diploma!thesis! - gfc.at · Erklärung an Eides statt Affidavit Wir erklären hiermit an Eides...

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

TABLE OF CONTENTS

1. ABSTRACT............................................................................................................................................ 3

2. AIM.....................................................................................................................................................13

3. INTRODUCTION..................................................................................................................................14 3.1 THE LIVER ............................................................................................................................................... 14 3.2 BIOCHEMICAL PROCESSES ......................................................................................................................... 15

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


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


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.


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.


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.


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.


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.


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.


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.


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


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.


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.


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).


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

upper abdomen. [16]

Figure 3: Structure of the liver

http://en.wikipedia.org/wiki/File:Anatomy_of_liver_and_gall_bladder.png (25.12.2010)


As seen in Figure 3 the liver can be divided in two big lobes. The right lobe is under the

phrenic and partly fused with it, the left lobe is smaller and extends to the left upper

abdomen. Two smaller lobes that are not shown in this picture (the quadratic and the

caudate lobe) are also part of the liver. [2]

3.2 BIOCHEMICAL PROCESSES

The functions of the liver can be sub-divided into:

3.2.1 REGULATIVE FUNCTIONS:

The liver plays an important role in the regulation of glucose, fat and protein-

metabolism. It can convert glucose into glycogen when a high glucose level is in the

blood and backwards at a low level. This keeps the blood glucose level constant. [2]

3.2.2 SYNTHESIS FUNCTIONS:

The gluconeogenesis (synthesis of D-glucose from glycerol, lactate or pyruvate),

the syntheses of ketones, cholesterine, bile and blood-proteins (albumin, globuline,

coagulation factors) take place in the liver. [2]

3.2.3 STORAGE-FUNCTION:

Glycogen, lipoproteins and vitamins are stored in the liver. There is also a high

amount of blood located in the liver. [17]


3.2.4 CATABOLISM OF BODY OWN SUBSTANCES:

The liver catabolizes bilirubine and steroid hormons. Firstly water insoluble

substances are modified to become water soluble and can then be eliminated by

the kidneys. Large compounds, that are retained by the kidneys are catabolized by

the liver itself. [2]

There is a difference in the decomposition of protein-bound and non-protein-bound

substances both at body own and foreign substances. Protein-bound substances

are decomposed by the liver while non-protein-bound substances can be removed

by the kidneys. [16]

3.2.4.1 Bilirubine-catabolism (protein-bound substance):

Figure 4: Chemical formula of the Hem group

http://de.academic.ru/pictures/dewiki/72/Heme_

b.png (25.12.2010)

Figure 5: Chemical formula of bilirubin

http://en.wikipedia.org/wiki/File:Bilirubin_ZZ.png (9.3.2011)


The figure below shows the catabolism of the red blood cells via bilirubin to

urobilinogen and sterkobilinogen that are excreted.[4]

Figure 6: decomposition of Hemoglobin in liver and kidneys

http://www.elmhurst.edu/~chm/vchembook/images/634bilimap.gif (25.12.2010)

When red blood cells lyse hemoglobin is released into the blood. During

degradation the Hem group and the globine group are separated. The globine part

is degraded to amino acids while Hem is converted to Bilirubine via Biliverdine.

During the transport bilirubine is reversibly bound to albumin which increases the

solubility and is transported into the liver.


In the cells of the liver (hepatocytes) the conjugation of bilirubine with glucuronic

acid to the water soluble bilirubinmonoglucuronid or –diglucuronid takes place by

way of the enzyme UDP-Glukuronyltransferase.

The water soluble products can be egested through the bile afterwards.

In the intestines bilirubine is reduced to urobilinogen and sterkobilinogen (the brown

colorant of feces). One part of this is reabsorbed by the intestine and egested

through the kidneys (yellow colour of urine).

If bilirubin is not removed by the liver an icterus can develop. [4] [17]

3.2.5 CATABOLISM OF FOREIGN SUBSTANCES:

Substances that are harmful for the body including ammonia or drugs are

catabolized in the liver. As an example the catabolism of ethanol is described. This

is an example of a non-protein-bound substance. [4] [17]


3.2.5.1 Alcohol-catabolism (non- protein-bound substance):

As can be seen in figure 7 the enzyme alcohol- dehydrogenase converts ethanol

into ethanal, then into the anion ethanoate, that reacts to Acetyl-Co-enzyme-A with

the Co-enzyme-A. [17]

Figure 7: Decomposition of ethanol

http://www.ganfyd.org/images/thumb/d/d4/Ethanol_metabolism.

gif/180px-Ethanol_metabolism.gif (25.12.2010)

As shown in the figure above the end product of the decomposition of ethanol is

acetyl-Co-enzyme A. This can enter the citrate circuit for subsequent degradation.

[2] [17]


3.3 LIVER FAILURE:

Liver failure can be caused by hepatitis- virus, drugs (e.g. with suicidal intention), alcohol

abuse or other toxins.

Symptoms of liver failure are icterus, blood coagulation diseases, a reduced albumin level,

endocrine diseases, kidney failure, hepatic coma (function diseases of the brain) and

death.

Liver dialysis can help patients to bridge the time between liver failure and a

transplantation or to support the liver and faciliate the regeneration of liver tissue. This is

no long-term solution and can just be used for temporary treatment.

The compensation of the liver´s synthesis functions cannot be done by now, the

detoxification system is just able to replace the catabolism. For that reason the processes

of syntheses are not described in more detail.

The liver removes substances that are too large for removal with normal dialysis that is

used to treat kidney failure. Examples are albumin-bound metabolites, like bilirubin, bile

acids, phenolic compounds, and aromatic amino acids.

There are two different systems of liver dialysis systems that remove both albumin bound

substances (e.g. unconjugated bilirubin or cholic acid) and non protein-bound substances.

[1] [2]


3.4 THE PROMETHEUS SYSTEM (COMBINED DIALYSIS-ADSORBER-TREATMENT):

The system combines normal dialysis, that is used for the removal of water soluble

substances with adsorber- treatment for water insoluble and protein-bound substances.

Figure 8 gives an overview of conventional dialysis.

Figure 8: Dialysis

http://kidney.niddk.nih.gov/kudiseases/pubs/hemodialysis/images/dialysis.gif

(25.12.2010)

In normal dialysis blood is pumped outside of the body and Heparin is added to prevent

coagulation. Afterwards the blood passes a Dialyzer where small molecules e.g. ethanol

are retained.


Blood cells and larger molecules do not enter the filter and are not retarded therefore.

Afterwards the purified blood passes an air trap and an air detector to prevent air from

entering the body. Afterwards the clean blood returns into the body.

Figure 9: Prometheus System

Kindly provided by Univ.-Prof. Dr. Viktoria Weber (28.3.2011)

As mentioned at the beginning of the chapter the Prometheus system combines

normal dialysis (circuit 2, Figure 9) with adsorber treatment (circuit 1, figure 9).


In circuit one the blood is pumped outside of the body(Fig. 9). Subsequently a

membrane module (Albuflow) is used for separation of plasma from blood cells.

Water-insoluble substances that are bound to albumin are removed from the

plasma in a the plasma circuit by adsorber particles. Blood cells that have been

separated before the plasma circuit are added again. In circuit 2 water soluble

substances are removed through a dialysis system.

The adsorber-particles used in the Prometheus system are cellulose microparticles

with diameter of >10µm. They have a low specific surface and a high amount of

blood is needed outside of the body. The particles are enclosed in capsules (Fig. 9-

“Adsorber Prometh 01”)

If a large volume of blood is outside of the body because of the treatment, the

patients suffer from similar symptoms as the ones after loss of blood: they feel very

dizzy and thirsty.

For that reason it is tried to reduce the needed amount of blood, which can be done

through a higher specific surface of the particles (smaller particles). This leads to

the development of the MDS-System. [1] [7]


3.5 THE MICROSPHERES DETOXIFICATION SYSTEM (MDS):

Figure 10: Correlation between volume and surface

http://www.uwgb.edu/dutchs/Graphics-

Geol/GEOMORPH/SurfaceVol0.gif (25.12.2010)

For blood purification with the MDS smaller cellulose particles (<5µm) are used because

they have a higher specific surface and thus can bind more toxins per volume. In contrast

to the Prometheus system the particles are not encapsulated and move freely in the

plasma.

The figure above shows that the surface-volume-relation of a particle increases if it is not

just one big particle but many small ones that altogether have the same volume.


The big advantage of smaller particles is that using the same volume of <5µm particles

instead of >10µm particles a smaller volume of blood is needed outside of the body.

In some of the first tests commercially available particles (so called Dynabeads®) were

used but they are too expensive for clinical application. Therefore cellulose microparticles

are utilized and show as good results when treated correctly.

Figure 11: MDS- System

http://www.donau-uni.ac.at/de/department/kmbt/forschung/biomedizinischetechnologie/projekte/id/14431/index.php (25.12.2010)


As can be seen in figure 11 the blood is pumped out of the body and citrate is added to

prevent coagulation. The blood enters a plasma-filter to remove blood cells. The plasma

enters a second circuit, where the microparticle suspension is added. Afterwards the

suspension passes a plasma filter again and the microparticles are retarded. Blood cells

and plasma are unified again.

Since particles can cause thrombosis and embolism in the human body they must not

reach the patient´s own blood circuit.

To protect the patient in the case of a malfunction of the plasma filter a microparticle

detector after the filter stops the pumps if particles are detected. Then the blood passes a

dialysis filter, where a dialysis is performed.

Finally calcium is added to reverse the effects of citrate and allow a normal coagulation

behaviour of the blood reentering the body.

[7] [8] [9]

3.6 SAFETY CONSIDERATIONS:

For systems that are used in medicine a double failure protection has to be guaranteed.

While the Prometheus system has two barriers between the cellulose microparticles and

the human body (filter membrane at the end of the capsule and the Filter of the Albuflow (

black bars, Fig .12a)) .

In contrast the particles in the MDS are only separated from the body by the Albuflow

membrane. To guarantee double failure protection a microparticle detector is attached

after the plasma filter. (black bars, Fig. 12b).


Figure 12 Comparison of Prometheus´and MDS´ safety

The microparticle detector is capable of detecting fluorescence signals. For that reason

1% (vol/vol) of the adsorber particles are fluorescent, magnetic cellulose microparticles.

In case of malfunction of the plasma filter the microparticle detector detects a fluorescence

signal and stops the pumps. To increase the sensitivity of the detector the fluorescence

particles are magnetized and can be concentrated in front of the detector by a magnetic

trap. [9] [10]


3.7 AIMS OF THIS STUDY:

Optimum suitable particles should have the following properties:

• Size <5µm

• No or only minor aggregations

• A similar density as non-magnetic celluloses to achieve an even distribution of the

magnetic particles all over the plasma circuit

• Accumulation beneath the magnet in less than five seconds [12]

The aim of our work was to improve the synthesis of the magnetic particles by trying

different types of coatings (Methylcellulose or Polyethylenglycol).

The variation of the washing process (use of albumin-solution, PBS-solution or water) to

reduce agglomerations was also an important influence on the results. The standard

procedure was modified as showed in the figure above. All of the synthesized particles

should be tested for magnetic properties, density and agglomeration.

Agglomerations can be detected through particle size distribution measurement with the

Mastersizer 2000. The Mastersizer 2000 is a device for the testing of microparticle size

distribution by laser light diffraction.

The magnetic behaviour is tested with a permanent magnet that is also used in the

magnetic trap assembled in the MDS. The time until the suspension has cleared has to be

less than 5 seconds otherwise in practical use particles could enter the body and cause

embolism.

It should also be tested whether an ultrasonic bath prevents particle aggregations.


Further analysis should be the determination of dry mass and density of magnetic

celluloses, unmodified celluloses and hydrophobic HPR10 adsorber.

Large differences in density could cause an unbalanced distribution of the magnetic

particles in the solution. As a result the magnetic particles could flow slower through the

circuit and pass the trap later than the normal particles. Because of this the detection could

be too late to prevent the trespass of some particles into the body.


4. Materials and Methods

4.1 MATERIALS

All Materials required for the experiments:

4.1.1 DEVICES

Centrifuges:

1) Hettich Universal 32 R

rotor nr: 1617

rotor radius: 120mm

maximum acceleration to: 1199g

2) Hettich Universal Rotanta 460 R

rotor nr: 5624

rotor radius: 196mm


inserts: 4839 for 15ml Greiner tubes

4840 for 50ml Greiner tubes

3) Heraeus Megafuge 10 R

rotor nr: 2702

rotor radius: 169mm



Mastersizer 2000, Malvern Instruments, Herrenberg, Germany

Pipettes:

10 – 100µl (Eppendorf research)

100 – 1000µl (Eppendorf research)

100µl (Eppendorf research)



Balances:

1) Kern&Sohn 770 analytical balance

2) Kern&Sohn K8 precision balance

magnetic stir sticks (20mm, 30mm, 40mm)

Vortex Mixer, LabMixer Ms2, IKA®

Magetic Stirrers:

1) yellow line MST basic

2) IKA® PCT basic

3) IKA® RT10 power

4) IKA® big squid froggy

5) IKA® MS1 Minishaker

Ultra Turrax Dispenser:

engine: yellow line DI 25 basic

dispersing tools: IKA® 1024200 S25N-8G

IKA® 0593400 S25N-18G


Glass Ware:

beakers (Schott Duran,100ml, 250ml)

erlenmeyer flasks (Schott Duran, 250ml)

petri dishes (ø 80mm)

graduated cylinder (Schott, 100ml, 250ml)

pasteur pipettes

Ultrospec 3300 pro UV/VIS–spectrometer, Amersham Bioscience,

Freiburg, Germany

Enviro Genie® shaking bench and incubator, Scientific Industries Inc.

Reverse-Osmosis-Supply:

device: euRO 20DI, SG Wasseraufbreitungs und Regnerier GmbH

article nr. 3001-DI, serial nr. 83275-01

modules: AMB Modul, prod. nr. 2057

VMD Modul, prod. nr. 2050

Permanent Magnet, field strength 1T

Nikon Coolpix L100, Nikon GmbH, Düsseldorf, Germany

4.1.2 SINGLE USE MATERIALS

centrifugation tubes (polypropylene, 15ml, 50ml)

Greiner Bio-One GmbH, Frickenhausen, Germany


pipette tips (polypropylene, 100µl, 1000µl, 5000µl)

Eppendorf AG, Hamburg, Germany

laboratory film (Parafilm® M)

Sigma-Aldrich, Fluka analytical, Buchs, Switzerland

4.1.3 CHEMICAL SUBSTANCES

substance consistence source product nr. LOT

cellulose microparticles in 20%(v/v) TU Dresden,

<5µm ethanol and

water

(Institute of Plant- and

Wood Chemistry)

PCKT 047 #1908

Germany

Dynabeads® M-280 Dynal Biotech

ASA,

Tosylactivated in water Oslo, Norway 301.01 H11102

osmosis water supplied by euRO 20DI reverse-osmosis-system


0,9%(w/w) Fresenius sodium chloride

in water Bad Homburg,

Germany

0698121/02A 14CK1004

iron-(II)-sulphate Sigma Aldrich,

heptahydrate Fluka analytical

purum p.a. Buchs,

Switzerland

44980 #0001320556

iron-(III)-chloride Sigma Aldrich,

hexahydrate Fluka analytical

purum p.a. Buchs,

Switzerland

31232 SZB91390

0,1 mol/L in water Sigma Aldrich,

f = 0,987 Fluka analytical sulfuric acid

Buchs,

Switzerland

38295 65596JJ

Sigma Aldrich,

Fluka analytical sodium hydroxide

purum p.a. Buchs,

Switzerland

71691 #72190


Sigma Aldrich,

Fluka analytical ammonium hydroxide

5,01 mol/L in water

Buchs, Switzerland

318612 22897JJ

albumin Sigma Aldrich,

(from bovines serum) Fluka analytical

purity: >96%(w/w)

Buchs, Switzerland

A3912 #028K0667

Sigma Aldrich,

Fluka analytical phophate buffered saline

tablet Buchs,

Switzerland

P4417 #107K8217

„One tablet dissolved in 200ml of deionized water yields 0.01M phosphate buffer.“

polyethylene glycol Sigma Aldrich,

(average mol weight 200g/mol) Fluka analytical

/

Buchs, Switzerland

P3015 # 029K0174

methylcellulose Sigma Aldrich,

(Methocel MC 1200-1800 mPas) Fluka analytical

(mol weight 204,22 g/mol)

/

Buchs, Switzerland

24645 #9004675

Table 1: chemicals used in the experiments


4.2 METHODS

Figure 13: synthesis chain [12] [13]


4.2.1 SYNTHESIS OF MAGNETIC CELLULOSE MICROPARTICLES

All experiments were carried out with reverse osmosis water (in further text: water) at room

temperature if there are no variations sighted.

4.2.1.1 Purification of Cellulose Microparticles

The particles were supplied in an ethanol suspension.

To remove the ethanol, the cellulose microparticles were washed with water by

shaking and an additional centrifugation step (2839g, 20 min). The supernatant was

decanted and the cellulose sediment was resuspended in fresh water. The

procedure was repeated until the supernatant was clear and foamless.

The cellulose microparticles were resuspended in water again and incubated over

night at 30°C on a shaking bench.

The suspension was centrifuged (2839g, 20min) again and the supernatant was

analyzed by UV/VIS-spectrometry at 280 nm (verification step of tensids). If no

peak was visible the sample was free of tensids as is should be

For further experiments, a homogeneous 50% (v/v) cellulose microparticle

suspension in water was prepared. [12]


4.2.1.2 Magnetization of Cellulose Microparticels by Alkaline (Precipitation of Magnetite in Cellulose Pores)

Fe2+ + 2 Fe3+ + 8 OH- Fe3O4 + 4 H2O

Preparation of Iron Solutions

4mol/L FeCl3 * 7H2O: 54g of FeCl37H2O were dissolved in 50mL of water to obtain

a 4mol/L ferric solution.

2mol/L of FeSO4 * 6H2O: 28 g of FeSO46H2O were dissolved in 50mL of

0.1mol/L H2SO4 to obtain a 2mol/L ferrous solution. [12] [13]

Soaking Procedure

2mL of the 50% (v/v) cellulose microparticle suspension (~1g wet microparticles, in

50mL centrifugation tube) were combined with 12mL

4mol/L ferric and 12mL 2mol/L ferrous solution. To remove oxygen from the

reaction mixture it was vented with gaseous nitrogen. The iron-soaked cellulose

suspension was gently mixed and incubated on a shaking bench over night at 30

°C.

After the incubation step, the suspension was centrifuged (2839g, 20min) and the

supernatant was decanted. The iron-soaked cellulose microparticle sediment was

used for further experiments [12][13]


4.2.1.2.1 Impregnation methods

To test if particle aggregation can be prevented, protective colloids were added to

the iron-soaked cellulose microparticle sediment before the precipitation step.

a) no protective colloid

For the comparison of the efficiency of different protective colloids, experiments

were carried out without any additives in the first test series.

b) methylcellulose

1mL of a 0.5%(w/v) methylcellulose solution was added to the iron-soaked cellulose

microparticle sediment. The mixture was homogenized before the precipitation step.

Figure 14: experimental setting for the oxygen removal (degassing)


c) polyethylene glycol

1mL of polyethylene glycol was added to the iron-soaked cellulose microparticle

sediment. The mixture was homogenized before the precipitation step. [13]

4.2.1.2.2 Precipitation of Iron-Soaked Cellulose Microparticles in Alkaline Medium

a) Precipitation in sodium hydroxide

In order to precipitate the magnetite in the cellulose pores 20mL of a 1mol/L sodium

hydroxide solution was added to the iron-soaked cellulose microparticles.

In one experiment the precipitation was supported with an Ultra Turrax dispenser

(“+UT”), in another the reaction mixture was simply homogenized with a vortex

labmixer (“-UT”).

b) Precipitation in ammonium hydroxide

In order to precipitate the magnetite in the cellulose pores 20mL of a

1mol/L ammonium hydroxide solution was added to the iron-soaked cellulose

microparticles.

In one experiment the precipitation was supported with an Ultra Turrax dispenser

(“+UT”), in another the reaction mixture was simply homogenized with a vortex

labmixer (“-UT”). [13]


4.2.1.3 Washing of the Magnetic Cellulose Microparticles

a) washing in water

The magnetized particles (obtained from 4.2.1.2) were aliquoted in portions of 5g

per 15mL centrifugation tube. The sediment was resuspended in 5mL of water

(eluent) and centrifuged (2839g, 10min). The supernatant was decanted. This

washing step was repeated three times.

Thereafter the sediment was resuspended in 5ml of water and incubated on a

shaking bench for 2 hours at 30°C.

The suspension was centrifuged (2839g, 10min.) and the sediment washed once

again with 5mL of water. The microparticle sediment was resuspended and

incubated over night (shaking bench, 30°C).

The tubes were centrifuged (2839g, 10min) again, and resuspended in 50mL of

water.

b) bovines albumin solution (1mg/mL)

The washing procedure with bovines albumin solution (1mg/mL) follows the

procedure described in 4.2.1.3 a), using bovines albumin solution (1mg/mL) instead

of water.

c) phosphate buffer solution (pH 7.4, 0.01mol/L)

The washing procedure with phosphate buffer solution (pH 7.4, 0.01mol/L) follows

the procedure described in 4.2.1.3 a), using phosphate buffer solution (pH 7.4,

0.01mol/L) instead of water.


To obtain a 100mg/mL suspension of magnetic microparticles in water ,bovines

albumin solution (1mg/mL) or phosphate buffer solution (pH 7.4, 0.01mol/L), 1mL of

the eluent was withdrawn and mixed with 4 mL of fresh eluent. [12] [14]

4.2.3 CHARACTERISATION OF MAGNETIC CELLULOSE MICROPARTICLES

4.2.3.1 Visual Characteristics

The color of the supernatant and of the microparticle sediment was determined after

the last centrifugation step in 4.2.1.3.

As another characteristic, the simplicity of the resuspension in the last washing step

of 4.2.1.3 was recorded.

To get further information about the particles aggregation behavior, the particles

that showed the best results in former analysis were sent to the TU Dresden where

they were imaged by scanning electron microscopy. [15]

4.2.3.3 Determination of the Particles Size and Size Distribution

The measurement was carried out with a “mastersizer 2000” device from Malvern

Instruments (www.malvern.de), using red, green and blue wavelength lasers

(focused light).

The particles were treated 0,5 and 10 minutes by ultra sound before the

measurement. [14]


4.2.3.2 Determination of the Magnetic Behavior

A permanent magnet with the field strength of 1T was attached to the centrifugation

tube containing the homogeneous 100mg/mL suspension obtained in 4.2.1.3.

The time span was recorded that the particles took to form first signs of

inhomogeneity in the suspension.

If the suspension showed no inhomogeneity within 10 seconds the measurement

was aborted. [14]

As a reference the “Dynabeads® M-280 Tosylactivated” (www.invitrogen.com)

which took 3 to 4 seconds to first remarkable inhomogeneity, were used.

Figure 15: magnetic cellulose microparticle suspension

Figure 16: assembly of measurement & time-stop-point

Figure 17: completely separated microparticles

(for the visualisation of this measurement see video:

measurement_mag_behavior.mov on the attached CD)


4.2.3.4 Determination of the Dry Matter Content

The homogeneous 100mg/mL suspension obtained in 4.2.1.3 was centrifuged

(2839g, 20min). The obtained particle sediment was transferred to a glass petri dish

(the particle weight recorded) and dried at 105°C until constant weight (max. 0,5mg

difference within one hour at 105°C). [14]

4.2.3.5 Determination of the Particles Density

The necessary pyknometers were dried with cotton swabs and left stand overnight.

This process was repeated after each measurement for further experiments.

At first the net weight ( mP ) of the pyknometer was measured.

Approximately 0,5g of the to-be-measured solid substance was weighted into the

pyknometer and the weight (pyknometer + solid substance) recorded. ( mP+S )

The solid substance (dry matter) for this analysis was obtained in 4.2.3.4.

The pyknometer was completely filled with water so that no bubbles rested in

between the solid or at the pyknometers inner wall. The weight (pyknometer + solid

substance + water) was recorded. ( mP+S+W )

The pyknometer was emptied and washed with water, at least it was filled up with

water and the weight (pyknometer + water) was determined. ( mP+W ) [14]


Figure 18: net weight of

pyknometer

Figure 19: pyknometer containing

the solid substance

Figure 20: pyknometer

containing the

solid filled up

with water

Figure 21: pyknometer filled

with water


As a reference the density of a commercially available adsorber microparticle

(hydrophobic HPR10) was recorded.

The density of the solid substance was determined by entering the recorded

weights into the formula:


5. RESULTS

5.1 GUIDELINES FOR INTERPRETATION OF THE RESULTS

5.1.1 THE MAGNETIC MICROPARTICLES WERE ANALYZED ACCORDING TO THE FOLLOWING ASPECTS:

• Colour (qualitative, indirect method)

• Particle size distribution (Mastersizer 2000)

• Magnetic properties (permanent magnet)

• Morphology ( scanning electron microscopy)

• For additional information:

o Density

o Dry matter content

[15]


5.1.2 OVERVIEW OF THE PROCESSES:

Figure 22: overview of treatments used for the preparation of magnetic cellulose

microparticles

“UT”... Ultraturrax

Figure 22 gives an overview of treatments that were used for the preparation of

magnetic microparticles. Cellulose microparticles were magnetized through

treatment with iron-solutions.

Afterwards they were protected with different impregnations (methylcellulose,

polyethylenglycol or no impregnation). The washing process was also variegated.

Water, albumin-solution or PBS were used for washing. [13]


5.1.3 AN OPTIMAL SUITABLE PARTICLE SHOULD HAVE FOLLOWING PROPERTIES:

• Size <5µm

• No or just minimal aggregations

• A similar density as non-magnetic celluloses to achieve an even distribution of the

magnetic particles all over the plasma circuit

• Accumulation beneath the magnet that is used in the magnetic trap in less than five

seconds. [14]

5.2. RESULTS

5.2.1 VISUAL CHARACTERISTICS- PRE TESTS

The visual characteristics were not decisive if a particle is suitable for use in the system,

they were just acting as a brief pilot test.

The color of the particles should be preferably dark. Reddish looking particles indicate

oxidations that decrease the magnetic properties.

Most of the particles had a slight shimmer of red. This is caused by the presence of

molecular oxygen during the precipitation which could have been cause by incomplete

removal of oxygen.

The resuspension behavior depended on the washing agent. When the particles were

washed with PBS, sucking and extruding with a syringe was necessary to resuspend the

particles completely.

Particles that were washed with water could mostly be resuspended with the vortex,

sometimes an initial mechanical help was required. The supernatant was slightly colored

but not as intensively as the one from washing steps with albumin. The particles that were

washed with albumin solution could be resuspended by shaking the tube gently. [10]


5.3.1 Uncoated Particles

The following tables give an overview of colour and behaviour of the particles

depending on the precipitation steps.

Washed with Albumin:

Precipitation

NaOH+ UT NH4OH+ UT NaOH- UT NH4OH- UT

supernatant yellow-orange-

brown;

Clear

yellow-orange-

brown

Clear

particle behavoir

easy to suspend

easy to suspend

easy to suspend easy to suspend

colour of resuspended

particles

dirty brown with

a shimmer of

red

black with a

shimmer of red-

brown

dirty brown with

a shimmer of

red

similar to

NH4OH+ UT,

but a little bit

lighter

Table 2: uncoated particles washed with Albumin


Washed with reverse osmosis water:

Precipitation


supernatant less coloured than with albumin but also visibly yellow- orange- brown

coloration

particle behavoir

particles easier to suspend than in PBS, but not as easy as in albumin


particles dirty brown with a shimmer of red

Table 3: uncoated particles washed with reversed osmosis water

Washed with PBS:

Precipitation


supernatant clear

particle behavoir

particles are hard to suspend, vortex needed


particles dirty brown with a shimmer of red

Table 4: uncoated particles washed with PBS


5.3.2 Particles coated with Methylcellulose


Precipitation


supernatant clear yellow-orange-

brown; clear

yellow-orange-

brown;

particle behavoir

hard to suspend, sucking with syringe required


particles dark brown

Table 5: MC coated particles washed with Albumin

Washed with reversed osmosis water:

Precipitation


supernatant clear

particle behavoir




Table 6: MC coated particles washed with reversed osmosis water


Washed with PBS:

Precipitation


supernatant clear

particle behavoir




Table 7: MC coated particles washed with PBS

As shown in table 2 to 7 all the color of all the particles was dark brown. The

supernatants were mostly clear, just two tubes that were washed with albumin had

a slightly orange supernatant. Their ability to resuspend them was similar with all

washing agents. All of them were very hard to suspend.


5.3.3 Particles coated with Polyethylenglycol


Precipitation


supernatant slightly brown

orange

slightly brown

orange

clear slightly brown

orange

particle behavoir

particles are

hard to dispend,

vortex and

sucking with

syringe required

particles are easy to suspend, a

small rest on the bottom had to be

suspended with a syringe

easy to suspend



Table 8: PEG coated particles washed with Albumin



Precipitation


supernatant slightly brown

orange clear slightly brown

orange clear

particle behavoir

particles are

hard to dispend,

vortex and

sucking with

syringe required

particles are

easy to

suspend, a

small rest on the

bottom had to

be suspended

with a syringe

particles are hard to dispend,

vortex and sucking with syringe

required



Table 9: PEG coated particles washed with reversed osmosis water


Washed with PBS:

Precipitation


supernatant clear

particle behavoir

particles are

hard to dispend,

vortex and

sucking with

syringe required

easy to

suspend, a

small rest on the

bottom had to

be suspended

with a syringe

particles are

hard to dispend,

vortex and

sucking with

syringe required

particles are

easy to suspend

with vortex



Table 10: PEG coated particles washed with PBS

As it can be seen in table 8 to table 10 the particles coated with PEG were dark

brown so it can be said that no oxidation had occurred. The particles weren´t as

compact as when coated with methylcellulose, but not as easy to suspend as

uncoated particles. It can also be said that the supernatants when washed with

albumin or water were colored in most cases, when washed with PBS they were

clear.


5.2.2 SCANNING ELECTRON MICROSCOPY PICTURES

The pictures below show whether agglomerations had occurred in the samples or

not. The scanning electron microscopy was performed at the Technical University

of Dresden. Results and interpretations were forwarded to us via E-Mail. [11]

Figure 23: difference between unaggregated and aggregated particles

Figure 23 shows the interpretation of the scanning electron microscopy. The difference

between aggregated particles and unaggregated particles can be seen clearly.


Sample 1: uncoated, precipitated with NH4OH with ultraturrax, washed with H2O

Figure 24: Sample 1: uncoated, precipitated with NH4OH with ultraturrax, washed with H2O


Figure 25: Sample 1: uncoated, precipitated with NH4OH with ultraturrax, washed with H2O


Sample 2: uncoated, precipitated with NH4OH without ultraturrax, washed with H2O

Figure 26: Sample 2: uncoated, precipitated with NH4OH without ultraturrax, washed with H2O


Figure 27: Sample 2: uncoated, precipitated with NH4OH without ultraturrax, washed with H2O


Sample 3: Coated with methylcellulose, precipitated with NH4OH with ultraturrax, washed with H2O

Figure 28: Coated with methylcellulose, precipitated with NH4OH with ultraturrax, washed with H2O


Figure 29: Coated with methylcellulose, precipitated with NH4OH with

ultraturrax, washed with H2O


Sample 4: Coated with methylcellulose, precipitated with NH4OH without ultraturrax, washed with H2O

Figure 30: Coated with methylcellulose, precipitated with NH4OH without ultraturrax, washed with H2O


Figure 31: Coated with methylcellulose, precipitated with NH4OH without ultraturrax, washed with H2O


Sample 5: Coated with polyethylenglycol, precipitated with NH4OH with ultraturrax, washed with H2O

Figure 32: Coated with polyethylenglycol, precipitated with NH4OH with ultraturrax,

washed with H2O


Figure 33: Coated with polyethylenglycol, precipitated with NH4OH with ultraturrax,

washed with H2O

The pictures from scanning electron microscopy (Fig. 24-33) show that almost all

particles are smaller than 5µm. The size varies from 0,5µm to 3µm. [15]


5.2.3 DETERMINATION OF THE MAGNETIC BEHAVIOUR

5.2.3.1 uncoated cellulose microparticles:

When the particles are concentrated by a permanent magnet the solution has to be

clear in less than 5 seconds, otherwise the particles aren´t suitable for clinical use. If

the system was leaky, too many particles could trespass into the body in more than

five seconds. [8] [9]


Precipitation

NaOH NH4OH

using UT > 10s > 10s 6s 5s

without


Table 11: magnetic behaviour of uncoated particles

washed with albumin


Precipitation

NaOH NH4OH


without


Table 12: magnetic behaviour of uncoated particles washed with reversed osmosis water


Washed with PBS:

Precipitation

NaOH NH4OH

using UT > 10s 9s 2s 4s

without


Table 13: magnetic behaviour of uncoated particles

washed with PBS

In general precipitation of the particles with NH4OH was the best (Table 11-13, right

side) and the use of the ultraturrax made it even better. The use of water and PBS

for washing showed the best results. As it can be seen in table 10 the use of

albumin for washing didn´t lead to suitable particles.

5.2.3.2 cellulose microparticles coated with methlycellulose:


Precipitation

NaOH NH4OH

using UT > 10s > 10s > 10s > 10s

without


Table 14: magnetic behaviour of MC-coated particles

washed with albumin



Precipitation

NaOH NH4OH

using UT 4s 5s > 10s > 10s

without

using UT 3s 2s 8s 10s


washed with reversed osmosis water

Washed with PBS:

Precipitation

NaOH NH4OH

using UT > 10s > 10s > 10s > 10s

without



washed with PBS


5.2.3.3 cellulose microparticles coated with PEG:


Precipitation

NaOH NH4OH

using UT > 10s > 10s > 10s > 10s

without


Table 17: magnetic behaviour of PEG-coated particles

washed with albumin


Precipitation

NaOH NH4OH


without



washed with reversed osmosis water


Washed with PBS:

Precipitation

NaOH NH4OH


without



washed with PBS


5.2.4 DETERMINATION OF THE PARTICLE SIZE AND SIZE DISTRIBUTION

The particle size distribution was determined by the Mastersizer and interpreted as follows:

• d(0,1) means that 10% of the particles are smaller than the given size. It can be

said that green labeled particles have no or just minimal aggregations

• d(0,5) means that 50% of the particles are smaller than the given size. Yellow

particles are also suitable for further analyses (e.g. ultrasonic bath) to test whether

the non-optimal size distribution has an influence on other important parameter

such as magnetic behavior.

• d(0,9) means that 90% of the particles are smaller than the given size. Red labeled

particles should not be used in the system if other (better) particles are available.

The d-values are the results that are printed out after measuring the samples with the

Mastersizer. The d(0,9)-value was taken for comparing the results. [14]


5.2.4.1 Guidelines for the colorous labeling:

If d(0,9) has a value of less than 20 the labeling is green, if it is less than 100, the

labeling is yellow, and if the value exceeds 100 it is labeled red. If the d(0,9) is low,

most of the particles are not aggregated. If the value is high, big particles

(aggregations) have been measured.

d(0,9)<20…green

Figure 34: Example for green labelling

(no significant aggregations)

d(0,9)<100…yellow

Figure 35: Example for yellow labelling

(some aggregations visible, particle can be used)


d(0,9)>100… red

Figure 36: Example for red labelling

(many aggregations visible)

[9] [10]

5.2.4.1.1 unmodified cellulose- microparticles:

d(0,1)= 0,996 µm d(0,5)= 1,465 µm d(0,9)= 2,183 µm

Figure 37: unmodified cellulose- microparticles

The figure above shows the size distribution of the unmodified cellulose

microparticles after conditioning with water.


5.2.4.2 uncoated cellulose microparticles

Precipitated with NaOH without ultraturrax, washed with albumin

d(0,1)= 1,358 µm d(0,5)= 3,255 µm d(0,9)= 11,866 µm

Figure 38: uncoated cellulose microparticles precipitated

with NaOH without ultraturrax, washed with albumin

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.


Precipitated with NH4OH without ultraturrax, washed with albumin

d(0,1)= 1,814 µm d(0,5)= 4,924 µm d(0,9)= 125,398 µm


with NH4OH without ultraturrax, washed with albumin

The figure above shows a high degree of agglomerations of the particles.

Therefore these particles cannot be used for further analyses.


Precipitated with NaOH with ultraturrax, washed with albumin

d(0,1)= 1,467 µm d(0,5)= 3,089 µm d(0,9)= 10,149 µm


with NaOH with ultraturrax, washed with albumin





Precipitated with NH4OH with ultraturrax, washed with albumin

d(0,1)= 1,448 µm d(0,5)= 3,368 µm d(0,9)= 140,947 µm

Figure 41: uncoated cellulose microparticles precipitated with NH4OH with ultraturrax, washed with albumin




Precipitated with NaOH without ultraturrax, washed with PBS

d(0,1)= 1,857 µm d(0,5)= 7,795 µm d(0,9)= 149,763 µm


with NaOH without ultraturrax, washed with PBS




Precipitated with NH4OH without ultraturrax, washed with PBS

d(0,1)= 1,428 µm d(0,5)= 3,543 µm d(0,9)= 87,400 µm


with NH4OH without ultraturrax, washed with PBS

In the figure above some agglomerations are visible but the particles can be

used for further analyses.


Precipitated with NaOH with ultraturrax, washed with PBS

d(0,1)= 1,616 µm d(0,5)= 4,569 µm d(0,9)= 70,036 µm

Figure 44: uncoated cellulose microparticles precipitated with NaOH with ultraturrax, washed with PBS




Precipitated with NH4OH with ultraturrax, washed with PBS

d(0,1)= 1,248 µm d(0,5)= 2,545 µm d(0,9)= 173,469 µm

Figure 45: uncoated cellulose microparticles precipitated with NH4OH with ultraturrax, washed with PBS




Precipitated with NH4OH with ultraturrax, washed with H2O

d(0,1)= 1,560 µm d(0,5)= 2,985 µm d(0,9)= 7,851 µm

Figure 46: uncoated cellulose microparticles precipitated with NH4OH with ultraturrax, washed with H2O





Precipitated with NaOH without ultraturrax, washed with H2O

d(0,1)= 2,111 µm d(0,5)= 5,279 µm d(0,9)= 58,183 µm


with NH4OH without ultraturrax, washed with H2O




Precipitated with NaOH with ultraturrax, washed with H2O

d(0,1)= 1,986 µm d(0,5)= 3,926 µm d(0,9)= 21,586 µm


with NaOH with ultraturrax, washed with H2O


to be undertaken further analyses to clarify whether the other parameters are

as good as the size distribution.


Precipitated with NH4OH without ultraturrax, washed with H2O

d(01,)= 1,555 µm d(0,5)= 3,371 µm d(0,9)= 42,569 µm

Figure 49: uncoated cellulose microparticles precipitated with NH4OH without ultraturrax, washed with H2O




5.2.4.3 Magnetic cellulose microparticles coated with methylcellulose


d(0,1)= 1,664 µm d(0,5)= 3,980 µm d(0,9)= 39,269 µm

Figure 50: MC-coated cellulose microparticles precipitated






d(0,1)= 1,941 µm d(0,5)= 9,399µm d(0,9)= 214,952µm


with NH4OH without ultraturrax, washed with albumin





d(0,1)= 1,731 µm d(0,5)= 4,737 µm d(0,9)= 147,388 µm

Figure 52: MC-coated cellulose microparticles precipitated with NaOH with ultraturrax, washed with albumin





d(0,1)= 1,778 µm d(0,5)= 5,007 µm d(0,9)= 227,287µm

Figure 53: MC-coated cellulose microparticles precipitated with NH4OH with ultraturrax, washed with albumin





d(0,1)= 1,572 µm d(0,5)= 4,017 µm d(0,9)= 17,442 µm


with NaOH without ultraturrax, washed with PBS






d(0,1)= 1,322 µm d(0,5)= 4,580 µm d(0,9)= 222,565 µm

Figure 55: MC-coated cellulose microparticles precipitated with NH4OH without ultraturrax, washed with PBS





d(0,1)= 1,470 µm d(0,5)= 3,547µm d(0,9)= 115,984 µm

Figure 56: MC-coated cellulose microparticles precipitated with NaOH with ultraturrax, washed with PBS





d(0,1)= 1,264 µm d(0,5)= 2,610 µm d(0,9)= 140,227 µm


with NH4OH with ultraturrax, washed with PBS





d(0,1)= 1,325 µm d(0,5)= 3,103 µm d(0,9)= 58,070 µm

Figure 58: MC-coated cellulose microparticles precipitated with NH4OH with ultraturrax, washed with H2O





d(0,1)= 1,826 µm d(0,5)= 5,214 µm d(0,9)= 118,138 µm

Figure 59: MC-coated cellulose microparticles precipitated with NaOH without ultraturrax, washed with H2O





d(0,1)= 1,749 µm d(0,5)= 3,862 µm d(0,9)= 39,849 µm







d(0,1)= 1,141 µm d(0,5)= 2,105 µm d(0,9)= 31,745 µm

Figure 61: MC-coated cellulose microparticles precipitated with NH4OH without ultraturrax, washed with H2O




5.2.4.4 Magnetic cellulose microparticles coated with PEG


d(0,1)= 1,410 µm d(0,5)= 3,331 µm d(0,9)= 82,532 µm

Figure 62: PEG-coated cellulose microparticles precipitated






d(0,1)= 1,453 µm d(0,5)= 4,635 µm d(0,9)= 163,063 µm

Figure 63: PEG-coated cellulose microparticles precipitated with NH4OH without ultraturrax, washed with albumin





d(0,1)= 1,413 µm d(0,5)= 3,446 µm d(0,9)= 75,438µm


with NaOH withultraturrax, washed with albumin





d(0,1)= 1,448 µm d(0,5)= 3,368 µm d(0,9)= 140,947 µm


with NH4OH with ultraturrax, washed with albumin





d(0,1)= 1,659 µm d(0,5)= 3,785 µm d(0,9)= 18,307 µm

Figure 66: PEG-coated cellulose microparticles precipitated with NaOH without ultraturrax, washed with PBS






d(0,1)= 1,181 µm d(0,5)= 2,476 µm d(0,9)= 96,500 µm


with NH4OH without ultraturrax, washed with PBS





d(0,1)= 1,535 µm d(0,5)= 3,430 µm d(0,9)= 22,212 µm


with NaOH with ultraturrax, washed with PBS





d(0,1)= 1,248 µm d(0,5)= 2,545 µm d(0,9)= 173,469 µm


with NH4OH with ultraturrax, washed with PBS





d(0,1)= 1,155 µm d(0,5)= 2,328 µm d(0,9)= 108,888 µm


with NH4OH with ultraturrax, washed with H2O





d(0,1)= 1,462 µm d(0,5)= 3,560 µm d(0,9)= 45,549 µm


with NaOH without ultraturrax, washed with H2O





d(0,1)= 1,472 µm d(0,5)= 3,937 µm d(0,9)= 51,322 µm







d(01,)= 1,447 µm d(0,5)= 2,613 µm d(0,9)= 4,795µm


with NH4OH without ultraturrax, washed with H2O





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]


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.


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]


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]


5.2.7 COMPARISON OF SIZE DISTRIBUTIONS AFTER 5MIN/10MIN AND WITHOUT AN ULTRASONIC BATH

Table 24: comparison of size distributions




Table 33: comparison of size distribution


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


b) MC-coated magnetic cellulose microparticles (MC-mcm)

Parameter: NH4OH / - UT / H2O

• before the precipitation: use of Methylcellulose


• 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


c) PEG-coated magnetic cellulose microparticles (PEG-mcm)

Parameter: NH4OH / - UT / H2O

• before the precipitation: use of Polyethylenglycol


• 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


Interpretation:


APPENDIX


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 )

[6] AMBERCHROM – HPR 10

(online in the internet: URL:

http://www.dow.com/products/product_detail.page?product=1120267&application=1011017,

24.05.2011 )

[7] Falkenhagen, Dieter; Brandl, Martin; Jens, Hartmann; Kellner, Karl-Heinz; Posnicek, Thomas;

Weber, Viktoria: Fluidized Bed Adsorbent Systems for Extracorporeal Liver Support – Center for

Biomedicals Technology & Christian Doppler Laboratoy for Specific Adsorption Technologies in

Medicine, Danube University Krems, Krems, Austria, 2006;

[8] Falkenhagen, Dieter; Brandl, Martin; Jens, Hartmann; Schildboeck, Claudia: Particle Leakage in

Extracorporeal Purification Systems Based on Microparticle Suspensions – Center for Biomedicals

Technology & Christian Doppler Laboratoy for Specific Adsorption Technologies in Medicine, Danube

University Krems, Krems, Austria, 2005;


[9] Brandl, M.; Hartmann, J.; Posnicek, T.; Ausenegg, F.R.; Leitner, A.; Falkenhagen, D.: Detection of

Flourescently Labeled Microparticles in Blood - Center for Biomedicals Technology, Danube University

Krems, Krems, Austria & Christian Doppler Laboratoy for Specific Adsorption Technologies in Medicine

& Institute of Experimental Physics, Karl Franzens University Graz, Graz, Austria, 2005;

[10] Falkenhagen, Dieter; Brandl, Martin; Ettenauer, Marion; Posnicek, Thomas; Weber, Viktoria:

Magnetic Fluorescent Microparticles as Markers for Particle Transfer in Extracorporeal Blood

Purification - Center for Biomedicals Technology, Danube University Krems, Krems, Austria, 2007;

[11] Fischer, Steffen; Thümmler, Katrin; Volkert, Bert; Hettrich, Kay; Schmidt, Ingerborg; Fischer, Klaus:

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


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


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


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


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

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