interactions of biodegradable drug carriers with
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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
2007
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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
9. Literature………………………………………………….......................................................49
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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
Polylactide Polyglycolide
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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);
• Shape and geometry of polymer body (degradation is a function of surface area);
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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 - -
PEU 2 50 50 BD HMDI
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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.
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
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
Valu
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.
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, %
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 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
Erosion degree (ED) (%) 34,81 6,560 3,571 5,279 6,813 6,108
-100
300
700
1100
1500
1900
Valu
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) (%)
Swelling degree (SD) (%) 267,88 355,14 613,31 987,54 541,15 515,76
Erosion degree (ED) (%) 24,699 23,416 13,525 17,812 21,631 13,559
1 DAY 3 DAY 7 DAY14
DAY
21
DAY 28
DAY
Valu
e, %
- 26 -
1 DAY 3 DAY 7 DAY 14 DAY 21 DAY 28 DAY
Swelling degree (SD) (%) 189,6 181,04 113,02 52,429 28,252 33,299
Erosion degree (ED) (%) 76,675 64,545 47,817 33,455 27,142 17,932
0
50
100
150
200
Valu
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.
- 27 -
1 DAY 3 DAY 7 DAY 14 DAY 21 DAY 29 DAY
Swelling degree (SD) (%) 56,862 185,03 485,07 488,32 805,16 650,04
Erosion degree (ED) (%) 82,422 51,673 21,276 30,354 24,758 25,467
0
200
400
600
800
1000
Valu
e,
%
Days
Swelling degree (SD) (%)
Erosion degree (ED) (%)
1 DAY 3 DAY 7 DAY14
DAY21
DAY29
DAY
Swelling degree (SD) (%) 13,321 23,211 86,338 51,998 48,642 27,845
Erosion degree (ED) (%) 95,372 95,922 80,318 58,375 31,695 26,520
0
50
100
150
Valu
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.
Figure 8. Time course of swelling and erosion characteristics. Carrier T3, Average of samples A
and B - 28 days placed in citrate-phosphate buffer pH 7,0
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 -
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
Va
lue
, %
Days
Swelling degree (SD) (%)
Erosion degree (ED) (%)
1 DAY 3 DAY 7 DAY14
DAY21
DAY29
DAY
Swelling degree (SD) (%) 136,53 207,64 203,88 187,84 155,39 285,79
Erosion degree (ED) (%) 91,237 82,291 53,230 21,262 11,199 5,3020
0
100
200
300
400
Valu
e,
%
Days
Swelling degree (SD) (%)
Erosion degree (ED) (%)
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, %
Swelling degree (SD) (%)
Erosion degree (ED) (%)
Swelling degree (SD) (%) 121,922 239,098 557,686 41,9279 59,6289 23,7270
Erosion degree (ED) (%) 87,5435 55,1164 24,3886 8,7514 5,5386 6,5626
1 DAY 3 DAY 7 DAY 14 DAY 21 DAY 29 DAY
-50
0
50
100
150
Days
Valu
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
- 30 -
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
Erosion degree (ED) (%) 88,503 92,230 84,198 86,850 69,935 60,207
0
20
40
60
80
100
Valu
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.
- 31 -
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
Erosion degree (ED) (%) 98,967 92,186 97,542 102,40 99,652 95,738
0
50
100
150
Valu
e, %
Days
Swelling degree (SD) (%)
Erosion degree (ED) (%)
1 DAY 3 DAY 7 DAY 14 DAY 21 DAY 29 DAY
Swelling degree (SD) (%) 1,7003 3,9106 8,1549 12,404 6,8137 6,1099
Erosion degree (ED) (%) 50,392 99,913 98,058 100,52 98,179 99,519
0
50
100
150
Valu
e, %
Days
Swelling degree (SD) (%)
Erosion degree (ED) (%)
Figure 14. Time course of swelling and erosion characteristics. Carrier PEA2, Average of samples
A and B - 28 days placed in citrate-phosphate buffer pH 7,0
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.
Figure 15. Time course of swelling and erosion characteristics. Carrier PEA3, Average of samples
A and B - 28 days placed in citrate-phosphate buffer pH 7,0
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
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 - -
- 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.
Day Sample Onset (ºC) Mid (ºC) Inflection
(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.
Day Sample Onset (ºC) Mid (ºC) Inflection
(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
Day Sample Tg Mid [ºC] ∆Cp 103 J/(gK)
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
Day Sample Tg Mid [ºC] ∆Cp 103 J/(gK)
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
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