Báo cáo khoa học: WORKSHOPS WS 1 Nanotherapeutics pdf

Kataoka The University of Tokyo, Materials Engineering, Tokyo, JAPAN Polymeric micelle, the self-assembly of block copolymers with core-shell architecture, is a promising nanocarrier for

WS 1 Nanotherapeutics

WS 1–1

Nanotherapeutics through polymer chemistry:

supra-molecular nanocarriers for gene and

drug delivery

K Kataoka

The University of Tokyo, Materials Engineering, Tokyo, JAPAN

Polymeric micelle, the self-assembly of block copolymers with

core-shell architecture, is a promising nanocarrier for drug and

gene delivery There are several relevant properties in polymeric

micelle as nanocarrier systems, such as longevity in blood

circula-tion, tissue-penetrating ability, spatial and temporal controlled

drug release, and reduced inherent toxicity of cytotoxic reagents

Also, engineering of the block copolymer structure allows the

preparation of polymeric micelles with integrated smart

func-tions, such as targetability as well as stimuli-sensitivity This

pre-sentation overviews the recent achievements as well as the future

perspectives of polymeric micelles as smart nanocarriers for drug

and nucleic acid delivery Notable anti-tumor efficacy against

hypovascular cancer, including pancreatic cancer and

diffused-type stomach cancer, of the doxorubicin-incorporated polymeric

micelles with pH-responding property was demonstrated to

emphasize a promising utility of the nanocarrier-modulated

che-motherapy for the treatment of intractable cancers Gene-loaded

polymeric micelles were applied as non-viral vectors in the fields

of regenerative medicine, particularly bone regeneration

Genera-tion of new bone in experimental animals was successfully

achieved by transducting genes encoding differentiation factors

using polymeric micellar carriers Gene therapy by polymeric

micelles was also demonstrated for intractable cardiovascular

dis-ease, pulmonary hypertension, by intratracheal transfer of

thera-peutic gene Further, the supramolecular assemblies, including

polymeric micelles, polymer vesicles, and photosensitive

dendri-mer assemblies, were utilized as nanodevices directing to the new

medical paradigm of smart nanotheranostic systems controlled

by external physical stimuli, particularly, photo illumination

(nano-photomedicine)

WS 1–2

Polymeric nanomedicines: intracellular

trafficking and subcellular fate

J Kopecek

University of Utah, Pharmaceutical Chemistry, Salt Lake City

Utah, USA

The concept of polymeric nanomedicines was developed to

design, synthesize, and evaluate a second generation of anticancer nanomedicines with enhanced efficiency The augmented effec-tiveness of novel conjugates will be derived from: longer intravas-cular half-life; controlled biodegradability of the main chain; and potential for double targeting: to tumor cells and to a crucial subcellular organelle The lecture will discuss recent results of cel-lular and subcelcel-lular targeting of N-(2-hydroxypropyl)meth-acrylamide (HPMA) copolymer – drug conjugates: structural factors influential in the kinetics of translocation of macromole-cules from the cytoplasm into the nucleus, the effect of the valence of HPMA copolymer – Fab fragment conjugates on the binding to B cell antigens, subcellular targeting of photosensitizer mesochlorin e6 to mitochondria mediated by lipophilic triphenyl-phosphonium cation, and subcellular targeting to nucleus facili-tated by glucocorticoid receptor – ligand recognition

WS 1–3 Multifunctional nanocarriers for delivery of drugs, genes, and diagnosticals in the body

V P Torchilin Center for Pharmaceutical Biotechnology and Nanomedicine Northeastern, University Boston, MA, USA

Various pharmaceutical nanocarriers, including liposomes and polymeric micelles, are frequently used for the delivery of a broad variety of both soluble and poorly soluble pharmaceuti-cals Using nanoparticulate pharmaceutical carriers to enhance the in vivo efficiency of many drugs is now well established Now, within the frame of this concept, it is important to develop multi-functional stimuli-responsive nanocarriers, i.e nanocarriers that, depending on the particular requirements, can circulate long; tar-get the site of the disease via both non-specific and/or specific mechanisms, such as enhanced permeability and retention effect (EPR) and ligand-mediated recognition; respond local stimuli characteristic of the pathological site by, for example, releasing

an entrapped drug or deleting a protective coating under the slightly acidic conditions inside tumors facilitating thus the con-tact between drug-loaded nanocarriers and cancer cells; and even provide an enhanced intracellular delivery of an entrapped drug Additionally, these carriers can be supplied with contrast moieties

to follow their real-time biodistribution and target accumulation Among new developments to be considered in the area of multi-functional pharmaceutical nanocarriers are: drug- or DNA-loaded delivery systems additionally decorated with cell-penetrat-ing peptides for the enhanced intracellular delivery; ‘smart’ multi-functional drug delivery systems, which can reveal/expose temporarily hidden functions under the action of certain local

alter their properties during the drug delivery process, to make

them most effective for the different extra- and intra-cellular

steps (1–3) For this purpose molecular chemical sensors (such as

bioreducible and pH-sensitive bonds or conformations) are

incor-porated able to respond to the relevant intracellular biological

(e.g endosomal acidification) or physical stimuli (light or

hyper-thermia) Targeting to cell surface receptors enhances specificity

and efficacy, as demonstrated for EGFR targeted polyplexes

con-taining therapeutic RNA poly-Inosine-Cytidine (4) or

transferrin-coated siRNA polyplexes (5) Delivery processes can be

moni-tored by innovate biooptical imaging tools including near

infra-red quantum dot in vivo imaging in mice

References:

1 Wagner E Expert Op Biol Ther 2007; 7: 587–593

2 Meyer M, Philipp A, Oskuee R, Schmidt C & Wagner E J

Am Chem Soc.2008; 130: 3272–3273

3 Knorr V, Russ V, Allmendinger L, Ogris M & Wagner E

Bio-conjug Chem.2008; 19: 1625–1634

4 Shir A, Ogris M, Wagner E & Levitzki A PLoS Med 2006; 3:

e6

5 Tietze N, Pelisek J, Philipp A, Roedl W, Merdan T, Tarcha P,

Ogris M & Wagner E Oligonucleotides 2008; 18: 161–174

WS 1–5

Polymer nanomaterials for CNS drug delivery

A Kabanov

University of Nebraska Medical Center, Pharmaceutical Sciences,

Omaha, USA

Neurodegenerative and infectious disorders including Alzheimer’s

and Parkinson’s diseases, amyotrophic lateral sclerosis, and

stroke are rapidly increasing as population’s age Alzheimer’s

dis-ease alone currently affects 4.5 million Americans, and more than

$100 billion is spent per year on medical and institutional care

for affected people Such numbers will double in the ensuing

dec-ades Currently disease diagnosis for all disorders is made, in

large measure, on clinical grounds as laboratory and

neuroimag-ing tests confirm what is seen by more routine examination

Achieving early diagnosis would enable improved disease

out-comes Drugs, vaccines or regenerative proteins present ‘real’

possibilities for positively affecting disease outcomes, but are

lim-ited in that their entry into the brain is commonly restricted

across the blood-brain barrier This presentation will review

high-lights how these obstacles can be overcome by polymer science

and nanotechnology Specific examples developed in our work

include (i) inhibition of drug efflux transport systems in the

blood-brain barrier by amphiphilic block copolymers, (ii)

chemi-cal modification of proteins with hydrophobic anchors groups

and amphiphilic polymers to enhance their transport across the

blood-brain barrier, (iii) development of nanogels capable of

car-rying antisense oligonucleotides and siRNA across the

blood-brain barrier, (iv) cell-mediated delivery of nanozymes to the

brain Such approaches may improve diagnostic and therapeutic

outcomes New developments in polymer science coupled with cell based delivery strategies support the notion that diseases that now have limited therapeutic options can show improved out-comes by advances in nanomedicine The work is supported by the United States National Institutes of Heath

WS 1–6 Immunotherapy of cancer with polymeric prodrugs: from dream to reality

B Rihova1, T Etrych2, M Kovar1, J Strohalm2, L Kovar1,

O Hovorka1, M Sirova1, D Plocova2, P Chytil2, V Subr2and

K Ulbrich2

1

Institute of Microbiology AS CR v.v.i., Immunology and Gnotobiology, CZECH REPUBLIC,2Institute of Macromolecular Chemistry AS CR v.v.i., Biomedical polymers, CZECH

REPUBLIC The majority of clinically approved anticancer drugs are charac-terized by a high systemic toxicity, including immunotoxicity Water-soluble polymeric therapeutics based on N-(2-hydroxypro-pyl) methacrylamide (HPMA) has been emerged as efficient ther-apeutics because they are able to not only directly destroy cancer cells but also elicit systemic tumor-specific anticancer responses Such polymeric drugs have been shown to be highly effective against experimental tumors induced in mice and rats It was possible to establish a protective anticancer immunity in normal mice with an intact immune system, but in immunodeprived nude (athymic) mice there were no long-term cancer survivors It could

be shown that the polymeric drug therapy generated a tumor-spe-cific long-lasting memory, which was based mainly an effectors cells of specific immunity, the CD8+ cytotoxic T lymphocytes (CTLs) Moreover, such resistance can be induced only within an optimal time frame: experimental mice treated either too early (i.e a few days after transplantation of cancer cells) or too late (i.e when the immune system is already exhausted) did not estab-lish systemic cancer resistance This suggests that treatment that might be presumed to be very effective, that is, treatment started either very early or with a highly aggressive dose, might actually eliminate cancer cells before they can supply the immune system with a sufficient amount of antigens for effector-cell activation The first clinical observation together with the laboratory data suggests non-toxic, anti-tumor and immunomobilizing character

of polymeric drugs based on HPMA similarly as it was docu-mented in experimental animals However, more data and addi-tional studies are needed in order to make a raaddi-tional and balanced conclusion and suggest them as a novel approach for synergized chemotherapy and immunotherapy of cancer Acknowledgement: This work was supported by grants from Academy of Sciences of the Czech Republic (no KAN200200651) and the Grant Agency of the Academy of Sciences of the Czech Republic (no.IAAX00500803)

WS 2 Glycomic Technologies

WS 2–1

Novel glycan-binding receptors identified by

genomic analysis

K Drickamer and M E Taylor

Imperial College London, Department of Life Sciences, London,

UK

Mammalian receptors that recognise specific glycans are

responsi-ble for glycoprotein trafficking in cells and organisms, cell–cell

adhesion and immunity to viral, bacterial and fungal pathogens

Glycomic and genomic analyses suggest that there are thousands

of glycan structures expressed on human cells and pathogens, but

only about a hundred different human sugar-binding receptors

have been described to date Using the structures of known

carbo-hydrate-recognition domains as templates, we have been

examin-ing the human genome for additional sugar-bindexamin-ing domains and

have identified several novel glycan-binding receptors One of

these has been examined in detail and is found to be expressed on

B cells Affinity chromatography, solid phase binding assays and

glycan array analysis reveal that the C-type

carbohydrate-recogni-tion domain in the extracellular domain of the receptor binds

gly-cans with terminal alpha-linked mannose or fucose residues

Compared to other known glycan-binding receptors, the receptor

contains an unusually large intracellular domain that consists of

multiple sequence motifs, including phosphorylated tyrosine

resi-dues, that allow it to interact with signalling molecules such as

Grb2 As the receptor is expressed on a specialized population of

proliferating B cells in germinal centres, we have suggested the

named prolectin This novel receptor has the potential to function

in carbohydrate-mediated communication between B cells and

other cells in the germinal centre and elsewhere

WS 2–2

NMR studies of free and protein-bound

glycomimetics

J Jimenez-Barbero

CIB-CSIC, Protein Science, Madrid, SPAIN

Molecular recognition by specific targets is at the heart of the life

processes In recent years, it has been shown that the interactions

between proteins (lectins, enzymes, antibodies) and carbohydrates

mediate a broad range of biological activities, from fertilization,

embryogenesis, and tissue maturation, to pathological processes

The elucidation of the mechanisms that govern how sugars are

accommodated in the binding sites of these receptors is currently

a topic of interest Thus, the determination of the structural and

conformational factors and the physicochemical features which

govern the molecular recognition of these molecules is of

para-mount importance This presentation is focused on the

applica-tion of NMR methods to the study of molecular recogniapplica-tion

complex mixtures of glycoproteins from biological extracts of cells and tissues to be screened thereby revealing the types of gly-cans present and, importantly, providing clues to structures that are likely to be functionally important Data emerging from some

of our glycomic programmes of collaborative research, which are helping to provide new insights into the functions of glycans in biological systems, will be described In addition our participa-tion in internaparticipa-tional glycobiology consortia will be discussed These include the Analytical Core of the NIH Consortium for Functional Glycomics, which is located at Imperial College, which is employing glycomic methodologies to study murine and human haematopoietic cell populations in order to provide a gly-comics data resource for the glycobiology community Informa-tion emerging from this programme will be highlighted together with progress on the development of informatic tools to manage the large volumes of data being acquired

WS 2–5 Protein engineering of carbohydrate binding proteins – understanding the recognition phenomena

M Wimmerova Masaryk University, National Centre for Biomolecular Reseach and Department of Biochemistry, Brno, CZECH REPUBLIC Carbohydrate-mediated recognition plays an important role in adhesion of parasitic organisms to the surface of the host cell as the first step of their invasion and infectivity Protein–carbohy-drate interactions are generally characterised by a low affinity for monovalent ligands that is balanced by multivalency resulting in high avidity for ligands with several potential epitops available, such as complex glycans or cell surface However, recent charac-terisation of bacterial lectins involved in pathogenesis has demon-strated their much higher affinity even towards monosaccharide than that observed for plant or animal lectins (1) The contribu-tion is focused on few examples where a combinacontribu-tion of experi-mental methods (isothermal titration microcalorimetry, surface plasmon resonance, X-ray crystallography) with computational approaches can bring a significant profit for better understanding

of protein-carbohydrate interactions on molecular level (2,3) Insight into structure correlation with function analysis of high-affinity lectins allows for construction of carbohydrate-binding proteins with tuned affinity and specificity for biotechnology and bioanalytical purposes

References:

1 Imberty A, Mitchell EP & Wimmerova´ M Curr Opin Struct Biol.2005; 15: 523–534

2 Adam J, Pokorna´ M, Sabin C, Mitchell EP, Imberty A &

WS 2–6

Identification and characterisation of lectins in

several tick species

J Sterba1, J Dupejova1, M Fiser1, M Golovchenko2,

N Rudenko2and L Grubhoffer1

1University of South Bohemia, Faculty of Science, Ceske

Budejovice, CZECH REPUBLIC,2Biology Centre of ASCR,

Institute of Parasitology, Ceske Budejovice, CZECH REPUBLIC

FReD (fibrinogen-related domain) proteins are lectins with

possi-ble functions in immunity of invertebrates Previously, a FReD

protein from the hemolymph of the tick Ornithodoros moubata,

Dorin M, was isolated and characterised In the present study,

FReD proteins with hemagglutination acitvity (HA) were

identi-fied in hemolymph of ticks Dermacentor marginatus,

Rhipicephal-lus appendiculatus, R pulchellus, and R sanguineus and

characterised Several FReD proteins were detected using

anti-bodies directed against Dorin M, Ixodes ricinus hemolymph lec-tin, and hemagglutination acitivity of O moubata and I ricinus ticks All identified FReD proteins are modified by truncated high-mannose glycans Hemagglutination activity of hemolymph

of the studied ticks was inhibited predominantly by N-acetylh-exosamines and sialoglycoproteins All tick species contained a hemelipoglycoprotein-like protein, described previously also in D variabilis Nucleotide sequence of novel protein containing fibrin-ogen-related domain was identified in ticks of species D margina-tus, Haemaphysalis punctata, and Hyalomma impeltatus FReD proteins of D marginatus localised in hemocytes, salivary glands, and gut cells

Acknowledgement: Supported by the KJB600960906 research project of the Grant agency of the Academy of sciences of the Czech Republic and by the LC06009 research centre project of the Ministry of Education, Sport and Youth of the Czech Republic

WS 3 Emerging Proteomic Technologies

WS 3–1

Mapping and measuring proteomes

R Aebersold

Institute of Molecular Systems Biology, Zurich, SWITZERLAND

The human genome project has taught us that a complete map –

in the case of the genome project the complete genomic sequence

– along with computational tools to navigate the map – represent

invaluable resources for experimental and theoretical biologists

A main consequence of such a complete map is that all the

bio-logical processes have to be explainable with the components that

constitute the map Proteomics has not reached the stage that

complete maps are available but the urgent need for their

genera-tion is now widely recognized In this presentagenera-tion we will discuss

experimental and computational challenges related to the

genera-tion of complete proteomic maps We will also discuss recent

technical advances towards complete proteome analysis and

describe software tools and data resources that will transform

proteomics from perpetual proteome mapping to accurate

prote-ome measurement

Further reading:

Kuster B, Schirle M, Mallick P & Aebersold R Nature Rev

Mol Cell Biol.2005; 6(7): 577–583

Brunner E, Ahrens CH, Mohanty S, Baetschmann H, Loevenich

S, Potthast F, Deutsch EW, Panse C, de Lichtenberg U, Rinner

O, Lee H, Pedrioli PG, Malmstroem J, Koehler K, Schrimpf S,

Krijgsveld J, Kregenow F, Heck AJ, Hafen E, Schlapbach R &

Aebersold R Nature Biotechnol 2007; 5: 576–583

Nesvizhskii A, Vitek O & Aebersold R Nature Methods 2007; 4:

787–797

Deutsch EW, Lam H & Aebersold R EMBO Reports 2008; 9:

429–434

Lange V, Picotti P, Domon B & Aebersold R Molecular Systems

Biology2008; 4: 222

Picotti P, Lam H, Campbell D, Deutsch E, Mirzaei H, Ranish J,

Domon B & Aebersold R Nature Methods 2008; 5: 913–914

Bodenmiller B, Campbell D, Gerrits B, Lam H, Jovanovic M,

Picotti P, Schlapbach R & Aebersold R Nature Biotechnol

2008; 26: 1339–1340

WS 3–2

Chalenges in guantitative proteomics and

posttranslational protein modifications

M Mann

Max-Planck Institute of Proteomics, GERMANY

WS 3–3

first applied to protein sequencing Brief discussion of the devel-opment of mass mapping strategies together with early structural studies including antifreeze glycoproteins and the discovery of the glycosylation of human Interleukin 2, will give an insight into current strategies for glycoprotein analyses These will then be illustrated with details of our developmental biology research, utilising ES Q-TOF and MALDI TOF-TOF technologies, with examples from human glycodelin and from the characterisation

of the O-glycans of gp273 from Unio elongatulus, believed to be the ligand for sperm-egg binding during fertilisation Such com-plex studies are aided by the recent development of powerful automated and interactive informatic tools such as Cartoonist, Peptoonistand GlycoWorkbench, and their use in unravelling the complexity of glycoproteomic interpretations will be described Overall, the advances in glycoproteomics methodology over the past four decades, building on the early concepts of biomolecular mass spectrometric strategy and tactics, are now reaching a point where despite the inherent complexity of the field, automated analyses, comparable to current proteomics methodologies, may

be only another decade away!

WS 3–4 Pathway proteomics using proteinarrays

J van Oostrum Zeptosens, a Division of Bayer (Schweiz) AG, Witterswil, SWITZERLAND

Molecular signalling pathways are frequently triggered by extra-cellular molecules binding receptors and activating relay systems inside cells, leading to processes that affect cellular behaviour and fate For many genetic disorders a link between disease and signalling pathways have been established consequently a system-atic analysis of dynamic cellular networks provides an opportu-nity for pharmaceutical discovery, by taking into consideration the complex biological context of drug targets, rather than observing the targets in isolation Such analyses are, perhaps, ide-ally suited for a systems biology approach that integrates experi-mental data with computational modeling with the aims of discovering and validating new drug targets and biomarkers, as well as predicting potential ‘off target’ effects of drug candidates Informing, calibrating and validating mathematical models with experimental data is a key component of an applied systems biol-ogy investigation and a number of genomic and proteomic

tech-WS 3–5

A human protein atlas

M Uhlen

School of Biotechnology – AlbaNova University Center, Royal

Institute of Technology (KTH), Stockholm, SWEDEN

The new version 4.0 of the Human Protein Atlas

(www.proteinat-las.org) have been generated with more than 6000 validated

anti-bodies corresponding to 5000 human genes The portal contains

more than 5 million high-resolution images generated by

immu-nohistochemistry and confocal microscopy Each image has been

manually annotated and curated by a certified pathologist to

pro-vide a knowledge base for functional studies and to allow

searches and queries about protein profiles in normal and disease

tissue A new structure has been implemented with the inclusion

of all predicted genes (approximately 20 400) with a visualization

of the encoded protein characteristics for all genes A new search

tool is also launched in which advance queries can be performed, including searches for chromosome location, protein class and/or tissue specificity We have used the protein atlas as a discovery tool to find potential biomarkers for cancer diagnostics Some examples of biomarkers in the field of breast cancer, colon cancer and prostate cancer will be discussed

Further reading:

Uhlen, Gra¨slunfd & Sundstro¨m Nature Methods 2008; 5(10): 854–855

Berglund, et al Protein Science 2008; 17: 606–613

Rockberg, Lo¨fbom, Hjelm, Uhlen & Stihl Nature Methods; 5(12): 1039–1045

Bjo¨rling, et al Mol Cell Proteomics 2008; 7(5): 825–844 Barbe, et al Mol Cell Proteomics 2008; 7(3): 499–508

Berglund, et al Mol Cell Proteomics 2008; 7: 2019–2202 Bjo¨rling & Uhlen Mol Cell Proteomics 2008; 7(10): 2028–2037