Interactions of biodegradable drug carriers with hydrophilic medium Master thesis

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Kaunas Medicine University

Faculty of Pharmacy

Department of pharmaceutical technology

Interactions of biodegradable drug carriers with hydrophilic

medium

Master thesis

Student: Kristina Miknevičiūtė

Supervisor: doc. RN Dr. Milan Dittrich,

department of pharmaceutical technology, Faculty of

Pharmacy in Hradec Kralove, Charles University in

Prague, Czech Republic; prof. V. Briedis, Kaunas

University of Medicine, Lithuania

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Acknowledgements

I would like to thank to Socrates Erasmus program and for Charles University in Prague for this wonderful opportunity to write my diploma thesis in Czech Republic. I am very grateful for my supervisor doc. Milan Dittrich for all his time and devotion, also to mgr. Eva Valentova for her personal care. I am very grateful for Kaunas University of Medicine and prof. Vitalis Briedis for their support from Lithuania, also my reviewer dr. Giedrė Kasparavičienė for her constructive thoughts. I am thankful for every person who helped me in this work from the first to the last word.

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CONTENT

1. Introduction………...………...5

2. Overview of literature………..………....6

2.1. Polymeric biomaterials……….……….………6

2.1.1. Linear polyesters and extended poly- (ester)- urethanes………...……….6

2.1.2. Linear polyester amides (PEA)………....…..7

2.1.3. Branched oligoesters………..………8

2.2. Interactions with hydrophilic medium………..……….8

2.3. Biodegradation……….….9

2.3.1. Hydrolytic degradation………..………...……10

2.3.2. Surface and bulk degradation ………..……10

2.3.3. Evaluation of erosion………..……..……11

2.4. Thermal analysis………..………11

2.5. Nano and micro particles……….………12

2.5.1. Particle size and zeta potential……….………13

2.5.2. Micro particulate system………..………14

3. Experimental part………...……….………16 3.1. Materials………...……….………..16 3.1.1. Objects………..………...………16 3.1.2. Chemicals………...…….18 3.1.3. Instruments………...………...18 4. Investigation………...……….19

4.1. Investigation of swelling and erosion kinetics of newly synthesized polymeric and oligomeric drug carriers in buffer medium………...….19

4.2. Thermal analysis – Tg measured by the DSC method of some degraded carriers…………...20

4.3. Experimental study concerning nano and micro particles. Preparation and evaluation……...21

5. Results and discussion………...….…..22

5.1. Biodegradable carriers interactions with hydrophilic media – swelling degree and erosion degree (figures 1- 15)...22

5.2. Conditions for thermal analysis for PLGA 30:70, PLGA 50:50 and PEU2 carriers (table 4)………...32

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5.3. Most typical plots of DSC (figures 16 - 21)………...…....33

5.4. Parameters of molecular weight and thermal behavior of carriers (table 5)………...…….39

5.5. Main characteristics of DSC- measurements (tables 6 - 8)………… ...………..40

5.6. The calculated averages of measured results in Tg and ∆Cp (tables 9 - 11)……...44

6. Conclusions………...…………..46

7. Summary………...……...47

8. Summary in Lithuanian………...………48

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1. INTRODUCTION

Prolonging and targeting the action of drugs, minimizing the necessity for surgical interventions leads to increasing need of biodegradable materials.

This work is a pilot study of new synthesized biodegradable drug carriers. Some of them possessed linear chain constitution, other ones were branched. The set of samples included not only traditionally studied polyesters, but also original polyester amides and one chain-extended polyester urethane. The purpose of this study is to evaluate a very important aspect of the behavior of the newly obtained carriers concerned their interactions with a hydrophilic medium which mimics the surroundings of implanted material’s piece. Interactions were studied as swelling and erosion kinetics. Other characteristics are represented as thermal behavior, mainly as glass transition temperature. For educational purposes it was decided to render some specific and basic theoretical aspects and practical methods of preparation and evaluation of nanoparticular and microparticular systems.

The main aims are:

1. Investigate swelling and erosion of new obtained carriers. 2. Evaluate swelling of new obtained carriers.

3. Evaluate erosion of new obtained carriers.

4. Evaluate differences in swelling and erosion between linear, branched, chain extended polymers and polyesteramides.

5. Look for possible predictable model of swelling and erosion common for certain group of materials.

6. Investigate glass transition temperature of certain carriers after swelling and drying. 7. Look for possible correlation between swelling and thermal behavior.

8. Evaluate theoretical possibility to prepare nano and micro particular systems using new obtained polymers.

Swelling and glass transitions are two phenomena whose mechanisms are substantiated by molecule chain relaxation. Comparison or differentiation of the two kinetics of random coil changes during carrier swelling and glass transition is one of numerous possible ways of comparing their mechanism. It gives us important knowledge about possible further experiments and ways of application.

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2. OVERVIEW OF LITERATURE

Biodegradable polymers are becoming more important not just in techniques, but also in medicine, where more and more attention is being paid to drug delivery. At the moment the greater priority is given to prolonging and targeting the action of drugs. In the topics of biomedical implants and drug delivery, biodegradable polymers occupy a unique position. They can be used for applications where only temporary implants are required, and where generally no surgical procedure is needed to remove the implant or drug delivery device at the end of its function. The polymer piece gradually degrades into harmless absorbable or resorbable fragments and small molecules, and hence is metabolized or excreted from the body (1).

In the context of medicine, biodegradation means disintegration, erosion, dissolution, breakdown and/or chain scission of the polymer into metabolizable or excretable fragments in the human body, in animal models, in ex vivo or in vitro test medium, which represent, mimic or approximate the body environment (1).

Applications of biodegradable polymers in medicine:

4. Tissue engineering - the repair, restoration or regeneration of natural tissue within biodegradable polymer scaffolds, with programmed degradation and resorption or elimination of the polymer scaffold (1).

5. Surgical devices.

6. Drug delivery devices (polymer-drug conjugates, implantable delivery systems). 7. Nano- and micro particulates drug delivery systems.

2.1. Polymeric biomaterials

Polymers are synthetic and natural macromolecules composed of smaller same or similar units called monomers. The molecular weight of polymers usually exceeds 10 000 g/mol. Oligomers are similar compounds, but with smaller molecules with molecular weight under 10 000 g/mol.

2.1.1. Linear polyesters and extended poly-(ester)-urethane

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Polyglycolide (PG) is a crystalline, biodegradable polymer which has the melting point (Tm) of ~225ºC and a glass transition temperature (Tg) of ~35ºC. The molecular and subsequent crystalline structure of PG allows very tight chain packing and thus afford some very unique chemical, physical and mechanical properties. The polymer is very insoluble in common organic solvents, but biodegradation by hydrolysis easily happens because of readily accessible and hydrolytically unstable aliphatic-ester linkages. The degradation time is generally just a few weeks though it depends on molecular weight, degree of crystallinity, physico-chemical environment, temperature and other factors.

Polylactides (PL) are quite different due to their chirality. The methyl group in PL causes the carbonyl of the ester linkage to be sterically less accessible to hydrolytic attack and that makes PL more hydrolytically stable than PG.

High-molecular-weight polymers and copolymers of glycolide and L- and DL-lactides are prepared by ring-opening addition polymerization of their respective cyclic dimmers. Copolymers having a wide range of physical and mechanical properties with varying rates of biodegradation can be prepared with glycolide and lactide and a variety of lactones, other lactides, cyclic carbonates, and lactams.

As with PG and PL homopolymers, the copolymers of lactide and glycolide are also subject to biodegradation because of the susceptibility of the aliphatic ester linkage to hydrolysis. However, biodegradation of the copolymers is normally faster than the homopolymers because copolymerization reduces the overall crystallinity of the polymer, thus giving the polymer a more open macrostructure for easier water penetration (2).

Extensive use has been found for polyurethanes in several in vivo biomedical applications such as blood catheters and artificial heart valves, due to their excellent blood contacting and mechanical properties. Poly-(ether-urethane) is the most widely used medical grade polyurethane, but for some purposes shorter and more predictable degradation is more desirable. Continuing this goal, biodegradable poly(ester-urethane) networks derived from lysine diisocyanate and degradable polyester blocks of lactide and glycolide have been synthesized (2).

2.1.2. Linear polyester amides (PEA)

The main reason for the synthesis of these biodegradable polyester amides is the introduction of reactive sites along the polymer chain. They can be readily modified with biologically active species (1). Unlike the ester bond, which can undergo hydrolysis under mildly basic conditions, such as the in vivo environment, the amide linkage is not easily hydrolyzed even under strong acidic

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or basic conditions. In vivo the only available route for cleavage of an amide bond is enzymatic. Attempting to improve the utility of polyamides in vivo biomedical applications hydrolysable bonds were incorporated as aliphatic ester linkages (mostly derived from lactides and glycolides) in such polymer backbone (1).

2.1.3. Branched oligoesters

For drug delivery low molecular weight star-like oligoesters are important. They are composed of lactic and glycolic acid branched with pentaerythritol (four arms), dipentaerythritol (six arms) and tripentaerythritol (eight arms) and even acrylic acid (15-20 branches). In today’s market only poly(ε-caprolactone) based star-shaped polymers and poly(lactide) based star-shaped polymers are available (3). The main advantage of branched polymers is the lower molecular weight of a single chain, which leads to a higher density of random coil and subsequently to a lower viscosity, which means better rheological properties and easier handling. Also they have a more suitable mechanism of the degradation process and less swelling, which is better for medical uses (4). At relatively low molecular weight, the viscosity of a star polymer is lower than its linear analog, however, the viscosity of the star polymer increases faster with molecular weight and exceeds that of the linear analog at some specific molecular weights. This molecular weight dependence occurs because the star polymer exhibits a reduced hydrodynamic volume compared to the linear polymer due to the higher segment density. However, a competing effect arises since the star polymer possesses restricted chain motion due to the constraint that one end of the arm is anchored to the star core (5). Practically, viscosity depends only on arm length, and is independent of the number of arms (5).

2.2. Interactions with hydrophilic medium

Swelling ratio is the ratio of extrudate diameter to die diameter in extrusion (6). It reflects the hydrophilicity of the polymer and is expressed by volume and bulk increase. Polymer hydrophilicity means compatibility with water, wetting and swelling in water (1). Swelling degree is evaluated using formula, which is given in experimental part.

In additional to chemical structure and hydrophobicity, swelling and degradation can be subject to either of following factors:

• pH of surrounding medium (usually the higher pH in alkaline regions, the faster polymer hydrolysis);

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• Permeability of polymer to water (porous materials degrade faster than nonporous ones) (1). Hydrophilic polymer surfaces generally enhance biocompatibility, whereas hydrophobicity may impart beneficial surface erosion properties to the polymer for controlled drug delivery application (1).

2.3. Biodegradation

Polymer properties relevant to biodegradation: • Molecular properties

Molecular weight (Mw) is an important parameter in polymer degradation and also the main criteria of assessing the extent of polymer degradation in its strict sense of chain scission. Also chirally regular polymers generally crystallize more readily than chirally irregular polymers (1).

• Crystallinity

Crystallinity is the long-range regular ordering of atoms and molecules in unit cells on a three-dimensional crystalline lattice (6). It generally improves polymer mechanical properties, but very high crystallinity leads to brittleness. Polymer chains within crystalline regions are tightly and regularly ordered, while chains contained in amorphous regions have lower chain density and greater degrees of randomness and free motion. For this reason, amorphous regions of semi crystalline polymers are generally more susceptible to degradation than crystalline regions.

• Macroscopic (bulk) properties

a) Polymer melting point (Tm) represents its phase transition from solid to liquid, but only crystalline regions undergo proper melt transition. Thus, the endothermic peak associated with melting transition is an indication of the degree of crystallinity.

b) Glass transition temperature (Tg) is the temperature at which polymers exhibit a transition between two specific polymer states – from glassy to rubbery. Below Tg polymer chains are less mobile, and the material is usually brittle. Above Tg the chains move, and the material is pliable, like rubber.

c) Polymer hydrophilicity is determined by its composition, and is a measure of how readily it is wetted by water (or swells or dissolves in water). Wetting by water is a key parameter in biodegradation, especially hydrolysis, because water is the universal solvent in biological medium. Thus, polymer surface hydrophilicity affects the rate of initial wetting (water adsorption), and bulk hydrophilicity governs the rate of water access to biodegradable bonds contained within the polymer bulk (1).

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2.3.1. Hydrolytic degradation

Hydrolytic degradation consists of three main steps:

1. Water adsorption. Its rate and extent is governed by hydrophilicity and porosity of polymer (1). Hydration of amorphous segments of the polymer occurs faster than with crystalline segments (2).

2. Water penetration. Water is absorbed by the polymer, and the hydrolysis begins. The rate of this process is influenced by polymer hydrophilicity, morphology, type and concentration of hydrolysable bonds. All these factors also determine whether degradation occurs primarily on the surface or throughout the bulk. This step is characterized by reduction of Mw, and concurrent changes in properties related to Mw such as viscosity and mechanical strength.

3. Erosion. Mass loss continues until the polymer bulk is completely broken down and disintegrated (1).

2.3.2. Surface and bulk biodegradation

Surface erosion occurs when water penetration into the polymer bulk is much slower than hydrolysis. In bulk erosion, penetration of water is much faster than the degradation reaction, hence degradation takes place at nearly equal rates throughout the polymer surface and bulk.

Dimensions of materials undergoing surface degradation are thus expected to decrease continually as degradation proceeds, but material properties should remain largely intact (if normalized to continuously diminishing dimensions). By contrast, materials undergoing bulk hydrolysis should show significant decrease in bulk Mw before any mass loss commences. Physical dimensions of the implanted device may remain constant, or more often increase due to swelling, until catastrophic disintegration occurs. This transition to mass corresponds to almost total loss of mechanical strength.

Bulk hydrolysis may be auto catalyzed by acidity of the degradation products, or changes in biodegradation environment. This effect may lead to faster degradation in larger devices, and inside the device bulk, due to slower release of soluble acidic degradation products in larger devices. The term “heterogeneous degradation” is used to describe this process, in contrast to “homogeneous degradation” which proceeds via uniform chain scission throughout the bulk (1).

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2.3.3. Evaluation of erosion

Erosion is the mass loss of a polymer matrix which can be due to the loss of monomers, oligomers or even pieces of non-degradable polymers. Erosion can be the result of biological, chemical or physical effects. From this definition it is obvious, that polymer degradation is a part of its erosion (2). The main model of evaluation of degradation involves the rate of Mw reduction, because Mw often decreases at a characteristic rate dictated by the nature and concentration of hydrolytically labile bonds, concentration of absorbed water, and morphology of the device (1).

Even though degradation is the most important aspect of the erosion (2), the main characteristic of erosion is mass loss. Evaluation of erosion is done using erosion degree (ED), which is calculated from formula given in experimental part.

2.4. Thermal analysis

Polymers are viscoelastic materials with strong time and temperature dependencies to their mechanical and diffusional properties (7). Thermal analysis refers to a variety of techniques in which a property of a sample is continuously measured as the sample is programmed through a predetermined temperature profile. Amongst the different techniques, differential scanning calorimetry (DSC) is widely used.

In a DSC experiment the difference in energy input to a sample and a reference material is measured while the sample and reference are subjected to a controlled temperature program. DSC requires two cells equipped with thermocouples in addition to a programmable furnace, recorder, and gas controller. A thermal analysis curve is interpreted by relating the measured property versus temperature data to chemical and physical events occurring sample.

In DSC the measured energy differential corresponds to the heat content (enthalpy) or the specific heat of the sample. It can be used for different measurements, also to determine the glass transition temperature to polymers (8). DSC defines the glass transition as a change in the heat capacity as the polymer matrix goes from the glass state to the rubber state. This is a second order endothermic transition (requires heat to go through the transition) so in the DSC the transition appears as a step transition and not a peak such as might be seen with a melting or crystallization transitions (9). Secondary transitions are generally attributed to one or more relaxation processes, such as rotation and/or oscillation of side chain, subgroups, and short segments of the main chain. The main or glass transition is thought to be due to the motion of longer segments of the main chain (10).

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The glass transition temperature (Tg) is the temperature at which a polymer chain possesses sufficient thermal energy that cooperative, segmental motion of the backbone occurs within the frequency domain of the experiment. Below the Tg, the polymer lacks mobility, but maintains the disordered state of the melt. As an amorphous polymer is cooled from the melt its free volume decreases since the thermal energy for chain mobility decreases. The Tg occurs, once the free volume shrinks such that cooperative motion of the backbone is prohibited. Thus, any variables that influence the polymer’s fractional free volume can affect the Tg. (5).

The glass temperature (Tg) of a given polymer depends on the rate of cooling, the pressure, molecular weight, structure and some other characteristics (orientation, crosslink density, impurity content, concentration etc.) (11, 7). Slower cooling rates in the DSC lead to lower measured values of the glass transition (7), because formation of an ordered system takes a certain amount of time (12). An increase of pressure on an amorphous material increases molecular crowding and interactions along with decreasing the entropy, so an increase of Tg is expected (11). For linear polymers Tg decreases regularly with increasing concentration of polymer chain ends (11). This Tg dependence on molecular weight is based on free volume theory, which declares that increased molecular motion is possible, when there is sufficient free volume, which is at Tg (7). Molecular structure also has a significant influence on Tg. For example, Tg increases with the size and number of substituents, the incorporation of ring structures in the chain raises the Tg value, etc. Polarity in the side group also increases Tg due to increased interchain and intrachain attractions. Crosslinking can cause a considerable increase in Tg too. The Tg value may be considerably lowered by plasticization (6). The branching polymer molecules dramatically influence the thermal properties. Long chain branching is utilized to control the rheological and processing properties, while short chain branching influences thermal behavior and mechanical properties (5).

2.5. Nano and micro particles

Nanoparticles are defined as being submicronic (<1 µm) colloidal systems generally made from polymers (biodegradable or not). They were first developed in the mid 1970s by Birrenbach and Speiser. Nanoparticles generally vary in size from 10 to 1000 nm. The drug is dissolved, entrapped, encapsulated, or attached to a nanoparticle matrix (13). Micro particles are particles between 1 and 100 µm in size. Nano and micro particles have a very high surface area to volume ratio, contributing to the powder's unique behavior (12). Drug nanoparticles consist of the drug and, optionally, of a biocompatible polymer, either biodegradable or non-biodegradable. Nanoparticles can be further classified into nanocapsules and nanospheres based on their structure. A nanocapsule particle consists of an oily core containing the lipophilic drug surrounded by a shell composed of

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the polymer. A nanosphere, however, has a matrix consisting of a random distribution of the drug and the polymer. The drug is either solubilised in the polymer matrix to form an amorphous particle or randomly embedded in the polymer matrix as crystallites (14, 4). In addition to physically stabilizing the drug nanoparticles, the polymers can also act as functional agents, leading to sustained release of the drug or drug release triggered by changes in environmental conditions, for example in pH level (14).

In recent years, biodegradable polymeric nanoparticles have attracted considerable attention as potential drug delivery devices, this is in view of their applications in controlling drug release, their ability to target particular organs/tissue, as carriers of oligonucleotides in antisense therapy, DNA in gene therapy, and in their ability to deliver proteins, peptides and genes through oral administration (13).

2.5.1. Particle size and zeta potential

Particle size and their zeta potential are the most important characteristics of nano particles. They both can be measured using Zetasizer Nano Series. The Zetasizer range of instruments provides the ability to measure three characteristics of particles or molecules in a liquid medium. These three fundamental parameters are:

1. Particle size; 2. Zeta potential; 3. Molecular weight.

By using the unique technology within the Zetasizer system these parameters can be measured over a wide range of concentrations. Particle size is the diameter of the sphere that diffuses at the same speed as the particle being measured. The Zetasizer system determines the size by first measuring the Brownian motion of the particles in a sample using Dynamic Light Scattering (DLS) and then interpreting a size from this using established theories. Brownian motion is defined as: “The random movement of particles in a liquid due to the bombardment by the molecules that surround them”. Particles suspended in a liquid are never stationary. An important feature of Brownian motion for DLS is that small particles move quickly and large particles move more slowly. The relationship between the size of a particle and its speed due to Brownian motion is defined in the Stokes-Einstein equation (15).

The results can be presented by intensity, volume and number distributions.

A potential exists between the particle surface and the dispersing liquid which varies according to the distance from the particle surface – this potential at the slipping plane is called the Zeta potential (15).

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Zeta potential is an important and useful indicator of surface charge which can be used to predict and control the stability of colloidal suspensions or emulsions, for example. The greater zeta potential the more likely the suspension is to be stable because the charged particles repel one another and thus overcome the natural tendency to aggregate. The measurement of zeta potential is often the key to understanding dispersion and aggregation processes in applications as diverse as water purification, ceramic slip casting and the formulation of paints, inks, cosmetics (15), pharmacy. Measured potential should be less than -30 mV or more than 30 mV for suspension or emulsion to be stable, in the ranges between -30 and 30 mV particles attracts each other and consequently aggregation occurs (16, 17).

Zeta potential is measured by using a combination of the two measurement techniques: 1. Electrophoresis;

2. Laser Doppler Velocimetry, sometimes called Laser Doppler Electrophoresis.

This method measures how fast a particle moves in a liquid when an electrical field is applied – i.e. its velocity. Once we know the velocity of the particle and the electrical field applied we can, by using two other known constants of the sample – viscosity and dielectric constant, work out the zeta potential (15).

2.5.2. Microparticulate systems

The term “microparticles” includes microspheres and microcapsules, the systems that differ in morphology and structure. They are in the range of size between 1 µm and 500 µm (4, 17, 18). Microspheres are broadly defined as (sometimes ideally spherical) particles, composed of one or more polymeric or other materials. Microcapsules are similar, but they are composed of a central core substance (active component or incipient) and a peripheral polymer wall (carrier, protective component) (18).

Microspheres may be formed from organic and inorganic starting materials, or from the corresponding organic- inorganic composites. Organic polymers encompass a much wider range of chemical structures compared with inorganic microspheres. From the view point of chemical structure, organic microspheres may be classified into naturally occurring and synthetic polymers, or biodegradable and nonbiodegradable polymers, and each category divided into different sub-divisions. In microcapsules, the protective layer (or wall) is usually an organic polymer (naturally occurring or synthetic), even for certain uses it may be an inorganic polymer or even a metal.

Among synthetic polymer microspheres, polystyrene, polyacrylamides and polymethacrylates are most widely used. In microcapsules a much wider range of polymers is used: polyamides, polyesters, polyanhydrides, polyurethanes, amino resins, polycyanoacrylates (18).

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Spray drying is an encapsulation technique employed mainly by the food and pharmaceutical industries. A substance to be encapsulated (the load) and a carrier (usually some sort of modified starch, mannitol etc.) are homogenized as a suspension in water (12). In the former process the core substance is dispersed in a solution of coating material, which is then atomized and the solvent dried off using heated air in a spray dryer. Heat-sensitive core substances can be coated by spray drying because exposure to elevated temperature is very short, normally ranging from 5 to 30 s (19). The drying time for droplets depends on the process conditions such as flow rate, pump rate, aspiration rate and heat. The temperature experienced by the droplets is considerably lower than the temperature of the drying air due to evaporative cooling. The dried powder is protected from overheating by rapid removal of solvent from the drying zone. The final product can be removed from the air stream by the use of cyclones or filters (20). Moisture-sensitive drugs can be encapsulated by using nonaqueous coating systems. However, the coating produced by spray drying tends to be rather porous which may make them adequate for taste-masking and other purposes but not for controlled release (19).

The mechanism of spray dryer function is quite simple. Air is blown into the drying chamber, having been preheated in the passage over a heat exchanger. When spray drying heat and mass transfer occur rapidly between the droplets and the surrounding hot air, it is because of the large surface area available for evaporation. The rate of drying is a complex function of feed rate, droplet size and distributions, coating solvent, inlet/outlet temperature, humidity, gas velocity, and other factors. As the solvent evaporates, it tends to deposit a spherical coating of solids as a skin around one or more core particles (19).

Microparticles are evaluated by these parameters: • Particle size, size distribution;

• Surface properties (opsonins, disopsonization); • Drug release kinetics (4, 17, 18).

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3. EXPERIMENTAL PART

3.1. MATERIALS

3.1.1. Objects

Preparation of drug carriers

All oligomers and polymers were synthesized in the laboratory of polymers and biomaterials of the department of pharmaceutical technology, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Czech Republic. They all were synthesized by the polycondensation method in the period August - October 2006. The composition of oligomers and polymers is different and is shown in tables 1 – 3. The composition and molecular weight of the polymers and oligomers was evaluated by Institute Synpo Ltd. in Pardubice, Czech Republic and is shown in table 5.

In original extent 16 polymers were chosen. One of them – acrylic acid branched polymer A1 was rejected from experiment, because it degraded on the very first day. It shows its high susceptibility for hydrolytic degradation. This feature may be useful for oral drug forms.

Table 1. Linear polyesters (PLGA 30:70, PLGA 50:50) and extended poly–(ester)-urethane (PEU2) – input reagents compositions.

Main chain Symbol

DLLA (mol %) GA (mol %)

Hydroxyl end

groups Chain extender

PLGA 30:70 30 70 - -

PLGA 50:50 50 50 - -

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Table 2. Linear polyester amides (PEA 1, PEA 2, PEA 3) – input reagents compositions.

Symbol SA (mol %) EA (mol %) AB (mol %) AMP (mol %) SnOct (mol %) PEA1 50 50 - - 0,02 PEA2 50 35 15 - 0,02 PEA3 50 35 - 15 0,02

Table 3. Branched oligoesters (P1, P3, P5, D0,5, D1, D2, T1, T3, T5) – input reagents compositions.

Branching monomer Symbol

Name Conc.

(%weight)

GA (% mol) DLLA (% mol)

P1 Pentaerythritol 1,0 100-P/2 100-P/2 P3 Pentaerythritol 3,0 100-P/2 100-P/2 P5 Pentaerythritol 5,0 100-P/2 100-P/2 D0,5 Dipentaerythritol 0,5 100-P/2 100-P/2 D1 Dipentaerythritol 1,0 100-P/2 100-P/2 D2 Dipentaerythritol 2,0 100-P/2 100-P/2 T1 Tripentaerythritol 1,0 100-P/2 100-P/2 T3 Tripentaerythritol 3,0 100-P/2 100-P/2 T5 Tripentaerythritol 5,0 100-P/2 100-P/2

Note: Used oligomers and polymers and their compositions. (Abbreviations: DLLA – DL- lactic acid, GA – glycolic acid, PEU - polyester urethane, BD – butandiol, HMDI – hexamethylene diisocyanate, PEA – polyester amides, SA – sebacic acid, EA – ethanolamine, AB – 2-amino 1-butanol, AMP – 2-amino 2-methyl 1-propanol, SnOct – stannous octoate).

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3.1.2. Chemicals

1. Citrate- phosphate buffer (pH 7,0) – preparation described in p.19

2. Poly(DL-lactide), made in Charles University, Faculty of Pharmacy in Hradec Kralove, Czech Republic.

3. Dichloromethane (DCM) p.a. Lachema a.s. Neratovice, M=84,93g/mol, ρ=23-24/25-36/37. 4. L-α lecithin – type II S, from soya beams, P-5638, Sigma Aldrich Prague, Czech Republic. 5. Polysorbate 80 – Lachema a.s., Czech Republic.

6. Terbinafine hydrochloride, Zentiva a.s., Czech Republic. 7. Mannitol pro infusiones, Roquette, France.

8. Polyacrylat-polyalcohol, Sigma-Aldrich, Prague, Czech Republic.

3.1.3. Instruments

1. Electronic balances KERN abs 220-4. 2. Piccolo ATC pH Meter HI 1280. 3. Biological Thermostat BT 120. 4. Vacuum drier SPT – 200.

5. Netzsch DSC Apparatus DSC 200 PC “Phox®” . 6. Automatic electro balances Cahn 26.

7. Zetasizer ZS Nano Series (Malvern Instruments Ltd., Worcestershire, Great Britain). 8. Homogenizator Diax 900 Heildolph max.8000-26000 rotation / min., 6.levels. 9. Magnetic stirrer Heidolph MR 3001 100-1250 rpm.

10. Ultrasound Sonorex super 10P Bandelin.

11. Mini Spray Dryer B-290 (Büchi Labortechnik AG, Flawil, Switzerland). 12. Microscope Olympus BX 51.

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4. INVESTIGATION

4.1. Investigation of swelling and erosion kinetics of newly synthesized polymeric and oligomeric drug carriers in buffer medium

Preparation of buffer solution

A citrate – phosphate buffer water solution was used for these standardized experiments. The pH value of 7,0 is intended to mimic slightly inflamed reaction, which naturally occurs when polymeric material is injected or implanted into the human body (4). The buffer was prepared using 0,1 M solution of monohydrate citrate acid and 0,2 M solution of dihydrate hydrogen phosphate disodium salt. For 1 liter of citrate acid- phosphate buffer was used 190 ml solution of citric acid (21,014 g/l) and 810 ml solution of hydrogen phosphate disodium salt (35,60 g/l). The pH of the prepared buffer was corrected by adding hydrogen phosphate disodium crystals to pH 7,0. The buffer’s pH was controlled by using pH-meter. 0,02 % sodium azide was used as an antimicrobial agent at the end of preparation of the buffer.

Preparation of samples

Samples for the study of swelling and erosion kinetics of newly synthesized polymeric and oligomeric drug carriers were prepared by weighing each of them using electronic balances KERN. All monolithic pieces of samples weighed 150 mg +/- 10% (in range from 135 mg to 165 mg). The shape of samples was irregular. 20 ml vials were also weighted by electronic balances KERN. Each sample was inserted into its vial and labeled.

Only three the most perspective samples were selected for thermal analysis. They all make one logical unit, are linear and consist from polylactic and polyglycolic acid copolymers. They all were prepared for thermal analysis by DSC Apparatus from 3, 7 and 14 days in buffer immersed samples after the swelling experiment. Three carriers were chosen for this research – PLGA 30:70, PLGA 50:50 and PEU2. Samples were weighted by automatic electro balances CAHN in the range of 0,2 mg – 1,0 mg. Weighted samples were inserted into also weighted Netzsch 100 DSC lids (made from aluminum) and sealed by special press. They were kept in dessicator in order to protect them from humidity.

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Investigation

The experiment was started by putting buffer (15 ml ± 1 ml) in the samples of carriers placed in the vials. Then the vials with samples were incubated at a physiologically relevant temperature 37 ºC in a biological thermostat for different time periods: 1, 3, 7, 14, 21 and 28 days. The weight changes were recorded in samples in swollen state and dried state. After weighing samples in the swollen state, samples were moved to a vacuum drier for 6-10 hours in 70 ºC, pressure about 38 mmHg (or 0,05 normal pressure 760 mmHg). Dried samples were weighed again and left in dessicators for further experiments.

The buffer of each sample was changed for a fresh one in periods of 1, 3, 7, 14 and 21 days. The weights are not presented in order not to complicate the results.

After weighting, the next step was to calculate the results in the form of swelling degree characteristic (SD) and erosion degree characteristic (ED), using these formulas:

1. Swelling degree (SD) was calculated using this formula:

MS– weight of sample in the swollen state (g); MD – weight of dried sample after swelling (g).

2. The degree of erosion (ED) was calculated using this formula:

MD – weight of dried sample after swelling (g); MO – weight of originally prepared sample (g).

The results of these calculations are shown in tables under figures 1 - 15. The experiments were doubled in variants a and b for each carrier, the average weight of samples a and b of the same carrier are shown in figures 1 - 15.

(%)

100 *

d d s

M

SD

=

−

(%)

100 *

1 _









−

=

o d o

M

ED

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4.2. Thermal analysis - Tg measured by the DSC method of some degraded carriers.

Thermal analysis was performed by Netzsch DSC Apparatus DSC 200 PC “Phox®” under given procedures. Temperature and heating rate ranges were selected under known characteristics of these polymers. Measurement conditions (table 5) were selected according to specific characteristics (melting temperature Tm, crystallinity etc.) for every carrier. Nitrogen was used as a protective gas, for cooling was used liquid nitrogen.

Values for PLGA 30:70, PLGA 50:50 and PEU2 are given in tables 6 - 8. The most typical plots are shown in figures 16 - 21.

The calculated averages of measured results in Tg and ∆Cp (change in heat capacity) are presented in tables 9 - 11.

4.3. Experimental study concerning nano and micro particles. Preparation and evaluation.

Nanosuspension was prepared taking 1 g of organic phase and 49 g of aqueous phase. 1g of organic phase contained 1 % polymer (0,01 g of poly(DL-lactide), made in Charles University, Faculty of Pharmacy in Hradec Kralove, Czech Republic). To some samples was added 10% active substance – terbinafine hydrochloride (0,001 g). The organic phase was prepared from 5 ml dichloromethane.

The aqueous phase was prepared by dissolving lecithin (75 %) and polysorbate 80 (25%) in water. Lecithin and polysorbate were used as emulsifying agents in this suspension.

Emulsion was made by using a homogenizer, and then the emulsion was diluted to 100 g by water and stirred for a few minutes until the organic phase was evaporated and the nanoparticles became solid.

Nanoparticles were analyzed by Zetasizer. Particle size, size distribution and zeta potential were detected by this apparatus.

Microparticles were prepared from one of the solutions used to analysis nanoparticles adding 5 % of mannitol to this solution. Mannitol was used as an inert carrier for making particles (17). Microparticles were prepared by mini spray drying B-290 by the mechanism mentioned above. Prepared microparticles were analyzed by microscope using analysis program FIVE. Microparticles size and size distribution are counted and evaluated by this program.

The purpose of these experiments was to get acquainted with apparatus, preparation of nanoparticles and microparticles, methods main principles of particulate systems analysis and evaluation of results. This experiment is very important also because it reflects possible abilities to use new obtain drug carriers as nanoparticles and microparticles.

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Results of this experiment are not given, because they don’t influence the conclusions or other results.

5. RESULTS AND DISCUSION

General considerations

The work was directed to characterize oligomeric and polymeric substances synthesized recently. Some of them were of original structure. Some items possessing similar molecular features were studied at Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Czech Republic in the past years. In numerous diploma theses the unusual behavior of linear and branched oligoesters has been documented. This behavior concerns the swelling time course characterized by one, two, or three extremes (picks) – maximal or minimal. Consequently the planned theme of this work was directed to verify this non-equilibrium swelling of recently synthesized oligomeric and polymeric potential drug carriers.

Swelling as a very important carrier property is accompanied with its degradation. Degradation, as the complex of various features, consists of molecular weight decrease by the chain scission mechanism and polymeric piece erosion. Erosion is usually defined as polymer body mass decrease. In some cases erosion is the main mechanism of drug prolonged release. Glass transition temperature is the unique thermal characteristic of each polymer in the amorphous physical state. In this temperature range molecule relaxation by chain segment motions commences. The polymer changes from the brittle to the plastic, viscoelastic or elastic form. For pharmaceutical and biopharmaceutical purposes it is important to be informed about abrupt increases of the diffusion coefficient in this polymeric material continuum.

The aim of this pilot study was to obtain sufficient amount of data about swelling and thermal characteristic time evolution. From this data it was our intention to be able to express a hypothesis about probable correlation between these two processes.

The nanoparticles and microparticles preparation and evaluation methods were in this thesis of informational and educational importance.

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1 DAY 3 DAY 7 DAY 14 DAY 21 DAY 28 DAY Swelling degree (SD) (%) 62,18929 134,8747 170,2820 370,9945 647,4137 833,7971 Erosion degree (ED) (%) 88,84830 62,15612 50,92275 32,86622 37,95811 26,96372

0 200 400 600 800 1000 V a lu e , % Days Swelling degree (SD) (%) Erosion degree (ED) (%)

5.1. Biodegradable carriers interactions with hydrophilic media –

swelling degree and erosion degree

Carriers with branched molecules

In the experiment in the whole extent 16 drug carriers was included. One of them, derived from polyacrylic acid was not suitable for this study because of its rapid solubility in buffer medium.

In this part figures and tables, where swelling and erosion are expressed in graphics and in percents, are presented.

Figure 1. Time course of swelling and erosion characteristics. Carrier P1, Average of samples A and B - 28 days placed in citrate-phosphate buffer pH 7,0

In figure 1 the results concerning swelling and erosion of the carrier signed as P1 are seen. This oligoester was branched in moderate degree. This was revealed by prof. Š. Podzimek from Synpo Institute, Pardubice, CZ, by the SEC-MALLS (Size-Exclusion Chromatography/ Multi-Angle Laser Light Scattering) analysis. Results of branching degree analysis are not presented here. Thermal characteristics data were also in higher values. This fact is an indication of the presence of linear molecules fraction in the blend of molecules differing in molecule constitution (architecture). Erosion proceeds very continuously, as well as swelling. On day 28 the sample pieces were swelled more than eight-times. The non eroded rest weight was of about one quarter of the whole initially measured weight. The erroneous value measured in the 21 day on carier P1-A-21 was probably influenced by instant disintegration of the sample. The fragmentation was caused probably by air bubbles expansion incorporated in the studied piece.

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1 DAY 3 DAY 7 DAY 14 DAY 21 DAY 28 DAY

Swelling degree (SD) (%) 13,579 32,677 41,969 27,17 8,5411 15,517

Erosion degree (ED) (%) 95,547 89,257 76,681 64,602 59,098 51,356

0 20 40 60 80 100 120 V a lu e , % Days Swelling degree (SD) (%) Erosion degree (ED) (%)

Figure 2 presents in whole very consistent results of unusual swelling behavior of carrier P3. This polymer is characterized by higher degree of branching (SEC MALLS study unpublished here) and lower glass transition temperature. The biphasic behavior is of interest, in the first phase proceeds swelling and after one week period interaction continues by the second deswelling phase. The mechanism of this unusual behavior is under study. Swelling degree is lower in comparison with previously described carrier. During 28 days two thirds of carrier eroded.

Carrier labeled as P5 contained unusually high concentration of the branching agent (5 % of pentaerythritol). This product of synthesis was branched in comparison with polymer P3 surprisingly in lower degree, parameters of molecular weight and glass transition were also in lower values. The results of swelling and erosion measurements are in the figure presented under number

0 20 40 60 80 100 120 140 Days V a lu e , % Swelling degree (SD) (%) Erosion degree (ED) (%)

Swelling degree (SD) (%) 12,064 29,568 132,96 60,39 35,871 32,753 Erosion degree (ED) (%) 98,218 96,183 84,744 57,297 45,146 33,833

1 DAY 3 DAY 7 DAY 14 DAY

21 DAY

28 DAY

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1 DAY 3 DAY 7 DAY 14 DAY 21 DAY 28 DAY Swelling degree (SD) (%) 193,2 541,3 1704, 910,7 338,7 377,2

-100 300 700 1100 1500 1900 V a lu e , % Days Swelling degree (SD) (%) Erosion degree (ED) (%)

6. The erosion was surprisingly slow, after 28 days only one half of initial mass eroded. Swelling degree values were very low also. In the time interval 7 days was detected sharp peek of maximum which had value of 42 %. After the second phase of rapid deswelling, the process continues to the 21 days interval, at which seems located non marketed minimum.

Figure 4. Time course of swelling and erosion characteristics. Carrier D0.5, Average of samples A and B - 28 days placed in citrate-phosphate buffer pH 7,0

Material named as D0.5 was synthesized from reagent agents mixture containing only 0,5% of dipentaerythritol. This low molecular weight oligoester was constituted of linear molecules, branched were presented in low concentration. Glass transition temperature was also low (18°C). The behavior of this sample is demonstrated in the figure 4. Erosion of this material was unusually rapid. Pieces eroded practically in three days, the rests persisted very long. This unique and very interesting behavior is influenced by the higher resistance of branched molecules fraction to the hydrolysis. At the interval of 7 days maximal extent of swelling was detected.

Figure 5. Time course of swelling and erosion characteristics. Carrier D1, Average of samples A and B - 28 days placed in citrate-phosphate buffer pH 7,0

0 200 400 600 800 1000 1200 Days Swelling degree (SD) (%) Erosion degree (ED) (%)

1 DAY 3 DAY 7 DAY 14 DAY 21 DAY 28 DAY V a lu e , %

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0 50 100 150 200 V a lu e , % Days Swelling degree (SD) (%) Erosion degree (ED) (%)

On the figure under number 5, swelling and erosion of the oligoester D1 with low value of glass transition temperature (16°C) is presented. The fraction of branched molecules was greater. This is evidenced via erosion course. Three quarters of material eroded quickly on the first day. The continuation of the process by slow erosion exhibited behavior of branched molecules. After 28 days the non eroded rest represents only 18 % of the initial samples weight. After two weeks maximum of the degree of swelling was revealed. At this point the samples had been imbibed by ten fold of initial weight by buffer medium. This behavior is typical for linear or in small extent branched molecules.

Figure 6. Time course of swelling and erosion characteristics. Carrier D2, Average of samples A and B - 28 days placed in citrate-phosphate buffer pH 7,0

Carrier D2 differs from the previously two in the carrier range (figure 6). The swelling degree decreased more gradually from 190% in the 1 day to 33% in 28 day. After very rapid swelling the process continued by slow deswelling (shrinking). This behavior was not founded in the literature. The erosion process was gradual. The first day was accompanied by rapid erosion of one quarter of material, then the kinetics approaches the pseudo zero order type. The rest represents only 18% of pieces after 28 days period.

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0 200 400 600 800 1000 V a lu e , % Days Swelling degree (SD) (%) Erosion degree (ED) (%)

1 DAY 3 DAY 7 DAY 14

DAY 21 DAY 29 DAY Swelling degree (SD) (%) 13,321 23,211 86,338 51,998 48,642 27,845

0 50 100 150 V a lu e , % Days Swelling degree (SD) (%) Erosion degree (ED) (%)

Figure 7. Time course of swelling and erosion characteristics. Carrier T1, Average of samples A and B - 28 days placed in citrate-phosphate buffer pH 7,0

This and following two polymers were branched by tripentaerythritol and were of higher molecular weight and high degree of branching. Figure number 7 concerns the behavior of polymer T1. For this polymer increasing trend of swelling degree is typical. At the 21 day interval was maximum representing eight fold weight body increase. Pieces eroded gradually, after 1 day burst periods when 18% of material eroded.

Sample evaluation of the T3 carrier was influenced by disintegration of one piece on the 3rd day. About this carrier is possible to declare typical two phase behavior. In the first 7 days was detected swelling to the maximum, then deswelling was revealed.

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1 DAY 3 DAY 7 DAY 14 DAY 21 DAY 29 DAY Swelling degree (SD) (%) 10,516 40,669 148,40 40,184 24,143 22,774 Erosion degree (ED) (%) 97,779 95,318 87,445 71,098 54,282 43,299

0 50 100 150 200 V a lu e , % Days Swelling degree (SD) (%) Erosion degree (ED) (%)

1 DAY 3 DAY 7 DAY 14

DAY 21 DAY 29 DAY Swelling degree (SD) (%) 136,53 207,64 203,88 187,84 155,39 285,79

0 100 200 300 400 V a lu e , % Days Swelling degree (SD) (%) Erosion degree (ED) (%)

The carrier T5 behaved very similarly from the view of swelling course. From 148% as maximal value of the swelling characteristic on the 7th day swelling degree decreased to 40% on 14th day and then on 24% in the following one week interval. In the comparison the erosion rate, sample T5 eroded slowly - 43% in the end of experiment against 27% at T3 polymer.

Carriers with linear molecule

Figure 10. Time course of swelling and erosion characteristics. Carrier Average PLGA(30:70), Average of samples A and B - 28 days placed in citrate-phosphate buffer pH 7,0

Linear oligoester PLGA 30:70 contained a very high concentration of more hydrophilic glycolic acid in copolymer with DL-lactic acid. This carrier swells to a higher extent, as is seen in

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figure 10. After first three days changes of swelling degree were small. After this near equilibrium stage the rests of oligomer after 21 days of experiment swelled more. Erosion rate of this low molecular weight carrier was very high, after 28 days the rests were only about 5%.

Figure 11. Time course of swelling and erosion characteristics. Carrier PLGA(50:50), Average of samples A and B - 28 days placed in citrate-phosphate buffer pH 7,0

Other linear oligoester PLGA 50:50 importantly differs from the mentioned above, as is seen in the figure 11. Molecular weight and glass transition temperature were lower than PLGA 30:70. After a 7 day period a sharp maximum with 558% of swelling degree value was revealed. This marked non equilibrium behavior is interesting from the theoretical point of view. Erosion of this oligomer lasts practically only two weeks, the rests of weight was under 10% of the initial mass value.

Figure 12. Time course of swelling and erosion characteristics. Carrier PEU2, Average of samples A and B - 28 days placed in citrate-phosphate buffer pH 7,0

0 200 400 600 Days V a lu e , % Swelling degree (SD) (%) Erosion degree (ED) (%)

-50 0 50 100 150 Days V a lu e , % Swelling degree (SD) (%) Erosion degree (ED) (%)

Swelling degree (SD) (%) 7,1410 -12,5170 25,7583 29,9101 43,9272 55,5843 Erosion degree (ED) (%) 96,3781 142,091 86,0376 68,5139 48,4820 36,1052 1 DAY 3 DAY 7 DAY 14 DAY 21 DAY 29 DAY

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1 DAY 3 DAY 7 DAY 14 DAY 21 DAY 29 DAY Swelling degree (SD) (%) 1,9263 28,455 30,878 49,814 70,573 21,540

0 20 40 60 80 100 V a lu e , % Days Swelling degree (SD) (%) Erosion degree (ED) (%)

The chain extension of PLGA oligomer by the two consecutive reactions with butandiol and hexamethylene diisocyanate with catalyst leads to very different swelling behavior. After 1 day oligomeric pieces from polyester urethane swells very slowly (figure 12). Swelling degree characteristics increased gradually to the value of 56% after 28 days of the hydrolysis period. Erosion rate was markedly slower in comparison with non extended oligoesters. After 28 days the erosion rate was of the value of 36%. Chain extension reaction may influence very significantly the interactions of modified materials with hydrophilic medium.

Polyester amides

Polyester amides are compounds which are very rarely used as drug carriers. Their degradation behavior is not sufficiently described. Oligomers PEA 2, PEA 3 and others not studied in this thesis had original structure. All of the polyester amides included in this study were semicrystalline. Parameters of their molecular weight will be studied, thermal behavior also.

Figure 13. Time course of swelling and erosion characteristics. Carrier PEA1, Average of samples A and B - 28 days placed in citrate-phosphate buffer pH 7,0

PEA1 swelled similarly to some other above mentioned carriers, as is presented on the figure 13. In the 21 day period a maximum peek was detected with a relatively high degree of swelling (71%). Erosion proceeds gradually, after 28 days of experiment eroded and disappeared about 40% of originally used samples.

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1 DAY 3 DAY 7 DAY 14 DAY 21 DAY 29 DAY Swelling degree (SD) (%) 2,8507 7,6740 9,4471 20,170 44,971 29,987

0 50 100 150 V a lu e , % Days Swelling degree (SD) (%) Erosion degree (ED) (%)

Drug carrier PEA2 behaved very differently from the PEA1 sample. On the figure 14 very low swelling degree and practically inertness in the aqueous medium is seen.

Copolymer PEA3 (figure 15) behaved differently. After a 7 day period swelling started to the maximum in the 21 day interval corresponding to the swelling degree value of 45%. Erosion rate of this material during 28 day period was very low. A possible solution in the future is interactions with enzymes.

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5.2. Thermal behavior (glass transition temperature) of degraded carriers

For this diploma work only three carriers were selected – PLGA 30:70, PLGA 50:50, and PEU2. These oligomers were typical by linear short chains. They had different values of glass transition temperature. They differ in degradation velocity in buffer medium very significantly. Table 4. Conditions for thermal analysis for PLGA 30:70, PLGA 50:50 and PEU2 carriers.

Carrier

code Stage Temperature (ºC) Time (mm:ss)

Heating/ Cooling rate (K/min) Initial +20 - - PLGA 30:70 PLGA 50:50 Cooling -20 - 30 Isothermal - 03:00 - Heating +60 - 10 Cooling -20 - 30 Isothermal - 03:00 - Heating +60 - 10 Final +70 - - PEU2 Initial +20 - - Cooling 0 - 30 Isothermal - 03:00 - Heating +110 - 10 Cooling 0 - 30 Isothermal - 03:00 - Heating +110 - 10 Final +120 - -

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The thermogram curve evaluation process is demonstrated in consequently fixed stages presented on figures 16 - 21.

Figure 16. Typical DSC-measurements recording. Full line – enthalpic changes of sample; dashed line – enthalpic changes of standard; sample – PLGA 50:50, 7A2

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Figure 17. Typical DSC-measurements recording. Full line – enthalpic changes of sample; dashed line – enthalpic changes of standard; sample – PEU-2, 14A1

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Figure 18. DSC-recording of heating steps – first and second run. Extract from DSC curve to indicate glass transition, sample – PEU2, 14 A2

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Figure 19. DSC-recording of heating steps – first and second run. Extract from DSC curve to indicate glass transition, sample - PLGA 30:70, 1B1.

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Table 5. Parameters of molecular weight and thermal behavior of carriers.

Note: Mn – number avarege molecular weight, Mw – weight average molecular weight, MP - molecular weight of fraction in the peak (maximum) of graphic plot from SEC (GPC) measurement, Mz – weight average of large molecules, Mz+1 – wight of the largest molecule, Polydispersity index express proportion Mw/Mn, Tg1- glass transition temperature, ∆Cp – change of heat capacity. Table 5 presents main characteristics of all examined drug carriers. The main aspect presented in this table is molecular weight, which illustrates that all polymers have smaller molecular weight than polymers, which now are used in practice. There is no data for polyesteramides, because these polymers were not soluble in the same solutions as others, so some other methods are needed for these polymers.

The various parameters of glass transition temperature used in description of the molecule relaxation process are presented in tables 6 – 8. The middle value was chosen. The averages of doubled measurements for each selected drug carrier and each time interval are presented in tables 9 – 11. Carrier Mn Mw MP Mz Mz+1 Poly dispersity Tg1 ∆Cp 1P 2944 8422 8466 14568 20771 2,86 26,6 0,600 3P 2440 5231 4659 8342 11778 2,14 22,2 0,416 5P 1711 2869 2610 4084 5397 1,68 12,7 0,453 0,5D 1679 4229 3132 7635 11042 2,52 17,7 1,443 1D 1889 5306 5850 9669 13825 2,81 16,1 0,912 2D 2645 6174 6287 9271 12391 2,33 17,9 1,618 1T 2877 12035 16849 26284 41062 4,18 25,3 0,656 3T 3355 13334 9074 27152 43224 3,97 21,7 0,508 5T 3016 8555 6379 15025 22625 2,84 17,2 0,471 PLGA 5/5 1833 4086 4049 6494 8818 2,23 17,0 0,902 PEU 2 2866 6664 5829 12212 18822 2,33 10,1 0,969 PLGA 3/7 2414 5611 6061 8532 11121 2,32 23,4 1,220 PEA 1 -3,1 4,057 PEA 2 -10,7 0,456 PEA 3 -8,5 4,531

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Table 6. Main characteristics of DSC- measurements. Carrier sample PEU2.

Day Sample Onset (ºC) Mid (ºC) Inflection

(ºC) End (ºC) ∆Cp 103 J/(gK) 1 A1/1 21,5 27,1 24,0 32,6 428 A1/2 21,5 27,1 24,8 32,6 298 A2/1 37,2 37,3 39,7 37,4 616 A2/2 21,0 27,6 22,7 34,1 474 B1/1 21,6 26,8 23,3 32,2 403 B1/2 21,1 26,5 25,0 31,9 464 B2/1 21,2 26,7 23,3 32,3 406 B2/2 21,1 25,7 25,0 30,2 394 AB/1 26,9 412 AB/2 26,7 444 3 A1/1 22,3 27,2 26,0 32,2 398 A1/2 20,4 26,6 23,1 32,8 578 A2/1 21,2 28,5 25,1 35,8 288 A2/2 20,8 28,3 25,1 35,7 296 B1/1 23,6 28,1 25,8 32,6 331 B1/2 21,3 27,0 23,6 32,8 435 B2/1 22,6 26,9 25,8 31,3 334 B2/2 20,1 27,1 23,6 34,1 741 AB/1 27,7 338 AB/2 27,3 513 7 A1/1 20,3 27,1 24,8 34,0 584 A1/2 20,4 25,9 23,4 31,4 506 A2/1 21,0 28,0 24,1 35,0 454 A2/2 20,1 26,8 23,2 33,5 538 B1/1 24,1 28,2 25,9 32,3 344 B1/2 22,2 26,6 23,6 31,0 443 B2/1 23,1 27,2 25,3 31,2 335 B2/2 22,2 27,8 24,8 33,3 491 AB/1 27,8 429 AB/2 26,8 495

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14 A1/1 22,3 27,5 25,3 32,8 445 A1/2 22,7 28,2 24,8 33,7 476 A2/1 21,5 27,4 24,5 33,2 429 A2/2 20,5 26,2 24,3 31,9 538 B1/1 23,7 28,2 26,7 32,8 327 B1/2 22,5 28,0 24,3 33,5 388 B2/1 22,6 27,4 25,1 32,2 388 B2/2 21,7 27,0 22,9 32,3 418 AB/1 27,6 397 AB/2 27,4 455

Table 7. Main characteristics of DSC- measurements. Carrier sample PLGA 30:70.

(ºC) End (ºC) ∆Cp 103_J/(gK) 1 A1/1 -10,9 -4,0 -5,7 3,0 418 A1/2 -8,5 -4,4 -4,6 -0,4 346 A2/1 -8,9 -3,7 -3,7 1,5 241 A2/2 -8,7 -3,9 -4,2 1,0 218 B1/1 -4,0 -0,9 -1,9 2,3 324 B1/2 -6,5 -1,6 0,6 3,4 343 B2/1 -7,2 -2,6 -0,8 2,0 281 B2/2 -7,6 -3,4 -5,3 0,7 275 AB/1 -2,8 316 AB/2 -3,3 296 3 A1/1 -9,2 -4,6 -5,3 0,0 304 A1/2 -8,6 -4,3 -5,8 0,0 283 A2/1 -7,0 2,1 4,3 11,1 218 A2/2 -8,8 -0,8 1,3 7,2 201 B1/1 0,6 4,7 4,9 8,8 208 B1/2 -8,3 -1,1 -5,6 6,1 217 B2/1 -7,5 0,6 -0,7 8,6 244 B2/2 -8.6 -2,7 -6,2 3,3 256 AB/1 0,7 243 AB/2 -2,2 239

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7 A1/1 -8,2 -3,9 -5,4 0,5 318 A1/2 -8,1 -3,8 -3,2 0,6 290 A2/1 -9,1 -4,6 -4,4 -0,1 326 A2/2 -9,1 -4,5 -4,3 0,0 342 B1/1 -7,0 -4,5 -4,4 -2,0 116 B1/2 -7,2 -5,0 -5,1 -2,9 104 B2/1 -8,3 -3,4 -5,2 1,5 183 B2/2 -8,1 -3,3 -4,4 1,6 161 AB/1 -4,1 236 AB/2 -4,2 199 14 A1/1 3,2 7,8 6,4 12,4 161 A1/2 2,9 7,4 6,4 11,9 162 A2/1 1,2 6,6 5,7 12,0 204 A2/2 2,9 7,0 6,1 11,2 230 B1/1 1,9 7,1 5,1 12,2 179 B1/2 0,6 5,7 4,4 10,7 206 B2/1 -0,4 5,3 2,6 11,1 323 B2/2 0,9 6,5 3,4 12,1 297 AB/1 6,7 217 AB/2 6,7 224

Table 8. Main characteristics of DSC- measurements. Carrier sample PLGA 50:50.

(ºC) End (ºC) ∆Cp 103_J/(gK) 1 A1/1 -7,0 -2,6 -3,6 1,8 208 A1/2 -8,3 -3,4 -5,1 1,5 246 A2/1 -10,9 -3,7 -6,6 3,4 235 A2/2 -8,5 -4,5 -5,7 -0,6 197 B1/1 -8,3 -2,6 -5,1 3,1 237 B1/2 -8,1 -2,7 -6,3 2,6 193 B2/1 -8,9 -2,4 -2,7 4,1 227 B2/2 -8,4 -3,4 -6,4 1,6 199 AB/1 -2,8 227

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AB/2 -3,5 209 3 A1/1 -9,3 -2,9 -6,8 3,5 291 A1/2 -8,4 -3,1 -5,1 2,2 332 A2/1 -8,3 -3,3 -5,3 1,6 204 A2/2 -8,4 -3,7 -4,6 1,0 191 B1/1 -29,5 (-29,4) -27,8 -29,3 (15) (10,9) (10,2) 12,0 9,5 (43) B1/2 -9,7 -2,7 -5,9 4,4 155 B2/1 -7,5 0,6 -0,7 8,6 244 B2/2 -8,6 -2,7 -6,2 3,3 256 AB/1 -1,9 246 AB/2 -3,1 234 7 A1/1 -9,3 -4,9 -6,6 -0,5 267 A1/2 -8,7 -2,8 -5,0 3,2 215 A2/1 -9,2 -3,1 -3,8 2,9 267 A2/2 -8,7 -3,2 -5,7 2,2 284 B1/1 2,7 4,7 3,9 6,6 121 B1/2 -2,3 0,8 -1,0 3,8 131 B2/1 -8,0 -2,3 -1,8 3,3 331 B2/2 -8,5 -4,2 -5,3 0,1 296 AB/1 3,4 288 AB/2 2,6 247

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Table 9. Carrier glass transition parameters (Tg and ∆Cp) time course; carrier PEU 2; sample placed in citrate-phosphate buffer pH 7,0

Table 10. Carrier glass transition parameters (Tg and ∆Cp) time course; carrier PLGA 30:70; sample placed in citrate-phosphate buffer pH 7,0

Table 11. Carrier glass transition parameters (Tg and ∆Cp) time course; carrier PLGA 50:50; sample placed in citrate-phosphate buffer pH 7,0

Day Sample Tg Mid [ºC] ∆Cp 103 J/(gK)

1 AB/1 26,9 412 AB/2 26,7 444 3 AB/1 27,7 338 AB/2 27,3 513 7 AB/1 27,6 429 AB/2 26,8 495 14 AB/1 27,6 397 AB/2 27,4 455

1 AB/1 -2,8 316 AB/2 -3,3 296 3 AB/1 0,7 243 AB/2 -2,2 239 7 AB/1 -4,1 236 AB/2 -4,2 199 14 AB/1 6,7 217 AB/2 6,7 224

1 AB/1 -2,8 227 AB/2 -3,5 209 3 AB/1 -1,9 246 AB/2 -3,1 234 7 AB/1 3,4 288 AB/2 2,6 247

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In tables 6-11 all parameters of measured values are presented. For evaluation of glass transition temperature middle temperature is taken. Changes in heat capacity reflects fixed change in heat flow.

In the tables 9 and 6 two repeated measures of the doubled samples of the carrier PEU2 are presented. It is seen that the two paralelly tested samples behaved in the standard way. The glass transition temperature was nearly constant during two weeks degradation process, while swelling parameter systemically rose.

Other situation is in the case of oligoester PLGA 30:70. As is presented in tables 10 and 7, the Tg value varied to a small extent and then abruptly raised between 7 days and 14 days from –4°C to +7°C. This change is not copied by the plateau course in the swelling of the same carrier. The repeated measurements of doubled samples behave reproducibly.

In the tables 11 and 8 glass transition temperatures of the oligoester PLGA 50:50 are presented. This copolymer made from equimolar mixture of glycolic and DL-lactic acids possessed constant glass transition temperature in the course of the first three days of experiment. Between 3 and 7 day glass transition temperature rose from –2.5°C to +3.0°C. It seems that this behavior is also different from swelling. The raising of Tg value is possible to explain by neutralization reaction of newly generated carboxyl end groups. It is obvious that polarization of polyelectrolyte leads to Tg value raising. The results from day 14 are not presented, because carrier was degraded too much. No correlation between glass transition temperature and swelling behavior was noticed. It might be a proove that even they both result in molecular relaxation, they are the manifestations of a different mechanism. Swelling extent is influenced by osmotic phenomena and glass transitions occur due to increasing heat flow and are influenced by molecular polarization.