3MiCRON summary of the activities M18-M33

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Oct 26, 2013 (3 years and 9 months ago)

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1


3MiCRON
s
ummary

of the activities M18
-
M33

The 3MiCRON project started on May 1st, 2010 with a smooth transition from its
predecessor, the FP6 project SIGHT.
All WPs were actively engaged
into s
cientific
work
and contribute to interdisciplinary discussions

and multi
-
university experiments to
validate project findings.

The
majority of the
scheduled activities during the

period

18
-
33

months have been achieved and preparations for the
closing

period have also been
performed. Presented below is an update on eac
h WP.

WP1 Project management

I
n response to the suggestion from
the
Project
Technical Advisor,

the 3MiCRON
management team decided to:
introduce
regular

telephone conferences
, on a monthly
basis,

followed by the distribution of the minutes
. In addition, we

increase
d the

number
of internal project meetings (g
eneral

assembly)
. Meetings took place as follows:

Project m
eetings
:
M25


May 2012 Rome
;
M29


Sept

2012 Stockholm
;
M30


Oct

2012

Genova
;
M34


Feb

2013 Brussels.

Dissemination meeting
s
:

M32


Dec 201
2
Athens, Workshop at Euroecho.

As a consequence the overall engagement of partners into internal research meeting
s

or
study visits was also incr
eased from 5 meetings (period 1
-
18M) to

10 (period 18
-
33M).

The recommendations provided
during the
Expl
oit
ation

Strategy Seminar (ESS) were

further evaluated and

a Business Plan D
evelopment (BPD) seminar provided through
ESIC2 (
Exploitation Strategy and Innovation Consultants
)

framework
and moderated by
representative from
META Group, Nina Mazgan
, and

Nicox, Elizab
eth Robinson
. A report
of BPD seminar including strategy for exploitation of foreground was provided.

WP2 Scientific coordination

This WP goes hand
-
in
-
hand with WP1 but include
s

as well specific points such as
r
epresent
ation of the
c
onsortium and the project
with
in
the
EU. In particula
r
Prof H.
Hebert was invited to
3
cluster meetings:
Paris Dec 2011, Hungary May 2012, Ireland
Sept 2012
. The
positive outcomes from these meetings were establishing new contacts
and collaboration (ex. NA
NOFOL project), submission of the n
ew application for EU
-
funding with partners from other FP7
-
projects

(ex.
EU FP7
-

CATAMARAN project (19
partners)
IMI6 initiative


Nano
-
LOGISTICS project (11 partners)

Uppsala Bio, BioX
framework



CADiLiC project (3 par
tners). In addition
the
consortium in general
,

and
KTH in particular
,

i
ncreased
its
awareness
of the

activity
of

the European Technology
Platform,

ETP, N
anomedicine. It is w
orth noting that most of the partners
have become
active members of this platform.

WP3 Synthesis of a new generation of polymeric microballoons

Two (2) deliverables ha
ve

been prepared during the period M18
-
M33:

D3.4
-

Microballoons with biodegradable polymeric shell

Major
findings are related to an
interesting interplay
between MB prod
uction and

ultrasound. Ultrasound can be used first to transform the microparticles with a liquid
core to the microballoons. After this pr
ocess has been accomplished the
microballoons
can be potentially used as ultrasound contrast agent. The polysaccharide

shell
of this
microballoons
is biodegradable with lysozyme.

Potentially u
sing this new type of balloon we can load drugs in the liquid, hydrophobic
core of the particle. Ultrasound irradiation can be used to burst the balloons to deliver
2


the drug in the v
icinity of the pathological tissue. The
overall
strategy to obtain the
above biodegradable microballoons

is a proof of concept

that can be applied to different
type of biodegradable polymers.

Patent application has been filed.

D3.5
-

Synthesis and surface
modifications of microballoons

Major findings are related to

the

development
of
the multimodal medical imaging
platform where targeting
imaging down to molecular level is considered.

UNITV

propose
d

the
approach
where

multimodality imaging
is supported by
the
implementation of PVA
-
MBs with superparamagnetic iron oxide nanoparticles for MRI
(reported in deliverable 3.3), with radionuclides for SPECT and with targeting
biomolecules for the specific visualization of inflamed endothelium. In this
perspective
,
t
he bifunctional macrocyclic ligand p
-
SCN
-
Bn
-
DOTA was conjugated to
the
microballoon
surface. Furthermore, targeted microballoons were synthesized anchoring, via biotin
-
streptavidin interaction, the monoclonal antibody anti
-
ICAM
-
1 onto MB shell.
Ultrasound
detection of acoustically active leukocyte adhesion molecule
-
targeted
microballoons might provide a means to noninvasively assess the functional status of
the endothelium.

Surflay

propose
d

to
introduce a

positive charge onto the MB surface

using

the
hydraz
ine
-
chemistry
, which enabled the

use
of

LbL
-
technology for derivatizing the
microballoons according to the partners


requirement
s
.
The f
ollowing substances were
coupled to the microballoons via LbL separately and also simultaneously in order to
ensure the
modalities:



magnetite (MRI)



NOTA complexation agent for technetium (SPECT), gallium (PET)



Fluorescence dyes fluoresceine, rhodamine and Cy3 (cell microscopy)



KODAK NIR dye or Perkin Elmer VivoTag 680XL (blue dye) for in vivo NIR tests (IVIS
imaging)



Streptavidin for biotine linkage



Anti
-
ICAM
-
1 antibodies and RGD
-
peptide (targeting)



manganese (MRI)

WP4 Structural/physical characterization of micro
balloons

WP4 consisting of UNITV, Surflay, KTH and UBT determined successfully the
concentration, size, sh
ell thickness, surface properties, micromechanics and ultrasound
response of microballoons including MBs with a standard PVA shell, modified with
super
-
paramagnetic ironoxde nanoparticles and coated with poyldopamine.

One deliverable has been submitted:


D4.1 Physical properties of biodegradable and magnetic MBs

The major findings reported are:

In conclusion the partners in WP4 were able to characterize
in depth
the magnetic MBs
that were identified by the consortium as the most promising candidates for a

multimodal contrast agent. KTH concluded from acoustic screening tests that that the
introduction of iron oxide nanoparticles (physically embed SPIONs) to the PVA shell
does not show any major differences related to the acoustic properties within the rang
e
from 3 to 13 MHz as compared to unmodified PVA balloons (MB
-
pH5
-
RT). Therefore,
both types of MBs can be recommended for further dual modality (US+MRI) testes from
the in
-
vitro performance as ultrasound contrast agent. Structural characterizations were
c
arried out by UBT and KTH. The major structural difference expected from the different
synthesis strategies between
Type A
and
Type B
MBs is the localization of the magnetic
nanoparticles, which KTH could successfully confirmed with their TEM studies.
Type A
MBs have the nanoparticles covalently to the shell surface and Type B have the
3


nanoparticles embedded in the PVA shell. The shell thickness was found by UBT to be
about 130 nm in dried state for both magnetic MBs. Hence no significant change of the
dried shell thickness was observed for the two synthesis strategies and also no change
of the MBs diameter. Mechanical tests carried out by UBT showed that MBs
Type A
are
softer than MBs
Type B
, which is in accordance with the results of the acoustic scree
ning
tests. Hence the two modification strategies did not affect geometric particle parameters
but changed the mechanical response of the shell for MBs (
Type B
and
Type A
). These
observed changes
also

impact on the acoustic in
-
vitro test
.

UNITV characteri
zed the free aldehydegroups on the shell surface, which are relevant to
quantitatively use the aldehyde functional groups present on the surface for further
modification of the shell. In particular this information is important for the assessment
of the am
ount of antibody necessary to target the balloons to the certain receptors.

Surflay investigated the stability, zeta
-
potenital and the

magnetization of magnetic MBs.
They compared
Type B
with
LBL MBs
, that could be a promising alternative for magnetic
MBs
Type A
and
Type B
. From zetapotential experiments Surflay could observe that MBs
Type B are slightly positively charged which should be taken into account for
biocompatibility experiments, because it is known that high positive charges are
cytotoxic for ce
lls. Also in terms of magnetization Surflay showed that the LBL approach
offers the possibility to adjust the magnetization properties of the particles in a
controlle
d manner. However these are preliminary results

performed in case that the
stability probl
ems become a severe issue for the current lead candidates.

WP5 Optimization and upscaling of microballoon preparation

One deliverable has been submitted during the period M18
-
M33:

D 5.3
-

Surface Modification

The major findings are related to the further
modification of
Type C2 magnetic bubbles,
developed
M18

and presented in the

Deliverable 5.2
. The key
feature
of this MBs is
a
charged surface, which makes them suitable for further

surface modification.

The bimodal
(ultrasound and NMR


nuclear magnetic
resonance)
Type C2 magnetic
microbubbles were further
developed

in order to provide

the third modality for SPECT
and targeting. The SPECT
-
ready NOTA
-
MBs were

produced using LBL technology.

Coating of the MBs with Streptavidine enables

targeting via

biotiny
lated antibodies.

WP6 Multimodal imaging approaches

This workpackage address the setting up of US, MRI and SPECT imaging modalities with
reference to the new

Contrast Agent (CA) developed under the 3MICRON project
framework.
Considering the main aim of the WP,
tuning of imaging systems started from
the development of a multimodal phantom. The in vitro study is preliminary and
a
fundamental step before the animal and phase I in vivo experiments.

Two deliverables have

been subm
itted during the period M18
-
33:

D6.3


Image analysis

The major findings are related to
the
development of
an improved tool of image analysis
for

the perfusion of a (multimodal) contrast agent. The development mainly addressed
CEUS analysis, namely Ultraso
und. The results underlined the accomplishment of the
main aims of allowing for a semi
-
automatic assessment of perfusion dynamic pattern
and a integration of information from different imaging modalities (with specific focus
on those more related to the 3M
ICRON project). Although further investigation is
necessary, we can state that our effort during the project is a major step toward data
4


integration and data quantification, which should increase sensitivity and specificity of
diagnosis and therapy decisio
n.

D6.4


Phantom study

The activity was fundamental to understand the properties of different MBs considered
within the project framework. The study started from an analysis of developed
phantoms and it was able to design and construct a multimodality ph
antom to be used
in MRI, US and SPECT. None of the components of the phantom caused any artefacts in
MRI
and SPECT.
During the project time the phantoms have been utilized successfully in
experiments regarding dosage finding, the evaluation of imaging prot
ocols in ultrasound
and to test for fusion imaging.

About US, the innovative magnetic microballoons (MB TYPE A, MB TYPE B 5 mg, 10 mg,
20 mg and 40 mg, MB TYPE C) have been analysed and compared considering a well
-
known (CPS3) and a non
-
conventional (not c
ommercially available) ultrasound signal
processing technique (Chirp CPS3). The measurement and calculation of the contrast
-
to
-
tissue ratio have shown that the structure of the magnetic bubbles can modify the
detection sensitivity and the performance of th
e technique. TYPE A and TYPE B bubbles
can be considered an effective contrast agent for ultrasound. It was observed that, when
the considered contrast agent is composed of polymeric bubbles with SPION trapped
into the shell (TYPE B, in particular TYPE B 2
0 mg and 40 mg), the CTR values are
reduced of roughly 6

8 dB in respect to the values obtained when bubbles with SPION
attached on the membrane (TYPE A) are used. With the SPION inclusion into the shell,
the bubble become stiffer than a bubble with SPION
attached on the shell. This fact
causes a loss of echogenicity. Anyway, even if TYPE B bubbles demonstrated lower
performance (i.e. lower CTR values), they can be satisfactorily detected. On the other
side, the stiffness and the thickness of the shell shou
ld be the cause of TYPE C poor
performance when contrast
-
enhanced ultrasound imaging techniques based on
nonlinear bubble response are assumed (no bubble detection with standard equipment
and human safe conditions set up of the system). Only pulse subtract
ion technique
(SINTEF) is able to partially detect type C bubbles, even if very high acoustic pressures
are required. Further experiments are necessary to better understand the possible
utilization of TYPE C as ultrasound contrast agent.

Shell modificatio
n seems to have an impact on the acoustic properties, as expressed by
the differences in CTR
-
values and acoustic shadowing. Significant shadowing was
observed for all contrast sequences (commercially available) at a concentration of 106
mL
-
1, indicating th
is concentration to be too high for a linear relation between MB
concentration and image intensity.


About MR (from low field (<0.5 T) to high fields (from 1.5 T to 9.4 T)) the phantom
study
allowed the in vitro characteriz
ation of potential contrast agents based on two
types of MBs (type A and B) by using different concentrations, and above all the
definition of a robust protocol for assessment of relaxometric properties of flowing
media, whose qualitative results were conf
irmed by quantitative measurements taken in
static experiments. Two pre
-
clinical in vitro environments were defined: flowing
bubbles in a liquid environment (simulating the presence of the CA in the blood flow)
and static bubble in a gel
-
like environment (
simulating the presence of the CA after
diffusion in an inflamed tissue); despite the strong differences, both in the CA
environment and in the experimental set
-
up used (flowing CA was investigated at 0.25T,
investigation in gel took place at field strengt
hs up to 9.4T), the detectable concentration
limit was independently set at the same level (around 106 mL
-
1), which confirms that
the T2/T2* relaxation process can be used as a nearly field
-
independent contrast
mechanism. The good results seen at low field

with this kind of contrast let us foresee
5


that further sequence parameter optimization at high field could result in even lower
detection threshold, therefore making high
-
quality in vivo examination possible with
moderate amounts of CA injected, reducing
toxicity issues.

Considering the SPECT, 99mTc
-
labelled type A MBs can be visualized using a clinical
SPECT/CT simulating MBs circulating in the rat aorta. The results indicate that the
concept of multimodal imaging using MRI and SPECT/CT to image magnetic

micro
-
bubbles in small animals using clinical MRI and SPECT/CT equipment is feasible.

The overall results proved significant advancements in knowledge about the develop
-
ment and the applicability of MBs as multimodality contrast agent (CA). Although
chal
lenge and further experiments are necessary to collect key data, the results obtained
within the WP6 proved the feasibility of use of such a complex CA.

WP7 Advanced bi
ocompa
tibility and biological impact

In vitro biocompability tests performed by UCD hav
e shown that all of the MBs
produced in the consortium, with the exception of LbL05 and LbL08, are stable and
monodispere after exposure to different biological fluids that mimicked the
in vitro/in
vivo

conditions (i.e. 6
-
10% and 100% human serum and plasm
a). Protein corona
assessment has shown that the protein interactions with the MBs' surface are relatively
weak suggesting that the proteins are easily displaced by other binders. Several
desopsonin proteins, such as serum albumin, have been identified as
major protein
corona binders while desosponin proteins bind to a less extent. This finding suggests
that MBs are not likely to be recognized by macrophages in the blood stream and thus
should have a prolonged circulation life in the system, which is an exc
ellent result in
terms of their potential for use as contrast agents where circulation time will be a
critical factor.

Based on CBNI, UC physochemical characterization and protein corona study all the MBs
used, with the exception of LBL05 and LBL08 that a
ppear highly aggregate, are likely to
be biocompatible.


In vitro toxicity studies performed in INT has shown a dose response cellular activity
after incubation with MBs after 3 and 7 days exposure and cell cultures were examined
for cell morphology and de
nsity by phase contrast microscopy.

MB

type and a c
lear outer layer composition have

a clear effect on the cellular
proliferation. This table

summarizes the in vitro biocompability findings (10% human
plasma) where MBs toxicity was considered where cell vi
ability was exciding 80%.

MBs

Name

Biocompability

in vitro

study

Cell growth inhibition

PVA



pH 5 RT



Not toxic



0

SPIONS:CHITOX

Type A (magnetic)

Toxic at high dose



20
-
40%

SPIONS



Type B (magnetic)

Toxic at high dose


20
-
50%

NOTA
-
PVA


TypeC
(magnetic)

Toxic





80
-
95%

LBL01



Unmodified


Modest toxicity



15
-
30%

LBL02



Cationic MBs


Toxic at high dose


15
-
40%

LBL03



AG
-
MB/PSS


Modest toxicity



15
-
30%

LBL04



AG
-
MB/PMAA


Not toxic



0

LBL05



AG
-
MB/Carrageenan

Not toxic



0

LBL06



AG
-
MB/Al
bumin

Modest toxicity




10
-
20%

LBL07



AG
-
MB/PSS/PAH

Toxic





30
-
40%

LBL08



AG
-
MB/PSS/Chitosane

Toxic




30
-
40%

LBL09



AG
-
MB/PSS/PEI


Toxic at high dose


20
-
40%

LBL10



AG
-
MB/PSS/PaOEt

Not toxic



0

LBL11



AG
-
MB/PSS/PDA

Modest toxicity




10
-
15%

6


1 del
iverable

has been submitted:

D7.1


consequences of main proteins: assessment

of the potential biological
consequences of

main proteins in corona

1 deliverable is in preparation D7.2
-

Identification of "new" proteins

WP8:
In vivo

testing of multi
-
modality MB:

Four

major topics such as contrast enhancement in ultrasound, MRI, SPECT/PET and
distribution of the MBs have been investigated.

1.

Contrast enhancement US


mouse

Clear increase of grayscale intensities after injection of 8
0μl 5x108
MB/ml in liver,
spleen and blood with all tested MBs has been observed. It was suggested, even though
In comparison to Sonovue and plain MB, blood half

live time of MBs Type C is shorter,
that
MB Type C might be used as a US/MRI liver and spleen
specific contrast agent for
the detection of certain types of tumors in liver or spleen

2.

Contrast enhancement MRI relaxometry


mouse,

rat, Type B and Type C MB

Strong decrease of T2 times in liver and kidney after injection of 70μl 1,12x109
Type B
MB a
nd 70 μl 5x108 of novel Type C MB

was reported. Moreover f
urther decrease

T2
times

in the first 3

4 days after injection of Type B and Type C in the liver

was observed.
We demonstrated that

MB or at least SPION stay in liver and kidney after injection in
h
igh amount for at least one week. Body clearance
was estimated to be
approx. 4 weeks.

3

Contrast enhancement SPECT

Regarding SPECT studies it was reported that l
abelling of chitosan and Nota coated MB
using 99mTechnetium is feasible with low efficiency
. Ho
wever d
ue to detection of
activity in the bladder, stability of
MB
-
99mTc
complexation remains disputable
.

Dynamic SPECT imaging shows the trapping

of MBs within very short time. Moreover


whole body distribution

was not observed
at any time

during the experiments.

4
Distribution of MB Histological analysis

.

4 types of tissue were inspected by histological analysis following by injection of MBs
Type B.

LUNG. A large number of MB B, distributed throughout the tissue section is present in
the
lung. The blue deposits are attributable to intact microbubbles as evidenced in
magnification. In particular, in some areas of the lung large aggregates of MB B are
shown and they probably could cause veno


occlusive disease.

LIVER. In the tissue section w
e observed a small number of monodisperse MB B. On the
contrary the results obtained in our laboratory indicated a great accumulation of MB in
liver of animals injected with naked FITC

MB.

SPLEEN. The blue signs are due to iron deposits and rarely to intac
t MB B. It is not
possible to conclude whether these deposits are due to iron released from MB B or to
the normal metabolic activity of the spleen as we did not receive a section of spleen of
control animals, not inoculated.

KIDNEY. We rarely found signs o
f intact MB B in the sections of kidney examined, but
frequently areas appeared with small deposits of iron in the cytoplasm of cells of the
kidney that may have incorporated fragments of MB B.

WP9:
In vivo

and
in vitro

detection of infl
amed tissue using m
icroballoons


1 deliverable
has been submitted:

7


D9.1


Behaviour in relation to surfaces and cells

The major findings related to the natural ability of macrophage cells to phagocytose
foreign matter. It is shown that plain, unmodified MBs are taken up to a

certain extent,
but only after minimum 3 h incubation, while the MBs modified with SPIONs are
phagocytosed faster. The modification where SPIONs are chemically attached to the
surface of the chitosan coated MB is taken up already after 30 min. The MBs wit
h
SPIONs physically adsorbed in the PVA
-
shell is taken up at a slightly slower fashion.

The experiments using endothelial cells show no significant uptake or adhesion of plain,
unmodified MBs. Endothelial cell models become more of a relevant model when t
hey
are stimulated to over
-
express certain adhesion molecules to mimic inflamed tissue. One
can then expect to see more adhesion of the balloons, especially if the balloons also
carry targeting moieties towards these adhesion molecules.

WP10:
Pre
-
clinical
testing of multimodality microballoons in animals
:


6 deliver
ables are scheduled for this WP
:

D 10.1 Visualization of microballoons

AIM:
The visualization of attachment of targeted microballoons using US, MRI, SPECT &
tissue samples using confocal
microscopy

STATUS:

a)
The biodistribution of numerous MB has been shown (WP8)
; b)
The
biological half
-
life has been shown (WP8)

D10.2 Accumulation of

magnetic balloons

AIM
: Visualization of accumulation of magnetic balloons targeted to specific areas in th
e
extremities by using external magnets will be shown using scintigraphy

STATUS:

a)
Verification of model ongoing
; b)
NOTA SPION MB will be used March
-

April

D10.3 Release of air and fluorescent material

AIM
: Visualization of local release of air and fluo
rescent material

STATUS:

Release of air shown by MB bursts
;
Release of fluorescent material


not solved

D10.4 Treatment effect

AIM
: The treatment effect will be shown using US, MRI, SPECT &
microscopy/

histo
-
pathology

STATUS
: a)
Verification of model ongo
ing
; b)
Treatment effect experiments planned
March and April using all modalities

D10.5 Improved Segmentation

AIM
: Endocardial border delineation using 3MiCRON MB


a comparison with the
commercially available contrast agent SonoVue®.

STATUS
:

a)
The highest mean score was observed for 5 ml 3MiCRON MB indicating this
injection to be preferable for endocardial border delineation. According to the blinded
observers, this injection also had the longest duration of clinically useful contrast. For
the i
njection of 5 ml 3MiCRON MB, both the mean score and the duration of clinical
useful contrast were comparable with the commercially available contrast agent
SonoVue®
. b)
When comparing the mean score from

different regions (apex, mid and
base) there were
no significant difference between SonoVue® and any of the 3MiCRON
MBs injections for the apex region. For the other two regions, Sonovue® showed better
performance then all 3MiCRON MB injections except for the mid region when injecting
1.5 ml 3MiCRON MB.

c
)
Injections of contrast agent do not affect the oxygen saturation,
heart rate or arterial pressure.

8


D 10.6 Perfusion studies

AIM:
Reproducibility of quantitative perfusion measurements in the myocardium.

STATUS:

a)
SonoVue® and 3MiCRON MB did not give
the same result for time and flow
variables during perfusion imaging. The difference was especially large in continuous
imaging.

b)
For repeated measurements in the same pig (continuous imaging), the
reproducibility of time variables appears to be better f
or SonoVue® than for 3MiCRON
MB, and in the same range for flow measurements.

c)

The difference between y
ROI X
(t)

and y
approx
(t) was significant higher for 3MiCRON MB
s

in all regions except two,
indicating better reproducibility for SonoVue®

than for 3MiC
RON.

d)
The suppression
of tissue signal and the enhancement of MB signals are important in contrast perfusion
imaging. Detecting small amounts of 3MiCRON MBs have shown to be difficult in
previous studies with commercially available contrast sequences. Sp
ecific contrast
sequences, optimized for polymer
-
shelled MBs, would probably yield a better result for
3MiCRON MB in future studies on myocardial perfusion.

WP11 Dissemination and exploitation promotion

The major objectives of WP11 are to ensure a wide

pu
blic

awareness

of

the results of
the project, to identify target industrial and/or academic sectors and/or clinical
institutions
,

and to develop effective strategies for partnership with industry and
research institutes. The main activities within WP11 dur
ing the time period M19
-
M33
are listed below:



3MiCRON workshop



Cluster meetings



Public activities (conference contributions,
external activities
, educational work, scientific
publications)



Business plan development seminar



Webpage and exchange of informa
tion

3MiCRON Workshop

The
3MiCRON workshop

with the title

Improving skills of contrast echocardiography
and new perspectives in multimodal contrast imaging


was the final deliverable (D11.4)
in WP11.

W
e decided to locate the 3MiCRON workshop at one of
the largest
echocardio
graphic meetings in the world,
EURO
ECHO & other Imaging Modalities, in
order to attract end
-
users of contrast agent such as medical do
ctors, sonographers and
nurses.
EUROECHO & other Imaging Modalities is the annual meeting of the Eur
opean
Association of Cardiovascular Imaging (EACVI) and the congress took
place in Athens,
Greece, 5
-
8th
December 2013.
Over the years, the meeting has grown more and more to
become one of the largest echo meetings in the world with over 3,000 attendances
and
has also expanded its focus to other imaging modalities.


During the
3MiCRON workshop
,
Reidar Winter

(KTH)
, Lars
-
Åke Brodin

(KTH)
, Kenneth
Caidahl

(KI)

and Gaio Paradossi
(UNITV) interactively
presented clinical benefits of
contrast
-
enhanced ultrasound

imaging as well as the 3MiCRON concept for end
-
users.
The workshop was in the format of an “imaging campus session”, meaning that the same
session
(1.5h)
was presented four times during the congress. The workshop attracted
persons from around 20 different

countries.


Public activities

Figures below demo
n
strate

all public activities per
formed within 3MiCRON during M18
-
M33. Compared with M1
-
18, the number of
submitted and papers in progress

has
increased.


9






Educational work



10


Business plan development

seminar

Throughout the project, i
nteresting results have been seen

and a technology platform
has been formed. However, the consortium was not convinced about how and in which
way to approach the market with these commercially ideas.
For this reason, a bus
iness
plan development seminar service was requested thought ESIC2 (Exploitation Strategy
and Innovation Consultants) initiative.
In general, the business plan development
seminar aims to support EU project partners in
developing

a real complete business
plan including a proper

market analysis, a reliable financial foresight and a convincing
market strategy
.

The seminar was held on the 17th of October 2012 in Genoa, Italy
,

at the premises of the
industrial p
artner of the project
Esaote S.p
.A.

3MiCRON was represented by
one
industr
ial partner (Dr. Sergio Paddeu, Esaote), two

academi
c

p
artners (Prof. Lars
-
Åke
Brodin and Dr
Dmitry Grishenkov
,

KTH, Prof. Gaio Paradossi,
UNITV
)

and
one clinical
partner (Dr. Torkel Brismar, KI
).

Two exp
erts, Nina Mazgan (Meta Group) and Elizabeth
Robinson (Nicox), were moderating the seminar in order to identify main critical issues
at this point and to recommend the next steps for exploiting the project results. The
following critical issues for further

business development were identified:



Low readiness to start a company



No clear product/service has been identified



Advantages of results has not been clearly identified



Market potential has not been specified



Potential next steps are not clear

However, a
fter fruitful and productive discussion
s

the next steps in the process towards
exploi
tation of the project results were

summarized:

1) Project partners have to agree on a common vision and next steps, and shape a small
focus group of interested partners

2)

Potentially develop an internal document identifying different options for
commerci
alization

3) Requesting a complete business plan development service could serve as an exercise
for the focus group

4) Organize focus group meetings to discuss further ste
ps

More detailed information regarding
the
ESIC2 initiative, business plan preparation,
financial schemes and investor readiness in general as well as specific to the 3MiCRON
project recommendations from the expert regarding composition of the research tea
m,
exploitable results and market needs, IPR and patenting strategy, technological and
commercial properties of the developed product is presented in the report delivered by
META Group.

Peer review
ed

publication
s:

1)

F. Saglimbeni, S. Bianchi, G. Bolognesi, G
. Paradossi and R. Di Leonardo

"Optical
characterization of an individual polymer
-
shelled microbubble structure via digital
holography"

Soft Matter 8 , 8822
-
8825 (2012)

2)

Torkel B. Brismar, Dmitry Grishenkov, Björn Gustafsson, Johan Härmark, Åsa Barrefelt,
Satya V. V. N. Kothapalli, Silvia Margheritelli, Letizia Oddo, Kenneth Caidahl, Hans Hebert,
and Gaio Paradossi

"Magnetite Nanoparticles Can Be Coupled to Microbubbles to Support
Multimodal Imaging"

Biomacromolecules 13 (5), 1390

1399 (2012)

3)

Trucco, C. Sc
iallero, G. Paradossi

"A preliminary in vitro assessment of polymer
-
shelled
microbubbles in contrast
-
enhanced ultrasound imaging"

Ultrasonics 52 (3) , 456
-
464 (2012)

11


4)

Barbara Cerroni, Ester Chiessi, Silvia Margheritelli, Letizia Oddo, and Gaio Paradossi

"
Polymer Shelled Microparticles for a Targeted Doxorubicin Delivery in Cancer Therapy"

Biomacromolecules, 12 (3), 593
-
601 (2011)

5)

Dmitry Grishenkov, Leif Kari, Lars
-
Åke Brodin, Torkel B. Brismar and Gaio Paradossi

"In
vitro contrast
-
enhanced ultrasound
measurements of capillary microcirculation: Comparison
between polymer
-

and phospholipid
-
shelled microbubbles"

Ultrasonics, 51(1), 40
-
48 (2011)

6)

Gaio Paradossi “Hydrogels Formed by Cross
-
Linked Poly(vinyl Alcohol)” in Polymeric
Biomaterials:Structure and Fu
nction, Vol 1; Eds S. Dumitriu, V. Popa.CRC Press January
2013.

7)

Surface Coatings Shape the Protein Corona of SPIONs with Relevance to Their Application in
Vivo. Ange

la Jedlovszky
-
Hajdu, Francesca Baldelli Bombelli, Marco P. Monopoli,§ Etelka
Tombacz, Kenn
eth A. Dawson

Submitted publications:

1)

R. Villa, B. Cerroni, L. Viganò, S. Margheritelli, G. Abolafio, L. Oddo, G. Paradossi, N.
Zaffaroni.
“Targeted doxorubicin delivery by chitosan
-
galactosylated modified polymer
microbubbles to hepatocarcinoma cells”
submitted, 2012

2)

Åsa Barrefelt, Gaio Paradossi, Silvia Margheritelli, Letizia Oddo, Peter Aspelin, Moustapha
Hassan, Torkel Brismar and Moustapha Hassan “Biodistribution and kinetics of magnetic
microbubbles containing SPIONs using MRI R2* and T1 mapping” s
ubmitted to BMC
Medical Imaging, 2013

3)

Åsa A Barrefelt, Torkel B Brismar, Gabriella Egri, Peter Aspelin, Letizia Oddo, Silvia
Margheritelli,

Annie Olsson, Kenneth Caidahl, Gaio Paradossi, Lars Dähne, Rimma Axelsson
and Moustapha Hassan.

“In vivo imaging of
ligand functionalized 99mTc
-
labeled
microbubbles using SPECT/CT

and MRI”. Submitted 2013

4)

Melanie Poehlmann DG, Satya V. V. N. Kothapalli, Johan Härmark, Alexandra Philipp,
Roland Höller, Maximilian Seuss, Gaio Paradossi, Andreas Fery. “On the interplay of
structural, mechanical and acoustic behaviour of multifunctional magnetic microbubbles”
Submitted to Soft matter 2012

5)

Malin Larsson, Anna Bjällmark, Matilda Larsson, Letizia Oddo,

Silvia Margheritelli,

Gaio
Paradossi, Jacek Nowak, Kenneth Caidahl,

Lars
-
Åke

Brodin “Acoustic response evaluation of
novel polymer
-
shelled contrast agents using clinical ultrasound systems
-
An experimental
study in a tissue mimicking flow phantom” Submitted to Ultrasound in Medicine and Biology,
2012.

Papers in preparation:

1)

Charac
terization of protein corona and dispersion properties of microballoons in biological
fluid. Sha Wan, Marco P Monopoli, Gaio Paradossi, Letizia Oddo, Iseult Lynch, Lars Dahne,
Gabriella Egri, Kenneth Dawson.

2)

Characterization of protein corona and dispersio
n properties of microballoons in animal tissue.
Sha Wan, Marco P Monopoli, Gaio Paradossi, Letizia Oddo, Iseult Lynch, Lars Dahne,
Gabriella Egri, Bjorn Gustafsson, Kenneth Caidhal, Nadia Zaffaroni, Raffaella Villa, Kenneth
Dawson.

Two review
-
type papers b
eing led by UCD with contributions from all partners:



Review of the current state of development of MBs as contrast agents and roadmap of next
steps to achieve commercial application



Lessons learnt from SIGHT and 3MiCRON regarding the design and clinical i
mplementation
of MB
-
based contrast agents.