ceramic materials as drug delivery systems
TRANSCRIPT
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 2087
Ceramic Materials as Drug Delivery Systems
AMINA VAZDA
ACTA UNIVERSITATIS
UPSALIENSIS UPPSALA
2021
ISSN 1651-6214 ISBN 978-91-513-1327-6 URN urn:nbn:se:uu:diva-456750
Dissertation presented at Uppsala University to be publicly examined in Polhemsalen, Lägerhyddsvägen 1, Uppsala, Friday, 10 December 2021 at 13:15 for the degree of Doctor of Philosophy. The examination will be conducted in English. Faculty examiner: Professor Santos Hélder (Faculty of Pharmacy, University of Helsinki).
Abstract Vazda, A. 2021. Ceramic Materials as Drug Delivery Systems. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 2087. 51 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-1327-6.
The aim of this thesis is to evaluate ceramic materials for drug delivery drugs applications. Three delivery routes were investigated; abuse-deterrent oral formulation, inhalation, and novel ceramic crystals with drug entrapped.
A geopolymer matrix was developed in order to withstand non-medical abuse such as crushing, snorting, extraction etc. An in vivo study was performed, where oral administration of zolpidem loaded geopolymer pellets was evaluated. The results showed a higher plasma concentration for immediate release formulations compared to the geopolymer, however the geopolymer formulation showed a prolonged release compared to the immediate release control group.
Pulmonary drug delivery offers a fast and non-invasive drug delivery. Releasing fentanyl from calcium sulfate for pulmonary drug delivery were investigated by applying the drug onto the ceramic and heating to 230-250°C for 1-10 minutes. The evaporated drug was collected and quantified which resulted in a total amount of 1.2µg released fentanyl.
Synthesis of ceramic materials containing drugs within the crystal structure has been investigated, both calcium carbonate and hydroxyapatite were tested. Hydroxyurea, a cytotoxic drug, was loaded into the crystal structure of calcium carbonate without altering size, crystallinity or morphology of calcite. Furthermore, human breast cancer cells were exposed to the obtained calcium carbonate crystals. The tested showed that pre-dissolved particles have a toxic effect on the cancer cells. Glycine, an amino acids, was entrapped into nano-sized hydroxyapatite by using a precipitation method where the effects of heat and pH was evaluated. The result showed a total of about 16 µg of glycine could be entrapped per milligram of HAp and that higher pH and higher temperature resulted in higher entrapment of the amino acid.
Keywords: Drug delivery, ceramics, Abuse deterrent, inhalation, single-crystals
Amina Vazda, Department of Materials Science and Engineering, Applied Material Science, Box 534, Uppsala University, SE-751 21 Uppsala, Sweden.
© Amina Vazda 2021
ISSN 1651-6214 ISBN 978-91-513-1327-6 URN urn:nbn:se:uu:diva-456750 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-456750)
List of Papers
This thesis is based on the following papers, which are referred to in the text
by their Roman numerals.
I Vazda, A., Xia, W., Engqvist, H. “Oral administration of
zolpidem tartrate in an abuse-deterrent formulation versus an im-
mediate release formulation in beagle dogs”, Scandinavian Jour-
nal of Material Science, 1 (2021) 1–6.
II Vazda, A., Xia, W., Engqvist, H. “The use of heat to deliver fen-
tanyl via pulmonary drug delivery.”, International Journal of
Pharmaceutics: X, Pharmaceutics, 3 (2021) 100096
III Vazda, A., Xia, W., Engqvist, H. (2021) “Evaporation of fenta-
nyl from ceramics for pulmonary drug delivery: a pilot study.”,
International Conference on Materials Science and Engineering.
(2021) In print.
IV Vazda, A., Xia, W., Engqvist, H. Entrapment of a Cytotoxic
Drug into the Crystal Structure of Calcite for Targeted Drug De-
livery. Under review.
V Vazda, A., Xia, W., Engqvist, H. Entrapment of Glycine Into the
Crystal Structure of Nanocrystalline Hydroxyapatite by a Con-
trolled Precipitation Method. Manuscript.
Reprints were made with permission from the respective publishers.
Author´s Contributions to the listed papers: Paper I Part of planning, experimental work, evaluation, and writing.
Paper II Major part of planning, all of the literature collection, evalua-
tion, and writing.
Paper III Major part of planning, experimental work, evaluation, and
writing.
Paper IV Major part of planning, experimental work, evaluation, and
writing.
Paper V Major part of planning, experimental work, evaluation, and
writing.
Contents
1. Introduction ............................................................................................... 11 1.1 Aim and objectives ............................................................................. 12 1.2 Drug delivery ...................................................................................... 13
1.2.1 Drug release mechanisms ........................................................... 13 1.3 Ceramics as drug delivery systems .................................................... 14
1.3.1 Geopolymer ................................................................................ 14 1.3.2 Calcium Sulfate .......................................................................... 15 1.3.3 Calcium Carbonate ..................................................................... 16 1.3.4 Hydroxyapatite ........................................................................... 16
2. Ceramics for opioid drug delivery ............................................................ 18 2.1 Motivation .......................................................................................... 18 2.2 Methods .............................................................................................. 19
2.2.1 Synthesizing geopolymer ............................................................ 19 2.2.3 In-vivo evaluation ....................................................................... 19
2.3 In –vivo properties of geopolymer in beagle dogs (Paper I) ............. 20
3. Ceramics for pulmonary drug delivery ..................................................... 22 3.1 Motivation .......................................................................................... 22
3.1.1 Lungs and the challenges of pulmonary drug delivery (Paper
II) ......................................................................................................... 22 3.2 Methods .............................................................................................. 24
3.2.1 Synthesis ..................................................................................... 24 3.2.2 Device and heating ..................................................................... 24 3.2.3 Analytical method ....................................................................... 25
3.3 Evaporation of fentanyl from ceramics (Paper III) ........................... 25
4. Ceramic hybrid crystals for drug delivery ................................................ 28 4.1 Motivation .......................................................................................... 28
4.1.1 Entrapment of Hydroxyurea into CaCO3 .................................... 28 4.1.2 Glycine in hydroxyapatite ........................................................... 28
4.2 Methods .............................................................................................. 29 4.2.1 Synthesis ..................................................................................... 29 4.2.2 Characterization .......................................................................... 29 4.2.3 Drug release ................................................................................ 30 4.2.4 Cell viability ............................................................................... 30
4.3 Entrapment of hydroxyurea into calcite (Paper IV) .......................... 31
4.4 Entrapment of glycine into the crystal structure of hydroxyapatite
(Paper V) ................................................................................................. 35
5. Concluding remarks .................................................................................. 39
6. Future outlook ........................................................................................... 41
7. Svensk sammanfattning ............................................................................ 43
8. Abstrakt na bosanskom jeziku .................................................................. 44
9. Acknowledgments ..................................................................................... 45
10. References ............................................................................................... 47
Abbreviations
1-PEP 1-Phenethylpyridinium salt
1-PPO 1-Phenethyl-1H-pyridin-2-one
1-SPO 1-Styryl-1H-pyridin-2-one
AA Amino Acids
BET Brunauer–Emmett–Teller (BET) theory
CaS Calcium sulfate
FDA U.S. Food and Drug administration
HAp Hydroxyapatite
HPLC High-Pressure Liquid Chromatography
LLOQ Lower limit of quantification
MCF-7 Human breast cancer cells. (Michigan cancer Foundation-7)
NRF Norfentanyl
OPA O-phthaldialdehyde
PRP Propionanilide
SEM Scanning electron microscopy
XRD X-ray Diffraction
11
1. Introduction
This thesis aims to evaluate the possibility of using ceramic materials as drug
delivery systems for various delivery routes. At the moment, drug delivery is
mainly based on conventional systems such as tablets, patches and inhalers
[1]. In recent decades, interest has risen in using ceramics as drug delivery
systems [2]. Tunable porosity, mechanical strength, and solvent-free manu-
facturing are some of the various advantageous properties that ceramics pos-
sess, making them an ideal candidate for various drug delivery systems. Three
main ways of drug delivery have been investigated in this thesis: abuse-deter-
rent formulation, inhalation, and ceramic hybrid crystals.
The aim and objectives are presented in the Introduction, where the neces-
sary background to the work presented in the thesis is also provided. The pur-
pose of drug release and different types of ceramics, along with their dissolu-
tion mechanism, are also described.
Three sections on Ceramics for opioid drug delivery, Ceramics for pulmo-
nary drug delivery, and Ceramic hybrid crystals will be presented, each stat-
ing the motivation and experimentation, and summarizing the major findings
and conclusions.
The section on Geopolymer formulation describes the inclusion of opioids
in an abuse-deterrent formulation, making it possible to decrease the risk of
abuse-related accidents. The section also presents methods for both in vitro
and in vivo methods. The main findings are summarized and discussed (Paper
I).
The section on Ceramics for pulmonary drug delivery presents the back-
ground, motivation, and a review of the field (Paper II), on how to use heat
to release drugs from ceramics for pulmonary drug delivery. Subsequently, a
study investigating the possibilities of using ceramics as drug delivery system
for releasing drugs when applying heat is presented (Paper III).
The section on Ceramic hybrid crystals describes a method to entrap a drug
molecule into the crystal structure of calcium carbonate and/or hydroxyap-
atite, and how it can be used for drug delivery. Furthermore, the current field
of hybrid crystals is briefly discussed. The main findings on entrapping drugs
into crystal structures is then summarized (Paper IV-V).
The Concluding remarks and Future outlook sections summarize the find-
ings of the thesis and suggestions for future work. Analytical techniques can
be found at the end of the thesis.
12
1.1 Aim and objectives
The thesis aims to evaluate the possibilities to use ceramics—i.e. geopolymer,
calcium sulfate, calcium carbonate, and hydroxyapatite—as drug delivery sys-
tems for different applications. The thesis is divided into three sections.
The first section covers geopolymers, which are inorganic aluminum sili-
cate-based materials that have been used in designing and developing an
abuse-deterrent formulation with controlled drug release and low abuse liabil-
ity. Previous studies have investigated both synthesis conditions and abuse-
deterrent properties for multiple geopolymer formulations [3–5]. In this thesis,
an in vivo study of beagle dogs is presented. The aim was to show that (1)
geopolymer is suitable for oral administration and (2) show a controlled re-
lease profile in blood plasma.
Calcium sulfates (CaS) or gypsum is an inorganic compound that under-
goes a hydration reaction. It has been extensively investigated for application
as a bone-filler material and transdermal drug delivery system, for both instant
or prolonged drug release [6,7]. In this thesis, an inhalation method is pre-
sented for pulmonary drug delivery, in which CaS acts as a drug carrier from
which the drug is evaporated when heat is applied. The aims for this section
were (1) to investigate the field of pulmonary drug delivery and the various
methods and techniques that are available on the market, and (2) to find a
method for evaporation and collection of evaporated drugs from a ceramic
material.
The final section in this thesis concerns the synthesis of calcium carbonate
and hydroxyapatite for targeted drug delivery. Both materials are extensively
used as biomaterials in healthcare. Recent studies have shown that they are
excellent drug carriers [8–13]. In this thesis, a drug was added during the syn-
thesis of the crystals, allowing the drug to be entrapped into the crystal struc-
ture. The aim was to (1) load the drug into the crystal structure, (2) evaluate
the drug loading efficacy, and (3) investigate in vitro behavior using human
breast cancer cells (MDF-7).
13
1.2 Drug delivery
The definition of drug delivery is to deliver a drug to achieve its therapeutic
effect [14]. When developing a drug formulation, it is crucial to adapt the ef-
fect and drug release to the therapeutic window. The therapeutic window is
the plasma concentration that is suitable for the patient, i.e. use of too low a
concentration will not have a pharmacological effect, and too high a concen-
tration will result in toxic levels and negative side effects. Different admin-
istration routes have different effects in relation to the therapeutic window, as
illustrated in Figure 1. It is important to give the patient the correct dose as a
function of time, e.g. intravenous administration gives a high dose instantly,
while oral administration gives a slower drug release and longer effect. Inha-
lation provides rapid drug delivery, whereas transdermal delivery is slow.
Figure 1. Illustration of plasma concentration with different formulations within the therapeutic window.
1.2.1 Drug release mechanisms
To better understand how and why a drug is released from its matrix, it is
crucial to study the release mechanism behind it. Diffusion, dissolution, swell-
ing and erosion are some of the biological and chemical principles of drug
release mechanisms. These mechanisms can act on their own or simultane-
ously, depending on the application.
The formulation described in Paper I is a low porosity geopolymer matrix
containing a poorly soluble drug. When the geopolymer is subjected to a wa-
ter-based liquid, several mechanisms act: (1) the uptake of water into pores in
the matrix, (2) dissolution of the drug, and (3) diffusion of the drug out of the
matrix and into the surrounding area.
When drug-loaded ceramics are subjected to erosion (dissolution), the ce-
ramic dissolves and the drug is exposed, leading to drug release. Drug diffu-
sion can occur at the same time as erosion. In Papers IV and V the ceramic
14
matrix is exposed to acidic media, leading to dissolution of the ceramic, which
can be described with both erosion and diffusion.
1.3 Ceramics as drug delivery systems
Historically, ceramics have been described as clay-based materials; today,
however, ceramics include a wide range of materials. Inorganic and non-me-
tallic materials are a more suitable name for contemporary ceramics. The wide
range of ceramics also means a wide range of synthesis methods, properties
and applications. Extensive research has been conducted in the field of drug
delivery from different ceramic materials [2,15–19].
The following sections present the four different ceramics used in this thesis:
geopolymer as an abuse-deterrent formulation, calcium sulfate for inhalation
of drugs, and calcium carbonate and hydroxyapatite as drug-loaded ceramics
for targeted drug delivery.
1.3.1 Geopolymer
Geopolymers are inorganic polymers with the basic unit of polysialates, con-
taining SiO4 and AlO4 tetrahedra (Figure 2). Aluminosilicate together with an
aqueous alkali solution forms a geopolymer. The choice of aluminosilicate
dictates the reaction between the aluminosilicate and the alkali solution. If
metakaolin—a commonly used aluminosilicate—is mixed with an alkali so-
lution, the alkali ions and hydroxyl ions will react on the outer surface and
interlayer spaces of the metakaolin. Siloxonate—another type of aluminosili-
cate—only reacts with the outer surface when mixed with an alkali solution.
By adjusting parameters for the synthesis of geopolymer, both chemical and
mechanical properties can be changed. Previous studies have been performed
to find the optimal synthesis route for advantageous abuse-deterrent behavior
in vitro [3–5]. In this thesis, an in vivo study on the safety and efficacy of using
geopolymer as an oral formulation is presented (Paper I).
Figure 2. Basic unit of geopolymer.
15
1.3.2 Calcium Sulfate
Gypsum—a calcium sulfate (CaS) salt—is a natural mineral that is the most
common form of calcium sulfate dihydrate (CaSO4 • 2H2O). Gypsum under-
goes calcination when heated over 110 °C, resulting in calcium sulfate hemi-
hydrate (Eq. 1).
CaSO4 • 2H2O → CaSO4 • ½H2O + 1½ H2O (1)
The obtained hemihydrate exists in two forms—α or β—which have the same
structure but different properties. The main difference between the two forms
is the morphology: alpha consists of rod-shaped crystals, while beta is less
organized, with aggregates and capillary pores (Figure 3). Moreover, beta re-
quires more water and therefore results in a more porous hardened material
than alpha. When mixed with water, calcium sulfate hemihydrate forms a di-
hydrate through an exothermic reaction (Eq. 2).
CaSO4 • ½H2O + 1½ H2O → CaSO4 • 2H2O + Heat (2)
CaS has been widely studied as a bone filler, replacement, and dental material
[6,7,20]. Its tunable porosity, biocompatibility, and high thermal resistance
make CaS an excellent candidate for drug delivery vehicles. In this thesis, CaS
is evaluated as a drug delivery system for the release of fentanyl by heat for
pulmonary drug delivery (Paper II).
Figure 3. Alpha-calcium sulphate (A) and beta-calcium sulphate (B). Reprinted with permission and adapted with modifications [21].
16
1.3.3 Calcium Carbonate
Calcium carbonate (CaCO3) is a versatile inorganic mineral that is used as
excipient in pharmaceuticals [11]. In nature, CaCO3 can be found in rocks, as
well as in eggshells and other animal shells, in which CaCO3 is used for stor-
ing ions and molecules[22]. Various methods, such as precipitation or carbon-
ation, can be used to synthesize CaCO3. Its synthesis can be controlled, mak-
ing it possible to adjust the desirable morphology, size, shape, and functional-
ity. Depending on synthesis parameters, three different polymorphs can be
obtained: calcite, aragonite and vaterite, with calcite being the most thermo-
dynamically stable [23] (Figure 4).
Calcite possesses pH responsiveness, meaning that when exposed to acidic
environments, such as tumors and inflamed tissue, it dissolves into Ca2+ and
CO32- ions[24]. By loading drugs into the crystal structure, calcite can be used
as a targeted drug delivery system with controlled release [8,10]. So far, one
study has demonstrated the encapsulation of an amino acid into the crystal
structure of calcite [25]. In this thesis, we present entrapment of cytotoxic drug
hydroxyurea into the crystal structure of calcite, and an in vitro study on hu-
man breast cancer cells, to study the behavior of the particles in a tumorous
environment (Paper IV).
Figure 4. Different polymorphs of CaCO3.
1.3.4 Hydroxyapatite
Hydroxyapatite (HAp) is a calcium phosphate with the stoichiometric formula
Ca10(PO4)6(OH)2 (Figure 5). HAp is the main inorganic component of bone
and teeth and is used as the gold standard for synthetic bone graft materials,
due to its properties and chemical resemblance of bone. Recent studies have
investigated the possibility of using HAp for drug delivery, with various drugs
and molecules having been investigated, including antibodies, growth factors,
17
and antibiotics [26–29]. Release rates have been proven to last up to several
days [30,31].
In this thesis, a novel precipitation method for incorporating amino acids
into the crystal structure of hydroxyapatite is described (Paper V).
Figure 5. Crystal structure of hydroxyapatite.
18
2. Ceramics for opioid drug delivery
2.1 Motivation
The increase in opioid consumption and misuse has led to a high death rate
due to overdose. In the United States, 70,630 deaths caused by drug overdose
were reported in 2019, 70.6% of which were due to opioids (49,860 people)
[32]. According to the Centre for Behavioural Health’s 2015 national survey
on drug use, approximately two million people misuse opioids, and it is now
considered a public health issue [33]. In order to solve the crisis, the Food and
Drug Administration (FDA) encourages the development of abuse-deterrent
formulations (ADF). An ADF is defined as having the ability to withstand
manipulation and tampering, e.g. crushing, snorting, or extraction in alcohol
and/or water. The most efficient way to decrease the possibility of tampering
is to add chemical barriers against extraction and physical barriers against
crushing, snorting, etc. Currently, eight commercial ADFs are approved by
the FDA [34]. The avaiable products are mainly based on physiochemical
barriers, which turn the tablet into a gel when exposed to crushing or presence
of a liquid [35]. Another form of ADF involves formulating agonist/antagonist
combinations, in which the antagonist can reduce the euphoric effect [36].
These formulations are polymer-based tablets. New efforts are focused on
how to make the matrix rigid against extraction, crushing or gelling [34,37].
In this part we present a tamper-resistant formulation based on opioids
loaded into a geopolymer matrix [5]. Geopolymers are inorganic polymers
consisting of a basic polysialate unit, containing SiO4 and AlO4 tetrahedra.
The chemical and mechanical properties—such as mechanical strength,
porosity and solubility—can be adjusted by altering synthesis conditions [38].
Previous studies have shown that drug-loaded geopolymer functions as a
controlled release formulation and exhibits tamper-resistant properties [3,39].
In this thesis, the geopolymer was subjected to crushing and extraction with
solvents: 40% ethanol, to simulate liquor, and 70 °C, water simulating hot
beverages. The results showed a low amount of drug extraction from the
matrix, demonstrating the abuse-deterrent properties for geopolymer
formulation (Paper I).
Although previous studies have investigated the behavior of drug-loaded
geopolymers in vitro, they have not been tested in vivo, therefore in this thesis,
an in vivo study is presented, in which zolpidem-loaded geopolymer was
administrated to dogs. Blood samples were collected for up to 24 hours and
the control group was an immediate-release formulation.
19
2.2 Methods
2.2.1 Synthesizing geopolymer
Waterglass was synthesized by mixing fumed silica and sodium hydroxide
with water into a homogenous and clear fluid. Metakaolin, Eudragit L100-55
(1 g per 6.5 g of metakaolin) and zolpidem (1 w/w%) were added to the
waterglass to form a geopolymer paste that was molded into 1.5 x 1.5 mm
pellets. The pellets were cured for 48 hours in 100% RH. After curing the
pellets were packed into capsules (Figure 6). The manufactured pellets
consisted of the following molar ratios: Si/Al: 1.94; H2O/Al2O3: 12.24; and
Na2O/Al2O3: 1.23.
Figure 6. Schematic view of synthesis of geopolymer and its packing into a mould.
2.2.3 In-vivo evaluation
The test facility SPM Biocameltec (Sidi Bou Othmane, Marocko) performed
the in vivo study on beagle dogs (approved by the local Animal Care
Veterinary Procedure, in accordance with animal care guidelines). The study
was also based on the OECD principles of Good Laboratory Practice
ENV/MC/CHEM (98) 17. The study consisted of two groups: one test group,
with drug-loaded geopolymer, and a control group, with a commercial product
(Table 1). The dogs fasted for 12 hours prior to dosing. Their body weight was
measured before dosing and 24 hours after the first dosing. Blood samples
were collected at the following time-points after administration: t=0, 0.5, 1, 2,
3, 4, 6, 9, 12, and 24 hours. Daily clinical observations were made, to detect
sedative effects, mortality and morbidity.
20
Table 1. Groups tested in the in vivo study.
Group
No.
n = Treatment Dose per dog
(mg)
1 4 Dose A: Stilnoct tablets in capsule
containing 5 mg zolpidem
5
2 4 Dose B: Geopolymer pellets in a
capsule containing 6 mg zolpidem
6
2.3 In –vivo properties of geopolymer in beagle dogs (Paper I)
In Group 1, one of the four tested dogs in the immediate release formulation
showed zero drug concentration in their blood plasma (Figure 7A). The
zolpidem plasma concentration reached a maximum at 17.4 ng/mL and went
back to LLOQ after 12 hours. In Group 2, two dogs showed zero plasma con-
centration and the other two dogs had zolpidem levels above LLOQ in plasma
(Figure 7B).
Figure 7. Zolpidem tartrate plasma profiles after oral administration to dogs. Group 1 received 5 mg Zolpidem tartrate IR tablets (A) and Group 2 received 6 mg Zolpidem tartrate geopolymer pellets (B).
The immediate-release tablet administration (Group 1) resulted in
significantly higher values for Cmax and AUC compared to geopolymer pellets
administration (Group 2). The geopolymer formulation, however, showed a
prolonged release of the drug (Table 2).
21
Table 2. Pharmacokinetic parameters for both groups, including average with standard
deviations. Abbreviations: D = Dog, ND = Non-detected, and NC = not calculated.
Cmax
(ng/mL)
Tmax (h) t1/2 elimination
(h)
AUC24h
(h*ng/mL)
Group 1
zolpidem
IR tablets
D1 23.7 0.5 0.2 43.7
D2 16.7 1 0.4 30.4
D3 ND ND ND ND
D4 22.9 0.5 0.4 26.5
Average 21.2±3.8 0.7±0.3 0.3±0.1 33.5±9.0
Group 2
zolpidem
pellets
D1 1.5 6.0 0.1 13.1
D2 0.1 1 NC 0.1
D3 ND ND NC 0.0
D4 1.0 1.0 0.5 2.9
Average 0.9±0.7 2.5±3.0 0.3±0.3 4.0±6.2
Clinical signs were normal for all dogs and no adverse effect could be detected.
In this study, we have presented the feasibility of delivering zolpidem from an
ADF.
22
3. Ceramics for pulmonary drug delivery
3.1 Motivation
Relieving acute pain has been one of the main goals in patient care, with the
gold standard for treating acute pain being intravenous delivery of opioids,
such as fentanyl or morphine [40]. The placement of an IV port can cause
infections and distress to the patient, and therefore other alternatives are con-
sidered [41]. Lungs have a large surface area, allowing drugs to be absorbed
and reach the site of action [42]. Moreover, pulmonary drug delivery is a fast
and non-invasive method for drug delivery. Ceramics have been studied as
drug delivery systems, due to their biocompatibility, tunable porosity, and me-
chanical strength. CaS has been widely studied for various applications, such
as local drug delivery in bone, and transdermal drug delivery systems, in
which both instant drug release and prolonged drug release have been inves-
tigated [6,7]. In this part, we will focus on the possibility of using heat to re-
lease fentanyl from calcium sulfate.
3.1.1 Lungs and the challenges of pulmonary drug delivery
(Paper II)
The adult human lung has a surface area of 70–140 m2, making them a bene-
ficial target for drug delivery [43]. By using the pulmonary route, the drug can
be used for both local and systematic delivery, without subjecting it to metab-
olism or extreme pH values [44]. The pulmonary route offers rapid and non-
invasive drug delivery. Additionally, side effects can be avoided due to rapid
onset action and the possibility of using a smaller dose [42]. The biggest lim-
itation with pulmonary drug delivery is that available devices require patient
compliance, and that doses can vary between patients, depending on lung ca-
pacity [45]. A new method of inhalation is emerging on the market, in which
the drug is evaporated by applying heat, making it possible to use small doses
of drug [46,47]. However, using heat to deliver drugs can result in drug deg-
radation [48].
23
3.1.1.1 Heat and degradation of fentanyl (Paper II)
New ways of delivering drugs using heat have been investigated, despite the
aforementioned challenges [49]. A crucial part of developing a drug delivery
system based on heat is to ensure the safety of patients, since degradation
products can either be more toxic or lack the desired pain relief effect.
Rabinowitz et al. studied forced degradation of fentanyl at 300 ºC on a hot
plate for five minutes, resulting in a degradation rate of 30% [50]. Another
degradation study was performed by Garg et al., who studied fentanyl degra-
dation in UV, acid, base, heat and oxidation [48]. To evaluate its heat degra-
dation properties, fentanyl was subjected to 350 ºC for five minutes, resulting
in five different fentanyl analogs (Table 3). Norfentanyl (NRF) and propi-
onanilide (PRP), are the most common degradation products of fentanyl. A
clinical study has been published describing the drug delivery of fentanyl by
generating a heated aerosol and applying it to healthy volunteers [46]. The
volunteers were given a preloaded handheld device, in which fentanyl was
dosed on a thin foil. The fentanyl was released when the foil was heated up to
350 ºC and the volunteers could inhale it during a five-second time period,
resulting in 100% bioavailability, which is comparable to an IV-administrated
drug. Additionally, the blood plasma analysis showed no presence of analogs
or degradation products.
Table 3. The detected fentanyl analogs when heated at 350°C for five minutes. Norfentanyl (NRF), propionanilide (PRP), 1-Phenethylpyridinium salt (1-PEP), 1-Phenethyl-1H-pyridin-2-one (1-PPO), and 1-Styryl-1H-pyridin-2-one (1-SPO).
Compound NRF
Mw 232
g/mol
PRP
Mw 149
g/mol
1-PPO
Mw 199
g/mol
1-SPO
Mw 197
g/mol
1-PEP
Mw 184
g/mol
Structure
24
3.2 Methods
3.2.1 Synthesis
Calcium sulfate rods were synthesized by mixing calcium sulfate with deion-
ized water at a power-to-liquid ratio of 0.5 into a homogenous paste, that was
packed into 6 x 12 mm rubber cylinders. The CaS rods were set aside to cure
for 24 hours at room temperature. The cured cement was crushed into pellets
(Figure 8) to further increase the surface area and was dosed with 100 µL of
5 mg/mL fentanyl in 20 mM hydrochloric acid or ethanol solution, by apply-
ing the solution onto the rods, resulting in a dose of 500 µg per rod.
Figure 8. Crushed pellets by mortal and pestle
3.2.2 Device and heating
A modified commercially available product called PAX 3 was used for this
study, as illustrated in Figure 9. A Teflon nozzle on top of a heating chamber
was manufactured to fit a syringe. Each sample was dosed with 100 µl of 5
mg/ml fentanyl in 20 mM hydrochloric acid or ethanol solution, resulting in a
dose of 500 µg per rod. Each sample was heated for 60 seconds at 230 ºC,
unless otherwise stated. After heating, vapor was collected by pulling the sy-
ringe (20 mL) containing the 1–5 mL of 20 mM hydrochloric acid at a speed
of 1mL/s for 15 seconds. When the vapor was collected, the syringe was re-
moved, shaken for 30 seconds, and then filtered (0.2 µm pore size). The heated
rods were immersed in 5 mL of 20 mM hydrochloric acid or methanol for
extraction (2 hours).
25
Figure 9. The desired temperature was set (1). When the temperature was reached, the sample was transferred to the heating chamber (2). An aliquot was taken out with a syringe that was filled with 20 mM hydrochloric acid (3-4). The syringe was shaken until the drug was dissolved into the hydrochloric acid and then quantified (5).
3.2.3 Analytical method
All samples were analyzed on High Pressure Liquid Chromatography (HPLC;
mobile phase 20/10/70 AcN/MeOH//H2O + 0.1% H3PO4 + 0.1% TFA) with
an ACE C18 column (100 x 21 mm, 2 µm). Isocratic method with a flow of
0.25 mL/min at 210 nm.
3.3 Evaporation of fentanyl from ceramics (Paper III)
In this study, we explored the possibility of using heat to release fentanyl from
a ceramic material. The first step was to evaluate the effect of the volume in
the syringe. Two different syringe volumes were tested: 1 mL and 3 mL, ac-
cording to Figure 8. The pellets were extracted in 5 mL of 20 mM HCl. It was
shown that by increasing the volume from 1 mL to 3 mL, the detected drug
increased by three times (Figure 10A). However, the evaporated drug was
very low in comparison to the amount loaded (500 µg). Moreover, condensate
was detected on the connector and was therefore rinsed with 1 mL of 20 mM
HCl and analyzed (Figure 10A).
26
Figure 10. Amount of fentanyl detected in the vapor by using different volumes in the syringe, n=1. The remaining fentanyl was extracted from the pellets and the conden-sate from the device was analyzed. Regarding fentanyl’s high potency and the safety of the person performing the test, only one sample was done (A). The recovery of fentanyl was investigated by applying the drug and extracting it without heating it, n=3, indicating that approximately 65 µg of fentanyl was missing (B). The extraction of fentanyl from heated pellets in the oven showed that approximately 120 µg had evaporated and/or degraded (C).
The recovery of fentanyl from the pellets was examined by dosing the pellets
with 500 µg of fentanyl and heating in the device for 10 minutes without col-
lecting vapor, showing a recovery rate of 80% (Figure 10B). The final step
was to investigate how higher temperature and longer heating time would af-
fect the release and degradation. The dosed ceramic was heated in a conven-
tional oven at 250 °C for 10 minutes. The extraction after heating indicated
that approximately 120 µg was missing (Figure 10C). Additionally, unknown
peaks were detected at retention times of 0.9 and 2.59 minutes, which are most
likely fentanyl analogs. The quantification of fentanyl was performed via
HPLC, which cannot identify unknown peaks. However, it has been reported
that the two most common fentanyl thermal analogs are NRF (Norfentanyl,
Mw=232) and PRP (Propionanilide, Mw=149). It can therefore be assumed that
27
the obtained peaks at retention times of 0.9 and 2.59 minutes are NRF and
PRP.
This study aimed to investigate the possibility of loading fentanyl in CaS
and releasing it by applying heat. Rabinowitz et al. reported that short periods
and lower temperatures would not cause degradation products or analogs,
which was confirmed in this study [50]. To conclude, it is possible to evapo-
rate fentanyl from ceramic material without forming analogs when heating at
a lower temperature and shorter time, but further testing needs to be done to
increase the effectiveness of drug release upon exposure to heat.
28
4. Ceramic hybrid crystals for drug delivery
4.1 Motivation
In this section, the focus will be on HAp and CaCO3, both of which are bio-
compatible and are used extensively in healthcare as bone void filler, drug
delivery vehicles, and excipients [11,26]. Since drug molecules are often sen-
sitive, mild manufacturing conditions for the ceramics are required. Precipita-
tion methods are the most common and mild way of synthesizing drug-loaded
ceramics, allowing the possibility to control their shape, size and functionali-
zation [51]. Previous studies have shown that it is possible to synthesize drug-
loaded nano- and microparticles, in which drugs are adsorbed on the surface
[8,10].
4.1.1 Entrapment of Hydroxyurea into CaCO3
Hydroxyurea (HU) is a small cytotoxic drug that is used to treat sickle cell
disease, leukemia and breast cancer [52–54]. Due to its size, it is an ideal can-
didate for entrapment into the crystal structure of materials such as CaCO3.
The pH surrounding tumors and inflamed tissue is often acidic, making pH-
responsive CaCO3 a promising vehicle for targeted drug delivery and con-
trolled drug release. By targeting a specific area, systematic toxicity can be
avoided. In this part, we present a diffusion method for entrapping HU into
single crystals of calcite (Paper IV). Additionally, the in vitro behavior of the
CaCO3 particles was investigated using MCF-7 human breast cancer cells.
4.1.2 Glycine in hydroxyapatite
HAp is highly biocompatible, osteoconductive, and mimics the mineral com-
position of bone and teeth. HAp synthesis can be performed using different
methods, such as precipitation or sintering [55]. When synthesizing drug-
loaded HAp, precipitation methods are preferred, since they are milder and do
not require high temperatures. Several studies have reported synthesis of drug-
loaded HAp with various sizes and properties [26,30,56]. Amino acids (AA)
have high affinity for HAp, and when introduced during synthesis, three forms
of bonding can occur: (I) adsorption effect, (2) inclusion effect, or (3) a com-
bination of both adsorption and inclusion. During synthesis, AA can bind to
the surface of Hap, with the amine group attaching to the calcium site and the
29
carboxylic group attaching to the phosphate site [57]. In order to only study
AA situated inside the crystal—i.e. not bound to the surface—an additional
step is needed, in which the particle is subjected to bleaching, in order to re-
move all surface-bound AA.
This study aimed to investigate the possibility of entrapping glycine into
the structure of HAp through a precipitation method, at different pH and tem-
peratures. The obtained powder was thoroughly washed and bleached, in order
to remove all surface-bound glycine.
4.2 Methods
4.2.1 Synthesis
Precipitation methods have been used in Paper IV and V to synthesize ceram-
ics while incorporating drugs. In Paper IV, synthesis was carried out in a
sealed box containing 2 L of free volume. Hydroxyurea was dissolved in a 20
mM CaCl2 solution, in order to obtain concentrations of either 400 mM or 800
mM. 40 mL of the drug solution was poured into a petri dish with a surface
area of 56 cm2, a small magnetic stirrer was added, and the petri dish was
sealed with parafilm, which was punctured with holes, allowing precipitation
to occur. Five grams of freshly crushed ammonium carbonate was added to
another petri dish. Both petri dishes were placed into a box, which was placed
on a magnetic stirring plate. Synthesis was terminated after 48 hours by filtra-
tion and washing with water and ethanol. The powder was then air dried, prior
to further analysis.
The synthesis in Paper V was carried out as an instant precipitation
method, in which HAp was synthesized with a modified precipitation method,
as described by Wang et al. [58]. Calcium citrate was added slowly (0.1 g/mL)
to diammonium phosphate solution (0.0335 g/mL) containing glycine (0.02–
0.07 g/mL). The pH was maintained at 8.5 or 12 via addition of sodium hy-
droxide solution (400 g/L). The obtained powder was washed and bleached
with sodium hypochlorite, in order to remove all surface-bound glycine. After
bleaching, the powder was once again washed with both water and ethanol.
4.2.2 Characterization
For both Paper IV and V, HPLC was used to quantify the amount of the drug
(Table 4). Since both HU and AA do not absorb UV, they had to undergo a
derivatization step prior to analysis. The derivatization step included the at-
tachment of an aromatic ring, making it possible to detect it on the UV spec-
trum.
30
Table 4. Parameters for quantification with HPLC. *OPA (o-phthaldialdehyde) is a
reagent that is used for the detection of amines.
Parameters Paper IV Paper V
Mobile phase A: 50% 20mM Ammo-
nium Acetate (pH = 6.9)
B: 50% acetonitrile
A: 40 mM NaH2PO4 (pH
= 7.8)
B: acetonitrile/metha-
nol/water (45/45/10
v/v/v).
Method Isocratic Gradient
0-1.9 min: 0% B
1.9-16.3 min: 53% B
16.3-24 min: 100% B
24-26 min: 0% B
Flow 2 mL/min 2 mL/min
Column Purospher® STAR RP-
C18 column (150 mm ×
4.6 mm, 5 μm)
Purospher® STAR RP-
C18 column (150 mm ×
4.6 mm, 5 μm)
Derivatization step 1 mL of sample mixed
with 700µl ethanol,
300µl 1 M hydrochloric
acid, and 50 µl 0.02 M
Xanthyrol in 1-propanol
750 µL of sample mixed
with 750 µl 0.4 M borate
buffer and 250 µl of
OPA reagent*.
Wavelength 213 nm 338 nm
4.2.3 Drug release
Prior to quantification, the particles need to be dissolved in order to expose
the entrapped drug. In Paper IV, CaCO3 particles were dissolved in 1 M hy-
drochloric acid before the derivatization step. In Paper V, before dissolving
the particles, all surface-bound glycine was removed by bleaching with so-
dium hypochlorite (50 µL per mg of hydroxyapatite). After bleaching, the par-
ticles were washed with water and ethanol. After bleaching and washing, the
powder was set to dry. After the particles were dry, they were dissolved in 0.1
N hydrochloric acid (10 µL per mg of hydroxyapatite), before the derivatiza-
tion step.
4.2.4 Cell viability
In Paper IV an in vitro test was conducted on MCF-7 human breast cancer
cells, purchased from ATCC. MCF-7 were subcultured in DMEM/F12 cell
culture medium (Gibco) containing 10% fetal bovine serum (FBS) and 1%
penicillin/streptomycin at 37 ºC in a humidified atmosphere of 5% CO2, with
31
complete medium replacement every 48 hours. The cells were subcultured at
80% confluence and used within six passages after thawing. Alamar Blue was
used to determine cell survival/proliferation. Cells were seeded at 1, 2 or 4 x
104 cells/well in 96-well tissue culture-treated plates. After 24 hours, the me-
dium was replaced with medium containing calcite particles, loaded with or
without HU, dispersed over a concentration range of 1–10 mg/mL. A stock
solution of particles in medium, dispersed at a concentration of 10 mg/mL in
PBS, was used to achieve the final treatment concentration, directly before
treatment, via a single dilution into DMEM/F12. After incubating cells in con-
tact with particles for 24 or 48 hours, the medium was replaced with 150 µL
of 10% Alamar Blue solution in fresh medium. The plates were incubated at
37 ºC for one hour, before transferring 100 µL to a black 96-well plate. Fluo-
rescence was detected at 570 nm excitation and 590 nm emission on a micro-
plate reader (Infinite M200, Tekan, Switzerland).
4.3 Entrapment of hydroxyurea into calcite (Paper IV)
Higher starting concentrations of hydroxyurea resulted in a higher amount of
entrapped drug (Figure 11); however, a higher starting concentration was not
as efficient at entrapping HU compared to the lower concentration (Table 5).
Drug release was performed at pH 7.4 for two weeks, showing that no drug
was released at pH 7.4, which confirms that drug release did not occur at neu-
tral pH.
Figure 11. Amount of drug recovered after washing with water and ethanol. The par-
ticles were dissolved in 1 M hydrochloric acid.
32
Table 5. Summary of collected data and calculations of loading properties for the two synthesis conditions (400 mM hydroxyurea and 800 mM hydroxyurea).
Synthesis Regimen Amount of drug
detected (µg)
Amount drug/cal-
cite (µg/mg)
Loading ef-
ficiency
(mol%)
400 mM HU 236.1±25.5 3.6±0.3 0.019±0.002
800 mM HU 408.0±46.5 6.7±0.7 0.016±0.002
An Alamar Blue assay was used to determine cell viability. Cell viability stud-
ies are typically conducted on cells that are actively dividing, in the linear
range of logarithmic division. However, tumors are typically dense, highly
populated, three-dimensional cultures. Often, the EC50 value for a given drug
differs between these two culture conditions, as lower density cultures are
more susceptible to drug-induced toxicity. Therefore, HU toxicity was vali-
dated using three different cell concentrations: 1 x 104, 2 x 104, and 4 x 104
cells/well, respectively (Figure 12). The observed EC50 values of HU in MCF-
7 cells was 0.2 mM for cells cultured at 1 x 104/well, 0.4 mM for cells cultured
at 2 x 104/well, and 0.8 mM cells cultured at 4 x 104/well.
0.01 0.1 1 10
0
50
100
Cell
via
bili
ty (
%)
Log concentration (mM)
10K
20K
40K
Figure 12. Cell viability assay for determination of EC50 of hydroxyurea at three dif-ferent cell concentrations (1x104, 2x104, and 4x104 cells/well).
33
Two different cell concentrations were treated with particles with and without the drug. Drug-loaded particles reduced viability by up to 50–70% (50 mg/mL, Figure 13). However empty calcite particles also showed reduced vi-ability, which could indicate that the toxicity is caused by the particles.
Figure 13. Cell viability after treating 20,000 cells/well (A) or 40,000 cells/well (B). The cells were treated with 50 mg/mL, 5 mg/mL, 10 mg/mL or 1 mg/mL. For each concentration, particles with (+) and without (-) hydroxyurea were tested.
To understand the effect of the calcite particles and toxicity, the cells were
treated with dissolved particles, with or without drug at two concentrations:
2.5 mg/mL and 5 mg/mL. The dissolved particles containing drugs were
shown to have lower cell viability compared to the particles without drugs
(Figure 14).
34
Figure 14. Cell viability after particle solution treatment for 10,000 cell/well (A), 20,000 cells/well (B) or 40,000 cells/well (C). The cells were treated with 2.5 mg/mL or 5 mg/mL. For each concentration, particles with (+) and without (-) hydroxyurea was tested. (Mean ± SD, n=3, * p < 0.05, **p < 0.01 compared to each control group.)
35
In this study, a precipitation method is presented, which makes it possible to
entrap hydroxyurea into the crystal structure of calcite. The particles were sub-
jected to PBS for two weeks, showing no release during that period. The par-
ticles were also investigated for their in vitro properties, where the cell viabil-
ity was measured on human breast cancer cells after treatment with different
particle concentrations. The cell medium was not acidic enough for the parti-
cles to dissolve, and together with the high EC50 value, the amount of hy-
droxyurea was not sufficient to kill the cancer cells. However, by pre-dissolv-
ing the particles and treating the cells with the solution, cell viability can be
lowered. These findings are of high importance for future studies involving
synthesis of drug-loaded single crystalline materials.
4.4 Entrapment of glycine into the crystal structure of hydroxyapatite (Paper V)
The effects of different starting concentrations (0.02–0.07 mg/mL), tempera-
tures (40 ºC or 70 ºC) and pH (8.5 or 12) were evaluated. Scanning electron
microscopy (SEM) images showed the formation of nanocrystalline Hap, with
no significant difference observed between the different concentration groups.
Therefore only representative samples are presented in Figure 15. Lower pH
showed slightly more distinguished particles. A difference could be seen in
the X-ray diffraction (XRD) results, in which high pH and high temperature
(pH 12, 70 ºC) resulted in the formation of portlandite; however, no portland-
ite could be seen in SEM results (Figure 16). Moreover, the specific area was
investigated by Brunauer–Emmett–Teller (BET) theory, showing a similar
surface area for all groups (Figure 17). One group that differed a little from
the rest was the one with both high pH and high temperature (pH 12, 70 ºC),
which might be an effect of the occurrence of portlandite.
36
Figure 15. SEM images for pH 12 and pH 8.5 at 40ºC and 70ºC for 0.07 mg/mL. The scale bar represents 1µm. Lower pH show more distinguished particles compared to pH 12.
Figure 16. XRD data for pH 12 and pH 8.5 at different temperatures show the for-mation of portlandite when increasing pH and temperature.
37
Figure 17. Surface area analysis with BET showing similar surface area for all sam-ples with an exception for pH 12-70ºC which showed a slightly lower area.
Finally, the drug loading properties were investigated, showing that a higher
pH and higher temperature resulted in a higher amount of entrapped gly-
cine/mg HAp (Figure 18). Moreover, a higher starting concentration of gly-
cine resulted in a higher quantity of entrapped drug. The biggest difference
could be seen between the two pH groups, with higher pH resulting in higher
entrapped drug quantity.
Figure 18. Drug release at different pH (8.5 and 12) and temperature (40ºC and 70ºC) with different starting temperatures of glycine.
38
In this study, a precipitation method is presented where an amino acid, Gly-
cine, was entrapped into the crystal structure of HAp. Different, methods and
techniques were used to confirm the presence of Glycine in the structure and
the effect on the crystal structure. Krukowski et al. showed that it is possible
to synthesize nanosized HAp particles containing glycine; however, the posi-
tion of the glycine was not assessed, since amino acids have a high affinity for
the surface of calcium phosphates, where the amine group is attached to the
calcium site and the carboxylic group is attached to the phosphate site [59]. In
order to remove all the surface-bound AA, it is crucial to bleach the HAp par-
ticle [25].
Prior to analysis, a derivatization agent was added for glycine to have UV-
absorbance for the HPLC analysis. When the derivatization agent (pH 10) was
added to the dissolved AA solution, reprecipitation of HAp occurred, due to
the increase in pH. This reprecipitation might have entrapped glycine once
again into the structure, therefore leading to a lower amount detected than ex-
pected. The XRD results showed the presence of portlandite, which might be
due to the addition of NaOH to maintain the high pH at a high temperature.
When incorporating other molecules into the crystal structure, peak shifts
should appear in the XRD spectra; however, no peak shifting could be ob-
served. This may be due to the broad peaks and the amount of glycine not
being sufficient to cause a peak shift.
To conclude, it is possible to entrap glycine, an amino acid into the crystal
structure of hydroxyapatite by having glycine present during the precipitation
reaction and by bleaching the crystals to remove all surface-bound glycine,
leaving only the entrapped glycine.
39
5. Concluding remarks
The focus of this work was to explore the possibilities of using ceramics as
drug delivery systems, since they are highly biocompatible materials with tun-
able properties. Three main tracks have been investigated: (1) geopolymers
for abuse-deterrent formulations; (2) calcium sulfate as a carrier for pulmo-
nary drug delivery of fentanyl; and (3) incorporating drug molecules into the
crystal structure of CaCO3 and HAp. The thesis contributes to an increased
understanding of using ceramics for drug delivery, as well as the synthesis,
use and functionality of hybrid ceramic crystals.
The first section explored the possibility of using geopolymer as an abuse-
deterrent formulation for oral drug delivery. Due to the opioid crisis, the need
for abuse-deterrent formulations is high. Currently available formulations are
mainly based on physiochemical barriers, which transform the tablet into a gel
and make it difficult to use a syringe. Other formulations are based on the
addition of an antagonist, which lowers the euphoric effect of the opioid. Ge-
opolymers can be designed to combine both abuse deterrence and prolonged
drug release, especially for oral or transdermal delivery. The geopolymer pro-
vides a physiochemical barrier, making it difficult to extract the drug. The in
vivo properties of the geopolymer were investigated in beagle dogs, in which
the control group received instant-release tablets (Stilnoct®). The geopolymer
showed a lower plasma concentration compared to the immediate-release tab-
lets. However, the geopolymer did demonstrate a controlled release plasma
concentration, indicating that geopolymers are suitable for oral controlled
drug delivery.
The second section explores the possibility of using heat to release fentanyl
from ceramics for pulmonary drug delivery. When using heat to release drugs,
analogs must be taken into consideration since they can be toxic or have no
effect at all. It was demonstrated that fentanyl could be released by heat from
calcium sulfate ceramics for pulmonary drug delivery. Fentanyl was loaded
onto ceramic rods and heated for 1–10 minutes at different temperatures (230
°C–250 °C). The results showed that it was possible to load and release fenta-
nyl (1.2 µg) without obtaining any analogs. However, longer heating time and
higher temperatures resulted in the formation of analogs.
The third section investigated the possibility of loading drugs into the crys-
tal structure of calcium carbonate and hydroxyapatite via precipitation meth-
ods. Incorporating a drug within a crystal structure allows formation of a hy-
brid crystal that can act both as the raw material and as a drug delivery vehicle
for targeted drug delivery. Hydroxyurea, a cytotoxic drug, was incorporated
40
into the crystal structure of calcium carbonate, resulting in entrapment of
6.7±0.7 µg of the drug. In vitro properties were investigated in human breast
cancer cells by exposing the cells to the crystals, which resulted in decreased
cell viability. This demonstrated that it is possible to use the crystals as drug
delivery vehicles. Glycine, an amino acid, was entrapped into the crystal struc-
ture of hydroxyapatite, resulting in entrapment of 16.3±2 µg of glycine per mg
of hydroxyapatite. The effect of pH and temperature on the loading efficacy
was evaluated, which showed that higher temperature and higher pH result in
a higher amount of entrapped glycine.
41
6. Future outlook
This thesis presents various methods for using ceramics (geopolymer, calcium
sulfates, calcium carbonate, and hydroxyapatite) as drug delivery systems.
The following sections present possible paths for continuation projects:
The effect of drug loading on drug release. In papers I, III, IV, V the meas-
ured doses were low and for many pharmaceutical substances higher doses are
required in order to give plasma concentrations resulting a therapeutic effect.
In paper I, an in vivo study was performed for an abuse-deterrent formulation,
resulting in a significantly lower plasma concentration compared to an imme-
diate-release formulation. A higher dose could be incorporated into the matrix,
in order to deliver sufficient dosage at the site of action. The effect of higher
drug loading on the drug release rate and on the abuse-deterrent properties
needs to be further studied. In paper III, heat was used to evaporate fentanyl
from a ceramic material. The amount of drug evaporated was low to deliver
an adequate dose (1.2 µg) and the release efficiency should be evaluated by
adjusting the porosity of the ceramic or by adding a higher dose of drug. In
addition, a vaporizing excipient (such as polyethylene glycol) could be added
to help fentanyl evaporate easier. Papers IV and V aimed to entrap a drug
molecule into the crystal structure of CaCO3 and hydroxyapatite. The loading
capacity is limited by free spaces within the crystal. However, the effect on
loading capacity with different synthesis methods, pH, and temperatures
should be further investigated.
The effect of heating method on drug evaporation. The drug evaporation
method presented in paper III was conducted using an existing inhalation de-
vice (PAX 3). Since heat was only applied for a short period (60 seconds), the
heat transfer within the ceramic may have differed and the release of the drug
might not have been very effective. It is therefore suggested to further study
different ways of heating, e.g. by using induction, which would result in faster
transfer and more even distribution of heat.
Drug dissolution rate from calcium carbonate (CaCO3). An in vitro study
was presented in paper IV, showing the release of hydroxyurea from CaCO3
in MCF-7 cells. The high EC50 for hydroxyurea and the low amount of en-
trapped hydroxyurea caused small changes in cell viability. However pre-dis-
42
solved particles caused a decrease in cell viability, meaning that the dissolu-
tion rate can be a limitation and needs to be further studied. A cell line with a
lower EC50 could be an alternative for in vitro studies, such as L1210 (mouse
lymphocytic leukemia cell line).
Further studies of the crystals structure. In papers IV and V, we present
the entrapment of drugs into the crystal structure of CaCO3 and HAp. The
experiments showed that the drug was entrapped in the crystal, but did not
reveal its exact location within the lattice. To learn more about the effect on
the crystal when incorporating a drug, TEM studies or solid-state NMR could
be performed. Moreover, theoretical simulations, such as molecular dynamics
simulations, could be performed, in order to model the lattice distortion that
arises when incorporating a drug molecule.
Future applications of hybrid crystals. The application of hybrid crystals in
drug targeting should be explored, due to their high biocompatibility and the
possibility to load various small drugs into different crystals. Another factor
is the structure of the crystal, i.e. a small drug can contain groups such as
phenol rings, which present challenges for its incorporation it into the crystal
structure, due to the three-dimensional network. The effect of the drug molec-
ular structure on loading in hybrid crystal should therefore be further studied.
This thesis opens the door to explore hybrid crystals as drug delivery systems,
raw materials, or biomaterial scaffolds.
43
7. Svensk sammanfattning
Keramer förknippas väldigt lätt med byggmaterial och sällan med läkemedel
och medicinska ändamål. Biokompatibla keramer används som både implan-
tat och som läkemedelsbärare. Keramernas egenskaper kan styras beroende på
tillverkningsmetod, vilket gör det möjligt att styra t.ex. porositet, egenskaper
och hållfasthet. I den här avhandlingen undersöktes tre olika fält för administ-
ration av läkemedel; missbrukssäkra läkemedelsformuleringar, inhalation och
keramiska hybrid kristaller.
Den första delen handlar on missbrukssäkra läkemedelsformuleringar.
Opioider är förstahandsvalet vid behandling av kronisk och svår smärta. Un-
dersökar har visat att missbruk av opioider har ökat markant på grund av den
euforiska känslan. Missbruket av opioider har lett till att myndigheter som
FDA har krävt utveckling av nya missbrukssäkra läkemedelsprodukter. I den
här avhandlingen undersöks geopolymerer som en missbrukssäker formule-
ring. Geopolymerer är oorganiska polymerer som har visat sig ha väldigt hög
mekanisk styrka och justerbar porositet. In vivo tester gjordes för att undersöka
plasma koncentrationer i beagle hundar vilket visade att geopolymerer gav en
fördröjd frisättning och upptag av läkemedlet.
Den andra delen handlar om att använda kalcium sulfat som läkemdelsbä-
rare. Standard metod för att behandla smärta är intravenös administration av
läkemdel vilket kan ge obehag till patienten men kan också leda till infektioner
vid installation av iv-port. I den här avhandling presenteras en alternativ ad-
ministrationsväg via lungorna som kommer att frisätta läkemedlet genom
värmning. Fentanyl doserades på keramen som sedan värmdes och ångorna
samlades upp och analyserades. Resultaten visade att det går att frisätta
fentanylen genom att värma keramen men om högre temperturer och längre
värmingstid användes så upkom biprodukter vilket man vill undvika.
Den sista delen av avhandlingen behandlar syntes av hybridkeramer där
läkemedel har inkorporerats in i keramernas kristallstruktur. Genom att inkor-
porera läkemedel i keramer så kan de användas för riktad läkemedelsleverans.
När keramerna exponeras för en sur miljö löser keramen upp sig och på så sätt
frigör läkemedlet på plats. I den här avhandlingen presenterars två olika typer
av synteser där läkemedel har inkorporerats i kristallstrukturen. Resultaten vi-
sade att det är möjligt att ladda signifikant mängd av läkemedel i strukturerna.
44
8. Abstrakt na bosanskom jeziku
Keramika se u praksi lako povezuje sa građevinskim materijalima, ali rijetko
sa lijekovima i ostalim medicinskim svrhama. Biokompatibilna keramika
koristi se i kao implantat i kao nosač lijekova. Svojstva keramike mogu se
kontrolirati ovisno o načinu proizvodnje, što omogućuje kontrolu npr.
poroznosti, svojstva i čvrstoće. U ovoj disertaciji ispitana su tri različita
područja primjene lijekova; formulacije lijekova sa sprječavanjem
zloupotrebe, inhalacijski i keramički hibridni kristali.
Prvi dio rada se bavi formulacijama lijekova sa sprječavanjem
nemedicinske zloupotrebe. Opioidi su prvi izbor u liječenju hronične i jake
boli. Studije su pokazale da se zloupotreba opioida značajno povećala zbog
osjećaja euforije. Zloupotreba opioida navela je vlasti poput FDA da
zahtijevaju razvoj novih lijekova koji izazivaju ovisnost. U ovoj disertaciji se
istražuju geopolimeri kao formulacije ovisnosti. Geopolimeri su anorganski
polimeri za koje se pokazalo da imaju vrlo visoku mehaničku čvrstoću i
podesivu poroznost. Provedeni su in vivo testovi za ispitivanje koncentracije
u plazmi beagle pasa. Testovi su pokazali da su geopolimeri rezultirali
odgođenim otpuštanjem i apsorpcijom lijeka.
Drugi dio rada govori o korištenju kalcijevog sulfata kao nosača lijeka.
Standardna metoda liječenja boli je intravenozna primjena lijeka koja može
uzrokovati nelagodu kod pacijenta, ali može dovesti i do infekcija pri ugradnji
IV porta. U ovoj disertaciji je predstavljan alternativni put primjene kroz pluća
gdje se lijek oslobađa zagrijavanjem. Fentanil je doziran na keramiku koja je
zatim zagrijana, a pare su sakupljene i analizirane. Rezultati su pokazali da je
moguće oslobađanje fentanila zagrijavanjem keramike, ali ako se koriste više
temperature i duže vrijeme zagrijavanja, nastaju nusproizvodi koji se nastoje
izbjeći.
Posljednji dio disertacije bavi se sintezom hibridne keramike gdje su
lijekovi ugrađeni u kristalnu strukturu keramike. Ugrađivanjem lijekova u
keramiku se može postići ciljano dopremanje lijeka. Kada je keramika
izložena okruženju kiselina, ista se otapa i tako se lijek oslobađa na licu
mjesta. U ovoj disertaciji su predstavljane dvije različite vrste sinteza u kojima
su lijekovi ugrađeni u kristalnu strukturu. Rezultati su pokazali da je moguće
ugraditi značajne količine lijekova u strukture.
45
9. Acknowledgments
First of all, I would like to thank my supervisors Håkan, Wei and Susanne.
Håkan, thank you for giving me this opportunity to conduct my PhD in your
group. You are an inspiration and you have taught me how to be a better re-
searcher. I will be forever grateful for these years. Wei, for your infinite sup-
port and your ability to see everything from the bright side. Susanne, for all
the proofreading and discussions we have had over the years, but mostly thank
you for being an amazing boss during our years together at Emplicure.
Camilla and Susanne Lewin, this journey would not have been possible
without you two; I am very grateful to have done this with you. Looking for-
ward to a lifelong friendship. Camilla, thank you for being my officemate
these years. I will always treasure our adventures. Susanne, thank you for put-
ting up with me and all of my stupid questions, for all your emotional support
and helping with my papers.
Michael, thank you for all the help and support during my PhD and espe-
cially the last year. You were always there and was so nice when I called in
on late evenings and weekends. Giannis and Salim, thank you for joining our
group and for making my last years more exciting. Estefania, thank you for
all the late nights in the cell lab; hopefully I won’t have to plate anymore cells
in my life. To all the present and former members of the MiM and BMS lab:
Cecilia, Caroline, Ana, Lisa, Andrea, Hanna, Alexandra, Yijun, Huasi, Lui-
mar, Lee, Anna Diez, Roger, Jun, Céline and the rest, thank you for your com-
pany during these years. Gabriel, thank you for all the fika sessions and the
long talks. Sara Galinetti, you are such an amazing and compassionate person,
thank you for always checking in on me. Henry, I wish you could move back
to Uppsala so that we could eat ice cream buckets (yes plural) for dinner.
Thank you for always being there and for proofreading.
Tack till alla mina vänner och familj. Malin, vilken tur att vi träffades på
nollningen för 11 år sen. Sen dess har du alltid varit vid min sida, hejat och
peppat och nu har jag fått äran att jobba tillsammans med dig, vilken tur jag
har! Sulejmen och Levan, tack för att ni har visat mig att det finns en värld
utanför akademin och alla gråa fyrkantiga ingenjörer. Tack mamma för din
analytiska förmåg som får mig att ta tag i saker på ett metodiskt sätt men
främst tack för alla varma kramar. Pappa, även om du inte altid riktigt förstod
vad jag håller på med så har du alltid varit där och stötta mig men också bi-
dragit med många matlådor. Nejra, tack för att få får mig att känna mig som
46
en pansionär. Även om vi är något otroligt olika så kommer du alltid att vara
min lillasyster.
Dževahidin i Berina, hvala za lijepa sjećanja, sijela do zore, ljetovanja i
zimovanja. Sad počinje nas ”odrasli život”. Berina, tebi posebno hvala na pre-
vodu. Nano, hvala ti za sve pite sto si kuhala, za svaku lijepu riječ sto si mi
pruzila i što si me uvijek podsjećala na ono što je ustvari najbitnije u životu.
Toliko sam zahvalna sto te imam i što ovo mogu podijeliti sa tobom.
Amar, hvala za svu podršku, ljubav i bezgranično strpljenje.
47
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Acta Universitatis Upsaliensis Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 2087
Editor: The Dean of the Faculty of Science and Technology
A doctoral dissertation from the Faculty of Science and Technology, Uppsala University, is usually a summary of a number of papers. A few copies of the complete dissertation are kept at major Swedish research libraries, while the summary alone is distributed internationally through the series Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology. (Prior to January, 2005, the series was published under the title “Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology”.)
Distribution: publications.uu.se urn:nbn:se:uu:diva-456750
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