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 Europeanlandscape needs some leading institutions that are able to coagulate the regional research potential inspecific 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 20111, followed by Al.I.Cuza University of Iasi.

1 http://www.ad-astra.ro/cartea-alba/articles.php?institution_id=5&topic_id=QE2 www.euchems.org D2. ObjectivesThe 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 proposalA1.1. 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 polymersA2.1. 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 polymersA3.1. 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 (Ca2+, Mg2+, Zn2+) 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 microspheresA4.1. 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 microspheresA5.1. 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 studiesA6.1. 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 LD50 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 projectObjectiveRisksBack-up solutions

O1O2O3 Medium reproducibility of particles preparation process Incorrect establishment of important variable parameters

T1T2T3 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 budgetThe 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 testsIn vitro release studies for active principles using transdermal model systems (on Franz cells)