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

1

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

2

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)

3

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

4

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)

5

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)

6

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

7

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)

8

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

9

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)

10

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).

11

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

12

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.

13

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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