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International Conference on Nanotheranostics

ICoN 2013

Conference Program and Summaries

26-28 September 2013

Golden Bay Beach Hotel Larnaca, Cyprus

1 | 2013 International Conference on Nanotheranostics (ICoN 2013)

Organizers

European Union http://europa.eu/index_en.htm

FP7 http://www.cordis.europa.eu/fp7

Marie Curie Actions - Industry Academia Partnerships and Pathways http://ec.europa.eu/research/mariecurieactions/about-mca/actions/iapp/

European Federation of Biotechnology http://www.efb-central.org

Cyprus Medical Association http://www.cyma.org.cy

EPOS-Iasis Research & Development Ltd http://www.epos-iasis.com

University of Cyprus http://www.ucy.ac.cy

Cyprus University of Technology http://www.cut.ac.cy

EUROPEAN FEDERATION OF

BIOTECHNOLOGY Section on Medicines Development

http://europa.eu/index_en.htm

http://cordis.europa.eu/fp7/home_en.html

http://ec.europa.eu/research/mariecurieactions/about-mca/actions/iapp/index_en.htm

http://www.efb-central.org/

http://www.cyma.org.cy/

http://www.epos-iasis.com/

http://www.ucy.ac.cy/

http://www.cut.ac.cy/

http://www.cyma.org.cy/


Contents Welcome .................................................................................................................................. 3

Committees .............................................................................................................................. 5

Keynote Speakers .................................................................................................................... 6

Program at a glance ................................................................................................................. 7

Sessions ................................................................................................................................... 8

Thursday 26/9/2013 ............................................................................................................................. 8

Plenary Session ................................................................................................................................ 8

Session 1 – Imaging and Characterization of Nanotheranostic Agents .......................................... 8

Session 2 – Novel Nanotheranostic Approaches ............................................................................. 9

Poster Session and Reception ......................................................................................................... 9

Friday 27/9/2013 ................................................................................................................................ 10

Session 3 – Biocompatibility and Toxicity of Nanotheranostic Agents ......................................... 10

Session 4 – Drug Delivery to Solid Tumors and Through Barriers ................................................ 10

Session 5 – Delivery of Nanotheranosis ........................................................................................ 11

Cultural Activities and Conference Dinner .................................................................................... 11

Saturday 28/9/2013 ........................................................................................................................... 12

Session 6 – Cancer Theranostics ................................................................................................... 12

Short Course: Nanotheranostics: all-in-one personalized medicine ............................................. 12

Session 7 – Materials and Tissues in Nanotheranostics ................................................................ 13

Round Table Discussion and Student Awards ............................................................................... 13

Abstracts ................................................................................................................................ 14

Author Index ........................................................................................................................... 67

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Welcome Dear Delegates of the ICoN2013 Conference, It is a great pleasure and an extreme honor to welcome all of you to Cyprus on the occasion of the 2013 International Conference on Nanotheranostics. In the vast field of nanomedicine, “Theranostics” combine therapeutics and diagnostics, aiming to provide a comprehensive platform for diagnosis, therapy and monitoring of the patient, leading to customized approaches and personalized treatment. Emerging nanotechnology discoveries provide a unique opportunity to design and develop such combination agents, permitting the delivery of therapeutics and concurrently allowing the detection modality to be used not only before or after but also throughout the entire treatment regimen, defining new supra-disciplinary fields in major clinical specialties such as Radiology, Surgery, Gynaecology and Oncology, to mention few. That is why a Nanotheranostics Conference is an important initiative and it is expected to provide the forum for idea exchange and discussion to create a potential high-impact nanomedicine paradigm. The ICoN 2013 Conference aims to provide the optimal venue to expand nanotheranostics research in a multidisciplinary environment which will bring together all the key researchers in the nanotheranostics field. International scientific events at the interface between cutting edge biotechnology and clinical translational research, through such an undertaking, are highly significant since they define in the most appropriate way the contribution of medical, pharmaceutical, scientific and academic entities towards the national and pan-European planning for knowledge- and innovation-based economies. Within the framework of the EC-FP7 Marie-Curie, Industry-Academia Pathways and Partnership Program, the technology transfer entity EPOS-Iasis, R&D, the University of Cyprus, the Cyprus University of Technology, the European Federation of Biotechnology and the Cyprus Medical Association, have joined forces towards a trans-disciplinary event that is expected to pave the way towards effective collaborations in translational nanomedicine. ICoN2013 brings together high-caliber researchers, pioneers in the field and a promising population of young scientists at the dawn of their career, from across Europe, USA, Asia and Africa. It combines thoughtfully topic-oriented plenary and keynote lectures with innovative research papers. Major thematic areas include (i) the roadmap of nanotheranostics development in a patient-oriented approach, (ii) emerging challenges for nanotheranostic applications, (iii) toxicology, regulatory aspects and ethics and, most importantly (iv) an emphasis session on cancer nanotheranostics. A specifically targeted and specially designed seminar for clinicians is featured, thus underlining the fact that nanotheranostics are currently mature enough for strategic clinical applications. It is, therefore, evident that the establishment of the International Conference on Nanotheranostics promises exciting new pathways towards clinical and research excellence and we expect that ICoN 2013 will set the grounds for the establishment of a lasting inter-sectoral and trans-disciplinary network on novel diagnostics and therapeutics both locally and on an international dimension. Cyprus, an island of nano-scale dimensions from a global prospective, but with tremendous potentials when it comes to creativity and effectiveness, is certainly the place for a Nanotheranostics Conference to be. I take the opportunity to wholeheartedly express the gratitude of the Organizing Committee and myself to His Excellency the President of the


House of Representatives of the Republic of Cyprus, Mr Yiannakis Omirou who, having acknowledged the significance and the anticipated impact of such an event, has put ICoN2013 under his auspices. This event would not have been possible without the endless efforts of all vice – chairs and members of the Organizing Committee nor without the input of the renowned members of the Scientific Committee. I feel deeply honored by their collaboration. Cyprus, a sunbathed island, the birthplace of Aphrodite and the key passage to Europe, has been an excellent example of hardships and endurance throughout its ten-thousand year history. Being at the crossroads of world’s civilizations it has assimilated all passing traits of culture onto its main Hellenic trunk. While at ICoN2013 you are strongly encouraged to take the unique opportunity to experience the turbulent, but still exciting, history of Cyprus and enjoy the warm hospitality of its people. I wish you all a rewarding experience and ICoN2013 and I am looking forward to welcoming you at ICoN2015. Andreani D Odysseos Costas Pitris George Potamitis General Chair Vice Chair Vice Chair


Committees General Chair: Andreani Odysseos, European Federation of Biotechnology, EPOS-

Iasis Research and Development Ltd Vice Chairs: Costas Pitris, University of Cyprus

George Potamitis, Cyprus Medical Association Local Organizing Committee Co-Chairs: Andreas Anayiotos, Cyprus Technological University

Triantafyllos Stylianopoulos, University of Cyprus Treasurer: Theodora Krasia, University of Cyprus Secretary: Costas Pitsillides, Cyprus Technological University Social Activities: Maria Kokonou, EPOS-Iasis Research and Development Ltd Scientific Committee Bogos (Pavlos) Agianian, Democritus University of Thrace, Greece Andreas Anayiotos, Cyprus Technological University Rena Bizios, University of Texas, USA Kenneth A. Dawson, University College Dublin, Ireland Haris Doumanidis, University of Cyprus, Cyprus Kostas Kostarelos, UCL School of Pharmacy, UK Theodora Krasia, University of Cyprus Jan Mollenhauer, University of Southern Denmark, Denmark Costas Pitris, University of Cyprus Claus Rebholz, University of Cyprus Jean-Michel Siaugue, Université Pierre et Marie Curie, France Alex Strongilos, Proactina SA, Greece Triantafyllos Stylianopoulos, University of Cyprus Chrysa Tziakouri-Shiakalli, Cyprus Medical Association


Keynote Speakers A number of keynote presentations will be delivered by distinguished speakers in field. Below is an initial partial list (in alphabetical order):

Bogos (Pavlos) Agianian Democritus University of Thrace Greece

Michael Averkiou University of Cyprus Cyprus

Rena Bizios University of Texas at San Antonio USA

Kenneth A. Dawson University College Dublin Ireland

George Kordas NCSR Democritos Greece

Kostas Kostarelos UCL School of Pharmacy United Kingdom

Jan Mollenhauer University of Southern Denmark Denmark

Jean-Michel Siaugue Université Pierre et Marie Curie France

Triantafyllos Stylianopoulos University of Cyprus Cyprus


Program at a glance

International Conference on Nanotheranostics (ICoN 2013)

Thursday 26/9/2013

Friday 27/9/2013

Saturday 28/9/2013

09.00-11.00

Welcome and Plenary Session

Session 3

Biocompatibility and Toxicity of Nanotheranostic Agents

Session 6

Cancer Theranostics

11.00-11.30 Coffee Break

11.30-13.30 Session1

Imaging and Characterization of Nanotheranostic Agents

Session 4

Drug Delivery to Solid Tumors and Through Barriers

Short Course

Nanomedicine

13.30-14.30 Lunch Break

14.30-16.45 Session 2

Novel Nanotheranostic Approaches

Session 5

Delivery of Nanotheranosis

Session 7

Materials and Tissues in Nanotheranostics

16.45-17.15 Coffee Break Cultural / Social Events

Coffee Break

17.15-18.30 Poster Session Roundtable Discussion Student Awards

20.00-23.00 Conference Dinner


Sessions

Thursday 26/9/2013

Plenary Session

09.00-09.15 Welcome

Andreani Odysseos, Conference Chair, EFB, EPOS-Iasis R&D Ltd

09.15-09.30 Welcome by the Cyprus Medical Association

Dr. Andreas Demetriou, CyMA President

09.30-09.50 European Commission’s Marie Curie Program in Horizon 2020

Dr. Audrey Arfi, European Commission Scientific Oficcer

09.50-10.00 Opening by the President of the House of Representatives

H.E. Mr. Yiannakis Omirou

10.00-10.45 Opportunities Offered by Nucleic Acid-Based Nano-Devices in Cancer Therapy and Theranostics

Jan Mollenhauer, Ines Block, Angela Riedel, Steffen Schmidt, Helle Christiansen, Birgitte Brinkmann Olsen, Helge Thisgaard, Poul Flemming Hoilund-Carlsen, Stefan Vogel and Jesper Wengel, University of Southern Denmark, Denmark (p. 15)

10.45-11.30 Nanoscale Interface Between Engineered Matter and Living Organisms: Understanding the Biological Identity of Nanosized Materials

Kenneth Dawson, UC Doublin, Ireland (p. 16)

11.30 – 11.45 Coffee Break

Session 1 – Imaging and Characterization of Nanotheranostic Agents

Session Chair: Pavlos Agianian, Democritus University of Thrace, Greece

11.45-12.35 Assessing biological specificity of nanoparticles in vitro: test tubes, chips or cells

Pavlos Agianian, Democritus University of Thrace, Greece (p. 17)

12.35-12.55 In-vitro release study and dynamic in-vivo imaging of pH- and Magnetic field sensitive hybrid microspheres

Eleni Efthimiadou, NCSR Demokritos, Greece (p. 18)

12.55-13.15 Preparation and characterization of new elements for the conception of nanohybrids for multicolored bioimaging

Morgane Rivoal and Andreani Odysseos, EPOS Iasis R&D Ltd, Cyprus (p. 19)

13.15-13.35 Size Dependent Biological Profiles of Pegylated Gold Nanorods for Biomedical Applications

Federica Scaletti, University of Florence, Italy (p. 20)

13.35 – 14.30 Lunch


Session 2 – Novel Nanotheranostic Approaches

Session Chair: Jean-Michel Siaugue, UPMC, France

14.30-15.20 Fluorescent and magnetic silica core shell nanoparticles for biomedical applications

Jean-Michel Siaugue, UPMC, France (p. 21)

15.20-15.40 Two different approaches for the synthesis of functionalized silica coated magnetic nanoparticles

Ana B. Davila Ibañez, Verónica Salgueiriño and Jean-Michel Siaugue, UPMC, France (p. 22)

15.40-16.00 Theragnosis combining ferrocenyl derivatives, carbohydrates and nanovectors

Jeremy Malinge, Jean-Michel Siaugue, Christine Ménager, Matthieu Sollogoub, Yongmin Zhang, Gérard Jaouen and Anne Vessières-Jaouen, UPMC, France (p. 23)

16.00-16.20 Chitosan – linear aldehyde nanoparticles obtained from reverse micellar method

Krzysztof Gawlik, Iga Wasiak and Tomasz Ciach, Warsaw University of Technology, Poland (p. 24)

16.20-17.00 New Routes Towards the Synthesis of Natural Products and Designed Derivatives

Elias Couladouros, Agricultural University of Athens, Greece (p. 26)

Poster Session and Reception 17.30-19.00

Session Chair: Dr. Costas Pitris, University of Cyprus, Cyprus

1 Hemocompatibility of Albumin microspheres as drug delivery system, in vitro study

Mohamed Elblbesy, University of Tabuk, Saudi Arabia (p.27)

2 Hemocompatibility of silver nanoparticles

Julie Laloy, Valentine Minet, Lutfiye Alpan, Bernard Chatelain, François Mullier and Jean-Michel Dogné, University of Namur, Belgium (p. 28)

3 From docking to synthesis and grafting: Preliminary results for new Anilinoquinazolines as potential EGFR inhibitors in multifunctional nanocarriers

Fotini Liepouri, Pavlos Agianian, Vassiliki Garefalaki, Elias Couladouros, Andreani Odysseos and Alexandros Strongilos, Proactina SA, Greece (p. 29)

4 Theoretical investigation of a new metal nanoparticle for combined imaging and therapy applications

Myria Angelidou and Costas Pitris, University of Cyprus, Cyprus (p. 30)

5 Surface Enhanced Raman Spectroscopy (SERS) for Point-Of-Care Diagnosis of Urinary Tract Infections

Katerina Hadjigeorgiou, Evdokia Kastanos and Costas Pitris, University of Cyprus, Cyprus (p. 32)

6 Activity, anti-cancer effect and nanodelivery of new anilinoquinazoline EGFR inhibitors

Eftychia Angelou, Fotini Liepouri, Maria Pavlaki, Andreani Odysseos, Jean-Michel Siaugue, Alexandros Strongilos and Pavlos Agianian. Democritus University of Thrace, Greece (p. 34)


Friday 27/9/2013

Session 3 – Biocompatibility and Toxicity of Nanotheranostic Agents

Session Chair: Cyrill Bussy, University of Manchester, UK

09.00-09.50 Toxicity and Safety of Carbon Nanomaterials for Biomedical Applications

Cyrill Bussy and Kostas Kostarelos, University of Manchester, UK (p. 35)

09.50-10.30 Guidelines proposal for studying hemocompatibility of manufactured nanoparticles and impact on fibrinolysis

Julie Laloy, Lutfiye Alpan, Valentine Minet and Jean-Michel Dogné, University of Namur, Belgium (p. 34)

10.30-10.50 Rat whole-body exposure model to nanoaerosol: Development with silicon carbide nanoparticles and study of their toxicity

Julie Laloy, Omar Lozano, Lutfiye Alpan, Valentine Minet, Olivier Toussaint, Bernard Masereel, Jean-Michel Dogné and Stephane Lucas, University of Namur, Belgium (p. 37)


Session 4 – Drug Delivery to Solid Tumors and Through Barriers

Session Chair: Triantafyllos Stylianopoulos, University of Cyprus, Cyprus

11.30-12.20 EPR-effect: a barrier to the effective delivery of large nanomedicines to solid tumors

Triantafyllos Stylianopoulos, University of Cyprus, Cyprus (p. 39)

12.20-12.40 Strategies to improve nanomedicine delivery to solid tumors

Konstantinos Soteriou, Eva-Athena Economides and Triantafyllos Stylianopoulos, University of Cyprus, Cyprus (p. 40)

12.40-13.00 Methods for delivery of living cells to the respiratory system via aerosol route

Tomasz R. Sosnowski, Ewelina Tomecka, Warsaw University of Technology, Poland (p. 42)

13.00-13.20 Enhancement of Drug Absorption Across Intestinal Membrane Using Magnetic Beads

Anjali Seth, David Lafargue and Christine Ménager, UPMC, France (p. 44)

13.30-14.30 Lunch


Session 5 – Delivery of Nanotheranosis

Session Chair: Mike Averkiou, University of Cyprus, Cyprus

14.30-15.20 Ultrasound and microbubbles for monitoring therapies targeting tumor vascularity

Mike Averkiou, University of Cyprus, Cyprus (p. 46)

15.20-15.40 Tunable magnetic/ICG small protocells as a platform for drug delivery

Jean-Sébastien Thomann, Gaëlle Corne, Didier Arl, Naoufal Bahlawane and Damien Lenoble, CRP Gabriel lippmann, Luxemburg (p. 47)

15.40-16.00 Ultrasound-induced temperature elevation for in-vitro controlled release of temperature-sensitive liposomes

Christophoros Mannaris, Jean-Michele Escoffre, Ayache Bouakaz, Marie-Edithe Meyre and Michalakis Averkiou, University of Cyprus, Cyprus (p. 49)

16.00-16.20 Biodegradable Dextran Nanoparticles as Potential Drug and Fluorescent Marker Carrier

Tomasz Ciach, Magdalena Janczewska and Iga Wasiak, Warsaw University of Technology, Poland (p. 52)

16.20-16.40 Engineering carbon nanotubes based scaffolds for the efficient delivery of new tyrosine kinase inhibitors

Davide Giust, Kostas Kostarelos, UCL School of Pharmacy, UK (p. 54)


Cultural Activities and Conference Dinner

17.15-18.30

20.30-23.00

Cultural Activities

Conference Dinner


Saturday 28/9/2013

Session 6 – Cancer Theranostics

Session Chair: Costas Pitsillides, Cyprus University of Technology, Cyprus

09.00-09.50 Folic Acid functionalized Quatro-NanoContainers as targeted agent: In vitro and In vivo study

George Kordas, NCSR Demokritos (p. 55)

09.50-10.10 Doxorubicin-loaded and Antibody-Conjugated Liposome-QD Hybrid Vesicles for Targeted Cancer Theranostics

Bowen Tian, Wafa Al-Jamal, Bowen Tian and Kostas Kostarelos, University of Manchester, UK (p. 56)

10.10-10.30 Monitoring tumor burden by multicolor in vivo flow cytometry

Costas Pitsillides, Konstantinos Kapnisis and Andreas Anayiotos, Cyprus University of Technology, Cyprus (p. 57)

10.30-10.50 Role of the Cell-Division Cycle on Nanoparticle Cellular Accumulation and Implications for Cancer Targeting

Christoffer Åberg and Kenneth A. Dawson, UC Doublin, Ireland (p.59)


Short Course: Nanotheranostics: all-in-one personalized medicine

Session Chairs: George Potamitis, Chrysa Tziakouri-Shiakalli, Cyprus Medical Association

This short course will provide an overview of the major concepts behind the newly created field of nanotheranostics. Nanotheranostic agents have a number of significant advantages over current approaches: (i) Nanotheranostic agents can be customized to the disease and personalized to the patient. (ii) Active targeting and localization allows for better treatment with much less intense side effects compared to current regimens. (iii) The integration of therapy and monitoring provides real-time information on whether or not the specific treatment regimen is working for the specific patient. Given these attributes, it is not surprising that the field of nanotheranostics is considered the future of treatment of highly inhomogeneous and variable diseases such as cancer and chronic inflammatory disorders.

11.30-11.45 Introduction

Going to the lower limits: nanotechnology and nanomedicine

Theranostics: all-in-one personalized medicine

Andreani Odysseos, EFB, EPOS-Iasis R&D Ltd, Cyprus

11.45-12.05 Theranostic Nanoparticles

Rena Bizios, University of Texas at San Antonio, USA

12.05-12.25 Clinical applicability of Optical Imaging

Costas Pitris, University of Cyprus, Cyprus

12.25-12.45 Nanotheranostics at the clinical fore

Image-guided therapy: paving the way of nanotheranostic agents to the clinic

Monitoring therapy by imaging

Andreani Odysseos, EFB, EPOS-Iasis R&D Ltd, Cyprus


12.45-13.00 Image-guided quantification of drug delivery: a revolution in Radiology (Positron Emission Tomography (PET) and Magnetic Resonance Imaging (MRI))

Radiation-based therapies

Costas Pitris, University of Cyprus, Cyprus

13.00-13.30 Discussion of Clinical Challenges and Prospects

13.30-14.30 Lunch

Session 7 – Materials and Tissues in Nanotheranostics

Session Chair: Theodora Krasia-Christoforou, University of Cyprus

14.30-15.20 Challenges in Nanotheranostics: A Materials Perspective

Rena Bizios, University of Texas at San Antonio, USA (p. 60)

15.20-15.40 Magnetoactive electrospun nanocomposite membranes in drug delivery and hyperthermia applications

Ioanna Savva, Andreani Odysseos, Loucas Evaggelou, Oana Marinica, Eugeniu Vasile, Ladislau Vekas, Yiannis Sarigiannis and Theodora Krasia-Christoforou, University of Cyprus, Cyprus (p. 61)

15.40-16.00 Electrospun PEO/PLLA Fibrous Meshes for Sustained Tyrosine Kinase Inhibitors Delivery in Situ

Maria Kokonou, Fotios Mpekris, Triantafyllos Stylianopoulos, Jean-Michel Siaugue and Andreani Odysseos, University of Cyprus, Cyprus (p.62)

16.00-16.20 Drug delivery through reconstructed bronchial mucus modified by functional carrier particles (FCPs)

Marcin Odziomek, Tomasz Sosnowski and Leon Gradoń, Warsaw University of Technology, Poland (p. 64)

16.20-16.40 Agglomeration of Theophylline Nanoparticles: a New Protocol for Pulmonary Drugs Administration

Heba Salem, Mohamed Abdelrahim, Kamal Abo Eid and Mohamed Sharaf, The University of Beni Suef, Egypt (p. 66)


Round Table Discussion and Student Awards

Session Chair: Andreani Odysseos, EFB, EPOS-Iasis R&D Ltd, Cyprus

17.15-18.15 Round Table Discussion: Challenges and Opportunities in Nanotheranostics

Moderator: Andreani Odysseos Panel Members: K. Dawson, J. Mollenhauer, H. Doumanides, K. Kostarellos

18.15-18.30 Best Student Paper Award


Abstracts


Opportunities Offered by Nucleic Acid-Based Nano-

Devices in Cancer Therapy and Theranostics

Jan Mollenhauer1, Ines Block

1, Angela Riedel

1, Steffen Schmidt

1, Helle Christiansen

1, Birgitte

Brinkmann Olsen2, Helge Thisgaard

2, Poul Flemming

2, Stefan Vogel

1, Jesper Wengel

1

1NanoCAN - University of Southern Denmark, Denmark

2Nuclear Medicine, Odense University Hospital, Denmark

[email protected]

Nucleic acids offer fascinating opportunities for the targeted design of nano-devices with therapeutical and

theranostics applications in cancer. Evolutionarily, RNAs are thought to have presented the first generation of

functional biomolecules and nowadays are considered for therapeutical applications as short interfering RNAs

(siRNAs), while, technically, immense progress has been achieved by DNA-based nano-engineering, commonly

known now as DNA-origami. In conjunction with the ability to create aptamers, nucleic acid-based targeting

molecules that function like nano-sized antibodies, the nucleic acid world essentially offers the building blocks

for the conception of nano-drugs and –theranostics.

Within our efforts, we concentrate on the identification of novel targets in breast cancer stem cells, based on

which we aim at designing nucleic acid-based nano-drugs, consisting of aptamers and siRNAs. These can also be

linked to imaging molecules in a simple fashion. Collectively, this may enable to develop personalized regimen,

in which a molecular trait of an individual patient`s tumor is targeted, causing selective (synthetic) lethality to

tumor cells but not to normal cells. On an individualized basis, tumor targeting could be assessed in advance and

therapy response can be monitored during treatment by implementation of imaging molecules. We reflect the

present state of the art of the individual research lines, i.e. of our efforts in identifying novel breast cancer genes,

systematic screening for active siRNAs, and experimental imaging devices.

[Back to Plenary Session]


Nanoscale Interface Between Engineered Matter and

Living Organisms: Understanding the Biological Identity

of Nanosized Materials

Kenneth Dawson

Ireland Centre for BioNano Interactions, School of Chemistry and Chemical Biology, University College Dublin, Ireland.

[email protected]

Nanoscale materials can interact with living organisms in a qualitatively different manner than small molecules.

Crucially, biological phenomena such as immune clearance, cellular uptake and biological barrier crossing are

all determined by processes on the nanometer scale. Harnessing these endogeneous biological processes (for

example in creation of new nanomedicines or nanodiagnostics) will therefore require us to work on the

nanoscale. This ensures that nanoscience, biology and medicine will be intimately connected for generations to

come, and may well provide the best hope of tacking currently intractable diseases.

These same scientific observations lead to widespread concern about the potential safety of nanomaterials in

general. Early unfocussed concerns have diminished, leaving a more disciplined and balanced scientific

dialogue. In particular a growing interest in understanding the fundamental principles of bionanointeractions

may offer insight into potential hazard, as well as the basis for therapeutic use.

Whilst nanoparticle size is important, the detailed nature of the nanoparticle interface is key to understanding

interactions with living organisms. This interface may be quite complex, involving also adsorbed proteins from

the biological fluid (blood, or other), leading to a ‘protein corona’ on the nanoparticle surface that determines its

“biological identity”. We discuss how this corona is formed, how it is a determining feature in biological

interactions, and indeed how in many cases can undermine efforts at targeting nanoparticles using simple

grafting strategies. Thus, nanoparticle interactions with living organisms cannot be fully understood without

explicitly accounting for the interactions with its surroundings, i.e. the nature of the corona.

References 1. Monopoli, M. P.; Aberg, C.; Salvati, A.; Dawson, K. A. Biomolecular Coronas Provide the Biological Identity of Nanosized Materials.

Nature Nanotechnology 2012, 7, 779-786.

2. Kim, J. A.; Aberg, C.; Salvati, A.; Dawson, K. A. Role of Cell Cycle on the Cellular Uptake and Dilution of Nanoparticles in a Cell

Population. Nature Nanotechnology 2012, 7, 62-68.

3. Monopoli, M. P.; Walczyk, D.; Campbell, A.; Elia, G.; Lynch, I.; Baldelli Bombelli, F.; Dawson, K. A. Physical-Chemical Aspects of

Protein Corona: Relevance to in Vitro and in Vivo Biological Impacts of Nanoparticles. Journal of the American Chemical Society

2011, 133, 2525-2534.

4. Cedervall, T.; Lynch, I.; Lindman, S.; Berggard, T.; Thulin, E.; Nilsson, H.; Dawson, K. A.; Linse, S. Understanding the Nanoparticle-

Protein Corona Using Methods to Quantify Exchange Rates and Affinities of Proteins for Nanoparticles. Proceedings of the National

Academy of Sciences 2007, 104, 2050-2055.

[Back to Plenary Session]


Assessing biological specificity of nanoparticles in vitro:

test tubes, chips or cells?

Pavlos (Bogos) Agianian

Department of Molecular Biology and Genetics Democritus University of Thrace, Dragana, 68100 Alexandroupoli, Greece.

e-mail: [email protected]

The hope of applying nanotherapy to cure human diseases has sparked a new era of collaboration between

biologists, chemist and medical doctors. Irrespective of disease or nanoplatform used, biological targeting and

specificity of action, combined with low toxicity, is commonly desired. To achieve this goal, a big number of

functionalized nanoparticles (NPs) are produced for each biological target, creating a need for efficient and

cost effective in vitro methods to screen the functionality of NPs, before they can be applied to animal models

for further biological studies.

I will critically present in vitro methods of assessing the biological specificity of NPs, emphasizing on

solution methods, on chip biosensor analysis using Surface Plasmon Resonance (SPR) and cellular imaging. I

will elaborate on results from functionalized NPs for targeted drug delivery of the anticancer drugs taxol and

ixabepilone and for theranostic applications targeting the EGFR receptor.

[Back To Session 1]

mailto:[email protected]


In-vitro release study and dynamic in-vivo imaging of pH-

and Magnetic field sensitive hybrid microspheres Eleni K. Efthimiadou

a*, Christos Tapeinos

a, Eirini Fragogeorgi

b,c, George Loudos

c and George Kordas

a*

a Sol-Gel Laboratory, Institute for Advanced Materials, Physicochemical processes, Nanotechnology & Microsystems, NCSR

“Demokritos”, 153 10 Aghia Paraskevi Attikis, Greece b Department of Medical Instruments Technology, Technological Educational Institute, GR 122 10 Athens, Greece

c Radiochemical/ Radiopharmacological Quality Control Laboratory, Institute of Nuclear and Radiological Sciences and Technology,

Energy & Safety, N.C.S.R. ‘Demokritos’, 15310 Aghia Paraskevi, Greece

[email protected]

This paper deals with the synthesis, characterization and property evaluation of drug-loaded magnetic

microspheres with pH-responsive cross-linked polymer shell. The synthetic procedure consists of 3 steps, of

which the first two comprises the synthesis of a Poly Methyl Methacrylate (PMMA) template and the

synthesis of a shell, using Acrylic Acid (AA) and Methyl Methacrylate (MMA) as monomers, and Divinyl

Benzene (DVB) as cross-linker. The third step of the procedure refers to the formation of magnetic

nanoparticles on the microsphere’s surface. AA that attaches pH-sensitivity in the microspheres and magnetic

nanoparticles in the inner and the outer surface of the microspheres, enhance the efficacy of this intelligent

Drug Delivery System (DDS), which constitutes a promising approach towards cancer therapy. A number of

experimental techniques were used to characterize the resulting microspheres. In order to investigate the in-

vitro controlled release behaviour of the synthesized microspheres, we studied the DOX release percentage

under different pH conditions and under external magnetic field. Hyperthermia caused by an Alternating

Magnetic Field (AFM) is used in order to study the Doxorubicin (DOX) release behaviour from

microspheres with pH functionality. The in vivo fate of these hybrid-microspheres was tracked by labelling

them with the γ-emitting radioisotope 99mTc after being intravenously injected in normal mice. According

to our results, microcontainers present a pH depending and a magnetic heating, release behaviour. As

expected, labelled nanocarriers were mainly found in the mononuclear phagocyte system (MPS). The

highlights of the current research are: (i) to illustrate the advantages of controlled release by combining

hyperthermia and pH-sensitivity and (ii) to provide non invasive, in vivo information on the spatiotemporal

bio-distribution of these microcontainers by dynamic γ imaging.

Scheme 1. In- vitro and in-vivo study of fabricated MCs

1. Ganta, S.; Devalapally, H.; Shahiwala, A.; Amiji, M., A review of stimuli-responsive nanocarriers for drug and gene

delivery. J Control Release 2008, 126, (3), 187-204.

2. Motornov, M.; Roiter, Y.; Tokarev, I.; Minko, S., Stimuli-responsive nanoparticles, nanogels and capsules for integrated

multifunctional intelligent systems. Progress in Polymer Science 2010, 35, (1-2), 174-211.

[Back To Session 1]


Preparation and characterization of new elements for the

conception of nanohybrids for multicolored bioimaging

M. Rivoala, A. D. Odysseos

a

aEPOS-Iasis, R&D, 5 Karyatidon street, Nicosia 2028, Cyprus

[email protected]

Currently, the development of organic/inorganic nanohybrid materials arouses the enthusiasm of many

researchers owing to their potential applications and in particular in bioimaging applications (Fig. 1).

Fig. 1: Principle of organic/inorganic nanohybrids for multicolored bioimaging

The aim of this work was to prepare and characterize new inorganic and organic components for the future

design of new multi-functional nanohybrids with properties responding to the current challenges. For this

purpose, we have prepared nanoparticles of zinc oxide (ZnO) as the inorganic component by laser ablation [1].

The surface of these nanoparticles can be modified by an organic component bearing the carboxylic group as an

anchor. First, we synthesized and characterized a number of viologen derivatives, well known as strong electron

acceptors, involving the anchoring groups. The nanohybrids of ZnO/viologens were prepared and characterized

by various spectroscopic techniques. In parallel, we recently have developed efficient synthetic routes toward a

series of new heterocycles (Fig. 2) possessing the electron donating properties: derivatives of

dibenzo[2,3:5,6]pyrrolizino[1,7-bc]indolo[1,2,3-lm]carbazole [2].

a

b c

Fig. 2: Molecular and crystal structure of a a) new fluorescent heterocycle substituted with b) -MeO and c) -COOH groups.

These new molecules exhibit high thermal stability and strong fluorescence in the visible range. Those

derivatives can also serve as the efficient electron donating moieties in two-photon absorbing (TPA)

chromophores. Their one- and two-photon (Near-infrared) absorption properties and electron donor ability were

investigated experimentally and by means of quantum mechanical calculations.

Here we report on the preparation and properties of inorganic ZnO NPs and organic components: a series of

original viologens and hitherto unknown parent heterocyclic system together with a novel series of derivatives

and their potential to be functionalized to highly selective, quinazoline-based Tyrosine Kinase Inhibitors

towards a new generation of fluorescent nanotheranostic agents for ERBB-related malignancies. The

experimental details, the results and the perspectives will be presented.

This work was supported by ANR (French Agency for National Research), project NEM, ANR-09-BLAN-0107. We thank the French

Ministry of Education and Ecole Doctorale des Sciences Chimiques (ED 250, Marseille) for a fellowship to M.R. This work has been partially

supported by the Marie-Curie IAPP project NANORESISTANCE- Grant agreement no.: 286125

[1] Chelnokov E., Rivoal M., Colignon Y., Gachet D., Bekere L., Thibaudau F., Giorgio S., Khodorkovsky V, Marine W. “Band gap tuning

of ZnO nanoparticles via Mg doping by femtosecond laser ablation in liquid environment”. Applied Surface Science, 2012, 258, 23,

9408- 9411.

[2] M. Rivoal, L. Bekere, D. Gachet, V. Lokshin, W. Marine, V. Khodorkovsky. “Substituted dibenzo[2,3:5,6]-pyrrolizino[1,7-

bc]indolo[1,2,3-lm]carbazoles: a series of new electron donors”. Tetrahedron, 2013, 69, 3302-3307.

[Back To Session 1]



Size Dependent Biological Profiles of Pegylated Gold

Nanorods for Biomedical Applications

F. Tatinia, I. Landini

b, F. Scaletti

c, L. Massai

c, S. Centi

d, F. Ratto

a S. Nobili

b, G. Romano

d, F. Fusi

d, L.

Messoric, E. Mini

b, R. Pini

a

aInstitute of Applied Physics "Nello Carrara" National Research Council, Via Madonna del Piano 10, Sesto Fiorentino, 50019, Italy. bDepartment of Health Sciences, Section of Clinical Pharmacology and Oncology University of Florence viale Pieraccini , 6, 50139

Florence , Italy.cDepartment of Chemistry, University of Florence , 500I9 Sesto Fiorentino, Italy.dDepartment of Clinical Physiopathology,

Viale G. Pieraccini 6, Firenze 50139, Italy

federica.scaletti@unift. it

Gold nanoparticles (GNP) have attracted a widespread attention due to their unique physicochemical

properties; they exhibit tremendous potential for biomedical applications from the detection of protein/DNA

interactions to drug delivery and cancer therapeutics and diagnostics. In particular gold nanorods (GNRs) are

attracting special attention due to their unique optical properties, which include a surface Plasmon absorption

band in the visible region and a second tunable absorption in the NIR, making them good agents for

photothermal treatment of cancers [1].

Crucial features of GNRs include their coating, shape and size. The kind of coating is critical to modulate the

interface between particles and biological systems, to enable the attachment of additional functional molecules

such as ligands for biochemical targets of interest and to determine the stability of the particles within culture

media and body fluids.

Most studies identified polyethylene glycol (PEG) as the coating of choice for GNRs because of lack of

toxicity [2], camouflage from immune systems, versatility for further functionalization and conferment of

stability in the blood.

An important requirement for the biomedical applications of GNRs is their solubility and stability in

physiological buffers. Previous studies [3,4] showed that gold nanoparticles exposed to biological fluids may

become coated with proteins. Inturn, adsorption of proteins on gold nanoparticles may modify their characteristic

conformation in solution, cause a loss of biological activity, elicit an altered immune response and modify

particle biodistribution and cellular uptake.

Here we present an extensive survey on the characterization, stability, proteins interaction, toxicity and

cellular uptake of PEG-coated GNRs of different size. In particular five distinct sizes of particles were

synthetized, characterized and studied.

Till now, the effect of size on the cytotoxicity and cellular uptake of PEG-coated GNRs has never been

reported. Therefore the understanding of such correlation provides useful hints for the selection of promising

products for biomedical applications.

Acknowledgements

We gratefully acknowledge Chiara Gabbiani for her work on the first stage of the project and Francesco Rugi for

the ICP-aes measurements, Beneficentia Stiftung (Vaduz, Liechetenstein) and Regione Toscana,

''NANOTREAT' project, for generous financial support.

References [I] F. Ratto, P. Matteini, F. Rossi, L. Menabuoni, N. Tiwari, S. K. Kulkarni, R. Pini; "Photothermal effects in connective tissues mediated

by laser-activated gold nanorods "Nanomed.: Nanotec. Bioi. Med.S, 143-151 (2009).

[2] A M. Alkilany, A Shatanawi, T. Kurtz, R. B. Caldwell, R. W. Caldwell "Toxicity and cellular uptake of gold nanorods in vascular

endothelium and smooth muscles of isolated rat blood vessel: importance of surface modification" Small. 8, 1270-1278 (20 12).

(3] S. Chakraborty, P. Joshi, V. Shanker, Z. A Ansari, S. P. Singh, P. Chakrabarti "Contrasting effect of gold nanopartic/es and nanarods

with different surface modifications on the structure and activity of bovine serum albumin " Langmuir, 27, 7722-7731(2011).

[4] T. T. Moghadam, B. Ranjbar, K. Khajeh, S. M. Etezad, K. Khalifeh, M. R. Ganjalikhany "interaction of lysozyme with gold nanorods:

c01![ormation and activity investigations" Int. J. of Bioi. Macromol., 49, 629-636 (2011).

[Back To Session 1]


Fluorescent and magnetic silica core shell nanoparticles

for biomedical applications

Ana B. Dávila-Ibañez1, Maria Kokonou

1,2, Efstathia Mylouli

1,3, Christine Ménager

1, Jean-Michel Siaugue

1

1PECSA laboratory (Physico-chimie des Electrolytes, Colloïdes et Sciences Analytiques), UPMC, 75005, Paris, (France)

[email protected] 2EPOS-Iasis Research and Development Ltd., 2028 Nicosia, Cyprus

3 pro-ACTINA S.A., GR-19400 Koropi Attikis (Industrial Zone) Athens, Greece

[email protected]

In the context of biomedical applications, we have developed a fluorescent and magnetic nanometric platform

made of a maghemite core embedded in a fluorescent and twice fonctionnalized silica shell. Synthesis,

functionalization and physico-chemical characterization of multifunctionalized magnetic core shell nanoparticles

(Fe2O3@SiO2(Fluorescent dyes)-NH2/PEG) were performed using either non size sorted or size sorted magnetic

nanoparticles and different fluorescent dyes like fluorescein, rhodamine and near-IR emitting dye.

This nanometric platform was used for the grafting of bleomycin, an antitumor antibiotic. The covalent

anchoring of bleomycin was performed thanks to reductive amination (A). Grafted bleomycin kept its capacity to

induce DNA cleavage (B). Moreover, selectivity and specificity of supported bleomycin to DNA were not

altered. Both bleomycin activated nanoparticles and nude nanoparticles internalization in human cancer cells

(HT1080) were studied at different times. Interestingly, confocal fluorescence microscopy and electronic

transmission microscopy indicated strong interactions between nanoparticles and cells nuclei, with major

accumulation near the nucleus (C,D). Some nanoparticles were observed too into the nucleus (E). Cells viability

assays were also carried out. Bleomycin activated nanoparticles were able to induce a cytotoxic effect whereas

nude nanoparticles did not thus demonstrating the role played by the grafted anticancer drug [1].

Bleomycin grafting (A). DNA cleavage (B). Nanoparticles translocation into human fibroblastom (TEM (C, E), fluorescent confocal

microscopy (D)).

Those core-shell nanoparticles were also used as a colloidal immunosupport in a homogeneous sandwich

immunoassay. PEG chains prevent non-specific adsorption and amino groups enable covalent grafting of α-

Lactalbumin (α-Lac) antigen, thus allowing the capture of the target model analyte (goat anti α-lac

immunoglobulin G (IgG)). In comparison with classic ELISA test, the incubation time for target analyte capture

was accelerated 200 fold and the limit of detection was 20 times lower. This nanometric homogenous

immunoassay was successfully applied for IgG determination in real matrix like serum with good agreement

with ELISA test and further integrated in a microsystem to develop an immunoassay in a lab on chip [2].

Those nanoparticles are also good candidates for multimodal imaging (MRI/fluorescence) applications. We

used them to decorate model and biological membranes. We obtained notably human red blood cells with core

shell nanoparticles adsorbed onto their surface, thus magnetic and fluorescent human red blood cells, which

could be usefull for the detection of hemorrhage [3].

References [1] T. Georgelin, S. Bombard, J.-M. Siaugue and V. Cabuil. Nanoparticle-Mediated Delivery of Bleomycin, Angew. Chem. Int. (2010), 49,

8897.

[2] B. Teste, F. Malloggi, J.-M. Siaugue, A. Varenne, F. Kanoufi, S. Descroix. Microchip integrating magnetic nanoparticles for allergy

diagnosis. Lab Chip (2011), 11, 4207.

[3] M. Laurencin , N. Cam , T. Georgelin , O. Clément , G.Autret , J.-M. Siaugue , C. Ménager, Human Erythrocytes Covered with

Magnetic Core–Shell Nanoparticles for Multimodal Imaging. Adv. Healthcare Mater ( 2013),, DOI: 10.1002/adhm.201200384

Acknowledgments

The authors gratefully acknowledge EU for funding through FP7 IAPP/NANORESISTANCE/Grant Agreement Number: 286125.

[Back to Session 2]



Two different approaches for the synthesis of

functionalized silica coated magnetic nanoparticles Ana B. Dávila-Ibañez

1, Verónica Salgueiriño


1

1PECSA laboratory (Physico-chimie des Electrolytes, Colloïdes et Sciences Analytiques), UPMC, 75005, Paris, (France)

2Departamento de Física Aplicada, Universidade de Vigo, 36310, Vigo (Spain)

[email protected]

Magnetic nanoparticles show great promise for a huge range of applications. The major advantages correspond

to their magnetic nature and ease of biofunctionalization. Herein will be presented the synthesis of

nanomaterials with magnetic properties that later will be functionalized in such a way to direct their possible

applications.

Magnetic materials were silica coated by two different approaches to provide them higher stabilities, as well

as to facilitate their functionalization. By the first approach we were able to synthesized silica coated

magnetic nanoparticles that later were functionalization with DNA[1, 2] to increase their biocompatibility,

follow by cytotoxicity studies that revealed the key role that surface functionalization plays in regulating the

mechanisms involved of the chemical degradation of the nanocoposites. By the second approach FIR

fluorescent dye-doped magnetic nanoparticles amino functionalized were synthesized providing magnetic

composites that could be used for therapeutic and diagnosis applications [3].

Different magnetic nanoparticles were synthesized, magnetite (Fe3O4), cobalt ferrite (CoFe2O4) and

maghemite (γ-Fe3O4) nanoparticles. They were characterized in terms of their magnetic behavior and

chemistry nature to later functionalize them. Magnetic nanoparticles were silica coated, firstly by reverse

microemulsion methods, providing silica coated magnetic nanoparticles with different shell thickness; and

secondly by a modified Stober method affording PEG and amino functionalized silica coated magnetic

nanoparticles. In terms of their functionalization two main approaches were carried on. In one hand the

synthesis of bio-functionalized magnetic composites by the deposition of DNA fragments on their surface. In

the other hand, the synthesis of dye-doped magnetic nanoparticles by the encapsulation of the dye into the

silica shell during the silica coating magnetic nanoparticles procedure[3]..

Fig 1. Image on the left shows the DNA functionalized silica coated magnetic nanoparticles once inside the cells where

morphological changes have started due cell environment. Images on the rigth shows dye-doped silica coated magnetic

nanoparticles (top) and the emission fluorescent spectrum (bottom) of these composites as function of the concentration of the

encapsulated dye.

References [1] A. B. Davila-Ibáñez, V. Salgueiriño, V. Martínez-Zorzano, R. Mariño-Fernández, A. García-Lorenzo, M. Maceira-Campos, M. Muñoz-

Úbeda, E. Junquera, E. Aicart, J. Rivas, F. J. Rodríguez-Berrocal, J. L. Legido, Magnetic Silica Nanoparticle Cellular Uptake and

Cytotoxicity regulated by Electrostatic Polyelectrolytes-DNA Loading at their Surface ACS Nano (2012), 6, 747.

[2] A. B. A. B. Davila-Ibáñez, Niklaas J. Buurma and V. Salgueiriño, Assesment of DNA complexation onto polyelectrolites- coated

magnetic silica nanoparticles, Nanoescale, (2013), 5, 4797.

[3] T. Georgelin, S. Bombard, J.-M. Siaugue and V. Cabuil. Nanoparticle-Mediated Delivery of Bleomycin, Angew. Chem. Int. (2010), 49,

8897.

Acknowledgments

The authors gratefully acknowledge EU for funding through FP7 IAPP/NANORESISTANCE/Grant Agreement

Number: 286125.

[Back to Session 2]



Theragnosis combining ferrocenyl derivatives,

carbohydrates and nanovectors

Malinge Jérémy, Siaugue Jean-Michel(1)

, Ménager Christine(1)

, Sollogoub Matthieu(2)

, Zhang Yongmin(2)

,

Jaouen Gérard(3)

& Vessières-Jaouen Anne(3)

. (1)-PECSA, UPMC ; (2)-IPCM, Equipe GOBS, UPMC ; (3)-Laboratoire Friedel, ENSCP

[email protected]

Over the past decades, theragnosis platforms have emerged as a powerful tool for the simultaneous

diagnostic and therapy of cancer. Gathering, in a single vector, an imaging agent, a targeting unit and an efficient

drug is still a challenging task and a tremendous amount of work is currently being published in the scientific

community.

Ferrocifen derivatives are specifically cancer-cell-activated prodrugs and have proven useful for untreatable

cancer cells [1]. However, with these polyphenol species, formulation and drug delivery remain challenging.

The strategy employed in our work is based upon the use of magnetic liposomes as the central scaffold

(Figure). This vector offers structural versatility in terms of membrane composition. For instance, the project

aims to functionalize the outer membrane with bioavailable units to target infected tissues and a fluorescent

phospholipids to provide bimodal imagery tools (fluorescence and MRI). In addition, the presence of iron

nanoparticles inside the liposome allows active targeting under magnetic field and possible hyperthermia [2].

Formulation of the drug, liposome synthesis and first results (in vitro and in vivo) will be presented.

Figure 1. a) Schematic representation of the aimed theragnostic platform; b) TEM image of an isolated liposome

[1] M. Görmen, P. Pigeon, S. Top, E. Hillard, M. Huché, C. Hartinger, F. de Montigny, M-A. Plamont, A. Vessières, G. Jaouen,

“Synthesis, Cytotoxicity, and COMPARE Analysis of Ferrocene and [3]Ferrocenophane Tetrasubstituted Olefin Derivatives against

Human Cancer Cells” ChemMedChem 5, 2039-2050 (2010).

[2] G. Béalle, R. Di Corato, J. Kolosnjaj-Tabi, V. Dupuis, O. Clément, F. Gazeau, C. Wilhelm, C. Ménager, “Ultra magnetic liposomes

for MR imaging, targeting, and hyperthermia” Langmuir 28, 11834–11842 (2012)

[Back to Session 2]


http://europepmc.org/search%3Bjsessionid%3D4WItB4QIYetsw1oiv7jt.10?page=1&query=AUTH%3A%22B%C3%A9alle%2BG%22

http://europepmc.org/search%3Bjsessionid%3D4WItB4QIYetsw1oiv7jt.10?page=1&query=AUTH%3A%22Di%2BCorato%2BR%22

http://europepmc.org/search%3Bjsessionid%3D4WItB4QIYetsw1oiv7jt.10?page=1&query=AUTH%3A%22Kolosnjaj-Tabi%2BJ%22

http://europepmc.org/search%3Bjsessionid%3D4WItB4QIYetsw1oiv7jt.10?page=1&query=AUTH%3A%22Dupuis%2BV%22

http://europepmc.org/search%3Bjsessionid%3D4WItB4QIYetsw1oiv7jt.10?page=1&query=AUTH%3A%22Cl%C3%A9ment%2BO%22

http://europepmc.org/search%3Bjsessionid%3D4WItB4QIYetsw1oiv7jt.10?page=1&query=AUTH%3A%22Gazeau%2BF%22

http://europepmc.org/search%3Bjsessionid%3D4WItB4QIYetsw1oiv7jt.10?page=1&query=AUTH%3A%22Wilhelm%2BC%22

http://europepmc.org/search%3Bjsessionid%3D4WItB4QIYetsw1oiv7jt.10?page=1&query=AUTH%3A%22M%C3%A9nager%2BC%22


Chitosan – linear aldehyde nanoparticles obtained from

reverse micelle method

Gawlik K. Wasiak I. Ciach T. Warsaw University of Technology, Faculty of Chemical and Process Engineering, Biomedical Engineering Laboratory,

Warynskiego Street 1, 00 -645 Warsaw, Poland

[email protected]

Introduction

Drug delivery systems can be defined as means to introduce therapeutic agents into the body. These systems

have been considered as the key to the clinical success of numerous drugs, they assure delivery of therapeutic

agent into the body. Ideal drug delivery system provides means to efficiently deliver active agent to the ill tissue

only, assuring efficacy and decreasing side effects. It is especially important in the case of cancer treatment,

when we sometimes administer very toxic drugs in nearly lethal doses. Recent researches suggest that

nanotechnology poses tolls that could overcome the disadvantages of current treatment. Nanoparticulate drug

delivery systems are purposely engineered and constructed objects that are measured in nanometer size. Their

size usually ranges from a few to several hundred nanometers. Nanoparticles can be made using a various raw

materials including polymer, lipids, viruses, noble metals, semiconductors, magnetic or organometalic

compounds. Biomedical application of nanoparticles include drug carriers, labeling and tracking agents, vectors

for gene therapy, hyperthermia treatments and magnetic resonance imaging contrast agents. This definition

includes monolithic nanoparticles in which the drug is adsorbed dissolved or dispersed throughout the matrix

and nanocapsules in which the active agent is confined to an aqueous or oily core surrounded by shell-like wall.

Alternatively the active agent can be covalently attached to the surface or into the matrix [1]. The unusual

property on nanoparticles in biological systems comes from the fact, that most of the interactions in cell and

between cells are performed in the same level of size. This can be a benefit but also a potential source of danger.

Nanoparticles can be biodegradable and nonbiodegradable. The first type will disappear from the body after

performing the desired action, due to hydrolysis or other biological process. The second type will probably stay

in our body for very long time or even forever, what pose a potential danger on such objects. That’s why

biodegradable – organic nanoparticles are frequently considered as safe, in comparison with inorganic. Perfect

materials for medical nanoparticle preparation are biodegradable polymers. They can be fabricated into various

shapes and sizes, with tailored, pore morphologies, mechanical properties and degradation kinetics to suit a

variety of application. By selecting the appropriate polymer type, molecular weight and copolymer blend ratio,

the degradation rate of nanoparticles can be controlled to achieve the desired type and rate of release of the

encapsulated drug. Biodegradable nanoparticles can be prepared from variety of materials such as non

biodegradable polymers, proteins, synthetic biodegradable polymers and polysaccharides. They protect

entrapped drug against degradation and control its site specific delivery. However, the main drawback of

conventional NPs is their non specific interaction with the cells and plasma proteins, leading to drug

accumulation in no target organs [2]. Therefore, polysaccharides coatings are attractive alternative to PEG once

their possess many recognition functions, allowing specific mucoadhesion or receptor recognition, as well as

providing neutral coatings with low surface energy, preventing non specific protein adsorption. The presence of

saccharide on the NP surface can also increase their uptake by cancer calls due to the Warburg effect – glucose

demand is about 200 times higher for cancer cells then for normal.

Chitosan is a biodegradable natural polymer with great potential for pharmaceutical applications due to its

biocompatibility, high charge density, non-toxicity and mucoadhesive propertiey [3]. Chitosan has been used as

a drug carrier for sustained release preparations and improvement of bioavailability for hydrophobic drugs, and

as a vehicle for directly compressed tablets, disintegrant, binder and granulating agent. Chitosan is a well-known

natural polysaccharide that is usually obtained from shells of crustaceans such as crab, shrimp, and crawfish. It

is a copolymer of 2-acetamido-2-deoxy-D-glucose (N-acetyl-glucosamine,GluNAc) and 2-amino-2-deoxy-D-

glucose (N-glucosamine,GluN) units randomly or block distributed throughout the biopolymer chain depending

on the processing method used to derive the biopolymer. Chitosan is a partially N-deacetylated derivative of

chitin. The term chitosan is usually used when glucosamine units predominate or the polymers become soluble

in a dilute acid solution. The biological properties of chitosan include biocompatibility, biodegradability, non-

toxicity, hemostaticity, antitumoral and antiviral activity. Chitosan is degraded by lysozyme present in the

various mammalian tissues and leads to production of N-acetyl-D-glucosamine and D-glucosamine, which also

plays an important physiological role in in vivo biochemical processes. In addition, chitosan has a special

feature of adhering to the mucosal surface and transiently opening the tight junction between epithelial cells [4].

Thus, chitosan nanoparticles are potential delivery system for hydrophilic drugs due to its outstanding

physicochemical and biological properties. Although chitosan should be useful for even more numerous

applications, its use suffers severe limitations because it is insoluble in neutral or alkaline media owing to its

rigid and compact crystalline structure and strong intra- and intermolecular hydrogen bonds.



Chitosan has both reactive amino and hydroxyl groups that can be used to chemically alter its properties

under mild reaction conditions. The presence of amino groups leads to the possibility of several chemical

modifications, including the preparation of Schiff bases (–RC=N–) by reaction with aldehydes and ketones. The

reaction of chitosan with aromatic aldehydes to produce the corresponding Schiff bases has been described [5].

Schiff base compounds containing an imine group, are usually formed by the condensation of a primary amine

with an active carbonyl. Its attractiveness as analytical reagents raises from the fact that they enable simple and

inexpensive determinations of various organic and inorganic substances. The imine linkage formed by this

reaction is fairly stable in neutral and alkaline solution, but it is rapidly hydrolyzed under acidic conditions.

Schiffe base can be easily converted into N-alkyl derivative by reduction with sodium borohydride. The aim of

this paper was to evaluated reverse micellar method for chitosan- linear aldehyde nanoparticles preparation

Methods

The present work describes the preparation of chitosan nanoparticles obtained by reverse micellar method.

Nanoparticles were prepared from chitosan (MW = 200 kDa with degree of deacetylation = 95%) and different

linear aldehydes (oktanal, dekanal and dodekanal). Briefly the solution of 0.01 M surfactant in hexane were

prepared. 50 -150 µl of chitosan solution and 10 µl of aldehyde and liquor ammonium were added to the

surfactant solution. After 24 h reaction, the solvent was evaporated, and the dry mass resuspended in Tris–Cl

buffer (pH 7.4) by sonicaton. To this, 1 ml of 30% CaCl solution was added drop wise to precipitate the

surfactant. The precipitate was pelleted by centrifugation. The size and concentration of the nanoparticles were

investigated as a function of the preparation conditions. The surfactant and aldehyde type, ammonium,

chitosan, and aldehyde concentration was evaluated.

Reasults

Proposed method allows for preparation of stabile chitosan nanoparticles. The size range of the obtained

nanoparticles was between 100 and 200 nm. Obtained results shows that there is no significant influence of

chitosan concentration in the range between 50 -150 µl on nanoparticles size. The diameter of nanoparticles

increase with linear aldehyde C atoms number and liquor ammonium concentration, even at to 40%.

Nanoparticles concentration decrease with C atoms number in aldehyde and bee not change with chitosan

concentration. However increasing ammonium concentration results in increasing nanoparticles concentration

up to 20 %. Use of anionic surfactant (SDS) is promoting nanoparticles preparation.

References

[1] C. Duncan, “The dawning era of polymer therapeutic,” Nat Rev Drug Disc 2, 347-360 (2003).

[2] A. Mahapatro, D.K. ASingh “Biodegradable nanoparticles are excellent vehicle for site directed in-vivo delivery of drugs and vaccines”

J.Nanobio.9, 55 (2011)

[3] Xiao-Ying Yinga, Dan Cuia, Lian Yub, Yong-Zhong Dua, “Solid lipid nanoparticles modified with chitosan oligosaccharides for the

controlled release of doxorubicin” Carbohydr Polym 84, 1357-1364 (2011)

[4] F. Qian, F. Cui, J. Ding, C. Tang, and C. Yin, “Chitosan graft copolymer nanoparticles for oral protein drug delivery: preparation and

characterization,” Biomacromolecules, 7, 2722–2727 (2006).

[5] X. Jin, J. Wang, J. Bai “Snthesis and antimicrobial activity of Schiff base from chitsan and citral” Cabohydr Res 344, 825-829 (2009)

[Back to Session 2]


New Routes Towards the Synthesis of Natural Products

and Designed Derivatives

E. A. Couladouros

Agricultural University of Athens, Chemical Labs, Iera Odos 75, Athens, 118 55, Greece. and

Pro-Actina S.A., Archimidous 59, Koropi Attikis, 19400, Greece.

[email protected]

Chemical synthesis is one out of the most important steps both for the discovery of a new pharmaceutical and

for its exploitation to become a drug. Bioactive natural products of complicated structure are the outmost

challenge in this field. Apart from the purely academic interest, such endeavours may lead to the development of

general and convergent routes for the preparation of designed and diversified libraries, thus facilitating their

further exploitation. In this respect, short reaction sequences having a high degree of diversification and a

suitable handle for solid phase application are most desirable.

New synthetic methodologies applied on five families of natural products will be presented:

a. An approach for the asymmetric synthesis of all naturally occurring tocotrienols.

b. Novel solid supported synthesis of “unnatural aminocyclitols”.

c. A short asymmetric synthesis of the highly potent antibiotic abyssomicin C.

d. Recent progress towards the development of a general method for the synthesis of polyprenylated

phloroglucinol derivatives.

e. The application of the “polymorphic scaffold” approach in the synthesis of sugars

[Back to Session 2]



Hemocompatibility of Albumin Microspheres as Drug

Delivery System, In vitro Study

Mohamed A. Elblbesy

University of Tabuk, Saudi Arabia

E‐mail: [email protected]

Purpose

The main objective of the present work is to evaluate the Hemocompatibility of albumin microspheres. Albumin

microspheres were prepared by coacervation method. The characteristics of Albumin microspheres such as

particle size, zeta potential, particle morphologie, entrapment efficiency and drug loading were evaluated. In

vitro release study prolonged duration (50% total cumulative percentage at the end of 24 hours, 85% at 72 hrs).

Conclusion

That coacervation method is well suited to produce albumin microspheres and the preparative variables of the

procedure can be fine-tuned depending on the clinical application.

[Back to the Poster Session]



Hemocompatibility of Silver Nanoparticles

Julie Laloy, Valentine Minet, Lutfiye Alpan, Bernard Chatelain, François Mullier and Jean-Michel Dogné

Department of Pharmacy, Namur Thrombosis and Hemostasis Center (NTHC), Namur Research Institute for LIfe Sciences (NARILIS),

University of Namur

[email protected]

Background

The presence of silver nanoparticles (Ag NPs) in consumer products like disinfectants, deodorants, antimicrobial

sprays, etc increased in the past few years. Ag NPs are also used for medical applications as antimicrobial agents

for wound dressings, catheters, and orthopedic and cardiovascular implants. As the clinical use of silver

nanoparticles increases, a better understanding of their safety when exposed to the bloodstream is needed. There

is a serious lack of information on the biological effects of Ag NPs on human blood cells.

Aim

The objectives of this study are to determine the impact of Ag NPs on erythrocyte integrity, platelet function and

blood coagulation.

Methods

Erythrocytes integrity was assessing based on measurement of haemoglobin release at 550 nm. Activation and

aggregation of the platelets was determined by optical aggregometry in combination with electron microscopy

observations. The calibrated thrombin generation test was used to study the impact of Ag NPs on coagulation

cascade. A particular attention was made on the potential interference of Ag NPs with the detection methods

used in these tests.

Results

With optical aggregometer, Ag NPs have no impact at on platelet function at low concentration. An impact on

platelet function could be observed at high concentration using electron microscopy. Ag NPs can also induce

hemolysis at concentration higher than 0.3 µg/ml. These NPs have also a procoagulant potential observed by an

increase in the thrombin generation.

Conclusions

Ag NPs have a high hemolytic potential, an impact on coagulation and on platelet aggregation. Maximal

precautions must be taken with the use of Ag NPs in medical applications.



From docking to synthesis and grafting: Preliminary

results for new Anilinoquinazolines as potential EGFR

inhibitors in multifunctional nanocarriers

Fotini Liepouri1, Pavlos Agianian

2, Vasssiliki Gerafalaki

2, Elias Couladouros

1,4 Andreani Odysseos

3,

Alexandros Strongilos1

1. Pro-Actina S.A., Archimidous 59, Koropi Attikis, 19400, Greece.

2. Democritus University of Thrace, Dragana 68100, Alexandroupoli Evros, Greece.

3. EPOS-Iasis, 5 Karyatidon Street, Suite 202, 2028 Nicosia, Cyprus.

4. Agricultural University of Athens,75 Iera Odos, 11855 Athens, Greece

[email protected]

Epidermal Growth Factor Receptor (EGFR) is a transmembrane receptor that constitutes a major biological target

for the development of anti-cancer drugs.

EGFR upon activation by its natural substrate EGF, mediates cell growth and proliferation pathways through

activation of its intracellular component which is a Tyrosine Kinase. EGFR’s overexpressions and mutant forms

found in cancer cells, make it a significant receptor whose inactivation would serve significantly to control cell

growth and proliferation.

Here in we present the roadmap towards optimized synthesis of EGFR kinase inhibitors as potential grafting

agents on nanoactuators, such as metal nanoparticles and carbon nanotubes. Bibliography search resulted in a

number of biologically active scaffolds that inhibit EGFR. Among the most potent and promising ones are

Anilinoquinazolines[1-3], of type I [Figure 1]. Furthermore, various Anilinoquinazolines carrying a Michael

acceptor inhibit the kinase irreversibly through formation of a chemical bond with a neighboring cysteine residue

in the active site of the kinase. Based in that rational, Anilinoquinazolines are selected as the most promising

scaffold for synthesis.

Figure 1: EGFR inhibitors carrying the Anilinoquinazoline moiety

In order to rationalize chemical synthesis of these derivatives in terms of their efficacy in EGFR binding and

inhibition, thus not compromising valuable resources, we initially aimed at in silico protein-ligand docking by

using Autodock 4.0. The advantage of this targeted approach is a higher chance to obtain functional EGFR

inhibitor derivatives in shorter times and most importantly, to be able to quickly reject molecules with significant

stereochemical issues that would prevent them from approaching the kinase’s binding cavity. The in silico

approach seems even more necessary if we consider that the constructs we finally aim at, contain large organic

parts.

After obtaining insight from our extended docking studies in a number of designed analogues, we moved on

to selected chemical synthesis of 12 new analogues. Determination of the EGFR- inhibitory activity and

assessment of their biological activity has been undertaken in order to identify their potential to serve as

promising grafting agents onto nanocarriers for drug delivery systems


Number: 286125

References [1] Gordon W. Rewcastle,†, Brian D. Palmer,†, Alexander J. Bridges,‡, H. D. Hollis Showalter,‡, Li Sun,‡, James Nelson,‡, Amy

McMichael,‡, Alan J. Kraker,‡, David W. Fry,‡ and, and William A. Denny*,† “Tyrosine Kinase Inhibitors. 9. Synthesis and Evaluation

of Fused Tricyclic Quinazoline Analogues as ATP Site Inhibitors of the Tyrosine Kinase Activity of the Epidermal Growth Factor

Receptor,”Journal of Medicinal Chemistry 39 (4), 918-928 (1996).

[2] Jeff B. Smaill,†, Gordon W. Rewcastle,†, Joseph A. Loo,‡, Kenneth D. Greis,§, O. Helen Chan,‡, Eric L. Reyner,‡, Elke Lipka,‡, H. D.

Hollis Showalter,‡, Patrick W. Vincent,‡, William L. Elliott,‡ and, and William A. Denny*,† “Tyrosine Kinase Inhibitors. 17. Irreversible

Inhibitors of the Epidermal Growth Factor Receptor: 4-(Phenylamino)quinazoline- and 4-(Phenylamino)pyrido[3,2-d]pyrimidine-6-

acrylamides Bearing Additional Solubilizing Functions” Journal of Medicinal Chemistry 43 (7), 1380-1397 (2000).

[3] Srivastava, Sanjay K.; Kumar, Vivek; Agarwal, Shiv K.; Mukherjee, Rama; Burman, Anand C., “Synthesis of Quinazolines as Tyrosine

Kinase Inhibitors” Anti-Cancer Agents in Medicinal Chemistry 9 (3), 246-275 (2009).




Theoretical investigation of a new metal nanoparticle for

combined imaging and therapy applications

Myria Angelidou, Costas Pitris KIOS Research Center for Intelligent Systems and Networks,

Dep. of Electrical and Computer Engineering, University of Cyprus

75 Kallipoleos St., 1678 Nicosia, Cyprus

[email protected], [email protected]

1. Introduction

Metal nanoparticles (NPs) have unique optical properties, due to the phenomenon of surface plasmon resonance

(SPR). By varying the size, shape, and material, the SPR wavelengths can range from visible to near-infrared

(NIR). [1] Most of the research effort focuses on noble metals, such as silver and gold, since they exhibit strong

absorption and scattering plasmon bands which can be exploited in both imaging and therapy applications [2].

The optical response, of metal nanoparticles, was extensively explored using computational methods. The noble

NPs were found to have absorption, or scattering or overlapping spectra [3, 4].

Tissue absorption in the NIR window (650-900nm) is minimal, and thus favorable to optimal light

penetration [5]. Imaging applications can significantly benefit from using NIR light, which minimizes absorption

from biomolecules and therefore allows deeper penetration of the incident light into living tissue. Photothermal

applications, on the other hand, could benefit from NPs having strong absorption with limited scattering, for

better efficiency. Here, we propose a new, complex metal nanoparticle which has the unique property of

distinctly separated absorption and scattering spectra. Its absorption peak is at the wavelength (λ) of 635nm, and

its scattering peak at λ = 785nm, both in the range of the optical window for biological applications. This could

be beneficial for combined imaging and therapy, since the laser wavelengths used for each application can be

decoupled for increased efficacy and safety.

2. Methodology

The Discrete Dipole Appoximation (DDA) method was used to calculate the optical response (absorption,

scattering, and extinction) of various metal nanoparticles. The DDA can address with any arbitrary shape,

composition and external medium as long as some criteria are satisfied [6]. The efficiency factors are used to

explore the various spectral characteristics, such as wavelength maximum and peak value. These factors are

defined as 2

abs sca abs sca effCQ r and ext abs scaQ Q Q where Cabs/sca is the absorption and scattering cross

sections, and reff is the effective radius, which represents the radius of a sphere having a volume equal to that of

the simple or complex nanoparticle. The complex dielectric function for the various metals was specified and

obtained from the E.D. Palik [7] tabutaled data. For small sized nanoparticles, such as nanospheres, the dielectric

function was modified to include the surface damping effect, as described in ref. [8]. All the calculations

assumed water as the external medium.

3. Results

3.1 Simple nanoparticles

First, the efficiency factors were calculated for various simple metal NPs, such as silver (Ag), gold (Au),

aluminum (Al) and nickel (Ni). The NPs had nanospheres, nanocubes and tetrahedral shapes, and various sizes.

From the calculations it was found that simple nanostructures have mainly scattering or overlapping spectra.

(Data not shown.) Since none of the simple nanostructures provided with distinct spectra, combinations were

considered for further exploration. The Au nanocube, with reff = 74.4nm, was chosen as the imaging

nanoparticle, since it provides the stronger scattering with less absorption in the NIR range even though

absorption and scattering overlap in the visible.

3.2 Complex nanoparticles

To obtain the desired property, of distinctly separated absorption and scattering spectra, small nanospheres and

the Au nanocube, chosen from previous calculations, were combined to create a complex nanoparticle. The small

nanospheres were chosen as the therapy nanoparticle since they provide with adequate absorption in the visible

wavelength range [2]. The small nanospheres were considered to be arranged on the front face of the nanocube,

perpendicular to the propagation x-axis, as also shown in Fig. 1(a, b). Parameters such as the number, size

(diameter), and material (Ag or Au) of the small nanospheres were varied in order to obtain the optimum

nanostructure with distinctly separated absorption and scattering spectra.




Figure 1. Example of a complex nanoparticle shape as viewed from (a) the side, and (b) front.

Figure 2(a) show the spectra of the Au nanocube chosen as the scattering nanoparticle. Figure 2(b) show the

optical response of the complex nanoparticle comprised of the Au nanocube combined with 16 (30nm) Ag

nanospheres. Other combinations, such as 4 (60nm), and 64(15nm) Ag nanospheres are not shown. It is clear,

from fig. 2 that the addition of a layer of small nanospheres, in front of the nanocube, affected the spectra. Table

1 provides with the ratios of absorption to scattering and vice versa for the various complex NPs, for better

comparison between them. We require high ratio for both wavelengths. From the two ratios, the combination

with 16 Ag provided with the highest values, and proved to exhibit the best separation between the spectra, and

at the desired wavelength range. The gold nanosphere combinations gave similar results, except in the case of 16

Au nanospheres, where the ratio at λ = 635nm was small compared to the counterpart.

Figure 2. Absorption and scattering spectra of (a) simple Au nanocube (reff = 74.4nm) and (b) the complex nanoparticle (nanocube plus 16

(30nm) Ag spheres).

Table 1. Ratio of absorption to scattering, and vice versa, for the two wavelengths of interest.

Qabs/Qsca, at λ = 635nm Qsca /Qabs, at λ = 785nm

Cube plus 4 Ag spheres 1.781 1.354

Cube plus 4 Au spheres 1.773 1.317





When the complex nanoparticle was rotated relative to the incident propagation and polarization, the separation

between absorption and scattering was lost and scattering was significantly enhanced in the visible range. Also,

when the small nanospheres covered all nanocube surfaces again the separation was again lost and replaced by a

flat absorption spectrum and a broad scattering spectrum having two peaks at visible and NIR wavelengths.

(Data not shown.)

4. References

[1] P. K. Jain, X. Huang, I. H. El-Sayed and M. A. El-Sayed, “Review of some interesting surface plasmon resonance-enhanced properties

of noble metal nanoparticles and their applications to biosystems,” Plasmonics 2, 107-118 (2007).

[2] N. G. Khlebtsov and L. A. Dykman, “Optical properties and biomedical applications of plasmonic nanoparticles,” J. Quant. Spectrosc.

Radiat. Transfer 111, 1-35 (2010).

[3] I. O. Sosa, C. Noguez and R. G. Barrera, “Optical properties of metal nanoparticles with arbitrary shapes,” J. Phys. Chem. B 107, 6269-

6275 (2003).

[4] P. K. Jain, K. S. Lee, I. H. El-Sayed and M. A. El-Sayed, “Calculated absorption and scattering properties of gold nanoparticles of

different size, shape, and composition: applications in biological imaging and biomedicine,” J. Phys. Chem. C 110, 7238-7248 (2006).

[5] R. Weissleder, “A clearer vision for in vivo imaging,” Nat. Biotechnol. 19, 316-317 (2001).

[6] B. T. Draine and P. J. Flatau, “Discrete-dipole approximation for scattering calculations,” J. Opt. Soc. Am. A 11, 1491-1499 (1994).

[7] E. D. Palik, Handbook of optical constants of solids, (Academic Press, Orlando, 1985).

[8] M. Angelidou and C. Pitris, “Investigation of nanostructure scattering and absorption for combined optical diagnostic and therapeutic

applications,” Proc. SPIE 8231, 8231081-8231087 (2012).



Surface Enhanced Raman Spectroscopy (SERS) for Point-

Of-Care Diagnosis of Urinary Tract Infections

Katerina Hadjigeorgioua, Evdokia Kastanos

b, Costas Pitris

a

a KIOS Research Center for Intelligent System and Networks, Department of Electrical and Computer Engineering, University of Cyprus, 75

Kallipoleos St, Nicosia, Cyprus, 1678 b University of Nicosia, Department of Life and Health Sciences, 46 Makedonitissas Avenue, Nicosia 1700, Cyprus.

[email protected]

1. Introduction

Urinary tract infections (UTIs) are very common, especially in women, resulting in billions of dollars in health

care costs every year in the US alone [1]. The current, standard, method of diagnosis for a UTI is by quantitative

urine culture which requires 24 hours to produce results. [2]. Antibiotic susceptibility testing requires an

additional 24 hour period. Due to the prolonged period of diagnosis, broad spectrum antibiotics are prescribed

before any definitive results are obtained. This practice leads to many undesirable consequences such as

unsuccessful treatments, chronic infections, rising health care costs, and, most importantly, increased antibiotic

resistance [2].

Surface Enhanced Raman Spectroscopy (SERS) has had a variety of bioanalytical applications in recent

years including the detection and identification of bacteria [3-7]. The aim of this work was the development of a

rapid identification and antibiogram method using SERS spectra obtained from bacteria incubated with various

antibiotics and colloidal silver nanoparticles. Bacteria were classified with an accuracy of ~ 94% and sensitivity

or resistance to antibiotics was determined with 81-100% accuracy using SERS spectra after just 4 hours of

exposure. These results may lead to the development of a fast and accurate diagnosis and antibiogram tool for

UTI based on SERS.

2. Methodology

Sample Preparation and Data Acquisition

Clinical bacterial isolates from patients with UTI were identified by biochemical tests. Liquid antibiograms were

used to specify the susceptibility of each bacterium to each of the 7 antibiotics used in this study. Bacterial

samples containing 2x105 cells/ml (determined optically) were used for the classification and antibiogram

studies. This concentration was chosen since it is equivalent to the minimum concentration found in urine

samples from UTIs.

For the purposes of this study, 16 bacterial strains were chosen, 7 E. coli, 4 K. pneumonia, and 5 Proteus

spp., and were treated separately with amoxicillin, amoxicillin/clavulanic acid (augmentin), cefaclor, cefazoline,

ceftriaxone, cefuroxime, and ciprofloxacin. 50 μl of each bacterial sample was mixed with an equal volume of

each antibiotic or PBS (as the control) and incubated for 0 and 4 hours at 37oC. 20 μl of the incubated bacteria-

antibiotic solutions (105 cells/ml) was mixed with 10μl of silver nanoparticles spotted on glass slides and allowed

to dry. SERS spectra were collected directly from the spots. SERS spectra were collected using the iRaman

system (BWTek, Inc) with a laser source at 532nm and 3.0cm-1 resolution. The power at the sample was

approximately 50 mW and the exposure time was 20s x 12 averages (4 min total.) The SERS spectra consisted of

data ranging from 300 cm-1 to 3000 cm-1 inclusive.

Analysis of SERS spectra

For classification of the bacterial species, the SERS spectra collected from the 16 bacterial strains at time 0

without any exposure to antibiotics were used. Spectra were pre-processed by removing cosmic spikes and

filtering to remove the background and the high frequency noise (Figure 1). The features used for the

classification were the spectrum itself and ratios of means of spectral segments (Figure 2). The classification

method used was Linear Discriminant Analysis. Principal component analysis (PCA) of the data preceded the

classification. A leave-one-out cross-validation (LOOC) procedure was performed and the results for each of the

classification exercises were recorded.

For the classification of bacteria as sensitive or resistant to any of the seven antibiotics, the SERS spectra

collected from the 16 bacterial strains after 4 hours of exposure either to each antibiotic or PBS (control) were

used. Pre-processing was performed as before. In addition, the corresponding control spectrum was subtracted

from the spectrum with antibiotic exposure. This procedure removes any background, bacterial, and/or antibiotic

contributions to the spectra leaving the data solely reflecting the changes due to the antibiotic activity. Each

sample was then classified as resistant, sensitive, or intermediate using the same feature creation, data reduction,

and classification techniques as before. A LOOC procedure was also performed.


3. Results

Following the methodology described above, the classification of the bacterial species yielded 93.75 % correct

classification rate (i.e. only 1 of the 16 samples was misclassified.) Figure 3 shows two such examples of the LOOC

process. Following the methodology described in section 2.2, each bacterium was classified as resistant,

intermediately sensitive, or sensitive to each of the 7 antibiotics used in the study. The spectra were correctly

classified between 81 and 100 % depending on the antibiotic (Table 1.) It is interesting to notice that the algorithm

is not only successful for antibiotics affecting the bacterial cell wall but also other types (e.g. ciprofloxacin which

acts on the bacterial DNA.)

4. Conclusions

The use of SERS for UTI diagnosis and antibiogram is presented in this study. The bacterial species can be

correctly classified with high accuracy (~94 % correct classification) even at low bacterial concentrations. In

addition, this method is able to distinguish the SERS spectra of bacteria that are treated and are sensitive to an

antibiotic from bacteria that resistant to an antibiotic, after a 4 hour incubation time with correct classification rate

of ~ 81-100%.

Experiments are currently under way to repeat this study using a larger number of samples belonging to a

greater number of species which will include both gram negative and gram positive bacteria. In addition, studies are

currently under way to determine what the shortest time of exposure. In order for this method to develop into a

point-of-care diagnostic the test time must be as short as possible. In addition, experiments have to be performed

directly on urine samples.

5. References [1] Litwin M. S., Saigal C. S., "Introduction", [Urologic Diseases in America], DHHS, PHS, NIH, NIDDK, NIH publication, Washington, DC,

07-5512, 3-7 (2007).

[2] Morgan, M. G., McKenzie, H., "Controversies in the laboratory diagnosis of community-acquired urinary tract infection," Eur. J. Clin.

Microbiol. Infect. Dis. 12, 491-504 (1993).

[3] Zeiri, L., Bronk, B. V., Shabtai, Y., Eichler, J., Efrima, S., “Surface-enhanced Raman spectroscopy as a tool for probing specific

biochemical components in bacteria”, Appl. Spectrosc. 58, 33-40 (2004).

[4] Guzelian, A. A., Sylvia, J. M., Janni, J. A., Clauson, S. L., Spencer, K. M., “SERS of whole-cell bacteria and trace levels of biological

molecules”, Proc. SPIE 4577, 182 (2002).

[5] Premasiri, W. R., Moir, D. T., Klempner, M. S., Krieger, N., Jones, G. II, Ziegler, L. D., “Characterization of the surface enhanced raman

scattering (SERS) of bacteria”, J. Phys. Chem. B; 109, 312-320 (2005).

[6] Jarvis, R. M., Goodacre, R., “Characterisation and identification of bacteria using SERS”, Chem. Soc. Rev. 37, 931-936 (2008).

[7] Walter,A.,Marz,A.,Schumacher,W.,Rosch,P.,Popp,J., “Towards a fast, high specific and reliable discrimination of bacteria on strain level by

means of SERS in a microfluidic device”, Lab Chip 11, 1013-1021 (2011).


Figure 1. Average and filtered SERS spectra of the bacteria used in

this study. The background was removed.

Figure 2. Ratios of means of spectral segments used as features for

species classification. The average for each species is presented

here.

Figure 3. Results of bacterial species classification. An uknown

E. Coli is correctly classified.

Table 1. Antibiotic sensitivity classification results.

Antibiotic %Correct

Classification

Amoxil 87.50

Augmentin 93.75

Cefaclor 81.25

Cefazolin 81.25

Ceftriaxone 93.75

Cefuroxime 100.00

Ciprofloxacin 87.50

Mean 89,29

500 1000 1500 2000 2500 3000

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

wavenumber (cm-1)

arb

itary

sta

cked c

ounts

E.Coli

Klebsiella

Proteus

Avg. E.Coli Ratios

Num

era

tor

Segm

ent

1 2 3 4 5 61

2

3

4

5

6Avg. Klebsiella Ratios

Denominator Segment

1 2 3 4 5 6

Avg. Proteus Ratios

1 2 3 4 5 6

E.Coli

Klebsiella

Proteus

Unknown

348 350 352 354 356 358 360 36245

46

47

48

49

50

Manova Score 1

Manova S

core

2

Unknown: Klebsiella (Misclassified)

254 256 258 260 262 264 266 268

88

90

92

94

Manova Score 1

Manova S

core

2

Unknown: E.Coli (Correctly Classified)

E.Coli

Klebsiella

Proteus

Unknown

A

B


Activity, anti-cancer effect and nanodelivery of new

anilinoquinazoline EGFR inhibitors

Eftychia Angelou1, Fotini Liepouri

2, Maria Pavlaki

1, Andreani Odysseos


4 ,

Alexandros Strongilos2, Pavlos Agianian

1

1. Department of Molecular Biology and Genetics, Democritus University of Thrace, Dragana, 68100 Alexandroupoli, Greece.

2. Pro-Actina S.A., Archimidous 59, Koropi Attikis, 19400, Greece.

3. EPOS-Iasis, 5 Karyatidon Street, Suite 202, 2028 Nicosia, Cyprus.

4. PECSA laboratory (Physico-chimie des Electrolytes, Colloïdes et Sciences Analytiques),

5. UPMC, 75005 Paris, France

[email protected]

Epidermal Growth Factor Receptor (EGFR) is a ubiquitous tyrosine kinase that modulates cell physiology (1).

Binding of physiological ligands, such as EGF, to EGFR alter cell differentiation, proliferation and migration and

control cell survival. In a number of malignancies, including those of the lung, breast, colon, and kidney, EGFR is

overexpressed or hyperactivated leading to aberrant cell signaling (2). Targeting EGFR with tyrosine kinase

inhibitors (TKIs) constitutes an important line of defense against EGFR-positive cancers, however, inevitably,

resistance develops. Recently discovered second generation TKIs, like the drugs gefitinib and lapatinib, are effective

against some resistant tumors (3). Chemically, they both comprise an anilinoquinazoline scaffold that specifically

recognize the ATP-binding pocket of the cytoplasmic, kinase domain of EGFR.

We aim to develop new anilinoquinazoline EGFR inhibitors and use them to direct nanodevices to cancer cells

and EGFR-resistant tumors for both targeted therapy and imaging (theranostics). A dozen of new type-I

anilinoquinazoline derivatives have been synthesized. We show their EGFR kinase inhibitory activity in vitro and

how they affect cell survival of HeLa and MCF-7 cells, untreated or after stimulation with EGF. Using confocal

microscopy, we present preliminary imaging data that demonstrate the interaction of promising fluorescently labeled

nanosystems, with living cells. We finally illustrate Surface Plasmon Resonance (SPR)- based approaches to quickly

screen the functional loading of the nanosystems in grafted anilinoquinazolines. The potential of using these results

for the construction of efficient, EGFR-specific, cancer theranostic applications is discussed.

Figure 1. Viability of EGF stimulated HeLa cells as a function of increasing concentrations (nM) of a new anilinoquinazoline

derivative

Acknowledgements

This work is supported from FP7-IAPP-NANORESISTANCE project (grant number 286125)

References 1. Yarden Y., Sliwkowski M. X. (2001) Nat. Rev. Mol. Cell Biol. 2, 127–137.

2. Rowinsky E. K. (2004) Annu. Rev. Med. 55, 433–457.

3. Rosa D.D., Ismael G., Lago L.D., Awada A. (2008) Cancer Treat. Rev. 34, 61–80.




Toxicity and Safety of Carbon Nanomaterials for

Biomedical Applications

Cyrill Bussy and Kostas Kostarelos The University of Manchester, Manchester, United Kingdom.

[email protected]

The dramatic development of nanoscience and nanotechnology in recent years has offered numerous opportunities

and innovative solutions in various fields and applications. Among the different types of novel materials discovered

at the nanoscale, carbon-based nanomaterials are a superfamily that includes amongst many others, fullerenes,

carbon nanotubes (CNTs), and graphene. In terms of scope of applications, two of these carbon nanomaterials seem

to be developed more widely and maturing faster than the rest: carbon nanotubes and graphene. Both CNT and

graphene materials have outstanding electronic, mechanical, electrical, and optical properties and a chemically

tunable surface that have made them attractive candidates for a broad range of applications, spanning from

composites and electronics to nanomedicine. Biosensors, tissue engineering, as well as components for the design

of various types of drug delivery and release systems are among the potential applications of graphene and CNTs in

biomedicine. However, questions have been raised regarding a potential toxicity upon human exposure. In this

context, the importance of toxicity profiling and physicochemical characteristics in relation to safety considerations

for carbon nanomaterials based products candidate for biomedical applications cannot be overemphasized.

We will focus on how the two most popular forms of carbon nanomaterials (carbon nanotubes, graphene) used

in biotechnology and medicine would fair toxicologically in terms of their drug development potential. We will

highlight key physicochemical parameters for safety considerations and will also emphasize the importance of

toxicological profiling during the development phase of biomedical products based on carbon nanomaterials. A

comparison with clinically used nanomedicines and nanomedicine products in clinical trials will be given in order

to understand where carbon nanomaterials stand.

[Back to Session 3]


Guidelines Proposal for Studying Hemocompatibility of

Manufactured Nanoparticles and Impact on Fibrinolysis

Julie Laloy, Lutfiye Alpan, Valentine Minet, Jean-Michel Dogné Department of Pharmacy, Namur Thrombosis and Hemostasis Center (NTHC), Namur Research Institute for LIfe Sciences (NARILIS),

University of Namur

[email protected]

1. Background

Nanosciences and nanotechnologies are in constant evolution. Development of new therapeutic and diagnostic

agents using nanotechnologies to reach their pharmaceutical target require the knowledge of biocompatibility of

nanoparticles (NPs) with the blood compounds. Hemostasis is the ensemble of physiological phenomena which

prevent and lead to stop bleeding; it maintains the vascular integrity. A dysfunction of the hemostasis can lead to

slow down or even to completely stop the circulation of the blood. It is therefore primordial to study the

hemocompatibility of NPs.

2. Aims

The aim of this study is the evaluation of the biocompatibility of manufactured NPs on hemolysis, platelet function

and coagulation.

3. Methods

Erythrocytes integrity was studied by determination of haemoglobin release. For platelet function, six platelet

function tests were investigated: light transmission aggregometry, whole-blood impedance aggregometry, platelet

function analyser-100 (PFA-100) and Cone-and-Plate(let) analyser (Impact-R®), transmission- and field emission

gun scanning electron microscopy (FEG-SEM). Several existing methods of clotting times and thrombin generation

assays were evaluated in human normal pool plasma for the impact of NPs on coagulation. Five NPs (carbon

nanotubes, carbon black, silicon dioxide, copper oxide and silicon carbide) with different physicochemical

properties were used.

4. Results

The Impact-R® with scanning electronic microscopy support are the reference methods to investigate the potential

impact of NPs on platelet function. For coagulation, the calibration thrombin generation test is the reference method

to investigate the procoagulant activity of NPs.

5. Conclusion

We suggest guidelines for testing NP hemocompatibility to respond to a request of scientific community due to lack

of recommendations for the evaluation of nanomaterial hemocompatibility.

[Back to Session 3]


Rat whole-body exposure model to nanoaerosol:

Development with silicon carbide nanoparticles and study of

their toxicity.

Julie Laloy, Omar Lozano, Lütfiye Alpan, Valentine Minet, Olivier Toussaint, Bernard Masereel, Jean-

Michel Dogné & Stéphane Lucas.

University of Namur, 61 rue de Bruxelles, Namur, 5000 Belgium

[email protected]

Background

Air represents the major route of human exposure to manufactured nanoparticles (NPs). Humans are exposed

every day to diesel exhaust particles containing NPs. In manufacturing industries, workers can also be

accidentally exposed to NPs due to the failure of ventilation system or individual protective equipment. The

assessment of the potential risks of NPs from air exposure on different tissues and organs, especially the

respiratory tract, is thus needed. The physicochemical property of NPs most considered for pulmonary tract

exposure is their size. The size and shape of particles determine their aerodynamic properties that govern entry,

depth of penetration, and deposition in the lung. Inhalation model is closer to the reality than instillation for

reproduction of working conditions, dose generated, and profile of deposition in the pulmonary system.

Inhalation is also more complex to implement and requires expansive equipments as aerosol generator and

analyzer.

Aims

The objectives of this work are: 1. The design, development, characterization and validation of a rat airborne

nanoaerosol whole-body exposure model. 2. To study the pulmonary impact of silicon carbide (SiC) NPs on rats

exposed to a dry aerosol in this whole- body exposure model in an acute toxicity protocol.

Methods

An airborne nanoaerosol whole-body exposure model was designed and developed to allow the simultaneous

exposure of six rats to a nanoaerosol and six rats to clean filtered air (Figure 1). The aerosol was generated using

a RBG-1000®

and analyzed with a detection system ELPI®

(Electrical Low Pressure Impactor) for real-time

measurement of the NP aerosol concentration and particle size distribution. Complete physicochemical

characterization of the generated aerosol was performed by ELPI®

, another aerosol analyzer (Aerotrak®

) and

scanning electron microscopy. SiC NPs are selected for the model development and the pulmonary toxicity

study. For the acute toxicity study, Sprague-Dawley rats were exposed during 6 hours to SiC NPs dry aerosol or

filtered air in the model.

Results

The aerosol generated in three individual experiments is stable and reproducible during 6 hours in the model.

A continuous monitoring of the NPs concentration in atmosphere during the experiment is however supported.

After rat exposure during 6 hours to SiC nanoaerosol, macrophages containing SiC NPs were observed in the

lungs with no histological toxicity observed.



Figure 1. Scheme of the whole-body exposure model.

Conclusion

This is the first model developed and validated that allows the integrated assessment of safety NPs on biochemical,

histopathological parameters and biopersistance in different tissues in acute but also subacute and chronic exposure.

[Back to Session 3]


EPR-effect: a barrier to the effective delivery of large

nanomedicines to solid tumors

Triantafyllos Stylianopoulos Department of Mechanical and Manufacturing Engineering, University of Cyprus, Nicosia 1678, Cyprus

[email protected]

The Enhanced Permeability and Retention (EPR) effect is based on the leakiness of tumor blood vessels that

enables nanoparticles to enter the tumor as well as on the absence of functional lymphatics in the tumor interior

that allows particles to stay in the tissue for a long time. The leakiness (i.e., hyper-permeability) of the tumor

vasculature is mainly due to large openings in the endothelial lining that comprises the neoplastic tumor vessel

wall [1]. These openings might be hundreds of nanometers in size, contrary to the openings of the normal vessel

wall, whose size is less than 10 nm [2, 3]. Therefore, the rationale for employing the EPR effect to treat cancer is

that particles with a size larger than 10 nm will not be able to extravasate to normal tissues - reducing adverse

effects - and would selectively pass through the openings of the tumor vessels. This reasoning, however, does not

guarantee that nanoparticles will reach tumors in amounts sufficient to cause cure.

The EPR was introduced by two independent studies more than two decades ago [4, 5]. The first study was

published by the research group of Hiroshi Maeda [4] and focused on the bright sight of the EPR, i.e., the selective

delivery of nanoparticle formulations to tumor and not to normal tissues. The second study was published by the

research group of Rakesh Jain [5] and apart from the great promise of EPR, they pointed out the potential barriers

that it might have to the effective delivery of the nanomedicines to the tumors. Since then the scientific community

was heavily based on the first study neglecting the barriers to the delivery of large size nanoparticles that are posed

by the abnormal tumor microenvironment.

Nowadays, despite the enormous scientific effort only three nanoparticle formulations have been widely

approved for treatment of solid tumors. These are: Doxil® a 100 nm pegylated liposomal doxorubicin that has

been approved for the treatment of HIV-related Kaposi's sarcomas, metastatic ovarian cancers and metastatic

breast cancers, DaunoXome® a 50 nm liposomal daunorubicin, approved for HIV-related Kaposi's sarcomas and

Abraxane® a 10 nm (following plasma disintegration) albumin-bound paclitaxel, which has been given approval

for metastatic breast cancers. These drugs are associated with significantly less adverse effects compared to

conventional chemotherapy, presumably due to the EPR effect. The increase in overall survival is, however,

modest in many cases [1, 6-8] largely because of limited delivery. Therefore, even though current nanomedicines

have succeeded in preventing delivery to normal tissues, they cannot ensure effective delivery in tumors.

In my talk, I will discuss how the EPR effect inhibits the effective delivery of large nanoparticles to solid

tumors and will provide strategies to overcome these barriers and improve intratumoral distribution. I will also

present design rules that optimize nanoparticle accumulation into tumor tissues.

References [1] Jain RK, Stylianopoulos T. Delivering nanomedicine to solid tumors. Nat Rev Clin Oncol 7, 653-64 (2010).

[2] Hobbs SK,Monsky WL, Yuan F et al. Regulation of transport pathways in tumor vessels: Role of tumor type and microenvironment. Proc

Natl Acad Sci U S A 95, 4607-12 (1998).

[3] Sarin H. Physiologic upper limits of pore size of different blood capillary types and another perspective on the dual pore theory of

microvascular permeability. J Angiogenes Res 2, 14 (2010).

[4] Matsumura Y, Maeda H. A new concept for macromolecular therapeutics in cancer chemotherapy: Mechanism of tumoritropic

accumulation of proteins and the antitumor agent smancs. Cancer Res 46, 6387-6392 (1986).

[5] Gerlowski LE, Jain RK. Microvascular permeability of normal and neoplastic tissues. Microvasc Res 31, 288-305 (1986).

[6] O'Brien MER, Wigler N, Inbar M et al. Reduced cardiotoxicity and comparable efficacy in a phase III trial of pegylated liposomal

doxorubicin HCl (CAELYX/Doxil) versus conventional doxorubicin for first-line treatment of metastatic breast cancer. Ann Oncol 15,

440-9 (2004).

[7] Gradishar WJ,Tjulandin S, Davidson N et al. Phase III trial of nanoparticle albumin-bound paclitaxel compared with polyethylated castor

oil-based paclitaxel in women with breast cancer. J Clin Oncol 23, 7794-803 (2005).

[8] Gill PS, Wernz J, Scadden DT et al. Randomized phase III trial of liposomal daunorubicin versus doxorubicin, bleomycin, and vincristine

in AIDS-related kaposi's sarcoma. J Clin Oncol 14, 2353-64 (1996).

[Back to Session 4]


Strategies to improve nanomedicine delivery to solid tumors

Konstantinos Soteriou, Eva-Athena Economides, Triantafyllos Stylianopoulos

Department of Mechanical and Manufacturing Engineering, University of Cyprus, Nicosia, Cyprus

[email protected]

Introduction

Failure of standard nanomedicine formulations to prolong the life of cancer patients is in large part due to the

inability of these drugs to penetrate deep into the tumor tissue and distribute homogeneously in the tumor

interstitial space. As a result cancer nanomedicines that have been approved for clinical use (e.g., Doxil®,

DaunoXome®) often provide modest survival benefits, despite the considerably less adverse effects compared to

conventional chemotherapy that they are associated with [1]. The leakiness of the tumor blood vessels has

served as the rationale for the use of nanoparticle formulations in cancer but at the same time it contributes to the

elevation of the interstitial fluid pressure (IFP), which hinders the transport of nanomedicines from the

vascular to the interstitial space (i.e., transvascular transport). Strategies to reduce IFP include anti-VEGF

treatment to improve pericyte coverage and thus, decrease the leakiness of the vessels, and anti-fibrotic treatment

to deplete extracellular fibers and decrease the resistance with which fluid can escape from the tumor interstitial

space [2]. Mathematical models were developed to investigate the potentials of both therapeutic strategies and

model predictions were validated with in vivo experiments in orthotopic murine mammary adenocarcinomas.

We found that anti-VEGF treatment improves the delivery of nanomedicines in a size- dependent manner,

favoring the transport of particles less than 20 nm in diameter. Anti-fibrotic treatment was able to improve the

transvascular transport even for large particles with sizes similar to that of currently used nanotherapeutics, such

as Doxil®.

Results

Fig. 1. Model predictions for the effect of anti-VEGF treatment on nanomedicine delivery. Anti-VEGF reduces the size of the

pores of the vessel wall, which improves the penetration of only small particles [2].

Equations

Vascular transport:

(1)

where v is the fluid velocity, cv is the intravascular concentration of the nanoparticle and Δcv is the

concentration difference that corresponds to a vascular length Δx.



Transvascular transport:

(2)

where Pe is the Péclet number across the vessel wall, P is the vascular permeability of the nanoparticle through the

pores of the wall, Lp the hydraulic conductivity, P the vascular permeability, and σ the reflection coefficient.

Interstitial transport:

(3)

where ci is the concentration of the nanoparticle in the interstitial space, D is the diffusion coefficient, and vi is the

interstitial fluid velocity.

References

[1] Jain RK, Stylianopoulos T, “Delivering nanomedicine to solid tumors,” Nature Reviews Clinical Oncology 7, 653-664 (2010).

[2] Chauhan VP, Stylianopoulos T, “Normalization of tumor blood vessels imporves the delivery of nanomedicines in a size-dependent

manner,” Nature Nanotechnology 7, 383-388 (2012).

[Back to Session 4]


Methods for delivery of living cells to the respiratory system

via aerosol route Tomasz R. Sosnowski

1, Ewelina Tomecka

2

1Faculty of Chemical and Process Engineering, Warsaw University of Technology, Waryńskiego 1, 00-645, Warsaw, Poland 2Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw, Poland

[email protected]

1. Introduction

Aerosol route is a convenient and widely accepted method of delivering therapeutics to the lung surface where

they act at a local level (e.g. in curing pulmonary diseases) but can be also transferred to the circulation and

produce systemic effects. As the lungs have a very huge surface area (up to 100 m2), designing a delivery

system as an air dispersion (aerosol) is rational since such a method allows to distribute a drug in different parts of

the lungs without employing excessive doses.

Respiratory tract is exposed to environment and its cellular structure can be damaged by accidental

inhalation of hot gases or irritants [1]. In such cases, the vital breathing function can be seriously defected

leading to life-threatening conditions. A possible acceleration in the recovery can be brought by recellurization of

the lung surface i.e. by seeding the progenitor cells which might at least partially substitute functions of

damaged epithelium. Similar healing method was successfully implemented in recovery from burns and ulcers of

the skin [2,3], and it can be also considered as a future strategy in the lung therapy, also with the application of

stem cells [1]. Our recent studies done with very simple biological models (bacteria and yeast cells) indicated that

spraying of biocolloids with conventional inhalers (nebulizers) is ineffective as the average droplets

produced by these devices are too small to carry whole cells. In addition, all tested methods were too destructive for

biological material due to high hydrodynamic stresses associated with liquid atomization [4]. Based on these

studies only two devices could be selected as potentially applicable for cell spraying, and in preliminary tests

they were demonstrated to maintain 65-95% survival rate of sprayed murine fibroblasts. In a very recent paper by

Kardia et al. [5] it was shown that sprayed fibroblasts proliferate after atomization done by the same devices as the

one employed in our research. This result indicated no cell inactivation due to spraying.

In the current study we present more comprehensive tests of technical possibilities of cell atomization in

order to suggest suitable methods for the generation and delivery of aerosols containing living cells to the lungs.

2. Materials and methods

The model cell line BALB/c3T3 (fibroblasts) was purchased from European Collection of Cell Cultures (UK).

The cells were suspended in DMEM (Dulbescco’s Modified Eagle’s Medium – Sigma Life Sciences, USA) at the

concentration of 4.35·105 ml-1. Biocolloid was sprayed from two mechanical devices: Microsprayer® Aerosolizer

model IA-1B (Penn-Century Inc., Wyndmor, PA, USA) and nasal atomizer (Coster, Italy) - Fig. 1. Collected

material was analyzed with three assays: (a) vital staining with trypan blue (0.4%) and subsequent cell counting

(Countess® Invitrogen, Korea), (b) fluorescent test in multi-well plate using calceine-AM (2 mM) with fluorescence

evaluation by SynergyTM Mx Multi-Mode Microplate Reader (BioTek, USA) at time = 0 and 24 hours after

spraying, and (c) direct fluorescence microscopic observations (IX7® inverted microscope, Olympus, Japan) after

cell staining with calceine-AM and propidium iodide.

Fibroblasts were also incubated in DMEM for 3 days at 37C at 5% CO2 (HERAcell® 150 – Thermo

Electron Corp. USA) for microscopic evaluation of cell proliferation.

sprayin

g tip

Fig.1. Atomizing devices: Microsprayer® Aerosolizer and nasal atomizer.

3. Results

The size of droplets emitted from both devices were characterized previously using Spraytec aerosol

spectrometer (Malvern, UK) and it was shown that aerosols were log-normally distributed with median diameter of

47-49 m and 79-82 m, respectively [4]. This allows to expect that the fibroblasts (with an average size of 10-

20 m in suspension) can be safely surrounded by a liquid inside a droplet generated from a nozzle. The

cumulative results of cell survival tests are presented in Fig. 2, where relative viability means the current

viability as a percentage of cell viability in the initial sample. It follows that at least 75% of cells are viable after

spraying with both methods, and the viability is maintained at similar level 24 hrs after spraying. Cells have

ability to proliferate during next 72 hrs what is demonstrated in Fig.3. This result suggests that biological



activity of cells is not significantly altered by atomization which is in agreement both with results of other

authors [5] and with our data from MTT tests presented recently [4].

Fig.2. Cell survival (a) and proliferation (b) after spraying with both atomizing devices.

4. Discussion and conclusion

Although two tested atomizing methods are suitable for generation of aerosols containing living cells, it should be

noted that they cannot be directly applied to deliver dispersed biocolloids to the respiratory system. The reason

is that the droplets are too big for their effective penetration to the bronchial tree. On the other hand, spraying

methods which allow to produce finer droplets (e.g. nebulization) cannot be used as such aerosols contain no

living cells [4]. Therefore it is proposed that cell delivery to the respiratory system should be accomplished

by a special techniques where the aerosol is generated either in situ (inside the trachea - e.g. by Microsprayer

device or similar atomizer) or outside the body but with specific inhalation maneuver to avoid droplets

deposition in the upper airways. Bioaerosol should be gently pushed into the bronchial tree e.g. via

endotracheal tube [6], allowing the droplets with cells at least partly penetrate the lower airways. In case of

intubated patients with massive lung damage, which might need this kind of treatment, both approaches of

aerosol delivery seem reasonable.

5. Acknowledgment

Work supported by governmental funds for science in the years 2010-13 (Project No. NN209 023339).

6. References [1] D.J. Angelini et al., "Chemical warfare agent and biological toxin-induced pulmonary toxicity: could stem cells provide potential

therapies?", Inhalation Toxicol. 25, 37–62 (2013).

[2] G. Gravante et al., "A randomized trial comparing ReCell ® system of epidermal cells delivery versus classic skin grafts for the

treatment of deep partial thickness burns", Burns 33, 966-972 (2007).

[3] R.S. Kirsner et al., "Spray-applied cell therapy with human allogeneic fibroblasts and keratinocytes for the treatment of chronic venous leg

ulcers: a phase 2, multicentre, double-blind, randomised, placebo-controlled trial", The Lancet 380, 977-985 (2012).

[4] T.R. Sosnowski et al., "Spraying of cell colloids in medical atomizers", Chem. Eng. Transact. 32, 2257-2262 (2013).

[5] E. Kardia et al., "Aerosol-based delivery of fibroblast cells for treatment of lung diseases", J. Aerosol Med. Pulm, Drug Deliv.

doi:10.1089/jamp.2012.1020 (2013).

[6] J. Mazela et al., "Aerosolized albuterol sulfate delivery under neonatal ventilatory conditions: in vitro evaluation of a novel ventilator

circuit patient interface connector", J. Aerosol Med. Pulm, Drug Deliv. doi: 10.1089/jamp.2012.0992 (2013).

[Back to Session 4]


Enhancement of Drug Absorption Across Intestinal

Membrane Using Magnetic Beads

Anjali Seth,1,2

David Lafargue,2

Cécile Poirier,2

Jean-Manuel Péan,2

Christine Ménager1

1University Pierre et Marie Curie, Laboratoire PECSA, UMR 7195 4 Place Jussieu, 75005 Paris, France 2TECHNOLOGIE SERVIER, Formulation Galénique 25-27 Rue Eugène Vignat, 45000 Orléans, France

[email protected]

Magnetic beads were prepared to study the impact of an external magnetic field on drug penetration across

intestinal membrane. Chitosan-alginate core-shell beads (Fig. 1.) were loaded with a low membrane

permeability drug and magnetic nanoparticles (MNPs). They were prepared by standard extrusion crosslinking

process.

Fig. 1. Magnetic core-shell chitosan-alginate beads.

Ex-vivo experiments were performed with Ussing chambers (Fig. 2.) using mice’s jejunum as model membrane.

A magnet was placed on the acceptor chamber wall to create a magnetic field. Apparent permeability was

measured on magnetic beads in comparison with different controls (free drug, magnetic beads without magnet,

drug loaded non-magnetic beads).

Fig. 2. Ussing chambers to measure drug apparent permeability.

Spherical beads with an average diameter of 1.5 mm were prepared with a drug experimental encapsulation ratio

of 4.5% w/w and a loading efficiency of 70%. The MNPs encapsulation ratio was 1.5% w/w. No MNPs release

from beads was detected during the experiments. Dissolution kinetics showed that 100% of the drug was

released within two hours without any influence of the magnetic field. When a magnetic field was applied,

magnetic beads were accumulated onto the intestinal membrane and the apparent drug permeability was

increased threefold in comparison with free drug or non-magnetic beads (Fig. 3.).



Fig. 3. Permeation studies of the drug across non-stripped rat jejunal membrane. Effect of magnetic beads maintained near the

membrane with a magnet (■) in comparaison with the controls, magnetic beads free in the donor compartment (□), non-magnetic

beads free in the donor compartment (●) and free drug (○).

The absorption enhancement was due to the local over-concentration of the drug close to its absorption site and

not to a membrane’s permeability change. The use of magnetic carriers to localize a drug near its absorption

window enabled to enhance significantly its permeation. This approach opens new perspectives in the field of low

permeable drugs for oral administration.

[Back to Session 4]


Ultrasound and microbubbles for monitoring therapies

targeting tumor vascularity

Mike Averkiou

Department of Mechanical and Manufacturing Enigineering, University of Cyprus, Nicosia 1678, Cyprus

[email protected]

Imaging is a key factor in the accurate monitoring of response to cancer therapies targeting tumor vascularity to

inhibit its growth and dissemination. Dynamic contrast enhanced ultrasound (DCE-US) is a relatively new

quantitative method with the advantage of being non-invasive, widely available, portable, cost effective, highly

sensitive and reproducible using microbubble contrast agents that are truly intravascular.

Advances in nonlinear imaging techniques have enabled ultrasound imaging to visualize the macro- and

microvasculature in real time. The image intensity of a region of interest (ROI) in the tumor is proportional to the

microbuble concentration. Metrics of blood flow and blood volume may be extracted from indicator dilution

models. The present talk will concentrate on the bolus injection method for contrast delivery and the analysis of the

wash-in and wash-out of the microbubbles in the ROI.

A review of current work in this area ill be presented and the issues described above will be discussed. Results

from clinical trials with liver cancer patients undergoing vascular targeted therapies will be presented.

[Back to Session 5]


Tunable magnetic/ICG small protocells as a platform for

drug delivery

Thomann Jean-Sébastien, Corne Gaëlle, Arl Didier, Bahlawane Naoufal and Lenoble Damien

Département Science et Analyse des Matériaux (SAM), CRP Gabriel Lippmann, 41, rue du Brill L-4422 Belvaux, Luxembourg

[email protected]

1. Introduction

Protocells have been reported as a promising drug delivery system due their high payload capability and their

versatility of usage [1]. Protocells are constituted by a mesoporous silica core (MSNPs) surrounded by a lipid

bilayer. The lipid bilayer plays the role of biocompatible gatekeeper which enables easy and efficient surface

functionalization. Brinker et al. [1] was the first to describe their morphological structure and to study their

physico-chemical stabilities. However, the size of reported protocells is typically in the range of 120 nm and

restricted to drug delivery function. The influence of MSNPs pores size on the protocell stability has not yet

been investigated. In our work, we propose to add imaging capability via building multi-phased small protocells

(MSNPs having diameter from 20nm up to 50 nm) incorporating a magnetic core and green indocyanine. The

pore size of the MSNPs will be carefully tuned and thoroughly characterized. This nanoparticle will offer

bimodal imaging capabilities combined with a drug delivery platform. The tailored inorganic carrier properties

that include size, pores geometry, magnetic and near-infrared imaging responses will be reported.

2. Methods

The synthesis of magnetic MSNPs is achieved using the silatrane route originally published by Möller et al [2].

The properties of the magnetic core are controlled by adjusting the concentration of Fe3O4 NPs and the used

surfactant CTACl (cetyl trimethyl ammonium chloride). The ratio between reactants and solvents is a key

parameter for tuning the size of the MSNPs [3]. ICG is grafted on MSNPs by using Aminopropyl tetraethyl

silicate (APTES) functionalization [4]. The pores size is engineered using a selective chemical etching with

NaBH4 [5]. The lipid coating is applied using ultra-sonication or mixing lipid vesicles with MSNPs. Physical

and chemical characterizations are carried out with Nanoparticle Tracking Analysis, Transmission Electronic

Microscopy (TEM), Scanning Electronic Microscopy (SEM), Fourier transform infrared microscopy, X-Ray

Diffraction.

3. Results

Figure 1: Small multi-phased protocells. A schematic view of the targeted protocells geometry is presented in the left-hand

side. Two sizes of NP (SEM pictures: A, B) were obtained by varying the dilution of reactant. The number of magnetic

nanoparticle loaded in MSNPs can be tuned by varying the ratio between CTAB, TEOS (triethylorthosilicate) and Fe3O4

nanoparticles (TEM picture C and D). After the synthesis of MSNPs, the pore size can be increased from 3 nm (TEM picture E)

up to 5 nm (TEM picture F) using NaBH4.



4. Conclusion

We have designed multifunctional nano-objects for enabling the deployment of theragnostic approach in cancer

therapy. By combining dual in vivo imaging and drug loading (via encapsulation of paclitaxel or cis-platinum), the

proposed new generation of protocells is particularly appealing to tackle the current challenges of cancer

nanomedicine. In particular, the MSNPs small size and porosity demonstrated in this study should enable the

design of multi-layers coating while keeping the overall size below 100nm. In one hand, this should favor the

passive targeting of diseases cells based on the EPR. On the other hand, this design should enable a precise

control of the drug payload. In last, the lipid bilayer would enable various bio-conjugation scenarii.

5. References

[1] Ashley, C. E.; Carnes, E. C.; Phillips, G. K.; Padilla, D.; Durfee, P. N.; Brown, P. A.; Hanna, T. N.; Liu, J.; Phillips, B.; Carter, M. B.;

Carroll, N. J.; Jiang, X.; Dunphy, D. R.; Willman, C. L.; Petsev, D. N.; Evans, D. G.; Parikh, A. N.; Chackerian, B.; Wharton, W.; Peabody,

D. S.; Brinker, C. J. The targeted delivery of multicomponent cargos to cancer cells by nanoporous particle-supported lipid bilayers. Nat

Mater 2011, 10 (5), 389-397.

[2] Moller, K.; Kobler, J.; Bein, T. Colloidal Suspensions of Nanometer-Sized Mesoporous Silica. Adv. Funct. Mater. 2007, 17 (4), 605-

612.

[3] Urata, C.; Aoyama, Y.; Tonegawa, A.; Yamauchi, Y.; Kuroda, K. Dialysis process for the removal of surfactants to form colloidal

mesoporous silica nanoparticles. Chem. Commun. 2009, 0 (34), 5094-5096.

[4] Quan, B.; Choi, K.; Kim, Y. H.; Kang, K. W.; Chung, D. S. Near infrared dye indocyanine green doped silica nanoparticles for

biological imaging. Talanta 2012, 99 (0), 387-393.

[5] Jia, L.; Shen, J.; Li, Z.; Zhang, D.; Zhang, Q.; Duan, C.; Liu, G.; Zheng, D.; Liu, Y.; Tian, X. Successfully tailoring the pore size of

mesoporous silica nanoparticles: Exploitation of delivery systems for poorly water-soluble drugs. International Journal of Pharmaceutics

2012, 439, 81-91.

[Back to Session 5]


Ultrasound-induced temperature elevation for in-vitro

controlled release of temperature-sensitive liposomes

Christophoros Mannaris1, Jean-Michele Escoffre

2, Ayache Bouakaz

2, M. E Meyre

3 and Michalakis

Averkiou1

1-Dept. of Mechanical and Manufacturing Engineering, University of Cyprus, Nicosia, Cyprus,

2-UMRS INSERM U930, CNRS ERL3106, Université François Rabelais-Tours, France,

3-Nanobiotix, Paris, France

1. Introduction

Drug loaded thermosensitive liposomes (TSL) release their payload with mild hyperthermia near their phase

transition temperature (Tm = 43-45 °C) [1]. Focused ultrasound may be used to non-invasively induce local mild

hyperthermia in a region of interest with high accuracy [2]. In combination, ultrasound induced temperature

elevation for localized drug delivery using TSL shows potential in improving the efficacy of drug delivery in a

lesion while at the same time reducing undesired side effects.

In spite of several reports for in-vivo drug delivery using ultrasound and TSL [3-5], reports for in-vitro work

are scarce mainly due to the difficulty in finding an appropriate in-vitro experimental setup. One of the main

problems often encountered is that cell culture media have a very low absorption coefficient and are thus unable to

be heated with ultrasound. Finding a biocompatible medium with high absorption coefficient has not been possible

thus far.

In the present work, an in-vitro method that allows activation of TSL using ultrasound is presented. A single

element focused transducer is used to induce the required temperature elevation in Opti-MEM® cell culture

medium via thermal conduction and activate drug loaded TSL. A significant release of doxorubicin from TSL is

achieved.


Experimental setup

Figure 1 shows a schematic of the experimental setup. A small volume sample holder containing Opti-MEM® cell

culture medium and TSL solution was placed inside a plastic cuvette filled with 99% glycerol that has a high

absorption coefficient (α=5.7 Νp/m/MHz). Mylar acoustic windows on the cuvette and holder allowed ultrasound

to propagate through thus avoiding heating of the plastic walls. A single element focused transducer (center

frequency 1.1 MHz, 50 mm diameter and 50 mm focus) was used to heat up the glycerol in the cuvette. The Opti-

MEM®/TSL solution in the sample holder reached the required temperature for activation of the TSL via thermal

conduction. The experiments were carried out in a 37°C water bath.

Figure 1: Schematic of experimental setup

Ultrasound Exposure

Detailed characterization of the ultrasound field was done in water using a 0.4 mm element membrane hydrophone

(Precision Acoustics Ltd, Dorchester, UK). The acoustic field experienced by the TSL in the sample holder was

also measured with the cuvette in place using a 0.5 mm needle hydrophone placed in the middle of the cuvette (the

rear Mylar window was removed). Any diffraction effects due to the presence of the cuvette were negligible. The

sample holder was placed at the focus of the transducer. Ultrasound was applied for 15 minutes at 1.1 MHz

frequency, 45% duty cycle and 1.4 MPa peak negative pressure.

Activation protocol

Thermosensitive lipososomes [DPPC:HSPC:Chol:DPPE-PEG (50:25:15:3)] from Nanobiotix were diluted in

OptiMEM® to a concentration of 3 μg/mL. The experiments were separated in the following categories:

1. OptiMEM + US: To check if US exposure alters the parameters of the medium.

2. Negative control: Samples kept at room temperature (0% release).

3. Positive control: Samples in 45°C water bath for 15 minutes (100% release).

Cuvette

Sample Holder

Mylar acoustic

windows


4. TSL + US (no heating): Same exposure conditions applied but without heating to check if US alone

induces any release from TSL. This was done by replacing the glycerol in the cuvette with water.

Since water has very low absorption, it does not heat up with US.

5. TSL + US + Heating: TSL activation experiment at p=1.4 MPa.

6. TSL + US + Heating*: TSL activation experiment at p=1.8 MPa.

Analysis of results

Analysis of the results was done using a 96-wells plate spectrofluorometer. The excitation wavelength, λex was 485

nm and the emission wavelength, λem was 580 nm. Each category was repeated at least three times and the results

are presented as mean ± standard deviation. The % release of doxorubicin was evaluated using equation 1 below

[1]:

(1)

where, Iexp, Ineg and Ipos are the fluorescence intensities of the experiment, negative control and positive control

respectively.

3. Results

A theoretical model based on the Pennes’ Bioheat equation was initially used to calculate the ultrasound

parameters required for temperature elevation in glycerol under conditions for drug activation (5-8°C). Fine-wire

(50μm) thermocouple readings were in close agreement with our theoretical predictions as shown in figure 2. A

temperature elevation of 6-7°C was obtained in the sample holder within 6 minutes before reaching a plateau.

Figure 2: Comparison of experimental with predicted values for temperature elevation in glycerol as a function of input

pressure (f=1.1 MHz, 45% DC)

The results for TSL activation and release are shown in Figures 3 and 4. Figure 3 shows the fluorescence

measurements from each category whereas figure 4 shows the calculated % release [using equation (1)]. It is noted

that ultrasound induced almost as much release as the positive control while further increasing the pressure (and

temperature) does not show any added benefit. It is also noted that ultrasound does not affect the medium

properties (OptiMEM = OptiMEM + US) and that US alone does not influence the TSL or cause any release (NEG

Control = TSL + US).

Figure 3: Fluorescence measurements of the different categories.

ΔΤ (

°C)

Input [MPa]

Experiment

Theory


4. Discussion

An ultrasound method for in-vitro drug release from thermosensitive liposomes has been developed. Water-based

cell culture media present difficulties in ultrasound heating but our method overcomes this issue. Temperature

elevation of 5-8 degrees (needed for the activation of the TSL) is reached with focused ultrasound at 1.1 MHz and

an 80% drug release was achieved with our method. Heating by thermal conduction approach mimics in-vivo

conditions where ultrasound is used to induce hyperthermia in tissue and the TSL (in blood) would be heated by

conduction.

Further improvements of the method will allow for faster and more uniform heating. A broader acoustic field

(very low focussing gain or even unfocused sources) will result in larger treatment areas. The current setup may be

used with cells in suspension; one possible modification is to allow testing of seeded cell cultrures (perhaps in

OptiCell).

Figure 4: Induced doxorubicin % release from thermosensitive liposomes.

5. References

[1] M. de Smet, et al., "Temperature-sensitive liposomes for doxorubicin delivery under MRI guidance," Journal of Controlled

Release, vol. 143, pp. 120-127, 2010. [2] M. O. Köhler, et al., "Volumetric HIFU ablation under 3D guidance of rapid MRI thermometry," Medical Physics, vol. 36, pp.

3521-3535, 2009. [3] S. Dromi, et al., "Pulsed-high intensity focused ultrasound and low temperature - Sensitive liposomes for enhanced targeted

drug delivery and antitumor effect," Clinical Cancer Research, vol. 13, pp. 2722-2727, 2007. [4] R. Staruch, et al., "Localised drug release using MRI-controlled focused ultrasound hyperthermia," International Journal of

Hyperthermia, vol. 27, pp. 156-171, 2011. [5] A. Yudina, et al., "Ultrasound-mediated intracellular drug delivery using microbubbles and temperature-sensitive liposomes,"

Journal of Controlled Release, vol. 155, pp. 442-448, 2011.

[Back to Session 5]

% D

OX

Re

leas

e


Biodegradable Dextran Nanoparticles as Potential Drug

and Fluorescent Marker Carrier

Janczewska M. Wasiak I. Pieczykolan J. Ciach T.

Warsaw University of Technology, Faculty of Chemical and Process Engineering, Biomedical Engineering Laboratory,

Warynskiego Street 1, 00 -645 Warsaw, Poland

1. Introduction

Cancer is one of the biggest challenges of contemporary medicine. Despite the enormous scientific and

financial effort undertaken in recent years cancer remains one of the major death cause in western civilization.

Thanks to this effort various new drugs have been developed, but still many types of cancer in metastasis are

virtually untreatable. A lot of hope for new treatment methods are set on nanotechnology. This is because all the

cellular machinery and transport phenomena are happening at this size range. Nanoparticles seem to be a perfect

drug carrier due to possible targeted cancer drug delivery, what is a key to minimize main side effects of

chemotherapeutic agents and reduce tumor expansion. They are defined as submicron structures that can

covalently or physically bind therapeutic agent and administrate it directly to the tumor cells [1]. Nanoparticles

can be biodegradable and nonbiodegradable. The first type will disappear from the body after performing the

desired action, due to hydrolysis or other biological process. The second type will probably stay in our body for

very long time or even forever, what pose a potential danger on such objects. That’s why biodegradable –

organic nanoparticles are frequently considered as safe, in comparison with inorganic. Nanoparticles designed as

drug carriers should have diameter in a range of over twenty up to even hundreds of nanometers. The exact size

can differ according to the administrated drug structure and nanoparticle destination. Size is one of the crucial

nanoparticle features as it strongly influence their fate after administrating into the bloodstream. Another

important characteristic is NPs surface charge that is defined by zeta potential. These two agents decide whether the

nanoparticle will manage to avoid opsonization and removal from the bloodstream shortly after intravenous

application. Thus nanoparticles having size below a hundred nanometers and slightly charged surface seem to be a

perfect tool for targeted therapy. Depending on nanoparticles preparation method it is possible to obtain

nanocapsules or nanospheres. Nanocapsules characterize with core – shell structure, where polymeric shell

encloses often liquid core where drug is dispersed, whereas nanospheres consist of covalently modified polymeric

chain where the active agent is attached.

Biocompatibility and bioavailability of nanoparticles is necessary for them to be effective and safe as a drug

carrier. Nanoparticles made from natural polymers such as chitosan, dextran, cellulose or starch are regarded as

more biocompatibile due to their low toxicity and high biodegradability. [2] Dextran is already widely used in

medical and pharmaceutical field. It is common component of eye drops and is often used as hematopoietic

replacement agent. It is naturally synthesized by lactic - acid bacteria such as Leuconostoc mesenteroides and

Streptococcus mutan and is enzymatically degradated. Because of easy degradation there is no risk of

cumulating polymer inside organs what is particularly important when it comes to nanoparticles. [3]

Preparation nanoparticles from polysaccharide gives an enhanced targeting properties thanks to increased

requisition for glucose molecules. As Otto Warburg proven, cancerous cells are producing energy mainly by

glycolysis. Following this thought, composed from the saccharide nanoparticles have a better chance to be up

taken by malignant cells. [4] Nanoparticles are mostly integrated by endocytosis, phagocytosis and

macropinocytosis, however there is still a lack of exact information about the actual uptake mechanism.

Generally, it is claimed that the most probable path of intracellular integration is either clathrin or caveolae

mediated endocytosis. [5]

Due to its polysaccharide structure dextran can be easily oxidized to polyaldehydedextran in the reaction

with sodium periodate in aqueous solution. In this reaction glucose rings are open and oxidized without breaking the

polysaccharide backbone. The oxidation of dextran chain opens further possibilities for its modification.

Aldehyde groups afterwards, can form covalent links with amines, creating Schiff base. Further Amadori

rearrangement can stabilize obtained compound. If the covalently linked groups are lipophylic we can observe a

molecular selfassembly leading to nanoparticle formation, what is the main topic of this paper. The same

chemical reaction was employed to attach anticancer drug and fluorescent probes. Attaching anticancer drug by

Schiff base assures that the drug will be released inside a tumor cells after internalization, due to decreased pH in

lisozomes.

The aim of this paper is to obtain and characterize dextran nanoparticles designed for drug delivery and

diagnosis. Nanoparticles were synthesized, adjusting proportions of reagents in order to obtain particles about

100 nm. The cytotoxicity was tested in vitro on A549 human lung carcinoma, MCF10A human breast cells and


MES-SA/Dx5 human uterine sarcoma cell lines. Futhermore, in vivo studies were conducted on mouse model of

multidrug resistant human uterine sarcoma (MES-SA/Dx5).

2. Methods

Nanoparticles were obtained from 40 and 70 kDa dextran (Nobilus Ent), which was oxidized in aqueous

solution with sodium periodate. The number of aldehyde groups in dextran chain was determined by the reaction

with hydroxylamine hydrochloride and titration with sodium hydroxide nominated solution. The product was

purified by dialysis against pure water for three days and dried overnight in 50ºC. Nanoparticles were prepared

by mixing solution of polyaldehydedextran (PAD), dodecylamine hydrochloride and common anticancer drug –

doxorubicin hydrochloride (Sequoia Research Products Ltd). In some cases fluorescent (9-aminoacridine) and

non fluorescent (trypan blue) dies were also covalently linked. Reaction was carried out in 30ºC in water bath

for 1,5 h. Product was purified by dialysis against pure water for an hour in order to remove non reacted

substances. The diameter of nanoparticles was measured using Nanoparticle Tracking Analysis (NTA) with

NanoSight. Subsequently, product was lyophilized and redissolved in order to examine if nanoparticles are

stable after dry storage. Cell lines were grown and nanoparticles were tested for cytotoxicity by test MTT. To

obtain fluorescent nanoparticles, fluorescent agent was covalently attached.

To improve fluorescent properties, fluorescent nanocristals were precipitated inside polysaccharide

nanoparticles. Fluorescent nanocrystals were precipitated by mixing 9-aminoacridine stearate tetrahydrofuran

solution with water containing polysaccharide nanoparticles.

3. Results

Developed nanoparticles are bioavailable and biodegradable. Size range of synthesized particles was about 70

to 100 nm, depending on polysaccharides and coiling agents used in synthesis, and was suitable for a drug

carrier. Developed method is quick and safe as it does not concerns usage of any organic solvents. Nanocarrier

gave no therapeutic response in cytoxicity tests, whereas nanoparticles with drug attached gave therapeutic

effect comparable to toxic influence of doxorubicin. To confirm in vitro experiments the in vivo studies were

conducted on the same type of carcinoma cell line. Too large dosage and too frequent administration turned out

to be fatal for representative group of mice. However a medium dosage of 7,5 mg/kg injected intravenously

every 6 days was not toxic for mouse organism in comparison with free doxorubicin administrated in the same

way. The Tumor Growth Inhibiton (TGI) was defined and calculated and it oscillated at 29% for NPs with

Doxorubicin (7,5 mg/kg) in 35 day of experiment. Free doxorubicin in the same dosage showed a fatal influence

on studied group.

Results of cancer cell staining with a use of fluorescent nanoparticles showed that polysaccharide

nanoparticles are efficiently entrapped by the cancer cells, even dies that never enters the intact cell (trypan

blue) can be efficiently transported into the cell interior. Organic fluorescent nanocristals precipitated inside

polysaccharide nano-shells can serve as an efficient cancer detection and staining technique. Fluorescent organic

nanocrystals due to its high quantum efficiency can be a safe and biodegradable alternative to quantum dots.

4. Acknowledgements

Presented research was partially supported by EU within the frame of uroNanoMed project FonDiag.

International patent pending by Nanovelos company (polysaccharide nanoparticles).

5. References [1] I. Brigger, C. Dubernet, P. Couvreur, “Nanoparticles in cancer therapy and diagnosis,” Adv. Drug. Del. Rev. 54, 631-651

(2002).

[2] A. Aumelas et al., “Nanoparticles of hydrophobically modified dextrans as potential drug carrier systems,” Coll. Surf. B: Bioint. 59

74- 80 (2007). [3] S. Daoud-Mohammed et al., “Spontaneous association of hydrophobized dextran and poly-β-cyclodextrin into

nanoassemblies. Formation and interaction with a hydrophobic drug,” J. Coll. Int. Sci. 307, 83-93 (2007).

[4] E. Christ, “The Warburg effect and its role in cancer detection and therapy” in Program in Biotechnology Department of Biological Sciences, (Columbia University, 2009).

[5] Ruthrotha B. Selvi et al., “ATP driven clathrin dependent entry of carbon nanospheres prefer cells with glucose receptors,” J.

Nanobio. 10:35, (2012).

[Back to Session 5]


Engineering carbon nanotubes based scaffolds for the

efficient delivery of new tyrosine kinase inhibitors

Davide Giust, Kostas Kostarelos UCL School of Pharmacy - 29-39 Brunswick Square - London - WC1N 1AX

[email protected]; [email protected]

Over-expression and/or co-expression of epidermal growth factor receptors (EGFRs) subtypes has been

found in numerous tumor types, including colon, breast, ovarian, non-small lung and other malignant

cancers [1]. Tyrosine Kinase Inhibitors (TKis) selective for the EGFR and inhibiting the related Tyrosine

Kinase activity have been approved for current therapy of different malignant forms. Nevertheless in

most cases, patients survival is strictly related to the decreased activity of these inhibitors due to receptors

mutation and/or decreased penetration inside cells [2]. In this sense, carbon nanotubes (CNTs) represent

good candidates to improve the delivery of Tkis. CNTs are allotropic form of carbon made of graphene

enrolled sheets with sp2 hybridized carbon, displaying unique chemical and physical properties useful for

the delivery of bio-active molecules [3]. The aim of this project is to use modified-CNTs to improve the

delivery of new synthesized TKis. This will achieve by the meaning of different functionalization

approaches. This involves the non-covalent attachment of Tkis to the side wall of MWNT upon π-π

stacking interactions, as well as the formation of covalent and covalent cleavable bonds [4-5] (Scheme 1).

All CNTs-Tkis will be further investigated in vitro as well as new synthesized Tkis. Human Glioblastoma

has been chosen as in vitro model among the new therapeutic challenges for these type therapeutic agents.

These malignant cells are in fact, one of the most challenging in cancer therapy due to the onset of

multi drug resistance and bad prognosis in all patients [6]. The project, still in progress, is at the half-

way between the synthesis and the in vitro screening of the CNT-TKis complexes. The most active

among the new synthesized TKis have been identified. By the non-covalent interaction between the CNT

and TKis, no improvement of the activity was found compared to that one of TKi alone. Some synthetic

trials are actually on going to get covalently conjugates CNTs-TKis and evaluate their the activity in vitro.

Scheme 1. Synthetic approach used to functionalized CNTs with the Tkis.

References [1] A. Arora and E. M. Scholar, “Role of Tyrosine Kinase Inhibitors in Cancer Therapy”, The Journal of Pharmacology and Experimental

Therapeutics, 315(3): 971-979 (2005).

[2] T. E. Taylor, F. B. Furnari and W. K. Cavenee, Curr Cancer Drug Targets, 12(3): 197–209 (2012).

[3] G. Pastorin, W. Wu, S. Wieckowski, J. P. Briand, K. Kostarelos, M. Prato, A. Bianco, “Double functionalisation of carbon nanotubes for

multimodal drug delivery”, Chem. Commun. 1182–1184 (2006).

[4] H. Ali-Boucetta, K. T. Al-Jamal, D. McCarthy, M. Prato, A. Bianco, K. Kostarelos, “Multiwalled carbon nanotube-doxorubicin

supramolecular complexes for cancer therapeutics”, Chem Commun (Camb), 4:459–461 (2008).

[5] C. Samori, H. Ali-Boucetta, R. Sainz, et al. “Enhanced anticancer activity of multi-walled carbon nanotube-methotrexate conjugates using

cleavable linkers”, Chem Commun (Camb), 46:1494–1496 (2010).

[6] A. D. Joshi, W. Loilome, I-Mei Siu, B. Tyler, G. L. Gallia, G. J. Riggins, “Evaluation of Tyrosine Kinase Inhibitor Combinations for

Glioblastoma Therapy”, PLOS ONE, 7 (10) (2012).

[Back to Session 5]



Folic Acid functionalized Quatro-NanoContainers as

targeted agent: In vitro and In vivo study Eleni K. Efthimiadou

a*, Christos Tapeinos

a , Eirini Fragogeorgi

b,c , George Loudos

b, and George Kordas

*a

a Sol-Gel Laboratory, Institute for Advanced Materials, Physicochemical processes, Nanotechnology & Microsystems, NCSR

“Demokritos”, 153 10 Aghia Paraskevi Attikis, Greece b Department of Medical Instruments Technology, Technological Educational Institute, GR 122 10 Athens, Greece

c Radiochemical/ Radiopharmacological Quality Control Laboratory, Institute of Nuclear and Radiological Sciences and Technology,

Energy & Safety, N.C.S.R. ‘Demokritos’, 15310 Aghia Paraskevi, Greece

Nanocontainers (NCs) can serve as highly versatile platforms for the controlled functionalization and delivery

in the last decade as DDS. The responsiveness of the functionalized NCs could be attained by choosing

monomers with specific sensitivities, such as, sensitivity in temperature NIPAAm, HPMA, DMAEMA),

pH (e.g. AA, MAA) and reductive-oxidizing (redox) environments, which are desired for selective

release in tumor area. Nanospheres present many advantages such as stability, presence of many active

groups that can be easily functionalized with specific molecules for targeted therapy and coated with

polymers improving their circulation time in bloodstream and inducing therefore, stealthy properties. For the

targeted therapy through the NCs, the small molecule of folic acid has been employed. Because folate

receptors are known to be overexpressed and actively internalized through folate receptor mediated

endocytosis in various types of cancer cells, folic acid has been considered to be a suitable ligand for

improving the cellular uptake of drugs and macromolecules into cells. The NCs were modified by

carbodiimide chemistry with FITC and/or Folic Acid. Ovarian cells, HeLa, which overexpress the folic acid

receptor, were incubated for 72 h with the empty and drug loaded NCs aiming at studying the targeted

internalization mechanism with and without folic acid.

Without Folic Acid With Folic Acid

Figure 1. Cellular trafficking of FITC-labeled and FA- FITC-labeled NCs in HeLa cells. The cells were incubated for

1h with the FITC-labeled NCs in the presence of LysoTracker Red (10 min) at 37 oC, followed by live cell imaging. In

all experiments cells were treated with either 3 μM folic acid functionalized NCs.

References 1. Brannon-Peppas, L.; Blanchette, J. O., Nanoparticle and targeted systems for cancer therapy. Advanced Drug Delivery Reviews

2012, 64, 206-212.

2. Brigger, I.; Dubernet, C.; Couvreur, P., Nanoparticles in cancer therapy and diagnosis. Advanced Drug Delivery Reviews

2012, 64, 24-36.

[Back to Session 6]


Doxorubicin-loaded and Antibody-Conjugated Liposome-

QD Hybrid Vesicles for Targeted Cancer Theranostics

Bowen Tian1, Wafa Al-Jamal

2, KostasKostarelos

2

1. University of Nottingham, United Kingdom

2. University College London, United Kingdom

[email protected]

Quantum dot (QD) have been extensively explored for in vitro and in vivo imaging due to their superior

fluorescence properties compared to organic fluorophores. The hydrophobic nature of QD hinders their

biomedical applications in biological milieu, therefore many efforts have been made to make-water soluble QD

by substituting the organic surface ligands with hydrophilic moieties. However such surface modifications

adversely affected the QD optical properties and colloidal stability. Previously, our group offered an alternative

approach to improve QD hydrophilicity by incorporating hydrophobic QD into the liposome lipid bilayer which

efficiently labelled cancer cells in vitro and in vivo[1]. In this study, we report the engineering of the multimodal

liposome-QD hybrids (L-QD) for targeted cancer theranostics. L-QD was loaded with doxorubicin (Dox) using

the osmotic gradient technique, achieving high loading efficiency compared to liposome alone[2]. Structural

elucidation using cryogenic electron microscopy (cryo-EM) clearly showed that QD were incorporated into the

lipid bilayer and Dox were encapsulated into the liposome aqueous core. Furthermore, the surface of Dox-loaded

hybrids were functionalized with anti-MUC-1 antibody for active targeting, using the post-insertion technique.

The specific binding of antibody-targeted hybrids was studied against MUC-1 epitope by surface plasmon

resonance (BIACORE) showing higher binding affinity compared to antibody alone due to multivalent effect. In

addition, cellular uptake studies of the antibody-targeted hybrids were conducted using confocal laser scanning

microscopy (CLSM). The antibody-targeted hybrids showed high binding and uptake by human breast cancer

cell lines (MCF-7) that overexpress MUC-1 receptors in contract to human pulmonary adenocarcinoma cells

(Calu-6) exhibiting low level of MUC-1 expression. Finally, cytotoxicity assays indicated higher toxicity of

antibody-targeted hybrids in MCF-7 compared to Calu-6 cells. In conclusion, MUC-1 antibody-targeted L-QD

hybrids encapsulating doxorubicin are thought to constitute a potential multimodal system for the simultaneous

delivery of therapeutic and diagnostic agents to cancer cells in vitro and in vivo.

References 1. Al-Jamal, W. T., Al-Jamal, K. T., Tian, B., Lacerda, L., Bornans, P. H., Frederik, P. M., Kostarelos, K., "Lipid-quantum dot bilayer

vesicles enhance tumor cell uptake and retention in vitro and in vivo", Acs Nano, 2008. 2(3): p. 408.

2. Tian, B., Al-Jamal, W. T., Al-Jamal, K. T., Kostarelos K., "Doxorubicin-loaded lipid-quantum dot hybrids: surface topography and

release properties", International Journal of Pharmaceutics, 2011. 416(2): p.443.

[Back to Session 6]


Monitoring tumor burden by multicolor in vivo flow

cytometry

Costas Pitsillides, Konstantinos Kapnisis and Andreas Anayiotos Department of Mechanical Engineering and Materials Science and Engineering, Cyprus University of Technology, Limassol, Cyprus

[email protected]; [email protected]; [email protected]

1. Introduction

In vivo measurement of tumor burden, both in cancer research models and in patients, is an important parameter

for the accurate assessment of disease progression and the response to therapeutic intervention [1]. Several in

vivo imaging modalities have been utilized in the assessment of tumor burden, including functional magnetic

resonance imaging, computer tomography and positron emission tomography [2, 3], fluorescence imaging [4, 5],

intravital microscopy [6] and bioluminescence imaging [7]. More recently, the detection/quantification of

circulating cancer cells has been explored as a method to evaluate tumor burden in the context of assessing

disease stage, prognosis as well as monitoring disease progression following therapeutic intervention in cancer

patients [8, 9]. Clinically, various ex vivo assays have been developed to detect cancer cells shed in circulation

by primary tumors, including breast cancer, prostate cancer and small-cell lung cancer [10, 11].

In vivo flow cytometry has been developed as a method for real-time detection of circulating cancer cells

injected into the circulation of experimental animals. The method does not require extraction of blood samples

and is therefore well suited for long-term monitoring of circulating tumor cells. In this report, we report on the

development of a multichannel in vivo flow cytometer to detect and quantify circulating cancer cells as a means

of assessing the tumor burden in animal models.


Development of a multichannel in vivo flow cytometer Multichannel in vivo flow cytometry combines the

principles of confocal detection and flow cytometry in order to enable the real-time detection of fluorescently

labeled cells circulating in a live animal. The system can be applied for the dynamic and simultaneous

monitoring of multiple populations of circulating cells, which can be targeted and labeled with multiple

fluorescent markers and probes. To accomplish this, light from up to three separate excitation lasers was focused

by a cylindrical lens and then imaged across a blood vessel to form an excitation slit. Figure 1 illustrates the

concept of the multichannel in vivo flow cytometer. When fluorescently labeled cells were flown through the

excitation slit, the emitted fluorescence signal emitted was confocally detected by a photomultiplier tube in each

of the detection channels of the system. The collected PMT signal was sent to an analog-to-digital converter to

be digitized and then stored on a PC to be analyzed by Matlab software in order to identify cell peaks and extract

quantitative information on the number of cells passing.

Figure 1. Schematic of the multichannel in vivo flow cytometer

The excitation lasers chosen were the 633 nm Helium-Neon laser, preferred due to its good penetrating

capability through tissue and blood, as well as the 561 nm (Cobolt AB, Solna, Sweden) and 488 nm (Coherent

Inc, Santa Clara, CA, USA) diode-pumped, solid-state lasers, chosen for their ability to excite fluorescence in a

host of commonly used fluorochromes such as green fluorescent protein (GFP), fluorescein, and the yellow/red

fluorescent protein variants (YFP/RFP). Arteries (with average diameter of 40-50 µm), rather than veins, were

chosen for experiments because of faster blood flow and absence of cell-endothelium interactions.


In vivo detection of circulating cancer cells

Approx. 106 cells/ml from the MDA-MB-231 breast adenocarcinoma cell line were incubated with the Vybrant

series of lipophilic fluorescent probes (each at a concentration of 5 µg/ml) and then separately injected through

the tail vein of anesthetized male, 10-12 week old, CD1 mice in order to demonstrate the in vivo capabilities of

the system. The anesthetized animals were placed on an imaging platform within 5 minutes after injection of the

cells and an appropriate arteriole in the ear was chosen from which to obtain circulating cell count.

Figure 2. Breast adenocarcinoma cells labeled with the Vybrant DiD fluorescent probe and detected in vivo

3. Results and discussion

We have developed the multichannel in vivo flow cytometer for the dynamic monitoring of circulating cells and

have demonstrated its capabilities in detecting and quantifying fluorescently labeled breast adenocarcinoma cells

directly injected in the circulation of experimental animals. The system was designed and built with the ability to

simultaneously assess the circulation kinetics of several distinct cell populations in a single animal. This will

allow for a more efficient in vivo investigation of complex biological processes by enabling the simultaneous

monitoring of the multiple cell populations that might be participating and interacting in such processes.

To better approximate the tumor environment in vivo, a mouse tumor model will be developed through the

adoptive transfer of fluorescent protein expressing cancer cells in immune-compromised mice. Once the cells

migrate to tumor growth areas and the tumors are established, the animals will be monitored long term using the

multichannel in vivo flow cytometer in order to quantify the fluorescently labeled tumor-shed cells in

circulation. Tumor burden will also be assessed via whole body reflectance imaging and the results will be

compared to data on circulating cancer cells in order to validate the method for the in vivo assessment of tumor

burden in animals. By quantifying circulating tumor cells, in cancer disease models that include a circulating cell

component, the in vivo flow cytometer can be used to non- invasively track tumor burden and thus assess

important cancer treatment parameters such as the tumor growth and the response to therapeutic intervention.

4. References [1] G. P. Schmidt, H. Kramer, M. F. Reiser, and C. Glaser, “Whole-body magnetic resonance imaging and positron emission tomography-

computed tomography in oncology,” Top. Magn. Reson. Imaging, vol. 18, no.3, pp. 193-202, Jun. 2007.

[2] T. Beyer, et al, “A combined PET/CT scanner for clinical oncology,” J. Nucl. Med., vol. 41, no. 8, pp. 1369-1379, Aug. 2000.

[3] H. U. Kauczor, C. Zechmann, B. Stieltjes, and M. A. Weber, “Functional magnetic resonance imaging for defining the biological target

volume,” Cancer Imaging, vol. 6, no. 1, pp. 51-55, Jun. 2006.

[4] C. S. Mitsiades, et al, “Fluorescence imaging of multiple myeloma cells in a clinically relevant SCID/NOD in vivo model: biologic and

clinical implications,” Cancer Res., vol. 63, no. 20, pp. 6689-6696, Oct. 2003.

5] H. Yamamoto, et al, “Quantitative assessment of small intraosseous prostate cancer burden in SCID mice using fluorescence imaging,”

Prostate, vol. 67, no. 1, pp. 107-114, Jan. 2007.

[6] J. Condeelis and J. E. Segall, “Intravital imaging of cell movement in tumours,” Nat. Rev. Cancer, vol. 3, no. 12, pp. 921-930, Dec.

2003.

[7] C. M. Deroose, et al, “Multimodality imaging of tumor xenografts and metastases in mice with combined small-animal PET, small-

animal CT, and bioluminescence imaging,” J. Nucl. Med., vol. 48, no. 2, pp. 295-303, Feb. 2007.

[8] M. Cristofanilli, “Circulating tumor cells, disease progression, and survival in metastatic breast cancer,” Semin. Oncol., vol. 33, no. 3,

pp. S9-14, Jun. 2006.

[9] J. B. Smerage and D. F. Hayes, “The measurement and therapeutic implications of circulating tumour cells in breast cancer,” Br. J.

Cancer, vol. 94, no. 1, pp. 8-12, Jan. 2006.

[10] Y. P. Sher, et al, “Prognosis of non-small cell lung cancer patients by detecting circulating cancer cells in the peripheral blood with

multiple marker genes,” Clin. Cancer Res., vol. 11, no. 1, pp. 173-179, Jan. 2005.

[11] K. Pachmann, et al, “Monitoring the response of circulating epithelial tumor cells to adjuvant chemotherapy in breast cancer allows

detection of patients at risk of early relapse,” J. Clin. Oncol., vol. 26, no. 8, pp. 1208-1215, Mar. 2008.

[Back to Session 6]


Role of the Cell-Division Cycle on Nanoparticle Cellular

Accumulation and Implications for Cancer Targeting

Christoffer Åberg, Kenneth A. Dawson Centre for BioNano Interactions, School of Chemistry and Chemical Biology, University College Dublin, Belfield, Dublin 4, Ireland

[email protected]; [email protected]

The role of nanoparticle size and surface properties on nanoparticle uptake by cells has been extensively studied,

both to investigate potential hazards of nanoparticles, but also to develop new and improved medicines. Of

particular importance in such studies is the so-called 'biomolecular corona' which denotes the biomolecules that

associate (often strongly) with nanoparticles in realistic biological milieux [1]. For example, in a realistic

biological milieu, such as in the presence of serum, nanoparticle cellular uptake is vastly reduced compared to in

the absence of serum [2]. Furthermore, the impacts are also altered significantly, with cytotoxic responses

essentially completely mitigated in the more realistic milieu [3].

Going beyond physicochemical properties of the nanoparticles and their dispersion, less attention has been

given to biological variables, for instance the evolution of the cell population. In vitro cell-nanoparticle studies

are typically performed on cell lines, where the cells continuously divide. We have shown experimentally how

this evolution of the cell population leads to a characteristic ranking during continuous exposure, where cells in

the G2/M phase have a higher intracellular nanoparticle load, followed by cells in the S phase, and finally cells

in G0/G1 phase with the lowest load [4]. Several other phenomena can also be traced back to an evolving cell

population.

The evolution of the cell population can be described by a simple model and also simulated numerically

[4,5]. Experimental data provides all parameters of the model and subsequent parameter-free studies can be used

to validate the model, to excellent agreement. Upon this basis, we extend the model to include nanoparticle

uptake and accumulation. The whole range of experimental observables can be calculated, and compared with

experimental data. We demonstrate notable agreement with several careful experiments.

The model can also be used when the nanoparticles cause functional impacts on the cells. We demonstrate

the approach by elucidating limits in targeting nanoparticles towards cancer cells [5]. The model places rather

stringent demands on the targeting efficiency required to reach therapeutic nanoparticle loads in dividing

(cancerous) cells compared to slow-dividing (healthy) cells, and can be used as a framework for optimizations.

References [1] M. P. Monopoli, C. Åberg, A. Salvati and K. A. Dawson, “Biomolecular coronas provide the biological identity of nanosized materials,”

Nature Nanotechnology 7, 779-786 (2012).

[2] A. Lesniak, A. Salvati, M. Santos-Martinez, M. Radomski, K. A. Dawson and C. Åberg, “Nanoparticle adhesion to the cell membrane

and its effect on nanoparticle uptake efficiency,” Journal of the American Chemical Society 135, 1438-1444 (2013).

[3] A. Lesniak, F. Fenaroli, M. P. Monopoli, C. Åberg, K. A. Dawson and A. Salvati, “Effects of the presence or absence of a protein corona

on silica nanoparticle uptake and impact on cells,” ACS Nano 6, 5845–5857 (2012).

[4] J. A. Kim, C. Åberg, A. Salvati and K. A. Dawson, “Role of cell cycle on the cellular uptake and dilution of nanoparticles in a cell

population,” Nature Nanotechnology 7, 62-68 (2012).

[5] C. Åberg, J. A. Kim, A. Salvati and K. A. Dawson, “Theoretical framework for nanoparticle uptake and accumulation kinetics in

dividing cell populations,” Europhysics Letters 101, 38007 (2013).

[Back to Session 6]


Challenges in Nanotheranostics: A Materials Perspective

Rena Bizios University of Texas at San Antonio, USA

[Back to Session 7]


Magnetoactive electrospun nanocomposite membranes in

drug delivery and hyperthermia applications

Ioanna Savva1, Andreani D. Odysseos

2, Loucas Evaggelou

1, Oana Marinica

3, Eugeniu Vasile

4, Ladislau

Vekas5, Yiannis Sarigiannis

6 and Theodora Krasia-Christoforou

1

1University of Cyprus, Department of Mechanical and Manufacturing Engineering, Nicosia, Cyprus

2 EPOS-Iasis, R&D, Department of Biomedical Research, Nicosia, Cyprus

3National Center for Engineering of Systems with Complex Fluids, University ‘‘Politehnica’’ Timisoara, Timisoara, Romania

4 METAV Research & Development, Bucharest, Romania

5Center for Fundamental and Advanced Technical Research, Romanian Academy, Timisoara Branch, Timisoara, Romania

6 Department of Materials Science, University of Patras, 26504 Patras, Greece.

Magnetoactive, polymer-based fibrous nanocomposites belonging to the broad category of stimuli-responsive

materials consist of magnetic nanoparticles (MNP) embedded within a polymeric fibrous matrix. The presence of

MNP within these materials allows for the manipulation of their properties by an externally applied magnetic

field, rendering them useful in numerous technological and biomedical applications including sensing, magnetic

separation, catalysis and magnetic drug delivery.

Electrospinning is a simple, versatile and low cost technique employed for the production of continuous

(nano)fibers of various materials with diameters from a few nm up to a few micrometers [1]. The fiber

morphology and dimensional characteristics depend on the polymer and solution properties as well as on

processing parameters. The large surface-to-volume ratio, the existing flexibility on the selection of the surface

functionality and the freedom on materials’ design, comprise representative characteristics that render the

electrospun polymeric (nano)fibers and the resulting membranes ideal for numerous applications.

The present work focuses on the fabrication of electrospun magnetoactive fibrous nanocomposite membranes

based on the water soluble and biocompatible poly(ethylene oxide) (PEO), the biocompatible and biodegradable

poly(L-lactide) (PLLA) and pre-formed oleic acid coated magnetite nanoparticles (OA.Fe3O4). Scanning and

transmission electron microscopy techniques, reveal the presence of continuous fibers of approximately 2 μm in

diameter, with the magnetic nanoparticles being evenly distributed within the fibers, retaining at the same time

their nanosized diameters (~5 nm). Assessment of the magnetic properties of these materials by vibrating sample

magnetometry discloses superparamagnetic behaviour at ambient temperature. For the first time the

biocompatibility and biodegradability of PEO/PLLA and the tunable magnetic activity of the OA.Fe3O4 are

combined in the same drug delivery system, with N-acetyl-p-aminophenol (acetaminophen) as a proof-of-

concept pharmaceutical (Figure 1). Drug release kinetic profiles showed that the presence of OA.Fe3O4 as well

as the protein content of the release medium as critical parameters for the release rate. Furthermore, their heating

ability under alternating current (AC) magnetic field conditions is evaluated using frequency of 110 kHz and

corresponding magnetic field strength of 25mT (19.9 kA/m), which is very close to the typical values of 100 kHz

and 20mT used in medical treatments.

Fig. 1. Schematic presentation of the drug release process and photograph of the drug-loaded

PEO/PLLA/OA.Fe3O4/acetaminophen membrane immersed in an aqueous solution.

References [1] Z. M. Huang, Y. Z. Zhang, M. Kotaki et al. Comp. Sci. Technol. 2003, 63, 2223.

[Back to Session 7]


Electrospun PEO/PLLA Fibrous Meshes for Sustained

Tyrosine Kinase Inhibitors Delivery in Situ

Maria Kokonou1,2

, Fotios Mpekris3, Triantafyllos Stylianopoulos


2 and Andreani

Odysseos1

1EPOS-Iasis Research and Development Ltd., 2028 Nicosia, Cyprus

2Physicochimie des Electrolytes, Colloïdes et Sciences Analytiques (PESCA), Université Pierre et Marie Curie Univ Paris 06, 75252 Paris

Cedex 05, France

3Department of Mechanical and Manufacturing Engineering, University of Cyprus, 1678 Nicosia, Cyprus

[email protected], [email protected]

1. Background

Tyrosine Kinase Inhibitors (TKI) comprise a promising targeted molecular therapeutic solution for tumors

associated with aberrant expression of mutated forms of Tyrosine Kinase Receptors (TKR). Gefinitib (Iressa) is

an epidermal growth factor receptor (EGFR) inhibitor, in particular the first selective inhibitor of EGFR tyrosine

kinase domain. Electrospun fibrous, biocompatible and biodegradable membranes [1] are a very promising

nanosystem for localized drug delivery in situ, due to their very high surface to volume ratio, their wide range of

materials, easy fabrication and tailored physical, chemical and mechanical properties.

In this work, electrospun polyethylene oxide (PEO)/poly(L-lactid) acid (PLLA) nanofibers are introduced as

nanosystems for sustained Gefinitib delivery in solid tumors in situ. Gefinitib is blended to the polymeric

solution prior to electrospinning procedure, in two different ways: pristine or grafted on functionalized

fluorescent magnetic nanoparticles (NPs) intended for targeted drug delivery [2,3].

2. Experimental

PEO/PLLA polymeric solutions were formed with variable ratio (100/0, 90/10, 70/30, 50/50, 30/70, 10/90), in

order to control their biodegradation rate, which is the main parameter that determines the drug release duration

and rate. A wide parametric study of the electrospinning process was realized to find the optimum parameters

that result in solid, fibrous meshes with cylindrical fibers isotropically oriented and uniform in diameter. These

characteristics are essential in achieving a homogeneous and controlled drug release. Then the polymeric

solutions were loaded with Gefinitib in one group of experiments, and with maghemite NPs in a second group of

experiments, and fibrous meshes were fabricated again under the optimum electrospinning parameters. These

meshes were characterized in terms of geometrical characteristics, mechanical properties and drug release rate by

scanning electron microscopy (SEM), dynamic mechanical analysis (DMA) and UV-visible spectroscopy

respectively. Mechanical characterization of electrospun membranes intended to be used as implants for

localized drug delivery is important, since elasticity of the membranes determine their applicability and

functionality.

3. Results

For low concentrations of PLLA there is great uniformity on the geometry of the fibers, which are cylindrical,

with fiber diameter at 3.7 ± 0.35 μm. Above 50% beads start to form (Fig. 1). The introduction of Gefinitib

reduced diameters to 2.12 ± 0.35 μm, improved the homogeneity of the meshes and prevented the formation of

beads at high PLLA concentrations, while introduction of the magnetic nanoparticles did not affect significantly

the geometry of the fibers.

Fig. 1 PEO/PLLA (90/10, 50/50, 10/90) electrospun fibers


The elastic modulus of the meshes was found to be 115.8±38.7 kPa (100/0), 98.3±23.2 kPa (90/10), 131.7

±11.6 kPa (70/30), 171.2 ± 54.2 kPa (50/50), 42.8 kPa (10/90), i.e. it was in the range of 42.8 to 171 kPa with the

70/30 and 50/50 meshes being the stiffest with statistically significant difference (p<0.05) compared to the 90/10

meshes but not the 100/0. This range is comparable with electrospun polyurethane meshes and an order of

magnitude lower than cellulose acetate meshes [4]. while drug release duration increased with increasing PLLA

concentration as expected, since PLLA does not hydrolyze, from 2 hours (pure PLLA) up to > 6 months (PLLA>

50%).

4. Conclusions

TKI-loaded PEO/PLLA fibrous membranes were successfully fabricated by electrospinning, with tailored drug

release durations from a few hours up to several months. Introduction of the drug took place with blending of the

drug in the polymer solution or by grafting the drug on functionalized nanoparticles and then blending in the

polymer solution, enhancing this way functionality of the membranes. None of the two ways of drug

introduction affected the structure of the membranes. Especially in the case of pristine drug blending,

improvement of the homogeneity of the fibers was observed. These membranes are important for localized drug

delivery or functionalized nanoparticles delivery on solid tumors in situ, where targeting of the nanoparticles is

not possible, allowing this way the local administration of multifunctional, both therapeutic and tracking

schemes.

3. References [1] A. J. Meinel, O. Germershaus, T. Luhmann, H. P. Merkle, L. Meinel, “Electrospun matrices for localized drug delivery: Current

technologies and selected biomedical applications”, Eur. J. Pharm. Biopharm. 81(1), 1-13 (2012).

[2] T. Georgelin, S. Bombard, J.-M. Siaugue and V. Cabuil, “Nanoparticle-Mediated Delivery of Bleomycin,” Angew. Chem. Int. 49,

8897¬8901 (2010).

[3] A. B. Davila-Ibanez, V. Salgueirino, V. Martinez-Zorzano, R. Mariño-Fernández, A. García-Lorenzo, M. Maceira-Campos, M. Muñoz-

Ubeda, E. Junquera, E. Aicart, J. Rivas, F. J. Rodriguez_Berrocal and J. L. Legido, “Magnetic Silica Nanoparticle Cellular Uptake and

Cytotoxicity Regulated by Electrostatic Polyelectrolytes – DNA Loading at Their Surface”, ACS Nano 6(1), 747-759 (2012).

[4] T. Stylianopoulos, M. Kokonou, S. Michael, A. Tryfonos, C. Rebholz, A. D. Odysseos and C. C. Doumanidis, “Tensile Mechanical

Properties and Hydraulic Permeabilities of Electrospun Cellulose Acetate Fiber Meshes”, J. Biomed. Mat. Res. PART B 100B(8),

2222¬2230 (2012).

4. Acknowledgements


Number: 286125

[Back to Session 7]


Drug delivery through reconstructed bronchial mucus

modified by functional carrier particles (FCPs)

Marcin Odziomek, Tomasz R. Sosnowski, Leon Gradoń Faculty of Chemical and Process Engineering, Warsaw University of Technology, 1 Waryńskiego Street, 00-645 Warsaw, Poland

[email protected], [email protected], [email protected]

1. Introduction

Mucus covering surface of the tracheobronchial tree is the part of natural biological barrier that protects

organism against inhaled foreign particles. Strange matter deposited on the mucus surface is removed towards

upper respiratory tract by flow induced by ciliated epithelial cells. As the result of this highly efficient

mechanism drug particles are removed before they start to interact with receptors localized in the membranes of

the epithelial cells. The importance of this problem is especially pronounced in diseased conditions when the

mucus thickness and its viscoelasticity is significantly increased [1]. The effectiveness of treatment can be

improved by altering mucus structure by mucoactive substances, e.g. mucolytics [2].

The aim of the research was analysis of the rheology and mass transfer through the mucus modified by, so

called, functional carrier particles (FCPs) and their individual components. FCPs are microparticles prepared by

spray drying technique, and they are expected potentially useful in aerosol therapy as carriers of inhalation drugs

[3]. They are composed of a mucolytic (N-acetylcysteine, NAC) capable of reducing disulphur bonds between

mucin molecules, and a low molecular-weight dextran which acts as an osmotic agent and the stabilizer of FCPs.

2. Methods

The bronchial mucus was reconstructed according to the modified procedure of McGill and Smyth [4]. Raw

mucin – main component of natural mucus (Sigma Aldrich, Germany) was dissolved in the phosphate buffer (7.4

pH) to the final concentration of 20 % w/w. A small amount (0,05% w/w) of sodium azide was added to protect

the samples from microbial contamination.

Rhodamine B (POCH, Poland) was selected as a model substance for mass transfer studies. Its molar mass

(479 g/mol) is similar to the molar mass of many drugs used in the treatment of respiratory tract diseases (e.g.

disodium cromoglycate: 468.4 g/mol). Moreover, Rhodamine B is a fluoresecent dye which significantly

simplifies selectivity and sensitivity of quantitative determination of transferred material by spectrofluorimetry.

The effective diffusion coefficient De of Rhodamine B in the mucus was estimated using made in-house

plexiglass diffusion chambers (Fig. 1).

Fig. 1.Schame of diffusion chamber.

A layer of mucus sample (thickness – 2 µm) was placed between two drug-permeable membranes (PVDF,

Millipore, USA) with a pore size 0.1 µm. The donor compartment of the diffusion chamber was filled with a

Rhodamine B solution (0,05 mg/ml), whereas the receiver part with the pure phosphate buffer (pH 7,4). In order

to reduce of the local mass transfer resistance at the membranes, both solutions were continuously agitated.

Samples for analysis were taken from the receiver compartment at regular time intervals during 6 hours. The

content of Rhodamine B in these samples was determined quantitatively using fluorescence spectrometer

(Lumina - Thermo Scientific, USA). The effective diffusion coefficient De was calculated using the solution of

Fick’s law, with the justified assumptions [5].

The viscosity of samples at physiological value of share rate was measured using rotational rheometer (Smart

- Fungilab, Spain).


3. Results and discussion

Viscosity and diffusivity results (Fig. 2) indicate that mucus structure is destructed by the mucolytic (reduction

of mucus viscosity) which has an influence on the mass transfer rate through the mucus even for a relatively

small molecules like Rhodamine B (molecular mass, Mr = 479). The effective diffusion coefficient of

Rhodamine B calculated for unmodified mucus (20-25•10-10 m2/s) is approximately 5 times lower than in a

pure buffer solvent (119•10-10 m2/s). De value increases to 42•10-10 m2/s for mucus modified by FCPs (1%

w/w content), which is slightly less than after modification of mucus structure by pure NAC (2% w/w content:

De = 55•10-10 m2/s). Presence of dextran (needed as FCPs stabilizer), caused only slight increase of mucus

viscosity and, in consequence, a small decrease of mass transfer rate.

Fig. 2. A – dynamic viscosity of mucus modified by FCPs and their components, B – effective diffusion coefficient of

rhodamine B in the model mucus

4. Conclusions

Our results indicate that FCPs may be an useful concept for inhalation drug delivery to diseased lungs.

Simultaneous reduction of viscosity and the increment of mass transfer across the mucus layer may help to

achieve a therapeutic effect quicker. A more comprehensive description of mechanisms of the investigated

phenomena should be obtained by incorporating the results of theoretical predictions from molecular dynamics

simulations, which are currently underway.

5. Acknowledgments

This work was supported by a grant from National Science Centre based on decision DEC-

2011/03/N/ST8/04912.

6. References [1] R. A. Cone, “Barrier properties of mucus”, Adv. Drug Deliv. Rev. 61, 75-85 (2009)

[2] M. Stern, N. J. Caplen, J. E. Browning, U. Griesenbach, F. Sorgi, L. Huang, D. C. Gruenert, C. Marriot, R. G. Crystal, M. G. Geddes,E.

W. Alton, “The effect of mucolytic agents on gene transfer across a CF sputum barrier in vitro. Gene Therapy. 5, 91-98 (1998)

[3] M. Odziomek, T. R. Sosnowski, L. Gradoń, “Conception, preparation and properties of functional carrier particles for pulmonary drug

delivery”, Int. J. Pharm. 433, 51-59 (2012)

[4] S. L. McGill, H. D. Smyth, “Disruption of the Mucus Barrier by Topically Applied Exogenous Particles”, Mol. Pharm. 7(6), 2280-2288

(2010)

[5] M. A. Desai, P. Vadgama, “Estimation of Effective Diffusion Coefficients of Model Solutes Through Gastric Mucus: Assessment of a

Diffusion Chamber Technique Based on Spectrophotometric Analysis. Analyst. 116, 1113-1116 (1991).

[Back to Session 7]


Agglomeration of Theophylline Nanoparticles: a New

Protocol for Pulmonary Drugs Administration

Salem HF1, Abdelrahim ME

1, Abo Eid K

2, Sharaf MA

3

1Faculty of pharmacy, University of Beni Suef, Beni Suef, Egypt

2Faculty of Science, Helwan University, Cairo, Egypt

3Department of Chemistry, the American University in Cairo, New Cairo, Egypt

[email protected]

The aim of this study is to formulate theophylline as a dry powder aerosol for treatment of pulmonary

obstruction. Dry powder aerosols provide improved stability, ease of administration, and higher bioavailability

compared to traditional dosage forms. Particles in a size range of 1 to 5µm are facilitating the deposition of the

drug into lung; however they retard its dissolution in lung due to limited dissolution medium there. Using

nanoparticles, which are agglomerated by addition of electrolytes, may act as an excellent way to deliver

theophylline. Therefore, theophylline was precipitated with stearic acid to form a nanosuspension of theophyllin

stabilized with stearic acid. This was followed by a quantitative addition of electrolyte to destroy the electrostatic

repulsion between the particles. Achieving agglomerated nanoparticles of a controlled size was the final results

of this process. Characterization of both nanoparticles and the agglomerates was carried out using SEM, PCS

and zetasizer for studying the morphology, the particles size and the surface charge of the particles consequently.

SEM revealed formation of well defined self assembled structures of hollow agglomerates. It also revealed

formation of nanoparticles in size range of 190 to 230nm with relatively low polydispersity index. The zeta

potential was measured and found to be 34mV. However, for agglomerates were in the size of 2 to 5 µm with

zetapotential much lower than that of the nanoparticles. The nanoparticle agglomerates revealed enhanced

dissolution in relative original drug species suggesting the efficiency of such formulation approach for enhancing

dissolution of poorly water-soluble pulmonary medicines. The aerodynamic characterization of agglomerated

nanoparticles was determined using Anderson cascade impactor. All of this indicated the efficiency of using

controlled agglomeration of the nanoparticles as a way for treatment pulmonary obstruction diseases.

[Back to Session 7]


Author Index Alexandros Strongilos, 10, 12 Ana B. Davila Ibañez, 10 Andreani Odysseos, 9, 10, 12, 13, 14 Andreas Anayiotos, 13 Angela Riedel, 9 Anjali Seth, 11 Anne Vessières-Jaouen, 10 Ayache Bouakaz, 12 Bernard Chatelain, 10, 11 Bernard Masereel, 11 Birgitte Brinkmann Olsen, 9 Bowen Tian, 13 Christine Ménager, 10, 11 Christoffer Åberg, 13 Christophoros Mannaris, 12 Costas Pitris, 10, 13 Costas Pitsillides, 13 Cyrill Bussy, 11 Damien Lenoble, 12 David Lafargue, 11 Didier Arl, 12 Eftychia Angelou, 12 Eleni Efthimiadou, 9 Elias Couladouros, 10 Eugeniu Vasile, 14 Eva-Athena Economides, 11 Evdokia Kastanos, 10 Ewelina Tomecka, 11 Federica Scaletti, 9 Fotini Liepouri, 10, 12 Fotios Mpekris, 14 François Mullier, 10, 11 Gaëlle Corne, 12 George Kordas, 13 Gérard Jaouen, 10 Heba Salem, 14 Helge Thisgaard, 9 Helle Christiansen, 9 Iga Wasiak, 10, 12 Ines Block, 9 Ioanna Savva, 14 Jan Mollenhauer, 9 Jean-Michel Dogné, 10, 11 Jean-Michel Siaugue, 10, 12, 14 Jean-Michele Escoffre, 12 Jean-Sébastien Thomann, 12 Jeremy Malinge, 10

Jesper Wengel, 9 Julie Laloy, 10, 11 Kamal Abo Eid, 14 Katerina Hadjigeorgiou, 10 Kenneth A. Dawson, 13 Kenneth Dawson, 9 Konstantinos Kapnisis, 13 Konstantinos Soteriou, 11 Kostas Kostarelos, 11, 13 Krzysztof Gawlik, 10 Ladislau Vekas, 14 Leon Gradoń, 14 Loucas Evaggelou, 14 Lutfiye Alpan, 10, 11 Magdalena Janczewska, 12 Marcin Odziomek, 14 Maria Kokonou, 14 Maria Pavlaki, 12 Marie-Edithe Meyre, 12 Matthieu Sollogoub, 10 Michalakis Averkiou, 12, 14 Mohamed Abdelrahim, 14 Mohamed Elblbesy, 10 Mohamed Sharaf, 14 Morgane Rivoal, 9 Myria Angelidou, 10 Naoufal Bahlawane, 12 Oana Marinica, 14 Olivier Toussaint, 11 Omar Lozano, 11 Pavlos Agianian, 9, 10, 12 Poul Flemming Hoilund-Carlsen, 9 Rena Bizios, 6, 7 Stefan Vogel, 9 Steffen Schmidt, 9 Stephane Lucas, 11 Theodora Krasia-Christoforou, 14 Tomasz Ciach, 10, 12 Tomasz R. Sosnowski, 11 Tomasz Sosnowski, 14 Triantafyllos Stylianopoulos, 11, 14 Valentine Minet, 10, 11 Vassiliki Garefalaki, 10 Verónica Salgueiriño, 10 Wafa Al-Jamal, 13 Yiannis Sarigiannis, 14 Yongmin Zhang, 10

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