Federal Training and Research Institute for Industrial Chemistry Secondary College for Chemical Technology educational focus: Biochemistry, Biotechnology and Genetic Engineering Höhere Bundeslehr- und Versuchsanstalt für chemische Industrie – Höhere Lehranstalt – Biochemie, Biotechnologie und Gentechnik diploma thesis (Diplomarbeit) Optimization of the Synthesis of Magnetic Cellulose Microparticles for the Extracorporeal Blood Purification conducted in the academic year 2010/11 by: supervised by: Prof. Dipl.Ing. Dr. rer. nat. tech. DominicPhilipp Klein 5AHCHB8 Veronika EBERT (internal supervision) Prof. Dipl.Ing. Dr. techn. Viktoria Maria Enk 5AHCHB4 Bibiana Meixner (examiner) Dipl.Ing. Dr. techn. Marion Ettenauer (external supervision) Vienna (Wien), 23 rd of May 2011 In cooperation with the „Department für Klinische Medizin und Biotechnologie – Zentrum für Biomedizinische Technologie“ at the „DonauUniversiät Krems“. A project sponsored by .
134
Embed
diploma!thesis! - gfc.at · Erklärung an Eides statt Affidavit Wir erklären hiermit an Eides statt, dass wir die vorliegende Diplomarbeit, ! We hereby declare that the following
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Federal Training and Research Institute for
Industrial Chemistry Secondary College for Chemical Technology
educational focus: Biochemistry, Biotechnology and Genetic Engineering
Höhere Bundeslehr- und Versuchsanstalt für chemische Industrie – Höhere Lehranstalt – Biochemie, Biotechnologie und Gentechnik
diploma thesis (Diplomarbeit)
Optimization of the Synthesis of Magnetic Cellulose Microparticles
for the Extracorporeal Blood Purification conducted in the academic year 2010/11 by:
supervised by:
Prof. Dipl.-Ing. Dr. rer. nat. tech.
Dominic-‐Philipp Klein 5AHCHB-‐8 Veronika EBERT (internal supervision) Prof. Dipl.-Ing. Dr. techn. Viktoria Maria Enk 5AHCHB-‐4 Bibiana Meixner (examiner) Dipl.-Ing. Dr. techn. Marion Ettenauer (external supervision)
Vienna (Wien), 23rd of May 2011
In cooperation with the „Department für Klinische Medizin und Biotechnologie – Zentrum für Biomedizinische Technologie“ at the „DonauUniversiät Krems“.
A project sponsored by .
Erklärung an Eides statt
Affidavit
Wir erklären hiermit an Eides statt, dass wir die vorliegende Diplomarbeit,
We hereby declare that the following diploma thesis,
„Optimization of the Synthesis of
Magnetic Cellulose Microparticles for the Extracorporeal Blood Purification“
selbstständig und ohne Hilfe verfasst, andere als die angegebenen Quellen nicht benutzt und die benutzten Quellen wörtlich oder inhaltlicher entnommenen Stellen als solche kenntlich gemacht haben.
was written without any exception by ourselves and without the use of any other than the sources, tools and all other explanations that we copied directly or in their sense are indicated as such.
Ort, Datum Viktoria Maria Enk Ort, Datum Dominic-Philipp Klein
Who is who?
This page serves the facility to the authors of this diploma-‐thesis to introduce themself.
I, Dominic-‐Philipp Klein, ... … was born on the 13th of December 1991 in Vienna where I grew up. I spend a lot of my leisure time traveling round the world what I would describe as my favorite hobby. I'm a very convivial companion and for that reason often "on the road" but I do also like spending my time with my family at home. In high school, I fell in love with chemistry and biology what encouraged me to leave the „Bernoulli Gymnasium“ in Wien-‐Donaustadt and sign up for the „Höhere Bundes-‐, Lehr-‐ und Versuchsanstalt für chemische Industie”. After leaving school I would like to study medicine, that’s why I’m that much amazed by our project.
Vienna, 17th of June 2010
I, Viktoria Maria Enk … was born on the 18th of September 1992 in Lilienfeld. I spent my childhood in Wiesenbach in Lower Austria and attended language school in Lilienfeld until I went to the Rosensteingasse in 2006. I moved to Vienna in 2009 because the exertion of the long way had become too hard. I like spending my time with my friends and my animals, travelling and having fun. Sometimes I even like going to school! The reason why I´ve convinced my diploma-‐thesis-‐partner to write the paper in english, is because I want to study in Ireland after school so I need to learn the technical vocabulary better.
Vienna, 22th of June 2010
… and now please enjoy reading the following pages!
Yours Viktoria and Dominic!
Acknowledgements
This thesis would not have been possible unless the support of many important people:
To write this thesis would not have been possible unless the support of our external supervision DI Dr. techn. Marion Ettenauer, who helped us whenever a problem occurred during the practical work.
We would also like to thank Priv. Doz. Dr. Viktoria Weber, and Univ. Prof. Dr. Dieter Falkenhagen the Head of the Department of Clinical Medicine and Biotechnology, who also helped us with answering our questions and providing us the literature we needed.
We would also like to show our gratitude to AV Prof. DI Dr. techn. Bibiana Meixner “Bibi” who not only supported us during the work on this thesis but also during the last few years at school. We are very grateful for making the experience of having such a caring teacher who was a little bit of a mum to all of us.
Prof. Dr. rer. nat. tech. Veronika Ebert has made available her support in a number of ways reading our thesis many times. We want to thank her for the good preparation for all the theses that may follow in our academic lives.
We are grateful for the contribution of Prof. Mag. Christine Raschauer-Andrecs who read all our text parts and corrected english language mistakes.
Another important person who is to be acknowledged here is Prof. DI Bert Sefcik “Onkel Bert”, our form teacher.
We also want to thank our parents, grandparents and other family members who are the ones who have been supporting us our whole lives long and made the education at this school possible.
An extraordinary “Dankeschön” goes to Dominics aunt, Rosa Krail, who provided accommodation and just the best he could wish of support in the time of the practical part of the thesis.
We would like to thank Chrisi Julius for her help and support with our graphics.
We want to show our gratitude to Johannes Theierling, for printing our final papers.
Last but not least we want to thank our friends,
Andi Dietrich, Daniel Theierling, Nici Golias, Mo Schöll, Simone Panholzer
for having endured all of our bad moods in our time together.
Viktoria Maria Enk page 1 of 130 Dominic-Philipp Klein
3.2.1 REGULATIVE FUNCTIONS:..................................................................................................................... 15 3.2.2 SYNTHESIS FUNCTIONS: ....................................................................................................................... 15 3.2.3 STORAGE-‐FUNCTION: .......................................................................................................................... 15 3.2.4 CATABOLISM OF BODY OWN SUBSTANCES: .............................................................................................. 16 3.2.5 CATABOLISM OF FOREIGN SUBSTANCES: ................................................................................................. 18
3.3 LIVER FAILURE: ........................................................................................................................................ 20 3.4 THE PROMETHEUS SYSTEM (COMBINED DIALYSIS-‐ADSORBER-‐TREATMENT): .................................................. 21 3.5 THE MICROSPHERES DETOXIFICATION SYSTEM (MDS):................................................................................. 24 3.6 SAFETY CONSIDERATIONS:......................................................................................................................... 26 3.7 AIMS OF THIS STUDY: ............................................................................................................................... 28
4. MATERIALS AND METHODS .................................................................................................................30 4.1 MATERIALS ............................................................................................................................................. 30
4.1.1 DEVICES ........................................................................................................................................... 30 4.1.2 SINGLE USE MATERIALS ...................................................................................................................... 32 4.1.3 CHEMICAL SUBSTANCES ....................................................................................................................... 33
4.2 METHODS.............................................................................................................................................. 36 4.2.1 SYNTHESIS OF MAGNETIC CELLULOSE MICROPARTICLES ............................................................................ 37 4.2.3 CHARACTERISATION OF MAGNETIC CELLULOSE MICROPARTICLES................................................................ 42
5. RESULTS .............................................................................................................................................47 5.1 GUIDELINES FOR INTERPRETATION OF THE RESULTS ..................................................................................... 47 5.1.1 THE MAGNETIC MICROPARTICLES WERE ANALYZED ACCORDING TO THE FOLLOWING ASPECTS: .................. 47 5.1.2 OVERVIEW OF THE PROCESSES:........................................................................................................... 48 5.1.3 AN OPTIMAL SUITABLE PARTICLE SHOULD HAVE FOLLOWING PROPERTIES:..................................................... 49
Viktoria Maria Enk page 2 of 130 Dominic-Philipp Klein
5.2. RESULTS .............................................................................................................................................. 49 5.2.1 VISUAL CHARACTERISTICS-‐ PRE TESTS .................................................................................................... 49 5.2.2 SCANNING ELECTRON MICROSCOPY PICTURES ................................................................................... 57 5.2.3 DETERMINATION OF THE MAGNETIC BEHAVIOUR................................................................................. 68 5.2.4 DETERMINATION OF THE PARTICLE SIZE AND SIZE DISTRIBUTION .................................................... 73 5.2.5 MEASUREMENT OF DENSITY .......................................................................................................... 112 5.2.6 DRY MATTER CONTENT.................................................................................................................. 115 5.2.7 COMPARISON OF SIZE DISTRIBUTIONS AFTER 5MIN/10MIN AND WITHOUT AN ULTRASONIC BATH .... 116
5.3 SUMMARY OF THE BEST PARTICLES AND INTERPRETATION .......................................................................... 120
APPENDIX.............................................................................................................................................124 INDEX OF FIGURES: ...................................................................................................................................... 127 INDEX OF TABLES: ........................................................................................................................................ 129 INDEX OF ABBREVIATIONS:............................................................................................................................ 130 COURSE OF THE PROJECT:............................................................................................................................... 130
Viktoria Maria Enk page 3 of 130 Dominic-Philipp Klein
1. ABSTRACT
ENGLISH
The liver is the central organ of the human metabolism so a complete breakdown would
cause a patients death. Due to the fact that the liver is a regenerative organ it can be
supported in its regeneration through supporting metabolic functions such as
detoxification.
If the liver cannot regenerate itself it has to be replaced by a donate organ. The time from
the failure of the organ to the transplantation has to be bridged. Because the body does
not have a detoxification unit during this time, sepsis is a possible risk. Because of this
reason the detoxification is supported by the MDS.
The MDS (Microspheres based Detoxification System) is a further development of the
Prometheus System that is already in clinical use.
For both systems blood is taken from the patient and the cells, that are recycled into the
body, are separated from the plasma. In the so called primary circuit blood and cells are
circulated while the secondary circuit contains just plasma.
The blood plasma contains coagulation factors that make the use of heparin necessary.
The plasma is purified in the secondary circuit of the Prometheus systems in capsules with
immobilized mircoparticles by the use of variable sorption-processes.
Viktoria Maria Enk page 4 of 130 Dominic-Philipp Klein
Medical applications have to be proved by double failure security. It has to be secured that
the patient cannot be harmed in case of breakdown of one security barrier. In the
Prometheus system this can be ensured by the immobilization in capsules.
In the MDS- System the specific surface is maximized through the use of smaller particles
(10µm in the Prometheus <5µm in the MDS). The efficiency is also increased by the
use of a particle suspension instead of capsules as a secondary circuit.
Figure 1: The MDS-system
If the particles enter the body they could cause embolism. Thus it has to be considered
that the safety barrier of the immobilization in capsules is not available in this system so it
has to be substituted with another security barrier.
Therefore a fluorescence detector with a magnetic trap was developed and 1-10% marker
particles are added to the adsorber or absorber particles.
The magnetically and fluorescence labeled particles are accumulated in front of the
detector to increase the emitted signal. If a fluorescence signal is detected the pumps are
stopped and the trespass of microparticles from the suspension into the human body is
prevented.
Viktoria Maria Enk page 5 of 130 Dominic-Philipp Klein
Currently just the so called Dynabeads® Tosylactivated (Invitrogen) are available
commercially. These are suitable for use in the system but are very expensive. For
medical application it is important to find a cost reduced version of the particles.
These particles are cellulose-beads in whose pores magnetite is precipitated in alkaline
media following the reaction below:
Fe2+ + 2 Fe3+ + 8 OH- → Fe3O4 + 4 H2O
The aim was to find an optimal way for the synthesis of the magnetic microparticles for the
extracorporeal blood purification.
The particles act as a carrier medium for covalently bound adsorbent and absorbent
material. They are also used for binding the fluorescence agent, cresyl violet on the
surface of the marker particles.
The synthesis was varied concerning impregnation, precipitation and removal of residual
components (washing) to synthesize ideal and cheap microparticles.
For impregnation steps the protective colloids Methylcellulose (MC) und Polyethylenglycol
(PEG) were used. To make a comparison possible one series of tests was made without
coating.
The two different alkali Sodium hydroxide and ammonia were used for precipitation. Each
precipitation was done one time with and one time without a disperser, the so called Ultra
Turrax.
In the last step of the synthesis all residuals of the precipitation steps should be removed
(for instance excess precipitation reagent, precipitate outside of the pores or destroyed
cellulose beads).
For this experiment reversed osmosis water, phosphate buffer (PBS) or an albumin solution were used.
Viktoria Maria Enk page 6 of 130 Dominic-Philipp Klein
Figure 2: synthesis chain
The suitability of the particles was tested with several analyses. The particles were
suspended in the washing agents to test their behavior in a suspension.
To test the properties of sedimentation the density which is an important parameter for the
sedimentation was measured.
The magnetic properties were determined through wandering of the particles in a magnetic
field from the suspension to a permanent magnet.
This value is measured in seconds that the suspension needs to become clear. This is
very important because the magnetic accumulation in front of the detector is the crucial
factor for the velocity of the signaling and stop of the pumps.
Viktoria Maria Enk page 7 of 130 Dominic-Philipp Klein
A further point is the homogeneity of the particle size which was determined with the
“Mastersizer” from Malvern Instruments.
The method is based on refraction and bending of bundled light on the surface of the
particles (Mie-theory)
For visualization the best particles were sent to the TU Dresden for analysis with scanning
electron microscopy. On these pictures suspicions of aggregations can be proved or
withdrawn.
Furthermore undesirable precipitation on the surface of the particles can be shown.
Magnetite on the surface of the particles also reduces the possible covalent binding sites.
The optimal way of synthesis that was determined in this study is:
• coating with Polyethylenglycol
• precipitation with ammonia
• with Ultra Turrax
• washing with reversed osmosis water
These particles have the best properties for further application.
Viktoria Maria Enk page 8 of 130 Dominic-Philipp Klein
DEUTSCH
Die Leber ist das Zentrum des Stoffwechsels im menschlichen Körper ein Ausfall dieses
Organs würde für den Patienten unbehandelt den Tod bedeuten. Da die Leber ein
regeneratives Organ ist, kann sie bei ihrem Wiederaufbau unterstützt werden indem ihr
Stoffwechselfunktionen, wie die Entgiftung des Körpers, abgenommen werden.
Im Fall eines kompletten Ausfalls des Organs muss dieses durch ein Spenderorgan
ersetzt werden. Die Zeit vom Organversagen bis zur Transplantation muss jedoch
überbrückt werden. Da der Patient keine funktionierende Entgiftungseinheit im Körper trägt
droht eine Sepsis. Diese Entgiftung wird durch ein extrakorporales System ersetzt, das
Microspheres based Detoxification System.
Das MDS (Microspheres based Detoxification System) ist eine Weiterentwicklung des
bereits in klinischer Anwendung befindlichen Prometheus Systems. Für beide Systeme
wird das aus dem Patienten entnommene venöse Blut von zellulären Bestandteilen
getrennt, welche direkt wieder dem Körper zugeführt werden. Es wird allgemein von einem
Primärzyklus (Blut mit zellulären Bestandteilen) und einem Sekundärzyklus
(Plasmakreislauf) gesprochen. Das Blutplasma (serale Blutkomponenten zuzüglich
Gerinnungsfaktoren) wird im Prometheus System mit in Kapseln immobilisierten
Mikropartikel, durch variable Sorptionsverfahren gereinigt.
Medizinische Anwendungen verlangen doppelte Fehlersicherheit, das bedeutet, dass im
Falle des Ausfalls einer Komponente der Patient nicht gefährdet wird. Diese doppelte
Fehlersicherheit ist im Prometheussystem durch die Immobilisierung der Micropartikel
gegeben.
Viktoria Maria Enk page 9 of 130 Dominic-Philipp Klein
Im MDS System wird die spezifische Oberfläche und damit die Sorptionskapazität erhöht
indem die Partikel noch kleiner (10µm im Prometh <5µm im MDS) synthetisiert werden.
Weiters wird der Phasenübergang durch die Verwendung einer Partikelsuspension als
Sekundärkreislauf massiv erhöht.
Figure 3: The MDS-system
Ein Eindringen der Partikel in den menschlichen Körper könnte fatale Folgen haben, als
eine von vielen drastischen Folgen lässt sich Embolie, also eine Ansammlung von
Partikeln in Gefäßen bis zur Verstopfung, nennen.
Bei der Anwendung von den Partikeln in Suspension fällt eine Sicherheitsbarriere
(Immobilisierung in Kapseln) weg welche durch eine andere substituiert werden muss. Für
das MDS wurde daher ein Fluoreszenzdetektor mit Magnetfalle entwickelt. Durch die
Magnetfalle sammeln sich die Partikel vor dem Detektionsfeld und erzeugen dadurch ein
höheres Signal, dieses Signal unterbricht die Blutentnahme aus dem Patienten und damit
auch die Rückführung. Diese Methode setzt jedoch den Einsatz von Fluoreszenz –
markierten, magnetischen Mikropartikeln voraus, welche zu ca. 1-10% der Summe an
Partikeln in Suspension zugegeben werden müssen.
Viktoria Maria Enk page 10 of 130 Dominic-Philipp Klein
Gegenwärtig sind nur die so genannten Dynabeads® Tosylactiviert von Invitrogen
kommerziell erhältlich und bieten sich für die Verwendung an. Diese Dynabeads zeichnen
sich jedoch durch einen sehr hohen Preis aus. Für die medizinische Anwendung gilt es
daher eine kostenreduzierte Version der Partikel zu finden.
Es handelt sich bei diesen Mikropartikeln um Cellulose-Perlen in deren Poren Magnetit
nach folgendem Schema in basischem Milieu präzipitiert wurde:
Fe2+ + 2 Fe3+ + 8 OH- → Fe3O4 + 4 H2O
Die Zielsetzung dieser Arbeit war es einen optimalen Syntheseweg für die Herstellung von
diesen magnetischen Mikropartikeln für die extracorporale Blutreinigung zu finden. Diese
Partikel dienen als Trägermedium für kovalent gebundene Adsorbenzien und
Absorbenzien. Weiters dienen diese Partikel als Grundlage für die kovalente Bindung des
Fluoreszenzfarbstoffes, Cresylviolett auf den Detektorpartikeln.
Die Synthese wurde in den Hauptpunkten Imprägnierung, Fällung und Entfernung von
Restbestandteilen aus der Fällung (Waschen) variiert um ideale und günstige Mikropartikel
zu synthetisieren.
Im Punkt Imprägnierung wurden die protektiven Kolloide MethylCellulose (MC) und
PolyEthylenGlycol (PEG) verwendet. Des Weiteren wurde aus Vergleichsgründen auch
eine Synthesereihe ohne Imprägnierschritt hergestellt.
Im Fällungsschritt wurden die verschiedenen Fällungsreagenzien Natriumhydroxid und
Ammoniak variiert. Um den Einfluss von Dispergiergeräten auf die Fällung auszutesten
wurde jede Versuchsreihe jeweils einmal mit und einmal ohne Ultra Turrax ausgefällt.
In der letzten Synthesestufe sollen alle Restbestandteile der Fällung entfernt werden, dazu
zählt beispielsweise überschüssiges Fällungsreagenz, Präzipitat außerhalb der
Celluloseporen und zerstörte Cellulosebeads. Für diesen Versuch wurde in einer Reihe
Viktoria Maria Enk page 11 of 130 Dominic-Philipp Klein
Umkehrosmosewasser verwendet in anderen Versuchen, Phosphatpuffer und eine
Albuminlösung.
Figure 4: synthesis chain
Die Eignung der Partikel wurde durch zahlreiche Analysen überprüft. Durch die Aufnahme
der Partikel in ihren Waschlösungen wurden deren Eigenschaften in einer Suspension
ermittelt.
Um unterschiedliche Sedimentationsverhalten abzuschätzen wurde die Partikeldichte
bestimmt, da die Dichte ein Einflussfaktor auf die Sedimentation ist.
Die magnetische Separation aus dem Träger wurde mittels der Wanderung im
magnetischen Feld ermittelt. Dieser Wert ist insbesondere wichtig weil sich die
magnetische Separation aus dem Medium bestimmend für Geschwindigkeit der
Signalgabe ist.
Viktoria Maria Enk page 12 of 130 Dominic-Philipp Klein
Ein weiterer Punkt ist die Gleichmäßigkeit der Größe der Partikel, um diese zu bestimmen
wurde die Mastersizing-Methode von Malvern Instruments verwendet. Diese Methode
basiert auf der Brechung und Beugung von gebündeltem Licht an der Partikeloberfläche
(Mie-Theorie).
Zum Zweck der Visualisierung wurden Partikel, welche in oben genannten Analysen die
besten Resultate aufwiesen, nach Dresden geschickt um dort Bilder im
Rasterelektronenmikroskop aufzunehmen. Auf diesen Bildern können eventuelle
Verdachte von Aggregation aus der Größenverteilungsmessung bestätigt oder verworfen
werden. Des Weiteren können auch unerwünschte Präzipitation an der Partikeloberfläche
dargestellt werden. (Magnetit an der Partikeloberfläche verringert die möglichen
kovalenten Bindungsstellen der Cellulose)
Aus der Arbeit geht als optimaler Syntheseweg hervor, dass Partikel welche mit
PolyEthylenGlycol vor der Fällung geschützt, mit Ammoniumhydroxid unter Verwendung
eines Ultra Turrax Dispergiergeräts gefällt und mit Umkehrosmosewasser gewaschen
wurden, die beste Eignung für die Weiterverwendung aufweisen.
Viktoria Maria Enk page 13 of 130 Dominic-Philipp Klein
2. AIM
The aim of this thesis was to optimize the synthesis of magnetic cellulose microparticles
for the extracorporeal blood purification.
The at present available magnetic microparticles on the global marked are settled on a
high price level so that mass application for extracorporeal blood purification methods is
too expensive.
The particle synthesis designed for the Microspheres based Detoxification System is a
cheaper alternative for the commercially available magnetic beads.
The optimization setup was to vary several steps of the synthesis (impregnation,
precipitation, reaction support, washing) provided by the Donau Universität Krems.
The impregnation was varied using different protective colloids before the precipitation.
Those protective colloids were MethylCellulose and PolyEthylene Glycol for comparison
another series was conducted without any protective colloid.
Precipitation was conducted with different precipitants (Sodiumhydroxide and
Ammoniumhydroxide). Both precipitations were realized twice, one time with a Ultra Turrax
dispenser as reaction support, the second one without any support.
The removal of residuals of the precipitation was also varied using different washing
agents (water, albumin solution, phosphate buffered solution).
Viktoria Maria Enk page 14 of 130 Dominic-Philipp Klein
3. INTRODUCTION
3.1 THE LIVER
The liver is the central organ of the metabolism and the biggest gland in the body of
vertebrates. A human liver weighs between 1500 and 2000g and is located in the right
In the figure above few agglomerations could be detected so the particle has
to be subjected to further analyses to clarify whether the other parameters
are as good as the size distribution.
Viktoria Maria Enk page 112 of 130 Dominic-Philipp Klein
5.2.5 MEASUREMENT OF DENSITY
The density is a very important measure because the density is a factor of sedimentation. To keep the plasma-particle-suspension of the secondary circuit homogenous the magnetic particles must not show a higher density than other particles in the cycle otherwise the magnetic celluloses would rather form a sediment than other particles. The requirement to the magnetic particles is that the density-difference to non-magnetic ones is not too high.
The variables from the pyknometric formula:
were substitued to the pariables given:
ρS ... Density [ρ]
mP ... m0
mPS ... m2
mPSW ... m3
mW ... m1
ρW ... density of water at measurement temperature
All measurements were conducted at 24°C the density of water at this temperature is 0,99729 g/cm3. [6]
Viktoria Maria Enk page 113 of 130 Dominic-Philipp Klein
5.2.5.1 Magnetic celluloses:
Table 20: Density of magnetic celluloses
The measurement of the Density of the magnetic celluloses led to an appoximate density of:
DENSITY (MAGNETIC MICROPARTICLES) : 1,57255 g/cm3
The measurement nr.2 was not included into the approximation, it was assumed an outlier.
Viktoria Maria Enk page 114 of 130 Dominic-Philipp Klein
5.2.5.2 HPR 10:
The HPR10 is commercially available particle and served as reference for our measurements.
Table 21: Density of HPR 10
The measurement of the Density of the HPR10 led to an appoximate density of:
DENSITY (HPR10) : 0,31054 g/cm3
5.2.5.3 Celluloses from the TU Dresden (in water)
Table 22: Density of Celluloses from the TU Dresden
The measurement of the Density of the non-magnetic celluloses led to an appoximate density of:
DENSITY (NON-MAGNETIC CELLULOSES) : 1,42727 g/cm3
As conclusion can be said that non-magnetic and magnetic microparticles do not distinguish in an inacceptable way so that they could both be used in the MDS. [12]
Viktoria Maria Enk page 115 of 130 Dominic-Philipp Klein
5.2.6 DRY MATTER CONTENT
Table 23: Dry matter content
As shown in table 23 above the dry matter of the unmodified cellulose
microparticles is the highest, followed by the magnetic celluloses
microparticles. The HPR10- adsorber- particles have the least dry matter of
all measured particles. [7] [8]
Viktoria Maria Enk page 116 of 130 Dominic-Philipp Klein
5.2.7 COMPARISON OF SIZE DISTRIBUTIONS AFTER 5MIN/10MIN AND WITHOUT AN ULTRASONIC BATH
Table 24: comparison of size distributions
Table 25: comparison of size distributions
Table 26: comparison of size distributions
Viktoria Maria Enk page 117 of 130 Dominic-Philipp Klein
Table 27: comparison of size distributions
Table 28: comparison of size distributions
Table 29: comparison of size distributions
Viktoria Maria Enk page 118 of 130 Dominic-Philipp Klein
Table 30: comparison of size distributions
Table 31: comparison of size distributions
Table 32: comparison of size distributions
Viktoria Maria Enk page 119 of 130 Dominic-Philipp Klein
Table 33: comparison of size distribution
Viktoria Maria Enk page 120 of 130 Dominic-Philipp Klein
5.3 SUMMARY OF THE BEST PARTICLES AND INTERPRETATION
a) Uncoated magnetic cellulose microparticles (U-mcm)
Parameter: NH4OH / + UT / H2O
• before the precipitation: no coating
• precipitation in NH4OH
• use of Ultraturrax
• use of H2O for washing
Table 34: magnetic properties of uncoated particles precipitated
with NH4OH with UT washed with
H2O
Figure 74: comparison of size distributions of uncoated particles precipitated with
NH4OH with UT washed with H2O
Viktoria Maria Enk page 121 of 130 Dominic-Philipp Klein
b) MC-coated magnetic cellulose microparticles (MC-mcm)
Parameter: NH4OH / - UT / H2O
• before the precipitation: use of Methylcellulose
• precipitation in NH4OH
• without Ultraturrax
• use H2O for washing
Table 35: magnetic properties of
MC-coated particles precipitated
with NH4OH without UT washed with H2O
Figure 75: comparison of size distributions of MC-coated particles precipitated with
NH4OH without UT washed with H2O
Viktoria Maria Enk page 122 of 130 Dominic-Philipp Klein
c) PEG-coated magnetic cellulose microparticles (PEG-mcm)
Parameter: NH4OH / - UT / H2O
• before the precipitation: use of Polyethylenglycol
• precipitation in NH4OH
• without Ultraturrax
• use of H2O for washing
Table 36: magnetic properties of PEG-coated particles precipitated
with NH4OH without UT washed
with H2O
Figure 76: comparison of size distributions of PEG-coated particles precipitated
with NH4OH without UT washed with H2O
Viktoria Maria Enk page 123 of 130 Dominic-Philipp Klein
Interpretation:
Viktoria Maria Enk page 124 of 130 Dominic-Philipp Klein
APPENDIX
Viktoria Maria Enk page 125 of 130 Dominic-Philipp Klein
BIBLIOGRAPHY
[1] Weber, Viktoria: Blut – Gerinnung und Reinigung einer besondern Flüssigkeit - Präsentation des
Zentrum für Biomedizinische Technologie Donau-Universität Krems – Krems: Club Biotech, 26.01.2010
[2] Wikipedia – Liver
(online in the internet: URL: http://en.wikipedia.org/wiki/Liver, 07.04.2011 )
[3] Wikipedia – Blood
(online in the internet: URL: http://en.wikipedia.org/wiki/Blood, 28.01.2011 )
[4] Wikipedia – Hemoglobin
(online in the internet: URL: http://en.wikipedia.org/wiki/Hemoglobin, 24.05.2011 )
[5] Wikipedia – Liver Failure
(online in the internet: URL: http://en.wikipedia.org/wiki/Liver_failure, 24.05.2011 )
Properties and Applications of Cellulose Acetate – Institute of wood and plant chemistry, technical
University of Dresden, Tharandt, Germany & Fraunhofer Institute for Applied Polymer Research
Potsdam-Golm, Potsdam, Germany, 2008;
[12] Gupta, Ajay-Kumar; Gupta, Mona: Synthesis and Surface Engineering in Iron Oxide Nanoparticles for
Biomedical Applications – Crusade Laboratories Limited, Southern General Hospital, Glasgow,
Scotland, UK & Division of Biochemistry and Molecular Biology, University of Glasgow, Glasgow,
Scatland, UK; SienceDirect; Elsevier Ltd, 2005;
[13] M. Yamaure et al.: Preparation and Coating Precedures for Magnetic Nanoparticles – published in the
Journal of Magnetism and Magnetic Materials, Nr. 279, pages 210 to 217, 2004;
[14] Ettenauer, Marion: Diplomarbeit – Untersuchungen zur Bioverträglichkeit von Polymeren auf
Kolenhydrat-basis für die extrakorporale Blutreinigung – Zentrum für Biomedizinische Technologie der
Donau Univerisät Krems, Krems, 2003;
[15] Stößer, Kathi: Diplomarbeit – Herstellung, Verwendung und Charakterisierung von Perlcellulosen zur
Immobilisierung von Magnetit – Hochschule für Technik und Wirtschaft Dresden, Dresden,
Deutschland, 2009;
[16] Fritsch, H.; Kuehnel, W.: Internal Organs, Color Atlas od Human Anatomy Vol.2, 5th edition – Thieme
Verlag, Stuttgart, Germany, 2008;
[17] Notations from the lessons of Prof. DI Dr. techn. Bibiana Meixner
Viktoria Maria Enk page 127 of 130 Dominic-Philipp Klein
INDEX OF FIGURES:
Figure 1: The MDS-system ...........................................................................................................................................................4 Figure 2: synthesis chain ..............................................................................................................................................................6 Figure 3: Structure of the liver....................................................................................................................................................14 Figure 4: Chemical formula of the Hem group ............................................................................................................................16 Figure 5: Chemical formula of bilirubin .......................................................................................................................................16 Figure 6: decomposition of Hemoglobin in liver and kidneys......................................................................................................17 Figure 7: Decomposition of ethanol ............................................................................................................................................19 Figure 8: Dialysis ........................................................................................................................................................................21 Figure 9: Prometheus System ....................................................................................................................................................22 Figure 10: Correlation between volume and surface ..................................................................................................................24 Figure 11: MDS- System.............................................................................................................................................................25 Figure 12 Comparison of Prometheus´and MDS´ safety ............................................................................................................27 Figure 13: synthesis chain [12] [13] .........................................................................................................................................36 Figure 14: experimental setting for the oxygen removal (degassing) .....................................................................................41 Figure 15: magnetic cellulose microparticle suspension.............................................................................................................43 Figure 16: assembly of measurement & time-stop-point ............................................................................................................43 Figure 17: completely separated microparticles .........................................................................................................................43 Figure 18: net weight of ..............................................................................................................................................................45 Figure 19: pyknometer ................................................................................................................................................................45 Figure 20: pyknometer ................................................................................................................................................................45 Figure 21: pyknometer filled........................................................................................................................................................45 Figure 22: overview of treatments used for the preparation of magnetic cellulose microparticles..............................................48 Figure 23: difference between unaggregated and aggregated particles.....................................................................................57 Figure 24: Sample 1: uncoated .............................................................................................................................................58 Figure 25: Sample 1: uncoated .............................................................................................................................................59 Figure 26: Sample 2: uncoated .............................................................................................................................................60 Figure 27: Sample 2: uncoated .............................................................................................................................................61 Figure 28: Coated with methylcel lu lose .............................................................................................................................62 Figure 29: Coated with methylcel lu lose .............................................................................................................................63 Figure 30: Coated with methylcel lu lose .............................................................................................................................64 Figure 31: Coated with methylcel lu lose .............................................................................................................................65 Figure 32: Coated with polyethylenglycol ...................................................................................................................................66 Figure 33: Coated with polyethylenglycol ...................................................................................................................................67 Figure 34: Example for green labelling .......................................................................................................................................74 Figure 35: Example for yellow labelling ......................................................................................................................................74 Figure 36: Example for red labelling ...........................................................................................................................................75 Figure 37: unmodified cellulose- microparticles..........................................................................................................................75 Figure 38: uncoated cellulose microparticles..............................................................................................................................76 Figure 39: uncoated cellulose microparticles..............................................................................................................................77 Figure 40: uncoated cel lu lose micropart ic les ...................................................................................................................78 Figure 41: uncoated cel lu lose micropart ic les ...................................................................................................................79 Figure 42: uncoated cel lu lose micropart ic les ...................................................................................................................80 Figure 43: uncoated cel lu lose micropart ic les ...................................................................................................................81 Figure 44: uncoated cel lu lose micropart ic les ...................................................................................................................82
Viktoria Maria Enk page 128 of 130 Dominic-Philipp Klein
Figure 45: uncoated cel lu lose micropart ic les ...................................................................................................................83 Figure 46: uncoated cel lu lose micropart ic les ...................................................................................................................84 Figure 47: uncoated cel lu lose micropart ic les ...................................................................................................................85 Figure 48: uncoated cel lu lose micropart ic les ...................................................................................................................86 Figure 49: uncoated cel lu lose micropart ic les ...................................................................................................................87 Figure 50: MC-coated cel lu lose micropart ic les ................................................................................................................88 Figure 51: MC-coated cel lu lose micropart ic les ................................................................................................................89 Figure 52: MC-coated cel lu lose micropart ic les ................................................................................................................90 Figure 53: MC-coated cel lu lose micropart ic les ................................................................................................................91 Figure 54: MC-coated cel lu lose micropart ic les ................................................................................................................92 Figure 55: MC-coated cel lu lose micropart ic les ................................................................................................................93 Figure 56: MC-coated cel lu lose micropart ic les ................................................................................................................94 Figure 57: MC-coated cel lu lose micropart ic les ................................................................................................................95 Figure 58: MC-coated cel lu lose micropart ic les ................................................................................................................96 Figure 59: MC-coated cel lu lose micropart ic les ................................................................................................................97 Figure 60: MC-coated cel lu lose micropart ic les ................................................................................................................98 Figure 61: MC-coated cel lu lose micropart ic les ................................................................................................................99 Figure 62: PEG-coated cel lu lose micropart ic les ...........................................................................................................100 Figure 63: PEG-coated cel lu lose micropart ic les ...........................................................................................................101 Figure 64: PEG-coated cel lu lose micropart ic les ...........................................................................................................102 Figure 65: PEG-coated cel lu lose micropart ic les ...........................................................................................................103 Figure 66: PEG-coated cel lu lose micropart ic les ...........................................................................................................104 Figure 67: PEG-coated cel lu lose micropart ic les ...........................................................................................................105 Figure 68: PEG-coated cel lu lose micropart ic les ...........................................................................................................106 Figure 69: PEG-coated cel lu lose micropart ic les ...........................................................................................................107 Figure 70: PEG-coated cel lu lose micropart ic les ...........................................................................................................108 Figure 71: PEG-coated cel lu lose micropart ic les ...........................................................................................................109 Figure 72: PEG-coated cel lu lose micropart ic les ...........................................................................................................110 Figure 73: PEG-coated cel lu lose micropart ic les ...........................................................................................................111 Figure 74: comparison of size distributions of uncoated particles ..........................................................................................120 Figure 75: comparison of size distributions of MC-coated particles.........................................................................................121 Figure 76: comparison of size distributions of PEG-coated particles......................................................................................122
Viktoria Maria Enk page 129 of 130 Dominic-Philipp Klein
INDEX OF TABLES:
Table 1: chemicals used in the experiments ...............................................................................................................................35 Table 2: uncoated particles washed with Albumin ......................................................................................................................50 Table 3: uncoated particles washed with reversed osmosis water .............................................................................................51 Table 4: uncoated particles washed with PBS............................................................................................................................51 Table 5: MC coated particles washed with Albumin ...................................................................................................................52 Table 6: MC coated particles washed with reversed osmosis water ..........................................................................................52 Table 7: MC coated particles washed with PBS .........................................................................................................................53 Table 8: PEG coated particles washed with Albumin .................................................................................................................54 Table 9: PEG coated particles washed with reversed osmosis water ........................................................................................55 Table 10: PEG coated particles washed with PBS .....................................................................................................................56 Table 11: magnetic behaviour of uncoated particles ..................................................................................................................68 Table 12: magnetic behaviour of uncoated particles ..................................................................................................................68 Table 13: magnetic behaviour of uncoated particles ..................................................................................................................69 Table 14: magnetic behaviour of MC-coated particles................................................................................................................69 Table 15: magnetic behaviour of MC-coated particles................................................................................................................70 Table 16: magnetic behaviour of MC-coated particles................................................................................................................70 Table 17: magnetic behaviour of PEG-coated particles..............................................................................................................71 Table 18: magnetic behaviour of PEG-coated particles..............................................................................................................71 Table 19: magnetic behaviour of PEG-coated particles..............................................................................................................72 Table 20: Density of magnetic celluloses..................................................................................................................................113 Table 21: Density of HPR 10 ....................................................................................................................................................114 Table 22: Density of Celluloses from the TU Dresden..............................................................................................................114 Table 23: Dry matter content ........................................................................................................................................................115 Table 24: comparison of size distributions................................................................................................................................116 Table 25: comparison of size distributions................................................................................................................................116 Table 26: comparison of size distributions...............................................................................................................................116 Table 27: comparison of size distributions...............................................................................................................................117 Table 28: comparison of size distributions...............................................................................................................................117 Table 29: comparison of size distributions...............................................................................................................................117 Table 30: comparison of size distributions...............................................................................................................................118 Table 31: comparison of size distributions...............................................................................................................................118 Table 32: comparison of size distributions...............................................................................................................................118 Table 33: comparison of size distribution.................................................................................................................................119 Table 34: magnetic properties of ..............................................................................................................................................120 Table 35: magnetic properties of .............................................................................................................................................121 Table 36: magnetic properties of ...............................................................................................................................................122
Viktoria Maria Enk page 130 of 130 Dominic-Philipp Klein
INDEX OF ABBREVIATIONS:
COURSE OF THE PROJECT:
1. day: washing of ethanol soaked particles 2. day: preparation of ferric and ferrous solutions and particle soaking 3. day: precipitation and washing 4. day: analysis of uncoated particles 5. day: planning of strategy and course, weekly report
6. day: impregnation with methylcellulose 7. day: precipitation and washing 8. day: analysis of MC-coated particles 9. day: Presentation of Prof. Weber „Blutreinigung – Basics“, analysis 10. day: planning, weekly report
11. day: impregnation with polyethylene glycol 12. day: precipitation and washing 13. day: analysis of PEG-coated particles 14. day: preparations for density measurement (dry matter) 15. day: density measurement of cellulose, planning, weekly report
16. day: density measurement of magnetic particles 17. day: density measurement of HPR-10 (reference) 18. day: Presentation of Jens Hartmann „Koagulation“ – analysis of best particles 19. day; analysis of best particles, cleaning of the laboratory 20. day: final meeting with supervision, final report