Dissertations / Theses on the topic 'Poly(glutamate de benzyle)'

Thesis (MSc)--Stellenbosch University, 2012.
ENGLISH ABSTRACT: The combination of natural and synthetic polymers allow for the synthesis of advanced hybrid copolymers. These hybrid copolymers have applications in biomedical areas, one such area being in drug delivery systems (DDS). In this study, a modular approach was used to prepare amphiphilic block copolymers with the ability to self-assemble into three dimensional structures. Reversible addition-fragmentation chain transfer (RAFT) was the synthetic tool used to mediate the polymerization of N-vinylpyrrolidone. RAFT is a versatile method to prepare polymers with control over molecular weight and dispersity. A xanthate chain transfer agent (CTA) was used to obtain the hydrophilic poly(N-vinylpyrrolidone) (PVP) block. An aldehyde functionality could be introduced due to the lability of the xanthate moiety, the procedure of which was effectively optimized to produce quantitative conversion. A dixanthate CTA was synthesized to produce a PVP chain which after the modification reaction, resulted in a α,ω-telechelic polymer. A polypeptide was synthesized via the ring-opening polymerization of Ncarboxyanhydrides (ROP NCA). The living and controllable ROP of NCAs is a method which results in polypeptides, but without a well-defined amino acid order. Poly(γ- benzyl-L-glutamate) (PBLG) was synthesized with a narrow dispersity (Đ = 1.10 – 1.15) using conditions that promote the retention of a terminal primary amine. A protected cysteine functionality was introduced via the terminal amine PBLG chain-end, using peptide synthesis techniques. This resulted in the conjugation of the aldehyde functional PVP and the cysteine terminal PBLG using a covalent, non-reducible thiazolidine linkage. The deprotection of the cysteine, more specifically the deprotection of the thiol was a non-trivial procedure. The thiol protecting acetamidomethyl (Acm) group could not be cleaved using traditional methods, but instead a modified procedure was developed to effectively remove the Acm group while inhibiting hydrolysis of the benzyl esters. It was determined that the conjugation reaction could effectively proceed in N,Ndimethylformamide (DMF) at a slightly elevated temperature and so continued to prepare the amphiphilic hybrid block copolymers, PVP-b-PBLG. A structurally different PBLG chain, namely PBLG-b-Cys was conjugated to the ω-aldehyde PVP and the conjugation efficiency was compared to our PBLG-Cys block. In the case of PBLG-b- Cys the in situ deprotection and conjugation as well as a two-step deprotection and conjugation reaction with PVP resulted in very low conjugation efficiency. The cysteine end-functional PBLG resulted in near quantitative conjugation with PVP. The critical micelle concentration (CMC) for PVP90-b-PBLG54 was determined to be 6 μg/mL, using fluorescence spectroscopy. Particle sizes were determined with TEM and DLS and found to range from 25 nm to 120 nm depending on the polymer block lengths as well as hydrophobic/hydrophilic block length ratios. Furthermore, when the micelles were subjected to an increased acidic environment, the labile benzyl ester bonds were hydrolyzed. This was observed with TEM where the particle sizes increased 10-fold to form vesicular structures. Hydrolysis was further confirmed with ATR-FTIR and 1H-NMR spectroscopy. Cytotoxicity tests confirmed that the copolymer micelles had good cell compatibility at high concentrations such as 0.9 mg/mL. Investigation into drug loading using a pyrene probe confirmed the viability of using PVP-b-PBLG as a responsive DDS.
AFRIKAANSE OPSOMMING: Die kombinasie van natuurlike en sintetiese polimere maak dit moontlik vir die sintese van gevorderde hibried kopolimere. Hierdie kopolimere het aanwending in biomediese gebiede, een so 'n gebied is in medisinale vervoer sisteme (MVS). 'n Modulêre benadering is in hierdie studie gebruik om amfifiliese blok kopolimere te berei. Omkeerbare addisie-fragmentasie kettingoordrag (OAFO) is gebruik as die sintetiese tegniek vir die polimerisasie van N-vinielpirolidoon (NVP). OAFO is 'n veelsydige metode om polimere te berei met beheer oor molekulêre gewig en dispersiteit (Đ). 'n Xantaat kettingoordrag agent (KOA) is gebruik om die hidrofiliese poli(N-vinielpirolidoon) (PVP) blok te sintetiseer. ‘n Aldehied endgroep was deur die terminale xantaat funksionaliteit berei, ‘n proses wat geoptimiseer is tot kwantitatiewe omsetting. 'n Di-xantaat KOA is gesintetiseer om, na modifikasie, 'n α, ω-telecheliese polimeer te produseer. Die polipeptied was gesintetiseer deur middel van ’n ringopening polimerisasie van Nkarboksianhidriede (ROP NKA). Die lewende en beheerbare ROP van NKAe is 'n metode wat lei tot polipeptiede sonder ’n gedefinieerde aminosuur volgorde. Poli(γ- benzyl-L-glutamaat) met 'n lae dispersiteit (Đ = 1.10 – 1.15), is gesintetiseer deur gebruik te maak van kondisies wat die behoud van 'n terminale primêre amien bevorder. 'n Beskermde sistien-funksionaliteit is ingebou via die terminale amien met behulp van peptiedsintese tegnieke. Die tiol beskerming van die asetamidometiel (Asm) groep kon nie gekleef word deur gebruik te maak van tradisionele metodes nie, maar ‘n nuwe proses is ontwikkel om die Asm groep te kleef sowel as om die hidrolise van die bensiel esters te inhibeer. Die koppelings reaksie het effektief verloop in DMF by 'n effens verhoogde temperatuur en sodoende is die amfifiliese hibried blok-kopolimere, PVP-b-PBLG berei. Twee verskillende PBLG kettings is gekoppel aan die ω-aldehied PVP en die koppeling doeltreffendheid is vergelyk. Daar is bevind dat net die sistien end-funksionele PBLG tot kwantitatiewe konjugasie kon lei. Die kritiese misel konsentrasie is bepaal vir PVP90-b-PBLG54 as 6 μg/mL met behulp van fluoressensie spektroskopie. Die deeltjie-groottes is bepaal met TEM en DLS en wissel van 25 nm tot 120 nm, afhangende van die polimeer bloklengtes sowel as hidrofobiese / hidrofiliese blok lengte verhoudings. Die miselle is blootgestel aan 'n verhoogde suur omgewing, wat tot die hidrolise van die bensiel ester groepe gelei het. TEM het getoon dat die deeltjie-groottes met 10-voud vergroot het tot vesikulêre strukture. Hidrolise is verder bevestig met ATR-FTIR en 1H-KMR spektroskopie. Sitotoksiese toetse het bevestig dat die miselle geen of min toksisiteit toon teenoor eukariotiese selle nie, selfs teen 'n hoë konsentrasies soos 0.9 mg/ml. Die medisinale behoud vermoë is met behulp van pireen bevestig en dus ook die potensiaal van PVP-b-PBLG as ‘n moontlike MVS.

Conférer simultanément de multiples fonctionnalités à des vecteurs nanoparticulaires polymères représente un objectif pharmacotechnique majeur dans le domaine de la vectorisation. Dans ce travail de recherche, quatre dérivés du poly (L-glutamate de γ-benzyle) possédant chacun une fonctionnalité spécifique ont été synthétisés par polymérisation anionique d'ouverture du cycle du γ-BLG-NCA: le poly(L-glutamate de γ-benzyle)-cyclodextrine, le poly(L-glutamate de γ--benzyle)-benzylamine, le poly (L-glutamate de γ-benzyle)-poly(éthylène glycol) et le poly (L-glutamate de γ-benzyle)-biotine. Les propriétés d'auto-assemblage de ces polymères ont permis d'obtenir, à partir de mélanges de ces polymères, des nanoparticules composites dégradables dont les caractéristiques ont été déterminées par DLS, MET, ITC, potentiel et activation du complément. De manière très prometteuse, cette stratégie a permis de développer des nanoparticules associant plusieurs fonctionnalités aisément modulables
To impart mutliple functionalities together into the same polymeric particulate carrier represents a real challenge for many applications in drug delivery. In the present work, four poly(γ-benzyl L-glutamate)-derivatives having a specific functionality: poly (γ-benzyl L-glutamate)-cyclodextrin, poly(γ-benzyl L-glutamte)-benzylamine, poly(γ-benzyl L-glutamate)-poly(ethylene glycol) and poly(γ-benzyl L-glutamate)-biotin, were synthesized by anionic ring opening polymerisation of γ-BLF-NCA. The self-assembling capacities of these polymers were used to prepare novel composite degradable nanoparticles based on mixtures of these derivatives were successfully prepared by the nanoprecipitation method. These nanoparticles were characterized by DLS, TEM, ITC, potential and complement activation. This strategy was successful in preparing nanoparticles combining simultaneously different functionalities, which moreover, can be easily modulated

En dépit de progrès considérables, le cancer reste l'une des principales causes de morbidité et de mortalité dans le monde. Actuellement, 90% des décès liés au cancer sont causés par la propagation de cellules cancéreuses vers des organes distants. Une fois implantées et disséminées, les métastases sont beaucoup plus difficiles à détruire par les moyens de la chimiothérapie.A la suite d’un processus d’intravasation, certaines cellules tumorales s’échappent de la tumeur primaire et empruntent les systèmes circulatoires avant d’être ensuite extravasées, puis distribuées et finalement disséminées dans divers organes. Ainsi, dans l’environnement circulatoire, ces cellules tumorales circulantes (CTCs) se trouvent particulièrement accessibles aux agents thérapeutiques. Dans ce cadre, nous avons imaginé d’utiliser des nanoparticules à architecture contrôlée, afin d’intercepter de manière sélective ces cellules dans l’environnement sanguin.Dans cet objectif, nous avons synthétisé par ouverture de cycle de la lactone correspondante des copolymères amphiphiles di- et tri-blocs du poly(glutamate de benzyle). Leur auto-assemblage a permis d'obtenir des nanoparticules amphiphiles de taille inférieure à 100 nm et de potentiel ζ négatif, dont la géométrie contrôlable va de la forme sphérique (rapport d'aspect 1.3) à la forme ellipsoïdale (oblats) (rapport d'aspect 2,6) et qui présentant en surface des chaînes de PEG sous des conformations et des densités de surface contrôlées.En raison de leur capacité de circuler dans le compartiment sanguin, ces nanoparticules ont une probabilité d’interaction optimale avec les CTCs.L’impact de la modification de leur architecture a été établi en étudiant les capacités d’interactions des différentes nanoparticules préparées, d’une part avec les protéines plasmatiques et d’autre part, avec les différents types cellulaires rencontrés dans le compartiment sanguin.Les résultats les plus marquants montrent que l’élongation des nanoparticules (oblats) et l’anisotropie de leur surface, caractérisée par leur balance hydrophile/lipophile, gouvernent profondément leurs interactions. De manière fort intéressante, il apparaît que l’élongation des particules dont la surface est uniformément hydrophile diminue l’intensité de leur capture par les différents types cellulaires modèles étudiés (HUVECs modèle de cellules endothéliales), cellules RAW 276.7 (modèle de macrophages) et cellules PC3 (cancer de la prostate) et B16 (mélanome). En revanche, lorsque ces nanoparticules présentent une anisotropie de surface, leur capture par ces différents types cellulaires est augmentée avec l’élongation des particules (facteur d’élongation de 2,1).Dans un dernier volet expérimental, ces nanoparticules ont été modifiées par greffage de la protéine MART1 à leur surface. Ces immuno-nanoparticules ont montré une certaine capacité de reconnaissance des cellules B16 (modèle du mélanome). Leur efficacité après injection intraveineuse devra toutefois être précisée in vivo
Despite the considerable progress, cancer remains one of the leading causes of morbidity and mortality worldwide. Currently, 90% of cancer deaths are caused by the spread of cancer cells to distant organs. Once implanted and disseminated, metastases are much more difficult to destroy by means of chemotherapy.Following a process of intravasation, some tumor cells escape from the primary tumor and migrate through the circulatory systems before being extravasated, then distributed and finally disseminated in various organs. Thus, in the circulatory environment, these circulating tumor cells (CTCs) are particularly accessible to therapeutic agents. In this context, we have imagined the use of nanoparticles with controlled architecture, in order to selectively intercept these cells in the blood environment.For this purpose, we have synthesized by ring opening of the corresponding lactone, amphiphilic di- and tri-block copolymers of poly (benzyl glutamate). Their self-assembly made it possible to obtain amphiphilic nanoparticles smaller than 100 nm in size and with a negative ζ potential, whose controllable geometry ranges from spherical (aspect ratio 1.3) to ellipsoidal (oblates) (aspect ratio 2, 6) and having PEG chains on the surface under controlled surface conformations and densities.Due to their ability to circulate in the blood compartment, these nanoparticles have an optimal probability of interaction with CTCs.The modification impact of their architecture has been established by studying the interaction capacities of the different nanoparticles prepared. On the one hand with the plasma proteins and on the other hand, with the different cell types encountered in the blood compartment.The most striking results show that the elongation of the nanoparticles (oblates) and the anisotropy of their surface, characterized by their hydrophilic / lipophilic balance, strongly govern their interactions. Interestingly, it appears that the elongation of particles whose surface is uniformly hydrophilic decreases the intensity of their capture by the different types of cell models studied (HUVEC model endothelial cells), RAW 276.7 cells (macrophage model) and cells PC3 (prostate cancer) and B16 (melanoma). Although, when these nanoparticles exhibit surface anisotropy, their capture by these different cell types is increased with the elongation of the particles (elongation factor of 2.1).In a final experimental part, these nanoparticles were modified by grafting the MART1 protein on their surface. These immuno-nanoparticles showed a certain recognition capacity of B16 cells (melanoma model). However, their efficacy after intravenous injection should be specified in vivo

Une mini famille constituée de copolymères possédant en commun un bloc poly (L-glutamate de γ-benzyle) hydrophobe mais portant chacun diverses fonctionnalités utiles à la vectorisation a été synthétisée. La capacité de ces copolymères à s'auto assembler et former des nanoparticules et à s'auto associer et conférer simultanément différentes fonctionnalités aux nanoparticules a été établie par les techniques de la physico-chimie. De plus, afin de pouvoir introduire des ligands de reconnaissance à la surface des particules, il a été fait appel au système biotine-avidine et l'efficacité de la bionylation a été démontrée. De manière fort intéressante, il est apparu possible de contrôler très finement la quantité de biotine présente à la surface des particules, et donc le nombre de molécules de ligands introduites, puisqu'il a été montré qu'un nombre discret (voire une unique molécule de biotine) de ces entités suffisait pour que la particule puisse être reconnue de manière spécifique
A mini family of copolymers with a common poly (γ-benzyl L-glutamate) hydrophobic block but each one carrying several useful functionalities to the vectorization has been synthesized. The ability of these copolymers to self-assemble and form nanoparticles and to self-associate and confer together different features to nanoparticles has been established by physico-chemical techniques. Moreover, in order to introduce target ligands on the surface of particles, it has been used the biotin-avidin system and the efficiency of biotinylation was demonstrated. Very interestingly, it became possible to finely control the amount of biotin present on the surface of particles, and therefore the number of molecules of ligand introduced ; because has been shown that a discrete number (even a single biotin molecule) of these entities is sufficient for the particle can be recognized specifically

Des nanoparticules multifonctionnelles polymères, préparées par auto-assemblage de plusieurs dérivés du poly (L-glutamate de gamma-benzyle) (PBLG), ont été conçues afin d’assurer le ciblage des tissus osseux et la libération contrôlée de molécules actives. Des propriétés d'attachement aux tissus osseux leur ont été conférées par la présentation en surface de différents ligands ostéotropes, l'alendronate et l' acide poly(glutamique), seuls ou en combinaison. Leur affinité pour les tissus osseux a été évaluée in vivo ainsi que leur distribution fine dans ces tissus. Par ailleurs, des propriétés anticancéreux ont été conférées aux nanoparticules grâce à un mécanisme originale d’association du cisplatin par complexation. Le procédé mis en œuvre permet d’obtenir des cinétiques de libération très progressives de dérivés actifs du platine et déclenchée par la présence des ions chlorure. Enfin, leur cytotoxicité a été mesurée. Cette stratégie constitue donc une approche prometteuse en vue d’améliorer le traitement des métastases osseuses
Multifunctional bone targeted polymeric nanoparticles prepared by self-assembly of several poly(gamma-benzyl-L-glutamate) (PBLG) derivates have been developed. Their bone binding properties were provided by two different osteotropic moieties, alendronate or/and poly(glutamic acid) exposed on the nanoparticle surface. Their affinity for bone tissues has been evaluated in vitro, ex vivo and in vivo, including their detailed distribution in bone tissues structures. Further, in view of bone cancer therapeutics, nanoparticles were provided with anticancer properties thanks to the complexation of cisplatin, which leaded to very well controlled release properties. Finally, cytotoxicity were studied. Therefore, this strategy constitute a promising approach for the improvement of bone cancer therapeutics

Blends of sodium poly(a,L-glutamate) (MW 49K), poly(pyrrole) and poly(ethylene oxide) (MW lOOK) were prepared by a solution-blending process. A series of blend compositions were studied and characterized to determine optimal parameters and conditions necessary for the encapsulation and stability of the peptide portion of this ternary system. The retaining of the conformational structure of the peptide is important for its activity in a delivery system. Results derived from Infrared Spectroscopy (lR) indicated that the helical conformation of the peptide was maintained. The solid-state morphological properties of the ternary blends were studied by Differential Scanning Calorimetry (DSC) and Scanning Electron Microscopy (SEM). Results suggest the formation of a solid-state system with miscible properties that are due to noncovalent interactions, such as, ion-dipole and hydrophobic interactions. Preliminary cell culture studies utilizing ovarian cancer cells indicate that these cells will proliferate and adhere to the surface of these polymer blends.

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Físicas e Matemáticas. Programa de Pós-Graduação em Quimica.
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Neste trabalho, foi realizada a síntese e a caracterização de copolímeros dibloco do tipo flexível-rígido formados pelo poli(estireno)-b-poli(g-benzil-L-glutamato) (PS-b-PBLG). A parte flexível foi constituída pelo PS, e a parte rígida pelo peptídio PBLG. Esse peptídeo apresenta uma estrutura secundária de a-hélice, que garante a rigidez desse bloco. O bloco de PS foi sintetizado via polimerização aniônica, enquanto que o bloco de PBLG foi sintetizado via polimerização por abertura de anel. Os homopolímeros e copolímeros foram caracterizados através das técnicas clássicas de ressonância magnética nuclear de próton e carbono (RMN 1H e 13C), cromatografia de permeação em gel (GPC) e análise térmica (calorimetria de varredura diferencial (DSC) e termogravimetria (TG)). A caracterização em solução foi realizada utilizando-se as técnicas de espalhamento de luz e de nêutrons. Nas análises de espalhamento de luz estático (SLS), foram determinados parâmetros como a massa molar ponderal média, o segundo coeficiente virial e a concentração crítica para os homopolímeros puros e para o copolímero PS100-b-PBLG330. Para esse copolímero as análises de espalhamento de luz dinâmico (DLS), indicaram a presença de dois coeficientes de difusão: um modo rápido atribuído à cadeia flexível (PS) e um modo lento devido à cadeia rígida do bloco de PBLG. O estudo da transição rígido ® flexível para o bloco de PBLG desse copolímero foi realizada por DLS. Essa transição foi confirmada através de cálculos utilizando-se a relação de Broersma. As análises de SANS foram realizadas para os copolímeros PS44d8-b-PBLG23, PS44d8-b-PBLG54 e PS44d8-b-PBLG72. As curvas de SANS apresentaram o pico de difusão característico para copolímeros dibloco (assinatura do copolímero). O copolímero dibloco PS44d8-b-PBLG72 apresentou uma transição do estado desordenado para o ordenado com o aumento da concentração e/ou diminuição da temperatura do sistema. Os outros copolímeros analisados não apresentaram diferenças nas curvas com a alteração da temperatura ou concentração do sistema. A transição rígido ® flexível para o bloco de PBLG foi estudada por SANS utilizando-se o copolímero PS44d8-b-PBLG54. Essa transição foi acompanhada por um deslocamento da posição do pico de difusão. Esse resultado foi concordante com o modelo teórico proposto. Essa transição foi confirmada através de análises de dicroísmo circular.

Les cnidaires sont un groupe-clé pour étudier l’origine et l’évolution des cellules nerveuses. Des marqueurs neuronaux de Bilateria sont exprimés dans le bulbe tentaculaire de la méduse de Clytia hemisphaerica, spécialisé dans la production massive de cellules neurosensorielles, les nématocytes. La dynamique de la nématogenèse du bulbe a été étudiée ; les stades de différenciation y sont répartis selon un gradient proximo-distal, corrélé à un étagement de profils d’expression de gènes. La capacité d’autonomie du bulbe in vitro en fera un modèle expérimental performant pour réaliser des études fonctionnelles. La pression de décharge de son organite, le nématocyste, est générée par du poly--glutamate. Différentes données suggèrent que la synthèse de ce polymère rare, intimement associé à l'histoire évolutive des nématocytes, est liée à la co-option d'un gène bactérien acquis par transfert horizontal, et au recrutement secondaire de gènes initialement utilisés pour une fonction nerveuse.

Les polyesters aliphatiques, comme le poly(acide malique) et ses dérivés, sont une famille de polymères aux propriétés de bio(comptabilité) et de bio(dégradabilité) remarquables, qui en font des candidats de choix pour l'élaboration de systèmes de vectorisation de principes actifs. Généralement, ces polymères sont synthétisés via des réactions de polymérisation utilisant des amorceurs, voir des catalyseurs, organiques, organométalliques ou métalliques. La présence de ces molécules, même à l'état de traces, peut être à l'origine d'une toxicité non souhaitée. Par conséquent, l'utilisation de biocatalyseurs, comme les lipases, se développe pour apporter une solution à cet inconvénient. Cependant, cette voie de synthèse enzymatique fait face à d'autres problèmes, tels qu'une polymérisation moins bien maîtrisée et des polymères de masses molaires faibles. Cette thèse a donc pour objectif de mettre au point une voie de polymérisation du malolactonate de benzyle utilisant la lipase de pancréas de porc (PPL) comme amorceur. Dans un premier temps, nous avons optimisé certains paramètres réactionnels permettant d'obtenir des poly(malate de benzyle) , PMLABe, de masses molaires suffisamment élevées pour que ces polymères puissent être utilisés dans la formulation de vecteurs de principes actifs, grâce à l'utilisation et l'extrapolation d'un plan d'expérience. Nous nous sommes ensuite intéressés à la compréhension du mécanisme réactionnel de la polymérisation enzymatique du malolactonate de benzyle, une β-lactone β-substituée. Les différentes études menées ont permis d'approfondir notre connaissance dans ce domaine. Deux mécanismes ont été proposés et des expériences sont en cours pour confirmer l'un d'entre eux. Finalement, comme l'objectif initial est de proposer une méthode de synthèse de dérivés du PMLA plus biocompatibles conduisant à des polymères sans résidus d'amorceurs chimiques toxiques, nous avons comparé les activités biologiques de nanoparticules préparées à partir de PMLABe synthétisés par voie chimique et par voie enzymatique. Pour cela, nous avons mesuré la captation de ces nanoparticules, encapsulant une sonde de fluorescence, par des cellules hépatiques HepaRG. Puis, nous avons évalué la toxicité aiguë et la toxicité chronique de ces nanoparticules vis-à-vis des cellules HepaRG. Ces études ont permis de mettre en évidence certaines propriétés des nanoparticules ayant une influence sur la survie cellulaire et le métabolisme des cellules HepaRG. De la compréhension théorique aux applications potentielles, cette thèse apporte des connaissances sur la polymérisation enzymatique des lactones substituées, un domaine peu décrit dans la littérature
Aliphatic polyesters, like poly(malic acid)and its derivatives, are a family of polymers with outstanding properties, such as bio(degradability) and bio(compatibility). Therefore, these polyesters can be considered as excellent candidates for the design of drug carriers. These kinds of polymers are usually synthesized thanks to polymerization reactions using organic, organometallic or metallic initiators or catalysts. The presence of such molecules, even in trace amounts, can cause undesired toxicities. Therefore, the use of biocatalysts, like lipases, is attracting more and more interest and research work to circumvent this problem. However, this enzymatic polymerization method has to face to other issues, such as a lower controlled of the polymerization process and polymers with lower molar masses. Therefore, this PhD research work aimed at setting up the enzymatic polymerization of benzyl malolactonate, using porcine pancreatic lipase (PPL). Firstly, we have optimized some reactional parameters allowing to obtain poly(benzyl malate), PMLABe, with molar masses adapted to their uses for the design of drug carriers, thanks to a Design of Experiments (DoE) and its extrapolation. We were then interested by the comprehension of the enzymatic polymerization mechanism of the benzyl malolactonate. The different studies we carried out allowed us to deepen our knowledges of such enzymatic polymerization. Two non-canonical mechanisms were proposed and further experiments are in progress to confirm the one which is the more probable. Finally, because our initial goal was to propose a more biocompatible polymerization method to obtain PMLABe free of traces of chemical initiator, we compared biologic activities of different nanoparticles prepared from PMLABe synthesized using chemical or enzymatic pathway. For that, we have first measured the uptake of these nanoparticles encapsulating a fluorescent dye, by the hepatic cells HepaRG. Then, we have studied the acute and chronic toxicity of the nanoparticles on the HepaRG cells. Results of these studies have highlighted that certain properties of the nanoparticles and/or of the polymers which constituted them have an influence on the cells viability and on the cells metabolism. From the theoretical mechanism to the probable applications, this thesis brings knowledge about the enzymatic polymerization of substituted lactone, a field poorly described in the literature

The synthesis of arborescent poly(gamma-benzyl L-glutamate) (PBG) molecules was achieved through successive grafting reactions of linear PBG chains. These linear PBG building blocks were obtained by the ring-opening polymerization of gamma-benzyl L-glutamic acid N-carboxyanhydride initiated with n-hexylamine. Cleavage of a fraction of the benzyl ester groups on a linear PBG substrate, followed by coupling with linear PBG side chains via standard peptide coupling techniques, yielded a comb-branched or generation zero (G0) arborescent PBG. Further cycles of partial deprotection and grafting reactions led to arborescent PBG molecules of the subsequent generations (G1-G3). Molecular weights reaching over 106 were obtained for G3 arborescent PBG, while maintaining narrow molecular weight distributions (Mw/Mn ≤ 1.06) for each generation. The arborescent PBG molecules displayed α-helix to randomly coiled chain conformation changes from N,N-dimethylformamide to dimethylsulfoxide. Amphiphilic copolymers were obtained by grafting the arborescent PBG substrates randomly with side chains of either poly(glycidol acetal), poly(ethylene oxide), or poly(γ-tert-butyl L-glutamate) via the same peptide coupling techniques used to generate arborescent PBG. Copolymers were also synthesized by a chain end grafting method, whereby the linear chain segments were coupled exclusively with the chain termini of the arborescent PBG substrates. Water-soluble species were obtained by removal of the acetal and tert-butyl protecting groups from the poly(glycidol acetal) and poly(γ-tert-butyl L-glutamate) side chains, respectively, while the copolymers with poly(ethylene oxide) side chains did not require further modifications. Dynamic light scattering (DLS) measurements on the arborescent copolymers in aqueous solutions revealed that unimolecular micelles were the dominant species for the chain end grafted arborescent copolymers, whereas the randomly grafted arborescent copolymers were either insoluble or displayed significant aggregation. The synthesis of arborescent copolymers with PBG cores was also achieved through “click” chemistry, using the copper-catalyzed azide-alkyne Huisgen cycloaddition (CuAAC) reaction. To that end, polyglycidol, poly(ethylene oxide), and poly(2-trimethylsilylethyl acrylate) chains terminally functionalized with azide groups were grafted onto either randomly or chain end alkyne-functionalized arborescent PBG substrates. DLS analysis revealed solubility trends similar to the arborescent copolymers obtained by the peptide coupling method. The CuAAC reaction enables the incorporation of a broader range of polymers into arborescent copolymer structures derived from PBG substrates.

Biosynthesis of monodisperse poly(α,L-aspartic acid) and optimization of poly(α,L-glutamic acid) production have been accomplished. Protein design having as ultimate goal the synthesis of monodisperse benzyl-ester derivatives of α-helical polypeptides by genetic engineering has been performed. This comprises design of a monomeric DNA cassette, cloning, multimerization, ligation into an expression vector, and expression of the target gene followed by purification of the resultant protein. Advantages of such techniques include control of molecular size, composition, sequence and stereochemistry of the synthesized polymers. 1H NMR (nuclear magnetic resonance), amino acid analysis, and MALDI-MS (matrix-assisted laser desorption mass spectrometry) analytical techniques were employed for characterization of monodisperse poly(α, L-amino acid)s. Modification of conventional purification and cyanogen bromide cleavage reactions are included as part of the strategy to obtain monodisperse poly(α, L-aspartic acid). Optimization of polymer production was achieved by means of utilization of a variety of Escherichia coli strains and plasmids, modification of large scale fermentation conditions, and alterations of purification procedures. Monodisperse poly(α,L-amino acid)s were benzylated by treating the acid forms of the polymers with phenyldiazomethane. Polarized light optical microscopy was used to observe textures from birefringent samples of poly(β-benzyl α,L-glutamate) and poly(β-benzyl α,L-aspartate). Small-angle X-ray diffraction data shows evidence of smectic ordering of monodisperse poly(β-benzyl α,L-glutamate) (DP = 76). poly(γ-4-(hexadecyloxy)benzyl α,L-glutamate) DP = 300, was synthesized by methods developed in our laboratories. Differential scanning calorimetry (DSC) and polarized light optical microscopy (POM) data is presented as evidence for thermotropic liquid crystal behavior.

碩士
國立中山大學
材料與光電科學學系研究所
100
In this study, tetraphenylthiophenen (TP) with aggregation-induced emission enhancement (AIEE) property is served as terminal and central fluorophores of poly(γ-benzyl-L-glutamate) (PBLG)-based polymers of TP1PBLG and TP2PBLG, respectively, to probe for the relationship between the secondary structure (α-helix) of polypeptides and the ALEE-operative fluorescence (FL). Intermolecular aggregation of the central TP unit in the di-substituted TP2PBLG is sterically blocked by the large α-helical PBLG chains, which lead to the reduced AIEE-oriented FL. On the contrast, the terminal TP units in TP1PBLG can easily approach each other to form aggregates with strong FL. Factor (e.g. solvent annealing) controlling the fraction of α-helix chain also varies the corresponding emission intensity. Conformational difference between TP1PBLG and TP2PBLG evaluated from the infrared and the X-ray (wide- and small-angle) diffraction spectra is also used to verify its influence on the AIEE-operative FL behavior.

Two types of monodisperse rod-like polymers, poly($\gamma$-benzyl $\alpha$,L-glutamate) (PBLG) and poly($\gamma$-4-(hexadecyloxy)benzyl $\alpha$,L-glutamate) with backbone sequences of have been prepared by chemical modification of monodisperse poly($\alpha$,L-glutamic acid) (PLGA) that was synthesized by recombinant DNA biosynthesis techniques. The monodisperse PLGA () was produced in Escherichia coli as a fusion to mouse dihydrofolate reductase and purified to homogeneity by metal affinity chromatography, CNBr digestion and ion-exchange chromatography.$$\rm GluAsp(Glu\sb{17}Asp)\sb{x}GluGlu\quad x = 3, 4, 5, 6\eqno {\bf1}$$ The monodisperse PBLG derived from showed smectic ordering both in solution and in films as characterized by polarized light optical microscopy and X-ray diffraction. The layer spacings of smectic order were nearly identical to the expected length of the rods, given the axial rise per residue of 1.5 A for the $\alpha$-helix. X-ray diffraction patterns of magnetically oriented films were consistent with the supramolecular structure in which helical rods are arranged in layers with their helical axes approximately perpendicular to the smectic layers. Transmission electron microscopy and electron diffraction on the PBLG films revealed a banded morphology with an approximately 120 nm period which provides strong evidence for helical rotation of the director field as in cholesteric order. Detailed examination of the relative orientation of the banding in the morphology image and the reflections in the electron diffraction pattern leads to the conclusion that the structure of the unoriented smectic PBLG is that of a smectic A*. The monodisperse poly($\gamma$-4-(hexadecyloxy)benzyl $\alpha$,L-glutamate)s exhibit strong melting transitions at around $40\sp\circ$C; however no ordered melt was observed due in part to the reduced aspect ratio brought about by the long alkyl side groups.

碩士
國立中山大學
材料科學研究所
90
Thermal behavior and molecular packing of a series of α-helical poly(L-glutamates), with n-alkyl side chain of various lengths (m(number of carbons in the alkyl group) = 1, 2, 6, 12,18), were studied by means of differential scanning calorimetry, polarizing light microscopy and X-ray diffraction. For polymers of m = 1 and 2, There is a pseudohexagonal structure below ca. 130 oC and above this temperature the stable phase is the hexagonal columnar phase. There exists a layered structure in the polymer of m = 6, as well as a solvent induced hexagonal columnar structure which formed during solution casting process. In the polymer of m = 12, a layered structure was formed in the temperature range between 20 to 255 oC. However, for longer side chain, m = 18, tendency of crystallization of alkyl long side chain forced the backbone to pack into layer structure. There are two distinct melting temperature at ca. 60 oC, and the enthalpy are ca. 53 and 19 J/g, which corresponding to the melting of hexagonal and monoclinic side chain crystallines. The polymers with longer side chain (m = 6, 12 and 18) tend to be lyotropic liquid crystalline phase within lamellar inter-rod distance of 1.25 nm in solution state, and the structure will remain after drying. However, the inter-rod distance will collapse at the temperature above ca. 200 ℃ and will not recover after cooling.

碩士
國立成功大學
化學工程學系碩博士班
98
Novel porous composite scaffolds for bone tissue engineering were prepared from water-based polyurethane (WPU), tricalcium phosphate (TCP) and poly(γ-glutamate) (γ-PGA). γ-PGA modified commercially available pellethane exhibit a smaller contact angle, and higher water absorption value than the pellethane without γ-PGA. In vitro platelet adhesion studies indicated that the addition of γ-PGA into pellethane leads to a reduction in platelet adhesion and activation. Results indicated that NCO/OH=1.8 and DMPA=6wt% of water-based polyurethane(WPU) had superior tensile strength, adequate particle size, swelling ratio, degradation ratio. The morphology and properties of the scaffolds were characterized by scanning electron microscope, X-ray diffraction, infrared absorption spectra, static contact angle analysis, porosity analysis, swelling ratio, compressive strength testing, and in vitro degradation measurement. The results indicated that the porous composite scaffolds had an interconnected porous structure. The compressive strength of the porous composite scaffolds showed lower enhancement with increasing γ-PGA content. The hydrophilic, swelling ratio, degradation raio, porosity and pore size of the porous composite scaffolds showed higher value with increasing γ-PGA content. X-ray diffraction and Fourier transform infrared spectroscopy were used to determine the crystal structure and chemical composition of scaffolds.

碩士
國立雲林科技大學
化學工程與材料工程系碩士班
100
In this study, poly-γ-glutamate-apatite (PGA-AP) particles were prepared for the adsorption of copper ions. They were synthesized by coprecipitation between calcium nitrate and diammonium phosphate in poly-γ-glutamate (PGA) solution with different feed ratios of PGA to calcium nitrate and diammonium phosphate. The theoretical weight ratios of poly γ-glutamate to apatite (PGA/AP) for PGA-AP particles are 1:20 (G1H20), 1:40 (G1H40) and 1:60 (G1H60). The PGA-AP particles were characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffractometer (XRD), energy dispersive spectrometer (EDS), thermo gravimetric analyzer (TGA) and particle analyzer. Adsorption experiments were carried out to investigate the effects of different adsorption parameters, such as different PGA/AP weight ratios, temperature and pH, on the adsorption capabilities of PGA-AP particles. The formation of PGA-AP particles were confirmed by the phosphate bands and amide bands at FTIR spectra. The XRD spectra of PGA-AP particles are similar to that of hydroxylapatite. The EDS shows that the Ca/P mole ratios of G1H20, G1H40 and G1H60 are 1.62, 1.54 and 1.72, respectively. The actual PGA/AP weight ratios of G1H20, G1H40 and G1H60 are 1:15, 1:24 and 1:44, respectively. The particle sizes of G1H20, G1H40 and G1H60 are 79.7±1.6、88.5±0.8 and 92.8±2.3 nm, respectively. The results of adsorption experiments show that initial adsorption rate is rapid and equilibrium is attained at 60 minutes. The adsorption capabilities increase with the increases of the PGA/AP weight ratios, temperature and pH. Kinetic data are well fitted by a pseudo second-order model and the equilibrium data are analyzed by Langmuir model very well with high correlation coefficient. From the kinetic data, G1H20 has the largest adsorption capability than those of G1H40 and G1H60. From the Langmuir isotherms, the maximum adsorption capacity of G1H20 towards copper ions is 85.47 mg/g when the experiments were carried out at 60℃ and pH 6. Thermodynamic parameters ΔH, ΔS and ΔG have been calculated. Positive value of ΔH and negative value of ΔG show endothermic and spontaneous nature of sorption, respectively.

碩士
國立臺北科技大學
化學工程研究所
101
The healing of wounds on skin is accelerated under a proper humid environment than a dry one. Nevertheless, immoderate free radicals will lead to excessive inflammation reactions and retard the healing processes. The motivation of this study is to prepare a cross-linked composite hydrogel with combining the advantages of gelation gelatin, humidifying γ-poly glutamate (γ-PGA) and antioxidative proanthocyanidins (OPCs). The natural polymer made hydrogel is proposed to be applied as a wound dressing. In this research, the hydrogel wound dressing was prepared by using sodium/calcium γ-PGA and gelatin cross-linked with OPCs. The optimized ratio of γ-PGA salt to gelatin was evaluated through swelling and degradation tests. Furthermore, the hydrogel was cross-linked with OPCs and then tested in vitro and in vivo. Results indicated that the cross-linked hydrogel using OPCs solution had stronger machine structure and improved antioxidant capacity significantly. The results of in vitro tests showed that the cross-linked hydrogel using 10% OPCs solution would be no bio-toxic for L929 cell. The results of in vivo tests showed that the cross-linked hydrogel using 10% OPCs solution would be of great potential to serve as a useful wound healing dressings.

碩士
國立嘉義大學
動物科學系研究所
99
The purpose of this study is to develop the organic γ-poly-glutamate -copper compound (γ-PGA-Cu) by chelating γ-poly-glutamic acid (γ-PGA) with Cu(II) ion and compare the relative bioavailability with copper sulfate (CuSO4‧5H2O), commercial organic copper (Bioplex® Cu) and microbial copper (Micro-Cu) in chickens. Different concentration of Cu(II) ion (10 mM, 20 mM, 50mM, 100mM and 200mM) was added into a γ-PGA solution (12.5 μM) to form different ratio of γ-PGA-Cu compounds to estimate the binding capability of γ-PGA with Cu2+. The ratio of binding Cu2+ in γ-PGA-Cu was raised with reaction Cu(II) ion and generate different γ-PGA-Cu precipate. Adding 20 mM Cu2+ showed a best binding rate. However, the lower binding rate was found when the reacting pH was decreaseing from adding higher Cu(II) ion. The resulted γ-PGA-Cu compounds was further analyzed by fourier transform infrared spectroscopy. The vibration frequency of carboxyl group of free γ-PGA at 1650 cm-1 was decreased since the ratio of chelating copper of γ-PGA-Cu was increased. A pH profile (adjusted to 5.0, 5.8, 6.0 and 6.6) showed the γ-PGA-Cu precipates was increased and the best binding affinity and precipitation of γ-PGA and Cu(II) ion at pH 6.0. The reaction of different concentration of γ-PGA (0.5、2.5、5、7.5、10%) and Cu(II) ion (0.12、0.62、1.24、1.78、2.50%) showed γ-PGA-Cu precipates was increased when higher reacting Cu(II) ion was used. Hower, the γ-PGA-Cu precipates found to be oxidative while the concentration of Cu(II) ion was more than 5% in the reaction. The best reacting concentration of γ-PGA was 2.5% with 0.62% Cu(II) ion. γ-PGA-Cu was manufactured following the condition: pH 6.0 and 2.5% γ-PGA. The animal test was examined using one-day old male chicks (Hendrix SV) to feed with Cu-deficient soy isolate-dextrose diet for seven days. On the age of 8 days, 108 chicks with similar body weight were chose and randomly assigned into control group in the absence of Cu(II) ion, and diets supplemented with 0.5 and 1 mg Cu/kg (in different source as CuSO4‧5H2O, Bioplex® Cu, γ-PGA-Cu and Micro-Cu) for a 15 days experiment. There were three replicates in each group and each replicate contained four chicks. Feed intake, weight gain, and copper intake were significantly increased (P<0.01) and the feed convertion ratio was improved (P<0.01) with Cu level. The interaction of Cu source and Cu level was existed in feed convertion ratio and weight gain (P<0.01). On carcass performance, relative liver weight, liver and bile copper content was increased with Cu level(P<0.05). The interaction of Cu source and Cu level was found in the liver copper content (P<0.05). When using the Cu intake as independent value, the coefficient of the multiple linear regression model was showed to be 0.53, 0.24 and 0.67 in weight gain, liver and bile copper content as dependent value. γ-PGA-Cu bioavailability was determined by slope-ratio methodology using CuSO4‧5H2O as a standard (100%) following the means of multiple linear regression model of copper intake and bile copper content and showed to be 139%. In conclusion, the optimal condition of γ-PGA-Cu production was pH 6.0 and 2.5% γ-PGA. The relative bioavailability of γ-PGA-Cu compound reached 139% in chicks.

碩士
國立中山大學
機械與機電工程學系研究所
102
In this study, the technology of near-field electrospinning (NFES) is developed to collect orderly poly (γ-methyl L –glutamate) (PMLG)/poly (vinylidene fluoride) (PVDF) fiber composites with enhanced piezoelectricity. PMLG solution was blended with PVDF solution uniformly to prepare PMLG/PVDF solution (30.68 wt%). When the droplet of polymer blend overcame the surface tension of PMLG/PVDF solution to form Taylor cone at high electric field of 1×107-1.6×107 V/m, a PMLG/PVDF piezoelectric fiber (diameter=17.25-7.62 µm) was spun from the tip of Taylor cone and collected on a rotating glass collector orderly at the tangential velocity of 1256.64 mm/s. Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC) were used to analyze structure interaction and secondary structure within the blends of PMLG/PVDF polymers. NFES process has a positive impact on the PMLG/PVDF piezoelectric properties, which can make dipole a better orientation. The ultimate stress (27.47 MPa) and Young’s modulus (2.77 GPa) of the optimum PMLG/PVDF fiber composites were measured by micro-tensile testing. Finally, PMLG/PVDF piezoelectric fiber composites were patterned on a PET-based structure with parallel electrodes as a flexible PMLG/PVDF energy harvester to capture ambient energy. By vibrating test, the maximum peak voltage (0.08 V) and power (637.81 pW) can be obtained. The electro-mechanical energy conversion efficiency of the PMLG/PVDF energy harvester is 1-3 fold higher than PVDF or PMLG energy harvester. PMLG/PVDF piezoelectric fiber composites with good piezoelectricity could promote the applications in various fields such as biomedical engineering, green energy, wearable sensors, and energy harvesters.

碩士
國立雲林科技大學
化學工程與材料工程研究所
99
In this study, poly-γ-glutamate-hydroxyapatite (PGA-HA) particles were synthesized by a co-precipitation method. Then, a series of PGA-HA/water-based polyurethane composite films and scaffolds with different amounts of PGA-HA particles were fabricated. The content of PGA-HA in the composite films and scaffolds was 0~43 wt% and 0~67 wt%, respectively. The results of wide-angle X-ray diffraction and energy dispersive X-ray spectroscopy (EDS) confirmed that the synthesized calcium phosphate was in the form of HA with the ratio of Ca/P = 1.67. The particle size of PGA-HA particles was about 110 nm. EDS mapping analysis revealed that the PGA-HA particles distributed homogeneously in the composite films. The composite film exhibited a higher Young’s modulus, tensile strength and elongation at break when the PGA-HA content was 33 wt%. Scanning electron microscope images showed that the pore size of the scaffolds decreased with increasing the concentration of polyurethane dispersion, but it increased with the increase of PGA-HA content. The swelling ratio and porosity of the composite scaffolds increased with the increase of PGA-HA content. The composite scaffold with 67 wt% PGA-HA had mean pore size of about 250 μm, porosity about 93% and swelling ratio of about 1060%. Moreover, the Young''s modulus and compressive strength of the composite scaffolds increased with the increase of III PGA-HA content.

碩士
國立中山大學
機械與機電工程學系研究所
103
The formation and application of fibers and films based on the near-field electrospinning (NFES) and electrospray (ESP) technology are investigated in this research. The poly [ (γ-methyl l-glutamate) , PMLG] was used as based material and then the Poly [ (ethylene oxide), PEO] and surfactant were mixed with PMLG to fabricate PMLG solution with 21.05 wt% concentration. Next, with the NFES and ESP processes, the high voltage electric field and numerical control (NC) controller (movement speed: 60 mm/sec, path spacing: 250, 500, 750 μm) were controlled to make the PMLG droplet to induce the repulsive force and form Taylor cone. Then, the ordered PMLG nanofibers (diameter: 4.15-6.25 μm) and thin films which deposited on the substrate were fabricated. The orientation of dipole in the PMLG solution could be enhanced with the applying of electric field due to the fact that NFES and ESP processed positive impact on piezoelectric properties. Furthermore, the experimental results showed that the maximum peak voltage was 0.056 V when the PMLG piezoelectric fibers were attached to the flexible electrical measuring device and measured by vibrating test. Finally, PMLG fibers and films which was non-toxic biological material and possessed excellent piezoelectric characteristics were stably fabricated on indium tin oxide (ITO) -coated glass substrate in order to observe the proliferation status of cell. According to the result of research, the PMLG with negative charge could weaken proliferation ability of cells. Therefore, the decrease of the distance between electrospun fibers (spacing: 250-750 μm) made the proliferation of mouse fibroblast cells (NIH3T3) slightly decrease (cell coverage with different spacing during 4 days: 66.42-88.38 %). Moreover, the diffusion of the NIH3T3 was significantly reduced or even halted (cell coverage with different films density during 4 days: 3.07-42.03 %) when density of ESP films increased (films coverage: 41.05-89.55%) which meant that it possessed inhibitory effect.