Interactions of biodegradable drug carriers with ...

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

2007

- 2 -

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.

- 3 -

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

- 4 -

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

9. Literature………………………………………………….......................................................49

- 5 -

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.

- 6 -

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

Polylactide Polyglycolide

- 7 -

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

- 8 -

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);

• Shape and geometry of polymer body (degradation is a function of surface area);

- 9 -

• 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).

- 10 -

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).

- 11 -

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).

- 12 -

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

- 13 -

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).

- 14 -

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).

- 15 -

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).

- 16 -

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 - -

PEU 2 50 50 BD HMDI

- 17 -

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



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



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).

- 18 -

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.

13. PC program analysis® FIVE (Soft Imaging System GmbH, Münster, Germany).

- 19 -

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.

- 20 -

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

M M SD

−=

(%) 100 * 1

− − =

o

d o

M

M M ED

- 21 -

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.

- 22 -

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.

- 23 -

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

Valu

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.

- 24 -

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



0

20

40

60

80

100

120

Valu

e, %

Days



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.

Figure 3. Time course of swelling and erosion characteristics. Carrier P5, Average of samples A and

B - 28 days placed in citrate-phosphate buffer pH 7,0

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

Valu

e, %





1 DAY 3 DAY 7 DAY14

DAY 21

DAY 28

DAY

Figure 2. Time course of swelling and erosion characteristics. Carrier P3, Average of

samples A and B - 28 days placed in citrate-phosphate buffer pH 7,0

- 25 -

1 DAY 3 DAY 7 DAY14

DAY21

DAY28

DAY

Swelling degree (SD) (%) 193,2 541,3 1704, 910,7 338,7 377,2


-100

300

700

1100

1500

1900

Valu

e,

%

Days



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


0

200

400

600

800

1000

1200

Days





1 DAY 3 DAY 7 DAY14

DAY

21

DAY 28

DAY

Valu

e, %

- 26 -




0

50

100

150

200

Valu

e,

%

Days



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.

- 27 -




0

200

400

600

800

1000

Valu

e,

%

Days



1 DAY 3 DAY 7 DAY14

DAY21

DAY29

DAY



0

50

100

150

Valu

e,

%

Days



Figure 7. Time course of swelling and erosion characteristics. Carrier T1, Average of samples A


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.

Figure 8. Time course of swelling and erosion characteristics. Carrier T3, Average of samples A


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.

- 28 -




0

50

100

150

200

Va

lue

, %

Days



1 DAY 3 DAY 7 DAY14

DAY21

DAY29

DAY



0

100

200

300

400

Valu

e,

%

Days



Figure 9. Time course of swelling and erosion characteristics. Carrier T5, Average of samples A and B - 28 days placed in citrate-phosphate buffer pH 7,0

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

- 29 -

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

Valu

e, %






-50

0

50

100

150

Days

Valu

e, %



Swelling degree (SD) (%) 7,1410 -12,5170 25,7583 29,9101 43,9272 55,5843



- 30 -




0

20

40

60

80

100

Valu

e, %

Days



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


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.

- 31 -




0

50

100

150

Valu

e, %

Days






0

50

100

150

Valu

e, %

Days





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.

- 32 -

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


Heating +60 - 10

Final +70 - -

PEU2 Initial +20 - -

Cooling 0 - 30


Heating +110 - 10

Cooling 0 - 30


Heating +110 - 10

Final +120 - -

- 33 -

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

- 34 -

Figure 17. Typical DSC-measurements recording. Full line – enthalpic changes of sample; dashed line –

enthalpic changes of standard; sample – PEU-2, 14A1

- 35 -

Figure 18. DSC-recording of heating steps – first and second run. Extract from DSC curve to indicate

glass transition, sample – PEU2, 14 A2

- 36 -

Figure 19. DSC-recording of heating steps – first and second run. Extract from DSC curve to indicate

glass transition, sample - PLGA 30:70, 1B1.

- 37 -

Figure 20. Result of DSC measurement – characteristics of glass transition. Sample – PLGA 50:50, 7A2

- 38 -

Figure 21. Result of DSC measurement – characteristics of glass transition. Sample – PLGA 30:70, 3A2

- 39 -

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

- 40 -

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

- 41 -

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

- 42 -

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

- 43 -

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

- 44 -

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

- 45 -

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.

- 46 -

6. CONCLUSIONS

Interactions of the set of fifteen recently synthesized biodegradable oligomeric and polymeric

drug carriers with hydrophilic medium were characterized from the view of point of swelling and

erosion. Conclusions cover swelling and degradation evaluation and model of these characteristics,

also deny correlation between glass transition temperature and swelling and furthermore give an

idea of possible further studies.

1. The gradual erosion course without burst-effect and lag-time was typical for the most of

polyester and polyester urethane carriers used. Some polyester amides eroded very slowly.

2. Branched oligoesters erode slower than linear ones, because of the branched parts of the

molecules.

3. Chain extended polyester resulted in polyester-urethane erodes by the slower velocity than

analogous polyester prepolymer. Degradation process was suppressed by urethane bonds

introduction.

4. Swelling behavior is a very sensitive parameter to the polymer-medium interactions. Small

modification in molecular structure lead to great changes in swelling degree values course.

5. Some of materials swell in initial period lasting from hours to weeks, then interaction with

the medium continue by deswelling of shrinking process. The mechanism of this unusual

behavior is currently under study.

6. Polyester amides differ very greatly in their possibility interact with hydrophilic medium.

Possible explanation is that steric hindrance or obstructing of ester bonds hydrolysis was

resulted by side methyl groups in the vicinity of these ester bonds.

7. Glass transition temperature measured by DSC method is parameter of oligomeric material

which does not corresponds to swelling behavior. The changes of this characteristic during

degradation process are mainly in relation with oligomer molecule polarization resulted

from neutralization of carboxyl end groups by buffer sodium cations.

8. Between glass transition temperature and swelling no potential correlation was revealed. It

shows that these molecular relaxation parameters are the manifestations of a different

mechanism. Swelling extent is influenced by osmotic phenomena, whilst glass transitions by

molecule polarization effects.

9. Introductory to nanoparticulate and microparticulate systems preparation and evaluation

methods were an integral part of the thesis and reveal possible application of new obtained

drug carriers.

- 47 -

7. SUMMARY

The aim of this thesis “Interactions of biodegradable drug carriers with hydrophilic medium”

is the study of polymer-hydrophilic medium interactions from the point of view of swelling, erosion

and glass transition. In the theoretical part attention was paid to basic information about polyester

amides and polyesters with molecule linear, branched, and chain extended. In this part, some basic

relations were used, concerning various aspects of biodegradation of polymers, thermal analysis of

amorphous phase of polymers and pharmaceutical particulate systems. The main part of the thesis is

focused on experiment. Fifteen recently synthesized oligomeric and polymeric carriers were studied

in the aspect of their swelling and erosion course. Some of these oligoesters with molecular linear

constitution were evaluated during in vitro degradation process via glass transition tested by DSC

method measurements. No correlation signs between swelling kinetics and glass transition

temperature values course were found. These two molecular relaxation parameters are the

manifestations of different mechanism. Swelling extent is influenced by osmotic phenomena, whilst

glass transitions by molecule polarization effects.

This work is one of the first steps examining these drug carriers. The results of this work will

be used in choosing perspective drug carriers and in further researches.

- 48 -

8. SANTRAUKA

Šio darbo „Biodegraduojančių vaistų nešėjų sąveika su hidrofiline terpe“ tikslas ištirti

polimero bei hidrofilinės terpės sąveiką atsižvelgiant į brinkimą, eroziją bei stiklėjimo temperatūros

pokyčius, galimą ryšį tarp polimero brinkimo ir stiklėjimo temperatūros, apžvelgti nano ir mikro

dalelių paruošimo bei įvertinimo teoriją. Literatūros apžvalgoje dėmesys skiriamas informacijai

apie poliesteramidus ir linijinius, šakotus ar praplėstos grandinės poliesterius. Šioje dalyje aptariami

įvairūs polimerų biodegradacijos, amorfinės fazės polimerų terminės analizės aspektai, taip pat

nano bei mikrodalelės kaip vaisto forma. Pagrindiniai darbo uždaviniai yra ištirti penkiolika

neseniai susintetintų oligomerinių bei polimerinių vaistų nešėjų atsižvelgiant į jų brinkimą bei

eroziją, įvertinti kai kurių linijinių oligoesterių degradacija in vitro atsižvelgiant į stiklėjimo

temperatūrą matuojant diferencinės skenuojančios kalorimetrijos metodu. Atkreiptas dėmesys į

galimą ryšį tarp stiklėjimo temperatūros ir brinkimo kinetikos, kurio nepastebėta; manoma, kad dėl

to, jog molekulės grandinės atsipalaidavimas pasireiškia dėl skirtingų mechanizmų. Brinkimo

laipsnis priklauso nuo osmotinių reiškinių, o stiklėjimo temperatūrai įtaką daro molekulės

poliarizacija.

Šis darbas yra pradinis etapas tiriant šiuos polimerus. Šio darbo rezultatai padės atrenkant

perspektyvias medžiagas tolimesniems tyrimams, prognozuojant galimas jų panaudojimo

galimybes.

- 49 -

9. LITERATURE

1. Arshady Reza: Biodegradable Polymers. London, 2003, PBM series vol. 2., pp. 2-385

2. Domb Abraham J., Kost Joseph and Wiseman David M.: Handbook of Biodegradable

Polymers. Harwood academic publishers, 1996, pp. 3-27, 129.

3. Anon.: Synthetic biopolymers catalog, JCS biopolytech, Toronto, Canada, Jan 2006.

4. Milan Dittrich: private communication, Charles University, Faculty of Pharmacy in

Hradec Kralove, Czech Republic.

5. McKee Mattew G., Unal Serkan, Wilkes Garth L., Long Timothy E.: Branched polyesters:

recent advances in synthesis and performance. Prog. Polym. Sci. 2005.

6. Alger Mark S. M.: Polymer science dictionary. London and New York, 1989.

7. Campbell D., White J. R.: Polymer Characterization, Physical Techniques, Academic

Publishers 1989, chapter 3.

8. Hatakeyama T., Zhenhai Liu: Handbook of Thermal Analysis. Wiley 1998, pp.17-24

9. Arlon Application Notes Measuring and Understanding Tg (glass transition temperature).

http://www.arlon-med.com/Everything%20You%20Wanted.pdf

10. Turley S. G., Keskkula J.: A survey of multiple transitions by dynamic mechanical

methods, J. Polym. Sci.:part C, No 14, 1966.

11. Mark James. E.: Physical properties of polymers handbook, American Institute of Physics,

Woodburry, New York, 1996, pp. 139-161

12. Wikipedia Dictionary http://en.wikipedia.org/

(http://en.wikipedia.org/wiki/Glass_transition; http://en.wikipedia.org/wiki/Nanoparticles).

13. Bogunia-Kubik Katarzyna, Sugisakaa Masanori: From molecular biology to

nanotechnology and nanomedicine, Biosystems, vol. 65, March 2002.

14. Eerikäinen Hannele: Preparation of nanoparticles consisting of methacrylic polymers and

drugs by an aerosol flow reactor method, VTT publications 563, Helsinki, 2005

15. Anon.: Zetasizer Nano Series User Manual, Worcestershire, UK, June 2003

16. Anon.: Brookhaven instruments corporation, New York, 2004.

http://www.bic.com/Zeta_Potential_overview.html

17. PhD thesis of Eva Valentova: Charles University, Faculty of Pharmacy in Hradec Kralove,

Czech Republic, 2007

18. Arshady Reza: Microspheres, microcapsules and liposomes, vol. 1, London, 1999, pp.11-

80

19. Deasy Patric R.: Microencapsulation and related drug process, vol. 20, New York, 1984,

chapter 8, pp. 181-191

- 50 -

20. Shoyele Sunday A., Cawthorne Simon: Particle engineering techniques for inhaled

biopharmaceuticals, Adv. Drug Deliv. Rev. 56, 2006

Interactions of biodegradable drug carriers with ...

Documents

Transcript of Interactions of biodegradable drug carriers with ...