GHENT UNIVERSITY
Master thesis performed at:
FACULTY
OF
PHARMACEUTICAL
CHALMERS UNIVERSITY OF TECHNOLOGY
SCIENCES
GOTHENBURG
Department of Pharmaceutics
Department of Chemical and
Laboratory of General Biochemistry &
Biological Engineering
Physical Pharmacy
Pharmaceutical Technology
Academic year 2011-2012
WATER PERMEABILITY OF PLGA AND PHB FILMS ENRICHED WITH ADDITIVES
Celine VAN VOOREN
First Master of Drug Development
Chalmers supervisors: Dr. Anna Bergstrand, Prof. Dr. Anette Larsson
UGent supervisor: Prof. Dr. Kevin Braeckmans
Commissioners: Dr. S. Soenen, Dr. B. De Geest
GHENT UNIVERSITY
Master thesis performed at:
FACULTY
OF
PHARMACEUTICAL
CHALMERS UNIVERSITY OF TECHNOLOGY
SCIENCES
GOTHENBURG
Department of Pharmaceutics
Department of Chemical and
Laboratory of General Biochemistry &
Biological Engineering
Physical Pharmacy
Pharmaceutical Technology
Academic year 2011-2012
WATER PERMEABILITY OF PLGA AND PHB FILMS ENRICHED WITH ADDITIVES
Celine VAN VOOREN
First Master of Drug Development
Chalmers supervisors: Dr. Anna Bergstrand, Prof. Dr. Anette Larsson
UGent supervisor: Prof. Dr. Kevin Braeckmans
Commissioners: Dr. S. Soenen, Dr. B. De Geest
COPYRIGHT
“The author and the promoters give the authorization to consult and to copy parts of this thesis
for personal use only. Any other use is limited by the laws of copyright, especially concerning
the obligation to refer to the source whenever results from this thesis are cited”.
June ..., 2012
Promotor
Author
Prof. Dr. Kevin Braeckmans
Celine Van Vooren
ACKNOWLEDGEMENT
First of all I would like to thank my supervisor Dr. Anna Bergstrand for the great guidance. Thank
you for all the advice and bringing ideas and discussions. Also your enthusiasm and positive
mood was a great support for me.
Thank you Prof. Dr. Anette Larsson for also being a great supervisor Thank you for sharing your
enthusiasm and ideas.
I also want to thank my supervisor from my home university, Prof. Dr. Kevin Braeckmans, for his
support and interest and for proposing an Erasmus programme at the Chalmers University of
Technology.
Many thanks to all the people from floor 8 of the department of chemical and biological
engineering in Chalmers for creating such a nice environment to work in.
Finally I would like to thank my family and especially my parents for supporting me in this
opportunity.
i
ABSTRACT
Biodegradable polymers are widely used in drug delivery systems which extend the release and
control the bioavailability of drugs administered to the body. Poly(D,L–lactide–co-glycolide)
(PLGA) and poly(hydroxybutyrate) (PHB) are both polymers which have been frequently used for
this purpose because of their biocompatibility, biodegradability and bioresorbability. The
production of high performance biodegradable composite materials with a favourable release
and erosion profile can be achieved by blending these polymers with additives. The aim of this
thesis was to formulate PLGA and PHB films with additives, characterize the water permeability
and correlate this to the film composition and microstructure. PLGA and PHB films, both with
included cellulose, and PHB films with poly(acrylic acid) (PAA) nanogels were prepared
according to a solvent casting method. The water permeability was studied using diffusion cells
and tritiated water. The microstructure of the PHB films was investigated through SEM analysis.
It was found that inclusion of higher amounts nanocrytalline cellulose (NCC) in PLGA films
promotes the water permeability. An increase in permeability was also observed with a higher
percentage of PAA nanogels in PHB films. This was in line with the results from the SEM analysis
which showed a more porous structure for higher concentrations PAA included. The
permeability of PHB films enriched with NCC decreased with higher amount of NCC, but this
trend may be attributed to the emulsifiers, which were included.
ii
SAMENVATTING
Biodegradeerbare polymeren worden vaak aangewend in systemen voor gecontroleerde
vrijgave. Deze systemen worden toegepast om de vrijstelling van de toegediende
geneesmiddelen te verlengen en de biologische beschikbaarheid te controleren. Poly(D,Llactide-co-glycolide) (PLGA) and polyhydroxybutyraat (PHB) worden vaak voor dit doel gebruikt
daar ze biocompatibel, biodegradeerbaar en bioresorbeerbaar zijn. Via toevoeging van
additieven aan deze polymeren kunnen materialen bekomen worden met een gunstig
vrijstellings- en erosieprofiel. Het doel van deze thesis was PLGA en PHB films met additieven
formuleren, de water permeabiliteit karakteriseren en dit in relatie brengen met de
filmcompositie en microstructuur. PLGA en PHB films met cellulose en PHB films met
polyacrylzuur nanogels werden aangemaakt via een solvent casting methode. De
waterpermeabiliteit werd bestudeerd door gebruik te maken van diffusiecellen en tritiumwater.
De microstructuur van de PHB films werd onderzocht via scanning elektron microscopie (SEM).
Uit de diffusie-experimenten kon afgeleid worden dat inclusie van grotere hoeveelheden
nanocrystallijne cellulose (NCC) in PLGA films de water permeabiliteit bevordert. Een toename
in permeabiliteit werd eveneens vastgesteld voor een stijgende concentratie van PAA nanogels
in PHB films. Dit kwam overeen met de resultaten verkregen via SEM, waarbij een meer poreuze
structuur merkbaar was voor hogere concentratie toegevoegd PAA. De permeabiliteit van PHB
films met NCC daalde met een hoger percentage NCC, maar deze trend is waarschijnlijk toe te
schrijven aan de emulgators toegevoegd aan de film.
iii
TABLE OF CONTENT
ACKNOWLEDGEMENT ....................................................................................................................... i
ABSTRACT ......................................................................................................................................... ii
SAMENVATTING .............................................................................................................................. iii
ACRONYMS ...................................................................................................................................... vi
1
INTRODUCTION ......................................................................................................................... 1
1.1 Biodegradable polymers for implant applications ........................................................................... 1
1.2 PLGA and PHB for implant applications ........................................................................................... 2
1.3 PLGA ................................................................................................................................................. 2
1.4 PHB ................................................................................................................................................... 3
1.5 Biodegradable polymers and additives ............................................................................................ 4
1.6 Microcrystalline cellulose and nanocrystalline cellulose ................................................................. 4
1.7 Poly(acrylic acid) nanogels ............................................................................................................... 5
1.8 Film casting ....................................................................................................................................... 6
1.9 Scanning electron microscopy.......................................................................................................... 6
1.10
Film permeability analysis ............................................................................................................. 6
2
OBJECTIVE ............................................................................................................................... 11
3
MATERIALS AND METHODS .................................................................................................... 12
3.1 Materials......................................................................................................................................... 12
3.2 Methods ......................................................................................................................................... 12
4
3.2.1
Film preparation .................................................................................................................. 12
3.2.2
Film characterisation ........................................................................................................... 15
3.2.3
Permeability analysis ........................................................................................................... 15
RESULTS AND DISCUSSION ..................................................................................................... 16
4.1 Formulation and casting ................................................................................................................. 16
4.1.1
PLGA enriched with NCC ..................................................................................................... 16
4.1.2
PHB enriched with NCC ....................................................................................................... 19
4.1.3
PHB enriched with PAA ....................................................................................................... 21
4.2 Morphology .................................................................................................................................... 23
4.2.1
PHB enriched with NCC ....................................................................................................... 23
4.2.2
PHB enriched with PAA ....................................................................................................... 23
4.3 Water permeability ........................................................................................................................ 26
4.3.1
PLGA enriched with NCC ..................................................................................................... 26
4.3.2
PHB enriched with NCC ....................................................................................................... 27
4.3.3
Comparison of permeability of NCC enriched films ............................................................ 29
4.3.4
PHB enriched with PAA ....................................................................................................... 30
5
CONCLUSIONS ......................................................................................................................... 33
6
FUTURE WORK ........................................................................................................................ 34
7
REFERENCES ............................................................................................................................ 35
8
APPENDIX ................................................................................................................................ 37
8.1 Materials......................................................................................................................................... 37
ACRONYMS
DCM: dichloromethane
EMIMac: 1-ethyl-3-imidazoliumacetate
LSC: liquid scintillation counting
MCC: microcrystalline cellulose
NCC: nanocrystalline cellulose
PAA: poly(acrylic acid)
PFA: perfluoroalkoxy
PHB: poly(hydroxybutyrate)
PLGA: poly(D,L-lactide-co-glycolide)
SEM: scanning electron microscope
Span: sorbitan monooleate
Tween: polyoxyethylene - sorbitan monooleate
vi
1
INTRODUCTION
1.1 BIODEGRADABLE POLYMERS FOR IMPLANT APPLICATIONS
Polymer materials and polymer film coatings are generally used for drug delivery systems. These
controlled release formulations are developed to extend the release and control the
bioavailability of drugs administered to the body. Many types of drug delivery devices exist:
microspheres, implantable rods, fibers, films, tablets, pellets, beads, nanoparticles, and others.
The requirements for the polymers depend on the application. Knowledge about material
properties is essential because of the influence on the biological result, biocompatibility and
toxicity of the device (2-4).
Parenteral depot systems for sustained release can be divided into degradable and nondegradable systems. Non-degradable systems require retrieval of the implant from the body
whereas degradable systems are converted into biologically compatible substances which
exclude a surgical procedure to eliminate the implant. It is of great importance that the
polymers as well as its degradation products are biocompatible and non-toxic. Degradable
systems are preferred for temporary applications because the retrieval of the implant has some
undesirable implications such as higher cost, increased infection risk and a lower patient
compliance (3).
A large range of natural and synthetic biodegradable polymers has already been developed and
tested such as: polyesters, poly( -caprolactones), poly(hydroxybutyrate), poly(phosphoesters),
polycarbonates, poly(amides), polyphosphazenes, poly(orthoesters) and polyanhydrides.
Comparing different degradable polymers, the way of degradation can be very divers. The most
important mechanisms are erosion and chemical degradation. Remarque that both mechanisms
can occur at the same time during the degradation of a drug delivery system. If a polymer
device degrades by erosion the chains dissolve away from the device after absorption of water
into the system. Erosion can progress through surface erosion, bulk erosion or both. In the case
of surface erosion the degradation is mainly located at the interface between water and the
polymer device. This means that the degradation and mass loss at the surface is faster than the
1
diffusion of water into the bulk of the material. The opposite happens when the device erodes
through bulk erosion. Then the water diffusion in the polymer device is faster and degradation
and mass loss takes place throughout the bulk of the material. The distinction between surface
and bulk erosion plays an important role in the choice of the right material for a particular
device. In controlled drug delivery, surface erosion is more advantaged because the possibility
of maintaining a stable near zero-order release. Degradation of a polymer resulting from
chemical changes is mostly due to hydrolysis of covalent bonds which results in the formation of
soluble oligomers or monomers. Studying the absorption and transport of water in the polymer
device is of great importance in order to understand and predict the drug release and
degradation process (2, 3, 5).
1.2 PLGA AND PHB FOR IMPLANT APPLICATIONS
PLGA is one of the most frequently used biodegradable polymers in drug delivery systems
because of its biocompatibility, biodegradability, mechanical strength and worldwide approval
for parental use (6-8). It is processed for example in microspheres, microcapsules, nanospheres
or nanofibers to control the delivery of encapsulated or absorbed drugs.
An additional advantage of PLGA is that there are several kinds commercially available which
differ for example in molecular weight or lactide:glycolide ratio. This makes it easy to select the
PLGA with the appropriate physico-chemical properties which is also reflected in the
degradation and drug release profile (7).
Due to the low degradation rate of PHB, this polymer is less favourable for controlled drug
release formulations (5).
Both PLGA and PHB are resorbable polymers and produce acids as the degradation progresses.
Their degradation products are resorbed and excreted and thus not accumulated in the body.
(7).
1.3 PLGA
PLGA or poly(D,L-lactide-co-glycolide) is a copolymer synthesized by ring opening
polymerisation of glycolide and lactide forming ester linkages (see Figure 1.1).
2
Figure 1.1: Structure of PLGA. x= number of units of lactic acid; y = number of units of glycolic acid
Different forms of PLGA can be distinguished depending on the ratio of the monomers. PLGA
50:50 contains 50 % lactic acid and 50 % glycolic acid whereas PLGA 75:25 is composed of 75 %
lactic acid and 25 % glycolic acid. All types of PLGA are more or less amorphous. The
degradation time is related to the ratio of lactide to glycolide units. The higher the content of
glycolide units, the lower the time required for degradation. PLGA 50:50 forms an exception for
it exhibits the fastest degradation. PLGA with less than 50 % glycolic acid is soluble in solvents
such as chlorinated hydrocarbons, tetrahydrofuran and ethyl acetate (2, 9).
Degradation of PLGA is due to bulk hydrolysis of ester bonds whereby the monomers lactic and
glycolic acid are formed. These can be eliminated from the body by metabolic pathways (10).
Bulkerosion of PLGA limits the possibility of significantly modifying the release rate from a drug
delivery system because water diffusion in, drug dissolution and diffusion out are not controlled
by the polymer degradation rate (5).
1.4 PHB
Poly(hydroxybutyrate) (PHB) is the most common polyhydroxyalkanoate (PHA) (see Figure 1.2).
Figure 1.2 Structure of PHB
This linear polyester is synthesized and catabolised by numerous organisms and therefore nonsurprising also biodegradable. PHB accumulates in several bacteria where it serves as an
intracellular reserve carbon and energy source and degrades in case of external carbon
3
shortage. In the trend of its biodegradability, this polymer is also biocompatible since the
breakdown product is 3-hydroxybutyric acid which is naturally occurring in the human body. For
those reasons this polymer is suitable for medical applications (11).
PHB has a narrow polydispersity and a high degree of crystallinity which is situated around 50%.
This high degree of crystallinity makes the polymer very brittle. PHB has a Tg around 5°C and the
melting temperature depends on the composition and has a value from 160°C to 180°C (2, 5).
Further, PHB is water insoluble, but soluble in chloroform and other chlorinated hydrocarbons
such as dichloromethane. Due to the chiral carbon atom, present in each monomer, PHB is an
optically active polymer (12).
1.5 BIODEGRADABLE POLYMERS AND ADDITIVES
Blending or co-polymerising PLGA or PHB with additives extends the possibility to obtain a
favourable drug release profile (7).
1.6 MICROCRYSTALLINE CELLULOSE AND NANOCRYSTALLINE CELLULOSE
Native cellulose is a polysaccharide consisting of repeating
(1→4)-linked D-glucopyranose
units. Intra- and inter-chain hydrogen bonding explains the occurrence of cellulose as
microfibrils in plants. Native cellulose has both amorphous and crystalline regions. This natural
occurring cellulosic fibre is both biodegradable and biocompatible (13-15). By using a strong
acid and mechanical force native cellulose can be converted into micro- or nanocrystalline
cellulose particles which are feasible to prepare high performance composite materials (13).
Microcrystalline cellulose (MCC) is a type of cellulose obtained by acid hydrolysis of high quality
wood pulp in order to break up and eliminate the amorphous regions. This commercially
available form of cellulose exists as a fine, white and odourless powder (16).
Nanocrystalline cellulose (NCC) is mostly obtained by acid hydrolysis using sulphuric acid,
hydrochloric acid or nitric acid (14). The hydrolysis with sulphuric acid leads to esterification of
the hydroxyl groups by sulphate ions. This provides the nanocrystals of a negatively charged
surface which has a stabilising effect in aqueous solutions. NCC nanocrystals have been
4
generated by a process starting from 5 g/100 mL MCC (particle size between 10 and 15 µm) (see
Figure 1.3), using 64.8 % (w/w) sulphuric acid, with a hydrolysis time of 120 minutes, a
temperature of 40°C and a ultrasonic treatment time of 30 minutes.
Figure 1.3: Transmission electron microscope image of
cellulose nanowhiskers obtained by sulphuric acid
hydrolysis; (1)
Figure 1.4 : Scanning electron micrograph at 5.0 kV
accelerating voltage showing MCC particles before acid
hydrolysis (Hitachi S-4300); (1)
These nanocrystals, also called nanowhiskers, have a length of 200 nm and a width of 10 nm
(see Figure 1.4) (1). Due to its nanoscale dimension and intrinsic physicochemical properties,
NCC is favored over cellulose fibres for its incorporation into polymer matrices (15).
1.7 POLY(ACRYLIC ACID) NANOGELS
Poly(acrylic acid) or PAA is a polymer consisting of acrylic acid (AA) monomer units (Figure 1.5).
AA is an important compound of a group pharmaceutical excipients known as Carbomers.
Figure 1.5 Molecular structure of PAA
PAA is a polyelectrolyte which is widely used in hydrogel applications, because acrylic acid is
cheap and easy to polymerize and PAA hydrogels absorb many times their own weight of water
and are able to swell in an aqueous environment. These hydrogels are formed when PAA is
loosely crosslinked to form a three-dimensional network (17-19). The nanometer-sized PAA
5
hydrogels are prepared through a reverse microemulsion polymerization technique based on
the method described in (20), neutralisation is performed with lithium hydroxide instead of
sodium hydroxide. The particles have a size of 50 to 60 nm as determined by dynamic light
scattering. The swelling of PAA is among others influenced by the type of ions in the medium
and decreases in the presence of divalent ions if comparing with monovalent ions. This is an
important implication since polyvalent ions are significantly present in body fluids (21, 22).
In superabsorbent applications, for example highly absorbing medical devices, the PAA has
mostly been partly neutralized with sodium ions. Hereby the acidic protons of the carboxylic
groups present in the monomers have been exchanged with sodium. This causes an increase in
osmotic pressure ,and thus swelling capacity of the gels (23). The PAA can also be neutralized
with lithium ions for example Lithium ions has been used typically for bipolar disorders. An
interesting side effect of ionic lithium, the stimulation of bone formation, initiated the search
for ways to administer lithium ionic locally (24). This could be established by incorporating
lithium ions in a polymer such as PAA for example in order to control the release rate.
1.8 FILM CASTING
One method to obtain polymer film is by solvent casting. Hereby a polymer solution in a solvent
is prepared and poured out on the casting material. An important fact to consider is whether
the casting vial, dish or mould is compatible with the solvent used. Also the drying conditions
need to be optimal in order to obtain a flat, homogenous film. In the end evaporation of the
organic solvent leaves a solid polymer network on the casting surface (25).
1.9 SCANNING ELECTRON MICROSCOPY
Scanning electron microscopy, SEM, can be used for the structural characterization of polymer
films. SEM also allows the examination of internal porous structures by focusing on the crosssections of the films (26).
1.10 FILM PERMEABILITY ANALYSIS
Small molecules or permeants diffuse through a polymer film when they are able to pass
through voids and other gaps between the polymer chains. The diffusion rate of a permeant
6
through a polymer film will be strongly influenced by the diffusion routes available in the
polymer matrix. The size of the gaps and the properties of the permeant itself determine if the
permeant is able to pass through a certain route or not. In this, the physical state of the polymer
plays an important role. Amorphous polymers can be in different states depending on the glass
transition temperature (Tg). Above the Tg, they are in the rubbery state and characterized by a
high mobility. Due to this mobility free volume is present in the mass of the polymer and
polymer fragments are also able to create space for the permeant to diffuse. Thus, permeants
will have a high diffusion rate in rubbery polymers. Below the Tg, in the glassy state, amorphous
polymers are less flexible and therefore the diffusion path will be more hindered. Crystalline
polymers show the highest resistance against diffusion because they are characterized by a
dense molecular packaging almost impossible for the permeants to pass. Diffusion is only
possible through the amorphous regions or structural imperfections (27).
A mathematical model to describe diffusion through films is based on “Van den Mooter” and
can be used to derive the permeability coefficient. The rate of diffusion is expressed by the first
law of Fick
(1)
where dM/dt is the rate of diffusion, D the diffusion coefficient, S the surface through which
diffusion takes place and dC/dx the concentration gradient. Assuming there is a linear fall of
concentration within the film, the concentration gradient can be written as
(2)
7
where C0 and Ch represent the surface concentration of the membrane at x = 0 and x = h,
respectively and h stands for the thickness of the film. Including equitation (2) in (1) gives the
following.
(3)
The partition coefficient K of the permeant can be expressed as
(4)
where Cd and Ca represent the respective solute concentrations in the donor and acceptor
compartment at a finite time. Since the amount of diffusing substance in the membrane is
negligibly small compared to that in the donor and acceptor compartment Cd and Ca can be
written as
(5)
and
(6)
where Md and Ma are the respective amounts of permeant in the donor and the acceptor when
t = 0, M is the mass change and Vd and Va represent the volumes of the two compartment. After
replacing C0 and Ch in equation (3), integration and rearrangement of the formula, the following
equitation is obtained.
8
(7)
where V is volume in the donor compartment, which is the same as the volume in the acceptor
compartment, t represents a finite time and Cd,0 denotes the solute concentration in the donor
compartment at t = 0.
Since the permeability coefficient is defined as (DK)/h = P, equation (7) becomes
(8)
–ln[(Cd,0-2Ca)/Cd,0] represents the diffused volume. When plotting the diffused volume versus
time, the permeability can be calculated from the slope (28).
Figure 1.6 Shows an example of such a plot.
1,00E-03
Diffused Volume (mL)
8,00E-04
6,00E-04
4,00E-04
2,00E-04
0,00E+00
0
100
200
300
400
500
Time (min)
Figure 1.6: Example of a plot showing the water diffusion (mL) through a PLGA film with 5 % (w/w) NCC versus time (min)
9
A diffusion chamber and tritium labeled water can be used to evaluate the water permeability
of a polymer film (see Figure 1.7).
Figure 1.7 Set up diffusion cells: 1. T20: tritiated water was added to the donor chamber; 2. Samples were taken from the
receiver chamber at predetermined times.
Tritium labeled water can be applied as a permeant and tritium (3H) is a very weak pure emitting radioisotope of hydrogen with a half life of 12 years. With liquid scintillation counting
the activity is measured in the samples. This radiometric technique is commonly used to
quantify the activity of 3H radionuclides (29, 30).
Before examination in the liquid scintillation counter, samples need to be dissolved in a
counting cocktail, such as Ultima GoldTM. This cocktail allows transformation of the emitted
energy into light and contains therefore an aromatic organic solvent and a scintillator. The
radioactive decay of tritium is accompanied by release of energy which causes excitation of the
aromatic solvent molecules. Then the energy is transferred to the scintillator of which the
electrons are excited. Decay of these electrons to the ground state generates a light pulse which
is ultimately detected by the photomultiplier tube (32).
10
2
OBJECTIVE
The vision is to design and produce high performance biodegradable composite materials with a
release and erosion profile that is suitable for controlled drug release systems.
The objective of this work is to formulate PLGA and PHB composite materials, characterize the
water permeability and correlate this to the film composition and microstructure in order to
better understand and predict the drug release and degradation process of such materials.
11
3
MATERIALS AND METHODS
3.1 MATERIALS
Poly-[(R)-3-hydroxybutyric acid], PHB; poly(D,L-lactide-co-glycolide) 50:50, PLGA 50:50, Mw
24000–38000; 1-ethyl-3-methylimidazolium acetate, EMIMac produced by BASF, ≥90%; acetone
and dichloromethane, DCM, grade puriss. p.a. ACS reagent ≥ 99,9% (GC) are purchased from
Sigma Aldrich, Germany. Tween 20 and span 80, sorbitan monooleate are purchased from
Sigma Aldrich, USA. Chloroform puriss p.a. is purchased from Sigma Aldrich, France. Tween 80,
polysorbate 80 is purchased from Sigma Aldrich, Switzerland. Poly(D,L-lactide-co-glycolide),
PLGA 50:50, RESOMER RG 504 H, Mw 45000 and poly(D,L-lactide-co-glycolide) 75:25, PLGA
75:25, RESOMER SAMPLE CR, Mw 45000 are purchased from Boehringer Ingelheim, Germany.
Microcrystalline cellulose, Avicel PH-101 NF is purchased from FMC BioPolymer, USA. Ultima
gold , high flash-point LSC-cocktail for counting aqueous and non-aqueous samples is used as
scintillation fluid and purchased from Perkin Elmer, USA. [3H]-water is also purchased from
Perkin Elmer, USA. Detailed information of the chemicals is included in 8.1.
3.2 METHODS
3.2.1 Film preparation
3.2.1.1 Selection of casting materials for PLGA films
350 mg PLGA is dissolved in 7 mL dichloromethane (DCM). The solution is poured out on glass
and telfon
1
dishes and left for drying in the fumehood covered by a filter paper to avoid dust
from the air ending up on the film. Films are also casted in PFA dishes, a material similar to
teflon. 450 mg of PLGA is dissolved in 4 mL organic solvent (DCM, chloroform) and casted on the
dishes. 20 % (w/w) Tween 80 is included in three films to evaluate the effect on the brittleness.
The PFA dishes are left for drying in the fumehood for 18 hours and then put in an oven for 60
hours at 35 °C.
1
Teflon is the trademark of polytetrafluorethylene (PTFE), which is a synthetic fluoropolymer
12
3.2.1.2 Casting PLGA films with solubilised microcrystalline cellulose
A 5 % (w/w) solution of MCC in 1-ethyl-3-imidazoliumacetate (EmimAc) is prepared. The MCC
powder is added to the Emimac and the mixture placed on a heating plate to accelerate the
dissolution process. The MCC-solution is added drop wise by a syringe to DCM in a beaker. A
homogeniser (DI18, IKA®, Brazil) is used to assure a fine dispersion of the droplets in DCM. 300
mg PLGA is added and the solution is stirred for a while during evaporation of the organic
solvent. When 40 mL of the solution remains in the beaker, it is poured out on a glass petri dish.
The dish is moved slowly while resting on the heating plate in order to avoid aggregation of the
cellulose. As soon as the content of the petri dish becomes slightly firm, the dish is removed
from heating and left at room temperature covered with a filter paper. The whole procedure of
film preparation, including drying of the films, is carried out in the fume hood.
3.2.1.3 Casting PLGA films with nanocrystalline cellulose
A nanocrystalline cellulose suspension in water with a concentration of 0,006 % (w/w) is a kind
gift by Anders Johnsson and prepared according to the procedure described in section 1.6. The
solvent is stepwise changed from water to DCM by switching from water to aceton and from
aceton to DCM according to the following. The nanocrystalline cellulose suspension is filled in
falcon tubes and subjected to centrifugation (Sigma Laboratory Centrifuges 4K15, UK) during 5
minutes at 5000 rpm and 20 °C. Aceton is added to the falcontubes to assure all NCC particles
sink to the bottom of the tube during centrifugation. The volume of suspension needed is
dependent on the desired percentage NCC in the final film, calculated as the weight of NCC on
the weight of both polymer and NCC. After centrifugation the supernatant is removed and the
NCC pellet resuspended in aceton, followed by centrifugation and removal of the solvent. The
falcon tube is vortexed well for approximately 30 seconds before each centrifugation step. The
washing step with aceton is repeated for 3 times. After the last washing step with aceton, the
solvent is changed to DCM and the falcon tube centrifuged again. The supernatant is removed
until 2 mL content is left in the falcon tube. Then the vortexed content is added to an amount of
polymer (450 mg). The falcon tube is rinsed with 2 mL of DCM and this is also added to the
polymer to obtain a total casting volume of 4 mL. The casting solution has been stirred with a
13
magnetic bar for 15 minutes, before it is poured out on a PFA-dish. The same drying procedure
is applied as for the pure PLGA films casted on PFA.
3.2.1.4 Casting of pure PHB films
All PHB films are casted on glass petri dishes. 560 milligram PHB is dissolved in 8 mL chloroform
in a bottle with cap and magnetic stirrer. The bottle is placed on a water bath during 30 minutes
at 58 °C and a thermometer is used to control the temperature. The bottle is cooled down at
room temperature before pouring the solution on a petri dish. Initially the films are left to dry
for 2 minutes without the lid and afterwards the lid is placed on the dish to avoid quick drying.
Tape on the border of the dish assures that it is not too tightly closed.
3.2.1.5 Casting of PHB films with nanocrystalline cellulose
PHB-films with cellulose are made in the same way as pure PHB-films, but after cooling NCC and
emulgator (Span 80 and Tween 20) is added. The same NCC suspension as for the PLGA films is
used for inclusion in PHB. The solvent is changed in an identical way from water to chloroform,
but with an extra washing step to switch to chloroform. Span 80 and Tween 20 are tested in
different concentrations.
3.2.1.6 Casting of PHB films with poly(acrylic acid) nanogels
Poly(acrylic acid) nanogels neutralized with lithium ions as mentioned in section 1.7 are a kind
gift from Dr. Mikael Larsson. PAA is available as a suspension in aceton. After resuspension in
chloroform, an appropriate amount of nanogels is added to the cooled PHB solution. The PHB
solution is made in the same way as described under 4.2.1.5. The appropriate amount of
nanogel suspension is dependent on the aimed percentage of nanogels in the final film [5 %
(w/w), 10 % (w/w) and 15 % (w/w)]. This percentage is calculated as the dry weight of nanogels
on the weight of both nanogels and polymer. After the PAA nanogels have been mixed in the
PHB solution with a homogeniser (DI18, IKA®, Brazil) during 15 seconds, the mixture is poured
on a petri dish and left for drying in the fumehood. Two minutes later, the lid is placed on the
dish which has tape on the edges to create some gaps between the dish and the lid.
14
3.2.2 Film characterisation
3.2.2.1 Scanning electron microscopy (SEM)
A scanning electron microscope (Leo Ultra 55 FEG SEM) is used to visualize the morphology of
the surface and cross section of PHB films. The polymer samples are covered with a thin layer of
gold before observation.
3.2.3 Permeability analysis
3.2.3.1 Diffusion chambers
A diffusion chamber is used to evaluate the water permeability of the film samples. Different
film pieces are cut out of one film and the piece of film is placed between a donor and receiver
compartment. 50 mL of Milli-Q water is added to both of the chambers. This happens
simultaneously in order to avoid pressure on the membrane. A rotating table is responsible for
stirring of the contents of the two chambers and the entire diffusion experiment is carried out
at room temperature. 10 µL of tritiated water (400 kBq) is added to the donor content.
At specified times samples are extracted from the medium and weighed. Each time 500 µL is
extracted from the receiver chamber and replaced by an equal quantity of Milli-Q water. At the
beginning of the experiment a sample is taken from the donor chamber as well as from the
receiver chamber. The volume taken from the donor chamber (10µL) is placed in a volumetric
flask to be diluted afterwards. Dilution of this sample is necessary because the tritium activity is
too high for analysis by the scintillation counter.
3.2.3.2 Liquid scintillation counting
A Liquid scintillation analyser (Tri-carb 2810 TR, Perkin Elmer, USA) is used to determine the
tritium activity of the samples extracted from the diffusion chambers. The tritium activity of the
samples taken from the receiver compartment at a specific moment is a measure for the
amount of radioactive labelled water that passes through the membrane at each time. With
these data, the film permeability can be calculated according to equation 8 described in 1.10.
15
4
RESULTS AND DISCUSSION
4.1 FORMULATION AND CASTING
4.1.1 PLGA enriched with NCC
4.1.1.1 Casting material
PLGA films casted on glass petri dishes show high affinity for the dish and are not removable
without deforming them. Removal of these films is achieved after washing them with water for
at least 20 sec., but they behave elastic and are stretched during this operation. The films are all
transparent and easy to bend without cracking (see Figure 4.1). After leaving the films for at
least one week in the desiccator they become more stiff compared to the state when they are
recently removed. The same results are observed for PLGA films casted on the teflon mould.
PLGA films casted on PFA dishes are easy removable without stretching and it is not needed to
immerse these films in water in order to remove them (see Figure 4.2). Unlike the PLGA films
casted on glass petri dishes or teflon mould, the films made in PFA dishes are very fragile and
break immediately when using little force. Casting with chloroform instead of DCM results in
slightly less fragile films. Pure PLGA films are more brittle than films where NCC is included.
Addition of 2 % (w/w) Tween 80 in the films shows no difference. Mostly, films casted on PFAdishes contain a few airbubbels which is never the case when using the other casting surfaces.
Figure 4.1: PLGA 50:50 casted on glass petri dish
Figure 4.2: PLGA 75:25 film with 3 % (w/w) NCC casted on PFA
with DCM used as solvent.
16
Initially glass petri dishes are used to produce PLGA films. This casting material does not meet
the expectations because the films are stretched during removal and therefore not reliable to
generate further experimental data. Teflon is chosen as an alternative casting material. This
choice is mainly based on the fact that teflon is like glass also compatible with the organic
solvent. Since no improvement has been made, this option is also abandoned and at last PFA is
of interest. PFA makes it possible to obtain removable films and is selected to generate films for
further analysis. The disadvantage of these films is that they are quite fragile which makes it
difficult to subject them to diffusion experiments without breaking. Optimisation of the
formulation is needed to reduce the brittleness. Another attempt to improve the formulation is
made with Tween 80. In the literature emulsifiers are often used in film preparation, though
the purpose is not always clearly stated. A try-out with inclusion of 2 % (w/w) Tween 80 does
not have any significant effect on the mechanical properties and for that reason no further tests
are made with emulsifiers.
4.1.1.2 Inclusion of cellulose
The method to formulate in EMIMac solubilised MCC in PLGA results in insufficient film
formation for every concentration of MCC [5, 15 and 25 % (w/w)]. Small film pieces are formed
surrounded by areas of residual solvent without polymer network. PLGA 50:50 with higher
molecular weight and PLGA 75:25 are more favourable towards film formation than PLGA 50:50.
Table 4.1 summarizes the results of different casting routes.
Table 4.1: Overview results of different casting routes
Method
Type PLGA
# films
Result
Inclusion of MCC
50:50
3
Insufficient film formation
50:50 higher Mw
2
One with no film formation
One with partly film formation, but brittle
75:25
2
One with no film formation
One with partly film formation, but brittle
Inclusion of NCC
50:50
3
Film formation
75:25
25
Film formation
17
The results show that formulating MCC solubilised in EMIMac is an inconvenient route to
include cellulose in PLGA films. This may be due to an incompatibility of DCM with the ionic
solvent EMIMac, resulting in phase separation during film formation. A reason for the more
favourable results obtained with PLGA 50:50 with higher molecular weight and PLGA 75:25
could be that the polymer network formation is more feasible than with PLGA 50:50.
Addition of NCC [3-20 % (w/w)] instead of solubilised MCC makes it possible to form intact films.
There is no difference observed between PLGA 50:50 and PLGA 75:25 concerning film formation
(see Table 4.1).
Emergence of NCC aggregates in PLGA films is influenced by the percentage of NCC included. All
the films containing 10 % (w/w) or 20 % (w/w) NCC are not homogeneous and have aggregates
of NCC particles. These aggregates appear less frequently in films with lower NCC percentage [3,
5 and 7 % (w/w)], but their presence is rather unpredictable and the films are not always
completely homogeneous. Figure 4.3 depicts a PLGA film with 3 % (w/w) NCC in the case where
aggregates have been formed.
Figure 4.3: Removed PLGA 75:25 film with 3 % (w/w) NCC casted on PFA dish
18
Because film formation is possible with NCC, this route is preferred to proceed film production
and PLGA 75:25 is selected as polymer for several reasons. The main reason is that better
network formation could counteract phase separation and aggregation of NCC particles. Next to
that PLGA 75:25 is expected to be beneficial in maintaining fully coverage of the PFA dish. This is
an important consideration since problems have occurred with contraction of the fluid on the
dish leaving empty spaces. Easier network formation with PLGA 75:25 could make the solution
sooner firm with a lower risk of gaps appearing. Increasing casting volume and polymer
concentration and optimizing drying conditions also contribute to maintain coverage of the dish.
Aggregation of NCC particles is never fully excluded and therefore only films with a
concentration up to 7 % NCC are used for diffusion experiments.
4.1.2 PHB enriched with NCC
Unlike PLGA films, PHB films disconnect spontaneously from the glass petri dish during the
drying process. These films are slightly opaque and rigid but not fragile (see Figure 4.4).
Figure 4.4: Pure PHB film without Span 80 and Tween 20
Addition of Span 80 and Tween 20 is necessary to obtain films with NCC included. Formulation
without emulsifiers results in white cracked films (see Figure 4.5 A). The same result is obtained
when 1.5 % (w/w) Span 80 is added. Improvement of the formulation is made by increasing the
concentration of Span 80 (see Figure 4.5 B). Acquisition of an integral film with 3-15 % (w/w)
NCC included is possible with 5.8 % (w/w) Span 80 and 5.8 %(w/w) Tween 20 (see Figure 4.5 C).
19
Figure 4.5: A: PHB with 5 % NCC only; B: PHB with 5 % NCC, 6.7 % (w/w) Span 80 and no Tween 20; C: PHB with 3 % NCC, 5.8
% (w/w)Span 80 and 5.8 % (w/w)Tween 20
Homogeneous PHB films with more than 10 % (w/w) NCC can be made (see Figure 4.6). Big clots
of NCC particles do not occur as seen in a few PLGA films. However several smaller white dots
are present in PHB films with NCC whereas they are absent in pure ones.
Figure 4.6: PHB film with 15 % NCC, Span 80: 5.8
% (w/w), Tween 20: 5.8 % (w/w)
Including NCC in PHB films results in phase separation. To overcome this, Span 80 and
Tween 20 are added. These amphiphilic molecules accumulate at the interface between the
NCC particles and the polymer phase and stabilize the random distribution of these particles in
the surrounding polymer material. The amount and ratio of Span 80 and Tween 20 are crucial
since not every combination results in smooth films.
20
4.1.3 PHB enriched with PAA
Emulsifiers are not needed to incorporate PAA nanogels in PHB films. PHB films with 5 % (w/w)
PAA nanogels and 10 % (w/w) PAA nanogels have the same appearance as pure PHB films
(Figure 4.7 A). The film with 15 % (w/w) PAA is more opaque then the films with lower
concentration (see Figure 4.7 B). An entirely opaque and white film is the result of including 25
% (w/w) PAA (see Figure 4.7 C). Small pieces of these films are submerged in water to see
whether the appearance and the dimensions would change. 7 hours later the pieces are
observed and they show no visible difference. The thickness is also measured after 15 minutes
and 5 hours. Only the films with 15 % (w/w) PAA and 25 % (w/w) PAA show an increase in
thickness after 15 minutes of approximately 8 % and 6 %, respectively. After 5 hours, the
thickness of the films with 5 % (w/w) and 10 % (w/w) has not changed. If the film with 15 %
(w/w) PAA is considered, the increase in thickness is higher comparing to the increase after 15
minutes and amounts 17 %. In case of the film with 25 % (w/w) PAA it has to be remarked that
after 5 hours the increase in thickness is lower than after 15 minutes and amounts 2 %.
Figure 4.7: PHB films with PAA; A: 5 % (w/w) PAA, B: 15 % (w/w) PAA, C: 25 % (w/w) PAA
The PHB films are more opaque with an increasing concentration of PAA nanogels. The
ascendant whitish appearance could be due to the presence of micro domains of PAA
nanoparticles which scatter the light significantly.
21
A higher degree of swelling after submersion in water is anticipated with increasing
concentration of PAA because PAA has a tremendous swelling capacity. However, the
surrounding polymer network could hinder the nanogels to increase in volume. The films with 5
and 10 % (w/w) PAA are quite transparent and show little light scattering which suggests that
the incorporated nanoparticles are finely dispersed throughout the film. However due to the
low amount they are probably not interconnected hence forming separate islands or particles.
This makes it more difficult for water to penetrate in the hydrophobic polymer network, which
explains the lack of swelling. In the more opaque film with 15 % PAA, channels of PAA particles
could have been started to form. In this way the water is allowed to penetrate through the PAA
particles causing them to swell in the surrounding PHB polymer. Because the swelling in the
filmpiece with 25 % (w/w) PAA decreases after 5 hours, the hypotheses is raised that PAA
nanogels are leaching out from the polymer network. An observation which could confirm this
idea is depicted in Figure 4.8. After freeze-drying the film piece placed in the surrounding
medium, a kind of net structure appears above. This expanded network could have been formed
from leached out PAA.
Figure 4.8: film piece PHB with 25 % (w/w) PAA
after freeze-drying
22
4.2 MORPHOLOGY
4.2.1 PHB enriched with NCC
PHB films without NCC have a very dense structure and there are no pores or channels visible
(data not shown). The same is observed for PHB films enriched with 7 % (w/w) NCC (see Figure
4.9) and 15 % (w/w) NCC (data not shown). These films are also homogenous and no aggregates
or particles are remarkable.
Figure 4.9: SEM Image of cross section PHB film
with 7 % (w/w) NCC; magnification: 500x
4.2.2 PHB enriched with PAA
Before exposure to water, PHB films with 5 %, 10 % and 15 % (w/w) PAA are inspected by SEM
(see Figure 4.10). The cross section of these films displays a fairly dense structure and slightly
more pores with increasing concentration of PAA.
Figure 4.10: SEM images of PHB films with PAA before submersion in water, magnification 500x; A: 5 % (w/w) PAA; B: 10 %
(w/w) PAA; 15 % (w/w) PAA
23
After submersion in water for about 7 hours and freeze-drying of PHB films with 5, 15 and 25 %
(w/w) PAA, SEM images are generated from the films (see Figure 4.11).
Figure 4.11: SEM images of PHB films with PAA after submersion in water, magnification 500x; A: 5 % (w/w) PAA; B: 15 %
(w/w) PAA; 25 % (w/w) PAA
When comparing these cross sections with the ones obtained before submersion in water, a less
dense structure is observed. This is best illustrated when taking the film with 15 % (w/w) PAA as
an example. Before submersion, a dense structure with virtually no pores is observed. After
submersion a few pores are visible and most of them have a diameter of approximately 3 µm.
The difference is less obvious when looking at the 5 % (w/w) PAA film. A few less dense areas
are remarkable after submersion, but the cross section shows largely the same dense structure
as before. When focussing on the percentage PAA incorporated in the film, an increase in pores
is visible with higher concentration. These pores are strongly present in the film with 25 %
(w/w) PAA.
Figure 4.12 depicts the porous structure of this film with a higher magnification. A lot of pores
with a wide range in size are noticeable and most of the pores have a diameter between 1 µm
and 10 µm. The figure also indicates the presence of channels due to the pores being connected
to each other.
24
Figure 4.12: SEM image of PHB film with 25 % (w/w) PAA; magnification: 3000x
After submersion in water, the porosity of the films increases as mainly illustrated by the PHBfilm with 15 % (w/w) PAA. Probably the swelling of the nanogels is responsible for emergence of
larger pores. An increase in porosity with higher concentration of PAA nanogels included, is also
clearly present. When observing the SEM image of the PHB film with 15 % (w/w) PAA after
submersion, some larger separate pores are present but they do not seem to form large
channels. On the other hand, the PHB film with 25 % (w/w) PAA has also larger pores but to an
extent where a percolating network has been formed. This becomes obvious by the SEM image
with higher magnification, which indicates a tridimensional porous network.
25
4.3 WATER PERMEABILITY
4.3.1 PLGA enriched with NCC
PLGA films included in the permeability analysis have all been casted with DCM on a PFA dish.
The permeability is determined for a pure PLGA 75:25 film and PLGA 75:25 films with 3, 5 and 7
% (w/w) NCC. Figure 4.13 shows a plot of the permeability values with increasing amount of
included NCC. Each datapoint represents the mean value of different film pieces derived from
one film, except for the pure film. In this case only one permeability value is generated due to
breakage of the other film pieces in the diffusion cell. The mean of the 3 % (w/w) and 5 % (w/w)
NCC film is calculated from 2 values and the 7 % (w/w) NCC film takes 3 values into account. All
error bars are based on the minimum and maximum value. The permeability for the films with
the lowest percentage NCC is similar, but the film with 7 % (w/w) NCC has a significantly higher
value which is 2 magnitudes higher than the others. This indicates that the permeability of NCC
enriched PLGA films increases with higher concentrations of NCC included.
1,60E-11
1,40E-11
1,25E-11
Permeability (m²/s)
1,20E-11
1,00E-11
8,00E-12
6,00E-12
4,00E-12
2,00E-12
8,49E-13
0
1
7,70E-13
5,48E-13
0,00E+00
2
3
4
NCC (%)
5
6
7
8
Figure 4.13: Water permeability of PLGA films with NCC with increasing percentage NCC included in the film
26
The high permeability of the 7 % (w/w) NCC film clarifies that the presence of NCC particles in
the film does not hinder the transport of water. The question now is if NCC makes the PLGA
network less dense and results in a higher chain mobility and free space for the water to pass.
Another possibility for higher permeability is that diffusion via the NCC particles is more
advantaged than diffusion via the PLGA because the NCC is expected to be more hydrophilic
than PLGA. Presumably, when diffusing through the film, the water does not take an alternative
route around the NCC particles slowing down its diffusion. If this is true, an even higher
permeability would be observed if NCC particles were connected forming a route of faster water
diffusion. However, this is highly unlikely in the analyzed films because the concentration of NCC
was too low in each case to form a connected structure. More information about this aspect
could be gained by permeability experiments on films with higher concentration of NCC.
4.3.2 PHB enriched with NCC
The water permeability is investigated for PHB films with 0, 3, 5, 7 and 15 % (w/w) NCC
included. The plot of the permeability versus the concentration NCC is given in Figure 4.14. In
this plot the average permeability of 3 to 5 film pieces from the same film is presented together
with a standard deviation error bar for each percentage NCC. All the permeability values have
about the same magnitude. A significant higher permeability is observed for the pure film with
emulsifiers than for the one without. No significant difference is noticed between the
permeability for the PHB films with 3, 5 and 7 % (w/w). The permeability of these films is lower
than the permeability of the PHB film with emulsifiers only. An even lower value is observed for
the film with the highest amount of NCC.
27
1,40E-12
1,20E-12
1,10E-12
Permeability (m²/s)
1,00E-12
8,70E-13
8,00E-13
6,63E-13
8,30E-13
6,34E-13
6,00E-13
4,00E-13
4,008E-13
2,00E-13
0,00E+00
0
2
4
6
8
NCC (%)
10
12
14
16
Figure 4.14: Water permeability PHB films with NCC plotted with increasing percentage of NCC included in the film; : with
Span 80: 5.8 % (w/w), Tween 20: 5.8 % (w/w); ●: without Span 80 and Tween 20
For the evaluation of the permeability, the influence of Span 80 and Tween 20 should not be
ignored. The permeability of the pure PHB film for example is considerably higher with
emulsifiers than without. These emulsifiers could have a plasticizing effect on the polymer. A
plasticizer is typically a low molecular weight component that lowers the Tg and melt viscosity of
the polymer. It occupies sites along the polymer chain providing more mobility for the chains
resulting in a softer, more easily deformable mass (31). Due to the higher flexibility of the
polymer, diffusing particles will be less hindered and thus the diffusion rate higher. Next to this,
the surprisingly low permeability of the PHB film with 15 % (w/w) NCC could also be explained
by the presence of emulsifiers. Due to their amphiphilic character they may be located on the
surface of the NCC particles. This could have a double effect. First, the plasticizing effect on the
polymer could be reduced, because depletion of emulsifiers in the polymer matrix. Second, the
emulsifiers could have modified the NCC making it more hydrophobic and creating in this way a
much more tortuous path for the diffusing water. The latter is because the water will avoid the
hydrophobically modified NCC and take an alternative route around the particles which leads
eventually to a lower diffusion rate.
28
4.3.3 Comparison of permeability of NCC enriched films
In order to compare the permeability values of PLGA films with PHB films, they are combined in
the same graph (see Figure 4.15). A log scale is used for the y-axis to distinguish the data points
of the different film types clearly. When considering films with 0 % (w/w), 3 % (w/w) and 7 %
(w/w) NCC, the permeability values of PLGA and PHB are located close to each other but
somewhat higher for PHB with emulsifiers. Until 7 percent NCC the same trend is followed for
both film types. The pure PHB film without emulsifiers has the lowest value of the pure films.
Permeability (m²/s)
1,00E-10
1,00E-11
1,00E-12
1,00E-13
0
2
4
6
8
10
12
14
16
NCC (%)
Figure 4.15: Water permeability of PLGA ( ) and PHB (with emulsifiers: , without emulsifiers:
increasing percentage NCC included in the film
) films with NCC plotted with
Due to its hydrophobic character and high degree of crystallinity, PHB may have a lower water
permeability than PLGA. This is true if pure PHB without emulsifiers is compared to PLGA. The
fact that the permeability of PHB with emulsifiers is higher than expected could be attributed to
the presence of emulsifiers. As also stated under 4.3.3, these could act as plasticizers which
soften the polymer and make it easier for the water to diffuse.
29
4.3.4 PHB enriched with PAA
For each concentration PAA [0, 5, 10, 15 and 25 % (w/w)] one film is made and subjected to a
diffusion experiment. The average permeability is plotted with increasing concentration of PAA
(see Figure 4.16). The error bars are based on the minimum and maximum values, because only
two film pieces are taken into account for the film with 25 % (w/w) PAA. Films with the lowest
concentration PAA [0, 5 and 10 % (w/w)] have a permeability with a magnification around 10-13.
A clear increase of permeability is noticeable for the films with 15 % (w/w) and 25 % (w/w) PAA.
When more than 10 % (w/w) PAA is included, the water permeability of PHB films enriched with
PAA clearly increases with the amount of included PAA.
1,00E-10
8,19E-11
Permeability (m²/s)
8,00E-11
6,00E-11
4,00E-11
2,00E-11
1,55E-11
6,63E-13
0,00E+00
0
5,50E-13
5
3,38E-13
10
15
20
25
30
PAA (%)
Figure 4.16 Water permeability PHB films with PAA plotted against percentage of PAA included in the film
The increase in permeability indicates that the water transport in the film happens preferably
through the PAA nanogels. This could be explained by the fact that PHB is very hydrophobic and
the nanogels are rather hydrophilic. It is likely that the nanogels influence the water transport,
but the water permeating through the films during the diffusion experiment possibly also
30
affects the nanogels. These nanogels have the feature of swelling when they are in contact with
water. The gels in the film will swell if the surrounding polymer matrix allows them to expand.
As described under 4.1.3, swelling is observed for the film with 15 % (w/w) PAA which indicates
that this film has absorbed some water, making it also more permeable. Though the increase in
thickness levels out again for the film piece with 25 % (w/w), an initial swelling is observed
indicating absorption of water. The higher absorption of water in the nanogels and the increase
in permeability with percentage PAA included, is in agreement with the results obtained under
4.2.2. A higher degree of porosity is related to an increase in water permeability. Especially the
film with 25 % (w/w) where a percolating network may have been formed, has a high
permeability value.
The film thickness was not only measured for film pieces submerged in a vial with water but also
for the pieces used in the diffusion cells. At the end of the diffusion experiment, after
approximately 7 hours, the pieces have all increased in thickness except the ones without PAA
nanogels. The average increase in thickness amounts 3 %, 9 %, 53 % and 44 % for the films
pieces with 5 % (w/w), 10 % (w/w), 15 % (w/w) and 25 % (w/w), respectively. The lower values
for the films with 5 % (w/w) and 10 % (w/w) are in line with the observations of the film pieces
submerged in a vial where little or no swelling was found. Remarkable here is that the film
pieces with 15 and 25 % (w/w) show a substantial final swelling of 53 % and 44 % in contrast to
a swelling of 17 % and 2 % for the corresponding film pieces submerged in water, as described
under 4.1.3. A reason for this may be that the PAA does not leach out from the film pieces when
mounted in the diffusion cells while the opposite is true if they are fully submersed in a vial with
water. In the diffusion cells water is not in direct contact with cross sections of the film and can
only enter the film through the surfaces facing the donor or receiver chamber. Adversely, the
film piece in the vial is fully exposed and water can also enter through the cross sections. These
observations indicate that leaching may occur less through the surfaces of the film than through
the cross sections. SEM pictures of the films after submersion in water support this idea
because the surface of the films (data not shown) despicts almost no pores while the cross
section displays a lot of pores in case of the films with 15 % (w/w) and 25 % (w/w) PAA. Next to
that, swelling of the film pieces in the diffusion cells has another implication. As seen under
31
1.10, (DK)/h = P, an increased film thickness would lead to a decrease in the observed
permeability if the other parameters were constant. The permeability value as calculated
according to “Van den Mooter” could be an underestimation in the case of swelling and it may
be required to take the swelling into account.
In a former thesis work (“Water diffusion through biodegradable PHB films” by Sanna
Uppström), porous PHB films with lithium sulfate included are created by a water in oil
emulsion. One of the findings is that if more water is included in the emulsion during film
preparation, more pores are developed during film drying. The permeability of these films is
evaluated for an increasing amount of water used during film preparation. Films prepared with
an emulsion containing less than 8 % of water, have a permeability around 10-14-10-13 m2/s. This
low permeability is attributed to a lack of pores. The highest value, 8.7 10-11 m2/s, is measured
for a film prepared with an emulsion containing 10 % water and the same film shows an
interconnected pore structure. If we compare the permeability values of these porous PHB films
with the values of PHB films enriched with PAA, hypothesizes about the structure of the last
ones can be made. The permeability of the PHB films with 0 % (w/w), 5 % (w/w) and 10 % (w/w)
PAA is of the same magnitude as the permeability of the porous PHB films made of an emulsion
with less than 8 % water. This suggests that the PHB films with PAA concentration below 10 %
(w/w) does not possess an interconnected structure. This is in line with the SEM images
observed. The permeability of the PHB film with 25 % (w/w) PAA closely resembles the
permeability of the PHB film prepared with an emulsion containing 10 % water. This points in
the direction of an interconnected network present in the PHB film with 25 % (w/w) PAA. The
SEM image indicates indeed a threedimensional porous network.
32
5
CONCLUSIONS
The permeability of PLGA films increases when a high amount of NCC is included. Thus, inclusion
of NCC in PLGA is an option to increase the water permeability. PFA is suitable as casting
material for PLGA films. NCC should be included instead of solubilised MCC to incorporate
cellulose.
The permeability of PHB films enriched with NCC decreases with higher amount of NCC. This
effect may be due to the presence of emulsifiers. A less plasticizing effect and hydrophobically
modified NCC in the polymer matrix could explain the decrease in permeability.
Addition of nanogels in PHB films gives the possibility to form gelfilled separate pores or
interconnected pores creating a percolating network in the polymer matrix which promotes the
water transport.
33
6
FUTURE WORK
Because only PLGA films with a concentration up to 7 % (w/w) NCC have been investigated,
future research with higher concentrations of NCC is needed. In order to get a reproducible way
of film preparation, time has to be spent to fully optimize the formulation and casting method.
Further experiments are needed for both PLGA and PHB films with NCC included to confirm the
results from the permeability analysis. Besides that, additional SEM to confirm the morphology
and DMA to evaluate the mechanical properties are advisable.
More extensive research on release of lithium ions, degradation, morphology, swelling and
mechanical properties of PHB films with PAA nanogels included could deliver interesting
information. The lithium release should not only be studied in a separate vial but also in the
diffusion chambers to see whether the release is the same in both chambers. Next to that,
swelling in water could be evaluated for different ionic strengths.
34
7
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8
APPENDIX
8.1 MATERIALS
Table 8.1: Materials with Lot number and CAS number.
Chemicals
Lot number
CAS
Manufacturer
Country
number
Acetone for pesticide residue analysis
67-64-1
Microcrystalline cellulose, Avicel PH- P111823018
Sigma-Aldrich
Germany
FMC Bio Polymer
USA
101 NF
Chloroform puriss p.a.
67-66-3
Sigma-Aldrich
France
Dichloromethane, DCM, grade puriss.
75-09-2
Sigma-Aldrich
Germany
1-Ethyl-3-methylimidazolium acetate, STBC 3627V
143314-17-
Sigma Aldrich
Germany
EMIMac produced by BASF, ≥90%
4
Poly(D,L-lactide-co-glycolide)
26780-50-7
Sigma Aldrich
Germany
Boehringer Ingelheim
Germany
Boehringer Ingelheim
Germany
p.a. ACS reagent ≥ 99,9% (GC)
50:50, STBC5398V
PLGA 50:50, Mw 24000–38000
Poly(D,L-lactide-co-glycolide),
PLGA 1046016
50:50, RESOMER RG 504 H, Mw 45000
Poly(D,L-lactide-co-glycolide)
75:25, Res-0340
PLGA 75:25, RESOMER SAMPLE CR, Mw
45000
Poly-[(R)-3-hydroxybutyric acid], PHB
S68924
29435-48-1
Sigma Aldrich
Germany
Tween 20
094K0052
9005-64-5
Sigma Aldrich
USA
Tween 80, polysorbate 80
1070172
Sigma Aldrich
Switzerland
Span 80, sorbitan monooleate
MKBD4327V
Sigma Aldrich
USA
Water [-3H], NET001B005MC
628113
Perkin Elmer
USA
Ultima gold
77-12151
Perkin Elmer
USA
1338-43-8
37