mri nanobiosensors part b · “mri_nanobiosensors” the marie curie actions international...

The Marie Curie Actions International Outgoing Fellowships FP7-PEOPLE-IOF-2008

STARTPAGE

PEOPLE MARIE CURIE ACTIONS

International Outgoing Fellowships (IOF)

Call: FP7-PEOPLE-IOF-2008

PART B

“MRI_Nanobiosensors”


Table of Contents

B1 SCIENTIFIC AND TECHNOLOGICAL QUALITY,

Scientific and technological quality, including any interdisciplinary and multidisciplinary aspects of the proposal Research methodology Originality and Innovative nature of the project, and relationship to the 'state of the art' of research in the field Timeliness and relevance of the project Host scientific expertise in the field Quality of the group/supervisors

B2 TRAINING

Clarity and quality of the research training objectives for the researcher Relevance and quality of additional scientific training as well as of complementary skills offered Host expertise in training experienced researchers in the field and capacity to provide mentoring/tutoring

B3 RESEARCHER

Research experience Research results including patents, publications, teaching etc., taking into account the level of experience Independent thinking and leadership qualities Match between the fellow's profile and project Potential to acquire new knowledge

B4 IMPLEMENTATION

Quality of infrastructure/facilities and international collaborations of host Practical arrangements for the implementation and management of the project Feasibility and credibility of the project, including work plan Practical and administrative arrangements and support for the hosting of the fellow

B5 IMPACT

Potential of acquiring competencies Contribution to career development or re-establishment Contribution to European excellence and European competitiveness

B6 ETHICAL ISSUES


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Dispersed State Clustered State

Large # of small NP’s Small # of large NP’s

analyte

T2

Figure 1. Magnetic Nanoparticles as Magnetic Relaxation Switch Based Biosensors. An analyte induces nanoparticles to switch between clustered and dispersed states and water's T2 (spin-spin relaxation time) changes [1-3].

B1 SCIENTIFIC AND TECHNOLOGICAL QUALITY

B 1.1 Scientific and technological Quality, including any interdisciplinary and

multidisciplinary aspects of the proposal

Executive Summary: The goal of this proposal is the development of a totally new type of reagent/instrumentation system based on a biosensor that Prof. Josephson and Prof. Weissleder have developed and termed “magnetic relaxation switches” (MRS). We propose to apply MRS for two different objectives: 1) detection of microorganisms through their specific sequences on their rRNA (outgoing phase); 2) identification of specific tumoral cells lines using MRS functionalized with specific lectins (return phase).

The principle used by MRS is depicted in Figure 1. MRS consists of magnetic nanoparticles (NP’s) to which biomolecules have been conjugated. Depending on the presence of target analytes the NP's switch between a dispersed and clustered state (Figure 1) [1-3]. The change in dispersion state decreases T2 because the clustered magnetic nanoparticles are more potent relaxation enhancing agents. T2 is the spin-spin relaxation time of water protons, a basic parameter determined in MR imaging. Each NP consists of a central core of magnetic iron oxide and coating of crosslinked dextran, represented as in Figure 1, to which multiple biomolecules, e.g., three oligonucleotides, are covalently attached (||||||||). MRS based assays have a series of unique properties that separate them from all biosensor systems. The assay is unique in employing radiofrequency-based radiation to sense the magnetic relaxation of water protons, rather than radiation that interacts with a labeled biomolecule. This allows the assay to be homogeneous (lacking a solid phase or separation step) and indifferent to interferences. MRS based assays can measure target oligonucleotides by hybridization reactions (Figures 2-4) or target proteins by antibody-antigen reactions. This offers the potential to design arrays where the presence of specific classes of bacteria are determined by immunochemical reactions and, using the same assay format and array design, the presence of genes causing pathogenicity are detected by hybridization reactions. The assay can be performed in a single tube T2 reader (MR spectrometer), see Figure 4 and [1], or in a high throughput fashion using MR imaging, see Figures 2, 3 and [3]. Additional advantages of the MRS assay method are its demonstrated sensitivity and specificity [3], and its use of a unique and well-proven magnetic nanoparticle [5-9]. During the outgoing phase we shall demonstrate the feasibility (sensitivity and specificity) of using MRS NP’s to determine the presence of three distinctly different microorganisms, based on sequences contained in their rRNA. A series of key parameters in NP design will be optimized, to maximize MRS assay performance for the detection of rRNA. The current MRS system, which has not been optimized, can detect approximately 30 attomolecules of mRNA. In a second phase of this research, we shall extend the MRS system to other, more pathogenic microorganisms and develop instrumentation specifically designed for the MRS system. During the return phase, we will analyse three types of tumoral cells lines using MRS functionalized with specific lectins, due to the unequivocal interactions between the lectins and the overexpressed carbohydrates on the surface of cancer cells. Because the design of MRS instrumentation will benefit from an environment where


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Non- complementary oligo,

Bottom row

T2 (msec)

Oligonucleotide (f moles)

0 0.5 1.0 1.3 1.7 2.7

f moles

70

50

30

0 0.5 1.0 1.5

Complementary oligo

Top row 3.5 mm

Figure 2. Reaction of MRS nanoparticles with a synthetic target oligonucleotide in a 384 well microtiter plate. T2 was determined using an MR imager as shown in Figure 2. Top row: increasing concentrations of an oligonucleotide joining nanoparticles, see Figure 1. Bottom row: non-complementary oligonucleotide.

the basic physics and principals of instrument design are widely known, this initial proposal focuses on the chemistry using currently available instrumentation. State of the art: Work with the MRS assay system has demonstrated many of the unique features of this biosensor system.

Sensitivity and Specificity: By designing nanoparticles that react with chemically different molecules, the MRS technology can accurately measure virtually any type of biomolecule with any type of bimolecular interaction. The determination of T2 (spin-spin relaxation time) can be accomplished with the assay run in single tube format using a tabletop MR spectrometer. Data from the assay run in this configuration is shown in Figure 4. This assay format serves as a model for the eventual development simple, portable, point of care instrumentation. The assay can also be run in a high throughput-screening format, by imaging microtiter plates. Here microtiter plates (384 well at 50 uL well or 1536 well at 10 uL/well) are stacked and the T2 values for hundreds to thousands of samples (wells) determined simultaneously from signal intensity data generated by the MR imager. Assay run in this format serve as model for the eventual development of extremely fast array based assays for the characterization and measurement of microorganisms, see Figures 2 and 3.

The reaction of oligonucleotide-nanoparticles (NP's) designed as shown in Figure 1 with

various concentrations of a target oligonucleotide is show in Figure 2. T2 was determined by imaging a 384 well microtiter plate, with 60 uL samples. Increasing concentrations of complementary (top row of wells) or non-complementary oligonucleotides were employed. Only complementary oligonucleotide changed T2. Assay sensitivity was approximately 0.2 fmole in this configuration with these reagents. Reduction of sample volume to 10 uL (1536 well plate), which has been accomplished, provides an assay sensitivity of 30 attamoles.

Prof. Josephson´s group designed a set (two twelve mer oligonucleotide) NP probes that hybridize to 24 bases of the mRNA from green fluorescent protein. Two new nanoparticle probes were then designed according to the principles shown in Figure 1, by attaching 12 mer synthetic oligonucleotides that would bind to a 24 base sequence of the mRNA of the green fluorescent protein (GFP). The NP's were then reacted with crude cell lysates from glioma cells transfected to express variable levels of GFP. As shown in Figure 3, selected wells gave changes in T2 indicative of the presence of mRNA for GFP. Well C3 contained a cell line expressing high levels of mRNA, well D4 expressed low levels of GFP and the remaining wells contained cells that expressed no GFP. The depression of T2 obtained correlated with the amount of GFP, determined as the GFP fluorescence.

A B C D E F

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T2 (msec)

3.5 mm

T2 (msec)70

6060

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Fluorescent Intensity

T2(msec)

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Figure 3. Detection of GFP mRNA in microtiter plate array fashion by MRI. T2’s of cell lines expressing GFP were obtained in a 384 well microtiter plate (60 uL/well, T2 weighted MRI pulse sequence). Cells in wells C3 and D4 showed high and low levels of GFP expression, respectively. Other wells contained cells lacking GFP mRNA.


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DTT

Oligo

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70

60

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Time (min)

0 100 200 300

DTT

Add TargetOligo

T2 (msec)

- oligo + oligo - oligo + oligo

100 200 300

Figure 4: MRS assay is indifferent to optical interferences. The addition of a target oligonucleotide causes dispersed nanoparticles to cluster and T2 decreases. Addition of DTT severs disulfide bonds used to conjugate oligonucleotide to nanoparticle and T2 returns to normal, see [1].

Homogeneous Assay Format and Indifference to Interferences: MRS assays involve mixing a single solution of NP's with a sample solution of target nucleic acid and recording the resulting T2. Despite the simplicity of the assay protocol, and presence of all potentially interfering substances when T2 is determined, the clustering of MRS nanoparticles is sensed by the radiofrequency radiation that penetrates biological samples. This occurs even with samples that are completely opaque to light. Figure 4 shows an assay for a target oligonucleotide is identical whether run in a clear buffer or an opaque lipidemic sample.

Universal Assay Format: The MRS assay provides a single assay format that can measure virtually any type of molecular interaction. This capability relies on the ability to attach virtually any type of biomolecule to the amino-CLIO nanoparticle with complete retention of biological activity such as oligonucleotides [1], peptides [9], proteins [20], and antibodies [21]. Moreover, consistent with our description of the nanoparticles as "relaxation switches" which implies controlled inter-conversions between the dispersed nanoparticle, low relaxivity state and the clustered nanoparticle high relaxivity state, well defined nanoparticle clusters have been synthesized. These clusters are held together with biopolymers, such as double stranded oligonucleotides or peptides, in such a manner that hydrolytic enzymes, such as nucleases or proteases, cleave them. For example, upon treatment with Bam H1 nuclease, which cleaves in a sequence specific fashion the tethering oligonucleotide, the nanoparticle is dispersed with an increase in T2. Thus the MRS system can assay for nucleic acids (Figures 2, 3, 4), enzyme activity, or antibody antigen interactions. Hence

the MRS system provides a universal assay format with a unique set of properties.

Use Of A Unique And Well Proven Nanoparticle: Though the ability to couple biological molecules to magnetic nanoparticles has been used for cell fractionation since the 1980’s [22, 23], the recent development of the amino-CLIO particle allows the full potential of superparamagnetic, crystal based MR contrast agents to be realized in magnetism based biosensor applications. Amino-CLIO has major advantages over earlier magnetic nanoparticles. First, the precursor of amino-CLIO is a monodisperse iron oxide nanoparticle that serves as a pharmaceutical. It is manufactured in large scale and well characterized [5-7]. Second, the dextran coating of amino-CLIO is remarkably stable. Amino-CLIO based materials can withstand heat stress, exposure to different ionic media, and alteration in their surface chemistry without suffering aggregation or a changes in size, see for example Figure 4 of [1]. Third, the presence of a primary amino group allows the use conjugation strategies that employ any one of a variety of commercially available, bifunctional crosslinking reagents (SPDP, SMCC, MBS, SIA from Pierce Chemical). Amino-CLIO based NP’s have been shown to be remarkably superior in direct comparison with other NP’s, see [20].

B 1.2. Research methodology

B 1.2.1 Outgoing phase: The critical tasks (specific aims) of the Outgoing Phase I research are:

1. Optimize Nanoparticle Design: While the current MRS system has been used to measure a variety of different analytes, it has not been used to measure rRNA from microorganisms. Hence the optimal design of the NP's for this application needs to be undertaken. Design factors evaluated will be (i) selection of either peptide nucleic acid (PNA) or oligonucleotide as polymeric backbones for the bases G, C, T and A, (ii) selection of the optimum


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polymer length (number of bases), (iii) selection of the conjugating reagent (linkage to nanoparticle). All nanoparticles will characterized by size, magnetic properties and the numbers of PNA or DNA attached per particle. We hypothesize that PNA will prove to be superior to DNA based oligonucleotides because it exhibits a higher affinity and greater specificity in base pairings and is preferred in biosensor applications [10-12]. However, given the substantial cost differential between PNA and DNA, the superiority and length of PNA needed must be established experimentally. For critical tasks below the use of PNA is assumed. We hypothesize that the long chain SPDP (lc-SPDP) will prove to be superior to other bifunctional conjugating reagents because the space arm will provide greater steric accessibility when oligonucleotide is attached to the NP.

NP’s will be evaluated using purified E. Coli rRNA as a target. MRI will be used to determine T2 on multiple samples simultaneously as shown in Figure 2 and [2-4]. Nanoparticles will be evaluated through the T2 changes they produce when exposed to various concentrations of E. Coli rRNA. Assay sensitivity will be defined in a standard PBS media, as the response (change in T2) versus the concentration of rRNA. The appropriate base sequences on RNA will be employed for probe design based on published studies where rRNA, species specific probes have been designed [13, 14]. The Tufts University Core Facility will synthesize RNA. Applied Biosystems will synthesize PNA. Nanoparticles will be synthesized at CMIR by published methods [1, 9]. We shall employ a series of bifunctional conjugation reagents (SMCC, lc-SPDP, SIA, MBS), available from Pierce Chemical. These will be employed to synthesize NP conjugates with differing linkages. The milestone will be selecting the NP design, and characterizing the resulting NP's, that produces the largest change in T2 at the lowest rRNA concentration.

2. Synthesize and characterize optimized oligonucleotide-NP’s targeted to the rRNA of

S. Cerevisiae and S. Aureus. To test the MRS for detection of microorganisms, we shall synthesize three pairs of NP's for the detection of the rRNA from three microorganisms (E.Coli, S. Cerevisiae, S. Aureus). These three organisms have been selected for initial studies for two reasons. First, their general lack of pathogenicity permits their handling in our current facility and permits investigation of whether the chemistry of the MRS system can be adapted to the detection of microorganisms at minimal cost. Second, the design of rRNA binding, organism specific PNA probes is well described [13, 14]. All NP's will be physically and chemically characterized as in critical task 1 above. Pair of NP's will be tested for their ability to detect rRNA from the appropriate species using the MRS system. The milestone for critical task 2 will be the synthesis and characterization of a series of NP's that detect the rRNA from all three organisms, E.Coli, S. Cerevisiae and S. Aureus.

3. Determine the optimal MRS assay of rRNA of E.Coli, S. Cerevisiae and S. Aureus. The benefit of the MRS assay system is its potential to act in a mix and read/homogeneous format. This requires the development of protocol where micro-organisms can be lysed, their rRNA denatured and released, and where the reaction between the rRNA and NP's will lead to the formation of NP clusters, see Figure 1. Moreover, a single media must be used to lyse our initial three microorganisms (E. Coli, S Cerevisiae and S. Aureus.) Proteases, enzymes for cell wall digestion (lysozymes), detergents, chelating agents and other materials will be used in combination to chemically disrupt microorganisms. We will use mechanical methods of lysis, such as bead beating, as a harsh reference method of cell lysis. We hypothesize that an assay media can be developed that will lyse microorganisms, denature the RNA, inhibit RNAases, and be compatible with the MRS assays system. The milestone will be the selection of media of a lysis/assay media capable measuring E. Coli, S. Cerevisiae and S. Aureus with the MRS method.

4. Assay performance and evaluation: Using optimized oligonucleotide-nanoparticles (see critical tasks 1 and 2 above), and the optimized lysis/assay media and protocol (critical task 3), we shall determine the sensitivity of detection for three microorganisms, E. Coli, S. Cerevisiae and S. Aureus. This will be accomplished by spiking microorganisms into samples (buffers, serum, urine) to assess assay sensitivity. Limits of detection for each microorganism will be determined in


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the presence of increasing numbers of the other two microorganisms, to determine assay specificity. Assay performance will be measured, not as the chemical detection of rRNA (see critical aims 1-3), but as the detection of viable microorganisms. The concentration of microorganisms will be determined by dilution plating. The milestone will be the determination of the number of viable organisms (E. Coli, S. Cerevisiae and S. Aureus) we can detect. Phase I will use existing instrumentation of all studies (MR spectrometer or a clinical MR imager). Phase II will consist of three general goals: 1) Expand our library of magnetic nanoparticle probes so that 50-500 organisms can be identified, including pathogens and/or organisms similar to pathogens. CMIR expertise in microbial diversity and phylogenetics will be employed in probe design; 2) Test specificity of detection of specific pathogens against high levels of background organisms such as would occur in real environment sampling; 3) Develop a prototype portable MR instrument, i.e. one using a permanent magnet, to measure the presence of rRNA in multiple samples. Phase III will consist of the full commercial development of a sample handling, reagent/instrumentation system.

B 1.2.2 Return Phase: In collaboration with Prof. Josephson and CMIR, and with the experienced gained by Dr. Alcantara in the outgoing phase, we propose the development of a new research line in Glycomics. Glycomics is the study of the structural and functional aspects of the various glycoconjugates present on proteins, cells and, in some cases, entire organisms. Compared with its counterparts, genomics [24] and proteomics [25] — which deal with nucleic acids and proteins, respectively — the field of glycomics is much less developed. Different natural products such as glycoproteins, glycolipids, glycosaminoglycans and glycosylphosphatidylinositol anchors are summarily known as sugars [26]. Overall progress in glycobiology has suffered from a lack of tools such as those that are readily available for studying nucleic acids and proteins, including automated sequencing [27, 28], automated synthesis [29, 30], high-throughput microarray screening, and detailed structure elucidation, including X-ray analyses. Given the structural diversity of glycoconjugates, different analytical approaches may persist for the analysis of different classes of sugar. The cell surfaces of bacteria, parasites and viruses exhibit oligosaccharides that are often distinct from those of their hosts. Specific types of glycoconjugate are often more highly expressed on the surface of tumours than on normal cells [31]. Such cell-surface carbohydrate markers are the basis for carbohydrate-based detection systems and vaccines. Several glycans, on both the tumour surface and host elements, have now been identified as mediating key pathophysiological events during the various steps of tumour progression. In the tumour environment, changes in glycosylation allow neoplastic cells to usurp many of the events that occur in development (for example, receptor activation, cell adhesion and cell motility), which allows tumour cells to invade and spread throughout the organism [26]. A massive potential for glycan diversity exists, but a relatively limited array of glycans correlates with invasion and metastatic potential across a wide range of tumours. Initial insight into the unique repertoire of glycans expressed on tumour cells emerged from the increased ability of tumours to bind a range of plant Lectins [27]. Lectins exhibit protein folds that define families of carbohydrate-binding proteins that can bind in a specific ‘lock-and-key’ fashion. Various endogenous animal lectins also exist, and these facilitate fundamental processes such as quality control of secreted proteins, cell–cell recognition, cell adhesion and motility, and pathogen–host recognition. Many lectins exist on the surface of immune cells and endothelial cells that line the vasculature, as ECM proteins and as soluble adhesion molecules such as mannose-binding proteins or galactose-binding lectins (galectins), and many of these can associate with tumour-cell-associated glycans. We therefore propose the use of magnetic nanoparticles functionalized with lectins to identify specific tumoral cells lines through their unique glycosilation patterns by means of MRI.

Regarding to these contexts, the first year of the return phase of this proposal will aim with three different objectives: 1) Establishment, adaptation and development the nanoparticle´s chemistry and MRI

methodology acquired in the outgoing phase (CMIR) on the host institution (INA). The same type of NPs prepared in CMIR will be prepared and adapted to incorporate different


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lectins. Bifunctional spacers will be developed to bioconjugate the lectin onto the NPs surface. Conventional proteins conjugation techniques will be applied (EDC, DCC, etc).

2) Determine the optimal MRS assay with NPs-lectins against tumoral cells lines. In this phase we pretend to extrapolate the MRS methodology to in vitro experimentation. The identification of living tumoral cells lines (starting with breast, lung and melanoma cells lines) will be carried out with a MRS assay. Microarrays of the above mentioned cells lines will be optimized and the different over expression of glycans for each cancer cells will be identified by the NPs-lectins and the MRS method. At this point we will need to improve efficiency of the NPs-lectins with the specific target carbohydrates. Protein orientation, density and flexibility in the NPs will also be optimized to improve the activity and sensitivity of the MRS.

3) Performance and extrapolation. Once the methodology was stated, novel microarrays with different tumoral cell lines will be prepared and tested. Full commercial development of these applications (with the establishment of a new spin off enterprise) will be carried out together with INA and CMIR group.

References 1. Josephson, L., J.M. Perez, and R.W. Weissleder. Angew.Chem. Int. Ed.,2001, 40, 3204-3206. 2. Perez, J.M., T. O’Loughin, J. Simeone, R. Weissleder, and L. Josephson. JACS, 2002 124:2856-7. 3. Perez, J.M., L. Josephson, D. Hogemann, and R. Weissleder. Nat Biotechnol.,2002, 20: 816-20. 4. Hogemann, D., V. Ntziachristos, L. Josephson, and R. Weissleder. Bioconjug Chem, 2002, 13, 116-121. 5. Jung, C.W. and P. Jacobs. Magn Reson Imaging,1995, 13, 661-74. 6. Jung, C.W. (1995). Magn Reson Imaging, 13, 675-91. 7. Shen, T., R. Weissleder, M. Papisov, A. Bogdanov, Jr., and T.J. Brady. Magn Reson Med,1993, 29, 599-604. 8. Josephson, L., J. Lewis, P. Jacobs, P.F. Hahn, and D.D. Stark. Magn Reson Imaging, 1988, 6, 647-53. 9. Josephson, L., C.H. Tung, A. Moore, and R. Weissleder. Bioconjug Chem,1999, 10, 186-91. 10. Thisted, M., T. Just, K.J. Pluzek, J.J. Hyldig-Nielsen, et al. Pept. Nucleic Acids,1999, 99-118. 11. Stender, H., M. Fiandaca, J.J. Hyldig-Nielsen, and J. Coull. J Microbiol Methods,2002, 48, 1-17. 12. Wang, J., E. Palecek, P.E. Nielsen, G. Rivas, X. Cai, H. Shiraishi, et al. J. Am. Chem. Soc.,1996 118, 7667-7670. 13. Perry-O'Keefe, H., S. Rigby, K. Oliveira, D. Sorensen, et al. J Microbiol Methods, 2001, 47, 281-92. 14. Perry-O'Keefe, H., H. Stender, A. Broomer, K. Oliveira, et al. J Appl Microbiol,2001, 90, 180-9. 15. Lee, G.U., S. Metzger, M. Natesan, C. Yanavich, and Y.F. Dufrene. Anal Biochem ,2000, 287, 261-71. 16. Baselt, D.R., G.U. Lee, M. Natesan, S.W. Metzger, et al. Biosens Bioelectron, 1998, 13, 731-9. 17. Chemla, Y.R., H.L. Grossman, Y. Poon, R. McDermott, et al. Proc Natl Acad Sci U S A,2000, 97, 14268-72. 18. Kotitz, R., W. Weitchies, L. Trahms, W. Brewer, and W. Semmler. J Magn Magn Mater, 1999, 194, 62-68. 19. Kriz, C.B., K. Radevick, and D. Kriz. Anal Chem, 1996, 68, 1966-1970. 20. Hogemann, D., L. Josephson, R. Weissleder, and J.P. Basilion. Bioconjug Chem, 2000, 11, 941-6. 21. Kang, H.W., L. Josephson, A. Petrovsky, R. Weissleder, and A. Bogdanov. Bioconjug Chem, 2000, 13, 122-7. 22. Abts, H., M. Emmerich, S. Miltenyi, A. Radbruch, and H. Tesch. J Immunol Methods, 1989, 125, 19-28. 23. Molday, R.S. and D. MacKenzie. J Immunol Methods,1982, 52, 353-67. 24. Insight: Human genomics and medicine. Nature 2004, 429, 439-481. 25. Insight: Proteomics. Nature, 2003, 422, 191-237. 26. Varki, A., Essentials of Glycobiology. 1999. 27. Hunkapiller, T.; Kaiser, R. J.; Koop, B. F.; Hood, L. Science, 1991, 354, 59-67. 28. Grant, G. A.; Crankshaw, M. W.; Gorka, J. Methods Enzymol. 1997, 289, 395-419. 29. Caruthers, M. H. Science 1985, 230, 281-285. 30. Merrifield, B. Methods Enzymol. 1997, 289, 3-13. 31. Hakomori, S. Adv. Exp. Med. Biol. 2001, 491, 369-402. 32. Raedler, A.; Schreiber, S. Crit. Rev. Clin. Lab. Sci. 1988, 26, 153-193.

B 1.3 Originality and Innovative nature of the project and relationship to the 'state of

the art' of research in the field

Our research objectives are the development of sensitive, robust, and versatile sensors for microorganisms in point-of-care or in-the-field type assays that can be used for the detection of micro-organisms in third world water supplies, in food products, or in medical applications such as the bedside identification of blood borne organisms causing sepsis. The advantages of magnetic relaxation switch (MRS) methodology for point-of-care assays include its use of radiofrequency


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radiation rather than light (indifference to light based interferences), and its use of solution phase chemistry (no solid phase, no separation of free and bound as in ELISA's). In addition, Dr. Alcantara's project will utilize ongoing efforts at CMIR to develop novel, portable MR relaxometers, which will employ small permanent magnets, micron sized coils, and low power requirements (battery operated). Current state of the art methods of identifying microorganisms are typically microscopy based or culture based and are not readily adaptable to such point of care uses. The attainment of the research objectives summarized on pages 3-5 will provide fundamentally new methods for the rapid detection and identification of microorganisms. The proposed research is highly interdisciplinary. Nanoparticle synthesis and surface chemistry with be employed to obtain nanoparticles recognizing sequences of rRNA that define the family of the microorganism. MR relaxometry, and the related physics, will be employed to measure nanoparticle aggregation state as the spin-spin relaxation time of water protons. Microbiology will be employed to obtain, characterize and quantitate the various microorganisms that will be detected. Finally, principles of magnetic and coil design will be used to develop a portable MR instrument capable of measuring solvent relaxation times.

B 1.4. Timeliness and relevance of the project

There is a wide recognition that the current laboratory based methods for the detection of microorganisms (largely culture and microscopy) needs to be replaced by rapid, user-friendly techniques based on the emerging fields of nanotechnology and microelectronics. However to date there are no consensus the specific biological molecules, such as base sequences of rRNA involved in this proposal, that should be detected to provide most definitive information on the presence of microorganisms. Second, there is no consensus on how the presence of the biomolecule (rRNA) to be detected can be interfaced with an electronic device. We propose this interface between the analyte (a microorganism) and microelectronic circuitry can occur through the use of an NMR based system, where magnetic nanoparticles react with a molecular target and effects on water relaxation will be measured. The proposed project will provide Dr. Alcantara with training in a variety of rapidly evolving fields including materials sciences (nanoparticles), surface chemistry, NMR and microbiology. These diverse fields will be central to evolution of the next generation of nanotechnology-based European chemists. On a long term the consequences and potential follow up projects in Spain and USA will put EU researchers in a more competitive position in comparison to others efforts in the field. On the community level this project will start the collaboration between the INA in Zaragoza (Spain) and the corresponding activities in northern USA (CMIR). Scientific contacts between individual researchers from this field of research have not already been established yet. This project will therefore establish successful ties and provides profitable “know-how” interchanges between the participants in a near future.

B 1.5 Host scientific expertise in the field

B 1.5.1 Outgoing phase: Dr. Alcantara will be mentored by Dr. Lee Josephson, a Chemistry Professor at Harvard Medical School, who has been a pioneer in the applications of magnetic materials to biology and medicine with over 30 patents and 80 publications. In conjunction with the head of the Center for Molecular Imaging Research, Dr. Ralph Weissleder, Dr. Josephson developed ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles for lymph node imaging, which have been commercialized in Europe by Guerbet. The nanoparticle has completed phase III trials (trade name Sinerem) and a dossier has been filed European Medicines Agency of the European Community. Dr. Josephson discovered that nanoparticle aggregation could be used to determine the concentrations of analytes, the physical principle that will be exploited to measure rRNA in the proposed research. In addition to MRS/MRI based assays, Dr. Josephson's interests include targeted magnetic nanoparticle MR contrast agents, nanoparticles for loading and tracking cells by MRI, implantable MRI based biosensors. Selected publications of Dr. Josephson in the nanoparticle field are provided in pages 18-19 of the present proposal.


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B 1.5.2 Return phase: The Instituto Universitario de Investigación en Nanociencia de Aragón (INA) was created in 2003. INA is one of the leading Centres on Nanoscience in Spain. INA is a multidisciplinary research institute where nanoscience is applied to a wide range of scientific disciplines. This multidisciplinary is shown in the different work subjects of the more than 70 scientists (see http://www.unizar.es/ina/ ) working on it, going from Chemistry, Physics, Biology, Engineering, Medicine and Pharmacology. The main research areas of INA are: 1) Nanostructured materials: Optimization of chemical processes (catalysis and microreactors), new materials, functional organic materials; 2) Physics of Nanosystems: coverage of thin films and micro- and nanocircuits; 3) Nanomedicine: Therapy (drug delivery) and diagnostic (contrast agents and nanobiosensors).

B 1.6 Quality of the group/supervisors

B 1.6.1 Outgoing phase: Dr. Alcantara will be provided space at the Center for Molecular Imaging Research (CMIR), which consists of about 75 scientists (see http://cmir.mgh.harvard.edu/) and approximately 20 principle investigators in the fields of chemistry, biology, physics, and medicine. CMIR is funded in part by two major "consortium" type grants that provide a wide array of collaborators in the fields of nanotechnology and material sciences. The first of these, the Translational Program of Excellence in Nanotechnology (TPEN), provides funds for principle investigators in cardiovascular related projects at the Massachusetts General Hospital, the Broad Institute, Massachusetts Institute of Technology (MIT), Harvard Faculty of Arts and Sciences (FAS), Harvard Medical School (HMS), and Brigham and Women's Hospital (BWH). The second of these, the Center of Cancer Nanotechnology Excellence (CCNE), provides funds for cancer related nanotechnology. Through these grants CMIR interacts with the materials sciences facilities and faculty at MIT including Dr. Robert Langer (MIT, Institute Professor) and Dr. Michael Cima (MIT, Professor, Department of Material Sciences and Engineering). This network of collaborators will provide Dr. Alcántara with a unique opportunity to interact, not only with Dr. Josephson and other scientists CMIR, but also with key leaders in the American academic community working in the fields of material sciences and nanotechnology. For more details, see pages 18-19

B 1.6.2 Return phase: The Instituto de Nanociencia de Aragon (INA) is a multidisciplinary research institute where nanoscience is applied to a wide range of scientific disciplines. The Group at the University of Zaragoza that coordinates this project is ranked by the local Government in the top 10 of Spanish groups in this field receiving for this reason the mention of “excellent group”. The members of this group are involved in different projects on new materials, including magnetic nanoparticles, polymers and dendrimers with applications in biomedicine. The group has, besides its high impact publication rate, several collaboration agreements with private companies.

Dr. Jesus M de la Fuente, Ph.D. Chemistry (2003) is a Senior Researcher in the Aragon Institute of Nanoscience (INA) since 2007, where he is leading the Biofunctional Nanoparticles Group. His research focuses on the development of new synthetic methodologies for the functionalization of gold nanoparticles, semiconductor nanocrystals and magnetic nanoparticles with biologically relevant oligosaccharides, peptides or proteins, for applications in Glycobiology, Biomedicine and Materials Science. He is author of more than 30 papers of SCI with more than 400 citations and he holds two international licenced patents. He is actually co-ordinator of the Nanobiotechnology and Nanomedicine Section of the Nanospain Network. For more details, see pages 20-21.

Dr. M.R. Ibarra, PhD in Physics (1983). Full professor at the University of Zaragoza since 1995 and Director of the Aragon Institute of Nanoscience (INA). He is leading the nanomagnetism group at the INA. His research focuses on magnetocaloric materials, magnetic oxides with CMR, thin film multilayers, nanostructured magnetic materials, encapsulated nanoparticles and nanostructured magnetic materials for biomedical applications. He is author of over 260 papers of SCI with more than 3000 citation and he holds ten patents. For more details, see pages 20-21.


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B2 TRAINING

B 2.1 Clarity and quality of the research training objectives for the researcher

Outgoing phase: Dr. Alcantara will be thoroughly trained in techniques of nanoparticle synthesis, surface chemistry, biomolecule conjugation chemistry, NMR proton relaxation (theory and practice) and microbiology. He will be trained in the characterization of nanoparticles including magnetic and surface charge measurements, and size determination (light scattering, light based and electron microscopy). All scientists at CMIR must give two seminars on their research per year and give two presentations on relevant academic literature per year. These seminars provide a young scientist with an opportunity to enhance his presentation skills and acquire information on work being conducted at CMIR and by other research groups around the world.

Return Phase: Training contribution to Dr. Alcántara from INA at the University of Zaragoza will be the synthesis, biofunctionalization and physical and magnetic characterization of different types of magnetic nanoparticles for their application in MRI.

Multidisciplinary training will include computational modelling of new materials, chemical synthesis and controlled chemical orientation, structural functional studies, physical characterization, magnetic characterization, biological modelling, molecular activity analysis, technology oriented samples and product escalation towards in line industry processes, MRI (working with radiologists), biosensing, supported by parallel research careers, combined and complementary formation, and cooperation key-events. All this together with the experience of the host groups and their own degree of initiative in organising the events will generate a scientist with unrivalled technical skills and the expertise to work in different environments and the ability to think outside his field.

B 2.2 Relevance and quality of additional scientific training as well as of complementary skills

offered

Outgoing phase: Dr. Alcántara will acquire a wide array of skills in diverse technical areas. He will be exposed to and participate in an extremely active intellectual environment at CMIR. He can use many research facilities in the Boston area, which include virtually any instrument that might be required in his research. He will interact with world-class scientists through a network of collaborations that is currently in place through consortium funding arrangements provided to CMIR. The skills he will acquire will complement and extend his core competencies in chemistry and glycobiology, adding training in material sciences, magnetic measurements, NMR, and nucleic acid (rRNA) chemistry. Within the seventh framework program of the EU the funding of projects from the Life Science and Chemistry areas with a significant impact for the improvement of health issues is a declared focus. Therefore this project enhances the efforts in this particular area.

Return phase: Dr. Alcántara is expected to participate in Ethics and Science/Research training, either at his own institution, or via network wide event. He will also attends (at least) two international scientific meeting per year. The host will make strong efforts to engage the wider public in sciences, for network wide activities via the Cordis site, but also through the website, local radio and web 2.0 types of activities.

Dr. Alcántara’s training will also include different topics: Team building/team work, negotiation skills, project management, career planning, proposal writing, design of research studies, ethical aspects of research with children and adolescents, how to write a paper and get it published, how to give a scientific presentation, effective communication (bringing science to non-scientists), technology transfer and IPR/Patents, enterprise start-up, communication strategies addressing childrens and adolescents. Some of these topics will be built into the host institutions; the others will be covered by regional and national short courses.


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Also at this stage, Dr. Alcántara will become involved with tutoring first year graduate students and Ph.D. students at the University of Zaragoza and INA who carry out research projects in the final year of their degree or start their doctorate program. As a researcher, it is vital to learn to supervise and direct students with an organised planned project and monitor their progress constructively. This will help Dr. Alcántara to improve his previously acquired oral and written communication skills to tutor and direct the students, providing training in project management skills. Dr. Alcántara will be encouraged to attend scientific meetings presenting his (or his team) research results enabling detailed discussion with other scientific researchers at a high level. Together with regular group meetings this will further develop his communication and presentation skills needed when preparing presentations to the foreign scientific public (at international meetings, conferences etc.).

B 2. 3 Host expertise in training experienced researchers in the field and capacity to provide

mentoring/tutoring

Outgoing phase: The vast major of the post-doctoral fellows trained by CMIR in the past or currently are not American citizens and return to their homeland after training. Among the European scientists trained by Dr. Josephson who have returned to positions in Europe are Dr. Eyk Schellenberger (current employment, Charite Hospital, Berlin), Dr. Annette Koch (Hoffman LaRoche, Basel, Switzerland), Dr. Patrick Wunderbaldinger (Radiologist, Vienna, Austria), Dr. Xavier Montet, (radiologist, Geneva University Hospital, Geneva, Switzerland) and Dr. Rachel Trehin (temporarily not working, raising daughter in Lausanne, Switzerland). The Massachusetts General Hospital, where CMIR is located, maintains an Office for International Scholars and Students to assist and advise scientists like Dr. Alcántara in immigration, visa, health insurance and related matters.

Return phase: The host group is involved in different Doctorate programs (Doctorate in Chemistry, Doctorate in Nanoscience and Nanotechnologies and Doctorate in Physics and Physics Technologies) plus 2 MsC degrees in Nanoscience. INA has more than 40 PhD students, 20 postdocs and 10 technicians working in different fields like Nanoscience, Physics, Magnetism, Nanofabrication, Electronics, Biomedicine and Organic Chemistry. The institute is part of the program project “MUNDIS” European Comision FP6/2004/IST/4 027827 (521.757€) and “CONSOLIDER-NANOBIOMED” MEC, CSD2006-00012 (4500000€) in collaboration with University of Vigo, University of Santiago, Autonomous University of Barcelona and several Spanish research Institutes (CSIC). Dr. Alcantara will interact within this community, executing new projects and reinforcing the EU-USA collaboration. In this context, and at the end of this fellowship, University of Zaragoza will accept Dr.Alcantara´s candidature to be part of INA scientific staff.


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B3 RESEARCHER

CURRICULUM VITAE David Alcántara Parra

•••• PERSONAL DATA

FIRST NAME: David

FAMILY NAME: Alcántara Parra

DATE OF BIRTH: 22nd of June 1977.

PLACE OF BIRTH: Malaga (Spain)

MARITAL STATUS: Common law marriage

PRESENT POSITION: Synthetic Chemist. Carbohydrate Group. Instituto de

Investigaciones Químicas, CSIC (Seville, Spain), since May

1st 2003.

ADDRESS: Centro de Investigaciones Científicas Isla de La Cartuja

Instituto de Investigaciones Químicas

Americo Vespucio, 49

41092 Seville (Spain)

Tel +34 954489568

Fax +34 954460565

[email protected]

•••• UNIVERSITY STUDIES AND DIPLOMAS

PhD in Chemistry (March 2008). Grupo de Carbohidratos, Instituto de Investigaciones Químicas (IIQ), University of Seville-CSIC. Supervisor: Prof. Soledad Penades. Mark: Summa Cum Laude

M.B.A. in the “Escuela de Organización Industrial” of Seville. “InnovaEmpresa” program. July 2007. Master in “Advanced Studies in Chemistry”, from the University of Seville. Seville. June 2005. Mark: “Sobresaliente” M.D. in quality management, UNED-FUE, 2003

Degree in Chemistry, University of Málaga. June 2001.

•••• DISSERTATIONS / THESES

Ph.D. degree: “Design and synthesis of Magnetic Glyconanoparticles: preparation and MRI applications”.

Dissertation for the Master in “Advanced Studies in Chemistry”: “Magnetic glyconanoparticles:

Synthesis, Characterization and MRI application”. Supervisor: Prof. Soledad Penades. University of Seville. Seville. June 2005


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•••• PROFESSIONAL /RESEARCH EXPERIENCE

08/2007-present Synthetic Chemist. “Synthesis of novel glycosidic antioxidants”. Grupo

de Carbohidratos, Instituto de Investigaciones Químicas (CSIC). (Dr.

J.C. Morales).

05/2007-08/2007 Fellowship of Fundación Renal Iñigo Alvarez de Toledo. Grupo de

Carbohidratos, Instituto de Investigaciones Químicas (CSIC).

(Prof. Soledad Penadés)

05/2003-05/2007 Ph.D. Fellowship (Ministerio Educación y Ciencia)

Grupo de Carbohidratos, Instituto de Investigaciones Químicas (CSIC).

(Prof. Soledad Penadés)

09/2004-11/2004 Training in Magnetic Resonance Imaging (MRI), Instituto de

Investigaciones Biomédicas (CSIC).

(Prof. Sebastián Cerdán)

05/2002-04/2003 Chemist. Iris-Spa S.L. Málaga (Spain)

06/2001-05/2002 Collaboration Fellowship from Malaga University

Dpto. Química Orgánica, University of Málaga

(Prof. F.Jorge López Herrera)

09/1999-11/1999 Undergraduate Fellowship from IAESTE,

Forchumgzemtrum Julich (Germany), Bioorganic Department

(Prof. M.Muller)

•••• RELEVANT SCIENTIFIC TECHNIQUES AND SKILLS

- Solution phase synthesis of phenolic derivatives with antioxidants properties. - Solution phase synthesis of oligosaccharides. - Synthesis of chelating agents as contrast agents for application in MRI. - Preparation of Gold Glyconanoparticles as water-soluble polyvalent models . - Preparation of magnetic and paramagnetic Glyconanoparticles as contrast agents. - Structural elucidation of complex oligosaccharides by spectroscopic methods such as NMR. - Experience working with Bruker NMR spectrometer, HLPC, ESI-MS, IR, UV, EDX, TEM,

HR-TEM, lyophilizer, etc. - Basic skills working with cellular and biological systems. - User of PharmaScan Magnetic Resonance Imaging Spectrometer, Paravision environment. - Working under Microsoft Windows environment: Office2000, Origin, ChemOffice2000,

Photoshop, etc. - Basic Linux skills (KDE, GNOME environment).

•••• PUBLICATIONS

- D. Alcántara, J. M. de la Fuente, M. Marradi, M. Garcia-Martin, S. Cerdan, and S. Penadés. “Paramagnetic Gold Glyconanoparticles as New Contrast Agents for Brain Tumour Imaging: the Sugars Control Relaxivities and in vivo Selectivity". Angew Chem Int

Ed, 2008, submitted - David Alcántara, Jesus M. de la Fuente. “Agentes de contraste positivos en imagen por

Resonancia Magnética para la diagnosis del cáncer”, revista eSalud, 2008, submitted.


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- Martina Fuss, David Alcántara, Jesús M. de la Fuente, Pedro M. Enríquez-Navas, Jesús Angulo, Soledad Penadés, Mónica Luna and Fernando Briones. “3D multilayer self-assembling of maltose neoglycoconjugate disulfides on hydrophobic graphite”, J.Chem.Phys.B, 2008, in press.

- Fuss, Martina; Alcántara, David; De La Fuente, Jesús; Luna, Mónica; Briones, Fernando; Penadés, Soledad. “Supramolecular self-assembled arrangements of maltose glyconanoparticles”, Langmuir, 2008, 24, 5124-5128.

- Soledad Penadés, J. M. de la Fuente, A. G. Barrientos, Caroline Clavel, O. Martínez-ávila and D. Alcántara. “Multifunctional Glyconanoparticles for application in biology and biomedicine” in Nanomaterials for Application in Medicine and Biology, 2008, pp 93-101.

- E. Grueso, D. Alcantara, J. Martinez, M. Mancera, S. Penades, F. Sanchez and R. Pradogotor “A kinetic approach for the study of non-covalent interaction between [Ru(NH3)5pz]2+ and gold nanoparticles” J.Chem.Phys.B, 2007, 111, 9769-9774.

- J.M. de la Fuente, D.Alcantara, S.Penadés “Cell response against magnetic glyconanoparticles: Does the carbohydrate matter?”, IEEE Trans.NanobioScience, 2007, 6, 275-281.

- de la Fuente, J.M, Alcántara D, Eaton, P, Crespo, P, Rojas TC, Fernandez A ,Hernando A, Penades S. “Gold and Gold-iron Magnetic Glyconanoparticles: Synthesis, characterization and magnetic properties” J.Chem.Phys.B, 2006, 110, 13021-13028

- P.Crespo, M.A.Garcia, E.Fernandez, M.Multigner, D.Alcántara, J.M. de la Fuente, S. Penadés, A.Hernando. “Fe impurities weaken the ferromagnetic behaviour in Au Nanoparticles”, Phys.Rev.Lett., 2006, 97, 17, 177203.

- de la Fuente, J.M.; Alcántara D.; Barrientos, A.G.; García, M.L.; Cerdán S.; Penadés, S. “Glyconanoparticles: New Nanomaterials for Glycoscience Studies”; Glycoconj.J.,2005, 22, 262-263

- F.Garcia Sanchez, A.Navas Diaz, A.F.Gonzalez Diaz, M.C.Torrijas, D.Alcántara, S.A.Eremin “Development of homogeneous phase-modulation fluoroimmunoassay for 2,4-dicholorophenoxyacetic acid”; Analitica Chimica Acta, 1999, 395, 133-142

•••• MEETING PRESENTATIONS

“Glyconanotechnology: A New Methodology to create Nanoparticles with Applications in

Biotechnology and Material Science” David Alcántara, Peter Eaton,Africa G.Barrientos, Jesus M.de la Fuente, Soledad Penadés,. VIII Congreso Nacional de Materiales, 2004, Valencia . Póster “Magnetic glyconanoparticles:preparations, characterization and potential applications”

David Alcántara, Africa G.Barrientos, Jesús M.de la Fuente, Soledad Penadés. 22nd International Carbohydrate Symposium, Julio 2004, Glasgow (U.K). Póster “Magnetic glyconanoparticles:preparation, characterization and MRI applications” David Alcántara,Jesús M.de la Fuente,M.L.García, Sebastián Cerdán and Soledad Penadés. NanoSpain, May 2005, Barcelona. Poster


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“Magnetic glyconanoparticles:preparation, characterization and Magnetic Resonance Imaging

applications” David Alcántara,Jesús M.de la Fuente,M.L.García, Sebastián Cerdán, Soledad Penadés. Trends in NanoTechnology, September 2005, Oviedo. Poster .

“Glyconanoparticles: New Materials for Glycoscience Studies” David Alcántara, Africa G.Barrientos, Jesús M.de la Fuente, M.L.García, Sebastián Cerdán, Soledad Penadés. Glyco XVIII: International Symposium on Glycoconjugates, Florencia (Italy), 2005. Poster

“Glyconanoparticles: a New materials for glyconsciences studies” Jesus M. de la Fuente, David Alcántara, Africa G.Barrientos,M.L.García, Sebastián Cerdán, Soledad Penadés. Simposio Severo Ochoa: " Ochoa 100 años : Mirando al futuro", Madrid, November 2005. Poster

“Water Soluble Glyconanoparticles: synthesis, Characterization, Cell Response and Applications

as Contrast Agents for MRI”. de la Fuente, J.M.; Alcántara D.; García, M.L.; Crespo, P.; Hernando, A.; Cerdán, S.; Penadés, S. NanoSpain2006, April 2006, Pamplona. Oral

“Gold and Gold-Iron Water Soluble Glyconanoparticles: Tools for the emerging Nanomedicine” David Alcántara, Jesus M de la Fuente, Patricia Crespo, Antonio Hernando, M.L.García, Sebastián Cerdán, Soledad Penadés. NanobioEurope2006 ,Grenoble (France). Poster.

“Water Soluble Glyconanoparticles: Synthesis, Characterization, Cell Response and Applications

as contrast Agents for MRI.” de la Fuente, J.M.; Alcántara, D.; García, M.L; Crespo, P.; Hernando, A.; Cerdán, S.; Penadés, S., ICN+T06, Basil (Swizerland), July 2006. Poster.

“Glyconanoparticle-DNA interactions: an AFM and TEM study” Peter Eaton, Jesus M de la Fuente, Africa G. Barrientos, David Alcantara, Raul V. Duran, Cristina T. Rojo and Soledad Penades. ICN+T06, Basil (Swizerland), July 2006. Poster.

“Water Soluble Magnetic Glyconanoparticles: Synthesis, Characterization, Cell Response and Applications as contrast Agents for MRI.” David Alcántara, Jesus M de la Fuete and Soledad Penadés, S., VIII Jornadas de Carbohidratos, Alcalá de Henares (Madrid), September 2006. Poster.

“Water Soluble Magnetic Glyconanoparticles: Synthesis, Characterization, Cell Response and Applications as contrast Agents for MRI.” Jesús M de la Fuente. David Alcántara, and Soledad Penades; III Nanospain Workshop, Pamplona (Spain), March 2006. Poster.

“Glyconanotechnology: A method for the preparation of biofuntional nanoparticles with

application in nanomedicine” M. Marradi, O. Martinez-Ávila, D. Alcántara, J. M. de la Fuente and S. Penadés. TNT2007, San Sebastián (Spain), September 2007. Oral.

“Synthesis of novel resveratrol and hydroxytirosol derivatives: evaluation as food antioxidants”

David Alcantara, José Manuel Gallardo, Isabel Medina and Juan Carlos Morales. XXII Bienal de Química Orgánica. Tarragona (Spain), June 2008. Oral


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Research results During my PhD I was engaged in the design and synthesis of magnetic and paramagnetic

glyconanoparticles. The glyconanoparticles (GNP) can be described as a nanoparticle covered with carbohydrates of relevant biologic importance (http://www.iiq.cartuja.csic.es/carbenglish/carbohydrates.htm). The GNP constitutes a good bio-mimetic model of carbohydrate presentation at the cell surface, and maybe, excellent tools for Glycobiology, Biomedicine and Material Science investigations. During this period (PhD) we have developed the strategy to obtain gold-iron oxide based magnetic GNP as a tool for Magnetic Resonance Imaging (MRI) of the brain. I have been successful achieving my PhD synthetic targets (GNP components and GNPs) through a long and complex total synthesis. As a part of a multidisciplinary group, I have also worked closely to the biologists. These GNPs have been tested in vitro and shows no citotoxicity. Also, we have studied the behaviour of the cell exposed to the different GNPs and we observed that the cell have different behaviour depending on the GNP constituting carbohydrate. The results of these investigations and the studies of their magnetic properties have been presented in 3 papers (included in my CV) and in several international meetings. The in vivo MRI experiments with these magnetic GNP are preliminary but showed good distributions among the brain in a tumoral mouse model. I have also been successful in the synthesis of paramagnetic GNPs as a tool for MRI. We have realized analogous MRI experiment with this system that shows that paramagnetic GNP can be proposed as a MRI contrast agent. The results of these investigations and the studies of their relaxation properties are being written and it will constitute at least 2-3 papers in international journals. On the other hand, the collaborations with other Spanish nanotechnology groups during my Ph.D (giving them the nanoparticles or/and training their students), have given us 4 papers in journals with high impact factors.

Since August 2008 I have been working with Dr. Juan Carlos Morales (Carbohydrates group, IIQ-CSIC) in a recently Spanish network (GLICOFEMUL) to synthesize and test (in vitro and in

vivo) novel glycosidic-phenolics antioxidants. I have prepared 18 new compounds that are being tested nowadays. The results of these investigations will constitute 2-3 papers that will be published in top-rate international journals.

B 3.2 Independent thinking and leadership qualities During my PhD I have deal with complex compounds like the 1,4,7-tris(carboximethyl)- 10-

(11-mercaptoundecyl)-1,4,7,10- tetraazacyclododecane, a macrocylic compound derived, that has required the improvement of the literature´s synthetic methods. I have developed a new strategy to attach these compounds to the glyconanoparticles (GNPs) platform in a stable way and opened a new application of GNPs as contrast agent for Magnetic Resonance Imaging (MRI). I have also demonstrated that our system was, at least, as good as the commercial contrast agent (Magnevist) widely employed in clinic.

I have also been opened to external collaboration in other projects during my PhD. With University of Seville I have been training in nanoparticles´ methodology to E. Grueso, a novel chemistry PhD student. I supervised all the work she done in our lab and trained her in all task related with Nanoparticles synthesis, purification and characterization. The result of this training

collaboration was 1 publication and a timeliness collaboration relationship between the participants.

Relating to international collaborations, I also received the task to train I. Gomes, a novel Portuguese PhD student (University of Lisboa), in carbohydrate methodology. The training

objectives: design, synthesis and characterization of different biologically relevant carbohydrates, was well accomplished by the student under my supervision. With this training we have opened a good relationship between the participants and opened the door for future collaborations and together publications.


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Relative to academic publications I have notice that there is a great lost of information due to not publication of “negative” results. At present, within the research community, more than 60% of

the experiments fail to produce results or expected discoveries. Even though, as in many cases this would be frustrating from an objective point of view, this high percentage of “failed “ research generates high level of knowledge. But generally, all these experiments have not been published anywhere as they have been considered unuseful for our research target. For that reason I manage the creation of a new family of academics journals so-called “The All Results Journals” that focuses on recovering and publishing these valuable pieces of science information. These experiments should be taken into account as a vital key for the development of science. In my opinion these “secondary” results are the catalyst for an empirical and real knowledge based on science. I have designed, created and configured a website (available in an operative provisional directory: www.arjournals.es/ojs) and coordinated an editorial team of eleven academic people that helps me to run this difficult project. I am the responsible too in asking for funding at national and international level, for coordinating all the legal aspect of the editing process and for marketing of the journals. I’ve planned to have all the permissions and start the journals at the beginning of 2009.

B 3.3 Match between the fellow's profile and project. Potential for reaching a position

of professional maturity

The two part of the project proposed is embedded into a scientific environment that is formed

by the presence of many internationally renowned scientists from the field of Chemistry and Magnetic Resonance Imaging (MRI). Dr. Alcántara is a researcher who is

a) Excellently skilled in a key vital component required for the proposed project: he possesses

excellent nanoparticles chemistry skills gained in Europe´s Glyconanoparticles pioneering group. His prior exposure and training to complex and challenging nanoparticles chemistry and it’s MRI applications, ideally equips him for the challenging, cutting-edge programme described above. This will allow him to expand these skills, to apply them to novel nanoparticles design and to gather additional complementary skills from the novel synthetic strategies employed by Prof. Josephson group. In summary, it would be difficult to find a more suitable researcher for both the synthetic phase and the applications of this project, yet the broad range of chemistry to be explored will further hone and improve Dr. Alcántara´s already excellent skills.

b) Driven to expand his training into Molecular Imaging aspects that will provide added value

to these synthetic skills. Dr. Alcántara has an enthusiasm to create not only the chemicals to attach to nanosensors but the Magnetic Resonance Switches (MRS) themselves. He possesses a good, theoretical understanding and powerful experience of the handling, creation and analysis of magnetic and paramagnetic nanoparticles. This project will capture and expand that enthusiasm by exposing him to all the modern techniques of Molecular Imaging and MRI.

B 3.4 Potential to acquire new knowledge

Dr. Alcantara´s balance of enthusiasm for additional knowledge in Molecular Imaging and MRS applications coupled with high level synthetic expertise in magnetic and paramagnetic Nanoparticles design makes him not only ideally suited as the researcher who will make this project but also for him to benefit from it dramatically. The aspect of MRS synthesis and their applications is not well developed yet in the applicant’s country and the knowledge acquired in the outgoing phase will assure bi-directional transfer of knowledge and significantly contribute to applicant’s professional maturity as a researcher.


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B4 Implementation

B 4.1 Quality of host organisations, including adequacy of infrastructures/facilities:

B.4.1.1.1 Outgoing host institution

Harvard University is one of the premier universities in the world. Depending of this University, the Center for Molecular Imaging Research (CMIR), a vital part of Massachusetts General Hospital (MGH), has an international reputation for excellence. Well-equipped with a unique combination of first-class laboratories for chemistry, biochemistry, molecular biology, cell culture and animal studies, together with a wide range of dedicated, state of the art imaging equipment, the CMIR at MGH is certainly leading the way in the most influential evolution of medicine and molecular imaging.

The long-term goal of the CMIR is to overcome current boundaries of conventional diagnostic imaging and develop techniques to image targets at subcellular and molecular levels in vivo. CMIR researchers have developed a number of key technologies, reagents and techniques for imaging as:

- Nanoparticle Chemistry and Bioconjugation. CMIR has long been a pioneer in the research and development of iron oxide based contrast agents. These materials are valuable probes to study specific molecular processes in animal research. Research at CMIR has helped pave the way for the ongoing clinical development and commercial use of this important class of contrast agents (US patent #5,492,814.; Radiology, 1988. 169(2): p. 399-403; Radiology, 1990. 175(2): p. 489-93; Magn Reson Med, 1993. 29(5): p. 599-604)

- Cell Labeling and Tracking In Vivo. The ability to label and track cells is an important capability for research in transplantation medicine, immunology and gene therapy. Magnetic, fluorescent and radioactive cell labeling techniques are used at CMIR to gather data on this important problem. CMIR has pioneered the development of materials that permit the labeling of cells for tracking by high resolution MRI (Bioconjugate Chemistry, 1999;10:186-191; Nature

Biotech, 2000;18:410-414). - Viral Labeling and Tracking In Vivo. To support gene therapy research, techniques to

image DNA, the expression of therapeutic genes, and the fate of gene therapy vectors are needed. Each of these techniques is a major area of research at CMIR. CMIR was the first to develop method to track the in vivo distribution of herpes simplex virions during gene therapy. These studies have resulted in important information regarding the kinetics of viral distribution and optimized administration routes (Human Gene Therapy, 1998; 9:1543-1549; Gene therapy, 2000; 7:1648-1655).

- DNA Labeling and Imaging. As part of CMIR’s effort to support gene therapy research, investigators have developed a method to directly label double stranded DNA with radioactive tracers. This permit an understanding of the rates of movement and eventual disposition of of DNA used gene therapy (US patent application filed 6/3/99;

- In Vivo MR Imaging of Gene Expression (Gene Transfer). We have demonstrated that transgene expression can be detected by MR imaging using mutated internalizing receptors or tyrosinase mutants. Some of this technology is now being explored to image endogenous gene expression (Nature Medicine, 2000; 6:351-355; Radiology, 1997; 204:425-429; Cancer Res, 2000. 60(23): p. 6656-62).

- Fluorescence Sensing of Enzyme Activity. Investigators at CMIR have pioneered and patented new types of optical probes, those that are activated to fluoresce in the near infrared portion of spectrum by specific enzymes. These smart optical imaging probes have been used to detect and quantitate a variety of enzymes in vivo. We have also developed tomographic imaging systems to detect the de-quenching of protease probes in vivo (Nature Biotechnology, 1999; 17:375-378; Nat Med. 2001;7:743-8; US patent 6,083,486).

- Polymers for Imaging and Drug Delivery. A number of different non-immunogenic drug delivery systems and gels have been pioneered in our laboratories over the years. The commercial


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nature of this area has led to an emphasis on patenting our advances (Bioconjug Chem, 1996. 7(1): p. 144-9; US patent 5,514,379.; US patent #5,605,672.; US patent, #5,871,710).

- Lymph Node Imaging and Detection of Nodal Metastases. CMIR has been at the forefront of development of lymphotrophic MR contrast agents and drug delivery systems. Monocrystyalline iron oxides nanoparticles (MION), a preparation developed at CMIR, has been used for experimental lymph node imaging in small animals (Radiology, 1990; 175: 494-498; N

Engl J Med, 2003, 348: 2491-9; PLoS Med, 2004, 1(3): e66). The scientist in charge of the training of Dr. Alcántara in the outgoing phase will be Prof.

Josephson. During recent years he has been supervising several postdoctoral fellowships. The work of Prof. Josephson`s group in CMIR has received financial support from the National Institutes of Health (NCI, NIBIB, NHLBI, NIDDK, NIAID), NSF, NASA, DOD, DOE, Dana Farber/Harvard Cancer Center (DFHCC), Reynolds Foundation, National Foundation for Cancer Research (NFCR), Breast Cancer Research Foundation (BCRF), Broad Foundation, Susan B. Komen Foundation, American Brain Tumor Association, American Heart Association (AHA), Charles Dana Foundation, Avon Breast Cancer Foundation, CapCure Foundation, Siemens Medical Systems, Schering AG, General Electric (GE), Advanced Magnetics Inc., and Millenium Pharmaceuticals among others. Prof. Josephson has published >40 papers in the last 5 years. Selected primary examples of relevance to this proposal are:

- Taktak S, Weissleder R, Josephson L. Langmuir. 2008;24 (14):7596-7598 - Koh I, Hong R, Weissleder R, Josephson L. Angew Chem Int Edit. 2008;47(22):4119-

4121 - Kim GY, Josephson L, Langer R, Cima MJ.. Bioconjug Chem. 2007;18(6):2024-8 - Pittet MJ, Swirski FK, Reynolds F, Josephson L, Weissleder R. Nat Protocols.

2006;1:73-9. - Sun EY, Weissleder R, Josephson L. Small. 2006;(2) 10:1144-1147. - Sun EY, Josephson L, Kelly KA, Weissleder R. Bioconjug Chem. 2006;17(1):109-13. - Weissleder R, Kelly K, Sun E, Shtatland T, Josephson L. Nat Biotechnol. 2005;23:1418-

1423. - Sosnovik DE, Schellenberger EA, Nahrendorf M, Novikov MS, Matsui T, Dai G,

Reynolds F, Grazette L, Rosenzweig A, Weissleder R, Josephson L. Magn Reson Med. 2005;54(3):718-724.

- Perez JM, Simeone FJ, Tsourkas A, Josephson L, Weissleder R. Nano Lett. 2004;4:119-122.

- Tsourkas A, Hofstetter O, Hofstetter H, Weissleder R, Josephson L. Angew Chem Int

Edit. 2004;43(18):2395-2399. - Grimm J, Perez JM, Josephson L, Weissleder R. Cancer Res. 2004;64:639-43. - Perez JM, Josephson L, Weissleder R. Chembiochem. 2004;5:261-4. - Zhao M, Josephson L, Tang Y, Weissleder R. Angew Chem Int Edit. 2003;42:1375-8. - Perez JM, Simeone FJ, Saeki Y, Josephson L, Weissleder R. J Am Chem Soc.

2003;125:10192-3. - Perez JM, Josephson L, O'Loughlin T, Hogemann D, Weissleder R. Nat Biotechnol.

2002;20:816-20. - Perez JM, O'Loughin T, Simeone F J, Weissleder R, Josephson L. J Am Chem Soc.

2002;124:2856-7. B.4.1.1.2 Quality of infrastructures.

Chemistry research at CMIR is focused on bringing the power of modern synthesis to bear on the development of molecular imaging agents. These agents are being developed to visualize specific molecular targets and pathways in live cells, tissues and organism (mouse to human). The


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specific aim of the chemistry groups is to a) synthesize new molecules, b) develop and use novel amplification schemes for the development of "next generation" agents, c) optimize pharmacokinetics and 'imagebility' and d) efficiently synthesize large collections of complex and diverse small molecules and test their ability as imaging agents. Because of the iterative nature of the development process, we collaborate very closely with biologists and imaging experts at CMIR.

The chemistry laboratory is specifically designed for the synthesis of novel imaging pharmaceuticals. This laboratory consists of 3,000 sq. ft. of modern research and production dedicated space equipped with acid resistant benches. This laboratory holds over 3,500 reagents and all necessary glassware and small equipment. In addition to bench space there are three walk in refrigerators for low temperature syntheses, and six Hoodaire airflow fume hoods, equipped with gas, vacuum, and water. The laboratory furthermore contains two modern spectrophotometers (Fluorolog 3, Varian Cary50) and two spectrofluorometers (Hitachi U-3000, Jobin Yuon) all connected to the network. There are four HPLC systems: 1) an analytic HPLC (Varian, ProStar) and 2) three preparative HPLC (Rainin, HPXL, Hitachi 7000) and an automated flash chromatography system (Biorad, Econo). Additional equipment includes a fully automated Rainin PS3 peptide synthesizer, an Advanced Chemtech Apex 396 combinatorial peptide synthesizer, two Buchi 011 Rotavapors, Precision and Thermoline incubators, Gyrotron G2 orbital shaker, pH meters (Orion), several stirrer/hot plates (Corning PC351, 510), several Amicon stirred cells (8010, 8050, 8200, 8400), an oven (Fisher Isotemp 200 series, model 230G) and fluorescence analysis cabinet for thin layer chromatography. A Bruker 120 Minispec pulse NMR spectrometer is available for T1 and T2 measurements. The lab furthermore contains a Branson sonifier 450, a RC5 high-acceleration centrifuge (Sorvall), a Sorvall ultracentrifuge, several Eppendorf microfuges, a submicron dynamic light scattering particle analyzer (Zetasizer 1000HS, Malvern), a lyophilizer (Virtis), a high speed, micro volume concentrater (Savant, SpeedVac AES1010), two high efficiency flow dialysis systems (Amicon, CH2 and A/G Technology, QuixStand), a fluorescence plate reader (Molecular Devises, Spectramax) and water purification system (MilliQ, Millipore).

All of the resources available in CMIR will be available, and much of this will be on a “hands-on” basis. A series of techniques lectures and training courses which covers all aspects of both chemical techniques and safety is available for all new workers in CMIR. In addition we possess extensive IT facilities and dedicated IT officers who run training courses in all aspects of IT that maybe relevant (modeling, database searching, etc). CMIR has extensive library facilities that are almost unrivalled in the USA.

B 4.2 Quality of return host Institution.

INA has one of the most complete laboratory facilities in Spain for the research in Nanoscience: lithography facilities (optical lithography and nanolithography); thin films growing systems like Pulsed Laser Deposition (Neocera Inc.), Molecular Beam Epitaxia (DCA, MBE600); scanning probe microscopies like Atomic Force Microscopy (AFM, Nanotec) STM); electron microscopies like Scanning Electronic Microscopy (SEM, Hitachi S2300) or High Resolution Trasmision Electronic Microscope (HRTEM, HAADF and STEM, Tecnai S-Twin30, 300KeV, GIF-TRIDIEM); biomedical applications (Hyperthermia laboratory capacity); a Bruker 60MHz Minispec pulse NMR spectrometer is available for T1 and T2 measurements; synthesis and functionalization of nanosystems (Nanoscience laboratory installations); nanostructure characterization techniques like XPS-Auger (Kratos, AXIS-ULTRA), X-Ray Difraction (Bruker D8 Advance), Vibrating Sample Magnetometer (VSM, ADE Technologies EV7) and other classical analytical techniques like IR, Calorimetry (DSC, etc.) or NMR (Bruker Advance 300 and 500 MHz). In 2009, INA will be also part of the new Institute of Biomedicine in Zaragoza. In this new research centre will be located two MRI (1.5 and 7 Ts) spectrometers, PET, as well as animal facilities.

The scientists in chargue of Dr. Alcantara in the return phase will be Dr. M. de la Fuente and Prof. Ibarra. Dr. M. de la Fuente has more than 27 articles and 2 patents. Relevant publications:


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- J.M. de la Fuente, D. Alcántara, S. Penadés. IEEE T Nanobiosci. (2007), 6, 275-281. - J.M. de la Fuente, C.C. Berry. IEEE T Nanobiosci.(2007), 6, 320 - J.M. de la Fuente, D. Alcantara, et al. J. Phys. Chem B 110 (2006) 13021. - P. Crespo, M.A. García, E. Fernández, M. Multigner, D. Alcántara, J.M. de la Fuente, S.

Penadés, A. Hernando. Phys. Rev. Let., 97 (2006) 177203 - J.M. de la Fuente, C.C. Berry, M.O. Wiehle, A.S.G. Curtis. Langmuir 22 (2006) 3286. - N.L. Mills, A. Nadia, S.D. Robinson, J. Davies, J.M. de la Fuente, N.A. Boon, W. MacNee,

A.M. Millar, K. Donaldson and D.E. Newby. Am. J. Resp. Crit. Care, 173 (2006) 426-431 - J.M. de la Fuente and C.C. Berry. Bioconj. Chem. 16 (2005) 1176. - J.M. de la Fuente, P. Eaton, A.G. Barrientos, M. Menéndez and S. Penadés. J. Am. Chem.

Soc., 127 (2005) 6192-6197. - S. Penades, M. Martin-Lomas, J.M. de la Fuente, T.W. Rademacher. (2004) Patent. No.

WO2004/108165 A3, - J.M. de la Fuente, C.C. Berry, M. Riehle, L. Cronin, A.S.G. Curtis. Patent. No

US11/409645. University of Glasgow. - A.G. Barrientos, J.M. de la Fuente, T.C. Rojas, A. Fernández and S. Penadés. Chem. Eur. J.

9 (2003) 1909-1921. - M.J. Hernáiz, J.M. de la Fuente, A.G. Barrientos and S. Penadés. Angew. Chem. Int. En. Ed.

41 (2002) 1554-1557. - C. Tromas, J. Rojo, J.M. de la Fuente, A.G. Barrientos, R. García and S. Penadés. Angew.

Chem. Int. En. Ed. 40 (2001) 3052-3055. - J.M. de la Fuente, A.G. Barrientos, T.C. Rojas, J. Rojo, J. Cañada, A. Fernández and S.

Penadés. Angew. Chem. Int. En. Ed. 40 (2001) 2257-2261. Dr. M.R. Ibarra, PhD in Physics (1983). Full professor at the University of Zaragoza since

1995 and Director of the Aragon Institute of Nanoscience (INA). He is leading the nanomagnetism group at the INA. His research focuses on magnetocaloric materials, magnetic oxides with CMR, thin film multilayers, nanostructured magnetic materials, encapsulated nanoparticles and nanostructured magnetic materials for biomedical applications. He is author of over 260 papers of SCI with more than 3000 citation and he holds ten patents. Relevant publications:

- Serrate, D; De Teresa, JM; Algarabel, PA; Galibert, J; Ritter, C; Blasco, J; Ibarra, MR. Phys.

Rev. B (2007), 75, 165109. - Arruebo, M; Fernandez-Pacheco, R; Irusta, S; Arbiol, J; Ibarra, MR; Santamaria, J.

Nanotechnology,(2006), 17, 4057-4064 - Magen, C; Algarabel, PA; Morellon, L; Araujo, JP; Ritter, C; Ibarra, MR; Pereira, AM;

Sousa, JB. Phys. Rev. Lett (2006) 96, 167201 - Yusuf, SM; De Teresa, JM; Ritter, C; Serrate, D; Ibarra, MR; Yakhmi, JV; Sahni, VC. Phys.

Rev. B (2006), 74, 144427. - Sikora, M; Kapusta, C; Borowiec, M; Oates, CJ; Prochazka, V; Rybicki, D; Zajac, D; De

Teresa, JM; Marquina, C; Ibarra, MR. App.Phys.Lett, (2006), 89, 62509. - de Teresa JM, P.A. Algarabel, C. Ritter, J. Blasco, MR Ibarra, L. Morellón, JI Espeso, JC

Gómez-Sal. Phys. Rev. Lett (2005), 94, 207205. The most recent relevant projects: Multiple Nanocontac Devices (MUNDIS) (521.757 €) (3

years) M.R. Ibarra Coordinator FP6/2004/IST/4 027827 and-Nanotechnologies in Biomedicine Program CONSOLIDER-INGENIO M.R. Ibarra Coordinator (2006-2010) (4.500.000 €)

B 4.3 Practical arrangements for the implementation and management of the project

Outgoing phase: Dr. Alcántara will be inducted and oriented within the CMIR including full awareness of safety and group protocols. This will include hands on training of all state-of-the-art


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equipment available within the centre. For each of the different fields that the project focuses on (see B 1.2) senior researchers are available in the institute who will introduce Dr. Alcántara into the different techniques lined out here. He will be trained in the characterization of nanoparticles including magnetic and surface charge measurements, and size determination (light scattering, light based and electron microscopy). He also will be trained in the MRI techniques necessary to the assessment of the project. The development of the project is described in B.1.2.1 (outgoing phase, pages 3-5) and summarised as: 1) Optimize Nanoparticle Design; 2) Synthesize and characterize optimized oligonucleotide-NP’s targeted to the rRNA of S. Cerevisiae and S. Aureus; 3) Determine the optimal MRS assay of rRNA of E.Coli, S. Cerevisiae and S. Aureus; 4) Assay performance and evaluation. The practical implementation of the project is guaranteed by the scientific infrastructure and staff of CMIR. The project will be regularly monitored and the progress assessed typically once a week with the project co-ordinator (Dr. Jesus Martinez) and scientist in charge Prof. Lee Josephson.

Return Phase: Dr. Alcántara will be fully integrated into the research groups of Dr. M. de la Fuente and Prof. Ibarra at the institute of Nanoscience. Laboratory space and access to all equipment mentioned above has already been provided. For each of the different fields that the project focuses on, synthesis and characterization of magnetic nanoparticles, senior researchers are available in the institute who will introduce Dr. Alcantara into the different techniques lined out. Dr. Alcantara will also participate in the infrastructure of the collaborative research grant “CONSOLIDER-NANOBIOMED” that comprises ca. 17 groups from different universities and the Research Institutes of Spain. The center grant comprises ca. 4.5 Mio € for a five year’s period and covers research topics ranging from synthesis, characterization and medical applications of magnetic nanomaterials. This will be very important for the training of Dr. Alcántara because within this center grant seminars on Nanotechnology and Biomedicine issues take place on a regular basis, and there is usually at least two international meeting on a specialized topic from Nanotechnology per year. The center grant will also be a platform for Dr. Alcántara to present the data that he is going to acquire during the project. The discussion with other experts from the field will significantly enhance the perspectives of this research project.

B 4.4 Feasibility and credibility of the project, including work plan

The project will be developed and implanted following the next scheme (in semesters):

GANTT workplan 1 year (Outgoing Phase)

2 year (Outgoing Phase)

3 year (Return Phase)

1-6 7-12 1-6 7-12 1-6 7-12

1. Optimize Nanoparticle Design (page3)

2. Synthesize and characterize optimized oligonucleotide-NP’s targeted to the rRNA (page 4)

3. Determine the optimal MRS assay of rRNA of E.Coli, S. Cerevisiae and S. Aureus (page 4)

4. Establishment, adaptation and development the NPs-Lectins (pages 4-5)

5.Determine the optimal MRS assay with NPs-lectins against tumoral cells lines (page 5)

6.Assay performance and evaluation (pages 3-6)


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The attainment of the research objectives summarized on pages 3-6 will provide fundamentally new methods for the rapid detection and identification of microorganisms in the outgoing phase and cancer cells in the return phase.

B 4.5 Practical and administrative arrangements and support for the hosting of the

fellow

Outgoing: The Massachusetts General Hospital, where CMIR is located, maintains an Office for International Scholars and Students to assist and advise scientists like Dr. Alcántara in immigration, visa, health insurance and related matters. Upon establishment of the start date, Dr. Alcántara will be given a list of contacts and/or possible accommodations, and will be helped to understand and sign the relative contract. As a rule, the foreign fellow is always supported by a native speaker in all the initial interaction with the USA institutions. CMIR has an induction week for all new researchers where they are introduced to and receive important and appropriate training in safety (COSHH, GLP, fire, handling skills, disposal practices) and core skills (eg. record keeping, data handling, experimental/factorial design, IT, databases, analytical facilities eg NMR, Mass Spectroscopy etc). CMIR encourages researchers to take, at appropriate times during their tuition, a comprehensive range of courses to develop general skills & awareness e.g., Scientific & Report Writing, IT Skills, Ethics & Science, Science & the Media, Library skills, Time

management, Teaching Practice, Careers, Research Management, Good supervisory practice. At the beginning of the project, in addition to standard departmental induction, the researcher will be orientated with respect to the teamworking of the research group, initiated in background reading and all laboratory procedures clearly explained. Practical work will start as soon as possible to promote his interaction with other department and group members.

Dr. Alcántara does not have children at the moment; however, CMIR has an internal childcare programme in which the applicant could be enrolled, if required.

Return: Dr. Alcántara will be integrated in the Biofunctional Nanoparticles Group (Dr. Jesus Martínez). He will be provided with lab space, students (Ph.D and undergraduate) and infrastructure to achieve with success his project. In parallel to this stage Dr. Alcántara will be encourage and supported to create a spin-off enterprise to apply his managing and scientific knowledge into the market place. He will be provided with lab space and money enough to start the activity and will be monitored by INA. INA have a specific administration helpdesk to facilitate Dr. Alcántara asking for European funding programs (scientific or enterprise form). Depending on his results and orientation, at the end of this fellowship, INA or University of Zaragoza will accept Dr. Alcantara´s candidature to be part of INA scientific staff.


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B5 IMPACT

B 5.1 Potential of acquiring competencies Outgoing phase: In CMIR the preparation of patents and papers for publication is a central

role for the researcher and ensures exposure to the peer review process. Researchers in the group actively participate in our collaborations in academia and industry as this fosters an appreciation of multidisciplinary and commercial approaches. Careers advice from the dedicated university centre is augmented in the group and by attendance at regional networking meetings. The training received by Dr. Alcántara in this proposal will give him independence and professional maturity. In this way he will become a truly, multidisciplinary researcher capable of readily crossing the vitally important Nanoscience-biology interface to direct and inform new avenues of research in these fields.

The central aim of this training in CMIR will be to enable Dr. Alcántara to initiate a

powerful, independent research career.

The Dr. Alcantara´s new acquired skills will be transferred to the return host by means of starting a new glycomic project as independent researcher in cancer area. The collaboration with Prof. Josephson and Prof. Weissleder in CMIR will be maintained in a timeliness scientist fashion during this and other future projects. The project provides the connection and support for long-term nanoscience research between USA and EU, to create novel nanotechnology applications.

Return phase: The fellow will acquire maturity and independence along this fellowship. He will be leading the return phase project, and the host group in INA will support his research. As it was previously appointed, Dr. Alcántara will be trained in negotiation skills, project management, career planning, proposal writing, design of research studies, ethical aspects and technology transfer and IPR/Patents. Some of these topics will be built into the host institutions and others will be covered by regional and national short courses.

The scientific contacts and training that Dr. Alcántara will acquire during the outgoing phase may prove essential for future collaborative research projects of the fellow. Apart from the skills in the field of Nanotechnology, CMIR partner centre (outgoing phase) provides excellent research in fields ranging from magnetic resonance imaging and biomedical application of magnetic nanobiosystems. In fact this group is an international reference in the field. It is not unlikely that the applicant will find his future area of research within the scope of these projects.

With the strong pharmaceutical background of the proposed project it is positioned right in the center of the EU main focus on Life Sciences, genomics, proteomics, glycomics and biotechnology for health. In particular, the project addresses novel approaches for the design of innovative techniques for biosensing based on nanotechnology and MRI tools. The approach heavily uses proteomics, genomics and glycomics tools in conjunction with magnetic nanomaterials and MRI spectroscopic methods. With this focus, the project is within the area of biosensing of microorganism, bacteria, viruses and tumoral cells applying genomics, glycomics and proteomics tools that has been put forward as one specific area of activity within the seventh framework program of the EU.

B 5.2 Contribution to career development or re-establishment

Outgoing phase: The array of skills Dr. Alcantara will acquire during his training at CMIR will make him uniquely trained for employment in a chemistry/material sciences/nanotechnology based university or company in Europe. At CMIR we often collaborate with European investigators who have been trained at CMIR, as a continuation of projects that have originated at CMIR, or with wholly original projects. A strong connection between CMIR and European scientists has long existed and is furthered by the fact that the head of CMIR, Dr. Ralph Weissleder is German born and educated.

Return phase: The training obtained by the fellow in the outgoing institution will be very beneficial to the host institution. The proposed project for the return phase is embedded into a scientific environment that is formed by the presence of many internationally renowned scientists


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from the field of nanobiotechnology. In particular, the integration of Dr. Alcántara into the collaborative research grant “CONSOLIDER-NANOBIOMED” that concentrates the activities of ca. 17 research groups in the area is of great benefit and guarantees that the training will not only be restricted to the topics lined out in the above research project. This collaborative project concerns the application and exploitation of magnetic nanodevices as biosensors, drug delivery and cancer treatment under the emerging field of Nanobiomedicine.

The collaborations and contacts of the Institute of Nanoscience of Aragon (INA) with industrial partners alongside with this project will also be beneficial to the project and to the perspectives for the applicant. Since the targets are of significant pharmaceutical relevance it is of interest for Dr. Alcántara to develop contacts with pharmaceutical firms. First steps into this direction may be undertaken during the course of the current research project.

The field of studying, magnetic resonance switches for biosensing by MRI is rather new and offers a multitude of different research directions in the future. Also, since this class of methodologies is of pharmaceutical interest, it can safely be assumed that future research projects of Dr. Alcántara target problems from within this area. These efforts would not only strengthen his own scientific standing but would also promote the research activities in the new field of Nanobiodiagnosis in Spain. Dr. Alcántara will have access to state of the art MRI equipment in the host center in Zaragoza. INA will take part of the new Institute of Biomedicine where two 1.5 and 7 Ts MRI facilities will be installed in 2009. Dr. Alcántara will be a fundamental human key for these new facilities in Zaragoza, and, once again, his obtained training in USA will be very beneficial for the host institution, open the possibility to establish new research areas using MRI.

INA has a Personal Career Plan for reintegration of Dr. Alcántara as permanent staff in the University of Zaragoza. This contract will be drawn up upon public recruitment and then updated annually. Dr. Alcántara will take part in the management of the Biofunctional Nanoparticles Group in INA, and he will also take part in the organisation of training events as Doctorate programs and summer courses. Training of students will be mainly aimed at making him more independent and providing him with the needed skills to become team leader in a near future.

B 5.3 Potential for creating long term collaborations and mutually beneficial co-

operation between Europe and the third country. Contribution to European excellence and

European competitiveness

The fellowship's main objectives presented in this proposal are to provide the connection and support for long-term nanoscience research between USA and EU, to create novel nanotechnology applications, to develop human and capital infrastructure needed for technology development, and to stimulate new nanotechnology research in advanced materials, like the Magnetic Resonance Switches presented in this proposal. It is hoped that such interactions will continue to build through such projects such as that described here.

On a long term the consequences and potential follow up projects between the host institution

in Spain and partner institution in USA will put EU researchers in a more competitive position in

comparison to others international efforts in the field. This project will start what we expect a fruitful and long collaboration between the Institute of Nanoscience of Aragon in Zaragoza (Spain) and the CMIR in Boston (USA). Scientific contacts between individual researchers from this field of research will be established by Dr. Alcántara and this project will therefore enhance these incipient ties. In this context, this action will contribute to improve European excellence due to first, the high level of training (and contacts for his future cooperation) received by Dr. Alcántara in USA and second, by the reintegration of the researcher in EU. The European competitiveness will

also be potentiated, through the creation of a new enterprise during the reintegration phase, giving high level EU employment and maintaining the contact with USA through CMIR staff. Thus this fellowship will significantly contribute to applicant’s professional maturity as a researcher and will assure valuable international and bidirectional transfer of knowledge.


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B5. ETHICAL ISSUES

There are no pressing ethical issues to be considered. ETHICAL ISSUES TABLE

YES PAGE

Informed Consent

• Does the proposal involve children? • Does the proposal involve patients or persons

not able to give consent?

• Does the proposal involve adult healthy volunteers?

• Does the proposal involve Human Genetic Material?

• Does the proposal involve Human biological samples?

• Does the proposal involve Human data collection?

Research on Human embryo/foetus • Does the proposal involve Human Embryos? • Does the proposal involve Human Foetal

Tissue / Cells?

• Does the proposal involve Human Embryonic Stem Cells?

Privacy • Does the proposal involve processing of

genetic information or personal data (e.g. health, sexual lifestyle, ethnicity, political opinion, religious or philosophical conviction)

• Does the proposal involve tracking the

location or observation of people?

Research on Animals

• Does the proposal involve research on animals?

• Are those animals transgenic small laboratory animals?

• Are those animals transgenic farm animals? • Are those animals cloning farm animals? • Are those animals non-human primates?

Research Involving Developing Countries

• Use of local resources (genetic, animal, plant etc)

• Benefit to local community (capacity building i.e. access to healthcare, education etc)

Dual Use

• Research having potential military / terrorist application

I CONFIRM THAT NONE OF THE ABOVE

ISSUES APPLY TO MY PROPOSAL

√


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Proposers confirm that the research presented in this proposal does not involve:

- Research activity aimed at human cloning for reproductives purposes; - Research activity intended to modify the genetic heritage of human beings which could

make such changes heritable; - Research activity intended to create human embryos solely for the purpose of research or for

the purpose of stem cell procurement, including by means of somatic cell nuclear transfer; - Research involving the use of human embryos or embryonic stem cells with the exception of

banked or isolated human embryonic stem cells in culture The information on ethics requirements and rules are given at the science and ethics website at http://ec.europa.eu/research/science-society/ethics/ethics_en.html and this research does not involve

any of these.


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ENDPAGE

PEOPLE MARIE CURIE ACTIONS

Marie Curie International Outgoing Fellowships (IOF)

Call: FP7-PEOPLE-IOF-2008

PART B

“MRI_Nanobiosensor”

mri nanobiosensors part b · “mri_nanobiosensors” the marie curie actions international...

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