive properties of polyester A2 plasticized by 40 % ethyl salicylate Sample Average±SD Fmax (N) ± 0.09 Area (N/s) ± 0.07 Time (s) ±

58 Figure 29: Record of tack test of polyester A2 plasticized by 50% ethyl salicylate Table 25: Adhesive properties of polyester A2 plasticized by 50 % ethyl salicylate Sample Average ± SD Fmax (N) ± 0.42 Area (N/s) ± 0.08 Time (s) ±

59 8 Discussion Copolymers of lactic acid and glycolic acid, labeled as PLGA, have their significant place in pharmacy and medicine. Unlike linear polyesters, star-like copolymers PLGA, branched on polyhydric alcohols or acids possess a low gyration diameter and low degree of swelling and the biodegradation rate runs continuously over several hours to several days depending on the molecular weight parameters. They are intended to employ as carriers of drugs in polymeric drug delivery systems with modified drug release. Very little is known about physical and physico-chemical properties of low molecular weight star-like polyesters although these characteristics are crucial in formulation of therapeutic systems, their application, drug release, and finally the resulting therapeutic effect of the preparations. In this diploma thesis, three types of polyesters originally synthesized at the workplace of supervisor were employed: i) linear PLGA, ii) star-like T3 branched on tripentaerythritol, iii) star-like A2 branched on polyacrylic acid. The step-growth polycondensation from equimolar mixture of lactic acid and glycolic acid, and branching monomer were carried out. The reaction ran at 160 C and 550 Pa for 75 h and was catalyzed by heterogeneous strongly acidic cation exchanger Dowex-50 W. The resulting reaction products were purified by double non-isothermal precipitation from methanolic solutions and vacuum-dried at 40 C for 48 h. Acquired oligoesters did not contain any catalyst 5. The nature of the branched molecules, in terms of their polarity, is conditioned by the ratio of terminal hydroxyl and carboxyl groups and is related to the molecular weight and degree of branching. The characteristics are shown in Table 2. 59

60 8.1 Flow properties of plasticized polyesters At ambient conditions, the tested polyesters are very hard and brittle materials (like a glass). Their flow properties must be modified accordingly, not only for a good processability and applicability via an injection needle, trocar applicator, or by spraying, but also to influence the drug release profile significantly. Sufficiently low viscosity is possible to achieve by heating which is exploited well during manufacturing but only limited use during application into the humans due to the maximal tolerable temperature without tissue necrotic changes 109. Another way to decrease the viscosity of the polymeric system is by plasticizing. Chemicals commonly used as plasticizers or solvents of polymers are limited by good manufacturing practice (GMP) or pharmacopoeial requirements. Demanded attributes of an ideal plasticizer depend on the intended application. For a medical or pharmaceutical use, biocompatibility and toxicity are the crucial criteria. Triethyl citrate, ethyl pyruvate, and ethyl salicylate were chosen as plasticizers for polyesters used in this work. All the tested plasticizers significantly reduced the viscosity but not always sufficiently (Figure 30). The plasticized polyesters tested are mostly Newtonian (Figure 8 to Figure 16). From the point of view of technological processing and application, this may be advantageous. It is possible to choose a definite plasticizer type and/or concentration with which to set a desired value of viscosity for the system. This value will not be influenced by shear stress or shear rate. The non-newtonian behaviour was evident only in two of the systems, polyester A2 plasticized with ethyl salicylate of 40 % and 50 % (Figure 17 and Figure 18). For these non-newtonian materials, the viscosity is not constant but decreases with increasing shear rate/stress, i.e. shear viscosity. This flow behaviour is called as shear thinning (pseudoplastic) for liquid materials or shear thinning with yield point (plastic) for semisolids, and mathematically described with Oswald-de Waele s equation (Power law) (1) or Herschel-Bulkely equation (2) respectively, = K D n (1) = y + K D n (2) 60

61 Dynamic viscosity [Pa.s] where denotes the shear stress, y is yield stress, K is the coefficient of consistency, D is the shear rate, and n is the power law index 110. The coefficient of consistency K represents the viscosity at the shear rate of 1 s -1, therefore serves as measure of consistency of a material. The power law index n is a measure of non-newtonianness or sensitivity to shearing. The value near zero applies for very shear thinning materials, Newtonian materials have n = 1. Our results show nearly Newtonian flow of polyester A2 plasticized with 40% ethyl salicylate (Table 13), as the value is closer to 1, and typically non-newtonian flow at plasticizing with 50 % ethyl salicylate (Table 14) as the value is further away from 1. Furthermore, we can say that the systems with the lowest viscosity are non-newtonian or tend to be non-newtonian. We can see from Figure 30 that A40%ES and A50%ES (the non-newtonian systems) happened to possess the lowest values of viscosity. Figure 30: Viscosity of polyesters plasticized with triethyl citrate (TEC), ethyl pyruvate (EP), ethyl salicylate (ES). The data are the average values obtained from three measurements

62 Based on our experience, the default value of viscosity required for better processability, ease of drug incorporation, easy spreading on the skin and application by injection was determined to be below 50 Pa s. We can expect these qualities from the following group of plasticized polyesters: PLGA30%EP, A30%EP, A40%ES and A50%ES (Figure 30) where the viscosity values ranges from 28 Pa s to 41 Pa s. We can also expect a lower adhesion and a faster release of incorporated drug from these systems. On the other hand, the group of plasticized polyesters with viscosity ranging from 93 Pa s to 276 Pa s (Figure 30) will require a further decrease in viscosity so as to facilitate handling and application of the system. This can be achieved by increasing the plasticizer concentration. As observed with ethyl salicylate, increasing the plasticizer concentration from 30% to 40% decreased the viscosity by up to 6 times. The highest viscosity 848 Pa s was seen in A30%TEC (Figure 30). It means that 30 % concentration of this plasticizer did not sufficiently decrease the viscosity of the system. However, it is possible to increase the concentration further, as triethyl citrate has unlimited miscibility with the polyesters. In 30 % concentration, the most effective plasticizer for PLGA and A2 polyesters is ethyl pyruvate. The resulting systems are Newtonian and the values of viscosity correspond with the required limit. In the polyester T3, the values of viscosity obtained by using ethyl salicylate and ethyl pyruvate plasticizer in 30 % concentration are quite close, 93 Pa s and 99 Pa s respectively. The right choice of plasticizer will however depend on the intended application of the plasticized system. The presence of 30 % triethyl citrate did not decrease the viscosity in any of the systems sufficiently. Ranking the plasticizers from most efficient to least efficient plasticizer, the order can be concluded: ethyl pyruvate > ethyl salicylate > triethyl citrate. 62

63 8.2 Adhesive properties of plasticized polyesters In the diploma thesis, the adhesive properties of plasticized polyesters were tested with the Kinexus rotational rheometer. It involves measuring the force required to separate two parallel plates having a defined volume of material between them from a stationary position with no initial pressure applied. Here the peak in negative normal force (tension) can be attributed to tack, the area under the force-time curve to adhesive or cohesive strength and the time taken for the peak force to decay by 90% a comparative measure of failure rate or time as illustrated in Figure 31. Figure 31: Annotated normal force-time profile showing key features for assessing adhesive/cohesive properties under tension