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)

Za dedu

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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