Academic year 2014-2015

Morgane FRANCK

First Master of Science in Drug Development

Promotor

Prof. Dr. S. De Smedt

Co-promotor

Prof. Dr. S. Aime

Commissioners

Prof. dr. F. De Vos

Dr. K. Kersemans

Design of theranostic PLGA-based nanoparticles loaded with MRI imaging agents

and boron/curcumin adducts to combine BNCT and chemotherapy

Master thesis performed at:

UNIVERSITA DEGLI STUDI DI TORINO

Department of molecular

biotechnologies and health sciences

Molecular imaging center

GHENT UNIVERSITY

FACULTY OF PHARMACEUTICAL SCIENCES

Department of Pharmaceutics

Laboratory for General Biochemistry and

Physical Pharmacy

Academic year 2014-2015

Morgane FRANCK

First Master of Science in Drug Development

Promotor

Prof. Dr. S. De Smedt

Co-promotor

Prof. Dr. S. Aime

Commissioners

Prof. dr. F. De Vos

Dr. K. Kersemans

Design of theranostic PLGA-based nanoparticles loaded with MRI imaging agents

and boron/curcumin adducts to combine BNCT and chemotherapy

Master thesis performed at:

UNIVERSITA DEGLI STUDI DI TORINO

Department of molecular

biotechnologies and health sciences

Molecular imaging center

GHENT UNIVERSITY

FACULTY OF PHARMACEUTICAL SCIENCES

Department of Pharmaceutics

Laboratory for General Biochemistry and

Physical Pharmacy

COPYRIGHT

"The author and the promoters give the authorisation to consult and to copy parts of this

thesis for personal use only. Any other use is limited by the laws of copyright, especially

concerning the obligation to refer to the source whenever results from this thesis are cited."

June 1, 2015

Promotor Author

Prof. Dr. S. De Smedt Morgane franck

ABSTRACT

In recent years, the use of nanoparticles (NPs) as medicine has become more and

more interesting to obtain specific drug delivery for formulations that have the possibility to

follow the treatment in real time. These particles are called theranostic nanoparticles,

because of their therapeutic and diagnostic effects. Most of the time they consist of

nanoparticles loaded with therapeutic agents and imaging reporters, and are functionalised

with a specific component to target the particle.

Biodegradable polymers like poly(lactic-co-glycolic acid) (PLGA) are widely used because of

the many advantages, such as low toxicity and easy surface functionalisation. Various types

of drugs and imaging agents can be incorporated in the PLGA-based NPs, depending on the

desired treatment and visualisation technique.

The aim of this thesis is to create PLGA-based NPs with an optimal action and toxicity

profile to cure cancer and follow-up the treatment using Magnetic Resonance Imaging (MRI).

The PLGA particles are loaded with rubrocurcumin (RbCur) and an amphiphilic Gd-

DOTAMA(C18)2 complex using an oil-in-water emulsion solvent evaporation method. Non-

targeted and folate-targeted particles are created to compare the difference in visualisation.

The therapeutic agent, RbCur, is made of curcumin, boric acid and oxalic acid. The purity of

the complex is verified by Nuclear Magnetic Resonance (NMR) and UV/VIS

spectrophotometry. Curcumin, a natural compound derived from Curcuma longa, possesses

different anti-cancer properties and boron is used for Boron Neutron Capture Therapy

(BNCT). The contrast agent, Gd-DOTAMA(C18)2, provides the particle with the necessary

relaxivity to obtain a good MRI visualisation.

The NPs are incubated in the cancer cell line IGROV-1, which are ovarian cancer cells with

95% overexpression of folate receptors (FRs). The difference in specificity and efficiency of

the therapy between the non-targeted and folate-targeted particles is evaluated by MRI.

Targeting specific cancers using folate looks successful and together with an

optimisation of the development, stability and action of the NPs it can lead to a big step

forward in the enhancement of cancer treatment.

SAMENVATTING

In de laatste jaren is het gebruik van nanopartikels (NPs) als medicijn interessanter

geworden om specifieke drug delivery voor formulaties te verkrijgen die de mogelijkheid

hebben om de behandeling in real time te volgen. Deze partikels worden theranostische

nanopartikels genoemd als gevolg van hun therapeutische en diagnostische effecten. Ze zijn

opgebouwd uit nanopartikels die geladen zijn met een therapeutische agent en een

beeldvormingsagent en gefunctionaliseerd zijn met een specifieke component om de

partikels op de gewenste plaats te verkrijgen.

Biodegradeerbare polymeren zoals poly(lactic-co-glycolic acid) (PLGA) worden veel gebruikt

omwille van de vele voordelen, zoals lage toxiciteit en eenvoudige oppervlakte

functionalisering. Verschillende geneesmiddelen en beeldvormingsagentia kunnen

geïncorporeerd worden in de PLGA-gebaseerde NPs, het type hangt af van de gewenste

behandeling en visualisatie techniek.

Het doel van deze thesis is het ontwikkelen van PLGA-gebaseerde NPs die een

optimaal werkings- en toxiciteitsprofiel bezitten om kanker te genezen en de behandeling op

te volgen met Magnetic Resonance Imaging (MRI). De PLGA partikels worden beladen met

rubrocurcumine (RbCur) en een amfifiel Gd-DOTAMA(C18)2 complex gebruik makende van

een olie-in-water emulsie solvent evaporatie methode. Folaat en niet-folaat bezittende

partikels worden aangemaakt om het verschil in visualisatie te vergelijken. Het

therapeutische deel, RbCur, bestaat uit curcumine, boorzuur en oxaalzuur. De zuiverheid van

het complex wordt nagegaan met Nuclear Magnetic Resonance (NMR) en UV/VIS

spectrofotometrie. Curcumine, een natuurlijk bestanddeel afgeleid van Curcuma longa, bezit

verschillende anti-kanker eigenschappen en boron wordt gebruikt voor Boron Neutron

Capture Therapy (BNCT). De contrast agent, Gd-DOTAMA(C18)2, bezorgt het partikel de

benodigde relaxiviteit om een goede MRI visualisatie te verkrijgen.

De NPs worden geïncubeerd in de kankercellijn IGROV-1, dit zijn ovarium kankercellen met

95% folaat receptor (FR) overexpressie. Het verschil in specificiteit en efficiëntie van de

therapie tussen de niet-folaat en folaat bezittende partikels wordt geëvalueerd met MRI.

Het gebruik van folaat om specifieke kankers te visualiseren ziet er succesvol uit en

kan samen met een optimalisatie van de ontwikkeling, stabiliteit en actie van deze NPs

leiden tot een grote stap voorwaarts in de verbetering van de behandeling van kanker.

ACKNOWLEDGEMENTS

First of all I would like to thank my supervisor Diego Alberti for the great guidance, advice,

support and ideas during the four months I worked in the laboratory. I would also like to

thank my supervisor Simonetta Geninatti Crich. Thank you for the enthusiasm, guidance and

support on the project.

Thank you to my promotor in Torino, prof. Dr. Silvio Aime and my promotor in Ghent, prof.

Dr. Stefaan De Smedt to give me the opportunity to discover the field of molecular imaging.

A big thank you to all the people of the molecular imaging department at the Molecular

Biotechnology Center for the great working atmosphere.

Finally I would like to thank my parents for the great support and to have given me the

possibility to go abroad.

TABLE OF CONTENT

1. INTRODUCTION ...................................................................................................................... 1

1.1 Cancer ............................................................................................................................... 1

1.2 Magnetic Resonance Imaging ........................................................................................... 2

1.2.1 The theory behind MRI .............................................................................................. 2

1.2.2 Contrast agents .......................................................................................................... 4

1.2.2.1 Gadolinium .......................................................................................................... 4

1.3 Nanoparticles .................................................................................................................... 5

1.3.1 PLGA ........................................................................................................................... 7

1.3.2 The use of folate as a targeting ligand for PLGA-NPs ................................................ 9

1.4 Therapeutic agents ........................................................................................................... 9

1.4.1 Curcumin .................................................................................................................... 9

1.4.2 Boron ........................................................................................................................ 10

2. OBJECTIVE ............................................................................................................................. 12

3. MATERIALS AND METHODS ................................................................................................. 13

3.1 Reagents ......................................................................................................................... 13

3.2 Methods .......................................................................................................................... 14

3.2.1 Preparation and characterisation of the RbCur drug complex ................................ 14

3.2.2 Preparation of PLGA nanoparticles .......................................................................... 15

3.2.2.1 Non-targeted nanoparticles .............................................................................. 16

3.2.2.2 Folate-targeted nanoparticles ........................................................................... 17

3.2.3 Characterisation of the particles .............................................................................. 17

3.2.3.1 Particle size ........................................................................................................ 17

3.2.3.2 Stability .............................................................................................................. 18

3.2.3.3 Relaxometric measurements ............................................................................ 21

3.2.3.4 Nuclear Magnetic Resonance Dispersion (NMRD) profiles .............................. 22

3.2.4 In vitro uptake and MRI visualisation ...................................................................... 22

4. RESULTS ................................................................................................................................ 25

4.1 RbCur complex ................................................................................................................ 25

4.2 Nanoparticles .................................................................................................................. 26

4.2.1 Characterisation ....................................................................................................... 26

4.2.2 Stability .................................................................................................................... 27

4.2.2.1 RbCur release .................................................................................................... 27

4.2.2.2 Relaxation rate, relaxivity and Gd concentration ............................................. 28

4.2.3 NMRD profile ........................................................................................................... 30

4.3 In vitro uptake and MRI visualisation ............................................................................. 30

5. DISCUSSION .......................................................................................................................... 33

5.1 RbCur complex ................................................................................................................ 33

5.2 Nanoparticles .................................................................................................................. 34

5.2.1 Size, relaxivity and RbCur incapsulation yield.......................................................... 34

5.2.2 Stability .................................................................................................................... 35

5.3 In vitro uptake and MRI visualisation ............................................................................. 35

6. CONCLUSION ........................................................................................................................ 37

7. REFERENCES ......................................................................................................................... 38

8. APPENDIX ............................................................................................................................. 41

8.1 Reagents ......................................................................................................................... 41

ACRONYMS

BNCT: boron neutron cancer therapy

CA: contrast agent

FBS: fetal bovine serum

FR: folate receptor

Gd: gadolinium

HBS: NaCl/Hepes buffer

ICP-MS: inductively coupled plasma mass spectrometry

MRI: magnetic resonance imaging

NMR: nuclear magnetic resonance

NMRD: nuclear magnetic resonance dispersion

NP: nanoparticle

PBS: phosphate buffered saline

PDI: poly dispersity index

PEG: polyethylene glycol

PLGA: poly(D,L-lactic-co-glycolic acid)

PVA: poly(vinyl alcohol)

R1: relaxivity

RbCur: rubrocurcumin

T1: longitudinal relaxation time

TLC: thin layer chromatography

1

1. INTRODUCTION

1.1 CANCER

Cancer is the general term used to describe all diseases provoked by quickly dividing

and invasive cells. These cells are characterised by an abnormal growth and have the

possibility to expand to other organs, this phenomenon is called metastasis. A lot of different

types of cancer are known. They are named after the organ in which they start, for example:

lung cancer, ovarian cancer, colon cancer,…(1)

A lot of factors are involved in the development of cancer. The most common risk factors are

smoking, an excess consumption of alcohol, an unhealthy diet, a lack of sport, infections,

radioactive radiation and genetic predisposition.

The symptoms show a great similarity with those of infection diseases. This complicates the

diagnosis of cancer, which leads to a late detection and more difficult treatment. The

symptoms will give rise to further investigation. If an early diagnosis is performed, the

possibility to obtain a successful treatment increases.

Different techniques are available to detect the presence of cancer cells. Detection

techniques, based on whole body imaging, give a clear visualisation of the tumour with

information about the localisation, size and possible phase of metastasis. The best known

visualisation techniques are based on x-rays (CT scan), radionuclides (PET scan) and magnets

(MRI scan). Blood and urine tests are necessary to confirm the presence of tumour cells, to

give an idea about the amount of cells and to measure the effect of the tumour on the body.

After a tumour is detected, treatment is needed. The therapy depends on the person,

tumour and phase of metastasis.

Chemotherapy is a widely used and strongly acting treatment, but has a lot of

adverse effects and a high toxicity. The therapy is based on the administration of chemical

agents which interact with a phase in the development of quickly dividing cells. Cancer cells

are killed, but other quickly dividing cells of the body, like the cells of the bone marrow, are

also influenced by these agents. This undesired effect induces the adverse effects known for

the traditional chemotherapeutic agents.

2

In the last ten years a lot of research has been done in order to decrease the adverse

effects and improve the selectivity of the imaging and therapeutic agents. Nanomedicines

are one of the best candidates recently proposed to enhance the specificity, delivery and

efficiency of these agents.

1.2 MAGNETIC RESONANCE IMAGING

Magnetic Resonance Imaging (MRI) is the preeminent methodology among the

various diagnostic modalities currently available. It offers a powerful way to map structures

and to function in soft tissues via a determination of the amount, flow and environment of

water protons in vivo. MRI is based on the principles of Nuclear Magnetic Resonance (NMR),

but the spatial information is recovered through the application of magnetic field gradients.

The resonance of water protons becomes dependent on the position they occupy in the

observed tissue.

The contrast in a MRI image is determined by a complex interplay between two

factors. First, the intrinsic factors such as the proton density and the longitudinal (T1) and

transverse (T2) relaxation times of tissue water protons. Second, the instrumental factors

such as the type of pulse sequence used, designed to enhance the contrast on the basis of

the differences in T1 or T2.

1.2.1 The theory behind MRI

MRI is based on the signal that is emitted by the nuclei of hydrogen atoms when they

are placed within a magnetic field. The nuclei of hydrogen atoms consist of one proton and

one electron. The protons are characterised by a positive charge and a rotation around their

own axis called spin. The rotating mass has an angular momentum and a magnetic moment

(B) when an electrical charge is added. Only the magnetic moment rotation is visible because

it has the possibility to generate a signal in a receiver coil.

When an external magnetic field (B0) is induced the proton will behave as a magnet.

The magnetic moment (or spin) of the proton will obtain the same direction as B0. This

change of direction is associated with a dissipation of energy. A longitudinal magnetisation

(Mz) arises, this represents the sum of the individual magnetic moments.

3

The axis of rotation also alters because of the external force induced by the

gravitational field of the earth (G). This process is called precession, the spinning top starts

to wobble and will fall over. The speed of this phenomenon, called Larmor frequency, is

related to the strength of the external magnetic field B0. “The Larmor or precession

frequency is the rate at which spins wobble when placed in a magnetic field.” (How does MRI

work?, Weishaupt, Dominik Köchli, Victor D, Marincek, Borut)

Introducing a radiofrequency pulse (RF) with the same frequency as the Larmor

frequency will provide energy to the spin system. This energy excites the proton with the

longitudinal magnetisation Mz moving away from the z-axis to the xy-plane. Transverse

magnetisation Mxy will arise. To obtain the transverse magnetisation a 90° RF pulse is

needed. This magnetisation rotates around the z-axis and provokes a voltage (MR signal),

that can be detected and translated into an image.

Figure 1.1: Transition from Mz to Mxy by a RF pulse

There are two important processes one should be aware of: the longitudinal

relaxation time (T1) and the transverse relaxation time (T2). These processes limit the

detection time of the MR signal and are responsible for the return of the spin to his stable

state. In this thesis only the longitudinal relaxation time, T1, will be of interest.

The longitudinal relaxation time or T1 recovery refers to a decrease in the transverse

magnetisation Mxy and the MR signal and to an increase in the longitudinal magnetisation

Mz. During this process the protons lose the energy that gave rise to their excitation. An

energy exchange occurs with a certain speed depending on the molecules. The speed is

associated with a certain time called T1. Free water will have a short T1 because of the small

molecular size, when the water is partially bound the speed can be altered.(2)

The knowledge of this T1 is of great value in the making of contrast agents to accomplish a

good MR image.(3)(4)

4

1.2.2 Contrast agents

The contrast in MRI can be further increased through the use of appropriate contrast

agents (CAs). The presence of CAs causes a significant increase in the longitudinal relaxation

time of water protons, allowing CAs to add physiological information to the morphological

image. Great remarkable effects on the relaxation times of tissue water protons are given

through the interaction with paramagnetic CAs containing unpaired electrons.

One of the most important classes of CAs for MRI consists out of polyaminocarboxylate

complexes of Gd3+.(5)

1.2.2.1 Gadolinium

Gadolinium (Gd) is a paramagnetic metal ion that possesses 7 unpaired electrons.

Most of the contrast agents used in clinical settings are polyaminocarboxylate complexes of

the Gd3+ ion, because the free ion has a high toxicity; it interferes with Ca2+ pathways. The

ligands are multidentate (seven or eight donor atoms) in order to form complexes with very

high thermodynamic and kinetic stability thus limiting the release of the toxic free metal ion.

In a proton MR image there is a direct proportionality between the observed relaxation

enhancement and the concentration of the paramagnetic MRI reporter (equation 1.2).

During the visualisation it is desired to have a large amount of agent in the intravascular

space, after visualisation a fast clearance of the agent is desired. These MR contrast agents

are called “blood pool” agents. A lot of research is done to obtain suitable agents.(5)

Gd3+ chelates improve sensitivity, specificity and tissue characterisation. The

gadolinium atom shortens the T1, T2 and T2* relaxation times of the surrounding water

protons.(6)(7)

This relaxation time is of great importance in the visualisation of the tissues where the

contrast agent is distributed.(8)

If a paramagnetic substance is dissolved in an aqueous solvent, the observed

longitudinal relaxation rates R1 (=1/T1) of the solvent protons can be described as the sum of

the paramagnetic (p) and diamagnetic (d) contributions. The diamagnetic contribution is the

water relaxation rate in the absence of the paramagnetic compound (equation 1.1).

5

(1.1)

where: T1,obs = observed longitudinal relaxation time (s)

T1,d = diamagnetic longitudinal relaxation time (s)

T1,p = paramagnetic longitudinal relaxation time (s)

(1.2)

where: T1,obs = observed longitudinal relaxation time (s)

T1,d = diamagnetic longitudinal relaxation time (s)

r1,p = relaxivity of the paramagnetic compound (mM-1s-1)

Gd(III) = concentration of gadolinium (mM)

Considering equation 1.2, the relaxivity of the paramagnetic compound (mM-1s-1) is

defined as the slope of the line that expresses the linear dependence between the relaxation

rate of the solvent (in absence of solute-solute interactions) and the concentration of

paramagnetic species.

1.3 NANOPARTICLES

When compared to other imaging modalities, the main advantage of MRI is its superb

spatial resolution whereas its major drawback is represented by the limited sensitivity of the

probes. To generate a detectable contrast in MR images, the realisation of highly efficient

contrast agents directed to specific molecular targets will expand the range of applications.

A combination of the non-invasiveness and the high spatial resolution of MRI with the

specific localisation of molecular targets is desired. For this purpose, nano-systems such as

PLGA nanoparticles, liposomes, micelles, micro-emulsions, polymers, etc. are used. They are

capable of carrying high amounts of contrast agents to the site of interest. The nano-carriers

are functionalised with suitable molecules that are able to recognise the selected cellular

1

T1,obs1

T1,d1

T1,p

1

T1,obs1

T1,d r1,p Gd III

6

target. For this reason the research for specific carriers, capable to deliver the contrast

agents within the cell, is a very important step, also in perspective of the simultaneous

delivery of therapeutic agents.(9)(10)

In recent years nanoparticles (NPs) became of great interest in the development of

medicines, especially in the field of cancer treatment. Nanoparticles are small structures that

have a size around 100 nm. A large surface is available to bind, adsorb and carry other

compounds. A lot of advantages are related to their specific characteristics. Depending on

the desirable application the composition of the NP may vary and natural or synthetic

materials are used.(11)

NP formulations may consist of more than one component, therefore they can be

used as theranostics; the particle functions as a diagnostic and therapeutic at the same time.

The diagnostic side refers to the visualisation and observation of the development of the

disease and permits to follow drug distribution in real time. The therapeutic side refers to

the incorporation of a drug to cure the disease.

Figure 1.2: Structure of a theranostic NP

Creating theranostic NPs permits to follow the drug delivery and the effect of the

therapy. Imaging contrast agents are bound to the NP. The internalisation rate of the agent

in the particle is of great importance to obtain a good contrast. Depending on the

visualisation technique different agents are used.(9)

The aims of research are to attain a more specific drug delivery, to reduce side effects

and to obtain a better biocompatibility.

7

In the drug delivery system development, biodegradable polymer formulations are

used. The degradation of the particle will result in release of the drug. The release must take

place in the cytosol of the cell to give an effect.

The NP size and surface characteristics influence the efficiency of the particles. A

small size ensures good uptake and distribution in the cells. Coating of the NP with a

polymer improves the endosome internalisation due to an alteration of the surface charge. A

positive charge enables the NP to escape the endosome and enter into the cytoplasm of the

cell.(10)

A reduction in the toxicity to the surrounding healthy tissues can be obtained by

developing target-specific NPs. Specific localisation and selective release is possible. These

NPs are very interesting to reduce the adverse effects and toxicity that are accompanied

with chemotherapeutic agents. As stated before, chemotherapeutic agents kill not only the

quickly dividing cancer cells but also normal cells. Internalisation of a chemotherapeutical

agent and a tissue specific compound in the same particle reduces the undesired effects. An

efficient targeting ligand for the tumour site is needed. Passive and active targeting can be

considered. Passive targeting is based on the tumour vasculature and poor lymphatic

drainage. These two factors give rise to an enhanced permeability and retention in the

tumour tissue. The use of this passive targeting method is limited by the unpredictable

nature of a tumour.(12)

Certain tumour cells overexpress specific receptors, this phenomenon gives rise to

the development of active targeting methods. The active targeting ligands must recognise

the receptors and bind to them. Today there are different studies in literature about these

receptors. Due to the many opportunities, the active targeting is under full research.(9)(10)

1.3.1 PLGA

PLGA or poly(D,L-lactic-co-glycolic acid) is a biodegradable copolymer. When

hydrolysis of this polymer occurs, the monomers lactic acid and glycolic acid are formed. The

two monomers are endogenous and metabolised by the body. These two characteristics

make sure that PLGA has a minimal systemic toxicity. The polymer can differ in composition

8

and molecular weight, which influences the degradation time. The form is described using

the monomer ratio.

Figure 1.3: PLGA hydrolysis n=number of units of lactic acid m=number of units of glycolic acid

PLGA has a lot of interesting characteristics for the use as a drug delivery system in

the form of NPs. PLGA-NPs are stable and easily internalised in the cell through endocytosis.

Endolysosomal release occurs with uptake of the NPs in the cytosol, which is needed to

obtain the desired therapeutic effect.

The PLGA particle is hydrophobic, this characteristic gives a signal to the reticulo-

endothelial system (RES) of the body to eliminate the particle. To obtain a better cell uptake

surface modification, by coating the particle with a compound that hides the hydrophobicity,

is applied to avoid the recognition by the RES. One of the most used coating polymers is

polyethylene glycol (PEG). This hydrophilic polymer increases the biocompatibility. Coating

the PLGA particle is also used to obtain a positive charged NP with a higher

internalisation.(12)

Different methods are possible to prepare PLGA-NPs. The mayor methods are

emulsification solvent evaporation, emulsification solvent diffusion, emulsification reverse

salting out and nanoprecipitation. They all consist of two steps. In the first step an emulsion

is formed, later in the second step formation of the nanoparticles takes place.(13) The

structure will depend on the used method and the particle size is determined by the

surfactant concentration and the sonication time.(14)

Due to the large surface area and the many functional groups to bind visualisation

and targeting agents, there is a great interest in these particles for the delivery of anti-cancer

drugs. The most common researched drugs until now are paclitaxel, docetaxel, curcumin,

doxorubicin and cisplatin.(13)

9

1.3.2 The use of folate as a targeting ligand for PLGA-NPs

Folate or folic acid (vitamin B9) is needed in the human body to catalyse one-carbon

transfer reactions. The vitamin is necessary to synthesise nucleotide bases. Proliferating

cells, like cancer cells, need folic acid in high amounts.(15)

Folate uptake is possible in several ways: via membrane associated proteins, via a

reduced folate carrier or through a folate receptor (FR). Normal cells use the two first

mechanisms because not so much folate is needed. The last mechanism is found on specific

cells like activated macrophages and polarised epithelial cells. Cancer cells also show a

higher expression of FRs due to their increased need for growth factors (nucleotide bases).

Different types of FRs are known. The two most important ones are FR-α and FR-β.

FR-α is found on 40% of the tumour cells. The FR has an affinity for folate and his conjugates.

Folic acid is small, stable and has a high binding affinity and low immunogenicity. This opens

the possibility to use folic acid in the development of targeted nanoparticles.(16) Uptake in

the cell is obtained with receptor-mediated endocytosis.(17) Folate conjugates bind to the

receptor and the cell membrane will develop into an endosome. The internalisation of folate

conjugates is low, only 15 to 25% of the total bound amount is released in the cell and

stands in correlation with the number of FRs expressed on the cell. The internalisation rate is

1-2 x 105 molecules/cell/h. The endosome acidifies and allows the release of folate in the

cytosol. In this way the NPs are delivered only in cancer cells overexpressing FRs.(15)(18)

1.4 THERAPEUTIC AGENTS

1.4.1 Curcumin

Curcumin or diferuloylmethane is a yellow polyphenolic compound which is present

in the rhizome of the plant Curcuma longa. The molecule is characterised by a low solubility

in water, a high sensitivity at physiological pH and a low bioavailability.

Figure 1.4: Structure of curcumin

10

The use of curcumin as a therapeutical is limited by its characteristics. Optimisation

of the bioavailability is desired. The bioavailability can be enhanced by the use of an

adjuvant or through the incorporation of curcumin into nanoparticles. An adjuvant is a

molecule that enhances the effect of another molecule. Piperine, quercetin or genistein can

be used. They interact with the metabolism of curcumin which leads to a decrease in

degradation and increase in bioavailability.

The incorporation of curcumin in nanoparticles has three mayor advantages. First,

the particle protects the compound from degradation. Second, a more specific delivery can

be obtained because the NPs have the possibility to bind with targeting ligands. Third, the

bioavailability will improve due to a lower hydrophobicity, a longer circulation time and a

higher permeability through membranes.(13)(17)

Once curcumin is taken up in the body it influences a lot of signalling pathways.

Curcumin is of high interest for the use as an anti-cancer drug. The natural compound

regulates multiple intracellular signalling pathways. The affected pathways play a role in the

inflammation process, cell proliferation and invasion, apoptosis and genomic modulations.

Curcumin induces or inhibits these processes in such a way that the cancer cells are not able

to grow anymore and die. The effects are structure-related: more methoxylation and

unsaturation and less hydrogenation give a higher anti-inflammatory and anti-tumour effect.

Using curcumin, as a single therapy, it has the potential to prevent cancer, in combination

with other anti-cancer drugs it can be used in cancer treatment therapies.(19)

1.4.2 Boron

Boron (B) is a chemical element that has an important role in the anti-cancer therapy

called Boron Neutron Capture Therapy (BNCT). The therapy is based on the irradiation of 10B

with neutrons generated by nuclear reactors; these are only available in the United States,

Japan, Argentina and several European countries. The neutrons are captured and give

induction to excited 11B atoms. The excited atoms fall apart in 4He2+ (α-particles) and 7Li3+

ions. Thus irradiation with low energy neutrons gives rise to a high linear energy transfer.

The release of high energy in the form of the ions induces cell death. Due to the short path

length (5-9 µm) of the ions only the cells containing boron experience the cytotoxic effect.

This selectivity gives a reduction in side effects.(20)

11

The effectivity of BNCT depends on the used boron delivery agents, which are still in

research for optimisation. The aim is to create an agent that consists out of all the

requirements. The requirements are: low uptake in normal cells, low systemic toxicity and

high uptake in tumour cells. Persisting levels in the tumour during the therapy are desired

with a fast clearance out of the blood and normal tissues after the BNCT. To obtain a

therapeutic effect the concentration needs to be around 20 µg of boron per gram of tumour

mass (20 ppm). This last requirement is the most difficult one and has to be reached first

before irradiation will be performed.

The biodistribution of these agents is different from patient to patient, with an

unpredictable boron uptake and distribution. Methods to measure the concentration in the

tumour are based on empirical data of tumour-to-blood, tumour-to-brain and brain-to-blood

concentration ratios. Real time detection of the concentration is possible with imaging

techniques as PET or MRI.(20)(21) This requires an extra imaging agent bound to the boron

agent.

12

2. OBJECTIVE

Nowadays the treatment of cancer knows a lot of side effects due to a non-specific

drug delivery. The introduction of nanoparticles creates new possibilities to develop specific

systems for drug delivery and diagnosis, with reduced toxicity and fewer side effects.

The aim of this research is to create and optimise PLGA-based theranostic

nanoparticles which can be used for specific drug delivery and real-time monitoring of

cancer treatment. The particles are loaded with rubrocurcumin (RbCur), a Gd-lipophilic

complex (Gd-DOTAMA(C18)2) and PEG-phospholipids functionalised with folate. The

particles need to have a good stability, a minimal toxicity, a good uptake and a specific

action in the cell.

First, the rubrocurcumin synthesis is optimised in order to obtain a pure complex

with a good yield. Curcumin and boron are combined in a stable adduct with the use of

oxalic acid. Thin Layer Chromatography (TLC), Nuclear Magnetic Resonance (NMR) and

UV/VIS spectrophotometry are used to check the purity.

The complex has a double action; boron gives the possibility to use BNCT and curcumin

possesses anti-cancer effects.

Second, optimisation of the NP process is needed. The development of the particles

consists of different steps that influence the size, toxicity and incorporation. The theranostic

effect depends on the incorporation of an amount of RbCur and Gd-complex sufficient to

obtain a therapeutic effect detectable by MRI. A fixed percent of Gd is loaded in the

particles. A calculation has to be made to obtain a sufficient amount to permit their

visualisation in vitro.

Third, the stability of the particles is checked by measuring the remaining Gd and

RbCur concentration and the relaxivity of the particle over several days of dialysis at 37°C in

an isotonic NaCl/Hepes buffer (HBS). The first 24 hours are the most important ones.

The last part of the study consists of the incubation of the nanoparticles in vitro in

cancer cells and the MRI visualisation.

13

3. MATERIALS AND METHODS

3.1 REAGENTS

For the RbCur complex synthesis curcumin, boric acid and oxalic acid were purchased

from Sigma Aldrich (St. Louis, Missouri, USA). The solvents dichloromethane, ethylacetate,

ethanol and acetone-d6 were purchased from VWR chemicals (Radnor, Pennsylvania, USA).

Toluene was obtained from Riedel-De Haën (Honeywell) (Seelze, Germany).

For the PLGA-NP preparation, PLGA 50:50 with an average molecular weight (Mw)

from 30000 to 60000 Da was purchased from Sigma Aldrich (St. Louis, Missouri, USA). Gd-

DOTAMA(C18)2 was purchased from Bracco Imaging (Milan, Italy). DSPE-PEG methoxy with a

Mw of 2000 and DSPE-PEG Folate were obtained from Avanti Polar Lipids (Albaster,

Alabama, USA). The solvents methanol and chloroform were purchased from VWR chemicals

(Radnor, Pennsylvania, USA). The 3% PVA solution was made out of Mowiol 4-88 from Sigma

Aldrich (St. Louis, Missouri, USA).

A 3% PVA solution was prepared dissolving 1.5 g Mowiol 4-88 (PVA) from Sigma

Aldrich (St. Louis, Missouri, USA) in 50 mL bidistillated water. The solution was heated until

70°C and stirred in a water bath for about 20 min until PVA was completely dissolved.

A NaCl/Hepes buffer (HBS) was prepared dissolving 1.5 M NaCl and 0.038 M Hepes

(4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid), both from Sigma Aldrich (St. Louis,

Missouri, USA), in bidistillated water. The solution was brought to pH 7.4 by adding sodium

hydroxide anhydrous pellets purchased from Carlo Erba reagents (Val de Reuil, France). A

1:10 dilution of this stock solution was used for all the methods.

Information about the LOT and CAS number is included in 8.1.

14

3.2 METHODS

3.2.1 Preparation and characterisation of the RbCur drug complex

A complex between boron, curcumin and oxalic acid was prepared using following

instructions:

“A suspension of curcumin (369 mg, 1 mmol), boric acid (61.8 mg, 1 mmol) and oxalic acid

dihydrate (126 mg, 1 mmol) in toluene is heated under reflux using a Dean-Stark trap for 16h.

After filtration the solid was recrystallized from ethyl acetate and acetonitrile.”

(Inhibition of the HIV-1 and HIV-2 Proteases by Curcumin and Curcumin Boron complexes,

Bioorganic & Medicinal Chemistry Vol. 1, No. 6, pp. 415-422, 1993)

Figure 3.1: Boron/curcumin complex with oxalic acid (RbCur)

The above described synthesis was not very clear and adjustments were introduced.

A suspension of curcumin (369 mg, 1 mmol), boric acid (61.8 mg, 1 mmol) and oxalic acid

dihydrate (126 mg, 1 mmol) was stirred and heated under reflux (120°C) for 16 hours in 50

mL of toluene. Then, 25 mL of the suspension was centrifuged for 10 min at 4000 rpm. The

purity of the surnatant and the precipitate was assessed by Thin Layer Chromatography

(TLC). TLC is a technique to separate non-volatile mixtures, based on the polarity of the

compounds. The mobile phase for the TLC was made out of a mixture of dichloromethane

and ethyl acetate, respectively in a ratio 7:3. The stationary phase consisted of a TLC Silica

gel 60 F254 plate (Merck KGaA, Darmstadt, Germany) containing silica gel. The TLC showed in

both the precipitate and the surnatant the stain of unreacted curcumin. The precipitate

consists of more impurities, for this reason it was not further used. The surnatant was

evaporated, dissolved in dichloromethane and stored at -20°C for one night (solution 1).

15

To the remaining 25 mL other 0.5 mmol oxalic acid and 0.5 mmol boric acid were added to

react with the remaining unreacted curcumin. The reaction was stirred and heated under

reflux for other 16 hours in toluene. After 16 hours a precipitate was formed. The precipitate

was separated from the surnatant and dissolved in dichloromethane (solution 2). A TLC of

the surnatant and precipitate was performed. The surnatant was evaporated (complex 3).

Both dichloromethane solutions (1 and 2) were filtered and TLC profiles of the

filtrates were compared. The TLC results showed a similar profile, so both filtrates were

evaporated together (complex 4). Finally, complexes 3 and 4 were obtained.

1H NMR profiles were performed at 600 MHz on a Bruker Avance NMR spectrometer

(Bruker Instruments, Milan, Italy) to check the purity of the obtained complexes. An amount

of the two complexes was dissolved in 0.6 mL acetone-d6 and put in a NMR tube. By

comparing the spectra of 3 and 4, it was possible to conclude that the complexes 3 and 4

were very similar in purity and so they were dissolved together in methanol.

The yield of the reaction was determined as follows: an empty round flask was

weighed (mass 1). The complex dissolved in methanol was added to the flask and the solvent

was evaporated. After evaporation the flask was weighed again (mass 2). The gain was

calculated using equation 3.1.

𝑚𝑎𝑠𝑠 2−𝑚𝑎𝑠𝑠 1

466 𝑚𝑔/𝑚𝑚𝑜𝑙∗ 100 (3.1)

The dry complex was collected into a bottle and stored at -20°C.

The characterisation of the complex was performed using an UV/VIS

spectrophotometer Jenway 6715 (Montepaone, San Mauro Torinese, Italy). A small amount

of complex was dissolved in ethanol and a spectrum was acquired in a range from 360 nm to

700 nm.

3.2.2 Preparation of PLGA nanoparticles

Two types of particles were prepared, one targeted with a folate derivate and the

other non-targeted to compare the difference in uptake in cancer cells overexpressing folate

receptors. Nanoparticles were obtained using an oil-in-water emulsion solvent extraction

method.

16

3.2.2.1 Non-targeted nanoparticles

Table 3.1: Formulation of non-targeted nanoparticles

Substance Amount

Phase 1

PLGA 25 mg

Gd-DOTAMA 3.2 mg

DSPE-PEG 2000 methoxy 2.2 mg

RbCur 6 mg

Phase 2 PVA 3%-3 mL

Two phases were prepared (Table 3.1). Phase 1 (PLGA, Gd-DOTAMA, DSPE-PEG2000

methoxy and RbCur) was dissolved in 540 µL chloroform + 60 µL methanol. The use of

methanol improved the solubility of the components. To facilitate the dissolution of PLGA,

the vial containing the phase 1 was sonicated in a sonication bath at 25°C for a few minutes.

The phase 1 solution was maintained in ice to minimize the evaporation of the solvent. The

cold solution of phase 1 was added drop by drop in the cold phase 2 solution (3 mL) and

sonicated (Bandelin electronic, Berlin, Germany) for 5 min at 100% power. After the

sonication step, the size of the particles was determined using a dynamic light scattering

(DLS) Zetasizer Nano series 3000HS (Malvern Instruments, Malvern, U.K.). When the

particles population was not homogeneous (more than one population with differently sized

particles was shown in the distribution profile), it was necessary to sonicate the emulsion

again for 5 min until only one population was obtained.

The final emulsion was transferred to a 50 mL round-bottom flask and put into a

rotary evaporator Laborota 4001 (Heidolph, Torre Boldone, Italy) for 120 min at 740 mmHg

and 30 rpm to remove the organic solvent. After the evaporation step, dialysis in a

membrane (molecular weight cut-off of 14000 Da) was carried out at 4°C using an isotonic

NaCl/Hepes buffer (HBS). The dialysis was performed in 1 L of HBS that was renewed after 4

hours and dialysis was continued for another 16 hours. The excess of PVA was removed by

washing the emulsion with a vivaspin 20 filter (Sartorius AG, Goettingen, Germany) (cut-off

of 1 x 106 Da) until the final concentration of 0.1% was reached: to get this PVA

concentration the nanoparticles suspension (3 mL), containing 3% PVA, was diluted to 20 mL

with HBS. Filtration was performed centrifuging the vivaspin at 6000 rpm until a volume of 4

mL was reached. This washing step was repeated twice until the final concentration of PVA

reached 0.09% (< 1%). These volumes were calculated using equation 3.2.

17

𝐶𝑖 ∗ 𝑉𝑖 = 𝐶𝑓 ∗ 𝑉𝑓 (3.2)

Where: Ci = initial concentration

Vi = initial volume

Cf = final concentration

Vf = final volume (20 mL)

3 𝑚𝐿 𝑃𝑉𝐴∗3%

20 𝑚𝐿= 0.45%

4 𝑚𝐿 ∗0.45%

20 𝑚𝐿= 0.09%

Finally, the NPs were sonicated for 5 min in a sonication bath to remove aggregates

that were formed during the filtration step and centrifuged 10 min at 6000 rpm.

3.2.2.2 Folate-targeted nanoparticles

Folate-targeted nanoparticles were prepared following the same procedure as

described above for the non-targeted ones with a few modifications (Table 3.2). First, the

DSPE-PEG Folate solution (1.2 mL) was evaporated in a 5 mL round flask. Then, PLGA was

dissolved in 0.6 mL of organic solvent (540 µL chloroform + 60 µL methanol) and this volume

was added to the flask containing the DSPE-PEG Folate. Finally, Gd-DOTAMA, DSPE-PEG2000

methoxy and RbCur were weighed and dissolved in the same flask.

Table 3.2: Formulation of folate-targeted nanoparticles

Substance Amount

Phase 1

DSPE-PEG Folate(1 mg/mL) 1.2 mg (1.2 mL)

PLGA 25 mg

Gd-DOTAMA 3.2 mg

DSPE-PEG 2000 methoxy 1 mg

RbCur 6 mg

Phase 2 PVA 3%-3 mL

3.2.3 Characterisation of the particles

3.2.3.1 Particle size

The hydrated mean diameter and the poly dispersity index (PDI) of the PLGA-NPs

were determined using a DLS Zetasizer Nano series 3000HS (Malvern Instruments, Malvern,

UK). All samples were analyzed at 25°C in filtered (cut-off of 100 nm) HBS (pH 7.4).

18

3.2.3.2 Stability

The stability was evaluated by measuring the relaxivity of PLGA-NP suspensions and

their release of RbCur following next procedure.

0.5 mL of the PLGA-NP suspension were diluted to 1.5 mL with HBS. The obtained solution

was dialyzed at 37°C in 1 L of HBS for several days. Parafilm and aluminium films were used

to cover the dialysis beaker containing the buffer. 200 µL of the suspension was taken out at

different times; 0, 3, 6, 24, 72 and 96 hours and transferred into an eppendorf. This amount

was used to determine the concentration of curcumin, RbCur and the relaxivity measuring

the Gd concentration and the relaxation rate (R1).

To determine the yield of encapsulation of RbCur in PLGA-NPs two calibration curves

were prepared, one for curcumin (Table 3.3, Figure 3.2) and one for RbCur (Table 3.4, Figure

3.3). For the curcumin curve, a 0.1 mg/mL solution was prepared by dissolving 5 mg of

curcumin in 50 mL ethanol and 2 mL of this solution was further diluted to 20 mL with

ethanol to obtain a concentration of 10 µg/mL. For the RbCur curve 2.8 mg of RbCur was

dissolved in 48 mL ethanol and 1 mL of this solution was diluted 10 times with 9 mL ethanol;

the obtained 10 mL had a concentration of 5.8 µg/mL. Out of these stock solutions, different

solutions with decreasing concentration were prepared and their absorbance was measured

with an UV/VIS spectrophotometer Jenway 6715 (Montepaone, San Mauro Torinese, Italy)

at λmax; 430 nm and 545 nm for curcumin and RbCur, respectively.

For the extraction of the encapsulated RbCur out of the PLGA-NPs, the

measurements were carried out, in duplicate, as follows. 50 µL of the NP suspension in the

eppendorf were dissolved in 1 mL ethanol and centrifuged for 10 min at 6000 rpm. The

extracted RbCur in the surnatant were transferred in a cuvette. As blank was used 1 mL

ethanol + 50 µL HBS. A spectrum from 360 nm to 700 nm was recorded and the absorbances

were observed at 430 nm and at 545 nm; at these wavelengths respectively curcumin and

RbCur showed their maximum peak. The concentration of RbCur was also measured with

inductively coupled plasma mass spectrophotometry (ICP-MS) (Thermo-Finnigan, Rodano,

Italy) measuring the amount of boron loaded in PLGA-NPs. The correlation between the two

techniques was evaluated.

19

Table 3.3: Calibration curve values for curcumin

Ethanol (µL) Solution of 10 µg/mL

of Cur (µL)

Concentration

(µg/mL)

Absorbance at 430

nm

1000 0 0 0

950 50 0.5 0.08

900 100 1 0.161

800 200 2 0.307

700 300 3 0.464

600 400 4 0.623

500 500 5 0.782

Figure 3.2: Calibration curve for curcumin at 430 nm

y = 0,1558x R² = 0,9999

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

0 1 2 3 4 5 6

Ab

sorb

ance

Concentration (µg/mL)

20

Table 3.4: Calibration curve values for RbCur

Ethanol (µL)

Solution of 5.8

µg/mL of RbCur

complex (µL)

Concentration (µg/mL) Absorbance at 545 nm

1000 0 0 0

900 100 0.58 0.041

700 300 1.74 0.094

400 600 3.48 0.196

200 800 4.64 0.276

0 1000 5.8 0.331

Figure 3.3: Calibration curve for RbCur at 545 nm

y = 0,0574x + 0,0011 R² = 0,9977

0

0,05

0,1

0,15

0,2

0,25

0,3

0,35

0,4

0 1 2 3 4 5 6 7

Ab

sorb

ance

Concentration (µg/mL)

21

Out of the calibration curves, equations 3.3 and 3.4 were obtained for curcumin and

RbCur respectively.

𝑦 = 0.1558𝑥 (3.3)

Where: x = concentration of curcumin (µg/mL)

y = mean absorbance at 430 nm

𝑦 = 0.0574𝑥 + 0.0011 (3.4)

Where: x = concentration of RbCur (µg/mL)

y = mean absorbance at 545 nm

3.2.3.3 Relaxometric measurements

The water proton longitudinal relaxation rates (1/T1) were measured with a

Spinmaster spectrometer (Stelar S.n.c., Mede, Italy) at 21.5 MHz and 25°C. 1H spin-lattice

relaxation times T1 were acquired by the standard inversion recovery (IR/S) method with a

typical 90° pulse width of 3.5 µs performing 16 experiments of 4 scans. The temperature was

controlled with a VTC-91 airflow heater (Stelar S.n.c., Mede, Italy) equipped with a copper–

constantan thermocouple (±0.1°C). The water proton longitudinal relaxation rates R1,w (1/T1)

were measured using 50-60 L of the NP samples in 5mm NMR glass tubes.

To determine the amount of contrast agent incorporated in the NPs, a glass vial

containing 100 µL of HCl 37% and 100 µL of the sample was prepared. The vial was

centrifuged for 3 min at 2000 rpm, closed and placed in an oven at 110°C for 16 hours. Upon

this treatment, all GdCl3 is solubilised as the free aquo-ion. The water proton 1/T1 (R1,HCl) of

these solutions were measured at 21.5 MHz and 25°C. The Gd3+ concentration was

determined using equation 3.5, that was obtained from a calibration curve using standard

GdCl3 solutions (0.01–2.00 mM) giving a Gd3+ relaxivity of 13.7 mM-1s-1. The method was

double-checked by ICP-MS element-2 (Thermo-Finnigan, Rodano, Italy) measurements.

[𝐺𝑑(𝐼𝐼𝐼)] = (𝑅1,𝐻𝐶𝑙−0.5

13.7) ∗ 𝛼 (3.5)

Where: [Gd(III)] = Gd3+ concentration in the sample (mM)

R1,HCl = relaxation rate in acid (s-1)

22

α = dilution factor

R1, p = (R1,w−0.38

[Gd(III)]) ∗ α (3.6)

Where: R1,p = millimolar relaxivity (mM-1s-1)

R1,w = relaxation rate in water (s-1)

[Gd(III)] = Gd3+ concentration in the sample (mM) (see equation 3.5)

α = dilution factor

3.2.3.4 Nuclear Magnetic Resonance Dispersion (NMRD) profiles

The nuclear magnetic relaxation 1/T1 dispersion profiles of water protons were

measured over a magnetic field strength continuum from 0.00024 T to 0.5 T (corresponding

to 0.01 MHz to 20 MHz proton Larmor frequency) on the fast field cycling Spinmaster FFC

2000 relaxometer (Stelar S.n.c., Mede, Italy) equipped with a silver magnet. The relaxometer

operated under complete computer control with an absolute uncertainty in the 1/T1 values

of ±1%. The typical field sequences used were the non-polarised sequence between 20 and 8

MHz and the pre-polarised sequence between 8 and 0.01 MHz. The observation field was set

at 13 MHz. Sixteen experiments of two scans were performed for the T1 determination for

each field. Water proton T1 measurements at fixed frequency were carried out on a

SpinMaster spectrometer (Stelar S.n.c., Mede, Italy) operating in the range from 20 to 80

MHz, by means of the inversion recovery method (16 experiments, two scans). The

reproducibility of the T1 data was ±0.5%. T1 measurements at 300 MHz were acquired on a

Bruker Avance 300 spectrometer (7 T) using a standard saturation recovery sequence.

3.2.4 In vitro uptake and MRI visualisation

A human ovarian carcinoma (IGROV-1) cell line was kindly provided by Dr. Claudia

Cabella (Bracco Imaging, Colleretto Giacosa TO, Italy). IGROV-1 cells were cultured in RPMI-

1640 medium (Lonza, Basel, Swiss) containing 10% (v/v) fetal bovine serum (FBS), 2 mM

glutamine, 100 U/mL penicillin, and 100 U/mL streptomycin. Cells were incubated at 37°C in

a humidified atmosphere of 5% CO2. This cell line was tested for mycoplasma with a

MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza, Basel, Swiss).

23

At 80% confluence, cells were detached with 1 mM EDTA (Lonza, Basel, Swiss). A cell

count was performed using a burker chamber and a microscope (Optika microscopes,

Ponteranica, Italy). 10 µL of trypan blue (0.09%) were mixed with 10 µL of cell suspension

and 10 µL of this mixture was put in the burker chamber. The trypan blue dye exclusion test

is used to determine the number of viable cells present in a cell suspension. It is based on

the principle that living cells possess intact cell membranes that exclude certain dyes, such

as trypan blue, eosin or propidium, whereas dead cells do not. In this test, a cell suspension

was mixed with dye and then examined by a microscope to determine if the cells took up or

excluded dye.

For in vitro uptake experiments, about 4.5 x 105 of IGROV-1 cells were seeded in 6 cm

diameter culture dishes. After 6 hours, the medium was removed and replaced with RPMI

without folate to increase the expression of folate receptors. After 24 hours the medium was

removed and substituted with 2 mL of fresh medium containing different concentrations of

non-targeted and folate-targeted NPs (Table 3.5). The amount of NP solution needed was

calculated using equation 3.2. The final volume here was 2 mL.

Table 3.5: Incubation conditions

Sample Gd concentration (µM) Time (hours)

CTRL / 24

Non-targeted 12.5 24

Folate-targeted 12.5 24

Non-targeted 25 24

Folate-targeted 25 24

Non-targeted 50 6

Folate-targeted 50 6

Non-targeted 100 6

Folate-targeted 100 6

After the incubation, the medium was removed from the cells and they were washed

3 times with phosphate buffered saline (PBS). Then, the cells were detached with 0.5 mL of

trypsin/EDTA and stored for 5 min in a cell incubator (Montepaone, San Mauro Torinese,

Italy) at 37°C. Trypsin, usually in combination with EDTA, causes cells to detach from the

growth surface. This method is fast and reliable but can damage the cell surface by digesting

24

exposed cell surface proteins. The destruction of proteins leads to the detachment of the

cells from the plate surface. Furthermore the trypsin solution contains EDTA as a Ca2+

chelator. Ca2+ is a fundamental ion for the adhesion of proteins.

The trypsin was inactivated by adding fresh medium containing FBS. The cells were

transferred in a 15 mL falcon tube and diluted with PBS to 10 mL. The cells were centrifuged

for 5 min at 1100 rpm. After centrifugation the surnatant was removed and 0.5 mL of PBS

was added to the cell pellet. The cell suspension was resuspended and 30 µL were

transferred in an eppendorf. A cell count was performed as described above. Then, 10 mL of

PBS were added to the cell suspension and the suspension was centrifuged again for 5 min

at 1100 rpm. After centrifugation PBS was removed from the cells and replaced with 40-50

µL of new PBS. These 40-50 µL were transferred into a glass capillary. The capillary was

centrifuged for 5 min at 1000 rpm and placed in an agar phantom for MRI analysis. MRI was

acquired on a Bruker Avance 300 spectrometer (7 T) equipped with a Micro 2.5 micro-

imaging probe (Bruker BioSpin, Ettlingen, Germany) using a standard T1-weighed multislice

multiecho sequence (TR/TE/NEX, 200:3.3:16; FOV, 1.2 cm; one slice = 1 mm; in-plane

resolution, 94 × 94 μm). T1 measurements of the cells were performed using a standard

saturation recovery sequence.

The Gd and B contents in the cell sample were determined using ICP-MS element-2

(Thermo-Finnigan, Rodano, Italy). Sample digestion was performed with concentrated HNO3

(70%, 2 mL) under heating with a MicroSYNTH Microwave labstation (Milestone, Shelton, CT,

USA) and the protein concentration was determined using a commercial Bradford assay

(Biorad, Hercules, CA, USA).

25

4. RESULTS

4.1 RBCUR COMPLEX

Figure 4.1 shows the obtained 1H-NMR spectrum at 600 MHz of complex 3, the

spectrum of complex 4 is not shown because of the similarity. The 1H-NMR spectrum of

curcumin (Figure 4.2) was also acquired to compare it with the neo-formed RbCur complex.

Figure 4.1: 1H-NMR spectrum of RbCur in acetone-d6

Figure 4.2: 1H-NMR spectrum of curcumin in acetone-d6

26

Figure 4.3: UV/VIS spectrum of RbCur in ethanol

The yield of the product was calculated according to equation 3.1.

36239.3 𝑚𝑔−36128.0 𝑚𝑔

466 𝑚𝑔/𝑚𝑚𝑜𝑙∗ 100 = 23.8%

4.2 NANOPARTICLES

4.2.1 Characterisation

Table 4.1: Characteristics of non-targeted and folate-targeted NPs

Gd-

DOTAMA

(Inc.%)

Relaxivity

(R1p)

RbCur

Inc.%

Size

(mean±SD)

PDI

(mean) Gd/B(RbCur)

NON-

TARGETED

NPs

3.2 mg

(46.59%) 25.47 18.93%

150.12±10.66

nm 0.11 1

FOLATE-

TARGETED

NPs

3.2 mg

(36.29%) 28.38 15.94%

143.1±2.83

nm 0.12 1

27

Figure 4.4: Correlation between B concentration measured by ICP-MS and RbCur concentration measured by

UV/VIS spectrophotometry in different PLGA-NPs preparations

4.2.2 Stability

The stability was evaluated by measuring the release of RbCur from the PLGA-NP

suspensions and their relaxivity (mM-1s-1) over several days of dialysis at 37°C in an isotonic

NaCl/Hepes buffer.

As an example, in Table 4.2, 4.3, 4.4 and 4.5 are only the results for one single NP

formulation reported because of the different dilutions that were used for each prepared

NP. In Figure 4.5 and 4.6 the media of the results of all the NPs made are reported.

4.2.2.1 RbCur release

Table 4.2: Mean absorbance after 0, 3, 6, 24, 72 and 96 hours of dialysis at 37°C in isotonic NaCl/Hepes buffer

Time (hours) Non-targeted NPsa Folate-targeted NPsb

430 nm 545 nm 430 nm 545 nm

0 0.532 0.268 0.485 0.247

3 0.3115 0.0995 0.322 0.1025

6 0.3125 0.099 0.287 0.0775

24 0.222 0.0615 0.2125 0.055

72 0.185 0.0415 0.2405 0.042

96 0.190 0.043 0.1695 0.017

a The showed values are of one NP formulation, diluted 1:5

b The showed values are of one NP formulation, diluted 1:4

28

According to the procedure described in chapter 3.2.3.2 and the equations 3.3 and

3.4 the following internalisation rate was calculated out of the measured absorbances.

Table 4.3: Total amount of curcumin and RbCur with the internalisation rate

Time (hours)

Non-targeted NPsa Folate-targeted NPsb

Total

amount

(mg)

Internalisation

rate (%)

Total

amount

(mg)

Internalisation

rate (%)

0 1.414 100 1.231 100

3 0.652 46.11 0.638 51.83

6 0.651 46.04 0.527 42.81

24 0.435 30.76 0.354 28.76

72 0.331 23.41 0.376 30.54

96 0.342 24.19 0.227 18.44

a The showed values are of one NP formulation, diluted 1:5

b The showed values are of one NP formulation, diluted 1:4

Figure 4.5: Stability of the RbCur complex in non-targeted and folate-targeted NPs. The mean and standard

deviation were obtained from all the prepared NPs in this research

4.2.2.2 Relaxation rate, relaxivity and Gd concentration

According to the procedure described in chapter 3.2.3.3 and the equations 3.5 and

3.6 the relaxivity and Gd concentration are calculated out of the measured R1 (in water and

in HCl) for each NP.

0

20

40

60

80

100

120

0 3 6 24 72 96

Inte

rnal

isat

ion

rat

e (

%)

Time (hours)

Non-targeted NPs

Folate-targeted NPs

29

Table 4.4: Relaxation rates, relaxivity and Gd conc. at different times for non-targeted NPsa

Time (hours) R1,w (s-1) R1,HCl (s-1) R1,p (mM-1s-1) [Gd] (mM)

0 5.146 1.704 27.11 0.879

3 4.531 1.682 24.05 0.863

6 4.603 1.668 24.75 0.853

24 4.653 1.654 25.37 0.842

72 4.379 1.507 27.2 0.735

96 4.129 1.458 26.82 0.699

a The showed values are of one NP formulation, diluted 1:5

Table 4.5: Relaxation rates, relaxivity and Gd conc. at different times for folate-targeted NPsa

Time (hours) R1,w (s-1) R1,HCl (s-1) R1,p (mM-1s-1) [Gd] (mM)

0 4.794 1.616 27.08 0.652

3 3.971 1.544 23.55 0.610

6 4.307 1.566 25.25 0.622

24 3.408 1.37 23.84 0.508

72 3.867 1.415 26.12 0.534

96 3.402 1.247 27.72 0.436

a The showed values are of one NP formulation, diluted 1:4

Figure 4.6: The stability of non-targeted and folate-targeted NPs was conducted measuring the relaxivity for

several days. The mean and standard deviation were obtained from all the prepared NPs in this research

0

20

40

60

80

100

120

140

0 3 6 24 72 96

Re

laxi

vity

(m

M-1

s-1)

(%)

Time (hours)

Non-targeted NPs

Folate-targeted NPs

30

4.2.3 NMRD profile

Figure 4.7: NMRD profile of non-targeted and folate-targeted NPs.

4.3 IN VITRO UPTAKE AND MRI VISUALISATION

Figure 4.8: Internalisation studies of targeted and non-targeted Gd-RbCur loaded NPs in IGROV-1 cells

incubated for 6 hours at 37°C in a Gd concentration range 25–200 μM.

31

Figure 4.9: Cell viability for folate-targeted NPs under different incubation conditions

Figure 4.9 shows the cell viability after incubation of IGROV-1 cells with different

concentrations of Gd and RbCur loaded folate-targeted NPs for 6 and 24 hours. After 24

hours of incubation, RbCur led to a high cytotoxic effect on the cells. For the 6 hours of

incubation the cytotoxic effect was lower.

Figure 4.10: Representative T1-weighed spin echo MRI (measured at 7 T) of an agar phantom containing

unlabeled cells (1) or cells incubated with non-targeted NPs: 25 µM 24h (2), 12.5 µM 24h (4), 50 µM 6h (6)

100 µM 6h (8) or folate-targeted NPs: 25 µM 24h (3), 12.5 µM 24h (5), 50 µM 6h (7) 100 µM 6h (9)

32

Table 4.6: T1 of cell samples under different conditions measured by MRI

Number Cell sample T1 (ms) measured by

MRI 1/T1 (s

-1)

1 CTRL 2420 0.413

2 Non-targeted 25 µM

Gd 24 hours 1698 0.589

3 Folate-targeted 25

µM Gd 24 hours 1596 0.626

4 Non-targeted 12.5

µM Gd 24 hours 1876 0.533

5 Folate-targeted 12.5

µM Gd 24 hours 1783 0.561

6 Non-targeted 50 µM

Gd 6 hours 1898 0.527

7 Folate-targeted 50

µM Gd 6 hours 1515 0.660

8 Non-targeted 100

µM Gd 6 hours 1708 0.585

9 Folate-targeted 100

µM Gd 6 hours 1123 0.890

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5. DISCUSSION

5.1 RBCUR COMPLEX

First, the RbCur complex was characterised by acquiring the 1H-NMR spectrum (at

600 MHz in acetone-d6). A 1H-NMR spectrum is of great importance to determine the

structure of a molecule, with respect to the hydrogen nuclei of the molecule. A NMR

spectrum is recorded in solution and solvent protons may not interfere. For this reason a

deuterated solvent is used to dissolve the substance. In the obtained 1H-NMR spectrum of

RbCur (Figure 4.1) the peaks at 2 ppm and 2.6 ppm are due to the solvents, respectively

acetone and water. Around 4 ppm the peak of methyl is found. The peaks from 6 ppm to 10

ppm concern the aromatic part of the molecule. By comparing these peaks with those

obtained for the same complex in literature(22) (3.84 (s, 6H, OCH3), 6.60 (s, 1H, CH), 6.88,

7.37 (2d, J = 8 Hz, 4H, Ar), 7.13, 8.04 (2d, J = 16 Hz, 4H, CH=CH), 7.48 (s, 2H, Ar), and 10.25

ppm (s, br, 2H, OH)), we observed a good similarity between the two spectra.

Second, the 1H-NMR spectrum of RbCur is compared with the spectrum of curcumin

(Figure 4.2). As shown in Figure 3.1 the interaction of boron with the diketonic group of

curcumin, will influence surrounding protons. A peak shift will arise; in the spectrum of

curcumin the chemical shift of the central proton (H1) is found at 6 ppm, in the RbCur

spectrum this peak is shifted to 6.6 ppm. The comparison between the obtained spectrum,

the spectrum found in literature and the curcumin spectrum demonstrated that the adduct

between the curcumin and boron was formed and thus RbCur was obtained.

Finally, the RbCur complex was characterised by acquiring an UV/VIS spectrum from

360 nm to 700 nm with a spectrophotometer (Figure 4.3). A single peak at 545 nm was

noticed, demonstrating that the RbCur complex was formed and no impurities of free

curcumin were present.

According to equation 3.1, the yield of the product was 23.8%. This value is much

lower with respect to the value mentioned in literature (75%) and further improvements of

the synthesis are desired. Despite this low yield, the synthesis method used was suitable for

this research because only 6 mg of RbCur are necessary to prepare one NP formulation. In

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fact, the obtained yield of 23.8% corresponds to a quantity of 111.3 mg, that allows the

preparation of more than 15 particles.

5.2 NANOPARTICLES

5.2.1 Size, relaxivity and RbCur incapsulation yield

Folate-targeted and non-targeted PLGA-NPs were prepared and characterised. The

selected methodology to obtain PLGA-NPs was an oil-in-water emulsion solvent extraction

method. The average hydrodynamic diameters of the PLGA-NPs were obtained by DLS

measurements (Table 4.1). The intensity distribution of the average PLGA-NP hydrodynamic

diameters were unaffected by the presence of the folate at the end of the PEG-2000 chain. A

single peak at 150±10 nm was showed with a PDI lower than 0.2 for all investigated

formulations. These results suggest that all the PLGA-NPs showed a homogeneous

population.

To perform MRI-guided drug delivery NPs were loaded with RbCur and the Gd-based

MRI contrast agent Gd-DOTAMA(C18)2. The millimolar relaxivity (at 21.5 MHz and 25°C) of

Gd-DOTAMA(C18)2 when incorporated in the NP formulation (non-targeted and targeted)

was ca. 27 mM-1s-1. The measured relaxivity is the consequence of the amount of Gd

complexes exposed to the external surface that can interact with the solvent water

molecules. The basic requirement to induce a significant increase in the water proton

relaxation rate is a dipolar interaction between the paramagnetic ion and the water protons.

This behavior was confirmed by analysing the NMRD profile (Figure 4.7). In a NMRD profile,

the relaxation rate (1/T1) is given as a function of the magnetic field or the Larmor frequency

on a logarithmic scale. This is an important tool for the characterisation of NPs because it

gives information about the efficiency (higher relaxivity) at different magnetic fields. The

profile shows a relaxivity hump around 20 MHz, this is typical for slowly moving systems.

The NMRD profiles, the amount of encapsulated RbCur complex (ca. 17%) and the

incorporated Gd-DOTAMA(C18)2 (ca. 40%) in the PLGA-NPs remained almost unchanged for

non-targeted and folate-targeted NPs.

The presence of RbCur in the PLGA-NPs was evaluated by measuring the absorbance

at 545 nm. The boron content was measured with ICP-MS. These two methods were

35

compared and a good correlation was obtained (adj R-square of 0,9931) (Figure 4.4).

Accordingly, the amount of encapsulated RbCur was directly proportional to the boron

content necessary to perform BNCT.

5.2.2 Stability

The stability of the nanoparticles was evaluated measuring the release of RbCur in

the PLGA-NP suspensions under dialysis at 37°C in an isotonic NaCl/Hepes buffer for several

days. Figure 4.5 shows that the RbCur complex was quickly released from the NPs. In fact,

only after 3 hours of dialysis, about 40% of RbCur was released in the buffer and after 24

hours, the amount released reached about 60%. The drug release of folate-targeted and

non-targeted NPs was compared but no significant differences of the trend were noticed.

This quite fast release of RbCur from the NPs could be the consequence of the relatively low

hydrophobicity of the RbCur complex that causes, after long incubation in water at 37°C, a

release from the internal PLGA core.

During the stability test also a degradation of the RbCur complex occured; the colour

of the NPs turned from red (mostly RbCur incorporated) to yellow (mostly curcumin

incorporated). The absorbance measurements showed that the amount of free curcumin

was higher than the amount of intact complex inside the particles. After 96 hours of dialysis

the complex was completely degraded. On the contrary, the release of Gd-DOTAMA(C18)2 is

significantly slower. It was evaluated by measuring the relaxivity (mM-1s-1) for several days of

dialysis at 37°C in an isotonic NaCl/Hepes buffer. As shown in Figure 4.6, the Gd-

DOTAMA(C18)2 concentrations remain the same in HBS for at least 4 days. The relaxation

rates during this period showed only a slight difference with respect to the starting point,

suggesting an overall stability of folate-targeted and non-targeted PLGA-NPs in these

conditions.

5.3 IN VITRO UPTAKE AND MRI VISUALISATION

PLGA-NPs were tested in vitro on IGROV-1 (human ovarian cancer) cells that

overexpress folate receptors. The Gd and the RbCur (containing boron) amount taken up by

cells after 6 and 24 hours of incubation were evaluated by MRI and ICP-MS analysis. The

results obtained using folate-targeted PLGA-NPs were compared with those using non-

36

targeted NPs. Figure 4.8 shows that the targeted NPs reach fast a complete saturation when

the concentration of folate-targeted and non-targeted NPs, in the incubation medium of

IGROV-1 cells, increases. This demonstrates a high affinity of folate-targeted NPs for the FRs.

The non-specific cell binding of non-targeted NPs is negligible and pegylated NPs are stable

under the experimental conditions. As shown in the T1 weighed MR images acquired at 7 T

for cells incubated for 6 and 24 hours with increasing concentration of RbCur NPs (Figure

4.10), the recorded Signal Intensity (SI) of targeted NPs is remarkably higher, with respect to

non-targeted ones, at an incubation time of 6 hours and at the highest Gd concentration

(100 µM). In fact, as shown in Figure 4.9, the cell proliferation was dramatically lower after

an incubation for 24 hours with 25 µM Gd (40%) with respect to 6 hours of incubation and

100 µM Gd (80%). The presence of a higher RbCur toxicity at longer incubation times can

influence the cell uptake by IGROV-1 cells. For these reasons the incubation time of 24 hours

was discharged. However, measuring the T1 of all the cell pellets (Table 4.6), the specific

accumulation of folate-targeted RbCur NPs in tumour cells with respect to non-targeted NPs

was noticed for every concentration and time of incubation.

Finally, the advantage of the folate to target IGROV-1 tumour cells, can be found only

after 6 hours of incubation using the highest Gd concentration (100 µM). Under this

experimental condition we obtained 1.4 x 10-9 moles Gd/mg protein (Figure 4.8) and a

concentration of internalised boron of 5 ppm (measured by ICP-MS) in tumour cells.

Unfortunately, the amount of boron is not the best to perform BNCT, however in

combination with the adjunctive curcumin chemotherapy, it may result in an improvement

in treatment outcome.

37

6. CONCLUSION

We can conclude that the PLGA-based nanoparticles proposed in this thesis delivered

a dual complex of boron and curcumin (RbCur) specific into tumour cells when targeted.

The aim was to perform Boron Neutron Capture Therapy (BNCT) in combination with

the adjunctive anti-proliferative effect of curcumin. Furthermore, loading PLGA NPs with an

amphiphilic MRI contrast agent (Gd-DOTAMA(C18)2) allowed the non-invasive determination

of the amount of boron internalised by tumour cells. By adding a pegylated phospholipid

functionalised with a folic acid residue to the formulation, PLGA NPs could be targeted

specifically to ovarian cancer cells (IGROV-1).

Both RbCur and the lipophilic Gd-complex were successfully encapsulated with an

incorporation yield of 17% and 40%, respectively, in folate-targeted and non-targeted PLGA

NPs. The amount of RbCur taken-up by tumour cells was high enough to give a significant

cytotoxic effect, even in the presence of a relatively fast RbCur release from the particles.

The relaxivity of the nano-system (28 mM-1s-1, at 21.5 MHz and 25°C) was high enough to

visualise IGROV-1 cells incubated with folate-targeted Gd-RbCur nanoparticles by MRI. This

demonstrated the specificity and the efficiency of the folate-targeted NPs with respect to

the non-targeted ones.

Measuring the local boron concentration is crucial to determine the optimal neutron

irradiation time and to calculate the delivered radiation dose. The boron amount

accumulated in cells (5 ppm) was not particularly high to perform BNCT. Despite this fact, a

significant therapeutic improvement is expected, due to the dual action of the RbCur

complex and the high specificity of the targeted nanoparticles for tumour cells.

Improvements to increase the boron concentration and to specifically target other

types of cancer are currently under investigation.

38

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8. APPENDIX

8.1 REAGENTS

Table 8.1: Used reagents with Lot number, CAS number and fabricant

Substance Lot number CAS number Fabricant

Acetone-d6 / 666-52-4 VWR Chemicals

Boric acid MKKBQ0638V 13813-79-1 Sigma Aldrich

Chloroform / 67-66-3 VWR Chemicals

Curcumin SLBH2403V 458-37-7 Sigma Aldrich

Dichloromethane / 75-09-2 VWR Chemicals

DSPE-PEG Folate / / Avanti Polar Lipids

DSPE-PEG(2000) methoxy 180PEG2PE-121 / Avanti Polar Lipids

Ethanol 96% / 64-17-5 VWR Chemicals

Ethyl acetate / 141-78-6 VWR Chemicals

Gd-DOTAMA(C18)2 RH391/96 / Bracco Imaging

Hepes SLBK4457V 7365-45-9 Sigma Aldrich

Methanol / 67-56-1 VWR Chemicals

Mowiol 4-88 (PVA) BCBL8261V 9002-89-5 Sigma Aldrich

NaCl SLBK9112V 7647-14-5 Sigma Aldrich

Oxalic acid 98% / 144-62-7 Sigma Aldrich

PLGA 50:50(30000-60000) SLBF6532V 26780-50-7 Sigma Aldrich

Sodium hydroxide

anhydrous pellets / 1310-73-2 Carlo Erba reagents

Toluene 80320 108-88-3 Riedel-De Haën

(Honeywell)

42