thermally based encapsulation based on calcium carbonate...
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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
2.1. Tables ........................................................................................................................... 3
2.2. Figures ......................................................................................................................... 3
3. Publications related to Master thesis ................................................................................... 7
4. Introduction ......................................................................................................................... 9
5. Literature ........................................................................................................................... 11
5.1. Micron and submicron particles ................................................................................ 11
5.1.1. CaCO3 particles .................................................................................................. 12
5.1.2. Particles of different shapes ............................................................................... 13
5.2. Encapsulation ............................................................................................................. 14
5.2.1. Poly(diallyldimetylammonium chloride) (PDADMAC) /Poly(styrenesulfonate)
(PSS) caspsules ................................................................................................................. 15
5.3. Loading and release ................................................................................................... 16
5.4. Enzymes ..................................................................................................................... 18
5.4.1. Asparaginase ...................................................................................................... 19
5.4.2. Insulin ................................................................................................................. 19
5.4.3. Human guanylate kinase .................................................................................... 20
5.5. Cellular uptake ........................................................................................................... 20
5.5.1. Shear stress on polymeric capsules .................................................................... 21
5.5.2. Influence of shape and size of particles for cellular uptake ............................... 21
5.5.3. Intracellular release of a capsule’s content......................................................... 21
5.5.4. Applications of cellular uptake of microcapsules .............................................. 22
6. Goals ................................................................................................................................. 25
6.1. Enzyme activity of hGMPk loaded on CaCO3 particles of different shapes and sizes
25
6.2. Encapsulation of enzymes based on thermal treatment of microcapsules ................. 25
6.2.1. Encapsulation into CaCO3 particles .................................................................. 25
6.2.2. Heat-shrinking method of encapsulation - obtaining hollow capsules .............. 25
7. Materials and methods ...................................................................................................... 27
7.1. Comparing the enzyme activity of hGMPk loaded on CaCO3 particles of different
size and shape ....................................................................................................................... 27
7.1.1. Loading hGMPk on CaCO3 particles of different shape and size ...................... 27
7.1.2. hGMPk standardized activity assay ................................................................... 27
7.1.3. Catalytic activity of the loaded hGMPk ............................................................. 29
7.2. Thermal treatment of capsules ................................................................................... 29
7.2.1. Preparing spherical CaCO3 particles .................................................................. 29
7.2.2. Encapsulation into PDADMAC/PSS capsules templated on CaCO3 particles . 29
7.2.3. Trial test of heating 8-layered capsules .............................................................. 30
7.2.4. Heating of capsules with even and odd number of layers at different
temperatures ...................................................................................................................... 30
7.2.5. Real-time heating of 8-layered capsules ............................................................ 30
7.3. Separation of polydisperse CaCO3 particles .............................................................. 31
7.3.1. First method ........................................................................................................ 31
7.3.2. Second method ................................................................................................... 31
7.4. Microscopy ................................................................................................................ 31
7.4.1. First method ........................................................................................................ 32
7.4.2. Second method ................................................................................................... 32
7.5. Thermal stability of hGMPk ...................................................................................... 32
7.5.1. Defining hGMPk concentration needed in assay ............................................... 32
7.5.2. Heating of the enzyme hGMPk and stabilisation ............................................... 32
7.5.3. Measuring activity of hGMPk ............................................................................ 32
8. Results and discussion ...................................................................................................... 33
8.1. Comparing the enzyme activity of hGMPk loaded on different sized and shaped
CaCO3 particles .................................................................................................................... 33
8.2. Heating of capsules .................................................................................................... 34
8.2.1. Trial test of heating 8-layered capsules .............................................................. 34
8.2.2. Heating of even and uneven layered capsules at different temperatures ........... 35
8.2.3. Real-time heating of 8-layered capsules ............................................................ 36
8.3. Separation of particles ............................................................................................... 38
8.3.1. First method ........................................................................................................ 38
8.3.2. Second method ................................................................................................... 40
8.4. Thermal stability of hGMPk ...................................................................................... 41
8.4.1. Concentration enzyme needed in assay .............................................................. 41
8.4.2. Heating of the enzyme hGMPk and stabilisation ............................................... 42
9. Outlook ............................................................................................................................. 44
9.1. Shrinkage of enzyme loaded capsules ....................................................................... 44
9.2. Shrinkage of capsules templated on CaCO3 particles of different size and shape .... 44
10. Conclusion ..................................................................................................................... 45
11. References ..................................................................................................................... 47
12. Attachments ................................................................................................................... 53
12.1. Script Fiji measuring particles with rhodamine ..................................................... 53
12.2. Significance test of the result of heating capsules ................................................. 54
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1. List with abbreviations
ADP Adenosine-5’-diphosphate
ATP Adenosine-5’-triphosphate
BSA Bovine serum albumin
EDTA Ethylenediaminetetraacetic acid
EG Ethylene glycol
GDP Guanosine-5’-diphosphate
GMP Guanosine-5’-monophosphate
hGMPk human guanylate kinase
LBL Layer-by-layer
LDH Lactate dehydrogenase
MF Formaldehyde
MHC Major histocompatibility complex
NADH Nicotinamide adenine dinucleotide reduced form
NP Nanoparticles
PAA Poly(acrylic acid)
PAH Poly(allylamine hydrochloride sodium salt)
PDADMAC Poly(diallyldimethylammonium chloride)
PEM Polyelectrolyte multilayer
PEP Phosphoenolpyruvic acid
PK Pyruvate kinase
PLA Poly(lactid acid)
PLL Poly(L-lysine)
PMA Poly(methacrylic acid)
PS Polystyrene
2
PSS Poly(styrenesulfonate, sodium salt)
PVA Poly(vinyl alcohol)
PVPON Poly(N-vinylpyrrolidine)
SPSS Statistical Package for Social Sciences
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2. List with tables and figures
2.1. Tables Table 1. Composition of the hGMPk activity assay with the end concentrations and the
volumes needed from a stockconcentration needed for 1 test. The buffer (Tris-HCl, KCl,
MgCl2) is made together in stock. At this mixture the enzyme and a volume of water to
1 ml will need to be added at the beginning of the activity measurement.
ATP: adenosine-5’-triphosphate, GMP: guanosine-5’-monophosphate, PEP:
phosphoenolpyruvic acid, NADH: nicotinamide adenine dinucleotide reduced form,
PK: pyruvate kinase and LDH: lactate dehydrogenase.
Table 2. Activity of the loaded hGMPK on particles of different size and shape per mg
particles and per mg enzyme
2.2. Figures Figure 1. Scanning electron micrographs of CaCO3 polymorphs: A: aragonite, B: vaterite,
C: calcite.(Volodkin, 2014)
Figure 2. Confocal fluorescence scanning images of a) spherical (ca. 3.6 mm), b) elliptical
(long axis 1.2 µm), c) star-like and d) cubic calcium carbonate particles loaded with
TRITC–dextran (70 kDA) molecules (the scale bar is 5 µm). Insets show scanning
electron microscopy images of the same particles (the scale bar is 1 µm). (Parakhonskiy
et al, 2014)
Figure 3. Chemical structure of poly(styrene sulfonate) (PSS) and
poly(diallyldimethylammonium chloride) (PDADMAC)(Chen et al, 2009)
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)
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)
Figure 6. Scheme of the principle of the proton sponge for endosomal release. (Agirre et
al, 2014)
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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)
Figure 8. Scheme of the work plan to encapsulate CaCO3 particles and shrink them by heat
treatment
Figure 9. Main scheme of separating CaCO3 particles by gravitation
Figure 10. 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’-monophosphate. PEP:
phosphoenolpyruvic acid, NADH: nicotinamide adenine dinucleotide reduced form,
PK: pyruvate kinase and LDH: lactate dehydrogenase.
Figure 11. Graph of the activity of loaded hGMPk on different sized and shaped CaCO3
particles. The absorbance of NADH at 340 nm is expressed as a function of time.
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)
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.
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.
Figure 15. Graph of the diameters of seven 8-layered capsules followed during 40 minutes
of heating as a function of time.
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 described in 2.5.1.
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.
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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.
Figure 19. Graph of the linearity of the enzyme assay with the activity of hGMPk as a
function of the concentration of hGMPk.
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.
6
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3. Publications related to Master thesis
The loading capacity versus the enzyme activity in new anisotropic and spherical vaterite
microparticles.
Senem Donatan, Alexey M. Yashchenok, Nazimuddin Khan, Bogdan Parakhonskiy, Melissa
Cocquyt, Bat-El Shani Pinchasik, Dmitry Khalenkow, Helmuth Möhwald, Manfred Konrad,
and Andre G Skirtach
ACS Appl. Mater. Interfaces, Just Accepted Manuscript
DOI: 10.1021/acsami.6b03492
Publication Date (Web): May 11, 2016
Copyright © 2016 American Chemical Society
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4. Introduction
This thesis deals with the development of carriers for proteins with the goal to deliver
therapeutic enzymes to the human body. Nowadays, not that many enzymes are used as drugs
because of their short half-life. This is why carriers based upon microparticles need to be
developed to protect these enzymes. Besides this, there are many other reasons why scientists
have put their interest in micro- and nanoparticles such as uses in chemical or physical industry
as well as the food industry. The research done in this thesis may also be a contribution to these
applications. Many materials can be used to make micro- and nano-carriers such as liposomes,
viral capsules, cells and polymeric electrolytes of which the last one will be used in this thesis
with the goal to ultimately use them as the delivery system of a therapeutic enzyme.
These polymeric capsules can be assembled on many core materials. The material that is mostly
used is silica because of its easy use in experiments. In this thesis however, the capsules will be
assembled on CaCO3 particles. Their advantage over silica is the porosity and biocompatibility
of the vaterite form. In addition, these particles can be made in different shapes and sizes. The
shape and size of particles influences the adsorption and activity of the adsorbed enzyme, which
will be tested.
The capsules protect the enzymes against proteases and the immune system and as such extend
the half-life of the drug. These capsules also need to be sturdy enough to survive the shear stress
caused by the uptake of particles by cells. To strengthen the capsule and reduce its size,
techniques are researched to accomplish this. These techniques rely on different modifications
like using adjusting of temperature or pH. The most important factor will be the size of the
capsules which will be measured with light and fluorescent microscopy. It is also important to
see what impact the method of modification of the capsules has on the enzymes. This is done
by measuring the activity of the enzymes using a spectroscopic assay.
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5. Literature
5.1. Micron and submicron particles Micron particles are particles within the range of a few micrometres in diameter, and can be
composed of several materials. Nanoparticles are much smaller with a diameter range between
two hundred and nine hundred nanometres, only particles of this size can be used for drug
delivery through blood circulation. That is why there is much interest to make particles smaller,
on the contrary larger particles are preferred for characteristics such as easy use of conventional
microscopy, prevention of aggregation, superior loading capacity and large surface area for
modification (Delcea et al, 2011).
The materials commonly used for assembling particles are weakly cross-linked melamine
formaldehyde (MF) (Sukhorukov et al, 1998) and polystyrene (PS) (Dejugnat & Sukhorukov,
2004; Elsner et al, 2004) cores, gold nanoparticles (NP) (Schneider & Decher, 2004),
poly(lactid acid) (PLA) particle (Shenoy et al, 2003), CaCO3 cores (Antipov et al, 2003a), silica
particles (Itoh et al, 2004) and cells (Moya et al, 2001; Neu et al, 2001). If hollow capsules are
to be made, these cores will need to be extracted from its capsules. For monodisperse MF
particles hydrochloric acid, dimethylformamide or dimethylsulfoxide can be used to dissolve
MF into oligomers (Dong et al, 2005; Sukhorukov et al, 1999). During this process ruptures of
the capsule can occur because of the high osmotic pressure caused by the slow diffusion of
these oligomers. Another negative point of these cores is the unfinished extraction of the
oligomers, some residue stays behind between the layers of the capsule. The same problem
occurs with PLA cores which are dissolved in organic solvents (Dejugnat & Sukhorukov,
2004). Organic solvents also used to dissolve PS cores are known to cause instability and
change the structure of capsules which includes swelling. Using cells as core material results in
capsules of a variety of shape and size (Neu et al, 2001). To dissolve these cells, they need to
be incubated in a basic hypochlorite solution that can cause oxidation to the capsules(Moya et
al, 2001).
These were all organic cores which are hard to dissolve in water contrary to inorganic cores
who furthermore dissolve into ions causing less rupture of the capsule. The use of silica as core
material results in monodisperse, smooth, spherical particles but can also give aggregation
problems and is not suitable for loading. This core can only be dissolved in highly corrosive
hydrogen fluoride, another disadvantage (Itoh et al, 2004). Gold particles have to be extracted
with a hazardous potassium cyanide solution (Schneider & Decher, 2004).
Because of all these disadvantages of certain materials, CaCO3 as core material will be chosen
due to its porosity and easy dissolving with ethylenediaminetetraacetic acid (EDTA). The
negative characteristics of CaCO3 such as irregular size and aggregation will be controlled as
much as possible.
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5.1.1. CaCO3 particles CaCO3 or calcium carbonate is very useful to make microparticles, it is not an expensive
material nor is it hard to make, it is very porous which makes it easy to load, it is biocompatible
and has mild decomposition conditions (Volodkin, 2014). The main problem with this material
shows in controlling the particles size and shape. These two parameters can be changed with
concentration, stirring, adding a solution with high viscosity, ultrasound and washing. A lower
concentration should result in smaller particles because there is less chance of aggregation. A
hard rhythm of stirring or the use of ultrasound should also reduce its size (Trushina et al, 2015).
There are three forms of anhydrous polymorphs of calcium carbonate (Figure 1), the form that
is needed for loading of molecules is vaterite with its high porous surface and spherical shape.
This form is metastable along with its other form aragonite. The thermodynamic stable form is
calcite with its smooth surface and cubical shape. Vaterite dissolved in water, recrystallizes in
a matter of hours into calcite (Volodkin, 2014).
Figure 1: Scanning electron micrographs of CaCO3 polymorphs: A: aragonite, B: vaterite, C: calcite.(Volodkin, 2014)
To obtain CaCO3 particles composed mostly of vaterite a supersaturated salt solution is
required, with an optimal temperature between twenty and fifty degrees Celsius. The pore size
will have a range of twenty to forty nanometre (Volodkin, 2014).
Controlling the size of vaterite particles is very important for further use in the biomedical
sector. The objective is to make particles small enough for blood circulation, therefor the size
of these particles needs to be pushed to a small size which can be obtained by the following
parameters. At first the particles are made by combining CaCl2 and Na2CO3 at equal or different
quantities dependent on the acquired shape (Parakhonskiy et al, 2014). From these two salts
CaCO3 is assembled from the formation of nuclei followed by vaterite crystal growth. A large
number of nuclei results in more and smaller particles which can be achieved by rapidly stirring
for a relatively long time during salt mixing (Volodkin, 2014; Parakhonskiy et al., 2014).
Another way of controlling size is by adding solvents such as ethylene glycol or glycerol
(Trushina et al, 2015). By adding ethylene glycol or glycerol to the salts the solution will
become more viscous, leading to a lesser movement of ions, thus reducing the size (Trushina et
al, 2015). According to the work done by Trushina and coworkers (Trushina et al, 2015), five
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hundred nanometre sized particles can be made from equal amounts of glycerol and salt solution
exposure for thirty minutes.
A third method consists of applying ultrasound during the formation of the CaCO3 particles. It
makes for a better distribution of the salts and more reaction of the ions.
5.1.2. Particles of different shapes The morphology of the CaCO3 particles can be adjusted by changing the precipitation
conditions. Next to spherical particles, elliptical, cubical and star-like CaCO3 particles can be
made. The cubical ones are formed from the re-crystallization of vaterite to calcite. To prevent
this and to create other morphologies, ethylene glycol was added to reduce the solubility of
calcium carbonate (Parakhonskiy et al, 2014). In doing so, more control on the precipitation is
achieved and different parameters on the precipitation of CaCO3 can be researched.
The influence of precipitation time, solvent and the ratio of the two salt concentrations NaCO3
and CaCl2 was determined by Parakhonskiy et al. (Parakhonskiy et al, 2014). The solvent being
water versus 80 % ethylene glycol. By adjusting these parameters, the shape of the CaCO3
particles changes as can be seen in Figure 2.
Figure 2: Confocal fluorescence scanning images of a) spherical (ca. 3.6 mm), b) elliptical (long axis 1.2 µm), c) star-like
and d) cubic calcium carbonate particles loaded with TRITC–dextran (70 kDA) molecules (the scale bar is 5 µm). Insets
show scanning electron microscopy images of the same particles (the scale bar is 1 µm). (Parakhonskiy et al, 2014)
To obtain spherical shaped particles, the two salts are mixed in equal amounts. When the
concentrations of the two salts are different from each other, elliptical CaCO3 particles are
created. At last the most special shape is the one of the star-like particles. They are believed to
be formed because of the aggregation of vaterite nuclei (Parakhonskiy et al, 2014). This is a
result of the competition of the forming of nuclei and crystal growth. The star-like particles
have a larger surface area than the spherical or elliptical ones.
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5.2. Encapsulation The core material will be covered with polymers to make capsules which can contain several
molecules. A lot of polymers can be used for this purpose varying of synthetic or functionalized
polyelectrolytes (Antipov et al, 2002; Li et al, 2004), biocompatible polyions (Itoh et al, 2004;
Shenoy et al, 2003), proteins (Caruso & Mohwald, 1999), DNA (Vinogradova et al, 2005),
lipids (Moya et al, 2000) or even viruses (Yoo et al, 2006).
The choice for one of these materials depends on the particles’ use. Mechanical stability,
elasticity, morphology, biocompatibility, permeability and surface characteristics will need to
be adjusted for the required properties (Delcea et al, 2011). For example the capsules of particles
who will need to be absorbed by cells will have to be able to withstand the resulting pressure.
In some cases the polymers would be required to have the ability to be manipulated for loading
or release of its contents.
The encapsulation can be done in several ways but the method that is mostly used is the layer-
by-layer (LBL) method which is based on alternating oppositely charged polymers resulting in
polyelectrolyte multilayer (PEM) capsules (Decher, 1997; Volodkin, 2014). For this method
the non-adsorbed polyelectrolyte needs to be removed by a cycle of centrifugation and
resuspension (Manning, 1969) or membrane filtration (Voigt et al, 1999) before applying
another layer. To prevent aggregation of the particles and capsules, ultrasound can be applied
to break ionic interactions so two particles are not coated as one. However high power
ultrasound, in the range of 100-500 W, is known to destroy PEM capsules (Shchukin et al,
2006; Skirtach et al, 2007). After dissolving the core, the capsules must be suspended in water
at all times. When capsules without their core are dried, they collapse. Large capsules show
many creases and folds while small capsules stay intact (Kohler et al, 2005).
Capsules of poly(acrylic acid) (PAA)/poly(vinyl alcohol) (PVA) (Yun & Kim, 2010),
chondroitin sulfate/poly(L-lysine) (PLL) (Zhao & Li, 2008), PAA/poly(methacrylic acid)
(PMA) (Kharlampieva et al, 2009), poly(ethylenoxide) (PEO)/poly(N-vinylpyrrolidine)
(PVPON) (Sukhishvili & Granick, 2000), poly(allylamine hydrochloride sodium salt) (PAH)/
poly(styrenesulfonate, sodium salt) (PSS) (Lvov et al, 2001) and
PSS/poly(diallyldimethylammonium chloride) (PDADMAC) (Gao et al, 2004) among others
were made.
The advantage of these capsules lies in the ability to affect them with certain stimuli thus
controlling the size of capsules and loading or release of molecules. This ability is a result of
the main properties of polymers like molecular weight, flexibility and density of charged groups
(Glinel et al, 2002). This last one is the reason of why external stimuli can be used. They are
affected by chemical factors such as ionic strength, pH and solvent but also by physical
parameters like light, magnetic field, ultrasound, mechanical action and temperature. At last
15
they also react with enzymes and receptors, which are biological stimuli. The LBL method is
also depended of these charged groups and its properties.
The manipulating of capsules as described above is for the most stimuli reversible and can come
in handy with controlled release and will be discussed further. A more irreversible change can
be made by exposing the multi-layered capsule to a temperature above the polyelectrolytes’
glass transition temperature (Kohler et al, 2005). Polyelectrolyte capsules are kinetically stable,
so it can be expected by increasing the temperature that there will be enough thermal energy
supplied to rearrange the polymeric shell. It was proven that an odd number of layers makes the
capsule swell opposite to an even number which makes it shrink (Kohler et al, 2005). This can
be explained by the charge balance. Heating the capsules also results in a rearrangement of the
added layers which makes the capsule more mechanically resistant (Buscher et al, 2002). This
method of heat treatment will be used to shrink capsules made of the polymers described below.
5.2.1. Poly(diallyldimetylammonium chloride) (PDADMAC)
/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).
16
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
17
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).
18
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.
19
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.
20
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.
21
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
25
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
27
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’-
monophosphate. PEP: phosphoenolpyruvic acid, NADH: nicotinamide adenine dinucleotide reduced form, PK: pyruvate
kinase and LDH: lactate dehydrogenase.
The substrates and their concentrations needed in the assay are viewed in Table 1. This solution
is quickly degraded so it needs to be made anew each day. A certain volume of the mixture in
Table 1 is prepared dependent on the amount of tests that will be done that day. In this case, a
volume of 562 µl of the mixture is needed for each test (Table 1). To obtain an end volume of
1 ml per test, 438 µl of water will be added together with the enzyme at the beginning of the
activity measurement. A cuvette with the assay mixture is prepared which is put in the
spectrophotometer at 340 nm with a time measurement program. The measurement is started
and the enzyme is added. The blank is composed of the assay mixture without NADH.
Table 1: Composition of the hGMPk activity assay with the end concentrations and the volumes needed from a
stockconcentration needed for 1 test. The buffer (Tris-HCl, KCl, MgCl2) is made together in stock. At this mixture the enzyme
and a volume of water to 1 ml will need to be added at the beginning of the activity measurement. ATP: adenosine-5’-
triphosphate, GMP: guanosine-5’-monophosphate, PEP: phosphoenolpyruvic acid, NADH: nicotinamide adenine
dinucleotide reduced form, PK: pyruvate kinase and LDH: lactate dehydrogenase.
Component Concentration in
assay
Concentration
stock
Volume stock in
1 ml assay (µl)
Tris-HCl (pH 7,5) 100 mM 200 mM
500 KCl 100 mM 200 mM
MgCl2 10 mM 20 mM
ATP 2 mM 100 mM 20
GMP 1 mM 100 mM 10
PEP 0,5 mM 25 mM 20
NADH 0,25 mM 25 mM 10
PK 4 U 4 U/µl 1
LDH 5 U 5 U/µl 1
29
7.1.3. Catalytic activity of the loaded hGMPk The activity of the enzyme loaded on pre-synthesized CaCO3 particles of different size and
shape was measured using the hGMPk standardized activity assay. All measurements were
performed at 25 °C. In these assays, the enzyme was replaced for 10 µl of hGMPK-loaded
particles from a suspension of 5 mg of the particles in a reaction volume of 1 ml. This however
resulted in a too steep slope for the activity measurement, as such the solution was diluted a
hundred times.
7.2. Thermal treatment of capsules
7.2.1. Preparing spherical CaCO3 particles The materials used for preparing spherical particles are: Na2CO3, CaCl2, ethylene glycol (EG),
70 % ethanol, a magnetic stirrer and a centrifuge. Spherical particles were made by three
methods. The first and simplest is by adding equal amounts of Na2CO3 and CaCl2 together
while stirring at 500 rpm for 1,5 minutes with a magnetic stirrer, this method should result in
particles around the size of 4 µm. The second protocol consists of adding 20 ml of EG to the
mixture while stirring for three hours at 500 rpm. These particles should have a diameter of 0,5
µm. The third kind of particles are formed while the solution is exposed to ultrasound for two
minutes, which should create particles of 1 µm diameter. Each sample of these particles is
washed three times with ethanol, centrifuged at 3000 rpm for 5 minutes and dried for storage.
7.2.2. Encapsulation into PDADMAC/PSS capsules templated on CaCO3
particles Solutions were made of 2 mg/ml polymer (PDADMAC and PSS) in 0,5 M NaCl. First 2 ml of
the PDADMAC solution was added to 20 mg of CaCO3 particles and exposed to ultrasound for
more or less 5 seconds to prevent aggregation which is very important to create single capsules.
Otherwise, the capsules will be formed around two or more CaCO3 particles together. The
polymers are left to be adsorbed for 15 minutes in a shaker. Before applying the second polymer
(PSS) for the second layer, the first polymer needs to be taken away. This was done by washing
the solution 3 times with water, breaking aggregation with ultrasound and centrifuged at 3000
rpm for three minutes. This procedure was repeated four times for each layer of polyelectrolyte
resulting in 6-layered capsules. The solution of 8-layered particles is then left in an 0,1 M EDTA
solution for half an hour so the CaCO3 core can be dissolved. After three more washing steps,
the capsules are stored in approximately 0,4 ml water. A scheme of these steps can be found in
figure 9. Throughout the washing steps some particles are lost but enough remain for further
tests.
30
Figure 9: Scheme of the work plan to encapsulate CaCO3 particles and shrink them by heat treatment.
7.2.3. Trial test of heating 8-layered capsules For the first test a solution of capsules was gradually exposed to 50 °C in a warm water bath.
The capsules were heated from 20 °C to 50 °C in 30 minutes and left at 50 °C for another 30
minutes. Afterwards the capsules were left at room temperature to cool down. A sample of the
initial capsules and the capsules after heating was put under the microscope and the size
difference was calculated by using a computer program and rhodamine.
7.2.4. Heating of capsules with even and odd number of layers at different
temperatures A second test was done in which the capsules were exposed to a series of different temperatures,
namely 35 °C, 50 °C and 60 °C. For this test, 7-layered particles were also included to prove
that the unbalanced, uneven layered particles will expand when exposed to a high temperature.
The same warm water bath was used beginning at 35 °C for the first part of the particles, leaving
them there for 30 minutes. Then increasing to 50 °C for the second part of the particles while
the first part cooled at room temperature. The same was done for the last part at 60 °C. The size
of the capsules of these samples were measured by hand on microscope pictures which is
described as the second method.
7.2.5. Real-time heating of 8-layered capsules The tests above both require a lot of data to prove the shrinkage of the capsules and statistical
analyses because the capsules before and after heating may come from the same batch but are
not the same particles. If the same particles can be followed during the heating process, the
shrinkage of capsules is beyond doubt shown. Therefor the hollow, unheated capsules will need
to be heated under the microscope and this is done in a heated chamber. The chamber “okolab
stagetop incubation system” works with a heat exchanger which allows heated water to go
through small tubes connected to the metal of the chamber, heating the chamber and the sample
within. In addition the lens is also heated to prevent cold bridges on the microscope slide.
31
7.3. Separation of polydisperse CaCO3 particles
To create a more monodisperse mixture of CaCO3 particles,
an effort was made to divide the original preparation into
some fractions. A first method was developed and adjusted
by prolonging the sedimentation time and ultrasound
application. The overall view of this method is illustrated in
figure 10.
7.3.1. First method 10 mg of dried CaCO3 particles was weighted and dissolved in 12 ml distilled water in an 15
ml tube and ultrasound was applied for 10 seconds. Some microliters were taken as initial mix
for comparison with separated fractions. The solution with particles was then left to rest for 5
minutes so gravitation could separate the particles. After 5 minutes, 1 ml of the surface was
taken and stored. The tube was shaken again and the former step was repeated, both steps are
taken as the first fraction. The next fraction is done in the same way for the next 2 ml. For
fraction 3 the sedimentation time was reduced to 3 minutes, again 2 ml was taking in two steps.
The same was done for fraction 4. The sedimentation time was again reduced, this time to 2
minutes for the next fraction of 2 ml. This leaves the rest as fraction 6.
7.3.2. Second method Again 10 mg of dried CaCO3 particles was weighted and dissolved in 12 ml H2O in an 15 ml
tube. Ultrasound was applied for 10 seconds. This time the sedimentation time for the first
fraction was expanded to 10 minutes. The upper 1 ml was taken, the tube was shaken, exposed
to ultrasound and this was repeated three times, as such this includes fraction 1 and 2. The next
two fractions, each existing of 2 ml, were taken with a sedimentation time of 5 minutes. Fraction
5 was already pipetted at 2,5 minutes and the sixth fraction after 1,25 minutes. Between each
sedimentation the tube is shaking and exposed to ultrasound for 10 seconds.
7.4. Microscopy Different methods of analysing the capsules were used in the duration of the different tests. At
first a method was developed that made it easy to analyse the particles on the microscopic
pictures with a computer program by using rhodamine to make the particles and capsules
fluorescent. Later on this became a problem when the dye began to leak from the particles and
after some failed attempts to resolve this the chose was made to manually measure the diameter
of the particles and capsules.
Figure 10: Main scheme of separating
CaCO3 particles by gravitation
32
7.4.1. First method To get images and measure the size of the particles and capsules a TI Widefield microscope
was used with a 100x oil lens. The particles and capsules were looked at before core dissolution,
before heating and after heating. 10 µl of the capsule solution was taken and 2 µl of 10-3 M
rhodamine 6G was added to dye the polymers for better images. The solution was diluted to
100 µl and centrifuged. The supernatants was replaced with 100 µl distilled water to wash away
the background fluorescence. The capsules were then exposed to ultrasound for five seconds to
break aggregation so single capsules can be measured. Sixteen pictures were taken on different
places inside the sample drop to create enough results for statistical analysis. With Fiji, a
computer program to analyse microscopic images, the size of the capsules was measured.
7.4.2. Second method Instead of dying the particles, the particles were simply observed by transmission with a 100x
oil lens. To reduce the analysis time of measuring by hand, only nine pictures were taken of
each sample. The diameter of the particles was then measured by drawing a line that indicates
the length over each particle.
7.5. Thermal stability of hGMPk Before applying the heat treatment of the capsules with enzyme inside, the thermal stability of
the enzyme needs to be checked. Therefor the activity is measured at certain points with a
standardized assay.
7.5.1. Defining hGMPk concentration needed in assay A range of concentrations of the enzyme were tested to determine the linearity of the enzyme
concentration. The helper enzymes cannot be the limiting factor otherwise their activity will be
measured instead of the activity of hGMPk which is indicated by the end of the linearity. The
concentrations used for this test were 1, 5, 10, 15, 20, 50 and 100 nM. The activity was measured
using the hGMPk standardized activity assay.
7.5.2. Heating of the enzyme hGMPk and stabilisation Samples of 10 µl of 5 µM enzyme were kept in an Eppendorf tube for 30 minutes at 25, 30, 35
and 40°C. After half an hour, the samples were put back on ice and diluted to an end
concentration of 15 nM in the assay in preparation of the activity measurement.
In addition the same was done but with adding 1 mg/ml bovine serum albumin (BSA) to the
enzymes to be heated. This will result in a higher chance that the enzyme wont destabilize.
7.5.3. Measuring activity of hGMPk 3 µl of the heated enzyme is diluted in the access of water from the standardized activity assay.
The diluted samples that were calculated to contain the amount of enzyme resulting in an end
concentration of 15 nM in the assay, were measured in the same way with the standardized
activity assay.
33
8. Results and discussion
8.1. Comparing the enzyme activity of hGMPk loaded on
different sized and shaped CaCO3 particles At first there was some trial and error to get the right amount of loaded particles in the assay
without crossing its boundaries. But after diluting the solution a hundred times, the activity of
the loaded enzymes on the different shaped and sized particles could be compared. This is
shown in Figure 11 where the absorbance per different sized and shaped particle is expressed
as a function of time.
Figure 11: Graph of the activity of loaded hGMPk on different sized and shaped CaCO3 particles. The absorbance of NADH
at 340 nm is expressed as a function of time.
Figure 11 shows a relative much bigger activity for star-like particles than for the others. The
cubical particles have the least activity which is expected if their loading characteristics are
taken into account. The cubical are the crystalized form of CaCO3 and not porous, as such the
absorption of enzyme should be less. From highest activity to lowest the list goes: star-like,
small elliptical, large spherical, small spherical and cubical.
To get a better understanding of the activity of each loaded particle, the specific activity and
the activity per mg particle was calculated. The results are viewed in Table 2.
0
0,2
0,4
0,6
0,8
1
1,2
0 500 1000 1500 2000 2500 3000 3500
Absorbance (340 nm)
Time (sec)
Cubical
Large spherical
Small spherical
Small elliptical
Star-like
34
Table 2: Activity of the loaded hGMPK on particles different size and shape per mg particles and per mg enzyme
Particle Activity U/mg particles Activity U/mg enzyme
Cubical 2,122 353,698
Large spherical 2,354 84,061
Small spherical 1,968 49,196
Small elliptical 1,736 57,878
Star-like 3,357 69,936
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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53
12. Attachments
12.1. Script Fiji measuring particles with rhodamine id = getTitle();
selectWindow(id);
run("Duplicate...", " channels=1");
close();
selectWindow(id);
run("Duplicate...", "duplicate channels=1");
run("8-bit");
run("8-bit");
run("Subtract Background...", "rolling=40 stack");
run("Gaussian Blur...", "sigma=1 stack");
setAutoThreshold("Li dark");
//run("Threshold...");
run("Set Measurements...", "area perimeter shape feret's redirect=None decimal=3");
run("Create Mask");
run("Watershed");
run("Analyze Particles...", " size=5-Infinity circularity=0.80-1.00 clear add slice");
54
12.2. Significance test of the result of heating capsules EXAMINE VARIABLES= InitialCapsules
/PLOT BOXPLOT STEMLEAF HISTOGRAM NPPLOT
/COMPARE GROUPS
/STATISTICS DESCRIPTIVES
/CINTERVAL 95
/MISSING LISTWISE
/NOTOTAL.
Testing Gaussian distribution InitialCapsules
Explore
Notes
Output Created 20-MAR-2016 11:43:03
Comments
Input Active Dataset DataSet0
Filter <none>
Weight <none>
Split File <none>
N of Rows in Working
Data File 547
Missing Value
Handling
Definition of Missing User-defined missing values for
dependent variables are treated as
missing.
Cases Used Statistics are based on cases with
no missing values for any
dependent variable or factor used.
Syntax EXAMINE
VARIABLES=InitialCapsules
/PLOT BOXPLOT STEMLEAF
HISTOGRAM NPPLOT
/COMPARE GROUPS
/STATISTICS DESCRIPTIVES
/CINTERVAL 95
/MISSING LISTWISE
/NOTOTAL.
Resources Processor Time 00:00:01,47
Elapsed Time 00:00:01,39
55
Case Processing Summary
Cases
Valid Missing Total
N Percent N Percent N Percent
InitialCapsules 215 39,3% 332 60,7% 547 100,0%
Descriptives
Statistic Std. Error
InitialCapsules Mean 6,18712 ,069885
95% Confidence Interval
for Mean
Lower Bound 6,04937
Upper Bound 6,32487
5% Trimmed Mean 6,19004
Median 6,11900
Variance 1,050
Std. Deviation 1,024716
Minimum 3,626
Maximum 8,549
Range 4,923
Interquartile Range 1,415
Skewness ,008 ,166
Kurtosis -,253 ,330
Tests of Normality
Kolmogorov-Smirnova Shapiro-Wilk
Statistic df Sig. Statistic df Sig.
InitialCapsules ,040 215 ,200* ,994 215 ,487
*. This is a lower bound of the true significance.
a. Lilliefors Significance Correction
56
InitialCapsules
InitialCapsules Stem-and-Leaf Plot
Frequency Stem & Leaf
4,00 3 . 6778
9,00 4 . 022223344
11,00 4 . 77888999999
32,00 5 . 00000000011112222333344444444444
41,00 5 . 55555566666666677777778888888899999999999
36,00 6 . 000000000111111122222233333334444444
35,00 6 . 55556666666667777777777788888899999
23,00 7 . 00001111122222223344444
14,00 7 . 55555666777778
7,00 8 . 0002234
3,00 8 . 555
Stem width: 1,000
Each leaf: 1 case(s)
57
EXAMINE VARIABLES=HeatedCapsules
/PLOT BOXPLOT STEMLEAF HISTOGRAM NPPLOT
/COMPARE GROUPS
/STATISTICS DESCRIPTIVES
/CINTERVAL 95
/MISSING LISTWISE
/NOTOTAL.
58
Testing Gaussian distribution HeatedCapsules
Explore
Notes
Output Created 20-MAR-2016 11:55:48
Comments
Input Active Dataset DataSet0
Filter <none>
Weight <none>
Split File <none>
N of Rows in Working
Data File 547
Missing Value
Handling
Definition of Missing User-defined missing values for
dependent variables are treated as
missing.
Cases Used Statistics are based on cases with
no missing values for any
dependent variable or factor used.
Syntax EXAMINE
VARIABLES=HeatedCapsules
/PLOT BOXPLOT STEMLEAF
HISTOGRAM NPPLOT
/COMPARE GROUPS
/STATISTICS DESCRIPTIVES
/CINTERVAL 95
/MISSING LISTWISE
/NOTOTAL.
Resources Processor Time 00:00:01,48
Elapsed Time 00:00:01,47
Case Processing Summary
Cases
Valid Missing Total
N Percent N Percent N Percent
HeatedCapsules 547 100,0% 0 0,0% 547 100,0%
59
Descriptives
Statistic Std. Error
HeatedCapsules Mean 4,18731 ,036220
95% Confidence Interval
for Mean
Lower Bound 4,11616
Upper Bound 4,25846
5% Trimmed Mean 4,16097
Median 4,09200
Variance ,718
Std. Deviation ,847120
Minimum 2,709
Maximum 7,143
Range 4,434
Interquartile Range 1,335
Skewness ,401 ,104
Kurtosis -,438 ,209
Tests of Normality
Kolmogorov-Smirnova Shapiro-Wilk
Statistic df Sig. Statistic df Sig.
HeatedCapsules ,071 547 ,000 ,972 547 ,000
a. Lilliefors Significance Correction
HeatedCapsules
60
HeatedCapsules Stem-and-Leaf Plot
Frequency Stem & Leaf
28,00 2 . 7778888899999
113,00 3 . 00000000011111111111222222222222333333333334444444444444
111,00 3 . 55555555555555666666666667777777888888888888899999999
95,00 4 . 0000000000011111222222222233333333344444444444
99,00 4 . 555555555566666666667777777778888888889999999999
68,00 5 . 000000011111122222222333334444444
24,00 5 . 5556667899
7,00 6 . 123&
2,00 Extremes (>=7,1)
Stem width: 1,000
Each leaf: 2 case(s)
& denotes fractional leaves.
61
NPAR TESTS
/WILCOXON=InitialCapsules WITH HeatedCapsules (PAIRED)
/MISSING ANALYSIS.
NPar Tests
Notes
Output Created 20-MAR-2016 12:06:16
Comments
Input Active Dataset DataSet0
Filter <none>
Weight <none>
Split File <none>
N of Rows in Working
Data File 547
Missing Value
Handling
Definition of Missing User-defined missing values are
treated as missing.
Cases Used Statistics for each test are based
on all cases with valid data for
the variable(s) used in that test.
Syntax NPAR TESTS
/WILCOXON=InitialCapsules
WITH HeatedCapsules
(PAIRED)
/MISSING ANALYSIS.
Resources Processor Time 00:00:00,00
Elapsed Time 00:00:00,00
Number of Cases
Alloweda 224694
a. Based on availability of workspace memory.
Wilcoxon Signed Ranks Test
Ranks
N Mean Rank Sum of Ranks
HeatedCapsules -
InitialCapsules
Negative Ranks 207a 111,34 23048,00
Positive Ranks 8b 21,50 172,00
Ties 0c
Total 215
a. HeatedCapsules < InitialCapsules
b. HeatedCapsules > InitialCapsules
c. HeatedCapsules = InitialCapsules
62
Test Statisticsa
HeatedCapsules -
InitialCapsules
Z -12,525b
Asymp. Sig. (2-
tailed) ,000
a. Wilcoxon Signed Ranks Test
b. Based on positive ranks.