Faculty of Bioscience Engineering Academic year 2015 – 2016 Thermally based encapsulation based on calcium carbonate particles for therapeutic enzymes Cocquyt Melissa Promotor: Prof. Dr. Ir. Andre Skirtach Tutor: Dr. Bogdan Parakhonskiy Master’s dissertation submitted in partial fulfilment of the requirements for the degree of Master of Science in industrial engineering: biochemistry
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Faculty of Bioscience Engineering
Academic year 2015 – 2016
Thermally based encapsulation based on calcium
carbonate particles for therapeutic enzymes
Cocquyt Melissa
Promotor: Prof. Dr. Ir. Andre Skirtach
Tutor: Dr. Bogdan Parakhonskiy
Master’s dissertation submitted in partial fulfilment of the requirements for the
degree of
Master of Science in industrial engineering: biochemistry
Faculty of Bioscience Engineering
Academic year 2015 – 2016
Thermally based encapsulation based on calcium
carbonate particles for therapeutic enzymes
Cocquyt Melissa
Promotor: Prof. Dr. Ir. Andre Skirtach
Tutor: Dr. Bogdan Parakhonskiy
Master’s dissertation submitted in partial fulfilment of the requirements for the
degree of
Master of Science in industrial engineering: biochemistry
“The author and the promoter give the permission to use this thesis for consultation and to
copy parts of it for personal use. Every other use is subject to the copyright laws, more
specifically the source must be extensively specified when using the results from this thesis”.
Promotor
Prof. Dr. Ir. Andre Skirtach
Author
Melissa Cocquyt
Ghent University 3 June 2016
Foreword
First of all I want to thank my promotor Prof. Dr. Ir. Andre Skirtach and my tutor Dr. Bogdan
Parakhonskiy for giving me this thesis, for guiding me and for having faith in me that I would
provide good results. Furthermore I thank them for giving me the opportunity to go to the Max-
Planck Institute in Göttingen, Germany.
In accordance with this I would like to express my gratitude to Dr Manfred Konrad who let me
into his group and helped me in my work with enzymes. From this group I would like to thank
Ursula Welscher-Altschäffel and Joanan Lopez Morales for helping me get familiar with the
lab and the equipment so quickly.
I also want to thank Ir. Tobias Corne, Ir.Tom Sieprath and Ir. Pieter Wuytens for the help they
gave me in microscopy. Without them I would never have collected the results I have now. My
gratitude goes to Geert Meesen as well for granting me access to the Widefield microscope and
trusting me with it.
At last I would like to thank my friends and family for their constant support.
Abstract
In this study the thermal behaviour of polyelectrolyte capsules of the polymers PDADMAC
and PSS assembled by the layer-by-layer method on CaCO3 particles was researched. More
specifically the shrinkage of even-layered capsules by heating, with the objective to create
smaller and more sturdier capsules, enhancing the mechanical strength for cellular uptake for
delivery of therapeutic enzymes. First, CaCO3 particles of different shape and size were loaded
with the enzyme hGMPk. This was done with the purpose to identify the most suited particle
with the highest enzyme activity. The star-like particles showed the most enzyme activity but,
because of their highly irregular surface, were not suitable to be used as core material for the
heating experiment. In spite of common believe that it would be very difficult to use the
shrinking method by heat treatment on capsules assembled on CaCO3 cores, because of its
porous structure, it was found to be possible to shrink the capsule even decreasing the diameter
of the capsule by one to two micrometres. In sight of using these capsules for the delivery of
therapeutic enzymes, these enzymes needed to be verified on thermal stability. It was found
that the enzyme alone wasn’t stable when heating above 40°C but when 1 mg/ml BSA was
added, the thermal stability of the enzyme increased with the overall activity. With this result,
there can be assumed that capsules themselves will provide the structure to protect the
encapsulated enzyme.
Samenvatting
In deze studie wordt het gedrag van poly-elektrolytische capsules samengesteld uit de
polymeren PDADMAC en PSS door de laag na laag methode op CaCO3 deeltjes onderzocht.
In het bijzonder het krimpen van even gelaagde kapsels door verwarmen met als doel het
produceren van kleinere en steviger kapsels, ter vergroting van de mechanische stabiliteit voor
het leveren van therapeutische enzymen. Eerst werden verschillende groottes en vormen van
CaCO3 deeltjes geladen met het humaan enzym GMPk met als doel het meest geschikte deeltje
te identificeren die de hoogste enzym activiteit vertoont. De stervormige deeltjes hadden het
meeste enzym activiteit maar waren ongeschikt voor het gebruik als kernmateriaal voor de
assemblage van de kapsels voor het verwarm experiment door hun heel onregelmatig
oppervlak. Ondanks de moeilijkheid om de verwarm methode toe te passen op kapsels
geassembleerd op CaCO3 kernen door hun hoge porositeit, werd er gevonden dat deze kapsels
konden krimpen met zo’n een tot twee micrometer. Met het oog op het gebruik van deze kapsels
voor het leveren van therapeutische enzymen, moet de thermische stabiliteit van deze enzymen
worden geverifieerd. Hieruit bleek dat het humaan enzym GMPk niet stabiel was boven de 40
°C maar wanneer 1 mg/ml BSA wordt toegevoegd, verhoogde de thermische stabiliteit samen
met de totale activiteit. Met dit resultaat kan er waarschijnlijk vanuit gegaan worden dat de
kapsels zelf de nodige structuur zullen bieden om het geïnkapsuleerde enzym te beschermen
tegen denaturatie.
Index
1. List with abbreviations ........................................................................................................ 1
2. List with tables and figures ................................................................................................. 3
/Poly(styrenesulfonate) (PSS) caspsules PDADMAC/PSS capsules are used as a model system and recent studies proved that they can
be shrunk by heating the hollow capsule made on silica particles (Kohler et al, 2005). Shrinking
or swelling of the capsules is depended on the charge balance within the layers of the capsules.
To shrink the capsules, they need to be assembled of an even number of layers so they have a
balanced ratio between the oppositely charged polymers. When an uneven number of layers is
applied the nonmatching charges will make the shell swell.
With the use of CaCO3 as core material which is slightly negatively charged, it is recommended
to start with PDADMAC as first layer because of its positive charge (Figure 3). As such, the
last layer will be of PSS. This is a positive outcome as PSS contains a rather large group that
will repel each other thus preventing more aggregation.
Figure 3: Chemical structure of poly(styrene sulfonate) (PSS) and poly(diallyldimethylammonium chloride)
(PDADMAC)(Chen et al, 2009)
The amount of adsorbed material added in the layer-by-layer process and thus the layer
thickness is influenced by the ionic strength and the type of salt of the solution, the polymer
charge density and concentration, the type of solvent, the deposition time and the temperature
(Kohler et al, 2005).
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In the experiment done by Köhler et al.(Kohler et al, 2005) capsules of eight layers were
prepared on a silica core. The size of particles was compared when heated at different
temperatures and different duration. When temperature is elevated on particles with their core
still inside, the capsule will not shrink. After core dissolution the capsules can shrink by
increasing temperature if dissolved in water. When polyelectrolyte multilayers are heated in the
absence of water, there will hardly be any difference. Hereby the conclusion that desorption of
water while rearranging the polymers, make the capsules shrink can be made. The higher the
temperature, the smaller they become until they stay at a constant size at 70°C. At this point a
diameter decrease of 70 % is observed. Besides the temperature, the incubation time is also of
importance. There is no known time period in which the shrinking procedure comes to a stop,
even after half a year at room temperature the shells showed smaller. The lower the temperature,
the longer it takes to shrink the capsules to a specified size (Kohler et al, 2005). This shrinking
process is irreversible.
5.3. Loading and release Loading particles consists of adsorbing molecules onto an adsorbents or putting it inside a
capsule. In the context of drug delivery, loading molecules inside a capsule is of more use.
Many molecules can be loaded, the most important factor to be accounted is their size. The
smaller they are, the harder it is to contain them. This can be done in several ways. Firstly they
can be loaded on a core material before coating, in which the molecule is administrated to the
core material or at the same time as the synthesis of the core, this last process is called co-
precipitation. Another method is for the capsule to be made first after which the coating can be
made more penetrable for putting the molecules inside by external stimuli, this process is known
as inclusion after fabrication (Lvov et al, 2001).
The ability of the capsules to become more penetrable because of certain stimuli is depended
on the characteristics of the used polymer for encapsulation. Some polymers like PAA, PAH
and PMA are weak polyelectrolytes (Burke & Barrett, 2003), which means they can be
protonated or deprotonated according to their pKa. As so, change in pH below or above their
pKa results in shrinking or swelling of the capsule because of less or more repulsion of charged
groups respectively. The stability of this sort of capsules also depends on the pH, too low or too
high and the attraction between the polymers will disappear (Dubas & Schlenoff, 2001). The
release of drugs by means of change in pH can be used in the human body for delivery to the
stomach for example or other organs with different pH.
The capsules can also be manipulated by ionic strength of the solution. By adding salt, the
electrostatic attractions inside layers of the capsule are reduced or defects or cavities are formed
(Antipov et al, 2003b), thus allowing loading or release of molecules in capsules. This
procedure can be used for loading PDADMAC/PSS capsules (Gao et al, 2004) by opening and
closing the polymeric shell. The efficiency of loading however is low (Delcea et al, 2011) and
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the use of release in the human body by increasing ionic strength is probably limited, so this
method is less appropriate for drug delivery. In contrast to swelling of capsules, polymers with
hydrophobic groups will shrink the capsule if exposed to an increase of salt concentration, for
example with PSS (Gao et al, 2004).
The use of different solvents can also cause opening and closing of capsules. It was shown for
PSS/PAH that, dissolved in 50% ethanol, the capsule was swollen while in water it was closed
(Figure 4) (Lvov et al, 2001). When molecules for loading, in this case urease, and these
capsules are brought into the first solution, the molecules will diffuse into the capsules. After
washing away the ethanol, closed capsules loaded with the molecules are left inside. The exact
reason why ethanol has this effect on the capsule is not known but it may be attributed to the
removal of water from the polyelectrolytes.
Figure 4:Schematic (top row) and CLSM images illustrating permeation and encapsulation of urease–FITC into polyion
multilayer capsules. Left, in water; middle, in water/ethanol (Lvov et al, 2001)
Another promising system to load and especially release molecules from capsules is the use of
ultrasound (Kolesnikova et al, 2010). The main advantage of this method applies in the medical
sector where ultrasound is already approved and commonly used for detection and imaging.
Capsules where ZnO nanoparticles are incorporated in their polyelectrolyte layers will open
when exposed to ultrasound enabling the loaded molecules to escape the shell. Also for loading
purposes, ultrasound can be very effective. For the drug rifampicin, the loading capacity was
up to 0,9 mg/ml while for the drug indomethacin it reached 19 mg/ml (Han et al, 2010).
Instead of using ultrasound the capsules can be brought into a magnetic field where they will
open up when magnetic nanoparticles are embedded in their shell. The problem with the use of
a magnetic field is the long exposure time and relatively strong magnetic field to permeate the
capsule will result in an increase in temperature (Lu et al, 2005).
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Release can also be achieved by mechanical deformation. This mostly happens when
mechanically unstable capsules are taken up by cells. They cannot hold the pressure of this
process and break, releasing its contents. This method could be interesting for intracellular
release (Javier et al, 2008).
The last of release stimuli is biological, in particular the use of enzymes which destroy the
capsule shell and so permanently releasing their molecules (Shu et al, 2010). This is very
promising for drug delivery as enzymes can degrade biodegradable polyelectrolyte multilayer
capsules. Premature capsule degradation should also be looked into to make sure capsules are
used that don’t release its contents to soon. Capsules can also be made that only enzymes inside
cells are capable of degradation, thus releasing intracellular.
Besides the usual method of loading and encapsulating as described above, there is another very
different way for loading enzymes. This method will be discussed further under enzymes.
5.4. Enzymes The molecules loaded in the particles for drug delivery have medical properties, among those
are therapeutic enzymes that are very important in certain therapies like leukaemia, where they
use L-asparaginase (Karamitros et al, 2013). Enzymes consist of amino acids linked together in
a polymer chain. These amino acids interact with one another by means of hydrogen bonds,
hydrophobic interactions and sulphite bindings thus creating a structure with α-helices and β-
sheets that help form the enzyme for catalytic purposes. Enzymes are produced by all kinds of
organisms and allow reactions needed for the survival of the organism to take place in less time.
So these enzymes act as catalyser for certain reactions by letting the target molecule bind with
a binding place on the enzyme and let it react with is catalytic centrum to create the product. A
reaction that could take hours to happen, can be done within a few seconds in the presence of
the right enzyme.
The use of therapeutic enzymes rather than chemical drugs has its advantages for the use of
drug delivery in the form of multi-layered capsules. First enzymes are more specific and
efficient than for example a chemical oxidants, thus resulting in less collateral damage.
Furthermore, most enzymes are rather big molecules that are much easier to contain in capsules
than small molecules.
As mentioned before, there is another way of loading and encapsulating certain enzymes by
using one of their properties. This method is somewhat more elegant than the classic method.
It all depends on the pH at which the particular enzyme crystallizes. This method can only be
used when the enzyme crystallizes at about the same pH where CaCO3 dissolves. If that is the
case then CaCO3 can be taken away before layers are added, resulting in smaller capsules
because of the absent volume of the core. The enzyme will not diffuse if it crystallizes together.
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When the crystallized enzymes are encapsulated and brought again in water with a more neutral
pH, the enzymes will return to their original state without any damage done.
The most interesting enzymes will be discussed below with their main properties, function and
ability to be used for this second, more elegant way of loading.
5.4.1. Asparaginase L-asparaginase as already mentioned can be used to treat leukaemia by hydrolysing the amino
acid asparagine to aspartic acid and ammonia (Karamitros et al, 2013). The reason for using
this enzyme lies in the fact that cancerous lymphoblasts, in contrary to healthy cells, do not
synthesize enough asparagine to grow and survive without the asparagine provided by food. If
all free asparagine in the bloodstream is hydrolysed, the cancer cells will eventually die. With
this knowledge, treating leukaemia seems easy by just administering L-asparaginase
intravenously or intramuscularly. The problem with this, is the lifetime of this foreign enzyme
inside the human body. High doses are required because of breakdown by proteases,
particularly trypsin and thrombin. If the enzyme is encapsulated where large molecules like
proteases cannot penetrate the capsule but small molecules like asparagine do, than the enzymes
will be protected while they can still carry out their function. Therefore, a much lower dose is
sufficient.
5.4.2. Insulin Insulin is very important for the treatment of diabetes. It regulates the glucose concentration in
the blood stream. When the concentration glucose is too high, insulin permits the absorption of
glucose into cells where it is stored as glycogen. Diabetes exists in two forms with two different
causes. The first you are born with, people with this type of diabetes are unable to produce
insulin. The second comes with age where the cells become resilient to the influence of insulin.
Insulin was proven suitable for this method of encapsulating crystalized enzymes by Volodkin
et al. In their research, they used hydrochloric acid to be titrated into a solution of CaCO3 and
insulin from pH 9,5 to pH 5,2 (Volodkin et al, 2010). The evolution of the CaCO3 and insulin
particles is shown in Figure 5.
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Figure 5: The fabrication of insulin microspheres by templating from CaCO3 microcores. a) The CaCO3 microcores with insulin
solution; b) insulin loading by isoelectric precipitation; c) dissolution of the CaCO3 template; d) shrinkage of the porous protein matrix to a compact sphere. Stability regions of insulin and CaCO3 cores are presented for the pH range of 9.0 to 5.0 shown.(Volodkin et al, 2010)
Around the isoelectric point of insulin, the enzyme becomes more insoluble because it becomes
more nonpolar in the polar solution. In the presence of CaCO3, flocculation only occurs inside
and on the surface which results in insoluble insulin agglomerates in the cores, not in the
solution (Volodkin et al, 2010). All the while, the CaCO3 is dissolving, leaving a shrunk insulin
matrix in its place. This shrinking is the result of water removal from the pores by hydrophobic
interactions between the proteins. To insure the formation and the survival of the insulin matrix,
decomposition of CaCO3 must be executed in mild conditions.
5.4.3. Human guanylate kinase The human guanylate kinase (hGMPK) is a phosphotransferase that transfers a phosphoryl
group from adenosine-5’-triphosphate (ATP) to Guanosine-5’-monophosphate (GMP)
resulting in adenosine-5’-diphosphate (ADP) and Guanosine-5’-monophosphate (GDP). As
such, the enzyme is important for the regeneration of GDP needed for the supply of guanine
nucleotides to signal transduction systems. In addition hGMPk plays an important role in the
intracellular activation of various antiviral and anticancer pro-drugs based on purine nucleoside
analogues (Jain et al, 2016; Sekulic et al, 2002).
5.5. Cellular uptake Enzymatic therapy usually takes place on a cellular level which implies that the protein needs
to be taken up by the cells. When this enzyme is encapsulated, the whole capsule will need to
enter the cell. Cellular uptake poses a big barrier for any particle or drug. The most common
uptake mechanism for an unidentified particle is by endocytosis. The mechanism of endocytosis
causes some shear stress on the particle that may break a drug loaded capsule. If this happens
the drug is unwillingly released before it enters the cell. A second problem of the uptake of
encapsulated drugs by endocytosis is the endosome it ends up in. Most endosomes end up
degrading their content dependent on the characteristics of the particle.
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5.5.1. Shear stress on polymeric capsules Usually capsules that are made for cellular uptake need to be reinforced to handle the shear
stress of this process. This can be done in a number of ways such as increasing the number of
layers of polymeric capsules or heating them above their glass transition temperature. The
contents of the capsules also has an influence on the mechanical stability (Delcea et al, 2010).
The heating technique has shown that incubation at high temperatures, more specifically
between fifty and seventy degrees Celsius, results in big changes of the morphology of the
polymeric capsules including an increase in stiffness. The cause of the increasing stiffness is
the densification of the polymeric shell by the shrinkage of the even layered capsule. It was
found that the capsules incubated at fifty degrees Celsius were ruptured upon cell uptake. From
this point more and more capsules survive when incubated at increasing temperatures till an
incubation temperature of seventy degrees Celsius where they almost all sustain the shear stress
of cellular uptake (Delcea et al, 2010).
5.5.2. Influence of shape and size of particles for cellular uptake The way particles are taken up by cells is dependent on their morphology, as is what happens
with the particles in the endosome. The parameters that influence the cellular uptake are size,
shape, charge, surface characteristics, the aggregation state, colloidal stability and stiffness of
the particle (Parakhonskiy et al, 2015). From all these parameters it is hard to decide what
influences a certain endosomal pathway. The aggregation state of the particles for example will
overrule their size and shape. It is therefore important that, to investigate the influence of size
and shape, the particles cannot be aggregated.
In the study of particles taken up by alveolar macrophages done by Gilbert et al.(Gilbert et al,
2014), it was shown that the angle of a particle to the macrophage influences the internalization
velocity. Spherical particles show a higher uptake than non-spherical particles. On the other
hand other studies show HeLa cells taken up non-spherical particles at a higher rate with a
different mechanism, as do brain and lung endothelial cells. Other tests attribute to the higher
uptake rate of non-spherical particles, however these contradicting results may also be caused
by the different experiment setup (Parakhonskiy et al, 2015).
Parakhonskiy et al. decided further research in this aspect was needed and conducted an
experiment of polymeric capsules templated on CaCO3 cores of different sizes and shapes being
taken up by HeLa cells. They concluded that the internalization rate was increased with
increasing aspect ratio with the exception of long fibres and wires (Parakhonskiy et al, 2015).
Meaning, that elliptical particles are more efficient in cellular uptake than spherical particles.
5.5.3. Intracellular release of a capsule’s content Once the capsule is taken up by the cell and the shell has survived the cellular uptake process,
the contents still needs to be released. In the work done by Palankar et al. (Palankar et al, 2009),
small peptides were released from microcapsules by using an infrared laser to break the with
22
metallic nanoparticles doped capsules. Other methods of releasing the contents of capsules were
tested with promising results such as using light-sensitive capsules (Volodkin et al, 2012), pH-
and redox-responsive polysaccharide-based microcapsules to make use of the different
environment between intra- and extracellular (Gao et al, 2012) and biodegradable capsules
(Dierendonck et al, 2011; Pavlov et al, 2011).
In addition to the release of the component from the capsule, the contents will also be acquired
to leave the endosome in which it ended up by the cellular uptake. Some techniques regarding
the capsule’s shell are developed. An important technique involves a mechanism called the
proton sponge in which a capsule is created capable of protonation (Boussif et al, 1995). Upon
cellular uptake, the particles end up in an endosome and the pH will drop to six. However,
because of the polymers that attract the H+ protons, the pH stays neutral. To try and get the
environment at a pH of six, the endosome will pump more and more H+ protons inside the
endosome. To compensate for the unbalanced charge inside the endosome, chloride ions will
also enter the endosome. The more and more ions enter the endosome, the greater the osmotic
force will get, allowing water to enter the endosome until it blows. The overall process is
presented in figure 6.
Figure 6: Scheme of the principle of the proton sponge for endosomal release. (Agirre et al, 2014)
A second method of endosomal escape is to incorporate fusogenic peptides into the capsules.
These peptides were first found in the influenza virus and provide the viral particle with a way
to escape the endosomal interior (Moore et al, 2008). At a change of pH environment the peptide
stretches out and fuses with the liposomal membrane of the endosome, thus escaping into the
cytoplasm.
5.5.4. Applications of cellular uptake of microcapsules There are tons of different applications for the uptake of microcapsules into cells, each one of
great importance mainly to a medical purpose. One of the uses is releasing a drug or a
therapeutic enzyme into the cell for a certain therapy. A more complex and elegant way is
releasing a small peptide for immunological therapies. Palankar and co-workers (Palankar et al,
2009) brought small, encapsulated, antigenic peptides inside the cell by electroporation and
released them with an infrared laser. They found a smart way to insert an antigenic peptide into
the cytoplasm and use the Major histocompatibility complex (MHC) class I-mediated antigen
presentation to present the antigen on the surface of the cell and mediate an immune response.
23
The inserted peptide becomes recognised as an intracellular peptide and is brought to the
endoplasmic reticulum where they bind to waiting MHC class I molecules. These are
transmembrane receptors who can bind peptides. After the binding of a suitable peptide, class
I molecules travel to the cell surface. When a virus-derived peptide in complex with a MHC
class I molecule at the surface of a cell is recognized, the cell will be killed by cytotoxic T-
lymphocytes (killer T cells). Figure 7 shows the mechanism of this intracellular delivery and
subsequent process.
Figure 7: Scheme illustrating the proposed mechanism of peptide induction of class I surface transport. Peptide-filled
microcapsules are introduced into the cell by electroporation (1) and opened by laser irradiation (2). Labeled peptides escape
from the capsules (3), are transported into the ER (4), bind to class I molecules (5) and induce their transport to the cell surface
(6). (Palankar et al, 2009)
24
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6. Goals
6.1. Enzyme activity of hGMPk loaded on CaCO3 particles of
different shapes and sizes First of we wanted to analyse the influence of size and shape of CaCO3 particles on the activity
of the loaded enzyme. This would give us an indication of which kind of particle is to be used
to deliver a therapeutic enzyme with the highest efficiency.
6.2. Encapsulation of enzymes based on thermal treatment of
microcapsules The thermal treatment of polymeric capsules has two main purposes. The first to increase the
durability of the capsules for shear stress by the uptake of cells. Second, the capsule will shrink
if it is composed of an even amount of layers.
6.2.1. Encapsulation into CaCO3 particles The capsules are to be made on CaCO3 cores because of its high absorption characteristics. This
however may bring a problem for the neat assemblage of the polymeric layers because of the
rough surface of the CaCO3 particles. To even have a chance of shrinking the capsule by heat
treatment, the layers need to be applied in a very orderly fashion.
6.2.2. Heat-shrinking method of encapsulation - obtaining hollow
capsules The ultimate goal is to produce capsules that can be shrunk by heat treatment preferably at the
lowest possible temperature. For this to work the CaCO3 core will need to be dissolved to create
hollow capsules. If the layers can be assembled in a way that results in perfectly balanced
capsules, chances are a high that the shrinkage and increased stiffness of these capsules can be
achieved.
26
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7. Materials and methods
7.1. Comparing the enzyme activity of hGMPk loaded on
CaCO3 particles of different size and shape Different sized and shaped CaCO3 particles can be made by adjusting the concentration ratio of
Na2CO3 and CaCl2 and by adding ethylene glycol. These particles also have different
characteristics when it comes to loading enzymes and their resulting activity.
The different shapes and sizes of the CaCO3 particles are defined by simple given names
according to their morphology. The particles that will be used are named cubical, large
spherical, small spherical, small elliptical and star-like.
7.1.1. Loading hGMPk on CaCO3 particles of different shape and size For loading hGMPK onto CaCO3 particles, 1 ml of 45 µM (1 mg/ml) hGMPK solution was
added to 5 mg of pre-synthesized CaCO3 particles of different sizes and shapes. After 30 min
of incubation, the particles were isolated from the hGMPK solution and washed three times by
centrifuging at 3000 rpm for 3 min.
The quantity of protein remaining in the supernatant was determined by the Bradford method
measuring optical absorption intensity at 600 nm. For estimation of the loading capacity in
weight percentage, the loading amounts were normalized by the weight of dry calcium
carbonate particles.
7.1.2. hGMPk standardized activity assay The catalytic activity of human guanylate kinase (hGMPK) can be determined by the standard
NADH-dependent enzyme-coupled assay using a JASCO V-650 UV-Vis spectrophotometer.
The activity measurement of hGMPk is based on the oxidation of NADH to NAD+ which results
in the lowering of the intensity of adsorbed light in the spectrophotometer at 340 nm (ε = 6.22
mM-1cm-1). This assay works in three steps with two helper enzymes namely pyruvate kinase
(PK) and lactate dehydrogenase (LDH) (Figure 8).
28
Figure 8: Reaction steps in the hGMPk activity assay. MgATP, MgGMP, MgADP, MgGDP: magnesium coupled to
respectively adenosine-5’-triphosphate, guanosine-5’-monophosphate, adenosine-5’-diphosphate and guanosine-5’-
The table shows a different result than the graph of the activity measurement. This is because
the loading capacity of each particle is different. The star-like particles still show the most
activity together with the large spherical ones but it is strange that the cubical ones show that
much activity.
It is difficult to compare the activity of the enzyme loaded on the particles of different size and
shape because of the different loading capacities of the particles. Comparing the activities of
the loaded enzyme per mg particles may be the best approach to overall compare every aspect.
As such the particle that shows the highest activity is the star-like particle probably because of
its high surface area.
8.2. Heating of capsules The heating of capsules was done by Köhler et al. (Kohler et al, 2005) on capsules assembled
on silica cores. It was never done on CaCO3 cores because of its irregular surface which could
provide problems for the shrinking hypothesis because of the probability of unbalanced layers
caused by the irregular surface. But thanks to the high loading capacity, the gentle dissolving
of the core and the biocompatibility of CaCO3, an effort to use the shrinking method by heating
the capsules is justified. The importance of this method lies in the strengthening of the capsules
and the reduction of size. The strengthening of the capsules is of great importance for cellular
uptake. Weak capsules will not survive the shear forces that come with the uptake of particles
in cells.
8.2.1. Trial test of heating 8-layered capsules The first set of experiments was analysed using the first microscopic method. The result can be
seen in figure 12 where it is clear that the size of the capsules has reduced after heating the
capsules by approximately one to two micrometres. The initial average diameter was 6,19 µm
with a standard deviation of 1,02 µm. The measured diameter of the capsules after heating them
for 30 min amounts to 4,19 µm with a standard deviation of 0,85 µm. A notable occurrence
here is the decrease of the polydispersity of the heated capsules compared to the initial non-
heated capsules. To be absolutely certain that the heated capsules are significantly smaller than
the initial capsules, because the distribution is wide, the results were statistically tested using
SPSS.
35
Figure 12: Heating capsules: pictures with fluorescence microscope 100x oil on the left and graphs depicting the amount of
capsules with a certain size on the right. A: Capsules before heating, B: Capsules after heating for 30 minutes at 50°C. The
diameter of the capsules was measured using a program written for Fiji (Attachment)
Because the sizes of the heated capsules was not of a Gaussian distribution, a two-related
sample non-parametric test (Wilcoxon rang) was used. This test resulted in a p-value of 0,000
which indicates a significant difference between the two samples of particles. Furthermore, the
z-value was negative indicating that the particles after heating are smaller. All the results of the
statistical test can be found in Attachments.
Because of the wide distribution of the particles, an attempt on creating more monodisperse
particles was done. The results of this experiment can be found in the next section.
8.2.2. Heating of even and uneven layered capsules at different
temperatures Because of the research done by Köhler et al. (Kohler et al, 2005), uneven and even layered
capsules respectively 7- and 8-layered capsules are heated for 30 minutes at different
temperatures. The result of this experiment can be seen in Figure 13.
36
Figure 13: Graph of the results of heating even and uneven layered capsules at different temperatures. The diameter of the
capsules is expressed as a function of their heating temperature 35, 50 and 60°C.
Not much can be said except that the capsules made on CaCO3 seem to follow the same trend
as the capsules made on silica in the experiment done by Köhler et al. (Kohler et al, 2005).
Furthermore, the 8-layered capsules seem to shrink more than the 7-layered capsules swell.
8.2.3. Real-time heating of 8-layered capsules To avoid further statistical testing and to see how the capsules change in function of time during
heating, a heating chamber was used on the microscope. That being said, it was still hard and
acquired constant vigilance to keep the same particles in the frame. Because of the heating of
the chamber a temperature gradient must have been created because the capsules in solution
were moving rapidly. Even the precipitated capsules moved slightly after waiting for them to
precipitate.
At first there was focused upon two particles but they became lost quite fast between
accumulating capsules. When they were not longer visible the focus was adjusted to a group of
seven capsules hurdled together. That is why there are no pictures of these seven capsules before
44 °C. Each couple of minutes a picture was taken of these particles. In total twelve pictures
were taken during a time set of 40 minutes. Out of each picture, the same seven particles were
cut out. These pictures were assembled and put next to each other as shown in Figure 14. Each
particle on the picture was measured twelve times for each picture, in which the diameter is
expressed in pixels.
3
3,5
4
4,5
5
5,5
6
20 25 30 35 40 45 50 55 60 65
Diameter (µm)
Temperature (°C)
8-layered
7-layered
37
Figure 14: Pictures from transmission microscope 100x oil of heating particles during 40 min, the numbers on the particles
indicate the widest diameter of the particle expressed in pixels. 1 µm resembles 10,24 pixels.
The diameter of the capsules measured in pixels were recalculated to be expressed in
micrometres. Each picture was composed of 2048 by 2048 pixels which resembled 200 by 200
µm under the microscope. So the recalculation was done by dividing each measurement by
2048 pixels and multiply by 200 µm. The evolution in diameter of each particle in time is shown
in Figure 15.
Figure 15: Graph of the diameters of seven 8-layered capsules followed during 40 minutes of heating as a function of time.
Overall it is clear that the capsules are shrinking by more or less 1 µm not including the
shrinkage done before the measurement at 44 °C. The fluctuations of the measurements during
the heating process are probably caused by temporally unfocused capsules and turning capsules
which can have a different widest diameter.
0
0,5
1
1,5
2
2,5
3
3,5
4
4,5
0 5 10 15 20 25 30 35 40
Diameter (µm)
Time (min)
particle 1
particle 2
particle 3
particle 4
particle 5
particle 6
particle 7
38
8.3. Separation of particles The size of the capsules was measured using the second method of microscopy. The reason for
this was because of the leakage of rhodamine from the particles which resulted in too much
background fluorescence. The results below are obtained from two experiments to part the
particles in six fractions.
8.3.1. First method
Figure 16: Graph of the distribution of the initial sample of CaCO3 particles and the fractions from 1 to 6 from the separation
by gravity of the initial CaCO3 particles with the first method..
39
In Figure 16, the results for the first separation test is shown. As can be seen, the fractions show
only a slight difference and are still polydisperse. This method does not work to separate the
particles in monodisperse fractions probably due to aggregation between the particles. That is
why there was chosen to apply ultrasound between the sedimentation steps to break up any
aggregated particles and to lengthen the sedimentation time in an attempt to separate the smaller
fraction from one another.
40
8.3.2. Second method
Figure 17: Graph of the distribution of the initial sample of CaCO3 particles and the fractions from 1 to 6 from the
separation by gravity of the initial CaCO3 particles with the first method described in 2.5.1.
The second method to separate the CaCO3 particles, with the addition of the application of
ultrasound and longer sedimentation times, also didn’t deliver the desired result. The graphs are
presented in Figure 17 and do not show much difference with the first experiment. So even with
the addition of ultrasound and longer sedimentation times, the particles cannot be separated by
simply letting them precipitate by gravity. A better method to separate the CaCO3 particles, is
41
probably by centrifugation. This however will not be tested because of the more complex setting
that is needed and the minimal value of it.
8.4. Thermal stability of hGMPk To have an idea of how the enzyme will react to the heating of the capsules with the enzyme
inside, the free enzyme was tested for thermal stability. First the linear range of the hGMPk
concentration in the assay needed to be characterised.
8.4.1. Concentration enzyme needed in assay Because of the limited linear range of the enzyme, the concentration of the enzyme that falls in
this linearity needs to be defined. There was chosen to test this with an enzyme concentration
between 1 nM and 100 nM. The activity measurements of this test are viewed in Figure 18.
Figure 18: Activity measurement of different concentrations of hGMPk ranging from 1 nM to 100 nM. The absorbance of
NADH at 340 nm is expressed as a function of time.
Of each activity measurement, the activity was measured by calibrating the slope divided by
the extinction coefficient of NADH at 340 nm (6,22 mM-1cm-1). The activity of each
concentration of hGMPk was then plotted as a function of the enzyme concentration (Figure
19).
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
1,8
2
0 100 200 300 400 500 600
Absorbance(340nm)
Time (sec)
1 nM
5 nM
10 nM
15 nM
20 nM
50 nM
100 nM
42
Figure 19: Graph of the linearity of the enzyme assay with the activity of hGMPk as a function of the concentration of hGMPk.
From the plotted activity of hGMPk as a function of its concentration, the linearity of the
activity assay can be determined. Figure 19 shows a linearity between the concentrations of 5
nM and 50 nM. From these results, a concentration of 15 nM hGMPk in the assay was chosen
as a good amount of enzyme to work with because of the linearity and the not too steep slope
of its activity measurement.
8.4.2. Heating of the enzyme hGMPk and its stabilisation Running the activity measurement with heated enzyme it became clear quickly that the enzyme
wasn’t at all thermally stable. At 40 °C, the enzyme had already lost almost all of its activity
and the goal of this test was to see if the enzyme could be heated to at least 45 °C.
That is why the test was done again but with the addition of a set of samples with 1 mg/ml BSA
added to the heating enzyme. The result of this test is shown in Figure 20.
Figure 20: Graph of the thermal stability of hGMPk with and without 1 mg/ml BSA from 25°C to 40°C. Activity measurement
is expressed as a function of the temperature at which the enzyme was exposed for 30 min.
y = 0,0032x + 0,0461R² = 0,9962
0
0,05
0,1
0,15
0,2
0,25
0,3
0 20 40 60 80 100 120
Activity (mM NADh/sec)
Concentration hGMPk (nM)
0
0,02
0,04
0,06
0,08
0,1
0,12
0,14
0,16
0,18
0,2
20 25 30 35 40 45
Activity (mM NADH/sec)
Temperature (°C)
hGMPk
hGMPk + BSA
43
As can be seen and what wasn’t anticipated, is that the sample with the BSA already has a
higher activity even without heating it. Until 35 °C the trend stays the same with or without
BSA, but at 40 °C the enzyme alone destabilizes enormously while in the presence of BSA the
activity stays constant.
The reason for this is probably the structural function of BSA and it may simulate the conditions
inside cells more than only the enzyme in the mixture. Another reason can be the higher
concentration of protein in the heating sample that may provide the stability.
From this result it may also be suspected that if the enzyme is encapsulated and heated, the
capsule itself will provide enough protection against the high temperature to prevent
denaturation. Even more so the high concentration of enzyme inside the capsules may stabilise
itself.
44
9. Outlook
9.1. Shrinkage of enzyme loaded capsules The next step to take in the research of this encapsulation method by heat treatment is to load
enzymes on the CaCO3 particles, encapsulate them in a similar way and heat them till they
shrink. Afterwards the activity of the enzyme will need to be tested and compared to the activity
of the initial non-heated capsules. This would be the ultimate test to what extend the capsule
will protect the enzyme against denaturation. If the protection is not sufficient, a stabilising
component can be loaded and encapsulated with the enzyme.
9.2. Shrinkage of capsules templated on CaCO3 particles of
different size and shape To broaden the application of this technique, the same experiment can be done for different
sized and shaped CaCO3 particles. It is possible that the process will become more difficult to
implement for smaller capsules and the result will be less clear. For the different shaped
particles it will be especially hard for irregular surface particles like the star-like structures
because of the difficulties to build a balanced capsule around these particles.
45
10. Conclusion
Overcoming significant challenges to load and measure the enzyme on CaCO3 particles
of different shape and size, a conclusion can be made which type of particles provides the
highest activity. The star-like particle can adsorb the most enzyme and is most active.
The star-like particles, however, are not ideal to test the capsule shrinkage, a widely used
method capable of enhancing mechanical strength of capsules, because of its highly irregular
surface. For this test spherical CaCO3 particles were used, and the thermal shrinking
encapsulation method is shown to be applicable for capsules templated on CaCO3 particles. To
the best of our knowledge such method of encapsulation has not been reported to-date. The
capsules used in this experiment were originally ~ 6,2 µm in diameter, decreasing the diameter
after shrinking to ~ 4,2 µm (as proven with the Wilcoxon Rang test).
To incorporate enzymes inside capsules and to subsequently apply the encapsulation methods
based on thermal shrinking, which is also used for enhancing mechanical stability of
microcapsules, the enzymes need to exhibit sufficient thermal stability. However, the human
GMPk was shown to be unstable even at 40°C. To improve the thermal stability of the enzyme,
a stabilizing agent, namely BSA, was added, which greatly increased the thermal stability of
hGMPk. It is expected that, in addition to concentrating of the enzyme that provides some
structure, this result should facilitate protection of enzymes at high temperatures.
At last, it was identified that the separation of the polydisperse CaCO3 particles into more
monodisperse fractions needs a more complex system, for example, centrifugation with
multiple stage separation steps.
46
47
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12. Attachments
12.1. Script Fiji measuring particles with rhodamine id = getTitle();