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Methods in Pharmacology
and Toxicology

Eleonora Patsenker Editors

Integrin Targeting
Systems for Tumor
Diagnosis
and Therapy
METHODS IN PHARMACOLOGY
AND TOXICOLOGY

Series Editor
Y. James Kang
University of Louisville
School of Medicine
Prospect, Kentucky, USA

For further volumes:

http://www.springer.com/series/7653
 Integrin Targeting Systems
for Tumor Diagnosis
and Therapy

Edited by

Eleonora Patsenker
Department of Gastroenterology and Hepatology, Universit€atsSpital Zürich, Zürich, Switzerland
Editor
Eleonora Patsenker
Department of Gastroenterology and Hepatology
Universit€atsSpital Zürich
Zürich, Switzerland

ISSN 1557-2153 ISSN 1940-6053 (electronic)

Methods in Pharmacology and Toxicology
ISBN 978-1-4939-7443-6 ISBN 978-1-4939-7445-0 (eBook)
https://doi.org/10.1007/978-1-4939-7445-0
Library of Congress Control Number: 2018941819

© Springer Science+Business Media, LLC, part of Springer Nature 2018

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Preface

The main aim of this book is to provide an update on the variety of techniques and
methodologies developed to facilitate research in integrin biology and to identify ideal
targets and approaches for the treatment of multiple organ diseases, with a focus on cancer
in particular. The chapters consecutively describe the tools for structural analysis, identifica-
tion, and detection of integrins as biomarkers, and include thorough laboratory and clini-
cally related methods on different strategies for generation, synthesis, and evaluation of
probes, carriers, peptides, or small particles for integrin targeting, imaging, and drug
delivery. This book is a good guideline for a broad spectrum of readers: (non)-researchers
out of the field and students in biological and medical sciences can get an overview on
existing developments in general, whereas professionals with high academic, clinical, and
industrial backgrounds will get familiar with innovative technologies in cancer research
dealing with integrins in detail.

Eleonora Patsenker

v
Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

Tumor Targeting by RGD-Grafted PLGA-Based Nanotheranostics Loaded

with Paclitaxel and Superparamagnetic Iron Oxides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Fabienne Danhier, Pierre Danhier, Nathalie Schleich, Chrystelle Po,
Sophie Laurent, Pierre Sibret, Christine Jérôme, Vincent Poucelle,
Bernard Gallez, and Véronique Préat
Fabrication of cRGD-Conjugated Dual-Responsive Micelles to Target
αvβ5 Integrin-Overexpressed Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Huacheng He, Remant Bahadur K.C., and Peisheng Xu
Copper-Free Click Chemistry Modification of Nanovectors
for Integrin-Targeted Cancer Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Chang-Fang Wang and Hélder A. Santos
Preparation, Characterization, and In Vitro and In Vivo Evaluation
of PEGylated Liposomal Doxorubicin Modified with Different cRGD Peptides . . . . . 51
Mohamadreza Amin and Mahmoud Reza Jaafari
Preparation of Heterobivalent and Multivalent Radiopharmaceuticals
to Target Tumors Over-Expressing Integrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Guillermina Ferro-Flores, Blanca Ocampo-Garcı́a, Clara Santos-Cuevas,
Nallely Jiménez-Mancilla, Myrna Luna-Gutiérrez, Flor de M. Ramı́rez,
Enrique Morales-Avila, Luis M. De Leo n-Rodrı́guez, and Erika Azorı́n-Vega
177
Lu-Labeled RGD-BBN Peptide for Targeting Prostate Cancer . . . . . . . . . . . . . . . . . 93
Lei Jiang and Zhen Cheng
Integrin-Mediated Targeting of Liposomally Bound siRNAs
to Tumor Vasculatures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Poulami Majumder and Arabinda Chaudhuri
Integrin αvβ3-Targeted Optical Imaging with Metal Oxide Nanomaterials:
Focusing on Zinc Oxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
Hao Hong and Weibo Cai
Integrin Targeting Using RGD-Based Peptide Amphiphiles . . . . . . . . . . . . . . . . . . . . . . 135
Poonam Saraf, Xiaoling Li, and Bhaskara Jasti
Cyclic-RGDfK-Directed Docetaxel Loaded Nanomicelles for Angiogenic
Tumor Targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Hitesh Kulhari, Deep Pooja, Shweta Shrivastava, T. Srinivasa Reddy,
Ayan Kumar Barui, Chitta Ranjan Patra, V.G.M. Naidu,
David J. Adams, and Ramakrishna Sistla
Targeting Glioma Cancer Cells with Fluorescent Nanodiamonds
via Integrin Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
Jitka Neburkova, Miroslav Hajek, Ivan Rehor, Jiri Schimer,
Frantisek Sedlak, Jan Stursa, Martin Hruby, and Petr Cigler

vii
viii Contents

NIR Imaging-Guided Photothermal Therapy by cRGD-Conjugated

Solid Lipid Nanoparticles Encapsulating IR-780 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
Ye Kuang, Kunchi Zhang, Min Liu, and Renjun Pei
Synergistic Active Targeting to B16F10 Tumors by αvβ3/CD44-Targeted
Nanoparticles Loaded with Docetaxel. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
Sanjun Shi

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
Contributors

DAVID J. ADAMS  Health Innovations Research Institute, RMIT University, Melbourne,

VIC, Australia
MOHAMADREZA AMIN  Biotechnology Research Center, Nanotechnology Research Center,
School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
ERIKA AZORÍN-VEGA  Department of Radioactive Materials, Instituto Nacional de
Investigaciones Nucleares, Ocoyoacac, Estado de México, Mexico
AYAN KUMAR BARUI  Biomaterials Group, CSIR-Indian Institute of Chemical Technology,
Hyderabad, India
WEIBO CAI  Department of Radiology and Medical Physics, School of Medicine and Public
Health, University of Wisconsin – Madison, Madison, WI, USA; University of Wisconsin
Carbone Cancer Center, Madison, WI, USA
ARABINDA CHAUDHURI  Biomaterials group, CSIR-Indian Institute of Chemical Technology,
Hyderabad, India; Academy of Scientific and Innovative Research (AcSIR), Chennai,
India
ZHEN CHENG  Molecular Imaging Program at Stanford (MIPS), Department of Radiology
and Bio-X Program, Canary Center at Stanford for Cancer Early Detection, Stanford
University, Stanford, CA, USA
PETR CIGLER  Institute of Organic Chemistry and Biochemistry of the CAS, Prague,
Czech Republic
FABIENNE DANHIER  Louvain Drug Research Institute, Advanced Drug delivery and
Biomaterials, Université catholique de Louvain, Brussels, Belgium
PIERRE DANHIER  Louvain Drug Research Institute, Laboratory of Biomedical Magnetic
Resonance, Université catholique de Louvain, Brussels, Belgium
GUILLERMINA FERRO-FLORES  Department of Radioactive Materials, Instituto Nacional
de Investigaciones Nucleares, Ocoyoacac, Estado de México, Mexico
BERNARD GALLEZ  Louvain Drug Research Institute, Laboratory of Biomedical Magnetic
Resonance, Université catholique de Louvain, Brussels, Belgium
MIROSLAV HAJEK  Institute of Organic Chemistry and Biochemistry of the CAS, Prague,
Czech Republic
HUACHENG HE  Department of Drug Discovery and Biomedical Sciences, South Carolina
College of Pharmacy, University of South Carolina, Columbia, SC, USA
HAO HONG  Department of Radiology, University of Michigan Health Systems, Ann Arbor,
MI, USA
MARTIN HRUBY  Institute of Macromolecular Chemistry of the CAS, Prague, Czech Republic
MAHMOUD REZA JAAFARI  Nanotechnology Research Center, School of Pharmacy, Mashhad
University of Medical Sciences, Mashhad, Iran
BHASKARA JASTI  Department of Pharmaceutics and Medicinal Chemistry, Thomas J. Long
School of Pharmacy and Health Sciences, University of the Pacific, Stockton, CA, USA
CHRISTINE JÉRÔME  Center for Education and Research on Macromolecule, Université de
Liège, Liège, Belgium
LEI JIANG  Department of Nuclear Medicine, Zhongshan Hospital, Fudan University,
Shanghai, China

ix
x Contributors

NALLELY JIMÉNEZ-MANCILLA  Instituto Nacional de Investigaciones Nucleares Cátedras

CONACyT, Ocoyoacac, Estado de México, Mexico
K. C. REMANT BAHADUR  Department of Drug Discovery and Biomedical Sciences, South
Carolina College of Pharmacy, University of South Carolina, Columbia, SC, USA
YE KUANG  CAS Key Laboratory of Nano-Bio Interface, Division of Nanobiomedicine,
Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou,
China
HITESH KULHARI  IICT-RMIT Research Centre, CSIR-Indian Institute of Chemical
Technology, Hyderabad, India; Medicinal Chemistry & Pharmacology Division,
CSIR-Indian Institute of Chemical Technology, Hyderabad, India; School of Applied
Sciences, RMIT University, Melbourne, Australia; Health Innovations Research Institute,
RMIT University, Melbourne, Australia
SOPHIE LAURENT  Department of General, Organic, and Biomedical Chemistry,
NMR and Molecular Imaging Laboratory, Université de Mons, Mons, Belgium
LUIS M. DE LEÓN-RODRÍGUEZ  School of Chemical Sciences, The University of Auckland,
Auckland, New Zealand
XIAOLING LI  Department of Pharmaceutics and Medicinal Chemistry, Thomas J. Long
School of Pharmacy and Health Sciences, University of the Pacific, Stockton, CA, USA
MIN LIU  CAS Key Laboratory of Nano-Bio Interface, Division of Nanobiomedicine,
Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou,
China
MYRNA LUNA-GUTIÉRREZ  Department of Radioactive Materials, Instituto Nacional de
Investigaciones Nucleares, Ocoyoacac, Estado de México, Mexico
POULAMI MAJUMDER  Chemical Biology Laboratory, National Cancer Institute, National
Institutes of Health, Frederick, MD, USA
ENRIQUE MORALES-AVILA  Faculty of Chemistry, Universidad Autonoma del Estado
de México, Toluca, Estado de México, Mexico
JITKA NEBURKOVA  Institute of Organic Chemistry and Biochemistry of the CAS, Prague,
Czech Republic; First Faculty of Medicine, Charles University, Prague 2, Czech Republic
BLANCA OCAMPO-GARCÍA  Department of Radioactive Materials, Instituto Nacional de
Investigaciones Nucleares, Ocoyoacac, Estado de México, Mexico
CHITTA RANJAN PATRA  Biomaterials Group, CSIR-Indian Institute of Chemical Technology,
Hyderabad, India
RENJUN PEI  CAS Key Laboratory of Nano-Bio Interface, Division of Nanobiomedicine,
Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou,
China
CHRYSTELLE PO  Louvain Drug Research Institute, Laboratory of Biomedical Magnetic
Resonance, Université catholique de Louvain, Brussels, Belgium
DEEP POOJA  Medicinal Chemistry & Pharmacology Division, CSIR-Indian Institute
of Chemical Technology, Hyderabad, India
VINCENT POUCELLE  Unité de Chimie organique et médicinale, Université catholique
de Louvain, Louvain-la-Neuve, Belgium
VÉRONIQUE PRÉAT  Louvain Drug Research Institute, Advanced Drug delivery
and Biomaterials, Université catholique de Louvain, Brussels, Belgium
FLOR DE M. RAMÍREZ  Department of Chemistry, Instituto Nacional de Investigaciones
Nucleares, Ocoyoacac, Estado de México, Mexico
 Contributors xi

T. SRINIVASA REDDY  IICT-RMIT Research Centre, CSIR-Indian Institute of Chemical

Technology, Hyderabad, India; Medicinal Chemistry & Pharmacology Division,
CSIR-Indian Institute of Chemical Technology, Hyderabad, India
IVAN REHOR  Institute of Organic Chemistry and Biochemistry of the CAS, Prague 6,
Czech Republic
CLARA SANTOS-CUEVAS  Department of Radioactive Materials, Instituto Nacional de
Investigaciones Nucleares, Ocoyoacac, Estado de México, Mexico
HÉLDER A. SANTOS  Division of Pharmaceutical Chemistry and Technology, Faculty
of Pharmacy, University of Helsinki, Helsinki, Finland
POONAM SARAF  Department of Pharmaceutics and Medicinal Chemistry, Thomas J. Long
School of Pharmacy and Health Sciences, University of the Pacific, Stockton, CA, USA
JIRI SCHIMER  Institute of Organic Chemistry and Biochemistry of the CAS, Prague 6,
Czech Republic
NATHALIE SCHLEICH  Louvain Drug Research Institute, Advanced Drug delivery and
Biomaterials, Université catholique de Louvain, Brussels, Belgium
FRANTISEK SEDLAK  Institute of Organic Chemistry and Biochemistry of the CAS, Prague 6,
Czech Republic; First Faculty of Medicine, Charles University, Prague 2, Czech Republic;
Department of Genetics and Microbiology, Faculty of Science, Charles University, Prague 2,
Czech Republic
SANJUN SHI  Department of Pharmacy, Institute of Surgery Research, Daping Hospital,
Third Military Medical University, Chongqing, People’s Republic of China
SHWETA SHRIVASTAVA  Department of Pharmacology, National Institute of Pharmaceutical
Education and Research, Hyderabad, India
PIERRE SIBRET  Center for Education and Research on Macromolecule, Université de Liège,
Liège, Belgium
RAMAKRISHNA SISTLA  Medicinal Chemistry & Pharmacology Division, CSIR-Indian
Institute of Chemical Technology, Hyderabad, India
JAN STURSA  Nuclear Physics Institute of the CAS, Prague, Czech Republic
V. G. M. NAIDU  Department of Pharmacology, National Institute of Pharmaceutical
Education and Research, Hyderabad, India
CHANG-FANG WANG  Division of Pharmaceutical Chemistry and Technology,
Faculty of Pharmacy, University of Helsinki, Helsinki, Finland
PEISHENG XU  Department of Drug Discovery and Biomedical Sciences, South Carolina
College of Pharmacy, University of South Carolina, Columbia, SC, USA
KUNCHI ZHANG  Shanghai University of Medicine and Health Sciences, Shanghai, China
Methods in Pharmacology and Toxicology (2018) 1–17
DOI 10.1007/7653_2015_43
© Springer Science+Business Media New York 2015
Published online: 11 August 2015

Tumor Targeting by RGD-Grafted PLGA-Based

Nanotheranostics Loaded with Paclitaxel
and Superparamagnetic Iron Oxides
Fabienne Danhier, Pierre Danhier, Nathalie Schleich, Chrystelle Po,
Sophie Laurent, Pierre Sibret, Christine Jérôme, Vincent Poucelle,
Bernard Gallez, and Véronique Préat

Abstract
Theranostic nanoparticles have the potential to revolutionize cancer diagnosis and therapy. Many groups
have demonstrated differential levels of tumor growth between tumors treated by targeted or untar-
geted nanoparticles; however, only few have shown in vivo efficacy in both therapeutic and diagnostic
approach. Herein, we first develop and characterize dual-paclitaxel (PTX)/superparamagnetic iron oxide
(SPIO)-loaded PLGA-based nanoparticles grafted with the RGD peptide, for a theranostic purpose.
Second, we compare in vivo different strategies in terms of targeting capabilities: (1) passive targeting
via the EPR effect, (2) active targeting of αvβ3 integrin via RGD grafting, (3) magnetic guidance via a
magnet placed on the tumor, and (4) the combination of the magnetic guidance and the active targeting
of αvβ3 integrin. In this chapter, we present the general flowchart applied for this project: (1) the
polymer and SPIO synthesis, (2) the physicochemical characterization of the nanoparticles, (3) the
magnetic properties of the nanoparticles, and (4) the in vivo evaluation of the nanoparticles for their
therapeutic and diagnosis purposes. We employ the electron spin resonance spectroscopy and magnetic
resonance imaging to both quantify and visualize the accumulation of theranostic nanoparticles into the
tumors.

Keywords: PLGA-nanoparticles, SPIO, Paclitaxel, Cancer therapy, Magnetic resonance imaging,

Tumor targeting, Nanotheranostic

1 Introduction

Theranostic nanoparticles have the potential to revolutionize can-

cer diagnosis and therapy. Theranostic refers to the combination of
a therapeutic and a diagnostic agent in a same unique vector. Such a
tool could be helpful in noninvasive assessment of the biodistribu-
tion, visualization of drug distribution, optimization of strategies,
and prediction and real-time monitoring of therapeutic responses
[1, 2].
Passive targeting is based on the so-called enhanced permeabil-
ity and retention (EPR) effect [3]. This strategy relies on the

1
2 Fabienne Danhier et al.

presence of fenestrations in the endothelium of tumor vessels,

allowing the entry of nanoparticle into the tumor tissue. Moreover,
the deficiency of the lymphatic system in tumor tissue prevents the
recapture of these nanoparticles leading to a retention effect [3–5].
Active targeting consists in coupling a ligand to the surface of
nanoparticles that can interact with its receptor at the target cell
site [6, 7]. Although angiogenesis is a physiological process by
which new blood vessels are formed, it is also at the root of
tumor growth and metastasis. Since angiogenesis is controlled
by the endothelial cells, it is of great interest to target tumor
endothelial cells. αvβ3 is an adhesion integrin overexpressed at
the surface of the endothelial cells of neo-angiogenic vessels
involved in the angiogenic process. Its expression is correlated
with the malignancy of tumor. Moreover, αvβ3 is also overex-
pressed at the surface of many tumor cells. Numerous studies
have shown that the tripeptide arginine–glycine–aspartic acid
(RGD) was able to bind preferentially to particular α vβ3 integrin
[6, 8]. More recently, magnetic drug targeting has been studied.
In this approach, magnetic nanoparticles are guided to tumor site
using magnetic fields [9].
We hypothesized that the combination of both active strategy
and magnetic guidance could enhance theranostic nanoparticle
concentration into the tumor tissue leading, therefore, to a better
anticancer efficacy (therapeutic purpose) and increased contrast
enhancement in magnetic resonance imaging (MRI) (diagnosis
purpose).
In this study, we aimed at developing RGD-grafted PEGy-
lated PLGA-based nanoparticles as an effective nanocarrier for
dual encapsulation of anticancer drug, paclitaxel (PTX), and
small paramagnetic iron oxides (SPIO) for a theranostic purpose
(Fig. 1). Hence, SPIO were prepared by the coprecipitation
technique and were encapsulated in PLGA-based nanoparticles.
The physicochemical properties of nanoparticles were character-
ized by different techniques such as transmission electron
microscopy (TEM), dynamic light scattering (DLS) method,
and electron spin resonance (EPR) spectroscopy. Their magnetic
properties were evaluated using relaxometry and magnetic reso-
nance imaging (MRI). Finally, we aimed at evaluating the
combination of the two targeting strategies (active targeting
and magnetic targeting) in vivo using two complementary
techniques: (1) the ESR spectroscopy [10] and (2) the 11.7 T
MRI [11].
 Tumor Targeting by RGD-Grafted Nanotheranostics 3

Fig. 1 Experimental flowchart of the current project highlighting the two main objectives: (a) the development
and the characterization of nanotheranostics and (b) the in vivo evaluation to demonstrate the benefit of the
combination of the active targeting and the magnetic guidance for both therapeutic and imaging purposes

2 Materials

1. Iron(II) chloride, Sigma-Aldrich (St. Louis, MO, USA).

2. Iron(III) chloride, Sigma-Aldrich (St. Louis, MO, USA).
3. Oleic acid, Sigma-Aldrich (St. Louis, MO, USA).
4. Nitrogen-gas.
5. Tetramethylammonium 11-aminoundecanoate, Sigma-Aldrich
(St. Louis, MO, USA).
6. PCL-b-PEG (MW ¼ 13,100–5000), synthesized by ring-
opening polymerization using triethylaluminum as the catalyst
[12].
7. O-succinimidyl 4-(p-azidophenyl) butanoate, Sigma-Aldrich
(St. Louis, MO, USA).
8. GRGDS, NeoMPS (Strasbourg, France).
9. PTX, Sigma-Aldrich (St. Louis, MO, USA).
4 Fabienne Danhier et al.

10. Poly(lactic-co-glycolic acid) (PLGA, lactide/glycolide molar

ratio of 50:50 MW: 7000–17,000), Sigma-Aldrich (St. Louis,
MO, USA).
11. PLGA-b-PEG (MW ¼ 10,040–4600) was synthesized as pre-
viously described [13].
12. PVA: Polyvinylalcohol (MW ¼ 30–70 kDa), Sigma-Aldrich
(St. Louis, MO, USA).
13. Sonicator: Digital Sonifier, Branson (Danbury, USA).
14. 1.2 μm filters, Acrodisc 32 mm Syringe filter with 1.2 μm Supor
membrane, Pall, Life Sciences (Zaventem, Belgium).
15. Thermogravimetric analysis (TGA): TA Instrument Q500
model (USA).
16. Transmission electron microscopy (TEM): Philips CM 100,
equipped with a Megaview G2 camera (Andover, USA).
17. Malvern Nano ZS, Malvern instruments (UK).
18. Bruker EMX ESR spectrometer, Bruker Biospin GmBh
(Germany).
19. Inductively coupled plasma mass spectroscopy (ICP-MS), Agi-
lent 7500ce instrument.
20. High-performance liquid chromatography (HPLC), Agilent
1100 series, Agilent Technologies (USA). The column used
was a CC 125/4 Nucleodur 100-5 C18, Macherey-Nagel
(Bethlehem, USA).
21. Fast Field Cycling Relaxometer, Stelar (Mede, Italy).
22. Minispec spin analyzers, Bruker (Karlsruhe, Germany).
23. SPIO, BioPAL (Worcester, UK).
24. 11.7 T animal Biospec MR system, Bruker Biospec (Ettlingen,
Germany).
25. 1.1 T neodyme-iron-bore external magnet, Webcraft GmbH
(Uster, Switzerland).
26. Isoflurane, Abbott (Ottignies, Belgium).
27. Ketamine (Ketalar®) and Xylazine, Sigma-Aldrich (St. Louis,
MO, USA).
28. CT26 cells, ATCC (Manassas, USA).
29. Freeze-dryer, Labconco (Kansas City, USA).
30. Haematocrit capillaries, ref 910 0175, Hirschmann Labor-
ger€a te (Eberstadt, Germany).
31. Haematocrit sealing compound, Cat. No. 7495 10, Brand
GmbH (Wertheim, Germany).
32. 18G needle, ref 304622, BD Microlance (Le Pont de Claix,
France).
 Tumor Targeting by RGD-Grafted Nanotheranostics 5

3 Methods

The methods described next follow the chronological steps of our

experiment flowchart for the development of the PLGA-based
nanotheranostics, shown in Fig. 1, and comprise (1) the synthesis
of SPIO, (2) the photografting of PCL-b-PEG with the RGD
peptide, (3) the preparation of the RGD-grafted PLGA-based
nanoparticles, (4) the physicochemical characterization of the
nanoparticles, and (5) the evaluation of the magnetic properties
of the nanoparticles.
After that, the combination of the two strategies (active target-
ing + magnetic targeting) will be evaluated in vivo: (1) antitumor
efficacy, (2) ex vivo biodistribution study, and (3) MR imaging.

3.1 Development of 1. Hydrophobic SPIO were synthesized using a classical copreci-

the PLGA-Based pitation technique of ferrous and ferric salts in alkaline
Nanotheranostics medium. 10 mmol iron(III) chloride and 5 mmol iron(II)
chloride were mixed together in 12 ml of a hydrochloride
3.1.1 Synthesis of aqueous solution (HCl 1 M).
Superparamagnetic Iron
Oxides Coated with Oleic 2. This solution was then added dropwise to an aqueous solution
Acid of NaOH 1 M containing 3.1 g of oleic acid with stirring on a
magnetic stir plate for 20 min under a nitrogen-gas atmosphere
at 80  C.
3. The black precipitate was separated using a magnet, washed
three times using absolute ethanol, and then dissolved in 50 ml
of dichloromethane (DCM).
4. The solution was then placed in an ultrasonic bath for 10 min
and centrifuged (4416 rcf, 10 min) to remove the undispersed
residue.
5. Hydrophilic SPIO used as SPIO aqueous solution (SPIO sol)
were also synthesized. 1 ml of DCM dispersion of SPIO coated
with oleic acid (40 mg/ml) was added to a suspension of
tetramethylammonium 11-aminoundecanoate in DCM
(40 mg in 2 ml). After 24-h magnetic stirring, the precipitate
was washed three times with DCM and dispersed in water [14].

3.1.2 Photografting of 1. PCL-b-PEG was solubilized in methylene dichloride or ace-

PCL-b-PEG with the RGD tonitrile (40 ml/g) with the molecular clip: O-succinimidyl
Peptide 4-(p-azidophenyl) butanoate (0.2 mmol/g), and the solution
was cast on clean plates (1 ml per plate).
2. After solvent evaporation, the polymer was dried under
vacuum to constant weight and was removed from the plates
as shaving.
3. The polymer sample was irradiated at 254 nm in a quartz flask
under an argon atmosphere for 20 min, using a homemade
6 Fabienne Danhier et al.

Fig. 2 Schematic representation of the RGD peptide grafting on the PCL-b-PEG copolymer [12]

reactor (rotating quartz flask of 15 ml; 3 UV lamps of 8 W

placed at a distance of 4.5 cm).
4. The samples were washed (to remove unreacted arylazide and
nonfixed reagent) with isopropanol:ethyl acetate (19:1, v/v)
(80 ml/g; three times) and dried under vacuum. The “acti-
vated” polymer was immersed in 1 mM solution of the ligand
GRGDS (80 ml/g) in phosphate buffer (0.1 M): acetonitrile
(1:1, v/v) at pH 8 and shaken for 24 h at 20  C.
5. The peptide solution was removed by suction and the sample
was then washed three times with 5 mM HCl, five times with
deionized water, shaken overnight in deionized water, rinsed
with MeOH, and dried under the vacuum at 40  C to a
constant weight (Fig. 2) [15].

3.1.3 Preparation of RGD-grafted nanoparticles loaded with SPIO and PTX (SPIO/
RGD-Grafted PLGA-Based PTX-RGD-NP) were prepared by an emulsion-diffusion-evapora-
Nanoparticles Loaded with tion method [14]. Chemical description of the polymers included
SPIO and Paclitaxel in the formulations is illustrated in Table 1.
1. PLGA (14 mg/ml), PLGA-PEG (3 mg/ml), and PCL-PEG-
RGD (3 mg/ml) were dissolved in 2 ml DCM containing
SPIO (Fe concentration: 15 mg/ml) and PTX (3 mg).
 Tumor Targeting by RGD-Grafted Nanotheranostics 7

Table 1
Chemical description of the polymers included in the formulations

Mn (SEC) Mn (NMR) g/mol Mol %

Polymer g/mola polyester-PEG glycolidelb
PLGA 7000–17,000 – 50
PLGA-b-PEG – 10,004–4600 26
PCL-b-PEG 22,400 13,100–5000 –
a
Polystyrene calibration
b
Determined by NMR by the following formula: ((I4.7/2)/(I5.2 + I4.7/2)) 100,
where I4.7 is the signal intensity of the glycolide unit at 4.7 ppm (CH2OC¼O) and
I5.2 is the signal intensity of the lactide unit at 5.2 ppm (CH(CH3)OC¼O)

2. This organic solution was then added to an aqueous solution

(4.5 ml) containing 3 % (p/v) PVA and emulsified using a
vortex for 2 min followed by sonication (2  30 s, 50 W).
3. The mixture was then added dropwise and under magnetic
stirring into an aqueous solution containing 1 % PVA and
stirred overnight to evaporate the organic solvent. Some con-
siderations around this procedure are gathered in Note 1.
4. To remove the non-encapsulated drug and the residual PVA,
the suspension was filtered (1.2 μm) and washed three times
with water using ultracentrifugation (11,000  g, 30 min,
4  C) and suspended in 2 ml water.
As a control, we used exactly the same method while PCL-
PEG-RGD was replaced by PCL-PEG.

3.1.4 Physicochemical 1. The coating percentage of SPIO with oleic acid was assessed by
Characterization of thermogravimetric analysis (TGA) on a TA Instrument Q500
Nanotheranostics model, under dry nitrogen flow, with a heating rate of 15  C/
min from RT to 600  C, in an open platinum pan.
2. The hydrodynamic particle size and size polydispersity of nano-
particles were assessed by photon correlation spectroscopy, using
a Malvern Nano ZS (Nano ZS, Malvern instruments, UK).
3. The morphology of the particles was achieved using transmis-
sion electron microscopy (TEM). TEM was carried out at a
voltage of 100 kV. Samples for TEM experiments were
prepared by spin coating a drop of nanoparticles in DCM on
a carbon-coated TEM grid.
4. The zeta (ζ) potential of the nanoparticles was measured by
laser Doppler velocimetry in KCl 1 mM with a Malvern Nano
ZS at 25  C.
5. Iron content was measured by ESR (Table 2) using a Bruker EMX
ESR spectrometer operating at 9 GHz validated by inductively
coupled plasma mass spectroscopy (ICP-MS) measurements.
8 Fabienne Danhier et al.

Table 2
Comparison between the electron spin resonance (ESR) spectroscopy and
the magnetic resonance imaging (MRI)

ESR spectroscopy MRI

Resonance method Electron spin Nuclear spin
Constant parameter Constant frequency Constant magnetic field
Frequency/magnetic 28 GHz/T 45 MHz/T
field ratio
Relaxation time Short (ns) Long (ms)
B amplitude 0.34 T 11.7 T
Frequency 9.5 GHz 500 MHz
Energy level Very high High
Sensitivity nM μM

Some considerations around this procedure are gathered in

Note 2. Typical parameters were selected for ESR measurements:
30 G modulation amplitude, 10.11 mW power, 3251 G center
field, and 4000 G sweep width field. Measurements were per-
formed at room temperature. Double integration (DI) of the
first derivative of iron oxides ESR spectra (Bruker WINEPR
software) was used to quantify signal intensity [16].
6. PTX content was determined using high-performance liquid
chromatography (HPLC) with UV detection at 227 nm, after
dissolution of the particles by acetonitrile. The mobile phase
consisted of acetonitrile and water (70:30 v/v, respectively) at a
rate of 1 ml/min. The column used was a CC 125/4 Nucleo-
dur 100-5 C18. The drug loading was defined as the amount of
drug (mg) loaded for 100 mg of polymer whereas the encap-
sulation efficiency was defined by the ratio of the encapsulated
drug compared to the initial amount of drug [6].

3.1.5 Magnetic 1. Nuclear magnetic relaxation dispersion (NMRD) profiles were

Properties of recorded at 37  C on a fast-field cycling relaxometer over a
Nanotheranostics magnetic field range from 0.01 to 40 MHz. Additional longi-
tudinal (R1) and transverse (R2) relaxation rate measurements
at 20 MHz and 60 MHz were, respectively, obtained on Min-
ispec mq 20 and mq 60 spin analyzers. The fitting of the
NMRD profiles by a theoretical relaxation model allows the
determination of the crystal radius (r) and the specific magne-
tization (MS). The proton NMRD curves were fitted using
data-processing software, including different theoretical mod-
els describing the nuclear relaxation phenomena [17].
 Tumor Targeting by RGD-Grafted Nanotheranostics 9

[Fe] mg/ml

0 5 10 15 20 25 30

BioPAL

0 5 10 15 20 25 30

SPIO sol

0 5 10 15 20 25 30

SPIO NP

Fig. 3 T2-weighted MR images (phantom MRI) of commercial SPIO (BioPAL, Worcester, UK), an aqueous
suspension hydrophilic SPIO (SPIO sol), and SPIO-loaded nanoparticles (SPIO NP) as a function of Fe
concentration (μg/ml, TE ¼ 30 ms). Adapted from Ref. [14]

2. Various concentrations of SPIO in aqueous solution (SPIO

sol) and PLGA-based nanoparticles loaded with SPIO
(SPIO-NP) ranging from 0 to 30 μg/ml were investigated
by T2-weighted MRI to assess their T2 enhancing capability.
Commercial SPIO (BioPAL) were used as a reference at the
same concentrations T2 relaxivity was obtained using a
11.7 T animal Biospec MR system. Phantom MRI of SPIO-
NP was carried out at various iron concentrations from
0 μg/ml to 30 μg/ml (0, 5, 10, 15, 20, 25, and 30 μg/ml)
in 10 % gelatin using a T2-weighted multi-slice multi-echo
(MSME) sequence (Fig. 3). The imaging parameters were as
follows: repetition time (TR) ¼ 2500 ms, echo time (TE)
¼ 30 ms, field of view (FOV) ¼ 3.00 cm, and flip angle
(FA) ¼ 180.0 .

3.2 In Vivo CT26 colon carcinoma was chosen because of its sensitivity to
Evaluation of the PTX [18] and angiogenic properties [19]. CT26 colon carci-
Combined Strategies: noma cells were inoculated subcutaneously in the right flank
Active and Magnetic (for antitumor efficacy and biodistribution studies) or in the
Targeting right leg (for MRI studies) of BALB/c mice (5  104 cells per
mouse) depending on the experiment (see Note 3) [20]. For all
3.2.1 Animal Tumor the experiments, mice were divided into four groups: Group 1:
Model control group (injected with PBS for in vivo antitumor efficacy
10 Fabienne Danhier et al.

study and SPIO-loaded nanoparticles for ex vivo biodistribution

and MRI), Group 2: mice treated by SPIO/PTX-NP grafted
with RGD peptide (active targeting or RGD), Group 3: mice
treated with SPIO/PTX-NP magnetic guided by placing a 1.1 T
neodyme-iron-bore external magnet on the surface of the
tumor during 1 or 4 h (magnetic guidance, MG) (see Note 4),
and Group 4: mice treated with SPIO/PTX-NP grafted with
RGD peptide using both magnetic and active targeting
(RGD + MG).
PTX and Fe doses were 5 mg/kg and 27.1 mg/kg, respec-
tively, for in vivo antitumor efficacy experiment and 2.5 mg/kg
and 13.5 mg/kg for other experiments. For tumor inoculation,
mice were anesthetized by intraperitoneal injections of a mixed
ketamine (100 mg/kg) and xylazine (10 mg/kg). For in vivo
MRI experiments, mice were maintained under anesthesia dur-
ing the entire experiment using 1–2 % isoflurane inhalation in
air.

3.2.2 Antitumor Efficacy The effect of SPIO/PTX-RGD-NP on tumor growth was assessed
by daily measurements of tumor volume with an electronic caliper.
CT26 cells (5  104 cells per mouse) were injected subcutaneously
in the right flank of the mice to allow easy and reproducible tumor
volume measurements. Mice were randomly assigned to a treat-
ment group when tumor reached a volume of 27  5 mm3. Treat-
ments were injected trough the tail vein. Four groups were defined
as aforementioned (n ¼ 6): PBS, RGD, MG, and RGD+MG (PTX
and Fe doses were 5 mg/kg and 27.1 mg/kg, respectively). In this
experiment, the magnet was placed on the surface of the tumor
during 4 h. The end point of the experiment was determined as the
moment when tumor reached 600 mm3. At this point, mice were
sacrificed.

3.2.3 Ex Vivo The biodistribution of the different treatments was assessed using
Biodistribution Study by ESR spectroscopy (X-band) (see Note 2). CT26 cells (5  104
ESR Spectroscopy cells per mouse) were subcutaneously inoculated in the right
flank of the mice. When tumor reached 50–100 mm3 in volume,
mice were randomly dispersed into four groups as aforemen-
tioned (n ¼ 6): PBS, RGD, MG, and RGD + MG. For mice
treated with magnetic guidance, the external magnet was main-
tained on the tumor until sacrifice. Treatments were injected in
the tail vein of the mouse. 1 or 4 h posttreatment, mice were first
taken a retro-orbital blood sample and were then sacrificed for
dissection in order to remove liver, lungs, and tumor. Thereafter,
samples were frozen in liquid nitrogen, dehydrated for 24 h in a
freeze-dryer, crushed into a fine powder, weighed, and analyzed
by ESR spectroscopy to determine iron concentration in each
tissue.
 Tumor Targeting by RGD-Grafted Nanotheranostics 11

3.2.4 In Vivo MR Imaging For these experiments, CT26 cells (5  104 cells per mouse) were
injected subcutaneously in the right leg of the mice to avoid res-
piratory artifacts (see Note 3). Mice were enrolled in the study
when tumor reached 50–100 mm3 in diameter (see Note 5).
Three groups were defined (n ¼ 5): RGD, MG, and RGD + MG.
Each mouse was imaged before and 1 h after treatment injection
in the tail vein in order to use each mouse as its own control.
MR experiments were performed using a 11.7 T Bruker Bios-
pec horizontal MR System (Table 2). RF transmission and recep-
tion were achieved with a quadrature volume resonator (inner
diameter 40 mm).
1. Mice were anesthetized by isoflurane inhalation 3 % in air and
they were placed in an MRI-compatible cradle.
2. The breathing rate was assessed via a breathing pillow, placed
under the thorax, and kept at 70 breaths/min by adjusting the
isoflurane concentration. The body temperature was main-
tained at 37  C by a warm waterbed and monitored using a
rectal probe. Vital functions were monitored during the whole
anesthesia period using SamPC Monitor (version 6.17, Small
Animal Instruments Inc.).
3. Anatomical images of the mice were provided by T2-weighted
axial images acquired with a rapid acquisition with relaxation
enhancement sequence (RARE; TR/TE: 2500/30 ms, RARE
factor: 6, 10 slices non-contiguous with a gap of 0.08 mm,
resolution: 125  125  800 μm3).
4. T2 maps were acquired with the same geometry than the ana-
tomical images using an MSME sequence (TR/TE: 2500/
10 ms; 16 echoes).
5. Quantitative T2 maps were calculated from the MSME multi-
echo trains and assuming mono-exponential decays, using Ima-
geJ (ImageJ version, 1.48 NIH).
6. The volume of interest (VOI) corresponding to the tumor
volume was manually delineated on a slice-by-slice basis on
the anatomical images acquired before and after treatment
injection, for each animal. These VOIs were applied on
corresponding maps to determine the T2 values. Relative stan-
dard deviations (RSD) were calculated as RSD ¼ SD/mean
T2, where SD is the standard deviation of the mean T2 value.
T2-weighted images, obtained with different TE from MSME
sequence, are illustrated in Fig. 4.
12 Fabienne Danhier et al.

Fig. 4 T2-weighted images, obtained from MSME sequence (TE ¼ 10 ms) of CT26-tumor bearing mice pre-
injection and 4 h after injection. Mice were treated with SPIO/PTX-NP grafted with RGD peptide using both
magnetic and active targeting (RGD + MG). The position of dark region in tumor was pointed by red-head
arrows (n ¼ 5). Adapted from Ref. [20]

4 Notes

1. For the last step of the nanoparticle formulation: “The mixture

was then added dropwise and under magnetic stirring into an
aqueous solution containing 1 % PVA and stirred overnight to
evaporate the organic solvent” (see section 3.1.3); it is impor-
tant to note that (1) the mixture should be contained in a glass
syringe with a 21 G needle. The size of the needle influenced
the size and the polydispersity index of the nanoparticles; and
(2) the mixture should be added dropwise in the vortex of the
liquid created by the magnetic stirring. This step is also impor-
tant for the size of the nanoparticles and to avoid aggregation
of the nanoparticles (Fig. 5).
2. The iron oxide content of the nanoparticles was determined by
ESR spectroscopy (also called electron paramagnetic reso-
nance, EPR). ESR is a spectrometric technique that is used to
study free radicals and (super) paramagnetic molecules. The
ESR method was shown to be sensitive and specific for the iron
oxide content determination in biological samples [16, 21].
SPIO present a typical broad ESR spectrum at room tempera-
ture that can be differentiated from free Fe+++ ion. Double
integration (DI) of the first derivative ESR spectrum is used
to quantify the amount of SPIO in a sample (Fig. 6). To obtain
a linear relationship between the DI values of ESR spectra and
the SPIO concentration, the baseline of ESR spectra must be
flat. Otherwise, baseline corrections are needed to improve the
accuracy of ESR measurements.
Previously, ESR has already been described for studying iron
oxide particles, mainly to characterize their physicochemical
Fig. 5 Preparation of RGD-grafted PLGA-based nanoparticles loaded with SPIO and paclitaxel. The mixture
was added dropwise with a glass syringe and a 23G needle under magnetic stirring into an aqueous solution
containing 1 % PVA and stirred overnight to evaporate the organic solvent

a
ESR signal intensity

ESR signal intensity

Double integration (DI)

Magnetic field (G) Magnetic field (G)

b
DI

[SPIO]

Fig. 6 Quantitative ESR. (a) Double integration (DI) of the first derivative of an SPIO ESR spectrum. For
quantitative ESR, the baseline of acquired ESR spectra should be flat. (b) Linear relationship between DI values
and the SPIO content in samples
14 Fabienne Danhier et al.

properties or to measure their distribution in tissues after sys-

temic injection [21]. For the characterization of new SPIO
formulations using ESR spectroscopy, a calibration curve
using several dilutions of iron oxides is mandatory to precisely
determine the iron oxide content of samples. Of note, iron
oxides used for the calibration must share similar physicochem-
ical properties than those contained in the samples [16, 21].
For this reason, inductively coupled plasma mass spectroscopy
(ICP-MS) was used to validate the ESR technique [16] (see
section 3.1.4). Unlike ESR, ICP-MS is not specific for SPIO
and measures the total iron content in samples [16, 21]. ESR
was used in several studies to measure in vitro and ex vivo the
iron oxide content in cells and rodent tissues such as the liver,
the brain, the lungs, the kidneys, and tumor tissues, while ICP-
MS cannot [21, 22].
For iron oxide quantification in aqueous solutions (Fig. 7),
samples are drawn into 75 μL hematocrit capillaries. Capillary
tubes are sealed using hematocrit sealing compound. Samples
are next placed into ESR quartz tubes. In order to obtain
reproducible ESR measurements (ESR spectrometer operating
at 9.4 GHz), it is crucial to keep the exact same location of the
samples in the ESR cavity (see section 3.1.4).
To quantify ex vivo the iron oxide content in rodent tissues
(Fig. 8), sample freeze-drying is mandatory to minimize the
non-resonant absorption of the electromagnetic radiation by

b
ESR quartz tube
a

ESR cavity

Capillary tube containing

iron oxides

ESR quartz tube Haematocrit capillary Sealing compound

Fig. 7 ESR measurements of aqueous samples containing iron oxides. (a) Material needed for ESR measure-
ments. (b) Schematic representation (upper panel ) and picture (lower panel ) of ESR tube positioning. The
samples should always be placed at the same position in the center of the ESR cavity
 Tumor Targeting by RGD-Grafted Nanotheranostics 15

Fig. 8 Ex vivo ESR measurements of freeze-dried tissue samples containing SPIO. (a) Material needed for ESR
measurements. (b) Picture showing a correct positioning of the sample in the center of the ESR cavity

the liquid water contained in tissues (see section 3.2.3). The

freeze-dried sample, crushed into a fine powder, is weighed and
then placed in the cap of an 18G needle. The cap containing the
sample is next fixed to a homemade ESR stand. As already
stated, the position of samples in the ESR cavity must be kept
constant for all measurements in order to obtain reproducible
data. For the SPIO calibration, liquid iron oxide standards are
dispensed in caps of 18G needles, briefly centrifuged, and next
heated for 72 h at 60  C to remove the water. After iron oxide
quantification, results are normalized to the dry weight of
samples.
3. For antitumor efficacy and biodistribution studies, CT26 colon
carcinoma was implanted subcutaneously in the right flank of
mice (see sections 3.2.1 and 3.2.4). Volume of spheroidal
tumor was measured as described previously using the formula
V ¼ l  w  h, where V ¼ volume, l ¼ length, w ¼ width,
and h ¼ height [14, 23]. These external tumors can be daily
measured using an electronic caliper. By contrast, for MRI
studies, because of its localization, respiratory artifacts
appeared, and correct acquisitions of tumors were impossible
to perform. For this reason, we decided to change the localiza-
tion of the tumor. We implanted thus CT26 tumor cells
16 Fabienne Danhier et al.

Fig. 9 Positioning in a bandage of a 1.1 T neodyme-iron-bore external magnet on

the surface of the tumor during 1 or 4 h on CT26 tumor-bearing mice

subcutaneously in the right leg of mice. The size of the tumor

was evaluated first using the electronic caliper and more pre-
cisely by MRI (see Note 5).
4. For the magnetic guidance of SPIO-loaded nanoparticles, use a
1.1 T neodyme-iron-bore external magnet on the surface of the
tumor during 1 or 4 h (see section 3.2.1). Under anesthesia, a
bandage was performed to avoid mice to remove their magnet.
First, the magnet was maintained with compresses, avoiding
injury. Second, the whole was fixed with a 3 M tape (Fig. 9).
5. As aforementioned in Note 3, for MRI studies, the size of
tumors for the study might be between 50 and 100 mm3. A
first approximate measurement was performed using the elec-
tronic caliper. We observed that MRI results and associated RSD
were impossible to compare due to the difference of tumor size
of mice. Hence, before each MRI acquisition of a mouse, the
volume of the tumor was measured and determined precisely,
using the delimitation of the VOI, as explained in section 3.2.4.

Acknowledgment

This work is supported by grants from the Université catholique de

Louvain (F.S.R.) and Fonds National de la Recherche Scientifique (F.
R.S.-F.N.R.S.). F. Danhier is a Postdoctoral F.R.S.-F.N.R.S.
Research Fellow. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the
manuscript.
 Tumor Targeting by RGD-Grafted Nanotheranostics
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Methods in Pharmacology and Toxicology (2018) 19–34
DOI 10.1007/7653_2015_42
© Springer Science+Business Media New York 2015
Published online: 12 August 2015

Fabrication of cRGD-Conjugated Dual-Responsive

Micelles to Target αvβ5 Integrin-Overexpressed Cancer
Huacheng He, Remant Bahadur K.C., and Peisheng Xu

Abstract
Decoration of nano-sized carriers with targeting ligands facilitates their cellular uptake in specific cells due
to the ligand-receptor interaction and is being widely applied to fabricate nanoparticles for tumor-targeted
therapy. In this chapter, we describe a strategy to covalently attach cyclo(Arg-Gly-Asp-D-Phe-Cys)(cRGD)
peptide to a pH and redox potential dual-responsive micelle to realize tumor targeting. The micelle
formation is based on the self-assembly of an amphiphilic polymer. The synthesis of the polymer and its
post-modification including PEG-SH grafting and cRGD conjugation are comprehensively described. The
fabrication of micelles and the investigation of its responsiveness to pH and redox potential are further
introduced. Finally, the study of the targeting effect of cRGD micelles to αvβ5 integrin-overexpressed HCT
116 cells is also described.

Keywords: Conjugate, cRGD, Integrin, Micelle, Polymer, Responsive, Stimuli, Target,

Tumor therapy

1 Introduction

Nano-sized carriers such as liposomes [1], micelles [2], and nano-

gels [3, 4] have been widely used to deliver anticancer drugs for
cancer treatment. These carriers can increase the water solubility of
the anticancer drugs [5], prolong their circulation time in the
bloodstream [6], and improve their accumulation in tumor tissues
due to the enhanced permeability and retention effect (EPR effect)
[7]. Upon accumulation in tumor, carriers should enter cancer cells
where anticancer drugs need to be released out and kill the cells [8].
Based on this scenario, high anticancer efficacy can be achieved by
increasing the accumulation and cellular uptake of carriers in tumor
as well as promoting enough drug release from the carriers. To
boost the drug release, stimuli-responsive nanoparticles are devel-
oped [9]. These carriers can greatly release anticancer drugs in
tumor by responding to internal or external stimuli that exerted
in tumors such as pH, redox potential, and temperature [10, 11].
To enhance tumor accumulation and cellular uptake, carriers deco-
rated with targeting ligands have been proved to be effective based
on the ligand-receptor interaction [12]. These ligands include small
19
20 Huacheng He et al.

molecules [13], peptides [14], and antibodies [15]. Among them,

cRGD, a cyclic arginine–glycine–aspartate (RGD) peptide, has
exhibited a great potential. cRGD shows high affinity to αvβ3 and
αvβ5 integrins [16, 17]. Nanoparticles modified with cRGD exhi-
bits significant tumor accumulation and cellular uptake both
in vitro and in vivo [18, 19].
According to above studies, we develop a cRGD-conjugated
pH and redox potential-responsive micelle to target αvβ5 integrin-
overexpressed cancer [20]. The micelles can efficiently enter cancer
cells by cRGD-integrin interaction. Upon cellular uptake, the anti-
cancer drug will be released out from the micelles in endosomes/
lysosomes due to the low pH there and in cytoplasm owing to the
high glutathione (GSH) concentration (1~10 mM). The micelles
are fabricated from a PEG-grafted polymer. The polymer is synthe-
sized by free radical polymerization following the grafting of PEG-
SH and conjugation of cRGD by thiol-disulfide exchange reaction.
In vitro, pH and redox responsiveness of the micelles is confirmed
by a dialysis method. And the targeting effect of cRGD-conjugated
micelles is verified in HCT 116 which overexpresses αvβ5 integrin.

2 Materials

2.1 Polymer 1. Aldrithiol-2 (Tokyo Chemical Industry Co., LTD, Portland,

Synthesis OR, USA). Stored at 4
C and avoiding light exposure.
2. 2-Mercaptoethenol (Sigma-Aldrich Chemical Co., St. Louis,
MO, USA).
3. Methanol (ACS reagent, Sigma).
4. Acetic acid (ACS reagent, Thermo Fisher Scientific Inc.,
Waltham, MA, USA).
5. Nitrogen (Airgas Inc., Radnor Township, PA, USA).
6. Silica gel (spherical, 100 μm) (Tokyo Chemical Industry Co.,
LTD, Portland, OR, USA).
7. Gravity column (33 cm  24 mm  28 mm, Sigma).
8. TLC plates (Sigma).
9. Acryloyl chloride (97.0 %, contains <210 ppm MEHQ as
stabilizer, Sigma). Store at 4
C and avoiding light exposure
10. 2,2-Azobisisobutyronitrile (AIBN) (purity 98 %, Sigma).
Stored at 20
C and recrystallization if necessary.
11. Anisole (purity > 99.0 %, Sigma).
12. Hexane (ACS reagent, Sigma).
13. Ether acetate (ACS reagent, Sigma).
14. Dimethyl sulfoxide (ACS reagent, Sigma). Stored in a dried
place and avoiding exposure in humidity environment.
Another random document with
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The Project Gutenberg eBook of Lord Lister
No. 0116: Een drama uit de groote wereld
 This ebook is for the use of anyone anywhere in the United
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eBook.

Title: Lord Lister No. 0116: Een drama uit de groote wereld

Author: Theo von Blankensee

Kurt Matull

Release date: October 25, 2023 [eBook #71959]

Language: Dutch

Original publication: Amsterdam: Roman- Boek- en Kunsthandel,

1910

Credits: Jeroen Hellingman and the Online Distributed

Proofreading Team at https://www.pgdp.net/ for Project
Gutenberg

*** START OF THE PROJECT GUTENBERG EBOOK LORD LISTER

NO. 0116: EEN DRAMA UIT DE GROOTE WERELD ***
[Inhoud]

[1]

[Inhoud]
EEN DRAMA UIT DE GROOTE
WERELD.
EERSTE HOOFDSTUK.
 Een hertogin in zicht.

Lord Lister, alias John C. Raffles, de Groote Onbekende, legde de

„Times” neer, nadat hij bijna een half uur lang de laatste nieuwtjes uit
Engeland had zitten lezen.

En terwijl hij zijn kop koffie aan den mond bracht, sprak hij tot Charly
Brand, zijn vriend en secretaris:

„Willy Montglad heeft weer eens tienduizend pond in zijn club

verloren.”

„Willy Montglad,” antwoordde zijn metgezel aan de ontbijttafel, die

inmiddels zijn derde sneedje geroosterd brood boterde en met een
lachje opzag naar zijn knappen overbuurman, „was altijd door den
speelduivel bezeten. Hij zal zichzelf en zijn ouden vader geheel
ruïneeren.”

„Als de oude man dat tenminste niet reeds lang is,” voegde lord Lister
eraan toe.

„Ik geloof niet,” meende Charly, „dat hier in Holland zoo grof gespeeld
wordt. Na Monte Carlo geloof ik wel, dat in de Londensche clubs.…..”

Hij voltooide zijn zin niet, want na een bescheiden tikken op de deur
was James, de vertrouwde huisknecht, binnengekomen en bracht op
een zilveren blad de pas aangekomen brieven.

Eén daarvan, gesloten in een groote gele enveloppe, was gericht aan
Charly, de andere waren bestemd voor graaf Van Haaren, onder
welken naam Raffles eenige maanden geleden een prachtige villa in
een der buitenwijken van Amsterdam had gehuurd.
Met een smal pennemesje sneed Charly voorzichtig het couvert open
en nadat hij den brief, dien het bevatte, vluchtig had doorgelezen,
riep hij op vroolijken toon uit:

„Goed nieuws, Edward! Raad eens wat?”

Glimlachend keek lord Lister zijn jongen vriend aan.

„Dat is moeilijk te raden, zelfs voor mij. Een erfenis soms?”

„Ja, waarempel, een erfenis! Ik ben erfgenaam van een oudoom, van
oom Willibald Brand!”

Dit zeggende overhandigde Charly den brief aan zijn vriend en terwijl
hij vol grappigen trots, de beide handen in de broekzakken en het
blonde, jongensachtige hoofd eenigszins achterovergeworpen, voor
lord Lister ging staan, vervolgde hij:

„Ik wist het wel, Edward, dat ik nog eenmaal schatrijk zou worden!
Vijfduizend pond heeft de oude baronet mij nagelaten; denk eens,
Edward, vijf duizend pond! Dat is in Hollandsche munt de kapitale
som van zestigduizend gulden! Ik weet niet, wat ik met al dat geld zal
doen!”

Glimlachend keek de Groote Onbekende op en zijn vriend de blanke,

smalle hand toestekende, antwoordde hij:

„Ik wensch je geluk met dat buitenkansje, boy! Allereerst zul je nu,
om aan de noodige formaliteiten te voldoen, even naar Engeland
moeten oversteken. Onderweg kunnen we dan eens overleggen, op
welke [2]wijze jij je onmetelijke rijkdommen het best kunt besteden.”

Charly lachte hartelijk.

„Dus je bent van plan om mij te vergezellen?”

„Ja, ik verlang er naar, mijn geliefkoosde Londen weer eens terug te
zien. Het is nu ruim een half jaar sinds we er voor het laatst een paar
weken waren.”

„Goed, Edward, wanneer zullen we gaan?” vroeg Charly, „dan kan ik

James alvast de noodige bevelen geven. Nemen we hem mee?”

„Laat hem nog vandaag vertrekken. Hij kan dan zorgen, dat we ons
vossenhol in Regentstreet een beetje behaaglijk vinden.

„Maar vertel eens, boy, had die oom zooveel geld, dat-ie jou een
kapitaaltje kon nalaten?”

„Ja zeker, Edward. Het was oom Willibald, de scheepskapitein. Heb

ik je nooit verteld van de merkwaardige weddenschap, waaraan oom
Willibald zijn kapitaal te danken heeft gehad?”

„Neen, mijn jongen, ik herinner mij niet, daarvan ooit iets te hebben
vernomen. Als het een interessante geschiedenis is, houd ik me zeer
voor dat verhaal aanbevolen.”

„Ja, het is interessant en hoogst grappig tevens,” antwoordde de

jonge secretaris. „Luister maar, Edward! Het is gauw verhaald.”

Lord Lister leunde op zijn gemak in zijn stoel achterover en Charly

begon zijn vertelling:

„Zooals ik je reeds zei, was oom Willibald scheepskapitein en het was

op een zijner reizen, welke hij naar Engelsch-Indië maakte, dat zich
onder zijn passagiers een Londensch geneesheer bevond, die in
opdracht van een vereeniging van zeer geleerde oudheidkundigen
zich naar Egypte begaf.

„De dokter moest daar een mummie zien machtig te worden.

„De vereeniging, die hem had uitgezonden, was samengesteld uit
zeer achtenswaardige mannen, die hun dagen en nachten aan
wetenschappelijke onderzoekingen hadden gewijd en zij hadden hun
geleerden broeder opgedragen om de mummie zoo mogelijk langs
eerlijken weg te bemachtigen.

„Hij mocht zich echter in geen geval door een verkeerde opvatting
van het begrip eerlijkheid in zijn taak laten belemmeren.

„De dokter had een blanco-chèque met de handteekening van den

secretaris er op, in zijn portefeuille en hij had volmacht van de
vereeniging om daar de som op in te vullen, die hem goed dacht.

„Maar ofschoon hij een geleerd man was, was de dokter zeer zuinig
van aard en hij vatte het stoute plan op om een mummie uit Egypte
mee te brengen, die hemzelf niets zou hebben gekost.

„Hij wist zeer goed, dat er een overvloed van mummies in de

catacomben lagen.

„„Niemand heeft iets aan die dingen,” zoo redeneerde hij tot zichzelf,
„en derhalve is het volstrekt geen inbreuk op de wet der eerlijkheid
om er eentje mee te nemen—natuurlijk in het belang der
wetenschap.”

„In den loop der jaren had de dokter ook het een en ander van het
Arabisch geleerd en het kostte hem heel weinig moeite om drie
Arabieren over te halen, hem in de berooving van een graftombe bij
te staan.

„Op een stikdonkeren nacht begaven zij zich op weg naar de

catacombe van een Pasha en het gelukte hun werkelijk hun plan ten
uitvoer te brengen.
„De mummie, die zij hadden weten te bemachtigen, was geen
bijzonder goed exemplaar.

„Het dreigde elk oogenblik uit elkaar te zullen vallen, als er niet
spoedig iets aan werd gedaan om het bijeen te houden.

„Maar dit was juist een voordeel voor den genialen dokter. Het was
een teeken van buitengewonen ouderdom en hij wreef zich van
genoegen de handen bij de gedachte, aan het aanzienlijke bedrag,
dat hij op de chèque zou invullen.

„„Dit is de mummie van den Pharao,” had een van de Arabieren den
dokter verteld.

„De dokter had te veel gezond verstand om een dergelijk verhaaltje te

gelooven, maar hij overlegde bij zichzelf, dat het hem niet al te veel
moeite zou kosten, zijn collega’s in het vaderland van die gewichtige
waarheid te overtuigen.

„De Arabieren waren zoo handig geweest om tegelijk met de

mummie ook de steenen kist mee te nemen.

„Deze kist was ontegenzeggelijk een prachtig voorbeeld van antieke

kunst en geheel bedekt met geheimzinnige hiëroglyphen.

„Bovenop het deksel van de kist was een ruw gelaat geschilderd—
een afbeelding van den dood.

„De kleur had de tand des tijds weerstaan en het gelaat grijnsde den
Engelschen dokter nog tegen, toen deze met onverholen voldoening
op het resultaat van zijn zonderling avontuur neerzag.

„„Een koningsmummie?” bromde hij in zichzelf, terwijl hij de steenen

kist rechtop zette.
„„Goed, dan zal het ook een echte koningsmummie wezen! Ik zal er
een inscriptie op maken, die in den [3]rook zwart laten worden en er
dan in Engeland mee voor den dag komen.

„„De geleerde heeren collega’s zullen verbaasd staan te kijken over

mijn belangrijke vondst.

„„Ha, ik zie den straal van vreugde al, die de gezichten van mijn
vrienden zal verlichten, wanneer zij dit staaltje van antieke kunst en
antiek bederf onder de oogen krijgen.””

Charly zweeg een oogenblik en keek zijn vriend glimlachend aan.

Toen vroeg hij:

„Nu, Edward, boeit mijn verhaal je of zal ik de rest maar liever vóór
mij houden?”

„Ik ben geheel en al aandacht, beste jongen,” antwoordde de Groote

Onbekende, „vertel verder, hoe het afliep met dezen oudheidkundige
en hoe die koningsmummie in verband staat met jouw erfenis?”

„Nog een oogenblik geduld, Edward, ik ben er bijna!” lachte Charly en

toen vervolgde hij:

„De dokter had dien nacht een aangenamen droom. Hij vulde
namelijk de blanco-chèque in voor een bedrag van honderdduizend
gulden. Dit was wel geen geringe prijs, doch hij was er zeker van, dat
zijn vrienden dat bedrag er met vreugde voor zouden betalen.

„Maar, helaas! Op dien aangenamen droom zou een wreed ontwaken

volgen.

„Hij nam een passagebiljet voor den terugtocht en verstopte de

mummie veilig in zijn hut.
„Het was wel geen erg pleizierig idee om zoo’n oud lijk in dezelfde
ruimte te hebben, waar hij moest slapen, maar hij was met dat idee
verzoend door de gedachte, dat het al zoo lang een lijk was geweest,
dat zijn macht om onheil te stichten wel niet heel groot meer kon zijn.

„Op een middag had er gedurende het diner een eigenaardig

onderhoud plaats.

„De kapitein—oom Willibald—vertelde van een zijner matrozen en

sprak tot den dokter, die naast hem zat:

„„U kent dien matroos Bilson toch wel, dokter?”

„„Zeker,” antwoordde de Engelsche geleerde.

„„Die kerel heeft een eigenaardigheid,” vervolgde de kapitein, „die

men zelden aantreft in zoo sterke mate als bij hem.”

„„En dat is?”

„„Hij kent absoluut geen vrees. Hij weet letterlijk niet, wat angst is!”

„„Ik wed met u om een hoog bedrag,” sprak de dokter, terwijl een
geheimzinnige glimlach op zijn gelaat verscheen, „dat ik dien held
van u bang heb gemaakt, eer we een dag verder zijn.”

„„Ik neem de weddenschap aan,” sprak de kapitein, die zeker van zijn
zaak meende te zijn, „maar dan ook om een flink bedrag.”

„„Uitstekend!” klonk het van des dokters lippen. „Laat ons zeggen, dat
we om tienduizend wedden.”

„„Drommels, dat is geen bagatel, maar top! Ik ga de weddenschap

met u aan, dokter. Dus u zult onzen Bilson vrees aanjagen vóór
morgen? Ik ben bang voor uw tienduizend gulden, dokter!”
„„Ik stel echter deze ééne voorwaarde, het moet een gepaste scherts
blijven! Ik weet maar al te goed, hoe dergelijke zoogenaamde
aardigheden soms ontaarden in roekeloosheid of baldadigheid.”

„„Zeker,” zeide de dokter, „ik zal niet te ver gaan, ik zal zelfs niets
tegen hem zeggen.”

„De weddenschap werd dus aangegaan en de belangstelling van alle

passagiers was op den uitslag gericht.

„Te drie uur ’s nachts liep de dokter op het dek rond met een glimlach
op het gelaat. Hij was er zeker van, zijn geld te zullen winnen en hij
maakte het plan om voor zijn vrouw een prachtigen juweelen
armband te koopen.

„Juist toen de maan vol door een dik gordijn van wolken heenbrak,
hoorde hij mijn oom, Bilson bevel geven, om den ankerketting uit het
ruim te halen.

„De dokter glimlachte en hij had al een opwekkend middel gereed,

om Bilson weer bij te brengen, als deze soms flauw mocht vallen van
schrik en angst.

„De matroos ging het ruim in, maar de ketting was nergens te vinden.
Hij zocht er op elke denkbare plaats naar, maar vond hem nergens.

„Toen hij den kapitein dit kwam vertellen, beval deze hem, om in de
raderkas te gaan kijken.

„De matroos opende de deur.

„De maan zond een stroom van zilverlicht naar binnen.

„Wat zag hij daar recht voor zich staan?

„Het was een zonderlinge gestalte, die daar in een soort van kist
tegen den muur stond—zoowaar een mummie.

„„O, ben jij de schurk, die den ankerketting verstopt heeft, zeg?” riep
hij uit.

„Zonder een woord meer te zeggen, of zich een oogenblik te

bedenken, greep hij een groote ijzeren staaf van den grond op en
sloeg er met zooveel geweld op [4]los, dat er van de koningsmummie
niets meer overbleef, dan een hoopje waardeloos stof.

„De dokter had ongeduldig op zijn triomf staan wachten, maar op

deze ontknooping had hij geen oogenblik gerekend.

„Dat was hem toch wel een beetje àl te erg!

„„O, mijn koningsmummie!” riep hij uit, terwijl hij over het dek rende.

„Hij wierp een wanhopigen blik op zijn vernietigden schat.

„Daarop schoot hem de gedachte te binnen, dat hij misschien zijn

tienduizend gulden toch nog wel zou kunnen redden en hij zeide tot
den matroos:

„„Hier heb je wat van mij mijn jongen, veeg dat stof weg!”

„Maar hij had nauwelijks uitgesproken, of de kapitein klopte hem op

den schouder en sprak:

„„Dokter, wilt u mij de tienduizend gulden uitbetalen? U zult moeten

bekennen, dat u de weddenschap verloren hebt!”

„Zeer onwillig gaf de dokter mijn oom een cheque over het
aanzienlijke bedrag en, als een beeld der wanhoop, sloop hij naar zijn
hut en bleef daar den geheelen nacht op den rand van zijn hangmat
zitten peinzen, hoe hij zich uit de moeilijkheid kon redden, waarin hij
geraakt was.

„Op welke wijze hij daarin geslaagd is, kan ik je niet vertellen, maar
zeker is het, dat het bedrag van die weddenschap de grondlegger is
geweest tot het aardige kapitaal, dat mijn oom bij zijn dood heeft
nagelaten.

„Zie je, Edward, dat was nu het verhaal van de koningsmummie! En

zeg me nu wanneer we naar Londen gaan, opdat ik de erfenis van
oom Willibald in ontvangst kan nemen.”

„Wel,” antwoordde Lord Lister, „laat ons overmorgen afreizen. James

heeft dan twee dagen om de noodige maatregelen te nemen.”

„Uitstekend, Edward! Dan behoeven wij morgen het avondfeest in

hotel de l’Europe en het bal ook niet te missen. Ik heb reeds verleden
week den eersten dans afgesproken met het jongste freuletje van
Verdooren, je weet wel, Edward, dat slanke meisje met haar
prachtige bruine oogen.”

„Waar ik je al eenige keeren mee zag praten in den foyer van den
schouwburg?”

„Ja, ik ben dolgraag in haar gezelschap, hoewel ik ook haar oudere

zuster charmant vind. Maar nu zal ik eerst James zeggen, dat hij zich
reisvaardig maakt.”

Charly drukte op het knopje der electrische bel en toen de oude

getrouwe weer in de kamer was gekomen, deelde hij dezen mede,
wat er voor de eerstvolgende dagen besloten was.

Eenige dagen daarna was de weelderig ingerichte villa in Regent

Park, die geruimen tijd onbewoond was geweest, weer in gebruik
genomen door de beide vrienden.
Gedurende zijn afwezigheid had Raffles de woning eenigszins laten
moderniseeren.

Overal was electrisch licht aangebracht en centrale verwarming

bevond zich nu door het geheele huis, alleen in de studeerkamer van
den Grooten Onbekenden was op diens uitdrukkelijk bevel de breede
haard gebleven.

Charly had reeds een conferentie gehad met den notaris, die belast
was met het ten uitvoer brengen van de laatste wilsbeschikkingen
van den overleden baronet en zijn verblijf in Londen zou slechts
gedurende eenige weken noodig zijn.

Dien avond had lord Lister zich naar de „Club of Lords” begeven,
waarvan hij reeds jarenlang lid was.

Hij stond bij de leden bekend als de schatrijke lord Aberdeen en was
zeer gezien bij de heeren, die het prachtig ingerichte clublokaal
geregeld bezochten.

Men wist, dat de knappe, elegante lord bijna de geheele wereld had
doorgereisd, dat hij zoowel Indië als Amerika kende en dat hij ook op
het gebied van litteratuur, muziek en schilderkunst uitmuntte boven
verreweg de meesten van hen.

Lord Lister had aan een der grootere tafels plaats genomen, waar
reeds een zestal heeren omheen zaten, die hem allen met blijdschap
hadden begroet na zijn lange afwezigheid.

De Groote Onbekende vertelde van een groot sportfeest, dat hij

eenige weken te voren in Brussel had bijgewoond en vol
belangstelling luisterden de aanwezigen naar zijn interessant verhaal,
toen hun aandacht werd afgeleid door het verschijnen van een der
jongste leden van de Club, Sir Basil Malwood, die eveneens aan het
tafeltje plaats nam en glimmend van genoegen vertelde, dat hij het
allerlaatste nieuwtje wist, heet uit den oven.

„Hebt gij het groote nieuws al gehoord, omtrent Silverton?” vroeg hij
aan graaf Simkins, dien hij het meest van nabij kende.

„Merkwaardig, wa-blief?”

„Wat is er dan gebeurd? Ik dacht, dat hij goed en wel in Amerika zat,”
luidde het antwoord van den [5]graaf, die nu een en al belangstelling
was geworden, want het gold hier een in de Londensche voorname
kringen veel besproken persoonlijkheid.

„Nu dat is ook zoo,” gaf Basil te kennen, „en daar is het ook gebeurd.
Je weet, dat hij al een jaar of drie heeft rondgekeken naar een
erfdochter, met voldoende fortuin om zijn hart, hand en schulden te
kunnen aanvaarden, maar dat het hem nog niet gelukt was om zoo’n
goudvischje aan den haak te slaan.”

„Ik herinner mij nog,” viel graaf Simkins hem in de rede, „dat hertog
Silverton indertijd een kansje heeft gewaagd bij die rijke Iersche
brouwersdochter, die zoo ordinair spreekt en zulk rood haar heeft,
maar dit dametje kon hem niet bekoren, misschien omdat zij zoo kort
aangebonden was of omdat zij zoo onaangenaam lachte of omdat er
iets niet in den haak was met de huwelijksvoorwaarden.

„Ik weet het rechte niet en ik geloof, dat niemand dit weet, maar een
feit is het, dat er iets in den weg kwam en dat het afraakte.”

„Ja”, vervolgde nu Malwood weer het verhaal, „daarna is hij nog aan
het hengelen geweest naar een fabelachtig rijk Indisch meisje, dat
met haar vader tijdelijk in Birmingham woonde.

„Een neusje, als een vuurspuwende berg. Toen hij haar niet kreeg,
hengelde hij achtereenvolgens naar de dochter van een
Sheffieldschen messenfabrikant; naar een meisje uit Glasgow, wier
vader in de reederij rijk is geworden; naar een dametje uit Zuid-Wales
met aandeelen in steenkolenmijnen en naar nog een half dozijn rijke
erfdochters.

„Maar bij geen van allen wilde het goed vlotten.

„Men zegt, dat Silverton te moeilijk was in zijn keuze en te veel geld
verlangde.”

„Dat hij veeleischend was, kan ik constateeren,” sprak de bejaarde

lord Crofton, een heer met een scherpen neus, sterk bijziende oogen
en een glimmenden, kalen schedel.

„Hij hechtte bijvoorbeeld veel aan uiterlijk en manieren,” vervolgde hij,

„en daarom kon hij het nooit met zichzelf eens worden omtrent de
rechte persoon.

„Hij zocht een ideaal meisje, dat alles en alles in zich vereenigde:
jeugd, schoonheid, voorname manieren, uitmuntende opvoeding en
een kapitaal, dat klonk als een klok.”

„En daarom is hij dus naar Amerika overgestoken op jacht naar

dollars?” opperde een der andere heeren.

„Juist,—precies als zijn lotgenooten,—en ik vind, dat hij groot gelijk

gehad heeft. Maar wat meer zegt, hij heeft ten langen laatste ook
gevonden wat hij zocht,—het moet juist iets van zijn gading zijn.”

„Hoe heet zij?” klonk het uit eenige monden tegelijk.

„Miss Ansberg heet zij,—het is de zuster van dien man van den
Ansberg-motor, herinnert ge u niet?—die machine, die zoo’n
enormen opgang heeft gemaakt.”