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D1. Problematica.

Se va justifica motivaia tiinific a temei proiectului prin delimitarea problemei


abordate n contextul tiinific actual. Se vor evidenia urmtoarele trei aspecte: (1) importana
problemei din punct de vedere tiinific, tehnologic, socio-economic sau cultural; (2) elementele de
dificultate ale problemei; (3) limitrile abordrilor curente, prin analiza stadiului actual al
cunoaterii legat de tematica proiectului.
D2. Obiective. Se va prezenta abordarea proiectului la nivel de principiu, cu evidenierea
urmtoarelor trei aspecte: (1) obiectivele concrete ale proiectului; (2) elementele de originalitate i
inovaie pe care implementarea obiectivelor le aduce domeniului, raportat la stadiul actual al
cunoaterii i raportat la proiectele derulate anterior de aplicant; (3) impactul preconizat al
proiectului n cadrul mai larg al domeniului tiinific.
D3. Metodologie. Se va prezenta n detaliu metodologia cercetrii, precizndu-se, pe ct posibil,
anumite inte intermediare cheie. n elaborarea acestei seciuni se vor evidenia urmtoarele aspecte:
(1) alegerea metodelor i instrumentelor de investigaie, prin raportare la cele mai noi abordri n
domeniul temei, precum i modul n care acestea vor fi integrate; (2) un plan de lucru, ealonat n
timp, ce va descrie modul de organizare i planificare al proiectului, n raport cu obiectivele
propuse; (3) descrierea potenialelor riscuri i abordrile prin care aceste riscuri ar urma s fie
adresate.
D4. Resurse i buget. Vor fi prezentate deopotriv resursele existente, relevante pentru desfurarea
proiectului, precum i cele necesare i care vor fi achiziionate n cadrul proiectului. Se vor adresa
n special urmtoarele aspecte: (1) argumentarea adecvrii infrastructurii de cercetare disponibile
pentru ndeplinirea obiectivelor proiectului n timpul alocat; (2) argumentarea necesitii
achiziionrii unor noi echipamente de mare valoare (peste 60.000 lei, pre ce include i TVA), prin
raportarea la obiectivele proiectului; (3) specificarea i justificarea distribuiei bugetului pe tipuri de
cheltuieli i pe ani calendaristici.

(20%) Please assess the overall solution described in the proposal in the context of the current state-of-the-
art and its potential future impact (see section D1, D2). Please comment on the following aspects: (1)
significance and the difficulty of the problem being addressed; (2) the originality of the proposed solution
and the appropriateness of the objectives; (3) the potential to advance knowledge in the field and to influence
the direction of thought and activity.
2.2 (20%) Please assess the method and work plan as defined by the proposal as a concrete approach to
reach the envisioned solution (see section D3). Please comment on how well selected are the methods,
design and investigation tools and on the effectiveness off the work-plan within the proposed timescale
and resources. Have potential problem areas been appropriately discussed, and have alternative
approaches been mentioned


D1. Problems
After harmonization of standard in the university education throughout the European
Community, including Romania by Bologna process, all research areas still require many top
researchers.



Chemistry as large research domain represents a priority of ANR-ANCS. The research
domains approached by the current project proposal are to be found within the eight major key areas
in the chemical sciences, where scientific breakthrough is required to meet the global challenges
2
.
Moreover, the proposal enrolls in the priority themes of European research area (ERA) by
interdisciplinary research, aiming to the development of advanced materials (nanomaterials and
nanotechnology) as delivery systems of active principles with applications in the human health
sector (population health improvement).




The integration of the Romanian chemical network in the European
landscape needs some leading institutions that are able to coagulate the regional research potential in
specific areas and to serve as best practice examples.


Colloids can be precursors to nanomaterials, composites, and other materials. They are used in inks,
paints, and other emulsions, pharmaceutical products, and are found naturally in clays, biological
fluids such as blood, natural organic matter colloids, and petroleum and geological processes.
Considering the research domain of Materials Engineering-Biomaterials, Petru Poni Institute
of Macromolecular Chemistry of Iasi, was the main deliverer of research articles in 2011
1
, followed
by Al.I.Cuza University of Iasi.

1
http://www.ad-astra.ro/cartea-alba/articles.php?institution_id=5&topic_id=QE
2
www.euchems.org

D2. Objectives
The main objective of this proposal is the developing and characterization of novel nano and
microparticulated biomaterials, based on natural and synthetic polymers, with application in
retarded, controlled and sustained drug release for the treatment of various health deficiencies.









D3. Methodology
The accomplishment of the project objectives implies multidisciplinary research and
collaboration with other researcher teams. The main objectives and associated activities of the
present proposal are described below:
O1. Thorough bibliography study concerning the breakthroughs in the research field of
the proposal
A1.1. Literature studies concerning the breakthroughs in the field of synthetic and natural
polymers applications in the development of biomaterials and also concerning the appropriate use
of these polymer based materials in the field of medicine and pharmacy.
A1.2. Literature studies concerning the latest and most feasible techniques used for polymer
nanocapsules, nanoparticles and microspheres preparation and characterization.
O2. Preliminary experimental studies on the preparation of nanoparticles based on
synthetic polymers
A2.1. Preparation of nanocapsules and nanospheres based on poly (-caprolactone), Eudragit
R100 and Eudragit E100 by nanoprecipitation method, using ionic or non-ionic surfactants
(benzalkonium chloride, sodium dodecyl sulphate, Span80, etc.).
A2.2. Preliminary characterization of synthesised polymer nanoparticles. Determination of
particle morphology (surface aspect, size, size polydispersity- scanning and transmission electron
microscopy), stability and surface charge (zeta potential).
A2.3. Selection of the appropriate materials (i.e. polymer) and technique for nanoparticles
preparation.
The preparation process of nanoparticles and nanocapsules by nanoprecipitation (also called
solvent displacement method) is a facile technique, usually employed for obtaining nanoparticles
based on synthetic polymers. It implies the instant formation of nanoparticles suspensions by slowly
adding the solvent phase to the non-solvent phase, under moderate stirring, followed by total
removal of the solvent (under reduced pressure). Essentially, the solvent phase consists in a solution
of synthetic polymer in a solvent or mixture of solvent (i.e. acetone, ethanol, hexane, methylen
chloride), oil (forming the nanocapsules core), a lipophilic surfactant and the active substance. The
non-solvent phase often (but necessarily) consists of an aqueous solution of one or more naturally
occurring or synthetic surfactants. The technique is simple and does not imply the use of toxic
organic solvents or complicated machinery; also, the good reproducibility and high yield of
encapsulation recommend this method for preparing nanoparticles as drug delivery systems.
Several synthetic polymers will be used as film (nanocapsules) or sphere (nanospheres)
forming substance: poly (-caprolactone), Eudragit E100 or Eudragit R100. Nanoparticle
suspensions will be stabilized by ionic (benzalkonium chloride, sodium dodecyl sulphate) and non-
ionic surfactants (Span80, Tween80). The choice of surfactant will be dictated by the charge of the
natural polymer which forms the microparticles.
The nanoparticles size and morphology will be evaluated by scanning and transmission
electron microscopy, confocal fluorescence microscopy, atomic force microscopy and laser
diffractometry. Particle stability will be evaluated by size measurements in time and determination
of zeta potential in different aqueous media.
O3. Preliminary experimental studies on the preparation of microparticles based on
natural polymers
A3.1. Preparation of microspheres based on chitosan, sodium alginate, pectin and pullulan,
by polymer crosslinking with ionic or covalent agents.
A3.2. Preliminary characterization of polymer microspheres. Determination of particle
morphology (surface aspect, size, size polydispersity), surface charge and chemical structure.
A3.3. Selection of the appropriate materials and technique for the preparation of
microspheres.
The polymer microspheres will be prepared by polymer crosslinking in O/W emulsion . The
method consists in emulsifying an aqueous phase (solutions of polymers) into an oily phase (non-
miscible with water), in the presence of appropriate surfactants, followed by polymer crosslinking
with ionic and/or covalent agents and the formation of stable polymer microspheres. Generally, the
use of ionic crosslinkers decreases the need for covalent crosslinking agents (often toxic). The
selection of ionic and covalent crosslinkers will be performed according to the polymer used:
sodium phosphate, tripolyphosphate, glutaraldehyde, genipin will be used for chitosan; salts of
divalent metals (Ca
2+
, Mg
2+
,

Zn
2+
) will be used for sodium alginate and pectin; epichlorohydrin will
be used for pullulan crosslinking.
The polymer microspheres will be characterized by determining particles size and size
polydispersity (scanning electron microscopy, confocal fluorescence microscopy, atomic force
microscopy, optical microscopy and laser diffractometry), surface charge (zeta potential in various
aqueous media) and chemical structure (elemental analysis and FT-IR spectroscopy).
O4. The designing of the experimental protocol for the preparation of polymer
nanoparticles and microspheres
A4.1. Optimization study concerning the nanoparticles preparation process, by variation of
certain important parameters (i.e. polymer nature and content, phase nature and ratio, etc.).
Synthesis and characterization.
A4.2. Optimization study concerning the polymer microspheres preparation process, by
variation of certain important parameters (i.e. polymer nature, phase ratio, type of crosslinker,
nature of non-solvent, etc.). Synthesis and characterization.
According to their final applications, nano and microparticles (as drug delivery systems)
require possessing certain morphological and biological behavioural characteristics, such as size
(from few nm for intravenous administration to few m for ocular formulations), toxicity or
bioavailability.
Various process parameters considered important for the preparation of nano and
microparticles with specific required characteristics will be varied and their influence will be
rigorously evaluated. Thus, in the case of synthesising nanoparticles, several important parameters
will be varied (nature and content of synthetic polymer, phases ratio and nature of solvent, type and
amount of surfactant, type of particles-capsules or spheres) and their influence on particle
characteristics (type, size, size polydispersity, stability in time, surface charge, swelling capacity in
simulated biological media, by thermogravimetric -TA method).
Optimization of microspheres preparation process implies the variation of certain parameters
such as polymer nature and amount, phase nature and ratio, type and amount of crosslinker, type
and amount of surfactant and emulsification related parameters (speed, time and apparatus) and the
evaluation of their influence on microspheres morphology (aspect, rugosity, size and size
distribution) by scanning electron microscopy, confocal fluorescence microscopy, atomic force
microscopy, optical microscopy and laser diffractometry, surface charge (zeta potential), swelling
behaviour in simulated biological fluids by thermogravimetric-TA method and their chemical
structure (by FT-IR spectroscopy and elemental analysis).
The indicators for successful fulfilment of O4 will be:
- The correct establishment of the process parameters allowing the preparation of polymer
nano and microparticles with the required characteristics, according to desired applications;
- Satisfactory reproducibility of particles characteristics.
O5. Preparation and characterization of nanoparticles entrapped into microspheres
A5.1. Nanocapsules and nanospheres preparation (by solvent displacement technique),
considering the parameters which influence the nanosystems properties.
A5.2. Preparation of microspheres containing nanospheres or nanocapsules (by polymer
crosslinking in emulsion), considering the parameters which influence the microparticles properties.
A5.3. Study of the influence of the overall varying parameters on the physical properties of
the microspheres containing nanoparticles (morphology, surface charge, swelling behaviour, etc.).
The confinement of polymer nanoparticles into microspheres will be performed in the
microparticles preparation step, by mixing the nanoparticles suspension into the aqueous phase (the
solution of natural polymer). The complex systems will be characterized by evaluating their
morphology (scanning and transmission electron microscopy, cryo-electron microscopy, atomic
force microscopy), charge (zeta potential), chemical structure (FT-IR spectroscopy, elemental
analysis), swelling behaviour in aqueous media (thermogravimetric-TA method).
The indicators for successful fulfilment of O5 will be:
- The preparation of complex polymer systems with adequate characteristics for the desired
application by good correlation between varying parameters and material properties;
- Satisfactory reproducibility of particles characteristics.
O6. Drug immobilization and release studies
A6.1. Selection of various hydrophilic and lipophilic drugs and model molecules (caffeine,
chloramphenicol, indomethacin, Sudan Red, Nile Red, vitamin E, 5-fluorouracil, etc.), according to
the desidered application.
A6.2. Drug loading from the formulation preparation step or by diffusion.
A6.3. In vitro evaluation of drug release kinetics in aqueous media simulating biological
fluids for all formulations. Comparative study.
A6.4. In vivo evaluation of drug release for all selected formulations. Comparative study.
Various hydrophilic and/or lipophilic active principles can be entrapped into
O7. Biological studies
A7.1. Toxicity tests on selected prepared formulations by determination of LD
50
on mice.
A7.2. Biocompatibility studies on selected prepared formulations (by evaluation of
hemolysis, prothrombin time and internation normalized ratio of blood in contact with the selected
formulations).
A7.3. Biodegradability studies on selected prepared formulations (evaluated in simulated
biologic fluids).
A7.4. Bioadhesion of selected formulations (on various types of tissues or mucous
membranes, according to their desired application).
The possible risks involved in project management and corresponding back-up solutions are
presented in Table1.

Table1. Scientific risks and back-up solutions of the project
Objective Risks Back-up solutions
O1
O2
O3
Medium reproducibility of particles
preparation process
Incorrect establishment of
important variable parameters




T1
T2
T3
Preparation process reproducibility of
the particles having certain characteristics
(with a maximal error limit under 10 %)
Increasing the number of experiments and
strict control of process parameters.
Obtaining of a narrow dimensional
polydispersity
Better controlling of the process parameters
(type and amount of surfactant, hydrodynamic
regime, phase ratio)
T4 Obtaining of particles with medium
toxic
Advanced purification of the particles
Obtaining particles with medium
biocompatibility
Advanced purification of the particles
Particles low drug loading capacity Using similar drugs with different solubility
T5 Obtaining a low accuracy model, due
to the errors affecting experimental data
or incomplete available data
Developing methodologies which combine
neural network with regression based method,
special for incomplete information of the
measurements.
T6 Difficulties at conversion of
mathematical model into experimental
variables
Difficult fitting procedure
Choice of the most appropriate mathematical
technique
T7 same as T5 inadequate model for
optimization procedure
Using models based on non-parametric
methods provided by machine learning.



Figure 2. Project structure and information flow

D4. Resources and budget
The project host institution is Petru Poni Institute of Macromolecular Chemistry (ICMPP), Iasi,
where numerous research platforms can support my research activity. Moreover, the priority
thematic areas of the new platform IntelCentre (Advanced Research Center for Bionanoconjugates
and Biopolymers) are focused on materials, products and innovative processes and health.
Regarding the infrastructure, ICMPP has a wide range of research equipment for the synthesis and
characterization of polymeric biomaterials, of which I would like to mention only the devices
necessary for the fulfilment of this project:
Equipment for synthesis/preparation of materials: magnetic, mechanical and high rotation
homogenizers; lyophilisation equipment Alpha 2-4LDplus; ultrasound Sonics VCX 750; millipore
water filtration system; ROTAVAP reduced pressure rotation evaporator; centrifuge Hettich
Universal 320R.
Equipment for characterization of materials: elemental analyser CHNS 2400 II
PerkinElmer; Titration system Titroline Alpha Plus; UV-vis spectrometer Specord 200Analytik
Jena; HPLC system Shimadzu; FT-IT spectrometer Bruker Vertex 70; NMR spectrometer Bruker
Avance DRX 400; fluorescence spectrophotometer Model LS 55 PerkinElmer; scanning probe
microscope (AFM) Solver Pro-M; optical microscope-fluorescence; scanning electron microscope
Tesla BS 301; particle size and charge analyzers-Mastesizer, Zetasizer and Nanosizer.
The biological tests will be performed by Intelcentre laboratories and by research teams
(physician and pharmacologists) from other institutes and universities.
The mentor, Dr. Silvia Vasiliu and all members of Laboratory of Functional Polymers have
access to all facilities of ICMPP and no particular equipment need to be purchased for achieving the
goals of the project.
The project overhead will be 20% of the direct expences, as result of a a mutual agreement
between with the host institution, a writen justification being presented withing the project proposal.








We will study the influence of several preparation process parameters on particles/capsules drug
loading and release characteristics for use the particles/capsules in biomedical applications: concentration of
polymer solution, phases ratio (aqueous / organic), surfactants amount, hydrodynamic regime, ratio between
the phases, crosslinking duration.



4. The last part of the project will concern the study of the analyzed systems from the point of view of biomedical potential
applications. Will be taken into account the following:
The toxicity of the liposomes-polymer systems by measuring median letal dose (DL 50)
The biocompatibility of the systems by their application / implantation to mice and then studying the body reaction (histology)
Cell bioadhesivity tests
In vitro release studies for active principles using transdermal model systems (on Franz cells)