Sessione 7 Biopolimeri - ictmp.ct.cnr.it · e l’immobilizzazione di cellule ed enzimi. Tali...

XVIII Convegno Italiano di Scienza e Tecnologia delle Macromolecole, Catania 16-20 settembre 2007

Sessione 7 Biopolimeri

XVIII Convegno Italiano di Scienza e Tecnologia delle Macromolecole, Catania 16-20 settembre 2007 S7 O 1

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SISTEMI MICROPARTICELLARI COME METODO INNOVATIVO PER LA RETICOLAZIONE CONTROLLATA DELL’ALGINATO

M. G. Cascone, P. Giusti, L. Lazzeri

Università degli Studi di Pisa, Dipartimento di Ingeneria Chimica, Chimica Industriale e Scienza dei Materiali, Via Diotisalvi 2, 56126, Pisa, Italy; e-mail: [email protected]

Introduzione L’alginato, commercialmente disponibile come sale sodico dell’acido alginico, è un polisaccaride lineare, estratto da vari tipi di alghe brune, le cui proprietà biologiche (biocompatibilità, biodegradabilità e non immunogenicità) lo rendono un ottimo candidato per applicazioni in vari settori dell’area biomedica (1). Idrogeli prodotti a partire da soluzioni acquose di alginato, vengono usati con successo per l’incapsulamento e l’immobilizzazione di cellule ed enzimi. Tali sistemi sono stati più recentemente presi in considerazione come “scaffold” per la rigenerazione tissutale (2) e potrebbero essere vantaggiosamente utilizzati per la sostituzione e la rigenerazione del nucleo polposo intervertebrale. Sfortunatamente il processo di gelificazione dell’alginato, generalmente basato sull’uso di sali di calcio, è difficile da controllare. Un controllo della cinetica di gelificazione è però molto importante non solo per ottenere materiali con uniformità strutturale ma anche per consentire la formazione di idrogeli “in situ” in seguito ad una semplice iniezione. Nel presente lavoro è stato messo a punto un nuovo metodo per la produzione di idrogeli di alginato, attraverso un processo di gelificazione controllata, da utilizzare per la sostituzione e rigenerazione del nucleo polposo intervertebrale. Tale metodo si basa sull’impiego di sistemi microparticellari caricati con calcio. Le particelle, prodotte a partire da biopolimeri, vengono addizionate alla soluzione acquosa di alginato, in cui si possono sospendere le cellule, e quindi gli ioni calcio vengono rilasciati in maniera controllata consentendo la formazione di un idrogelo. Come polimero naturale per la produzione delle particelle è stata inizialmente utilizzata la gelatina, sia da sola che addizionata con acido poli(acrilico) (PAA). La scelta della gelatina è fortemente legata al fatto che le presenza di particelle di gelatina all’interno dell’idrogelo, una volta esaurita la funzione di rilascio, contribuisce a creare un ambiente favorevole per le cellule. L’aggiunta del PAA, noto polianione, ha lo scopo di migliorare l’intrappolamento dei cationi Ca2+. Microparticelle di gelatina e microparticelle gelatina/PAA sono state prodotte attraverso una procedura di emulsione di tipo acqua-in-olio (W/O). Sono state preparate sia particelle precaricate con calcio che particelle caricate con calcio per assorbimento. E’ stata condotta una analisi morfologica, mediante microscopia elettronica a scansione (SEM) e una analisi dimensionale delle particelle prodotte. La cinetica di rilascio del calcio è stata studiata mediante test in vitro.

Recentemente un altro polimero biologico, l’agarosio è stato selezionato per la produzione di microparticelle in quanto offre il vantaggio di poter essere reticolato termicamente evitando l’impiego di un reticolante chimico, come la glutaraldeide, usata nel caso della gelatina. Microparticelle di agarosio precaricate con calcio sono state prodotte anch’esse mediante una procedura di emulsione di tipo W/O, specificamente messa a punto, operando alla temperatura di 60°C, superiore a quella di gelificazione dell’agarosio. Le particelle di agarosio sono state sottoposte ad analisi morfologica, mediante microscopia ottica, e ad analisi dimensionale. Test in vitro sono in corso per valutare la quantità di calcio intrappolato e la cinetica di rilascio. Idrogeli di alginato, sono stati prodotti, addizionando soluzioni acquose del polimero con quantità note di particelle di gelatina, e ne sono state valutate le caratteristiche morfologiche e la capacità di supportare l’adesione e crescita cellulare mediante test in vitro condotti utilizzando fibroblasti embrionali ovini. Risultati Dall’analisi morfologica è risultato che le particelle di gelatina, prodotte in forma di polvere fine, hanno una superficie liscia e una forma perfettamente sferica (Fig. 1).

Figura 1 – Immagine al SEM di microparticelle di gelatina La distribuzione dimensionale è risultata piuttosto uniforme e il diametro medio è risultato di 12 µm per le particelle precaricate con calcio e di 8 µm per quelle caricate per assorbimento. Dall’analisi dei dati relativi al rilascio del calcio, determinato mediante misure di assorbimento atomico, si è dedotto essenzialmente che esiste un limite nella quantità massima di calcio che può essere caricata nelle particelle di


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gelatina. Tale limite sembra essere indipendente dalla procedura utilizzata per il caricamento e potrebbe dipendere invece dal tipo di equilibrio che si instaura fra l’interno e l’esterno delle particelle. Sulla base di ciò si è proceduto con la preparazione di particelle impiegando, in aggiunta alla gelatina, un polimero con caratteristiche anioniche più spiccate quale il PAA per favorire l’intrappolamento e quindi il successivo rilascio di una quantità di calcio idonea all’ottenimento di idrogeli di alginato con un adeguato grado di reticolazione. Diversi esperimenti sono stati condotti al fine di trovare la quantità ottimale di PAA da addizionare alla gelatina senza alterare la morfologia delle particelle prodotte Particelle gelatina/PAA, precaricate con calcio, aventi un diametro medio di 11 µm sono state prodotte in forma di polvere fine e sono risultate in grado di intrappolare e quindi rilasciare quantità di calcio maggiori rispetto a quelle di gelatina pura. La percentuale di intrappolamento del calcio nel caso delle particelle gelatina/PAA è risultata pari al 54% quindi decisamente superiore rispetto a quella delle particelle di gelatina pura che era del 36%. Per quanto concerne la microparticelle di agarosio, la procedura appositamente messa a punto ha permesso l’ottenimento di particelle, in forma di sospensione acquosa aventi una distribuzione dimensionale piuttosto omogenea e un diametro medio attorno a 70 µm (Fig. 2).

Figura 2 – Immagine al microscopio ottico di microparticelle di agarosio.

Dati molto preliminari relativi alla quantità di calcio che queste particelle sono in grado caricare e quindi di rilasciare sembrano essere molto promettenti. Esperimenti sono attualmente in corso per verificare e confermare questi risultati. Conclusioni Microparticelle a base di gelatina e microparticelle di agarosio capaci di intrappolare e quindi di rilasciare ioni calcio sono state prodotte utilizzando procedure di emulsione appositamente messe a punto. Le microparticelle di gelatina pura hanno mostrato un limite relativo alla quantità di calcio che sono capaci di intrappolare. Una strada per superare tale limite, dovuto probabilmente alla natura stessa della gelatina, è rappresentata dall’aggiunta di un componente, come il PAA, con spiccate caratteristiche anioniche e quindi capace di intrappolare una quantità maggiore di ioni calcio. Interessante alternativa sembra essere rappresentata dalle microparticelle di agarosio che oltre ad offrire il vantaggio di una semplice reticolazione per via termica, evitando l’uso di reticolanti chimici potenzialmente tossici, sembrano essere in grado di caricare quantità di calcio maggiori e quindi appropriate per una efficace reticolazione dell’alginato. Idrogeli prodotti utilizzando i sistemi microparticellari sopra descritti sono risultati, sulla base di test preliminari, capaci di supportare l’adesione e la crescita cellulare. Riferimenti 1. I.W. Sutherland, In Biomaterials: Novel materials from Biological Sources. (D. Bryon ed.), Stockton press, New York, 309 (1991). 2. C. K. Kuo, P.X. Ma, Biomaterials, 22, 511-521 (2001)


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A NOVEL BIOACTIVE BIOMATERIAL FOR CHONDROCYTE ENCAPSULATION BASED ON ALGINATE/LACTOSE-MODIFIED CHITOSAN (CHITLAC) HYDROGELS

I. Donati1, E. Marsich1, M. Borgogna1, I.J. Haug2, K.I. Draget2, B.L. Strand2, G. Skjåk-Bræk2,

S. Paoletti1 1 Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, 34127 Trieste,

Italy. E-mail: [email protected] 2 Inst. of Biotechnology, Norwegian University of Science and Technology (NTNU), N-7491 Trondheim, Norway

Introduction Polysaccharides constitute major components of that part of the biological milieu which is cumulatively referred to as Extracellular matrix (ECM). Together with the whole matrix biopolymers, they augment the mechanical stability through the formation of a three dimensional network, ensure appropriate dynamic response to stresses and create highly swollen environments with controlled permeability. Therefore, polysaccharides from different sources represent appealing candidates to obtain three-dimentional scaffolds, typically hydrogels, acting as analogues of the natural extracellular matrix. Among the family of polysaccharides, alginate and chitosan play an important role, from the point of view of the industrial applications, since they are obtained from abundant natural sources and their production on large scale is cost effective. Alginate is a negatively charged polysaccharide widely used both industrially and in biotechnological field1,2 due to its ability to form stable hydrogels when in contact with solution of divalent cations such as calcium.3 This important feature is however counterbalanced by its biological inertness, i.e. the lack of interaction with biological material.4 Chitosan is a positively charged polysaccharide obtained from crustaceans. It is characterized by the presence, on the polymer chain, of amino groups that render it particularly appealing for the chemical introduction of cell-specific ligands such as oligosaccharides and/or peptides to improve interaction with cells and to stimulate specific responses. As an example, chitlac, a lactose-modified chitosan, induced chondrocyte aggregation and stimulated the production of both type-II collagen and of glycosaminoglycans (GAGs).5,6 Results Prompted by the considerations reported in the Introduction section, we attempted developing a bioactive biomaterial based on a mixture of the polysaccharides above mentioned. It was found that soluble complexes between the two oppositely charged polysaccharides, namely alginate and chitlac, could be obtained both in dilute and semi-dilute condition. The presence of such soluble aggregates induced a remarkable synergistic effect on the viscosity of the binary solution. The main features of the polymer mixture were assessed prior and after the treatment with cross-linking divalent cations, namely calcium. Once

more, a synergistic effect on the mechanical properties of the hydrogels was detected as a consequence of the presence of soluble complexes containing oppositely charged polysaccharides. Finally, the mixed system containing alginate (a gel forming polysaccharide) and chitlac (a bioactive polysaccharide) was used to encapsulate pig articular chondrocytes. These analyses performed on the encapsulated cells revealed the strong influence of the bioactive polysaccharide in stimulating the proliferation of chondrocytes.7,8,9

Conclusions The novel biomaterial presented in this communication is composed of a mixture of oppositely charged polysaccharides. Due to the gel forming ability of one of the components of the binary mixture (alginate) and to the bioactivity of the other (chitlac), the present system can be proposed as promising intelligent scaffold for tissue engineering and cartilage repair. References 1 Soon-Shiong, P.; Feldman, E.; Nelson, R.;

Komtebedde, J.; Smidsrød, O.; Skjåk-Bræk, G.; Espevik, T.; Heints, R.; Lee, M.; Transplantation; 1992; 54; 769

2 Rokstad, A.M.; Holtan, S.; Strand, B.; Steinker, B.; Ryan, L.; Kulseng, B.; Skjåk-Bræk, G.; Espevik, T.; Cell Transplant.; 2002; 11; 313

3 Smidsrød, O.; J. Chem. Soc., Faraday Trans. 1; 1974; 57; 263

4 Lee, K.Y.; Mooney, D.J.; Chem. Rev.; 2001; 101; 1869 5 Donati, I.; Stredanska, S.; Silvestrini, G.; Vetere, A.; Marcon, P.; Marsich, E.; Mozetic, P.; Gamini, A.; Paletti, S.; Vittur, F.; Biomaterials; 2005; 26; 987

6 Marcon, P.; Marsich, E.; Vetere, A.; Mozetic, P.; Campa, C.; Donati, I.; Vittur, F.; Gamini, A.; Paoletti, S.; Biomaterials; 2005; 26; 4975

7 Donati, I.; Haug, I.J.; Scarpa, T.; Borgogna, M.; Draget, K.I.; Skjåk-Bræk, G.; Paoletti, S.; Biomacromolecules; 2007; 8; 957

8 Donati, I.; Borgogna, M.; Turello, E.; Cesàro, A.; Paoletti, S.; Biomacromolecules; 2007; 8; 1471 9 Marsich, E.; Borgogna, M.; Donati, I.; Mozetic, P.; Strand, B.L.; Vittur, F.; Gomez Salvador, S.; Paoletti, S.; J. Biomed. Mater. Res. Part A; 2007; in press


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SURFACE MODIFICATION OF CELLULOSE BY UV-GRAFTING OF AN ORGANO-POLYSYLAZANE OLIGOMER

P. M. Serafini1, R. Bongiovanni1, E. Zeno2

1 Politecnico di Torino, Dipartimento di Scienza dei Materiali, Corso Duca degli Abruzzi, 24, 10129, Torino, Italy; e-mail : [email protected]

2Centre Technique du Papier (CTP), Domaine Universitaire BP 251, 38044, Grenoble Cedex 9, France

Introduction During the last decade, sustainable development of human technology has gained an ever-increasing interest for both fundamental and applied research, whatever the industry involved.In this context, the exploitation of vegetal biomass and of its main constituent, cellulose, plays a key-role. Modifying cellulosic fibers in order to improve the process of paper making or to have a final product with specific properties (resistance to water penetration, hydrophobic behavior etc....) is very attractive. In particular the modification of the cellulose at the fiber surface by grafting specific functions could open the way to the enhancement of fibres of modest quality or to the use of paper for the development of new products. In this work we describe the functionalisation of the cellulose with an organo-polysilazane oligomer by means of a UV-induced process. Interesting properties were imparted to the material and are described here. Results The modification was performed onto Whatman 42 filter paper by a UV-induced process (sketched in Figure 1), in the presence of benzophenone as a photo-initiator, using a polysylazane oligomer having structure

Figure 1: Procedure for cellulose functionalisation The reactions that take place under irradiation are: formation of radicals on the paper surface induced by the photo-initiator once irradiated by UV light; grafting of the oligomer onto the reactive sites present on the paper through the oligomer double bonds[1]. The modified paper shows interesting properties (see Table 1), such as high hydrophobicity, i.e. the contact angle is higher than 90°. These high values are constant over a long time of observation, while also the volume of drop does not change, meaning that there is no water penetration. The

treatment improved also the tensile strength of the paper (measured by the Zero Span Test) and enhanced its internal cohesion (the Mullen Test Test) Table 1: Characterisation of the UV-grafted paper Sample

Oligomer Concentration

[g/l]* Contact angle

ZERO SPAN [kN/m]

Mullen Test [kPa]

1 1 in acetone 118° 8 152 2 5 in acetone 124° 9,45 146 3 50 in acetone 136° 7 207

untreated ≈ 0 5,19 151 *conc. of the oligomer used; irradiation time = 4 min. Figure 2 shows the TGA analyses performed on the treated samples (in air). Organopolysilazanes are well known for their thermal resistence (purple curve). Sample 3 (red curve) gives the highest residue (7.7% w/w) and a better resistence to high temperature compared to the untreated paper ( green curve).

Figure 2: TGA analyses of UV-grafted papers Conclusions The results obtained show that the modification of cellulosic materials can be performed by a UV-induced process. of the modified material. This leads to interesting perspectives, considering that the UV-grafting technique is extremely versatile for the variety of the functionalising agents and of the procedures that can be adopted. Using the polysylazane as a modifier, the paper is made highly hydrophobic and more resistant to mechanical stresses. Acknowledgement Dr. Can Wu (Clariant) is acknowledged for providing us the organopolysilazane. Reference 1. C.Decker, A.Jenkins Macromolecules 18(6), 1241 (1985)


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SINTESI DI BIOMATERIALI ALTAMENTE POROSI ATTRAVERSO LA TECNICA DEL GAS FOAMING

A. Gumiero, A. Barbetta, M. Dentini

Università degli Studi di Roma “La Sapienza”, Dipartimento di Chimica, piazzale Aldo Moro, 5 00185 Roma; email: [email protected]

Introduzione Nell’ambito delle biotecnologie, materiali altamente porosi a base biopolimerica, vengono impiegati per colture cellulari sia nel campo del tissue engineering che nello studio sulla crescita e sul differenziamento cellulare. Tali supporti vengono prodotti con differenti tecniche. Nel nostro laboratorio da anni viene utilizzata con successo la tecnica cosiddetta “emulsion templating: high internal phase emulsion” (polyHIPEs). I supporti si ottengono da emulsioni in cui la fase dispersa è prevalente in volume rispetto al mezzo disperdente ed è suddivisa in microgocce circondate dalla fase continua che contiene i reattivi monomerici o polimerici. Mediante polimerizzazione radicalica o di altro tipo si ottiene la formazione di reticoli che immobilizzano la fase continua e fissano stabilmente la struttura dell’emulsione. In questo modo si producono scaffolds monolitici altamente porosi, caratterizzati da un elevato grado di interconnessione. Le polyHIPEs a base biopolimerica così ottenute hanno, tuttavia, limiti morfologici. Difficilmente si è riusciti a sintetizzare matrici con pori ed interconnessioni di dimensione superiore ai 100 µm e 30 µm rispettivamente. Inoltre l’impiego di solventi organici nel processo di preparazione delle emulsioni può pregiudicare la biocompatibiltà del materiale prodotto. La necessità di superare i limiti connessi alla sintesi in emulsione di scaffolds, ci ha indotto alla ricerca di una nuova metodologia. Il gas foaming è una tecnica che viene utilizzata per la sintesi di materiali polimerici cellulari, che trovano impiego nell’industria dell’imballaggio, come materiali insonorizzanti, isolanti termici, ecc. Si è pensato perciò di applicarla ai nostri sistemi biopolimerici per produrre scaffolds. Viste le difficoltà tecniche relative alla realizzazione di schiume per insuflaggio di gas, si è proceduto alla realizzazione di queste mediante sviluppo di gas in situ, impiegando reazioni chimiche, ed in presenza di un tensioattivo appropriato. In dettaglio, la sintesi delle foams è stata eseguita sfruttando lo sviluppo di due gas, Anidride Carbonica ed Azoto rispettivamente. L’Anidride Carbonica è stata ottenuta per acidificazione di una soluzione di Bicarbonato di Sodio (reazione 1.) a mezzo di D-Glucono-δ-Lattone; quest’ultimo, pur non essendo un acido, in ambiente acquoso, come noto, subisce idrolisi generando acido gluconico (HX nella reazione 1.). L’Azoto, invece, è stato ottenuto per reazione di ossidoriduzione tra Acido Sulfamico e Nitrito di Sodio (reazione 2.). 1. HXNaHCO +3 � NaXOHCO ++ 22 2. →+ 232 NaNONHSOH OHNaHSON 242 ++

Nel contesto del presente lavoro, sono stati preparati supporti a base di due polimeri, Destrano e Gelatina; la scelta di questi due polimeri è stata in primo luogo, dettata dalla loro comprovata biocompatibilità ed economicità. Destrano e Gelatina, derivatizzati o meno, d’altra parte, sono già stati diffusamente impiegati nel nostro laboratorio per la sintesi di scaffolds con la tecnica delle polyHIPEs e ciò ha permesso di avere un confronto diretto, delle caratteristiche morfologiche e della biocompatibilità, tra i precedenti supporti e quelli di più nuova fattura, studiati in questo lavoro.

Risultati L’impiego della tecnica del gas foaming ha permesso la sintesi di supporti con dimensioni medie di pori ed interconnessioni dell’ordine di 250 µm e 100 µm rispettivamente, assolutamente innovative rispetto a quanto da noi precedentemente ottenuto; tale tecnica inoltre si è rivelata anche semplice e riproducibile, in grado, anche se in modo ancora limitato, di facilitare la modulazione delle caratteristiche morfologiche dei supporti sviluppati. Inoltre gli scaffolds ottenuti sono risultati validi per la crescita di cellule staminali mesenchimali: su di essi, infatti, tali cellule hanno proliferato subendo anche un

Micrografia SEM di uno scaffold a base di Gelatina ottenuto con la tecnica del gas foaming attraverso sviluppo di N2

Micrografia SEM di uno scaffold a base di Destrano metacrilato ottenuto con la tecnica del gas foaming attraverso sviluppo di CO2


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parziale differenziamento; quest’ultimo aspetto non è emerso invece coltivando le stesse cellule in identiche condizioni su supporti ottenuti in emulsione. Conclusioni In conclusione è stata messa a punto una nuova metodologia per la sintesi di materiali porosi per la crescita cellulare, applicando la tecnica gas foaming a sistemi biopolimerici. Gli scaffolds ottenuti hanno dimensioni massime dei pori e delle interconnessioni (finestre) di uno o due ordini di grandezza superiori rispetto a quelle riscontrate per le polyHIPEs (60-80 µm e 20-30 µm rispettivamente). Tali risultati ci permettono di estendere il campo di applicazione dei nostri scaffolds a base di biopolimeri alla coltura di cellule di qualsiasi dimensione, dalle più piccole (quelle staminali) a quelle più grandi (ad

esempio osteoblasti), portando quindi un contributo agli studi non solo nel campo della rigenerazione dei tessuti ed organi, ma anche agli studi sul differenziamento di cellule staminali in qualsivoglia tessuto. Riferimenti 1. A. Barbetta, M. Dentini, M. S. De Vecchis, P.

Filippini, G. Formisano, S. Chiazza, Ad. Funct. Mater., 15, 118 (2005).

2. A. Barbetta, M.Dentini, E. M. Zannoni, M. E. De Stefano, Langmuir, 21, 12333 (2005).

3. A. Barbetta, M. Massimi, L. Conti Devirgiliis, M. Dentini, Biomacromolecules, 7, 3059 (2006).


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STRUCTURE-FUNCTION RELATIONSHIPS IN CEPACIAN BIOFILM FORMATION

P. Cescutti1, Y. Herasimenka1,#, G. Impallomeni2, R. Rizzo1 1Dept Biochemistry, Biophysics and Macromolecular Chemistry, Univ. Trieste, Via Licio Giorgieri 1, 34127 Trieste,

Italy; e-mail: [email protected] 2Inst. of Chemistry and Technology of Polymers, CNR, Viale A. Doria 6, 95125 Catania, Italy.

Cepacian is the exopolysaccharide (EPS) produced by the majority of the strains belonging to the Burkholderia cepacia Complex [1-6]. This is a group of nine closely related bacterial species. Some of them are involved in serious pulmonary infections in patients affected by cystic fibrosis. The persistence of bacterial infection is also due to the formation of a biofilm on the lung epithelia which protects the micro-organism from the host immune response. The chemical structure of cepacian repeating unit is rather complex including seven sugar residues four of which are located in three short lateral chains as reported in the figure 1: β-D-Galp-(1→2)-α-D-Rhap 1 ↓ 4 [3)-β-D-Glcp-(1→3)-α-D-GlcpA-(1→3)-α-D-Manp-(1→]n 2 6 ↑ ↑ 1 1 α-D-Galp β-D-Galp Figure 1. Primary structure of cepacian. In addition, NMR spectra showed a variable number of acetyl groups depending on the growth medium utilised for bacterial cultures. The EPS macromolecular properties, which explain its role in the biofilm structure formation, has been investigated by means of capillary viscosity, molecular mass determination, atomic force microscopy (AFM), and NMR NOE investigation. Intrinsic viscosity measurements carried out in water as well as in water-dimethylsulphoxide (DMSO) mixed solvent indicated the presence of a multiple stranded polymer association in water. This characteristic was also confirmed by evaluating the polymer molecular mass both in water and in DMSO by means of HP-SEC coupled with low angle light scattering detection. The morphology of cepacian polymer chains was investigated by AFM both in diluted and concentrated solutions in water and in DMSO. Micro-images from diluted solutions allowed the evaluation of EPS chain diameters, while those from concentrated solutions gave interesting images of chain aggregation. The diameter obtained for the native polymer in water was

consistent with the presence of double stranded polymer aggregates. On the contrary, the lateral dimension obtained in water/DMSO solutions was consistent with the presence of single stranded chains. Interestingly, AFM microimages of de-acetylated cepacian showed the presence of mixtures of single and double stranded structures thus revealing that acetyl groups are relevant for the formation of a suitable polymer chain network to be used as biofilm skeleton. EPS chain stiffness was studied resorting to NMR NOE experiments pointing at the localisation of non-bonded short intra-chain interactions responsible for reduction of saccharidic conformational mobility. The data obtained led to conclude that biofilm formation is promoted by cepacian throughout the formation of an extended polymer network where double stranded structures interact with each other in a random fashion and are stabilised by the presence of acetyl groups substituents. 1) P. Cescutti, M. Bosco, F. Picotti, G.Impallomeni, J.H. Leitão, J. Richau, I. Sá-Correia. Biochem. Biophys. Res. Comm. 273, 1088-1094 (2000). 2) C. Lagatolla, S. Skerlavaj, L. Dolzani, E.A. Tonin, C. Monti Bragadin, M. Bosco, R. Rizzo, L. Giglio, P. Cescutti. FEMS Microbiol. Lett. 209, 89-94 (2002). 3) P. Cescutti, G. Impallomeni, D. Garozzo, L. Sturiale, Y. Herasimenka, C. Lagatolla, R. Rizzo. Carbohydr. Res. 338, 2687-2695 (2003). 4) Y. Herasimenka, M. Benincasa, M. Mattiuzzo, P. Cescutti, R. Gennaro, R. Rizzo. Peptides 26, 1127-1132 (2005). 5) P. Cescutti, S. Scussolin, Y. Herasimenka, G. Impallomeni, M. Bicego, R. Rizzo. Biochem. Biophys. Res. Comm. 339, 821-826 (2006). 6) J. Bylund, L.A. Burgess, P. Cescutti, R.K. Ernst, D.P. Speert. J. Biol. Chem. 281, 2526-2532 (2006). #) Present address: FB Biologie/Chemie, Universität Osnabrück, Barbarastrasse 11, 49069 Osnabrück, Germany.


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NONWOVEN BIOPOLYMER MEMBRANE BY ELECTROSPINNING

M. Roso, A. Lorenzetti, M. Modesti Università degli studi di Padova, Dipartimento di Processi Chimici dell’Ingegneria, Via Marzolo 9, 35100, Padova,

Italy; e-mail: [email protected]

Introduction Emerging market requires more and more to invest on environmental-friendly materials especially focusing on biopolymers production as an alternative to petroleum-based polymers (traditional plastics). (Bio)polyesters, such as Polylactic acid (PLA), Polycaprolactone (PCL), Polyhydroxyalkanoates (PHA), to cite as examples, have properties similar to traditional polyesters and starch-based polymers (Mater-Bi) are often a blend of starch and other plastics (e.g PE), which allows for enhanced environmental properties. Electrospinning has gained more and more interest in the last decades as the scientific community recognizes it as one of the most versatile technique for nanofibers processing. The process itself is very simple to perform, though a whole understanding on the basic principles and on the reciprocal interactions among the different parameters is still going in progress. When a high voltage is applied to a polymer solution/melt charges are induced within it and when their amount overcomes a threshold value, a jet is ejected from the droplet at the tip of the needle forming a cone structure noted as a Taylor cone. Then, the jet goes towards the grounded collector and different instabilities can occur [1]. The controlling parameters of the process are hydrostatic pressure in the capillary tube, external electric field, viscosity, conductivity, dielectric permeability, surface tension and ambient parameters. This powerful technique provides a huge range of applications, such as high performance filters media, protective textiles, catalysis and sensors, advanced composites, wound dressing and as scaffolds in tissue engineering [2]. Results As previously mentioned, several forces play a fundamental role on nanofibers formation by electrospinning. In order to get nanofibers, it is necessary to find a good balance in the system so that the morphology, the structure, and the dimensions of the nanofibers respect the aimed requirement. According to Design of Experiments theory (DoE), screening experiments were planned to test which is the effect of the main parameters on the process, in term of change in nanofibers diameter. At the same time this method is useful to minimize the experiments run. We report the results concerning the effect of the main solution properties (concentration, surface tension, conductivity) and operative parameters (voltage, needle-collector distance, feed rate) on the dimension and morphology of the fibers. Moreover nanocomposite electrospun biomembranes have been produced and antibacterial test results are reported.

Mater-Bi and Polylactic acid (PLA) has processed by electrospinning and all the samples have been characterized by Scanning Electron Microscope (SEM) analysis. Effect of biopolymer concentration. Mater-Bi concentration ranging from 7 to 10 wt% has been considered, other things being equal. At 7 wt.% aerosol and beads formation controlled the process, then at 8.5 wt% a beads on string morphology was revealed. Finally, at 10 wt% fiber formation without any defect occurred. It can be explained because the enhanced concentration affect the viscosity of the solution so that an increase on entanglement of the molecule chains prevent the electrically driven jet from breaking up, maintaining a continuous solution jet. Polylactic acid (PLA) concentration has been varied from 5 to 12 wt%, other things being equal. At 5 wt.% aerosols was obtained, then rising at 8 wt.% beads on string morphology has been observed. Going up to 10 wt% smooth nanofibers were obtained with diameter ranging from 90nm and 250nm Effect of solvent. The choice of the solvent is very important since the whole surface tension and conductivity of the solution depends on it. In order to encourage the fiber formation rather then aerosols or beads, the surface tension should be as low as it possible and the conductivity as high as possible, compatibly with the system. For Mater-Bi solutions, pure CHCl3 (σCHCl3 =27.5 mN/m), CHCl3:DMF=3:1 (σDMF =37.10 mN/m) and CHCl3:EtOH=3:1 (σEtOH =21.97mN/m), were tested respectively as a solvent. In the first electrospun sample, a lot of beads were noticed. Only the ethanol addition promoted the fibers formation because it affects the surface tension of the solution, leading a decrease from 27.5 to 25 mN/m. However the nanofibers surface is still not completely smooth and further investigation are necessary. In spite of DMF addition enhances the solution conductivity (κDMF = 1.090 mS/m), the fibers had a lot of beads and it means that probably the surface tension is a control factor. Firstly Polylactic acid (PLA) solvent used was DMF. Then, in order to reduce the surface tension, an equal mix ratio DMF/Aceton (Ac) was tested and the nanofibers morphology was definitely smoother and beads-free. Anyway, for the latter system PLA/DMF/Ac a study on the polymer concentration (from 8 to 10wt%) effect was also carried out. If compared to the results showed in the previous section, where pure DMF was used as a solvent, it has been noticed an increase on nanofiber diameter in the range of 360 -760nm and 400 nm-1µm for 8 and 10wt% samples respectively. So, even the nanofibers are smoother, the reduction of the conductivity due to the acetone addition is more significant and the ongoing work is on the optimization of the ratio DMF/Ac.


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Effect of applied voltage. Usually, whenever the applied voltage grows up, the ejected jet is accelerated and more stretched resulting in a decrease of the nanofibers diameters. The experiments performed on Mater-Bi were carried out rising the voltage from 10 kV to 15 kV. As revealed by SEM analysis, the mean diameter go down and the fibers showed a reduction of the defects number. As regards PLA, the applied voltage values ranging from 7 kV to 15 kV. In this case it has been more evident the change of morphology going towards completely smooth nanofibers formation. Moreover, deposition time is found to be directly proportional to the applied voltage, dispensed solution being equal. Effect of the feed rate and needle-collector distance. The feed rate of the PLA solution was varied from 5µl/min to 20µl/min and, according to the theory, the nanofibers diameters of the electrospun samples increased from 300 -700nm to 1.1µm-1.8µm respectively. As regards the distance between the electrodes, it has been proved that a distance of 5 cm is not enough to allow the solvent to completely evaporate before the jet hits the collector; consequently the fibers tends to merge each other. In Table I the results of the process optimization for both Mater-Bi and PLA are summarized. Table I: process parameters optimization results Biopolymer Parameter Mater-Bi

Polylactic acid

Solvent CHCl3/EtOH=3/1 DMF/Ac=1/1 Concentration (wt%) 10 8 Voltage (kV) 15 15 Feed rate (µl/min) 5 10 Distance N-C (cm) 15 15 The SEM (scannino electron microscopi) image of such PLA nanofibers is shown in Fig. 1. Nanocomposite electrospun membrane Both Mater-Bi and PLA were electrospun at the conditions reported in Table1 adding to the feed solution the 5 wt% of silver nitrate (AgNO3) The first result observed regards the morphology of the fibers: since Ag+ ions from AgNO3 dissolution has the effect to increase

the conductivity of the solution, the nanofibers were smoother. After photoreduction of Ag+ to metallic Ag (partially occurred before electrospinning), Ag antibacterial activity was tested on Staphilococcus Aureus and Escherichia Coli coltures. A strong inhibition has been noticed in all the samples with AgNO3 based electrospun membrane. These results can suggest the use of this kind of biomembrane as a bioactive and biodegradable filter media.

Fig. 1. PLA nanofibers electrospun according to optimised conditions reported in Table 1.

Conclusion First, Mater-Bi was successfully electrospun into nonwoven mats thanks to a consistent and controllable electrospinning process. In order to achieve fiber formation a mix of two solvent, CHCl3 and EtOH, has been found to be effective. Nanofibers diameter of both biopolymer processed, strongly depends on the process and solution parameters and it can be tuned playing on a different combination of them. We proposed these biopolymer membranes as a good candidate as antibacterial biodegradable filter media, as well as a scaffold for catalyst immobilization. References 1. M. M. Hohman, M. Shin, G. Rutledge, M. P: Brenner, Phys. Fluids, Vol.13, pag. 2201/20 (2001) 2. S. Ramakrishna, K. Fujihara, An introduction to Electrospinning and nanofibers, (2005) World Scientific Publishing Co. Pte.Ltd.


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EFFECT OF POLYMER MATRIX ON MORPHOLOGY AND PROPERTIES OF QUERCETIN MICROSPHERES

P. Scarfato1, E. Avallone1, V. Speranza1, P. Iannelli2, R.P. Aquino2, D. Acierno3

1Università degli Studi di Salerno, Dipartimento di Ingegneria Chimica e Alimentare, Via Ponte don Melillo, 84084 Fisciano (SA), Italy; e-mail: [email protected]

2Università degli Studi di Salerno, Dipartimento di Scienze Farmaceutiche, Via Ponte don Melillo, 84084 Fisciano (SA), Italy

3Università degli Studi di Napoli “Federico II”, Dipartimento di Ingegneria dei Materiali e della Produzione, P.le Tecchio 80, 80125 Napoli, Italy

Introduction Microencapsulation technology allows to isolate an active product from the environmental conditions and/or to release the active agent in a controlled manner into a surrounding medium. The process is very interesting from a scientific, industrial and commercial point of view and is used in many applicative sectors. In particular, pharmaceutical applications of micro-encapsulated systems for controlled drug delivery occupy an unique position since these carriers offer numerous advantages compared to conventional dosage forms, which include reduced toxicity, improved dosage capability and efficacy of the active product. In these last years, several methods were developed to prepare micro-particulate systems. Among them, the emulsion solvent evaporation is one of the most versatile and advantageous technique. In fact, it requires only mild conditions, such as ambient temperature and controlled stirring; therefore, a stable emulsion can be formed without compromising the activity of core material. Moreover, using such methodology both hydrophilic and liophilic substances can be encapsulated, with a good size distribution control a high reproducibility of the produced systems. However, the processing and formulation variables can greatly affect not only the physicochemical characteristics of the prepared microspheres (i.e. mean size, porosity, roughness, drug loading, etc.) but also the release properties (i.e. release kinetics and targeting). In this field, we are performing a study with the aim to ensure gastric protection and better deliver of poorly bioavailable drugs by microencapsulation in gastro-resistant controlled delivery systems. The active molecule selected for the experiments was the flavonoid quercetin (Q, 3,5,7,3’,4’-pentahydroxyflavone). Q, a common dietary component occurring in various edible plant and herbal medicines marketed in Europe, has shown numerous beneficial biological effects (i.e. anti-oxidant and anti-inflammatory properties, adjuvant for cardiovascular chronic pathologies, etc.), in vitro as well in vivo. However, its bioavailability is an important unsolved problem: in fact, it is very slightly soluble and has inherently limited ability to permeate the gastric mucosa from oral dosage forms and to reach the bloodstream in efficacious quantity. In the attempt to get over these limitations, we prepared, by water in oil (W/O) emulsion solvent evaporation, gastro-resistant microspheres of Q using several biocompatible polymers as matrix materials. In particular, two different cellulose

derivatives (cellulose acetate phthalate, C-A-P, and cellulose acetate propionate, CAP) and a copolymer based on methacrylic acid and methacrylic acid methyl ester (Eudragit L100) were tested. The prepared microspheres were characterized by means of several techniques (light scattering, fluorescence microscopy, SEM, AFM, X-ray diffractometry, DSC, UV-Vis) to evaluate their morphology and physicochemical properties and to investigate the effect of the polymer matrix nature on the drug solubility. Results The obtained samples were preliminary characterized in order to evaluate the drug encapsulation efficiency, the microspheres yield and the particle size distribution. The results of these analyses showed that all systems were produced with yield higher then 80%, the microencapsulation efficiency was always close to the theoretical value (20 wt%) and the particle size distributions were unimodal, with mean sizes ranging from approx. 25 to 50 µm (Table I). Table I.

Sample Dispersed Phase Solvent

Dispersed Phase Viscosity [cP]

Actual Drug Content [%]

Mean size (DV0.5) [µm]

C-A-P acetone 10.4 - 32.4 CAP acetone 10.9 - 35.1 Eu acetone/ EtOH 5:1 6.6 - 47.4 C-A-P+Q acetone 10.5 19.3 24.6 CAP+Q acetone 11.0 17.9 27.1 Eu+Q acetone/

EtOH 5:1 7.9 20.9 48.5 Microscopy investigations revealed that the microspheres were free-flowing and spherical in shape, without coalescence (Figures I-II). AFM measurements showed that the surfaces roughness and the mechanical resistance of the particles are affected by the polymer kind used as matrix: samples based on Eudragit L100 were slightly more rough and stiff then those based on cellulose derivatives C-A-P and CAP. Correspondingly,


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the X-ray diffractograms reveal that, in our experimental conditions, Q incorporation into the C-A-P and CAP matrices reduces its crystallinity to practical insignificance, giving amorphous drug/polymer blends, whereas Q incorporation into the Eudragit L100 matrix produces a physical dispersion of microcrystalline drug into the polymer.

Figure 1. Fluorescence microscopy image of C-A-P microspheres loaded with 20wt% of Q.

Figure 2. SEM image of C-A-P microspheres loaded with 20wt% of Q.

Preliminary in vitro dissolution studies, carried out using a pH change method showed that the samples exhibit a typical biphasic drug release trend, due to the pH dependent solubility of the enteric polymers used. In fact, at pH 1 all polymers partially protect quercetin, limiting the release at about 25% or less. Nevertheless, at pH 6.8 the amount of released drug depends upon the polymer matrix kind: it is about 90% in 30 min after pH change in the case of C-A-P based microspheres and decreases up to 75% and 50% for Eudragit L100 and CAP based samples, respectively. These results were related to the different internal structure of the prepared microspheres. Conclusions The drug delivery systems prepared in our experimental conditions were demonstrated to be an efficient way to target quercetin to the intestine following oral administration, since they have adequate properties to protect the flavonoid in the gastric medium and to incorporate it in a more bioavailabile form. References 1. Arshady R., Polymer Engineering and Science, 30, 915-924 (2004). 2. Edgar KJ, Cellulose, 14, 49-64 (2007). 3. Kühnau J, World Review of Nutrition and Dietetics, 24, 117-191 (1976). 4. Lauro MR, Maggi L, Conte U, De Simone F, Aquino RP, J. Drug Del. Sci. Tech., 15, 363-369 (2005). 5. Varde NK, Pack DW, Expert Opinion on Biological Therapy, 4, 35-51 (2004).


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NANOPARTICLES OF PCL-PEI AS NON-VIRAL VECTORS FOR GENE DELIVERY

G. Gomez d’Ayala1, P. Laurienzo1, M. Malinconico1, A. Calarco2, G. Peluso2, O. Petillo2 1Istituto di Chimica e Tecnologia dei Polimeri, CNR, Via Campi Flegrei 34, Pozzuoli (Napoli), Italy;

e-mail: [email protected] 2Istituto di Biochimica delle Proteine, CNR, Via P. Castellino 111, Napoli, Italy

Introduction Gene delivery has numerous potential applications, both clinical (therapies for cancer and several vascular, monogeneic and infectious diseases), as well as for basic science research. These applications employ both viral and non-viral vectors; however, immune response problems arising from viral vectors open the doors to development of non-viral vectors based on biomaterials (1). The term non-viral vector indicates a formulation consisting of a vector backbone, usually a cationic lipid or polymer able to form stable complexes with the plasmid, modified by incorporation of functional groups that may stabilize the vector and interact with the environment to overcome the various barriers to gene transfer. Cationic polymers can be either natural (polysaccharides, proteins) or synthetic, as poly- ethylenimine (PEI). PEI spontaneously associates with phosphates of DNA through electrostatic interactions due to protonable amine groups (2), forming stable DNA/PEI complexes. Nevertheless, PEI is cytotoxic and non biodegradable. To prevent aggregation of complexes and reduce toxicity, PEI can be combined with biopolymers. Examples of the realization of vector based on nanoparticles of PEI and biodegradable polymers are reported in literature (3). Polymer particles with diameter less than 250 nm have the advantage of easy uptake into the cells by endocytosis. In nanoparticles gene delivery systems, DNA can be encapsulated inside the nanoparticle or adsorbed onto nanoparticle surface. This last system has the advantage of ease binding with DNA, DNA protection, avoidance of the direct contact with organic solvents during particles preparation, and, furthermore, a rapid release of DNA inside the target cells is facilatated. In the present work, we report on a new gene delivery system based on polycaprolactone (PCL) and PEI. PEI has been chemically bound onto PCL, in order to overcome cytotoxicity problems related to free PEI after the dissociation of the DNA/PEI complex. The synthesis and characterization of PCL-PEI copolymer as well as preparation and characterization of nanoparticles are described. Cytotoxicity tests and a preliminary characterization of DNA absorption on the nanoparticles are also discussed. Results PCL (Mw=50,000-60,000 Da) has been functionalized with glycidyl methacrylate (GMA) before the reaction with PEI. The functionalization has been performed in bulk in a Rheometer apparatus, in presence of GMA and benzoyl peroxide, following a previously described procedure. The obtained PCLgGMA can react with PEI (highly branched, Mw=25,000 Da) by condensation of

glycidyl groups with amines, using triethylamine as catalyst, in chloroform solution:

O CO

C CH3CCH2

OO CH2 C CH2

O

H+

H2Nor

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OO CH2 C

OH

Nor

Crosslinking with formation of an unsoluble gel may arise; a careful control of T, PEI concentration and reaction time allows to obtain a grafted, uncrosslinked PCL-PEI copolymer. The reaction time was therefore fixed to one hour and the temperature to 60°C. After this time, the reaction is stopped by cooling down to room temperature, and the solution is homogenized with a 0.5% PVA aqueous solution with an ultrasonicator (mod.,45s, 15). The particle suspension was stirred at 200 rpm rate for 24 hrs for chloroform evaporation. The nanospheres were collected by centrifugation, rinsed three times with water and lyophilized. After recovering, the polymer particles are no more soluble, suggesting a further enhancement of reaction during the particle preparation. FTIR spectroscopy has been used to confirm the reaction. Diagnostic band of PEI at 1580 cm-1 (C-N stretching) and 3357-3289 cm-1 (primary and secondary amines N-H stretching, respectively) are detected, along with typical ester band of PCL (1727cm-1), and an additional band at 3446 cm-1, attributed to stretching of –O-H which are generated from the reaction (Fig.1).

Fig. 1 – FTIR spectrum of PCL-PEI copolymer

DSC analysis shows an increase in PEI’s Tg from -50° (free PEI) to -31°C, consistent with the formation of a chemical network, and the endotherm of PCL melting at 60°C, followed by evolution of water adsorbed by PEI at around 100°C. (Fig.2).

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Fig. 2 – DSC trace of PCL-PEI copolymer Nanoparticles have been characterized by scanning electron microscopy (SEM, Fig. 3).

Fig. 3 - SEM micrograph of nanoparticles

Particles are small and regular in size (around 200 nm) with smooth surface; although a certain agglomeration is detected, particles were dispersed easily in water by simple agitation. Murine fibroblast cell line (L929) was used for direct contact test at 24h (MTT test). The results were obtained referring the absorbance values at 570nm (O.D.570) of cell exposed to synthesized material with that of control cells cultured on the film and TCPS. Our results showed that the material supported the proliferation and growth of cells and did not elicit any noticeable cytotoxic effect. Cells adhesion and morphology were observed by SEM after 48 hrs of incubation on PCL-PEI film (Fig. 4). L929 seeded on polymer showed better attachment and spreading in comparison to those on control (PCL, not shown). DNA binding capacity was assessed with respect to PEI amounts by a gel shift assay. A 25mM HEPES plasmid solution (pH 7.4) was mixed with an aqueous suspension containing various amounts of PCL-PEI particles dispersed inside. The nanoparticles were then loaded with ethidium bromide and electrophoresed on agar gel. Bands corresponding to DNA were detected under UV light.

Fig. 4 –SEM observation of fibroblasts cultured on PCL-

PEI after 48h incubation. The band of pDNA was retarded as the amount of N/P ratio increased, suggesting that PCL/PEI forms a complex with DNA (Fig. 5). pDNA was completely retarded at a 3:1 N/P ratio. This result indicates that the negative charges of DNA are completely shielded by the PEI.

Fig. 5 Agarose gel electrophoresis of PCL-PEI/DNA complexes at various N/P ratios.

Conclusions A novel PCL-PEI copolymer was prepared by chemically binding PEI onto a PCL modified by grafting of glycidyl methacrylate molecules. Nanoparticles of PCL-PEI copolymer of appropriate dimension have been successfully obtained. The MTT tests has proved that the material is not cytotoxic, so indicating a shielding effect of the PCL in masking the positive PEI charge that is harmful for cellular membranes. Nanoparticles have been proved to be able to bind DNA plasmid onto their surface by simple mixing with plasmid solution. Experiments are in progress to characterize the ability of the biomaterial to delivery plasmid DNA and to transfect the cells which are in contact with nanoparticles. References 1. L. De Laporte, J. Cruz Rea, L.D. Shea, Biomaterials 27, 947 (2006) 2. R. Kircheis, L. Wightman, E. Wagner, Advanced Drug Delivery Review, 53, 341 (2001) 3. C.G. Oster, N. Kim, L. Grode, L. Barbu-Tudoran, A.K. Schaper, S.H.E. Kaufmann, T. Kissel, J. contr. rel. 104, 359 (2005)

N/P ratio DNA 1 2 3


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BIODEGRADABLE PLASTICS AND VALUE ADDED PRODUCTS FROM LEATHER INDUSTRY WASTE THROUGH POLYMER DERIVATIZATION

L. Sartore, A. Sassi

Dipartimento di Chimica e Fisica per l’Ingegneria e i Materiali, Via Valotti 9 - 25123 Brescia, Italy e-mail: [email protected]

Introduction The increasing use of plastics and their non-biodegradability, have raised environmental awareness and hence the need for the development of environmentally friendly degradable materials. Environmentally degradable polymers and polymer systems are important for modern technology as ecologically safe materials. Considering this problem, all polymer materials that undergo biodegradation become preferable. One of the ways to reach this goal consists of the development of new polymeric materials, and introduction of bio-based components in the formation of blends and composites [1]. A synthetic polymer present in these materials is often needed to impart necessary physical properties to the blends. Blending bio-based components with non-biodegradable commodity polymer can reduce the total amount of plastic wastes, but often it couldn’t be an ultimate solution to the environmental problem caused by the disposal of plastic wastes. Many of the candidates for biodegradable polymers, however, have some limitations in their mechanical properties or manufacturing or costs. Among bio-based polymers, proteins have shown to be versatile materials that combine many valuable characteristics for technical applications such as good reactivity and processability, both in solution [2] and in the melt [3]. Collagen hydrolysate (CH) is composed of a mixture of oligopeptides originating from enzymatic or chemical hydrolysis, of solid waste generated downstream to chrome tanning (leather shavings). Thus, CH is easily available at low cost and it is readily biodegradable. One of the prospective possibilities of adding value to the hydrolyzate is its use in the form of an additive to some synthetic polymers aimed at improving and possibly ‘‘directing’’ their biodegradation properties. Polymer blending is one of the easiest and most cost efficient ways to produce new material with the desired properties from each component. However, a great number of polymer blends are thermodynamically immiscible in makeup because of the low entropy of mixing. Therefore, many researchers express considerable interest in compatibilization and in reactive compatibilization of immiscible polymers. Reactive compatibilization is used to overcome agglomeration problems and the weak adhesion between phases by creating chemical bonds across the interface[4]. The polyester family has the possibility to create chemical reactions because of its ester bonds and because the transesterification is the major reaction in polyesters. Researches about the exchange reaction of polymer blends commonly concentrate on polyester blends whose constituent ester bonds exist in their backbones. On the contrary, there are a few polymer investigations whose reactive groups exist in branching chain, such as EVA.

EVA, is a modification of polyethylene (PE) with vinylacetate as comonomer which reduces the crystallinity of PE. Thus, it has many characteristic of thermoplastic elastomers, which depends on percentage of vinyl acetate content. EVA provide good mechanical properties, excellent ozone resistance, good weather resistance and relatively lower material cost. In addition, EVA is an example of a synthetic petrochemical thermoplastic that is slowly biodegraded particularly where there is a high percentage of vinyl acetate in the copolymer.[5] Results Table 1. Composition of individual blend and Tgs

Sample CH (%)

EVA (%)

Tg (°C)

EVA(1) 100 -32,18 CH35%_EVA(1) 36 63 43,17 CH50%_EVA(1) 49,42 49,55 CH60%_EVA(1) 59,78 39,2 * CH70%_EVA(1) 69,27 29,67 * CH35%_EVA(2) 34,94 64,07 CH50%_EVA(2) 49,58 49,40 (1) EVA:VA content=40 wt%; (2) EVA: VA content=18 wt%

The research activity is focused on the development of new polymeric biodegradable materials and their physical, chemical, thermal and mechanical characterisation. The compatibility of CH and EVA were obtained by reactive blending in a Brabender mixer [3]. Preliminary investigations were carried out preparing CH/EVA having different composition. EVA with different vinyl acetate content as well as CH having different hydrolysis grade were considered (Table 1). Table 2. Mechanical properties of CH/EVA blends

DMTA Tensile test Sample E’

(MPa) E

(MPa) σr

(MPa) ∆lr (%)

EVA (1) 2,39 >1,6 >700 CH35%_EVA(1) 7,84 10,93 >1,7 >700 CH50%_EVA(1) 34,95 1,6 237 EVA(2) 13,21 >5 >700 CH35%_EVA(2) 41,92 3,21 58,26 CH50%_EVA(2) 256 2,38 6,1 CH-PEG 491,81 797,92 14,5 150

(1) EVA:VA content=40 wt%; (3) EVA: VA content=18 wt%


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Differential scanning calorimetry (DSC), mechanical properties, as well as morphology of protein hydrolysate /EVA blends were investigated. Besides, the Brabender torque-time graph provides information on the effectiveness of the mixing or compounding, rheological behaviour, thermal and shear stability and the effect of different additives on the processing behaviour. The stabilization torque reached at the completion of melt mixing are used to assess the processability in the blend. The stabilization torque is an indicator for viscosity of the final blend. Rheological behaviour and biodegradability investigation are in progress.

Figure 1. Swelling of CH-PEG samples in distilled water. Polyethylene glycol (PEG) derivatives were also prepared in water solution following a synthetic procedure which involves the reaction between collagen hydrolysate aminogroup and an appropriate group of end functionalised PEG (i.e. acrylate, vinyl ether, glycidyl ether, methacrylate) [2] (Table 2). After grafting, a preliminary investigation was carried out to characterize the CH-PEG cast films in terms of thermal and mechanical behaviour, water sensitivity and propensity for degradation (Fig 1- 2).

Figure 2. Weight loss of CH-PEG samples in aqueous medium

Also in this case encouraging results were obtained: different CH-polymeric formulations resulted promising for industrial applications in several fields such as packaging, agriculture (as transplanting films with additional fertilizing action of CH), and leather industry. Acknowledgements This research was financially supported by SICIT Chemitech S.p.A. – Arzignano (VI). Reference [1] J.P.H. Van Wyk, Biotechnology and the utilization of biowaste as a resource for bioproduct development, Trends in Biotechnology 19 (2001) 172–177. [2] L. Sartore, M. Penco, A. Sassi, M.C. Candido, "Nuovi derivati polimerici biodegradabili” Italian Patent n° RM 2006A000682 (2006) - Sicit Chemitech S.p.A. [3] L. Sartore, M. Penco, A. Sassi, M.C. Neresini, M.C. Candido, "Blends biodegradabili a base di idrolizzati proteici e copolimeri etilenici funzionalizzati” Italian Patent n° MI2007A762 (13/4/2007)- Sicit Chemitech S.p.A. [4] M.M.Coleman, J. Graf, P.C. Painter, Specific Interaction and the Miscibility of Polymer Blends, Technomic: Lancaster, 1991. [5] D.L. Kaplan, J. M. Mayer, D. Ball, J.McCassie, A.L.Allen, P. Stenhouse, Fundamentals of biodegradable polymers, in “Biodegradable Polymers and Packaging”, eds. C. Ching, D.L. Kaplan, E.L. Thomas, Technomic Publishing CO. Inc. and reference therein.

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MATERIALI FUNZIONALI BIOISPIRATI A STRUTTURA MELANICA CONTROLLATA:POLIMERIZZAZIONE OSSIDATIVA DEL 5,6-DIIDROSSIINDOLO

IN POLIVINILALCOL/TAMPONE FOSFATO V. Ambrogia, V. Baronec, C. Carfagnaa,d, M. d’Ischiab, A. Napolitanob, L. Panzellab, A. Pezzellab,

R. Savaresea a Department of Materials and Production Engineering, University of Naples Federico II, Piazzale Tecchio 80125

Naples, Italy, e-mail: [email protected] b Department of Organic Chemistry and Biochemistry, University of Naples “Federico II” Complesso Universitario

Monte S. Angelo, Via Cintia 4, I-80126 Naples, Italy c Department of Chemistry, University of Naples Federico II, Complesso Universitario Monte S. Angelo, Via Cintia

4, I-80126 Naples, Italy d Institute of Polymer Chemistry and Technology of CNR, Via Campi Flegrei 34, 80078 Pozzuoli (Italy)

Intoduzione L’importanza delle eumelanine è riconosciuta da oltre un secolo e deriva dalla rilevanza che questi pigmenti hanno nella colorazione della pelle, nella fotoprotezione e nella cura di alcuni disturbi della pigmentazione come albinismo, vitiligine, melanoma. Più recentemente le eumelanine derivanti dalla polimerizzazione ossidative delle 5,6-diidrossiindolo (DHI) hanno attratto l’attenzione della comunità scientifica a causa delle loro proprietà chimico-fisiche già peraltro interessanti negli stessi oligomeri del DHI che presentano caratteristiche quali fluorescenza, semiconducibilità, atropoisomeria, suggerendo possibili impieghi nella realizzazione di materiali cosiddetti intelligenti1. Nonostante l’intensa attività di ricerca negli ultimi cinquant’anni, la struttura delle eumelanine e le sue proprietà fisiche risultano scarsamente definite a causa del carattere intrattabile di questi pigmenti, della loro eterogeneità chimica e della mancanza di proprietà chimico-fisiche ben definite che impedisce un’applicazione efficace delle tecniche spettrali moderne. In tale contesto risulta quindi evidente l’interesse verso la definizione di un quadro unitario della chimica ossidativa dei precursori indolici fino alla formazione della melanina. Inoltre il presente lavoro trova motivazioni anche nell’esigenza di chiarire il meccanismo di polimerizzazione ossidativa del DHI. Studi precedenti2, 3 hanno consentito l’individuazione di condizioni ottimali, relativamente a consumo di substrato e rese dei prodotti, in cui condurre l’ossidazione biomimetica del 5.6-diidrossiindolo. La difficoltà principale che si incontra nello studio di tali processi è essenzialmente legata alla maggiore ossidabilità dei prodotti di reazione rispetto allo stesso precursore per cui un maggiore consumo di reagente non sempre si riflette in un aumento delle rese dei prodotti. La reazione di ossidazione del DHI, condotta in condizioni ottimali, procede inizialmente con formazione di una fase cromoforica blu-viola, attribuita ad un ipotizzato intermedio chinonoide denominato melanocromo, che tende gradualmente a scomparire con concomitante formazione di un precipitato bruno scuro, melanico. La riduzione della miscela di reazione, nel momento di massima concentrazione del melanocromo, seguita da acetilazione della frazione estraibile in acetato

di etile ha consentito di isolare dei prodotti di natura dimerica e trimerica 2-6:

NH

OHOH

NHOHOH

NH

OH OH

NHOHOH

NHOHOH NH OH

OH

NH

OH OH

NHOHOH

NH OHOH

NH

OH OH

NHOHOH

NHOHOH

NH

OH

OH

2

5

43

6

DHI (1)

L’approccio biomimetico, pur consentendo progressi significativi nella conoscenza dei meccanismi di polimerizzazione dei 5,6-diidrossiindoli e circa la struttura dei costituenti molecolari del pigmento, presenta il limite di non consentire un facile accesso a strutture oligomeriche ad alto peso molecolare. Tale limite è fondamentalmente connesso con l’elevata reattività degli intermedi, in particolare la maggiore ossidabilità degli oligomeri rispetto al precursore monomerico, e il notevole moltiplicarsi di specie regioisomeriche al progredire del processo di polimerizzazione ossidativa. Qui riportiamo una possibile strategia modello per superare simili difficoltà basata sull’impiego di un polimero idrofilico quale il PVA che consenta di modificare le caratteristiche di viscosità del tampone fosfato e quindi possa modulare e rallentare il processo di accoppiamento ossidativo. Risultati e discussione In una serie di esperimenti preliminari le ossidazioni di DHI sono state condotte in soluzione di PVA a diversi pesi molecolari (Mn = 27000 Da e Mn = 61000 Da) e a differenti titoli (2, 4, 10 %(peso)) in condizioni biomimetiche, ovvero in soluzione acquosa a pH neutro in utilizzando come sistema ossidante perossidasi/H2O2. La procedura generale prevede l’aggiunta del sistema ossidante alla soluzione di indolo in tampone il che da luogo ad un rapido sviluppo della colorazione viola tipica della fase cromoforica denominata melanocromo. Nel momento di massimo sviluppo della colorazione


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viola la miscela di reazione è ridotta con una soluzione di ditionito di sodio, quindi acidificata a pH 5.0 ed estratta con acetato di etile, gli estratti organici sono seccati ed acetitati a dare una miscela di oligomeri 2-6. Le ossidazioni di DHI sono state ripetute variando natura del tampone di reazione e pH, ed i prodotti delle reazioni nelle varie condizioni sono stati analizzati mediante TLC analitica ed identificati per confronto delle proprietà cromatografiche con quelle di prodotti oligomerici noti e infine sono satati realizzati spettri di massa in modalità LC-ESI (+). Conducendo le reazioni in una soluzione al 10 % wt di PVA 27000 in un tampone fosfato 0.1 M a pH 7 ad una concentrazione di DHI di 15 mg/20 mL e di H2O2 rispettivamente di 20mM ed in presenza di 36U/mL di perossidasi si osserva la formazione del dimero 3, come prodotto principale, e in rese minori del dimero 2. Le miscele dei prodotti ottenuti in tali condizioni sono state analizzate mediante HPLC e sono state confrontate con quelle ottenute in assenza di PVA (vedi figura 1)

0

0,5

1

1,5

2

2,5

3

rese %molari

tampone FOSFATO/PVA tampone FOSFATO

2,4'-biindol ile 2,7'-biindol ile

Figura 1: Rese percentuali molari dei prodotti 2-3 ottenuti dall’ossidazione di DHI promossa da per ossidasi /H2O2 in: a) tampone fosfato 0.1M pH 7.0 PVA; b) tampone fosfato Esperimenti successivi sono stati finalizzati alla determinazione dell’effetto del PVA sulla velocità del processo ossidativo. Il decorso delle reazioni di ossidazione di DHI, promosse dal sistema perossidasi/H2O2, è stato quindi seguito spettrofotometricamente a 530 nm la lunghezza d’onda di assorbimento del massimo del melanocromo.

Il decorso nei due casi ovvero in solo tampone ed in presenza di PVA, si è mostrato molto simile in termini dell’aspetto del cromoforo tuttavia notevolmente differente dal punto di vista dell’evoluzione degli assorbimenti. La presenza del PVA nel mezzo di reazione ha provocato un rallentamento delle fasi di formazione e il decadimento del massimo a 530 nm come riportato in tabella I. Tabella I: Assorbanza e tempi di decadimento del massimo a 530 nm del melanocromo in fosfato/PVA e in fosfato

ABS 530 nm tempo Fosfato/PVA Fosfato 10’’ 0.740 0.800 30’’ 0.880 0.960 1’ 0.890 0.870 5’ 0.880 0.760 10’ 0.820 0.680 20’ 0.780 0.540

Conclusioni I risultati ottenuti sebbene preliminari sono di particolare interesse in quanto dimostrano la possibilità di utilizzare un “holder” di oligomeri del DHI che non solo stabilizzi la forma ossidata prevenendone la decomposizione ma consente anche di indurre un certo grado di controllo regiochimico nel processo di accoppiamento ossidativo. Da un punto di vista pratico questo può tradursi sia nella messa a punto di un processo di preparazione selettiva di oligomeri del DHI (i.e.2,7 biidolile 3) sia nella possibilità di una analisi dettagliata del cromoforo delle specie coinvolte nella ossidazione. Riferimenti 1. P. Meredith, B.J.Powell, J. Riesz, S.P.Nighswander,

M.R.Pederson and E.G.Moore, Soft Matter, 2, 37-44 (2006)

2. d’Ischia, M.; Napolitano, A.; Tsiakas, K.; Prota, G. Tetrahedron 1990, 46, 5789-5796

3. Pezzella, A.; Vogna, D.; Prota G, Tetrahedron Asymmetr., 2003, 15, 681-690


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CARATTERIZZAZIONE STRUTTURALE DI COPOLITIOESTERI BATTERICI MEDIANTE NMR E ESI-MS

G. Impallomeni,1 A. Steinbüchel,2 T. Lütke-Eversloh,2,3 T. Barbuzzi,4,5, A. Ballistreri 5

1 Istituto di Chimica e Tecnologia dei Polimeri, Consiglio Nazionale delle Ricerche, Viale A. Doria 6, 95125 Catania, Italy, 2 Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität

Münster, Corrensstraße 3, D-48149 Münster, Germany; 3Present Address: Massachusetts Institute of Technology, Department of Chemical Engineering, 77 Massachusetts Avenue 56-422, Cambridge MA 02139, USA; 4 Present Address: ST Microelectronics, Stradale Primo Sole, 95121 Catania, Italy; 5 Dipartimento di Scienze Chimiche,

Università di Catania, Viale A. Doria 6, 95125 Catania, Italy; e-mail: [email protected]

Introduzione I politioesteri (PTE) di origine microbica sono nuovi e interessanti biomateriali con proprietà fisiche e chimiche diverse da quelle mostrate dai loro analoghi poliossoesteri (PHA)1,2. Il loro impiego come materiali biocompatibili per varie applicazioni biomediche è previsto in quei settori dove è necessario sfruttare la loro più alta stabilità termica e le proprietà antibatteriche. In questo lavoro viene presentato uno studio sulla caratterizzazione strutturale di alcuni PTE utilizzando la spettroscopia NMR per i campioni non degradati e la spettrometria di massa ESI degli oligomeri generati dalla loro degradazione parziale. Inoltre, i campioni sono stati frazionati per GPC e le frazioni analizzate mediante 1H-NMR per la determinazione della composizione. Risultati Cellule del ceppo Ralstonia eutropha H16 furono coltivate in medium di sali minerali contenenti gluconato di sodio e 3-mercaptopropionato (MP) o 3-mercaptobutirrato (MB). Mentre questi ultimi tioderivati servirono come substrati precursori per l’incorporazione dei 3-mercaptoalcanoati (MA), il gluconato servì come sorgente di carbonio ed energia per la loro crescita. Le composizioni comonomeriche di quattro copolimeri 3-idrossialcanoati/3-mercaptoalcanoati (HA/MA) ottenute dall’analisi 1H-NMR vengono riportate in Tabella 1. Table 1. Contenuto delle unità monomeriche, lunghezza media dei blocchi e grado di randomness dei campioni Sample 3HB/3MP/3HP

(mol%) 3HB/3MB

(mol%) L3HB

L3MP

L3MB

DR

1

62/35/03 10.1

4.0

0.35

2 42/55/03

4.1 4.3 0.483 28/72

1.8 4.4 0.79

4 32/68

2.3 4.7 0.64 L’intensità dei segnali delle diadi relative alle zone dei carbonili negli spettri 13C-NMR ci permise di calcolare la lunghezza media dei blocchi ed il grado di randomness esposti in Tabella 1.

I campioni dei politioesteri furono sottoposti a metanolisi e pirolisi parziale e l’analisi successiva mediante spettrometria di massa-elettrospray (ESI-MS)3 permise l’identificazione dei vari oligomeri ottenuti. L’analisi statistica eseguita sui relativi spettri di massa mostrò che il modello della sequenza random non spiegava i risultati sperimentali. Ciò era dovuto alla parziale selettività delle due reazioni di degradazione: la metanolisi risultava più veloce al legame tioestereo che non a quello ossoestereo, mentre la pirolisi avveniva con meccanismi diversi alle unità 3MP e 3MB. I dati MS furono ugualmente utili, perché la maggior parte dei picchi presenti rivelò l’esistenza di specie contenenti tutte le unità ripetitive presenti nel campione, dimostrando che erano stati ottenuti veri copolimeri o terpolimeri. Infine furono registrati gli spettri 1H-NMR delle frazioni raccolte dopo la separazione GPC dei singoli campioni e fu determinato il contenuto delle unità monomeriche delle singole frazioni. Si osservò una significativa variazione nella composizione comonomerica delle varie frazioni, rivelando la presenza di blends di P(3HB-co-3MP-co-3HP) o P(3HB-co-3MB) con P(3HB). Conclusioni L’analisi ESI-MS dei prodotti generati dalla metanolisi o pirolisi parziale dei PTE studiati ha mostrato che essi sono veri copolimeri o terpolimeri. Tuttavia, la loro distribuzione di sequenza studiata mediante 13C-NMR dei campioni non degradati non segue il modello statistico Bernoulliano. Avendo trovato, mediante analisi 1H-NMR di frazioni GPC, che i campioni contengono anche omopolimero P(3HB), abbiamo calcolato le intensità teoriche delle diadi per miscele costituite da un copolimero random con P(3HB) ed abbiamo trovato una soddisfacente corrispondenza tra i valori calcolati ed i dati sperimentali (13C-NMR). Questa analisi riconcilia la microstruttura dei PTE con quella dei più studiati PHA: entrambi i gruppi di biopolimeri possiedono una sequenza random delle unità ripetitive Riferimenti 1. T. Lütke-Eversloh, K. Bergander, H. Luftmann, A. Steinbüchel, Microbiology, 147, 11 (2001). 2. T. Lütke-Eversloh, J. Kawada, R. H. Marchessault, A. Steinbüchel, Biomacromolecules, 3, 159 (2002). 3. T. Barbuzzi, M. Giuffrida, G. Impallomeni, S. Carnazza, A. Ferreri, S. P. P.Guglielmino, A. Ballistreri, Biomacromolecules, 5, 2469 (2004).


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NEW HYALURONAN SOLUBLE AND CROSSLINKED DERIVATIVES HAVING BIOMEDICAL POTENTIAL

C. Di Meo1, L. Panza2, L. Cornelio1, S. Nardecchia1, D. Capitani3, L. Mannina3, 4 and

V. Crescenzi1 1Department of Chemistry, University of Rome “La Sapienza”, p.le Aldo Moro 5, 00185 Rome, Italy;

email: [email protected] 2Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, University of Piemonte Orientale

“A. Avogadro”, via Bovio, 6, 28100 Novara, Italy 3Institute of Chemical Methodologies, CNR, Research Area of Rome, Via Salaria km 29,300, 00016 Monterotondo

Stazione (Rome), Italy 4Dipartimento di Scienze e Tecnologie Agro-Alimentari Ambientali e Microbiologiche, University of Molise, 86100

Campobasso, Italy Intrduction Hyaluronan (HA) is a biopolymer especially suited for formulations aimed at providing controlled and “targeted” drug delivery as well as at obtaining special scaffolds for tissue engineering. In this context, relevant examples, recently worked out in our laboratory, will be afforded pointing out the usefulness of both conventional chemical procedures and of novel approaches based on “click chemistry”. “Click chemistry” includes a few reactions compliant to a set of stringent criteria: rapidity, regioselectivity, stereospecificity and high yields. In addition, by-products (if any) must be easily removable by non-chromatographic methods. In this work, we applied a nowadays popular type of click chemistry reaction, i.e. the Huisgen 1,3-dipolar cycloaddition between an azide and an alkyne to form a triazole ring. In this way, novel HA soluble or crosslinked were obtained. Results a) Several hyaluronan derivatives bearing carborane rings potentially suitable for application in the boron neutron capture therapy (BNCT) and capable of targeting CD44 antigen, the main hyaluronan receptor widely expressed by several tumor histotypes, were prepared. The first derivative is composed by n-propyl carborane linked to hyaluronan via ester linkage for a degree of substitution of about 30%, leading to a water soluble derivative. The structure and the main physico-chemical characteristics of the new HA derivative were determined by means of FTIR, fluorescence and 1H, 13C, 10B NMR analyses1. In vitro biological experiments showed that the bioconjugate product was not toxic for a variety of human tumor cells of different histotypes; it specifically interacts with CD44 as the native unconjugated HA, and undergoes an uptake by tumor cells, leading to accumulation of amounts of boron atoms largely exceeding those required for a successful BNCT approach.

Other two water soluble macromolecular intermediates bearing free azido and alkyne functionalities in the side chains, respectively, were prepared by a partial derivatization of hyaluronan. These intermediates were separately reacted in water/N-methylpyrrolidone with, respectively, propargyl carborane, prepared with a new efficient synthesis, and with azidoethyl carborane in the presence of catalytic amounts of Cu(I) ions at room temperature. In both cases, the Huisgen 1,3-cycloaddition reaction took place smoothly. The polymeric samples were thoroughly characterized by means of NMR spectroscopy techniques that fully confirmed the hypothesized structures; the degrees of substitution resulted to be 17% and 8%, respectively2. b) We have applied the same type of click-reaction between an azide derivative and a propargyl-derivative of HA, both aqueous soluble, to obtain a new class of networks, i.e. HA-based “click-gels”3,4. In brief, water soluble polysaccharide derivatives bearing side chains endowed with either azide or alkyne terminal functionality were prepared. When the two types of derivatives are mixed together in aqueous solution, they give rise to 1,3-dipolar cycloaddition reaction resulting in a fast gelation (in the presence of catalytic amounts of Cu(I)) at room temperature. Gel formation was characterized rheologically and was followed qualitatively by means of IR spectroscopy. The resulting gels were studied in terms of swelling properties and, in particular, of 1H HR-MAS NMR spectral features4. Carrying out the gelation process in aqueous solutions of benzidamine and of doxorubicin, the polysaccharide networks acted as drug reservoirs. The doxorubicin release resulted well controllable acting upon the gels degree of cross-linking. Finally, the formation of the click-gels using aqueous suspensions of Saccharomyces cerevisiae yeast cells allowed us to obtain the scaffolds inside which cells were homogeneously distributed and smoothly adhered to the inner pores surfaces, according to SEM analysis. After 24 hrs about 60% of the entrapped cells exhibited proliferating activity.


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Conclusions a) Biological studies disclosed that all the HA-carborane derivatives are biocompatible and well tolerated by several tumor cell lines: they retain the capacity to specifically interact with CD44 receptor and to be internalized in high amounts, leading to an elevated boron atom content within cells. Therefore, they may be considered as new promising BNCT agents, endowed with both tumor-targeting characteristics and high 10B atom carrier capacity; these are important features that should increase the therapeutic efficacy and reduced side-effects. b) Click gels prepared as detailed herein do have a number of positive features that make them, in perspective, materials of choice for drug release and tissue engineering manipulations.

References 1. C. Di Meo, L. Panza, D. Capitani, L. Mannina, A. Banzato, M. Rondina, D. Renier, A. Rosato and V. Crescenzi Biomacromolecules,; 8; 552-559 (2007); 2. Di Meo, L. Panza, F. Campo, D. Capitani, L. Mannina, A. Banzato, M. Rondina, A. Rosato, V. Crescenzi Bioconjugate Chemistry (submitted); 3. Crescenzi, V.; Di Meo, C.; Galesso, D. Italian Patent n° M12006A001726, Dep. 11.09.2006. 4. V. Crescenzi, L. Cornelio, C. Di Meo, S. Nardecchia, R. Lamanna Biomacromolecules, ASAP article, web release date: 25 may 2007


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SPECIFIC INTERACTIONS VERSUS COUNTERION CONDENSATION IN THE CASE OF POLYURONATES. THEORETICAL TREATMENT WITHIN THE COUNTERION

CONDENSATION THEORY

I. Donati, S. Paoletti Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, Via Licio Giorgieri

1, I-34127, Trieste, Italy. E-mail: [email protected] Introduction The most relevant application of alginate and pectate in the biotechnology and health-related fields is connected with their ability to form stable gels when in contact with solutions of divalent cations such as calcium. Pectins are natural polysaccharides composed of sequences of α-D-galacturonate interrupted by a series of defects of natural sugars, including rhamnose, galactose and methyl-galacturonate ester.1 Alginate is a collective term for a family of polysaccharides produced by brown algae2 and bacteria.3,4 Chemically they are linear copolymers of 1→4-linked β-D-mannuronic acid (M) and α-L-guluronic acid (G) arranged in a blockwise pattern along the chain with homopolymeric regions of M (M blocks) and G (G blocks) residues interspersed with regions of alternating structure (MG blocks). As an example of relevant applications, different cells have been suggested as candidates for gel immobilization in microcapsules for cell therapy including parathyroid cells for treatment of hypocalcemia,5 dopamine-producing adrenal chromaffin cells for treatment of Parkinson’s disease,6 and endostatin-producing cells for treatment of brain tumors.7 Major interest has been focused on insulin producing cells for the treatment of type 1 diabetes, and alginate-poly-L-lysine capsules containing pancreatic islets of Langerhans have been shown to reverse diabetes in large animals.8 The ionotropic gelation of alginate and pectate has been described, based on X-ray fiber diffraction data from dehydrated specimens,9,10 using the so-called “egg-box” model.11,12 According to this model, chain-chain associations are induced by the presence of calcium and junction zones are formed between 2/1 helical chains of Guluronate (alginate) or Galacturonate (pectate) sequences that present cavities suitable to accommodate divalent cations in a chelate type of binding. Results In the present contribution, the characteristics of the interaction between non-gelling divalent cations (typically Mg2+) and polyuronates have been explored.13 In particular, three polyuronates mimicking separately guluronan (polyguluronate, polyG), mannuronan (polymannuronate, polyM) and polyalternating (polyMG), the three block-components of natural alginate samples, have been considered. Despite the absence of a site-specific chemical bonding between the two, as confirmed by circular dichroism spectroscopy, a substantial deviation of the experimental enthalpy of mixing from the theoretical

behavior, as predicted by the classical Counterion Condensation (CC) theory, was observed. Such deviation has been interpreted in terms of a “generic” non-bonding affinity of the condensed divalent counterion for the polyelectrolytes. The mathematical formalism of the CC theory was extended to include a contribution to the (reduced) free energy and enthalpy arising from the counterion affinity, gaff,0 and haff,0 respectively, and allowed the parametrical calculation of the fraction of divalent counterions condensed as function of the reduced thermodynamic quantity gaff,0. The theoretical predictions have been confirmed by means of viscosity measurements and 23Na-NMR spectroscopy. References 1) Rees, D.A.; Morris, E.R.; Thom, D.; Madden, J.K.; in The polysaccharides; Aspinall G.O. Ed.; Academic Press, New York; 196 (1982) 2) Painter, T. J. In The Polysaccharides; Aspinall, G. O., Ed.; Academic Press: New York; 1983, Vol. 2;195 3) Gorin, P. A. J.; Spencer, J. F. T. Can. J. Chem. 1966, 44; 993 4) Govan, J. R. W.; Fyfe, J. A. M.; Jarman, T. R. J. Gen. Microbiol. 1981, 125, 217 5) Hasse, C.; Boher, T.; Barth, P.; Stinner, B.; Cohen, R.; Cramer, H.; Zimmermann, U.; Rothmund, M. World J. S urg. 2000, 24, 1361 6) Winn, S. R.; Tresco, P. A.; Zielinski, B.; Greene, L. A.; Jaenger, C.B.; Aebischer, P. Exp. Neurol. 1991, 113, 322 7) Rokstad, A. M.; Holtan, S.; Strand, B.; Steinkjer, B.; Ryan, L.; Kulseng, B.; Skjåk-Bræk, G.; Espevik, T. Cell T ransplant. 2002, 11(4), 313 8) Soon-Shiong, P.; Feldman, E.; Nelson, R.; Komtebedde, J.; Smidsrød, O.; Skjåk-Bræk, G.; Espevik, T.; Heintz, R.; Lee, M. Transplantation 1992, 54, 769 9) Atkins, E. D. T.; Nieduszynski, I. A.; Mackie, W.; Parker, K. D.; Smolko, E. E. Biopolymers 1973, 12, 1865 10) Atkins, E. D. T.; Nieduszynski, I. A.; Mackie, W.; Parker, K. D.; Smolko, E. E. Biopolymers 1973, 12, 1879 11) Grant, G. T.; Morris, E. R.; Rees, D. A.; Smith, P. J. C.; Thom, D. FEBS Lett. 1973, 32, 195 12) Morris, E. R.; Rees, D. A.; Thom, D.; Boyd, J. Carbohydr. Res. 1978, 66, 145 13 Donati, I.; Cesàro, A.; Paoletti, S.; Biomacromolecules; 2006; 7; 281


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STRUCTURAL ANALYSIS OF THE POLYSACCHARIDES FROM ECHINACEA ANGUSTIFOLIA RADIX

R. Cozzolinoa, P. Malvagnaa, E. Spinaa, A. Giorib, N. Fuzzatib, A. Anellib, D. Garozzoa*,

G. Impallomenia * a CNR Istituto di Chimica e Tecnologia dei Polimeri, Viale R. Margherita 6 95125 Catania , Italy

b INDENA S.p.A. Via don Minzoni, 6 20090 Settala, Milano, Italy. Introduction Echinacea is a hardy perennial plant indigenous to North America, which belongs to the Asteraceae or Compositae plant family and includes nine different species (McGregor, 1968). Of these species E. purpurea, E. angustifolia, and E. pallida have medical properties (Schulthess, Ginger, & Baumann, 1991), and are commercially traded as medicinal plants. From the research conducted during the last 20 years Echinacea appears to have strong anti-inflammatory activity, wound-healing action, stimulates the immune system and may be effective against some viral and bacterial infections (Schulthess et al., 1991; Bauer, Reminger, & Alstat, 1990; Burger, Torre, & Warren, 1997; Cox, 1998 ). Generally, Echinacea is thought to create activity in the immune system by stimulating T-cell production, phagocytosis, lymphocytic activity, cellular respiration, activity against tumour cell (thought its application is debatable) and inhibiting hyaluronidase enzyme secretion (Brauning, Dom, Linburg, & Knick, 1992). Some experts believe that the polysaccharides are primary active ingredients for immune modulating effects (Tubaro, Tragni, Del Negro, Galli, & Della Loggia, 1987; Wagner, Stuppner, Schafer, & Zenk, 1988). It appears that the immune-stimulating effects of Echinacea result from polysaccharides surrounding tissue cells and thereby providing protection from bacterial and pathogenic invasion (Newall, Anderson, & Phillipson, 1996). The polysaccharide components have also been shown to promote tissue regeneration by stimulating fibroblasts and inhibiting the enzyme hyaluronidase, which breaks down the intracellular cement called hyaluronic acid (Enbergs, & Woestmann, 1986). Barman has reported about the HIV killing activity induced by E. angustifolia (Barman, See, See, Justis, & Tilles, 1998). Despite its popularity, the scientific understanding of how Echinacea extracts work on the immune system is incomplete and so is also the knowledge of the active components. Three different polysaccharides with immune-stimulating properties were isolated from E. purpurea and characterized: two neutral fucogalactoxyloglucans with mean molecular mass (Mr) of 10000 and 25000 and an acidic arabinogalactan with a mean Mr of 75000 (Wagner et al., 1988; Wagner, & Proksch, 1987), while the structure of polysaccharides from E. angustifolia is still unknown, even though the immune-stimulating properties of the polysaccharidic fractions is known since 1987 (Tubaro et al., 1987). In this paper we report the characterization of the polysaccharides extracted from E. angustifolia radix, by monosaccharide and linkage analyses and by NMR

spectroscopy and size exclusion chromatography. In addition, polysaccharidic fractions obtained by enzymatic degradations were characterized by MALDI-TOF mass spectrometry. The data obtained permitted to determine the distribution of methyl groups and degree of esterification over the homogalacturonan segments, and some structural features of the hairy region. Results and Discussion SEC analysis of the carbohydrate fraction extracted from Echinacea angustifolia radix showed that it is constituted by two polysaccharides with molecular weight (MW) of about 128.000 and 4.500 Daltons. NMR and MALDI TOF MS characterization of the low molecular weight fraction (data not shown) allowed to identify this sample as inulin. In fact 1H NMR and MALDI TOF MS spectra of a commercial sample of inulin were performed and appeared identical to those obtained from this fraction. Monosaccharide analysis, conducted on the high MW polysaccharide and performed both by HPAEC and GC showed that the monosaccharides present are GalA, Gal, Ara, Rha with a molar ratio of 20.5, 3.5, 5.0 and 1.0 respectively. Linkage analysis was conducted accordingly to Lindberg (Lindberg, 1972) and the results are reported in Table 1. Table 1. Linkage analysis results Partially methylated alditolacetates Terminal rhamnopyranosyl 2-O-rhamnopyranosyl Terminal arabinofuranosyl Terminal arabinopyranosyl 5-O-arabinofuranosyl 3,5-O-arabinofuranosyl Terminal galactopyranosyl 3-O-galactopyranosyl 6-O-galactopyranosyl 3,6-O-galactopyranosyl Terminal galacturonosyl 4-O-galacturonosyl In the 500 MHz 1H spectrum of the polysaccharide, reported in figure 2, the methyl group of Rha is visible as a broad multiplet at about 1.3 ppm. Comparing the area of this multiplet with that of the H4 of GalA (a broad resonance at 4.46 ppm) an abundance of 5% of Rha respect to GalA can be calculated, confirming the sugar analysis results. At 2.17 and 2.06 ppm there are two singlets that can be assigned to acetyl groups. The acetylation degree calculated respect to the H4 of GalA is 9%.


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In the spectrum of figure 2 there is also an intense singlet at 3.80 ppm which suggests the presence of –OCH3 groups belonging to the esterified GalA.

In the 13C spectrum, not shown, two signals at 100.19 and 100.93 can be readily assigned to the anomeric carbons of GalA and galacturonic acid methyl ester (GalAMe), respectively. The presence of GalAMe is confirmed by a resonance at 53.66 ppm, characteristic of methyl ester groups. In the carbonyl region of the spectrum, signals at 174.0 and 171.4 correspond to C6 of GalA and GalAMe, respectively. From the integration of the areas related to anomeric and carbonyl resonances it can be calculated the same degree of esterification of 60%. Treating the polysaccharide with 0.1 M NaOD at room temperature for 24 h and recording again the 1H and 13C spectra, after neutralization of the base with DCl, the results reported above can be confirmed. In particular, the proton spectrum, presented in figure 2, now appears simplified, as the heterogeneity due to partial methylation is absent.

Consequently, the resonances of GalA from H1 through H5 can be assigned. Furthermore, the intense singlet at 3.80 ppm due the –OCH3 of GalAMe present in the spectrum of the native polysaccharide is now absent, and in its place a singlet is found at 3.34 ppm. This peak is due to methanol formed after the saponification of the

ester, and from its area the degree of esterification of60% can be verified. The two O-acetyl signals of the native polysaccharide have been substituted by one singlet at 2.06 ppm that can be assigned to AcONa. Indeed, this chemical shift coincides with one of the two acetyl resonances in the spectrum of the native polysaccharide, but recording the NMR spectrum after saponification and before neutralization this signal goes to 1.90 ppm. In fact, at lower pH AcONa is partially converted to AcOH, bringing the methyl chemical shift to lower field. Again the area calculation of this signal confirms a degree of acetylation of 9%. From the data illustrated above it is possible to assert that the polysaccharide is a high methoxy (HM) pectin in which the backbone structure of the smooth region is constituted by β-(1-4) polygalacturonan partially carboxymethylated (60%) and acetylated (9%) and with the hairy regions containing 2-O and 2,4-O-rhamnopyranose, 5-O and 3,5-O-arabinofuranose and 3,6 galactopyranose and terminal rhamnose, arabinofuranose, arabinopyranose, galactopyranose and galacturonopyranose. In order to have some information about the GalAMe distribution along the homogalacturonan backbone, the polysaccharide was subjected to enzymatic degradations utilizing endo-pectin lyase and endo-pectate lyase and analysing the oligomers produced by MALDI MS. Figure 3 presents the MALDI mass spectrum of a high molecular weight fraction obtained from isopropanol precipitation of the products derived from degradation with endo-pectate lyase. This spectrum, containing oligomers of GalA units up to 20 and more, is particular suitable for statistical estimate.

By comparing theoretical spectra obtained using statistical calculation with the MALDI mass spectrum presented in figure 8, it was possible to verify a random distribution of the methyl ester groups according to a Bernoullian law and to calculate a methylation degree about 60% confirming the datum reported above. The data obtained during this study allow to conclude that Echinacea angustifolia radix contains two polysaccharides: inulin, and a high molecular weight polysaccharide corresponding to high methoxy (HM) pectin in which the backbone structure of the smooth region is constituted by k-(1-4) polygalacturonan partially carboxymethylated (60%) and acetylated (9%) and with the hairy regions containing 2-O and 2,4-O-rhamnopyranosyl, 5-O and 3,5-O-arabinofuranosil and 3,6 galactopyranosyl and terminal rhamnosyl.

500

250

2000

1000 1800m/

2497.4

2509.7

2523.75

2538.1

2551.8 2565.5 2671.6

2686.2

2700.2

2714.1

2727.6 2742.2

2755.3

2861.61

2876.1

2890.1

2903.6

2918.6 2931.1 2944.7 3052.1

3065.9

3079.9

3094.1 3108.5 3121.8 3136.4

(ppm) 1.4 2.2 3.0 3.8 4.6 5.4

HO

GalA H4

GalA-OCH3

O-Acetyl -CH3

(ppm) 1.2 2.0 2.8 3.6 4.4 5.2

GalA 1

GalA 5

GalA 3

GalA 2

GalA 4

CH3OH AcONa

Rha-CH3

HOD


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SINTESI E CARATTERIZZAZIONE DI DERIVATI RETICOLATI DELL’ACIDO IALURONICO

A. La Gatta, I. Marzaioli, A. De Rosa , C. Schiraldi

Dipartimento di Medicina Sperimentale, Seconda Università degli Studi di Napoli, Via L. De Crecchio 7, 80138 Napoli, Italia. E-mail: [email protected]

Introduzione L’acido ialuronico (HA) è un polisaccaride naturale a catena lineare costituito da unità ripetitive di N-acetil-D-glucosammina e acido D-glucuronico. La sua eccellente biocompatibilità, biodegradabilità e le sue proprietà chimico-fisiche (elevata idrofilia e viscoelasticità) hanno portato ad un suo massiccio impiego in campo medico e cosmetico, in particolare nel trattamento di problemi articolari e nella “tissue augmentation”. [1,2] Per tutte le applicazioni è da considerare che l’acido ialuronico esogeno è rapidamente degradato in vivo ad opera della ialuronidasi (HAsi): la sua emivita nei tessuti è di circa 12-24 h.[2] La ricerca è quindi impegnata nella sintesi di derivati dell’HA che siano più resistenti alla degradazione enzimatica in modo da aumentarne la permanenza in situ e di modularla a seconda del tipo di applicazione. Le strategie chimiche impiegate a tale scopo prevedono la reticolazione del polimero attraverso l’uso di svariati agenti reticolanti.[3] Una delle strategie più interessanti in termini di biocompatibilità del prodotto è l’autoreticolazione dell’HA attraverso la formazione di legami esterei tra i gruppi carbossilici e i gruppi ossidrilici della stessa molecola di HA. Tra le sostanze impiegate per l’attivazione del carbossile dell’HA, la 1-etil-3-(3-dimetilamminopropil) carbodiimmide (EDC) è quella più studiata.[3] Il presente lavoro consiste nella reticolazione di HA mediante l’uso di EDC in diversi rapporti stechiometrici e nella caratterizzazione dei prodotti risultanti sia sotto l’aspetto chimico-fisico sia in termini di studi in vitro di degradazione ad opera di ialuronidasi testicolare bovina. Materiali e Metodi La reticolazione dell’HA è effettuata sospendendo l’HA in una miscela di solvente organico/H2O in cui è disciolta la carbodiimmide in quantità molari pari al 2.5, 5, 20 e 100% rispetto ai gruppi carbossilici dell’HA. La sospensione è lasciata in agitazione a temperatura ambiente per 2 h. Il prodotto di reazione è sottoposto a

lavaggi con il solvente di reazione e, quindi, portato a secco. La modifica chimica apportata è stata indagata mediante analisi FT-IR. La solubilità in acqua a 37°C dei prodotti di reazione è stata determinata quantificando il glucuronico ritrovato nella frazione solubile mediante test del carbazolo. La degradazione dei prodotti di reazione è stata studiata in tampone fosfato pH 6.3 a 37°C e in HAsi 1 U/ml in tampone fosfato pH 6.3 a 37°C determinando, a vari tempi di incubazione, la frazione solubile di prodotto. Risultati e Conclusioni Gli spettri IR dei prodotti di reazione mostrano una nuova banda a 1700 cm-1, non presente nell’HA nativo, imputabile ad un C=O estereo. [3] I prodotti di reazione presentano una bassa solubilità in acqua a conferma dell’avvenuta modifica chimica. La solubilità aumenta al diminuire del grado di modificazione. Le prove di degradazione hanno rivelato che i prodotti si degradano anche in solo tampone fosfato pH 6.3 a 37°C divenendo completamente solubili dopo 28gg di incubazione. L’incubazione in presenza di HAsi non ha portato a differenze nei tempi di solubilizzazione ma un’analisi dei pesi molecolari dell’HA presente nella frazione solubile è in corso. I risultati ottenuti dimostrano che la strategia chimica adottata risulta in derivati dell’HA reticolati, insolubili, più stabili rispetto all’HA nativo e potenzialmente utilizzabili in applicazioni che prevedono tempi di degradazione dell’HA compatibili con quanto trovato sperimentalmente. Riferimenti 1. Y. Luo, R. Kirker Kelly, D. Prestwich Glenn, J Control Release, 69:169-184 2000. 2. Seth L. Matarasso, Aesthetic Surg J, 24:361-364 2004. 3. K. Tomihata, Y. Ikada, J Biomed Mat Res, 37:243-251 1997.


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MECHANICAL PROPERTIES OF WOOD FLOUR FILLED BIOCOMPOSITES: A STATISTICAL APPROACH

F. P La Mantia, R. Scaffaro, M. Morreale

Università degli Studi di Palermo, Dipartimento di Ingegneria Chimica dei Processi e dei Materiali, Viale delle scienze Ed. 6, 90128, Palermo, Italia; e-mail: [email protected]

Introduction Wood-flour filled polymer composites have several advantages they can assure, in comparison to mineral filler-polymer composites. These advantages regard mainly the low cost of wood based fillers, the lower specific weight, reduced hazards for production workers in case of inhalation, special aesthetic features, environmental issues [1]. According to scientific literature data, many studies have been done regarding polymer matrices (especially polyolefins) in combination with different natural-organic fillers [1]. Nevertheless, due to the matrix bioresistance, a limitation of these composites is represented by the absence of a complete biodegradability. To overcome this limit, it is necessary to replace the traditional, non-biodegradable polymer matrices with biodegradable ones like, for instance, those belonging to the Mater-Bi® family. Statistical analysis can be a very powerful tool to investigate on the properties of polymer composites, especially when both the polymer matrix and the filler are intrinsically heterogeneous (like in the case of Mater-Bi and natural organic fillers) [2, 3]. In this work, a full-factorial design [4] was carried out, in order to assess the statistical significance of several process variables (filler size, filler content, humidity content, processing temperature, processing speed) on the main mechanical and morphological properties of Mater-Bi® / wood flour biocomposites. Procedures and Results The Mater-Bi® used in this work is of unknown chemical composition but it is likely to contain a starch-based fraction and a synthetic biodegradable polyester. The wood flour was kindly supplied by LA.SO.LE. (Italy) in two different types: the “35” (average particle size 350-500 microns) and “150/200” (average particle size 150-200 microns) indicated respectively as “SDC” and “SDF”. The materials were mixed in a Brabender PLE330 batch mixer, thus compounding several blends with each of the five main process variables (filler content, filler size, treatment, mixing speed, mixing temperature) assuming two possible levels: for instance, filler content levels were 15 and 30 % by weight, filler sizes were the “SDC” and “SDF” previously described. Wood flour was always thermally treated before mixing, by drying it in a oven at 70°C overnight. Some samples were prepared with the matrix without any treatment (“humid”), while other (“dry”) were characterized by a thermal treatment on the polymer prior to processing, performed in a vacuum oven at 70°C for one night, plus one hour at 90°C. A number of different mixing conditions were

adopted, with temperature being 140°C or 160°C, rotating speed 30 or 60 rpm, mixing time 4 minutes. The blends were compression-molded in a Carver laboratory press at 180°C for 2-3 minutes in order to obtain the specimens for tensile, impact, and Heat Deflection Temperature tests. Tensile tests according to ASTM D882 were performed by an Instron mod. 3365 universal machine. Impact tests according to ASTM D256 were carried out by a Ceast mod. 6545 universal apparatus on notched samples in Izod mode. Heat Deflection Temperature (HDT) tests according to ASTM D2990 were performed by means of a Ceast mod.6505 apparatus. Finally, in order to get a deeper understanding of some phenomena emerged during the characterization, SEM analysis of specimens was carried out by a Philips XL30 equipment. In order to interpret the results obtained upon changing the operative conditions, a two-level full factorial design was performed according to the methods described by Box and Hunter [4]. This analysis allows investigating on the effects of multiple operative variables on the observed properties, and in particular the contribution of each variable, their optimal combinations and the possible interactions. The analysis of elastic modulus has been developed through a 25 factorial design with the five variables being filler content, filler size, mixing speed, temperature, polymer treatment. The analysis of variance and the t-test highlighted that the most important factor is filler content, which has a positive effect on the elastic modulus. Humid materials revealed to be stiffer than the dry ones, therefore this suggests that the adopted pre-treatment was not optimal. Speed had a moderate statistic significance, but showed to be practically not influent from an applicative point of view. The other two variables did not have any statistical significance. The statistically significant binary interactions revealed to be filler content/mixing speed, filler content/temperature, filler size/pre-treatment, mixing speed/temperature, mixing speed/pre-treatment; this was confirmed by the binary plots of the elastic modulus as a function of each individual couple of variables. An example is reported in fig. 1, which shows the filler content/temperature interaction. Interaction occurs in a very evident way since the two lines intersect each other at a filler wt% equal to 22. This means that up to this critical concentration, the materials mixed at 160°C are stiffer than those processed at 140°C, but this trend radically changes at higher concentrations.


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200250300350400450500550

15 18 21 24 27 30filler, wt%

E [M

Pa]

Fig. 1 – Filler content/Temperature Interaction. The filler content/mixing speed and the filler content/filler size interactions, though statistically significant, were not practically significant, in fact the two lines were basically coincident. The filler size/pre-treatment interaction is significant, and SDC is more sensitive to the drying of the matrix; possible explanations can be found in structural variations of the polymer matrix after the treatment and in the intrinsic variations of lignocellulosic fillers. The temperature/mixing speed interaction is significant, and in particular the critical temperature is 156°C, over which the higher mixing speed allows obtaining better mechanical properties, while 30 rpm gives better outcomes with lower temperatures. The mixing speed/treatment interaction is significant as well, suggesting also that the humid composites are better than the dry ones, even though it must be pointed out that at speeds higher than 65/70 rpm this trend should be reversed, since the stiffness of humid composites decreases with increasing the speed. A micro-scale confirmation of a certain degree of superiority in the humid composites was observed also by SEM analysis on the fracture surface morphology of humid and dry samples. All the remaining binary interactions were not significant, with parallel lines in the binary interaction plots. Statistical analysis of the impact strength was performed following the same procedures. The analysis of variance and the t-test provided that the major statistical weight is assumed by filler size and mixing speed, while only a moderate statistical significance should be attributed to temperature and pre-treatment, and no statistical significance to filler content. The statistically significant interactions revealed to be filler content/temperature, filler size/mixing speed, filler size/temperature, mixing speed/temperature, temperature/pre-treatment. The temperature/pre-treatment one was, in particular, the strongest interaction. Another significant interaction exists between temperature and speed. The composites prepared at 30 rpm are not practically sensitive to temperature variations, while those prepared at 60 rpm increase their impact strength upon increasing the temperature.

The temperature/filler size interaction plot (not reported) showed a superiority of SDC if compared to SDF, furthermore the dispersion of SDC in the matrix is improved through a temperature increase, which in turn improves the impact strength. The filler size/velocity interaction highlighted that SDC, probably thanks to his higher aspect ratio [1], grants higher values of impact strength if compared to SDF, an effect which increases with increasing the velocity because the latter allows a better mixing. An other significant interaction is the filler content/temperature one: the samples processed at 160°C have a higher impact strength, but this tendency seems to change at a predicted filler concentration of about 31 wt%. The remaining interactions had low or no statistical significance. The full factorial design adopted for the HDT analysis was a 23 rather than a 25 because two variables ( temperature and mixing speed) were maintained at 160°C and 60 rpm, respectively. The justification lies on the practical difficulties encountered to correctly produce the HDT test samples coming from the blends processed at low temperature and low mixing speed. The analysis of variance provided that only filler content and filler size have a statistically significant weight, while no statistical significance was attributed to the treatment and the binary interactions. The filler concentration exerted a positive influence on the HDT, and the influence of filler size was in accordance with the results of elastic modulus. The pre-treatment did not have any practical significance. Conclusions Statistical analysis can be a very useful approach to deepen the characterization of complex systems like polymer composites, allowing to handle huge amounts of experimental data and to draw important information from them, in order to assess the optimal combinations of the processing parameters. In this work, a full factorial design has been performed on the mechanical properties (elastic modulus, impact strength, heat deflection temperature) of Mater-Bi®-wood flour biocomposites. It has been found that the most influencing variable on elastic modulus and HDT is represented by filler content, while the ones which most influence impact strength are filler size and mixing speed. References

1. F.P. La Mantia, M. Morreale, Polym. Eng. Sci. 46, 1131 (2006)

2. M. Johnson, Ind. Cro. Prod. 22, 175 (2005) 3. R.M. Johnson, N.Tucker, S. Barnes, Polym.

Test. 22, 209 (2003) 4. G.E.P. Box, J.S. Hunter, W.G. Hunter, Statistics

for experimenters, 2nd edn, J.Wiley&Sons, Hoboken, NJ (2005)


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IMPIEGO DI POLIIDROSSIALCANOATI IN MISCELE POLIMERICHE BIODEGRADABILI: PROPRIETA’ E APPLICAZIONI

R. Scaffaro1, R. Marino1,2, G. Marchese1,2, F. Salemi2, F.P. La Mantia1,

1Università degli studi di Palermo, Dipartimento di Ingegneria Chimica, dei Processi e dei Materiali, Viale delle Scienze, 90128, Palermo, Italy; e-mail: [email protected]

2Parco Scientifico e Tecnologico della Sicilia S.c.p.a., Z. I. Blocco Palma I Stradale V. Lancia 57, 95030 Catania, Italy; e-mail: [email protected]

Introduzione I Poliidrossialcanoati (PHA) sono dei poliesteri termoplastici sintetizzati e accumulati a livello intracellulare da diversi microrganismi durante la loro crescita. Possiedono proprietà comparabili alle plastiche convenzionali ed il vantaggio di essere biodegradati molto facilmente, infatti non necessitano di ambienti e condizioni particolari per essere idrolizzati [1]. In questo lavoro vengono riportati risultati relativi a miscele polimeriche costituite da ZI01U/C (ZI), prodotto e commercializzato da Novamont S.p.A, PHA e Biomassa (BIO) batterica liofilizzata contenente il 30% (in peso) di PHA, forniti dal Parco Scientifico e Tecnologico della Sicilia S.c.p.a. (PSTS). Nello specifico ZI01U/C è un polimero termoplastico biodegradabile avente le stesse caratteristiche chimico-fisiche dei polimeri convenzionali, ma completamente biodegradabile una volta abbandonato nell’ambiente e il PHA è sintetizzato dal ceppo batterico Pseudomonas corrugata IPVCT A.1. [4]. L’obiettivo di questo lavoro è quello di studiare le proprietà [3] igroscopiche, meccaniche e reologiche di queste miscele e valutare il loro eventuale impiego in processi industriali. Risultati I materiali oggetto di questo studio sono stati realizzati in estrusore bivite controrotante avente L/D=7, con velocità delle viti di 60 rpm e temperatura di testa di 110 °C, nelle composizioni ZI/PHA 95,5/4,5 e ZI/BIO 85/15. E’ stata fatta un’indagine sulla capacità di adsorbimento e di desorbimento di umidità relativa delle miscele realizzate: i campioni da sottoporre alle prove di trazione, con lo spessore di circa 1 mm, sono stati prima introdotti in una camera climatica a 25 °C e 50% di umidità e poi, mantenendo la stessa temperatura, in una stufa sottovuoto. In accordo con le curve di cinetica di assorbimento, qui non riportate, i campioni raggiungono le condizioni di equilibrio igroscopico dopo circa 60 ore nella prova di adsorbimento, nella prova di desorbimento dopo 25-30 ore. Le prove reologiche sono state effettuate tramite un reometro rotazionale a piatti piani e paralleli ((Rheometrics RDAII, avente diametro dei piatti 25mm a 110°C nel campo di frequenze 0,1-500 rad/sec con il 5% di deformazione). In figura 1 è rappresentato l’andamento della viscosità adimensionalizzate ottenuta dividendo la viscosità delle miscele per la viscosità del materiale puro (ηad), in funzione della frequenza delle oscillazioni dei piatti. Le prove sono state eseguite a 110°C e il campo di

frequenze indagato va da 0,1 a 100 rad/sec Il materiale contenente la biomassa, in tutto l’intervallo di frequenze indagato, ha una viscosità più alta rispetto al materiale che contiene il PHA e negli intervalli di frequenza tipici per le principali lavorazioni ηad < 1, soprattutto nel materiale contenente il PHA. Tale occorrenza, evidenziando una diminuzione della viscosità nelle miscele rispetto alla matrice pura, indica un miglioramento della lavorabilità.

0,8

1

1,2

1,4

1,6

1,8

2

0,1 1 10 100 1000Freq (rad/s)

Viscosità adimensionale

Z I/PHAZ I/BIO

Fig.1 : Curve di flusso adimensionalizzate I provini (12,7x70x3 mm) in equilibrio igroscopico (camera climatica a 25°C e a 50% di umidità per 80 ore) ed essiccati in stufa sottovuoto (a 25 °C per 30 ore), sono stati sottoposti a prove ad impatto (Izod, ASTM D256). La prova è stata eseguita con un pendolo da 2 Joule ed i risultati sono illustrati in figura 2.

84

66,8

118

65,4

34,0

52,943,1

80,0

57,1

81,890,1

60,9

0

20

40

60

80

100

120

140

ZI ZI/BIO ZI/ PHA

R (J/m) Umidi (int)Secchi (int)Umidi (non int)Secchi (non int)

Fig.2: Prove ad impatto

Il materiale preparato con il 15% di Biomassa presenta una riduzione della resilienza rispetto al materiale vergine, questo è probabilmente dovuto al fatto che la biomassa favorisce la formazione di difetti e di discontinuità nel materiale che si propagano, per effetto della sollecitazione impulsiva, più facilmente di quanto non succeda nel materiale vergine. Nei campioni contenenti il 4,5% di PHA si presenta una notevole differenza tra la resilienza dei campioni intagliati e di quelli non intagliati; la presenza di PHA


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sembra determinare un significativo aumento dell’energia di innesco della frattura e ciò costituisce un miglioramento di questa proprietà. I materiali estrusi sono stati successivamente lavorati tramite stampaggio ad iniezione. Tale processo assicura un’elevata produttività, la possibilità di adattarlo ad un buon livello di automazione e di applicabilità. Le prove di stampaggio sono state effettuate su tutte le miscele utilizzando diverse condizioni operative fino al raggiungimento di un optimum in termini di stampabilità: profilo di temperatura 120°C-120°C-120°C , tempo di mantenimento di 1 sec, pressione di mantenimento 900 bar, dosatura di 60 mm e velocità di iniezione 6 cm3/sec. Le miscele in presenza del PHA risultano migliori, in termini di attitudine allo stampaggio, rispetto al materiale vergine. Sul materiale estruso e sui provini ottenuti per stampaggio ad iniezione (10x90x2 mm) sono state realizzate prove meccaniche (Instron 3365 secondo la norma ASTM D882). Per il materiale estruso i provini sono stati ottenuti per stampaggio a compressione in una pressa Carver alla temperatura di 110°C. I relativi risultati sono riportati in tabella 1. Tabella 1: Prove meccaniche

Modulo elastico ,MPa

Sforzo a rottura, MPa

Allungamento a rottura,%

ZI Iniezione 212 10,8 21 ZI/PHA Iniezione 190 9 46 ZI/BIO Iniezione 201 6,7 75 ZI Compressione 314 10,5 7 ZI/PHA Compressione 294 10,2 8 ZI/BIO Compressione 220 9,8 140 Dai risultati delle prove di trazione si evince che il PHA, per tutte e due tipologie di provini, determina una modesta riduzione del modulo elastico e dello sforzo a rottura rispetto al materiale vergine. Tali risultati possono essere spiegati considerando una diversa degradazione nei due processi legata a differenze dei tempi di residenze a temperature di processo lievemente più elevate nello stampaggio ad iniezione. Peraltro, le prove morfologiche, qui non riportate,confermano nei campioni stampati ad iniezione la presenza di microvuoti probabilmente generati da prodotti di degradazione durante la lavorazione. Per quanto riguarda l’allungamento a rottura la presenza di biomassa ne provoca un aumento notevole: nel campione stampato ad iniezione si passa dal 21 al 75%, e nel campione stampato a compressione da 7 a 140%. Per il campione ZIO/BIO ottenuto per stampaggio ad iniezione si ha una diminuzione del 40% dello sforzo a rottura rispetto al materiale puro; questa diminuzione risulta trascurabile se si considera lo stesso campione stampato a compressione. Ciò è ascrivibile alla diversa

morfologia raggiunta dal materiale nei due processi (prove non riportate), alla presenza, nel campione stampato ad iniezione, di bolle di gas e di una degradazione maggiore. Allo scopo di indagare più a fondo sulle possibili cause legate all’aumento dell’allungamento a rottura nel campione ZI/BIO preparato per compressione, si sono effettuate indagini di tipo calorimetrico tramite calorimetria a scansione differenziale. Da tali prove si evince un aumento del calore di fusione per il campione ZI/BIO rispetto alla matrice pura, a conferma che durante la prova di trazione il materiale con il 15% di Biomassa è soggetto a fenomeni di cristallizzazione sotto stiro che contribuiscono a favorire l’aumento dell’allungamento a rottura. Dall’analisi morfologica condotta al S.E.M. si è ipotizzato che il significativo aumento dell’allungamento a rottura potrebbe essere dovuto allo scorrimento tra le molecole della matrice polimerica e gli agglomerati della Biomassa favorito dal PHA in essa contenuto. Conclusioni In questo lavoro si è valutata la possibilità di migliorare le performance meccaniche e reologiche del Mater-Bi ZI01U/C, utilizzato come matrice, introducendo PHA e Biomassa. La caratterizzazione reologica ha messo in evidenza un miglioramento della lavorabilità del materiale. Dalle prove d’impatto si è dedotto che l’introduzione del PHA nella matrice polimerica sembra avere un doppio effetto: da un lato determina l’aumento dell’energia d’innesco della frattura del materiale, dall’altro, sotto forma di Biomassa, può favorire la propagazione delle microcricche. Le indagini effettuate in questo lavoro dimostrano che esiste la possibilità di migliorare le proprietà del Mater-Bi ZI con il PHA e la Biomassa, per applicazioni industriali. La caratterizzazione meccanica ha permesso di valutare l’effetto dell’aggiunta di PHA e Biomassa sul comportamento meccanico sul materiale stampato per compressione e per iniezione e di apprezzare il miglioramento nella duttilità di tali materiali in presenza di Biomassa. Riferimenti 1. R.J. Sanchez, J. Schripsema, L.F. da Silva, M.K.Taciro, J. G. C. Pradella, J.G. C. Gomez, European Polymer Journal, vol. 39, pag.1385-1394 (2003) 2. G. de Koning, B. Witholt, Materials Science and Engineering C, vol. 4, pag.121-124 (1995) 3. Parco Scientifico e Tecnologico della Sicilia S.c.p.a., Processo di fermentazione per la produzione di poliidrossialcanoati e lo smaltimento di oli esausti mediante l’impiego di ceppi di Pseudomonas produttori di lipasi, Brevetto Italiano No. RM2005A000190 (2005)


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SMART ALGINATE MICROSPHERES BY COATING WITH A CATIONIC THERMOSENSITIVE BLOCK COPOLYMER

G. Mascia, P. Matricardib, , T. Coviellob, F. Alhaiqueb

aChemistry Department, bFaculty of Pharmacy, “Sapienza” University of Rome, p.le A. Moro 5, I-00185 Rome Italy e-mail: [email protected]

Introduction Alginate is a well known polysaccharide widely used due to its gelling properties in aqueous solutions related to the interactions between the carboxylic acid moieties and bivalent counter ions, such as calcium1. In the pharmaceutical field, alginate is widely used as excipient in tablets, with modulated drug delivery dosage forms being developed from its gelled forms. Numerous applications of calcium alginate gel beads or microspheres have been proposed, in particular for drug/protein delivery or cell entrapment. In order to optimize the modulation of drug release from such systems, alginate gel matrix surface was modified by means of macromolecules, such as chitosan, chitosan derivatives, and poly-L-lysine, which are able to establish ionic interactions with the alginate carboxylate ions, thus forming a shell around the alginate gel systems that, in turn, become more resistant and suitable for numerous applications. In order to impart thermo-sensitive properties to calcium alginate microspheres, a new thermosensitive polycationic block copolymer (PCT) showing a Lower Critical Solution Temperature (LCST) is synthesized and used as microparticle coating agent. Materials and Methods PCT is a new cationic block copolymer of poly(N-isopropylacrylamide) (NIPAAM) and poly(3-acrylamidopropyl) trimethylamonium cloride) (AMPTMA) obtained via Atom Transfer Radical Polymerization (ATRP), with 100 and 50 repeating units of AMPTMA and NIPAAM, respectively. Calcium alginate microspheres (200-500 µm) are prepared by percolation of a water alginate solution into a CaCl2 solution using an “electrostatic microbeads generator”. The microparticles are then soaked in a PCT water solution at 25 °C. The obtained coated microspheres are tested for their thermo-sensitive behaviour. In particular adhesion and drug carrier properties at different temperatures are investigated.

Results PCT, above a critical concentration, shows a LCST in water, with core-shell micelles formation above 40°C for the presence of the thermosensitive NIPAAM

block.2,3 In the adopted experimental conditions, at room temperature, the cationic moiety of PCT interacts preferably with the negatively charged carboxyl groups of alginate lying on the surface, leading to freely fluctuating microspheres showing thermo-sensitive behaviour. Above 40 °C they aggregate due to hydrophobic interactions among the pNIPAAM blocks of the chains located on the surface of the microspheres. The effect of the presence of the thermo-sensitive block copolymer below and above the LCST on the delivery of drugs and model compounds from the calcium alginate microspheres is reported. Conclusions The surface modification of calcium alginate beads leads to a system that appears to be suitable as a new drug delivery system that can be very useful in applications where thermo-sensitive microparticles and/or hydrophobic interactions are needed. References 1. Coviello T., Matricardi P., Marianecci C., Alhaique

F., J. Control. Release 119, 5 (2007). 2. Yoshida R., Uchida K., Kaneko Y., Sakai K.,

Kikuchi A., Sakurai Y., Okano T., Nature 374, 240 (1975). 3. Masci G., Giacomelli L., Crescenzi V., Macromol. Rapid Commun. 25(4), 559 (2004).

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POROUS BIOPOLYMERS BY SCCO2/W TEMPLATED EMULSIONS

C. Palocci, A. Barbetta, M. Dentini Università di Roma “La Sapienza”, Dipartimento di Chimica, Piazzale Aldo Moro 5, 00185 Roma, Italy; email:

[email protected] Introduction The preparation of porous monolithic biomaterials as separation media or support for cell (or tissue) cultures is an emerging area of research activity. These materials can be easily produced by oil-in-water emulsion templating and give rise to matrices suitable in tissue engineering as a result of their high porosity and fully interconnected morphologies. More recently, the authors demonstrated that the reaction conditions can be specifically modified to generate well-defined macroporous biopolymer monoliths by using SCCO2 as the internal phase. No organic solvent is required in this process: just water and CO2. Results As a model biopolymer for the preparation of concentrated C/W emulsions it was chosen dextran, an anhydroglucose polymer consisting mainly of α-(1-6)-glucosidic linkages with occasional branches at C(3). In order to lock-in the structure of the external phase, once the concentrated emulsion is formed, the dextran chains were previously functionalised (DMA) with methacrylic moieties (Figure 1). We employed a perfluoropolyether surfactant (PFPE) characterized by a low molecular weight, very soluble in water and able to stabilise C/W emulsions. An aqueous solution of DMA (20-25% w/v) and PFPE was employed as external phase of the emulsion together with a radical initiator in order to make possible the radical polymerization among the vinylic functionalities. The novelty of this approach was the use of SCCO2 as the internal phase of the emulsion. The morphology of the templated polymeric matrices depend on several physico-chemical parameters of the parent emulsion: concentration of the polymer dissolved in the external phase, type and concentration of the surfactant employed to stabilize the emulsion, volume fraction of the internal phase (φ), method used for the preparation of the emulsion. In the case of SCCO2 emulsions, an additional parameter was represented by CO2 density which in turn can be modulated by pressure and temperature variations. To find a possible correlation between the physico-chemical composition parameters of the parent emulsions and the morphology of the ensuing porous biomaterials we decided to vary only the volume fraction of the dispersed phase (φ) and the surfactant concentration (Cs) while the other variables were keep constant. Foam morphologies of the resulting porous materials were investigated with scanning electron microscope (SEM) and morphometry of the voids and interconnects were carried out on micrographs of the specimens sections obtained by light microscopy. In the first instance Cs was kept constant at a value of 3.5% w/v with respect to the volume of the dispersed

phase and φ was varied in the range between 0.75-0.95. In Figure 2 SEM and LM micrographs are displayed according to increasing φ. The qualitative inspection of both SEM and LM micrographs of the porous structures characterised by a nominal φ in the range between 0.75-0.92 (Figure 2a, b, c) shows that there is a significant increase in both the void and interconnect sizes. Above 0.90 such an increase is relatively less pronounced. The increase in φ offers a limited tool to change over a wide range the morphological features of the matrices. On the contrary, the possibility to tailor the size of voids and interconnects according to those required by applications represent a very important issue. In order to obtain porous matrices characterised by pores and interconnects of adequate dimension for tissue engineering applications it was though to induce a controlled coalescence among the droplets of the dispersed phase. Above a certain concentration, PFPE molecules organize into micelles of ~ 100 nm in dimension. These aggregates promotes a phenomenon known in emulsion science as depletion flocculation. This phenomenon consists of the exclusion of the micelles from the interstitial space between two neighbouring droplets. The concentration of non-adsorbed entities increase outside of the gap and create an osmotic pressure gradient between the interstitial space and the solution at the plateau borders. As a consequence the droplets of the dispersed phase are pushed into contact. The interface in such circumstances may become very weak and more prone to coalescence phenomena. The implementation of such an approach was achieved by simply increasing the Cs from 3.5 to 5% w/v while φ was set at 0.75, and 0.90. In Figure 4 the SEM and LM micrographs of 75-5.0 and 90-5.0 are shown. While the morphology of 75-5.0 underwent only minor changes with respect to 75-3.5, that of 90-5.0 changed dramatically. All these features indicate that the parent emulsion of 90-5.0 underwent coalescence to some extent On the overall 90-5.0 porous structure may be a good candidate as a scaffold for tissue engineering applications. Conclusions This paper presents one of the very few attempt to synthetise porous biomaterials through the employment of natural polymer such as dextran in combination to a pore generating procedure based on SCCO2/W emulsion templating. Moreover, this methodology does not involve any volatile organic or toxic solvents either in the synthesis or in the purification steps. It was shown that the matrix morphology can be tailored by varying the volume fraction of the dispersed phase. In order to increase both voids and interconnections size a partial destabilization of the parent emulsion was induced by


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modulating the relative surfactant concentration. At a Cs=5% w/v the parent emulsion characterised by a relatively high φ (0.90) underwent partial coalescence enabling the formation of a high percentage of large voids in the ensuing solid foams. Future work will be focused on the possibility to improve further the morphology of the matrices in terms of size and interconnects and to extend this methodology to the processing of other biological polymers such as gelatin, and hyaluronic acid. References (1) (a) Hautal, W.H. Chemosphere 2001, 43, 123-135. (b) Cruz, F. J.; Szwajcer, D.E. Acta Microbil.Pol. 2003, 52, 35-43. (c) Williams, J.R.; Clifford, A.A.; Al-Saidi, S.H. Mol. Biotechnol. 2002, 22, 263-86. (2) (a) Cooper, A.I. Adv. Mater. 2003, 15, 1049-1059. (b) Hebb, A.K.; Senoo, K; Bhat, R.; Cooper, A.I. Chem. Mater. 2003, 15, 2061-2069. (c) Butler, R.; Davies, C.M.; Cooper, A.I. Adv. Mater. 2001, 13, 1459-1063. (d) Butler, R.; Hopkinson, I.; Cooper, A.I. J. Am. Chem. Soc. 2003, 125, 14473-14481. (3) (a) Howdle, S.M.; Watson, M.S.; Whitaker, M.J.; Popov, V.K.; Davies, M.C.; Mandel, F.S.; Wang, J.D.; Shakesheff, K.M. Chem. Commun. 2001, 109-110.

Figure 2. Photograph of a low-density C/W templated dextran-methacrylate based solid foam.

Figure 3. Scanning electron (1) and light (2) micrographs of dextran-methacrylate solid foams characterized by increasing nominal volume fraction of the dispersed phase (φ): (a) 75%, b) 90%, c) 92%, d) 95%, Cp=25% w/v, Cs=3,5% w/v

Figure 4. Scanning electron (1) and light (2) micrographs of dextran-methacrylate solid foams characterized by increasing nominal volume fraction of the dispersed phase (φ): a) 75%, b)90%. Cp=25% w/v, Cs=5% w/v


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COMPOUNDING LDPE-LAYERED SILICATE NANOCOMPOSITES: EFFECT OF PROCESS PARAMETERS

M. Scatto1, L. Andreotti1, S. Coiai1, M. Sterner1, M. Bertoldo2, E. Passaglia3, L. Conzatti4

1 Centro Italiano Packaging (CIP), Via delle Industrie 25/8- 30175 Venezia – Italy e-mail: [email protected]

2 PolyLab-INFM, Largo Pontecorvo 3, 56127 Pisa, Italy 3 CNR-ICCOM Pisa Section, Department of Chemistry and Industrial Chemistry, University of Pisa, Via

Risorgimento 35, 56126 Pisa, Italy 4 ISMac-CNR, Division of Genoa, Via De Marini 6, 16149 Genova, Italy

Introduction In recent years, polymers filled with low amounts of high aspect ratio layered silicates, dispersed at nanoscale level, have been the subject of intense research efforts due to the possibility to obtain materials with very promising properties. Particularly interesting is the application of nanocomposites as novel food packaging materials because of their several benefits such as enhanced mechanical, thermal and barrier properties. However to completely benefit from all these advantages it is necessary to achieve exfoliation or delamination of the large stacks of silicate nanoplatelets into single layers or tactoids of a small number of layers. Only then the enormous aspect ratio of the platelets can fully contribute to the nanocomposite property profile. In this perspective it has been found that processing conditions, promoting high shear stress and shear rate, may aid in clay dispersion along with the use of proper organic clay modifiers and compatibilizer [1,2,3]. In this work low density polyethylene (LDPE, Riblene FL34 Polimeri Europa)/organoclay (Dellite 72T, Laviosa) nanocomposites were prepared via direct melt intercalation in a co-rotating twin screw extruder (Thermo) with a screw diameter of 24 mm and a length-to-diameter ratio (L/D) of 40. The objective was to investigate the effects of feed and processing conditions upon the formation and properties of LDPE nanocomposites. Figure 1 – Extruder and process parameters Maleic anhydride grafted very low density polyethylene (VLDPE-g-MAH Compoline, Auserpolimeri) was used as compatibilizer to improve the dispersion of the clay. Nanocomposites were obtained by previous preparation of a masterbatch by mixing the compatibilizer and organo-clay with 90/10 as feed ratio (by adding the clay through a side feeder), operating at 180-200 °C and different screw rates in co-rotating mode. Later, nanocomposites with mass fraction composition equal to 90/10/1 and 70/27/3 (LDPE/VLDPE-g-MA/Dellite 72T) were prepared by mixing the pure LDPE

and masterbatch in the twin screw extruder at 180-200 °C and 90 and 150 rpm respectively. The effect of operating conditions on the nanocomposite was established by on-line capillary rheological measurements, thermo gravimetric analysis (TGA), X-ray diffraction (XRD) and transmission electron microscopy (TEM). Results The XRD analysis of the masterbatch showed that the (001) basal reflection of the organoclay was shifted toward a lower angle indicating the increase in interlayer spacing after the extrusion process (from 25.2 Å to 41.1 Å, fig.2). In addition the absence of the basal reflection of the original organo-clay (at 2θ=3.5°) suggests that an intercalated structure was obtained. A slight improve of the intercalation level was achieved for the highest screw speed (90rpm). Direct morphological observations by transmission electron microscopy (TEM) analysis allow to conclude that in the master-batch coexist both disordered exfoliated platelets and intercalated tactoids with a very good dispersion degree. The XRD analysis of both LDPE/VLDPE-g-MAH/D72T composites (90/9/1 and 70/27/3) evidenced a further increase of the clay intergallery spacing with respect to the master-batch. Moreover the peak intensity significantly decreased indicating a promotion of clay exfoliation in the LDPE matrix. Apparently the shift of the (001) peak as well as the intensity decreasing seem more evident for the nanocomposite 70/27/3 characterized by higher concentration both of clay and compatibilizer. The (001) peak did not change appreciably in position and size by changing the extruder’s screw rate. TEM micrographs for Nanocomposite 90/9/1 and 70/27/3 confirmed the XRD results: the samples are mainly composed of exfoliated clay layers, even if still exist a very small amount of intercalated tactoids. Figure 2 - XRD patterns of master-batches prepared at different screw speeds (λ=0.15406, 1.5–10° 2θ region, 0.016 degree/sec) and at same feed rates.

2 3 4 5 6 7 8 9 10

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0

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0 50000 100000 150000 200000shear stress (Pa)

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LDPE/VLDPE-g-MAH70/27 (150 rpm)Composite 70/27/3(90rpm)Composite 70/27/3(150 rpm)

The thermal stability of pure VLDPE-g-MAH, of LDPE/VLDPE-g-MAH blends and of their composites with D72T was tested by thermo-gravimetric analysis (TGA) carried under air flow at 10°C/min from 25 to 900°C. Degradation starts before 400°C in all samples except in master-batch. Both master-batch and nanocomposites start to degrade at temperature higher than pure VLDPE-g-MAH and LDPE/VLDPE-g-MAH blends, showing to be more stable than the pure polymers. In order to understand the potential processability of layered silicate nanocomposites, the steady shear response at high shear flow conditions was investigated with on line capillary rheometer equipped with capillary dies having a diameter of 1 mm. It is possible to observe that at high shear rates the viscosity and the shear thinning behavior of the nanocomposites are comparable with the unfilled polymer, with a slight trend in decreasing viscosities values at higher silicate loading (3% D72T) and at higher screw speed (150rpm) as reported in Fig.3 at low shear stress. Figure 3 - Rheological behavior of nanocomposite 3% D72T, compounded at 90 and 150 rpm and of the pure matrix (LDPE/VLDPE-g-MAH 70/27)

Moreover increasing the organoclay content led to increase of the barrier properties. Oxygen transmission rate (OTR) at 23°C, 0%RH of Composite 70/27/3 is decreased of 21% compared to OTR of LDPE/VLDPE-g-MAH (70/27wt-%/wt-%) matrix . In conclusion, the proposed process seems to be very efficient to develop LDPE-Montmorillonite nanocomposites, in particular: -WAXD results and TEM micrographs indicated a good dispersion of platelets in LDPE matrix. -Rheological results suggest that nanoclay particles do not significantly impair the LDPE processability. -The nanofiller exerts a positive effect on the thermal stability and barrier properties (permeability) indicating that these materials are promising systems for food packaging applications. Acknowledgments: The authors gratefully acknowledge Prof. Francesco Ciardelli for his contribution to the discussion of results. This research is co-funded by the Italian National Program MIUR-NANOPACK FIRB 2003 project D.D.2186 Prot. N. RBNE03R78E References

J.W. Cho, D.R. Paul, Polymer, 42, 1083 (2001) H.D. Hunter, D.L.Chang, S. Kim, J.L. White, J.W.

Cho, et al. Polymer, 42, 9513 (2001) W. Lertwimolnun, B. Vergnes, Polym. Eng., 46, 314

(2006)

Sessione 7 Biopolimeri - ictmp.ct.cnr.it · e l’immobilizzazione di cellule ed enzimi. Tali...

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