DEVELOPMENT OF PRINTED DOSAGE FORMS WITH BIOACTIVE …
Transcript of DEVELOPMENT OF PRINTED DOSAGE FORMS WITH BIOACTIVE …
GHENT UNIVERSITY Master thesis performed at:
FACULTY OF PHARMACEUTICAL SCIENCE ÅBO AKADEMI UNIVERSITY
Department of Pharmaceutical Analysis Department of Biosciences
Laboratory of Process Analytical Technology Laboratory of Pharmaceutical Science
Academic year 2013-2014
DEVELOPMENT OF PRINTED DOSAGE FORMS WITH BIOACTIVE
MOLECULES
Elise BUGGENHOUT
First Master of Drug Development
Promotor
Prof. Dr. T. De Beer
Co-promotor
Prof. Dr. N. Sandler
Supervisor
MSc. Mirja Palo
Commissioners
Prof. J.P. Remon
Prof. B. De Geest
GHENT UNIVERSITY Master thesis performed at:
FACULTY OF PHARMACEUTICAL SCIENCE ÅBO AKADEMI UNIVERSITY
Department of Pharmaceutical Analysis Department of Biosciences
Laboratory of Process Analytical Technology Laboratory of Pharmaceutical Science
Academic year 2013-2014
DEVELOPMENT OF PRINTED DOSAGE FORMS WITH BIOACTIVE
MOLECULES
Elise BUGGENHOUT
First Master of Drug Development
Promotor
Prof. Dr. T. De Beer
Co-promotor
Prof. Dr. N. Sandler
Supervisor
MSc. Mirja Palo
Commissioners
Prof. J.P. Remon
Prof. B. De Geest
Deze pagina is niet beschikbaar omdat ze persoonsgegevens bevat.Universiteitsbibliotheek Gent, 2021.
This page is not available because it contains personal information.Ghent University, Library, 2021.
Abstract
Development of personalized medicines is necessary to meet the patients’
individual needs. The search for new methods to produce controlled and accurate dosage
forms is in progress. The applicability of inkjet printing technique in which drug formulations
are printed on an edible substrate has been investigated for this purpose.
In this work the inkjet printing technique was used to develop stable protein systems.
It has been reported that disaccharides in their amorphous solid state can stabilize proteins.
The activity of the printed protein lactase was examined and stabilization by the sugars
sucrose and trehalose dihydrate was investigated.
Ink formulations of the disaccharides and the enzyme were prepared and their
printability was evaluated. A 20% w/V and 10% w/V in 20:80 vol% PG:water solutions for
sucrose and trehalose dihydrate, respectively, were chosen for ink formulations. For the
enzyme ink formulation a 1% w/V lactase solution in 10:90 vol% PG:water mixture was used.
Inkjet printing was done onto two substrates with two sugar:protein ratios.
Printed formulations were visualised by optical microscopy and the solid state of the
printed disaccharides was analysed by ATR-IR and DSC. Based on the obtained results it was
found that the disaccharides printed without the protein occurred at least partially in their
amorphous solid state. However, the disaccharides on the sugar:protein systems showed
crystallization.
The activity of lactase was examined by a standardized UV-method 48h after printing
of the protein. The enzyme activity was decreased for all the samples but the sugar:protein
samples showed lower degradation compared to the protein sample. Sucrose was shown to
be a better stabilizer than trehalose dihydrate and the higher sugar:protein ratios gave less
change in the enzyme activity over time.
This study indicates that development of dosage forms by the printing technique
gives many opportunities for the future. However, prepared printed protein formulations
with sugars did not result in stable systems.
Samenvatting
Ontwikkeling van gepersonaliseerde medicatie is nodig om aan de noden van de
patiënt te voldoen. Het zoeken naar nieuwe methoden om gecontroleerde en accurate
dosissen te produceren is in volle gang. De inktjet printtechniek waarbij
geneesmiddelenformulaties geprint worden, zou zo een nieuwe methode kunnen zijn.
In dit onderzoek werd de inktjet printtechniek gebruikt om een gestabiliseerd
proteïne systeem te ontwikkelen. Het is geweten dat suikers in hun amorfe toestand
proteïnen stabiliseren. De activiteit van het geprinte proteïne, lactase, werd bepaald en
stabilisatie door de suikers sucrose en trehalose dihydraat werd onderzocht.
Inktformulaties van de disacchariden en het proteïne werden bereid en de
printmogelijkheid werd geëvalueerd. De 20% m/V en 10% m/V in 20:80 vol% PG:water
inktformulaties voor sucrose en trehalose dihydraat respectievelijk gaven de beste
printeigenschappen. Voor de enzym inktformulatie werd een 1% m/V lactase oplossing in
10:90 vol% PG:water gekozen. Er werd geprint op twee substraten en gewerkt met twee
suiker:proteïne ratio’s.
De geprinte disacchariden werden gevisualiseerd met optische microscopie. De vaste
toestand werd geanalyseerd door ATR-IR en DSC. Gebaseerd op de resultaten kon besloten
worden dat de geprinte suikers zich in afwezigheid van het proteïne op zijn minst
gedeeltelijk in de amorfe toestand bevonden. De disacchariden van de suiker:proteïnestalen
vertoonden echter kristallisatie.
De activiteit van lactase werd bepaald door een UV methode 48u na het printen. De
enzymactiviteit daalde voor alle stalen maar de suiker:proteïnestalen gaven minder
degradatie dan enkel het geprinte proteïne. Sucrose gaf betere stabilisatie dan trehalose
dihydraat en de hogere ratio’s gaven een hogere enzymactiviteit.
Dit onderzoek duidde aan dat het ontwikkelen van formulaties door de inktjet
printtechniek veel mogelijkheden geeft naar de toekomst toe. De geprinte
proteïneformulaties met suiker resulteerden echter niet in stabiele systemen.
ACKNOWLEDGMENT
I would like to thank my promoter, Professor Thomas De Beer for giving me
the opportunity to go abroad for my Master thesis and work on a very interesting
topic. I would also like to express gratitude to Professor Niklas Sandler to cooperate
in the laboratory of pharmaceutical science of Åbo Akademi. My deepest
appreciation goes to my supervisor, MSc. Mirja Palo, who supported me endlessly
and who taught me how research has its insurmountable obstacles but that
perseverance rewards. In addition, a thank to Henrika Wickström, who was always
willingly to answer questions or give suggestions.
Furthermore, my sincere thanks to Karen Rijckaert who was always there for
me in tough moments and made my Erasmus stay in Finland unforgettable. Last but
not the least, I would like to thank my sisters and parents for their support during this
period.
Table of Contents
1. INTRODUCTION ....................................................................................................................... 1
1.1. INKJET PRINTING TECHNIQUE .................................................................................. 1
1.2. STABILITY ENHANCEMENT OF PROTEINS .................................................................... 4
1.2.1. β-galactosidase .............................................................................................. 5
1.3. DISACCHARIDES AS STABILIZERS ............................................................................... 6
1.3.1. Sucrose ......................................................................................................... 7
1.3.2. Trehalose dihydrate ........................................................................................ 8
1.3.3. Characteristics of the sugar solutions................................................................. 8
1.4. CHARACTERIZATIONS OF PRINTED FORMULATIONS ..................................................... 9
1.4.1. Attenuated Total Reflectance Infrared Spectroscopy ............................................ 9
1.4.2. Differential Scanning Calorimetry ................................................................... 10
2. OBJECTIVES .......................................................................................................................... 11
3. METHODS AND MATERIALS ................................................................................................. 13
3.1. PREPARATION OF INK FORMULATIONS .................................................................... 13
3.2. INKJET PRINTING TECHNIQUE ................................................................................ 13
3.2.1. Printed formulations ..................................................................................... 14
3.3. CHARACTERIZATION OF THE PRINTED SAMPLES ........................................................ 14
3.3.1. Optical microscopy ....................................................................................... 14
3.3.2. ATR-IR ....................................................................................................... 14
3.3.3. DSC ............................................................................................................ 15
3.4. DETERMINATION OF THE ENZYME ACTIVITY ............................................................. 15
4. RESULTS ............................................................................................................................... 18
4.1. PRINTING OF THE DISACCHARIDE INK FORMULATIONS ............................................... 18
4.2. CHARACTERIZATION OF THE PRINTED DISACCHARIDE SAMPLES ................................... 19
4.2.1. Optical microscopy images of the printed disaccharide samples........................... 19
4.2.2. ATR-IR spectra of the printed disaccharides samples .......................................... 19
4.2.3. DSC thermograms of the printed disaccharides samples ..................................... 22
4.3. PRINTING OF THE SUGAR:PROTEIN INK FORMULATIONS ............................................. 24
4.4. CHARACTERIZATION OF THE PRINTED SUGAR:PROTEIN SAMPLES ................................. 25
4.4.1. Optical microscopy images of the printed sugar:protein samples ......................... 25
4.4.2. ATR-IR spectra of the printed sugar:protein samples .......................................... 27
4.5. DETERMINATION OF THE ENZYME ACTIVITY ............................................................. 29
5. DISCUSSION ......................................................................................................................... 31
5.1. PRINTING OF THE DISACCHARIDE INK FORMULATIONS ............................................... 31
5.1.1. Cleaning process of the PIJ printer .................................................................. 31
5.2. CHARACTERIZATION OF THE PRINTED SUGAR SAMPLES .............................................. 32
5.2.1. Optical microscopy images of the printed disaccharide samples........................... 32
5.2.2. ATR-IR spectra of the printed disaccharide samples ........................................... 32
5.2.2.1. ATR-IR spectra of sucrose samples.............................................................................33
5.2.2.2. ATR-IR spectra of trehalose dihydrate samples..........................................................34
5.2.3. DSC thermograms of the printed disaccharide samples ...................................... 35
5.3. PRINTING OF THE SUGAR:PROTEIN INK FORMULATIONS ............................................. 36
5.4. CHARACTERIZATION OF THE PRINTED SUGAR:PROTEIN SAMPLES ................................. 36
5.4.1. Optical microscopy of the printed sugar:protein samples .................................... 37
5.4.2. ATR-IR spectra of the printed sugar:protein samples .......................................... 37
5.4.2.1. ATR-IR spectra of sucrose:lactase samples.................................................................37
5.4.2.2. ATR-IR spectra of trehalose dihydrate:lactase samples..............................................38
5.5. DETERMINATION OF THE ENZYME ACTIVITY ............................................................. 39
6. CONCLUSION ........................................................................................................................ 41
7. BIBLIOGRAPHY ..................................................................................................................... 43
List of abbreviations
API Active pharmaceutical ingredient
ATR-IR Attenuated total reflectance infrared
β-gal β-galactosidase
DoD Drop on demand
Dpi Droplets per inch
Gal-DH Galactose dehydrogenase
HPC Hydroxypropyl cellulose
IR Infrared
NAD+ Nicotinamide adenine dinucleotide
NADH The reduced form of NAD+
ONPG o-nitrophenyl-β-D-galactopyranoside
PG Propylene glycol
PIJ QF RT
Piezoelectric inkjet Quality factor Room temperature
Tg Glass transition temperature
UV Ultraviolet
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1. INTRODUCTION
Individual therapy and the production of personalized medicines have recently
gained more attention. Over the years studies have shown that various factors such as body
weight, body composition, age, gender, metabolizing enzymes and hormone concentrations
affect the pharmacodynamics and pharmacokinetics of medications. (1)
Therapeutic drug monitoring is the most widely used technique for individual
therapy. The term itself says that drug concentration in the blood of the patient is monitored
and used for fine-tuning of doses. This clinical practice is especially preferred for drugs with a
narrow therapeutic range. This approach allows to keep the treatment of diseases under
better control and to minimize the risk of severe adverse reactions. (2)
The inkjet printing technique, where a drug solution or suspension can be printed
directly onto an edible substrate, is an alternative method in the development of novel
dosage forms. This technique can be used to produce accurate and homogeneously
distributed doses in low amounts. Tailored medicines allow the administration of the active
pharmaceutical ingredient (API) in a required therapeutic dose for individual patients. The
inkjet printing technique could potentially be more cost-effective and less time-consuming
compared to the conventional manufacturing methods. Furthermore, it could contribute to
the increased stability of bioactive molecules. (1, 3)
In the present work a model sugar:protein system was prepared by the inkjet printing
technique. Evaluation of the printed samples was done by different analysing techniques
and the activity of the protein was investigated.
1.1. INKJET PRINTING TECHNIQUE
There are two main inkjet printing techniques. First, there is the ‘drop-on-demand’
(DoD) technique. Second, there is the ‘continuous’ inkjet printing technique. The latter – as
the name says – ejects ink droplets continuously. A high-pressure pump ensures that the ink
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passes through an orifice and a continuous stream of ink droplets is formed by a
piezoelectric crystal. This technique will not be discussed more in detail because it is not
relevant for this thesis. The DoD printing technique that is described below was used in this
research. (1, 4)
In the DoD inkjet printing method ink droplets are only ejected onto the substrate
where needed. The working principle of the DoD inkjet printer is presented in Figure 1.1.
There are two main types of printers using the DoD technique. These printers have different
transducer elements. A thermal inkjet printer has a thin film resistor as a transducer. The
current heats the resistor whereby a vapour bubble is formed. The bubble enlarges and the
ink droplets are pushed out through a nozzle. The diameter of the nozzles varies in the range
of 20-50 µm which assures that the small droplets are formed. The other type is a
piezoelectric inkjet (PIJ) printer, which has a piezoelectric element as a transducer. An
adjustable current passes through the element and changes the shape or size of the element.
The deformation of the element applies pressure to the liquid and droplets are ejected from
the nozzles onto the substrate. (4)
Figure 1.1: Principle of the DoD inkjet printing technique. (5)
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The ink solutions that are used in inkjet printing of drug formulations consist of the
API and the excipients. These are dissolved either together or separately depending on the
solubility properties of the substances. The viscosity and surface tension are the main ink
properties that need to be modified to ensure an optimal and successful droplet formation
during printing and overall printability. Therefore viscosity enhancers, such as propylene
glycol (PG), are often added to the ink solution. Normally a viscosity of 1–20 mPa•s is
desirable. Surface tension between 25–45 mN/m is preferred (6). The choice of solvent is
important. A solvent that evaporates quickly during printing affects the recrystallization of
the API at the nozzle, which can cause nozzle clogging. Additionally, an appropriate substrate
has to be chosen depending on the final goal. (6)
There are several substrates whereupon the ink can be printed. A non-impermeable
transparency film is an adequate substrate for evaluation of the printed formulations due to
lack of interactions between the substrate and the API. Several commercially available and
in-house-made edible substrates are suitable for pharmaceutical applications. Those
substrates are mostly based on starch or different cellulose derivatives. Starch based rice
paper and hydroxypropyl cellulose (HPC) film are examples of these edible substrates. The
porosity of rice paper and HPC film can affect the solid state properties of the printed drug
dosage forms. (3, 7)
The PIJ printer has several operational parameters that have to be adjusted for every
ink in order to get round and uniform droplets. The most important parameters are
temperature of the printing unit, voltage, wave form and ink pressure. The voltage drives a
current that causes the deformation of the piezoelectric element. The wave form has three
adjustable time settings. The first time setting is to determine how long it takes for the
current to reach its maximum. The second time setting indicates the time in microseconds
that the current stays at its maximum and the last time setting determines how long it takes
for the current to reach its minimum. The ink pressure is set to exerted pressure on the ink
to make sure that all nozzles are filled with the ink solution. The voltage and wave form can
be set differently for the even and odd nozzles. The temperature and ink pressure are the
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same in every nozzle. In order to obtain the flexibility of precise dosing of the API, printing
of multiple layers gives additional opportunities. (8)
During the past ten years a lot of research and studies have been carried out on the
properties and benefits of inkjet printing for pharmaceutical applications. A recent study by
Genina et al. (2013) (9) analysed printing of a model drugs, loperamide hydrochloride. In this
study it was found that loperamide hydrochloride occurred in the amorphous or the
molecularly dispersed form by the printing technique. This affected the dissolution
properties of the drug and thus could printing be advantageous for poorly soluble drugs. The
differences in the existing substrates have also been investigated (7). Porous substrates
showed better features in terms of the printing. Meanwhile, more research is needed to
obtain knowledge and understanding of the printing technique and its prospects.
1.2. STABILITY ENHANCEMENT OF PROTEINS
A lot of proteins undergo chemical and physical degradation. The quaternary, tertiary
and secondary structures of proteins break down under stress conditions. Heat, pH, heavy
metals and agitation are factors that influence the denaturation. The primary structure is
sensitive to hydrolysis. For this reaction water and heat are required. In the development of
pharmaceutical products, containing proteins, the stability of the protein is a major
stumbling block. In the pharmaceutical industry freeze-drying and spray-drying of protein
formulations are favourable techniques to increase the shelf life of proteins. During freeze-
drying, also called lyophilisation, products are first frozen and then low pressure encourages
frozen water molecules to sublimate. Although this method assures preservation of these
freeze-dried products, removal of water can destabilize and inactivate the proteins because
of structural changes. Spray-drying, on the other hand, has the main purpose to obtain dry
pharmaceutical powders starting from a solution, a suspension or an emulsion. Fine droplets
are formed by atomizing the solution and evaporating the solvent with hot gas, mostly air, to
produce a stable fine powder. This technique has the same disadvantage as freeze-drying
thus the properties of the biomolecules can be lost. (10-12)
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1.2.1. β-galactosidase
β-galactosidase (β-gal) is a hydrolase enzyme that can be used as a model protein to
investigate protein stability. This enzyme cleaves β-galactosides into monosaccharides.
Lactase, belonging to the β-gal family, hydrolyses the β-glycosidic bond of lactose into
glucose and galactose as illustrated in Figure 1.2. Much attention has been paid to this
protein because lactose intolerance is caused by deficiency of this enzyme. The addition of
the enzyme allows lactose intolerant people to consume dairy and low lactose products. (13)
The activity of β-gal can be measured with ultraviolet (UV) spectrophotometry. There
are two main methods to measure the activity of this enzyme. The first method is based on
the reactions that are represented in Figure 1.2. The principle of this assay is the enzymatic
hydrolyse of lactose into D-glucose and D-galactose in aqueous medium by β-gal.
Subsequently D-galactose is oxidized by nicotinamide-adenine dinucleotide (NAD+) to D-
galactonic acid in the presence of β-galactose dehydrogenase (Gal-DH). The reduced form of
NAD+ (NADH) absorbs light at 334, 340 or 365 nm and the absorbance intensity corresponds
to the amount of lactose that is converted. Therefore it allows to calculate also the activity
of β-gal that is exploited during the hydrolyse reaction. (14)
Figure 1.2: Principle reactions in the determination of lactose and D-galactose by UV-method. (14)
The second method is based on a reaction with o-nitrophenyl-β-D-galactopyranoside
(ONPG). This molecule resembles lactose and it is converted by β-gal into yellow o-
nitrophenol and colourless galactose. The light absorbance intensity of the yellow product
corresponds to the activity of the enzyme. (15)
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1.3. DISACCHARIDES AS STABILIZERS
Several studies have investigated the effect of sugars on proteins. These studies have
shown that disaccharides, such as sucrose and trehalose dihydrate, are good protein
stabilizers during freeze-drying and spray-drying (16, 17). However, the mechanisms of
stabilization are not yet fully understood. It is most likely to assume that the property of
those lyoprotectants to proceed to the glass transition state plays an important role in the
bioprotective effect of disaccharides. The sugars have a high viscosity below their glass
transition temperature (Tg) and adopt an amorphous structure. The molecules of an
amorphous solid do not have a long-order molecular arrangement as seen for crystalline
structures. Amorphous molecules have a very high viscosity making them act like solids. By
heating it softens at the Tg but does not show a melting point. Biomolecules that are
enclosed by disaccharides in their amorphous state become immobile. This mechanism
protects the proteins from degradation. The Tg must be high above the storage temperature
so that the reaction between molecules is prevented. (16, 18)
On the other hand, disaccharides in their amorphous state can form strong hydrogen
bonds with water. This interaction results in two effects. The water activity decreases,
because there is less free water. Secondly, the biomolecules can also bind with the water
molecules, which leads to an indirect interaction between the sugars and the proteins. The
strong sugar-water network ensures that the protein is less mobile. Direct bonds between
disaccharides and biomolecules also take place. Thus the loss of hydrogen bonds during
freeze-drying is compensated by the new hydrogen bonds between the sugar and the
protein. The degree of stabilization depends on the concentration of the disaccharide
matrices in the protein formulation. In Figure 1.3 the three main mechanisms of the
stabilization of the proteins by the disaccharide trehalose are illustrated. (16, 18)
In a study by Ken-ichi et al. (2011) (19) the effect of sugars, such as sucrose and
trehalose dihydrate, on β-gal during freeze-drying was measured with the UV
spectrophotometer. This study revealed that sucrose and trehalose dihydrate maintained
the activity of the protein during freeze-drying and storage, whereas the enzyme without
these sugars had 15% loss in the activity. Other sugars, such as glucose and fructose, did not
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protect the protein from damage during storage although there was no loss in the activity of
the protein during freeze-drying. (19)
Figure 1.3: Mechanisms of stabilization of the proteins. A. glassy matrix of the sugars; B. sugar-water interaction; C. indirect sugar:protein. (16)
1.3.1. Sucrose
Sucrose, also called saccharose, consists of two monosaccharides, glucose and
fructose. These two monosaccharides are linked with an ether bond (α,β-1,2), called the
glycosidic linkage. The formed disaccharide, with a molecular formula C12H22O11, is a white
crystalline powder. It is mostly known for its use as table sugar. The molecular weight of
sucrose and its solubility in water (at 20°C) are 342.30 g/mol and 342 mg/mL, respectively.
(20)
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1.3.2. Trehalose dihydrate
Trehalose dihydrate, consisting of two glucose units, is widely spread in nature. The
α,α-1,1 glycosidic bond makes trehalose dihydrate, like sucrose, a nonreducing sugar. The
main function of trehalose dihydrate (C12H22O11· 2H2O) is its cryoprotectant effect in cell
structures. The molecular weight of trehalose dihydrate and its solubility in water (at 20°C)
are 378.33 g/mol and 50 mg/mL, respectively. Compared to sucrose, trehalose dihydrate can
form a stronger interaction with water. In Figure 1.4 the molecular structure of sucrose and
trehalose dihydrate are shown. (20)
Figure 1.4: Molecular structure of sucrose (left) and trehalose dihydrate (right). (20)
1.3.3. Characteristics of the sugar solutions
The properties of sugar solutions can be defined with several techniques. The
characteristics of the solid state of sugars as well as the viscosity and the surface tension of
the sugar solutions are all important factors with respect to printing of sugar solutions.
1.3.3.1. Viscosity of the sucrose and trehalose dihydrate solutions
The viscosity of sugar solutions shows a Newtonian behaviour. In other words, the
viscosity of the sugar solution does not depend on the applied shear stress and force.
However no fluid is strictly Newtonian. Newtonian behaviour can be observed usually only in
a small shear rate interval, where the viscosity remains constant. Viscosity can be measured
with a rotational viscometer. This device measures the viscosity at a fixed rotation speed.
Because of this it is also called a single point measurement. The measurement tool is a rod
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that is immersed in the fluid. This technique is simple to use and good for quick quality
control. In Figure 1.5 the effect of the concentration and the temperature on the viscosity is
illustrated. The viscosity of sucrose and trehalose increases exponentially when the
concentration increases. As previously mentioned, the viscosity of the ink solution has to be
in the range of 1-20 mPa•s. Also Newtonian behaviour of ink formulations is preferred. (21,
22)
Figure 1.5: Relationship between the concentration of the sucrose (left) and trehalose
(right) solutions and the viscosity at different temperatures. ♦ 20 °C; □ 27 °C; ▴ 34 °C. (23)
1.4. CHARACTERIZATIONS OF PRINTED FORMULATIONS
1.4.1. Attenuated Total Reflectance Infrared Spectroscopy
Attenuated total reflectance infrared spectroscopy (ATR-IR) is a good method to
identify sugars and distinguish the crystalline and amorphous state of sugars from each
other. The ATR-IR contains an ATR crystal through which an infrared (IR) beam passes. The
sample that does not need any preparation is placed on the surface on top of the crystal.
The ATR-IR beam passes through the crystal and by total reflection an evanescent wave is
formed. The evanescent wave extends through the sample and absorption of energy by the
sample can occur. In that case the energy of the evanescent wave debilitates. All waves are
returned to the infrared beam that goes to the detector. The obtained spectra can be
compared with reference spectra in the literature. (24)
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1.4.2. Differential Scanning Calorimetry
Solid state properties of sucrose and trehalose dihydrate can be investigated by
differential scanning calorimetry (DSC). This technique provides elucidation on the behaviour
of molecules during a temperature controlled program. It measures the differences in heat
flow associated with endothermic or exothermic processes of the substance sample
compared to a reference sample. Endothermic processes, such as melting, require energy
while exothermic transitions, like crystallization, release energy. These energy changes occur
in the form of heat. Thermocouples, attached to the substance and reference sample detect
differences in temperature between both samples. In Figure 1.6 a scheme of the DSC
measuring cell is illustrated. (25)
Figure 1.6: Scheme of a DSC measuring cell. (25)
Thermograms obtained by DSC show endo- and exothermic phase transitions and the
temperature whereby these reactions take place. Glass transitions can also be observed by
chances in the baseline of the thermogram. In other words, with DSC the glass transition,
melting, crystallization, dehydration and other temperatures of the samples can be
obtained.
Earlier thermal analyses of sucrose reported melting points from 160 to 191°C (26).
The recrystallization of freeze-dried sucrose was detected at roughly 110 °C and the Tg of
pure amorphous solid sucrose is normally seen around 60°C (27, 28). The thermal profile of
trehalose dihydrate gives two endothermic peaks, the dehydration at 101 °C and the melting
point of the anhydrous trehalose at 212 °C (29). Amorphous solid trehalose is characterized
by a Tg at 119 °C (28).
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2. OBJECTIVES
There is a high demand for new methods to fulfil the need for individual therapy. The
applicability of the inkjet printing has been proposed as a novel alternative method to obtain
personalized medications. The main advantage of this technique is its feasibility to print the
formulations of low drug content with high dose precision. Currently the most important
focus in this research area is to gain more understanding of the printing technique and its
limitations.
The main aim of this work was to develop stabilized sugar:protein systems by novel
PIJ printing. Two disaccharides, sucrose and trehalose dihydrate were attached to a model
protein lactase by PIJ printing. The effect of sucrose and trehalose dihydrate on the enzyme
activity was studied.
The viscosity of the ink solutions is very important to ensure the optimal conditions
for the inkjet printing. Therefore different solutions of sucrose, trehalose dihydrate and the
protein were prepared and tested as ink formulations. After optimizing the ink formulations
the printing parameters were adjusted for each of the chosen solutions. Optical microscopy
was one of the techniques to characterize the printed sucrose and trehalose dihydrate
samples. This technique was used to examine the distribution uniformity of the printing and
to visualize potential crystallization.
The sugar:protein systems were printed on the transparency film and an edible HPC
substrate. The amorphous form of the disaccharides enables the stabilization of the protein
systems. Therefore, the applicability of the PIJ printing technique to obtain the amorphous
form of sugars was examined. ATR-IR spectra were taken to identify and investigate the solid
state of the disaccharides. The thermal analysis of the printed sugar samples was done with
DSC.
The activity of the enzyme lactase was examined after printing by a
spectrophotometry method. The goal was to determine whether the protein activity
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remained the same in the presence of the disaccharides. Also the difference between the
disaccharides and the used substrates on the enzyme activity was examined.
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3. METHODS AND MATERIALS
3.1. PREPARATION OF INK FORMULATIONS
In total twelve solutions with sucrose (Sigma-Aldrich, Switzerland) and trehalose
dihydrate (Sigma-Aldrich, USA) were tested as ink solutions. Sucrose and trehalose dihydrate
were dissolved in purified water (Milli-Q), with or without PG (Sigma-Aldrich, Germany) as
viscosity enhancer. The sugars were dissolved at room temperature (RT) under stirring.
The protein solutions were prepared similarly to the sugar solutions. The
concentration of lactase (Amano Enzyme, USA) was 1% w/V in 30:70 vol% and 10:90 vol%
mixture of PG and water, respectively. The enzyme activity was not less than 14 000 units/g.
The protein was dissolved at RT under stirring.
3.2. INKJET PRINTING TECHNIQUE
The printing of drug formulations was performed with the printer PixDro LP50 (OTB
Solar – Roth & Rau, The Netherlands). Isopropanol (Rathburn, of Walkerburn, Scotland) was
used to clean the ink reservoir and printhead before printing. Thereafter the reservoir was
filled with the ink solution. 0.2 μm syringe filters were used to filter all inks before printing in
order to avoid clogging of the nozzles. The resolution (500x500 droplets per inch (dpi)), firing
frequency (1400 Hz) and the quality factor (QF 8) were held constant throughout the
printing procedure. However, before the actually printing on the substrate some parameters
had to be optimized to obtain a suitable formation of the droplets. As mentioned the
temperature, the voltage, the ink pressure and the wave form were those changeable
parameters. Droplets were evaluated after ejection from the nozzle based on a snapshot
with a dropview camera that was incorporated in the printing system. Once droplets with an
acceptable shape and volume were achieved the printing was performed. The substrate was
placed on the substrate holder with a vacuum pump to ensure the substrate was fixed
tightly. The temperature of the printhead was set on 25 °C for all solutions.
14
3.2.1. Printed formulations
Printing of the ink solutions was done on transparency film (DatalineTM, Espoo,
Finland) and on the in-house produced HPC film. The latter was made by solvent casting of a
5 wt% HPC (Klucel LF PHARM, Ashland, Wilmington, USA) solution in water. The solution was
casted onto a transparency film with 36 mL of the solution per 216 cm2 of the film. The
sugar:protein samples were obtained by printing the disaccharide and protein ink solutions
separately. All the protein layers were printed in between the sugar layers. In total five
protein layers were printed on each substrate. The disaccharides layers were printed in two
concentration ratios, 20:3 and 40:3 sugar:protein respectively. A printing pattern of 5x3
squares with each square of 1 cm2 was used.
3.3. CHARACTERIZATION OF THE PRINTED SAMPLES
3.3.1. Optical microscopy
An optical microscope (Evos XL, AMG, USA) was used to visualize the dry printed
samples. Images were taken at magnifications of 4x, 20x and 40x. The printing uniformity of
the ink and potential crystallization was examined.
3.3.2. ATR-IR
An ATR-IR (PerkinElmer, UK) was used to obtain IR spectra of printed formulations.
The spectra of the raw materials of sucrose and trehalose dihydrate, PG and the substrates
were taken as a reference.
The dry samples were placed on the sampling window of the spectroscope and a
force of 140 N and 75 N was applied for printed samples and raw materials, respectively. The
obtained spectra were subject to baseline correction, normalization and data tune-up pre-
treatment prior to comparative analysis with the Spectrum 10.03 software (PerkinElmer,
UK).
15
3.3.3. DSC
The thermal analysis of the raw materials and printed samples was performed with a
DSC (DSC Q2000, TA Instruments, USA). Four layer printed disaccharide squares were
scraped off the transparency film. The disaccharide samples on the HPC film were cut into
little pieces before the analysis. The sample amount in closed Tzero aluminium pans was in
the range of 1-10 mg. The measurements were conducted under nitrogen purge with a flow
rate of 50 ml/min. The samples with sucrose and trehalose dihydrate were measured with a
heating rate of 10°C/min in a temperature range of 20-220°C and 20-250°C, respectively.
3.4. DETERMINATION OF THE ENZYME ACTIVITY
The enzyme activity was determined by a standardized spectrophotometry method.
The measurements were performed using a lactose/D-galactose UV-method test kit
(Boehringer Mannheim, Germany). All reagents of the UV-method test kit are listed in Table
3.1.
Table 3.1: The components of the lactose/D-galactose UV-method test kit
Bottles Compounds
1 Lyophilizate: citrate buffer (pH~6,6), NAD+, MgSO4
2 β-galactosidase suspension
3 Potassium diphosphate buffer (pH~8.6)
4 Galactose dehydrogenase suspension
5 Lactose assay control solution
The content of bottle 1 was dissolved in 7 mL purified water (= solution 1) before
analysis. All other reagents did not require further preparation. Bottle 2 was replaced by the
printed sugar:protein samples. A 0.2% m/V lactose solution in purified water was prepared
(= solution A). In order to obtain the calibration curve four enzyme standard solutions with
concentrations 0.25, 0.5, 1.0 and 2.0 mg/mL in 10:90 vol% PG:water were prepared. The
16
printed samples were measured 48h after printing the protein layers. The disaccharide
layers on top of the protein were printed the same day as the enzyme activity assay was
performed. As a reference the same amount of the protein ink solution was printed on both
substrates without the disaccharides. The enzyme activity of the samples without the sugars
was measured directly after printing and 48h after printing. Prior to the measurements two
squares of each printed samples were dissolved in 0.5 mL purified water. Table 3.2 describes
the procedure of the assay.
Table 3.2: Procedure of the enzyme activity method
Blank Sample
Solution 1 200 µL 200 µL
Solution A / 500 µL
Enzyme solution / 50 µL
Mixed and incubated for minimum 20 min at RT
Bottle 3 1000 µL 1000 µL
Purified water 2050 µL 1500 µL
Mixed, after 2 min: read the absorbance at 340 nm
(Ablank1, Asample1)
Bottle 4 50 µL 50 µL
Mixed, after 30min: read absorbance at 340 nm
(Ablank2, Asample2)
The equation for absorbance difference, ΔA = (A2 – A1)sample – (A2 – A1)blank > 0.100
had to comply to achieve reliable results.
The absorbance intensity at 340 nm was in relation to the amount of the formed
NADH and thus the amount of D-galactose as illustrated in the first reaction in Figure 1.2.
The concentration of D-galactose was calculated by the following equation. The calculated
D-galactose concentration was put into relation with the enzyme activity expressed in
concentration.
17
C =
C = D-galactose concentration (g/L)
V = final volume (3.3 mL)
MW = molecular weight of D-galactose (180.16 g/mol)
A = difference in absorbance (Ablank2 – Ablank1) – (Asample2 – Asample1)
ε = extinction coefficient of NADH at 340 nm (6.3 L x mmol-1 x cm-1)
d = light path (1 cm)
v = volume of the lactose solution (0.500 mL)
18
4. RESULTS
4.1. PRINTING OF THE DISACCHARIDE INK FORMULATIONS
In total twelve disaccharide solutions were tested as ink formulations and printed on
transparency films. In Table 4.1 the composition of the sugar ink formulations is described.
Table 4.1: The composition of the sugar ink formulations
The operational parameters to optimize the printing of the chosen sugar ink
formulations are illustrated in Table 4.2. The temperature of the printhead was set on 25 °C.
Table 4.2: Printing parameters for the sugar ink formulations; A = even nozzles, B = odd nozzles
Sucrose 20% w/V in 20:80 vol% PG:water
Trehalose dihydrate 10% w/V in 20:80 vol% PG:water
Ink pressure -19 mbar -20 mbar
Voltage A/B 70/78 V 75/75 V
Wave form A/B 2 - 10 - 3.5 / 2.5 - 10 - 3 µs 2.5 - 10 - 3.5 / 2.5 - 10 - 3.5 µs
Sucrose concentration (w/V)
PG:water vol% Trehalose dihydrate concentration (w/V)
PG:water vol%
20% 20:80 10% 20:80
20% 10:90 10% 10:90
20% 0:100 10% 0:100
25% 0:100 20% 20:80
30% 0:100 20% 10:90
40% 0:100 20% 0:100
19
4.2. CHARACTERIZATION OF THE PRINTED DISACCHARIDE SAMPLES
4.2.1. Optical microscopy images of the printed disaccharide samples
The disaccharide samples were analysed with the optical microscopy. In figure 4.1
images of the printed sucrose and trehalose dihydrate on the transparency and the HPC film
are illustrated.
A B C
D E F
Figure 4.1: Images of two layers of sucrose and trehalose dihydrate samples taken by the optical microscopy, 4x magnification. A: sucrose on transparency film; B: trehalose
dihydrate on transparency film; C: transparency film; D: sucrose on HPC film; E: trehalose dihydrate on HPC film; F: HPC film.
4.2.2. ATR-IR spectra of the printed disaccharides samples
Spectra of the disaccharides printed on the transparency and HPC film are illustrated
in Figure 4.2, 4.3, 4.4 and 4.5. The spectra are compared to the spectra of PG, the pure
disaccharides and the substrates.
20
Figure 4.2: IR spectra of PG, pure sucrose, sucrose printed on the transparency film and the transparency film in the range of 4000-400 cm-1.
Figure 4.3: IR spectra of PG, pure sucrose, sucrose printed on the HPC film and the HPC film in the range of 4000-400 cm-1.
21
Figure 4.4: IR spectra of PG, pure trehalose dihydrate, trehalose dihydrate printed on the transparency film and the transparency film in the range of 4000-400 cm-1.
Figure 4.5: IR spectra of PG, pure trehalose dihydrate, trehalose dihydrate printed on the HPC film and the HPC film in the range of 4000-400 cm-1.
22
4.2.3. DSC thermograms of the printed disaccharides samples
DSC thermograms of the printed sucrose on the transparency and HPC films are
shown in Figure 4.6 and Figure 4.7. DSC thermograms of the printed trehalose dihydrate on
the transparency and HPC films are presented on Figure 4.8 and Figure 4.9.
Figure 4.6: DSC thermogram of sucrose printed on the transparency film.
23
Figure 4.7: DSC thermogram of sucrose printed on the HPC film.
Figure 4.8: DSC thermogram of trehalose dihydrate printed on the transparency film.
24
Figure 4.9: DSC thermogram of trehalose dihydrate printed on the HPC film.
4.3. PRINTING OF THE SUGAR:PROTEIN INK FORMULATIONS
Two protein solutions with 1% w/V lactase concentration in 30:70 vol% and 10:90
vol% PG:water were prepared. The printing parameters of the chosen protein ink
formulation are described in Table 4.3. The temperature of the printhead was set on 25 °C.
Table 4.3: Printing parameters of the chosen protein ink formulation; A = even nozzles, B = odd nozzles
In total eight printed sugar:protein samples were obtained. The average volume of
the droplets (in pL) of the printed protein solution with respect to the substrate and
sugar:protein ratio is listed in Table 4.4.
Protein 1% w/V in 10:90 vol% PG:water
Ink pressure -21 mbar
Voltage A/B 85/80 V
Wave form A/B 4 – 12 – 4 / 12 – 12 – 12 µs
25
Table 4.4: Average droplet volumes of the protein ink formulation
Sucrose 20% w/V in 20:80 vol% PG:water
Trehalose dihydrate 10% w/V in 20:80 vol% PG:water
SUBSTRATES Transp. film HPC film Transp. film HPC film
sugar:protein 20:3 40:3 20:3 40:3 20:3 40:3 20:3 40:3
Average droplet volumes (pL)
15.9 15.4 15.7 16.6 14.5 14.5 14.1 14.8
4.4. CHARACTERIZATION OF THE PRINTED SUGAR:PROTEIN SAMPLES
4.4.1. Optical microscopy images of the printed sugar:protein samples
The sugar:protein samples were visualized with the optical microscopy and are
illustrated in Figure 4.10, 4.11 and 4.12.
A B
Figure 4.10: Images of the protein samples by optical microscopy, 4x magnification. A: five layers of lactase on transparency film; B: five layers
of protein on HPC film.
26
A B
C D
Figure 4.11: Images of the sucrose:protein samples taken by the optical microscopy, 4x magnification. A: sucrose:lactase on transparency film
with 20:3 ratio; B: sucrose:lactase on HPC film with 20:3 ratio; C: sucrose:lactase on transparency film with 40:3 ratio; D: sucrose:lactase
on HPC film with 40:3 ratio.
A B
27
C D
Figure 4.12: Images of the trehalose dihydrate:protein samples taken by the optical microscopy, 4x magnification. A: trehalose dihydrate:lactase on transparency film with
20:3 ratio; B: trehalose dihydrate:lactase on HPC film with 20:3 ratio; C: trehalose dihydrate:lactase on transparency film with 40:3 ratio; D: trehalose dihydrate:lactase on
HPC film with 40:3 ratio.
4.4.2. ATR-IR spectra of the printed sugar:protein samples
The ATR-IR spectra of the sugar:protein samples with ratios 40:3 are illustrated in
Figure 4.13, 4.14, 4.15 and 4.16. The spectra are compared to the spectra of lactase, the
pure disaccharides and the substrates.
Figure 4.13: IR spectra of lactase, pure sucrose, lactase-sucrose printed on the transparency film and the transparency film in the range of 4000-400 cm-1.
28
Figure 4.14: IR spectra of lactase, pure sucrose, lactase-sucrose printed on the HPC film and the HPC film in the range of 4000-400 cm-1.
Figure 4.15: IR spectra of lactase, pure trehalose dihydrate, lactase-trehalose dihydrate printed on the transparency film and the transparency film in the range of 4000-400 cm-1.
29
Figure 4.16: IR spectra of lactase, pure trehalose dihydrate, lactase-trehalose dihydrate printed on the HPC film and the HPC film in the range of 4000-400 cm-1.
4.5. DETERMINATION OF THE ENZYME ACTIVITY
Linearity for the UV-method was proven for enzyme concentration range of 0–2.0
mg/mL with a correlation factor of R2=0.99. The theoretical amount was calculated based on
the average droplet volumes of lactase, listed in Table 4.4, and the dpi. The theoretical and
experimental protein amounts of the printed samples on the transparency film are listed in
Table 4.5. The experimental amount of lactase was measured with the UV-method.
Table 4.5: The theoretical amount compared to the experimental (exp.) amount of lactase (µg) printed per one square
SAMPLES ON TRANSPARENCY FILM
Theoretical amount (µg)
Exp. amount (µg) day 0
Exp. amount (µg) day 2
Sucrose:lactase 20:3 30.8 54.2
Sucrose:lactase 40:3 29.9 57.4
Trehalose dihydrate:lactase 20:3
28.0 36.7
Trehalose dihydrate:lactase 40:3
28.1 46.2
Lactase 26.7 70.1 22.3
30
In Figure 4.17 the enzyme activity in percentage in the function of the time is
illustrated for all the samples on the transparency film.
Figure 4.17: Activity of lactase (%) of all the samples on the transparency film measured after two days.
0
20
40
60
80
100
0 2
en
zym
e a
ctiv
ity
%
Days
sucrose 40:3
sucrose 20:3
trehalose dihydr 40:3
trehalose dihydr 20:3
Lactase
31
5. DISCUSSION
5.1. PRINTING OF THE DISACCHARIDE INK FORMULATIONS
The twelve sugar solutions listed in Table 4.1 were tested as ink formulations.
Sucrose solutions in water with concentrations between 20-40% were prepared. The 40%
and 30% w/V sucrose solutions could not be printed, because the obtained droplets did not
exhibit good shape and volume. This was probably due to too high viscosity of the ink that
hindered the ink to get through the nozzles. The 25% and 20% w/V sucrose solutions showed
good droplet formation and were printed on transparency film. These ink solutions showed
smearing upon printing, therefore PG was added to the 20% w/V sucrose solution. The
sucrose solution in 20:80 vol% PG:water showed the best printability.
Trehalose dihydrate solutions were prepared with lower concentration than the
prepared sucrose solutions due to its lower solubility (50 mg/mL) in water. All 20% w/V
trehalose dihydrate solutions showed unsuitable droplet formation due to high viscosity of
those solutions and the recrystallization of the sugar in the nozzles. The 10% w/V trehalose
dihydrate solutions gave better droplets. Based on the printing quality of the squares on
transparency film the 10% w/V trehalose dihydrate in 20:80 vol% PG:water was selected as
the best printable trehalose dihydrate ink solution. Several layers from one to four of the
two best sugar ink formulations were printed on transparency films and HPC films. In Table
4.2 the operational parameters for printing are presented. These parameters were also
applied for printing of the sugar layers in the sugar:protein samples.
5.1.1. Cleaning process of the PIJ printer
It was found that especially high concentrations of the disaccharides clogged the
nozzles of the PIJ printer. The cleaning process where only isopropanol was used did not
remove the sugars from the nozzles. Therefore cleaning of the printhead was changed.
Before and after printing, the nozzles were put in a warm water bath (40°C), thereafter
water at 60°C was purged through the nozzles because heating increases the solubility of the
32
sugars and also decreased the viscosity of the ink. At last the nozzles were cleaned with
isopropanol at RT.
5.2. CHARACTERIZATION OF THE PRINTED SUGAR SAMPLES
The solid state of the sugars was investigated after printing. It is known from earlier
studies (18) that the sugars in their amorphous state stabilize proteins. The samples with
only printed sugars were kept at ambient conditions after printing and the dry samples were
subject to optical microscopy imaging, ATR-IR and DSC analysis. X-ray diffraction is also a
good technique to determine the solid state of the substances, however in the formulations
with a low amount of printed material the substrates have been shown (9) to give too much
interference thus this method was not exploited in this study.
5.2.1. Optical microscopy images of the printed disaccharide samples
In Figure 4.1 images of two layers of sucrose and trehalose dihydrate printed on
transparancy and HPC films are illustrated. The printing pattern of the disaccharides on
transparency films was clearly seen on the images A and B. Droplets could be distinguished
from each other and merging of droplets was also observed for both disaccharides when
higher number of layers was printed (data not shown). Crystallization of both disaccharides
on transparency film was not seen. Similar images were obtained for the sugars on the HPC
films. Images D and E show sucrose and trehalose dihydrate, respectively, printed on the
HPC film with a 4x magnification. Again the printing pattern was seen and crystals were not
detected in the samples on the HPC film.
5.2.2. ATR-IR spectra of the printed disaccharide samples
In Figure 4.2, 4.3, 4.4 and 4.5 the spectra of the disaccharides on both substrates are
illustrated. The aim was to investigate in which solid state the printed disaccharides occurred.
Spectra of amorphous disaccharides show relatively broader bands in the mid-IR
region compared to the spectra of crystalline molecules. This is due to the fact that the
ordered structure and intermolecular interactions in the crystalline form lead to a lower
33
dispersion of vibration levels. Hereby the selectivity increases and sharper and more intense
absorbance bands in the spectra of crystalline disaccharides are observed. (30)
5.2.2.1. ATR-IR spectra of sucrose samples
In Figure 4.2 the ATR-IR spectrum of sucrose printed on the transparency film is
illustrated. A broad band that is typical for amorphous disaccharides was seen at 3305 cm-1.
This band is located in the O-H stretching vibration region (3600-3000 cm-1) (31). The little
peak at 3500 cm-1 observed for crystalline sucrose was not seen in the ATR-IR spectrum of
the printed sucrose. This could give an indication for the presence of an amorphous solid
state but the band could also originate from the overlapping band for PG in that region. The
six peaks between 3000-2800 cm-1 (C-H stretching vibration (31)) in the ATR-IR spectrum of
crystalline pure sucrose disappeared and one peak at 2929 cm-1 was seen. Again an
overlapping with the spectrum of PG was seen. The IR spectrum of PG showed three peaks in
that region thus it could not be confirmed that the peak derived from the amorphous solid
state. A small peak at 1650 cm-1 was detected and could be attributed to the H-O-H
scissoring mode, indicating that water molecules were present in the sample (31). In the C-H
deformation (1500-1200 cm-1) region less and broader absorbance peaks were observed for
the printed sample compared to the crystalline sucrose. Also in the region of 1200-800 cm-1
broad absorbance bands in the ATR-IR spectrum of the printed sucrose were observed.
Those peaks could be assigned to the C-OH bending vibration and C-O stretching vibration
(31). The broader absorbance bands followed the same pattern as the sharp peaks of the
ATR-IR spectrum of the crystalline sucrose but they were shifted to lower wavenumbers.
The ATR-IR spectrum of sucrose printed on the HPC film is illustrated in Figure 4.3.
The observed peaks were very similar to the ones seen in the ATR-IR spectrum of sucrose
printed on the transparency film. The peak in the region of 3000-2900 cm-1 could be derived
from the sugar, but the PG and also the HPC film gave peaks in that wavelength range.
Compared to the reference spectrum of amorphous sucrose taken from literature, it was
assumed that the amorphous state of sucrose was present in the formulations on both films
(31).
34
5.2.2.2. ATR-IR spectra of trehalose dihydrate samples
In Figure 4.4 the ATR-IR spectrum of trehalose dihydrate printed on the transparency
film is illustrated. Typically in the spectrum of amorphous trehalose dihydrate there is a
broad absorbance band in the O-H stretching region. In the ATR-IR spectrum of trehalose
dihydrate on the transparency film this broad band was seen at 3299 cm-1. The sharp peak at
3500 cm-1 that was seen in the spectrum of crystalline trehalose dihydrate was missing in the
obtained spectrum of the printed trehalose dihydrate. It is assumed that this peak is derived
from the O-H stretch vibration of two crystalline water molecules in the dihydrate form (30).
In the C-H region there was only one broad band seen whereas in the spectrum of crystalline
trehalose dihydrate six peaks were identified in this region. Again the spectrum of PG
showed overlapping in the region of 3600-2800 cm-1. At the wavelength of 1646 cm-1 there
was a small peak indicating the H-O-H scissoring vibration of residual water. For trehalose
dihydrate in its crystalline state, this peak was seen at 1685 cm-1. Shifting of that peak to a
lower wavenumber has been noted before in the spectrum of amorphous trehalose
dihydrate (30). Less and broader bands were seen for the printed sample in the C-H
deformation, C-OH bending and C-O stretching vibration regions compared to the spectrum
of pure crystalline trehalose dihydrate.
The obtained ATR-IR spectrum of trehalose dihydrate printed on the HPC film is
illustrated in Figure 4.5. Although the characteristic peaks described above were also seen in
this spectrum, sharper absorbance peaks in the fingerprint region (below 1500 cm-1) were
observed compared to the absorbance peaks seen for trehalose dihydrate on transparency
film. The spectrum was shown to be more similar to that of amorphous trehalose dihydrate.
Based on the ATR-IR spectra it was concluded that trehalose dihydrate was present in its
amorphous form on both substrates.
35
5.2.3. DSC thermograms of the printed disaccharide samples
5.2.3.1. DSC thermograms of sucrose
In Figure 4.6 the thermogram of sucrose on transparency film obtained by DSC is
illustrated. Two endothermic peaks were observed. The peak at 139.47 °C was not found in
literature to be characteristic for sucrose. Although an endothermic peak at 135 °C has been
reported for sucrose and therefore the temperature shift was assigned to the change in
viscosity (32). The endothermic peak at temperature 183.34°C was attributed to the melting
of sucrose. Pure crystalline sucrose showed melting at 189.97 °C. Neither Tg nor an
exothermic transition was observed. Based on this thermogram it can be concluded that the
printed sucrose was present in the crystalline form and that the amorphous state was absent.
The thermal analysis of sucrose printed on the HPC film is shown in figure 4.7. There
was only one endothermic peak which could be assigned to the melting of sucrose (33). This
peak was seen at 188.89 °C and it deviated less from the melting peak of pure solid
crystalline sucrose compared to the melting point of sucrose printed on transparency film.
The exothermic peak at 113.25°C was due to the recrystallization of amorphous sucrose
molecules (27). Like in the thermogram of sucrose on transparency film the Tg was not
observed. These results from the thermal analysis allowed to conclude that sucrose printed
on the HPC film was at least partially in amorphous state.
5.2.3.2. DSC thermogram of trehalose dihydrate
In Figure 4.8 the obtained thermogram of trehalose dihydrate printed on
transparency film is illustrated. The thermogram showed a similar pattern as the
thermogram of solid crystalline trehalose dihydrate. Two endothermic peaks were observed
at 89.04 °C and 210.44 °C. The first one with the highest enthalpy value can be assigned to
the dehydration of trehalose dihydrate. Thereafter anhydrous trehalose was formed and the
melting occurred at 210.44 °C. The thermal analysis of the samples gave no indication that
trehalose dihydrate appeared in the amorphous form.
36
Finally, a DSC scan of trehalose dihydrate printed on HPC film was done. The
thermogram did not differ much from the thermogram of trehalose dihydrate on the other
substrate. The dehydration and melting peaks were seen at 98.86 °C and 212.09 °C,
respectively. As in the thermogram of sucrose on HPC film the melting temperature, and
here also the dehydration temperature, diverged less from the temperatures seen in the
thermogram of pure solid crystalline trehalose dihydrate compared to the trehalose
dihydrate printed on transparency film. The two endothermic peaks for pure solid crystalline
trehalose dihydrate were observed at 98.66 °C and 212.05 °C.
The thermal analysis gave opposite results compared to the microscopic images and
the ATR-IR spectra. However, the sample preparation for the DSC analysis could have
affected the solid state of the sugars and thus explains, why the thermograms of the printed
sugar samples did not show the presence of amorphous state. In addition, the low content
of the disaccharides in the samples could have hindered the detection of Tg and exothermic
peaks.
5.3. PRINTING OF THE SUGAR:PROTEIN INK FORMULATIONS
The protein ink solution with the lower concentration of PG (10:90 vol%) had better
printability than the ink with 30:70 vol% PG:water. In Table 4.3 the optimal parameters for
printing of the protein are listed. Table 4.4 shows the average droplet volumes of the protein
ink formulation on each sample. It was taken into account that the printed amount of the
protein had to be high enough to reach the detection limit of the UV-method. Therefore five
layers of lactase ink were printed in between the disaccharide layers for each sample.
5.4. CHARACTERIZATION OF THE PRINTED SUGAR:PROTEIN SAMPLES
As for the sugar samples, the solid state of the disaccharides on the sugar:protein
samples was investigated. After printing the sugar:protein samples were kept at ambient
conditions and the dry samples were examined by the optical microscopy and ATR-IR.
37
5.4.1. Optical microscopy of the printed sugar:protein samples
Microscopic images were taken of all the dry samples. In Figure 4.10 images of the
printed protein taken with the optical microscopy are shown. The printing pattern was
visible and no unusual behaviour could be reported. The images of the sucrose:protein
samples illustrated in Figure 4.11 on the other hand showed prominent solid structures.
Visible crystals were observed in all the sucrose:lactase samples. In the image D for
sucrose:lactase with 40:3 ratio on the HPC film widely distributed crystallization was seen.
In Figure 4.12 the trehalose dihydrate:lactase samples are shown. Crystals of
trehalose dihydrate were present in all the samples. Again, higher concentration of the
disaccharide resulted in an extensive crystallization. A distinctive blue colour of the trehalose
dihydrate crystals was observed for the trehalose dehydrate:lactase samples on the
microscopic images.
5.4.2. ATR-IR spectra of the printed sugar:protein samples
5.4.2.1. ATR-IR spectra of sucrose:lactase samples
In Figure 4.13 the spectrum of the printed sucrose:lactase on the transparency film is
illustrated. It was apparent that sharp absorbance peaks were presented. The small peak at
3500 cm-1 was observed which is typical for crystalline sucrose. Also shoulder peaks instead
of one broad band were seen in the O-H stretching vibration region. In the fingerprint region
a similar pattern to the pure crystalline sucrose was seen. The obtained peaks were not as
sharp and that could probably be assigned to the overlapping of the absorbance bands of
lactase and the substrate. It was speculated that sucrose was presented in the crystalline
form since there were no indications of the amorphous solid state. The identification of
lactase was intricate in this spectrum. A lactase absorbance band at 1635 cm-1 was partially
overlapping with the absorbance peak of the transparency film and therefore gave a peak
with a wide shoulder on the spectrum of the printed sample.
38
In Figure 4.14 the spectrum of sucrose:lactase on the HPC film is illustrated. The
typical crystalline peak at 3500 cm-1 was not observed in the obtained spectrum. The broad
band was seen without shoulder peaks at 3300 cm-1. In the fingerprint region the
absorbance peaks were very similar to the peaks seen for the sucrose sample on the HPC
film as described in chapter 5.2.2.1. Based on this data it seemed that amorphous sucrose
was present. However, spectra were taken at different places of the printed samples and
thus also on places where no crystallization was seen. Obviously the spectra of the
crystalline areas showed the characteristics of crystalline sugar whereas the other spectra
were more similar to those of the printed sugar samples. The presented spectrum in Figure
4.14 was taken from a place without any visibly detectable crystals. Images taken by the
optical microscopy showed already the presence of crystals. The lactase was identified by
one peak in the amide I band region (1600-1700 cm-1) at 1635 cm-1. This peak was attributed
to the conformation of the β-sheet of lactase (34).
5.4.2.2. ATR-IR spectra of trehalose dihydrate:lactase samples
The ATR-IR spectrum of the trehalose dihydrate:lactase sample on the transparency
film is illustrated in Figure 4.15. The ATR-IR spectrum showed a very typical spectrum of
crystalline trehalose dihydrate. Two characteristic peaks for crystalline trehalose dihydrate
were seen at 991 cm-1 and 943 cm-1 indicating the α,α-1,1 glycosidic bond stretch. A peak in
this spectrum that could be assigned to the protein was not found. This was expected due to
the extensive crystallisation of trehalose dihydrate seen on the microscopic images as
illustrated in Figure 4.12, C.
The spectrum of trehalose dihydrate on the HPC film, that is illustrated in Figure 4.16,
showed similarly to the spectrum of trehalose dihydrate on the transparency film sharp and
intensive absorbance peaks. Compared to the obtained spectrum of crystalline trehalose
dihydrate similar peaks were observed. Again, the protein could not be identified in this
spectrum due to the overlapping of the absorbance bands of the other components in the
sample.
39
It could be concluded that crystallization occurred in all the sugar:protein samples.
One possible explanation why crystals were observed on these samples and not on the
samples without the protein, is that the top layer of the sugars in the sugar:protein samples
was not in contact with the substrate. Hence it was concluded that the substrate also affects
the crystallization behaviour of the molecules.
5.5. DETERMINATION OF THE ENZYME ACTIVITY
The theoretical content of lactase per one printed square compared to the
experimental values is listed in Table 4.5. Determination of the protein amount directly after
printing was done only for the lactase sample. It was expected that the printed lactase
amount would be the same for every printed sample. The protein sample gave a remarkably
higher experimental amount than the theoretical value indicated. This behaviour, where the
content of the printed sample is higher than the calculated value has been observed before
in other printing experiments. However, data on this matter have not yet been published. It
was speculated that one of the reasons for this behaviour could probably be the deviation of
the droplet volumes that were used in the calculation for the theoretical values.
The protein amount of the samples on the transparency film in Table 4.5 was used to
express the enzyme activity as seen on the graph in Figure 4.17. The enzyme activity was
reported in amount of enzyme present in the sample that was able to convert the reaction
during the lactase determination assay. As illustrated in Figure 4.17 the enzyme activity
decreased for all the samples after two days. Higher activity was seen for all sugar:protein
samples compared to the lactase sample. Sucrose gave higher stability than trehalose
dihydrate. The stabilization was also better for the samples with higher sugar:protein ratios
compared to the lower sugar concentration.
Lactase activity measurements of the HPC samples were not possible. The UV-
method was not compatible with the HPC film and sedimentation was observed during the
incubation period of the reaction. This was probably due to interactions between the HPC
film and the reagents of the enzyme activity assay.
40
Unfortunately, due to the low protein content a degree of uncertainty has to be
taken into account when drawing conclusions from the results of the enzyme activity assay,
since the recommended absorbance difference of at least 0.100 was not fulfilled.
41
6. CONCLUSION
The main goal of this study was to develop stabilized sugar:protein systems. Firstly,
different ink formulations were prepared and their printability was tested. The 20% w/V and
10% w/V in 20:80 vol% PG:water ink formulations for sucrose and trehalose dihydrate,
respectively, were the best printable sugar ink formulations. For the enzyme formulation, a 1%
w/V lactase solution in 10:90 vol% PG:water was chosen. The sugar:protein systems were
prepared by printing the disaccharide and protein ink formulations separately on top of each
other onto two different substrates with the protein in between.
Characterization of the sugar samples to investigate the solid state of the
disaccharides was done by the optical microscopy, ATR-IR and DSC. Based on the obtained
images by the optical microscopy and the ATR-IR spectra the sugars on both substrates were
present in their amorphous solid state. The thermal analysis gave opposite results and did
not support the ATR-IR findings. Microscopic images and ATR-IR spectra revealed that
crystallization occurred in the sugar:protein samples on both substrates, possibly due to loss
of interaction with the substrates.
Even though the disaccharides did not occur in the amorphous solid state on the
sugar:protein samples less degradation of the protein was still observed for the printed
sugar:protein formulations on the transparency film. The higher sugar:protein ratios gave
higher enzyme activity and sucrose turned out to be a better stabilizer than trehalose
dihydrate. However, prepared printed protein formulations with sugars did not result in
stable systems. The enzyme activity of the printed formulations on the HPC film could not
be determined due to an incompatibility of the HPC film and the reagents of the UV-method.
To increase the reliability of the enzyme activity assay alternative options would be
to increase the area of the printed formulations or the amount of the printed layers of the
protein. Different compositions of the ink solutions depending on the protein should also be
evaluated.
42
In conclusion, the inkjet printing technique shows potential as a new method for the
stabilization of biomolecules. However, limitations of the analysing techniques were seen
because of the low printed amounts and the interference from the substrates. Future
studies could be targeting on the development of more suitable printed amounts and the
use of different compositions of the printed formulations as well as other substrates.
43
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