PhD thesis of Fabio Fenili R07830 Tutor: Prof. Elisabetta Ranucci Coordinator: Prof. Dominique Roberto NOVEL BIOACTIVE AND BIODEGRADABLE BIOMATERIALS OF POLY(AMIDOAMINE)S STRUCTURE Biomaterials are any materials designed to interface with biological system to evaluate, support or replace any tissue, organ or body function. Metals, ceramics, polymers and composites are biomaterials commonly used every day for biomedical applications in the form of sutures, vascular grafts, heart valves, intraocular lenses, dental and orthopaedic implants, pacemakers, biosensors, drug delivery systems, gene carriers, etc. Compared to the other types of biomaterials, polymeric biomaterials offer the advantages that can be prepared in different compositions with a variety of structures and properties. In particular, in the case of biodegradable polymers, their physical, chemical, and biological characteristics as well as degradation rate can be tuned to meet specific applications. Among polymeric biomaterials, poly(amidoamine)s (PAA), a family of biodegradable and biocompatible polymers, can be considered as promising materials for different pharmaceuticals and biotechnological applications. The present PhD project aimed at the design of novel poly(amidoamine)s biomaterials of different architecture for diversified biomedical applications, each representing a distinct research sub-objective. Poly(amidoamine)-gelonin conjugates as potential anti-cancer drugs. Two PAAs bearing pendant 2-ethenyldithiopyridine (ISA1-SSPy and ISA23-SSPy) were used to investigate their ability to mediate intracellular delivery of the type I ribosome-inactivating gelonin. The strategy adopted consists of a three step synthesis. In the first step, a cystamine crosslinked PAA was synthesised generating a hydrophilic tri-dimensional network. In the second step, the hydrogel obtained was de-reticulated by direct disulfide exchange reaction with dipyridyl disulfide. Finally, the linear and soluble PAA-SSPy thus obtained was conjugated via thiol-disulfide exchange reaction to a thiol-containing gelonin, a ribosom inactivating protein. Two different recombinant protein, encoding gelonin with an N-terminal 6xHistidine (H or His) and V5 epitope tag (6H-V5 Gelonin) and gelonin encorporating a C-terminal HA and 6xHis tag flanking a cysteine residue (Gelonin HA-Cys-6H) were prepared introducing the required tag on a gelonin plasmid by polymerase chain reaction (PCR), isolated and purified from E. Coli. In vitro experiments were performed on B16F10 cells using non-toxic concentration of gelonin and polymeric samples up to 2 mg/ml. ISA1-SSPy promoted the intracytoplasmic delivery of gelonin more efficiently than the parent non-functionalized ISA1 with IC50 values of 100 µg/ml. The results obtained for ISA1-SSPy-HA-Cys-6H, designed to have a covalent bound between the polymeric vector and the toxin, and ISA1-SSPy-6H-V5 Gelonin were the same, suggesting a non specific conjugation to the thiol-groups in the Gelonin HA-Cys-6H. This findings could be attributed both to the ability of ISA1-SSPy to react with protein’s disulfide groups and to the interactions between the ethenyl-dithiopyridine pendants and the hydrophobic domains of the protein, giving stable complexes. ISA23-SSPy was unable to mediate toxin delivery. Tricarbonyl-rhenium complexes of a thiol-functionalized amphoteric poly(amidoamine). In this work we investigate the possibility to design new polymeric radiopharmaceuticals using poly(amidoamine)s polymers (PAA)s and [Re(CO)3(H2O)3]+. An amphoteric thiol-functionalized ISA23 copolymer (ISA23SH10%) has been obtained following a two step pathway: the first was the synthesis of polymers containing cystamine-m-Boc pendants, the second one was the reduction of the disulphide bond in the presence of dithiotreitol (DTT) as reducing agent. The copolymer was able to tightly bind up to 0.8 equivalents of Re(CO)3+ fragments, with respect to the thiol groups. The polymeric complexes, containing 0.5 or 0.8 equivalents of rhenium, respectively, were easily obtained by reacting ISA23SH10% with [Re(CO)3(H2O)3](CF3SO3), in aqueous solution, at pH 5.5. The complexes maintained the water solubility of the parent polymer and were stable in physiological conditions and also in the presence of cysteine. The coordination of the Re(CO)3 fragment involves the cysteamine-deriving moiety. A detailed 1H, 13C and 15N NMR characterization of ISA23SH10% and of complex with 0.8 equivalents of Re(CO)3+ provided a clear evidence that the binding of rhenium occurs by chelation through the S and N atoms of the -amino-thiolate fragment. A morphological evaluation by TEM analysis showed that both complexes form nanoparticles with a regular spherical morphology and a narrow size distribution. In vivo toxicological tests showed that ISA23SH10% is highly biocompatible, with a maximum tolerated dose of 500 mg/kg. Preliminary biological studies in vitro and in vivo have been performed also on both complexes. No hemolytic activity was observed, up to a concentration of 5 mg/mL, neither citotoxicity effect was observed on Hela cell after 48 h of incubation. No toxic side effects were observed after the intravenous injection in mice of the two complexes in doses up to 20 mg/kg. Poly(amidoamine)-based hydrogels as scaffold for tissue engineering applications. In this work a new synthetic method has been developed leading to PAA hydrogels with improved mechanical strength and good biological properties for tissue engineering applications. In particular a two-step pathway has been followed: in the first step an acryloyl end-capped linear Agma1 oligomer was synthesised using a controlled excess of the bisacrylamide; in the second step the oligomer was photopolymerized by UV irradiation producing hydrogels with the required mechanical characteristics. Using this new synthetic procedure, PAA-UV made hydrogels with different form and shape were prepared. In particular, tubular scaffolds with 1 mm inner diameter were tested in vivo as conduit for nerve regeneration in a rat sciatic nerve cut model. The implants were analyzed at 30, 90 and 180 days post-surgery and resulted particularly promising in many important respects, such as biodegradability, biocompatibility, lack of inflammatory reaction upon degradation and capability of promoting optimum morphological and functional nerve regeneration. The regenerated nerves showed several interesting signs of morphological improvements even at 30 days post-surgery. At 180 days the scaffold was almost completely reabsorbed and the regenerated nerve morphologically comparable to the control animals. Cationic PAA as non-viral vector for gene therapy. Recently, a new linear and amphoteric but prevailing cationic PAA, nicknamed Agma1 (Scheme 4) has been successfully tested as trasfection promoters in vitro without any signs of toxicity is despite its polycationic behavior. Based on this premise, in this work, three samples of Agma1 having different molecular weights, named AGMA5 ( =5100), AGMA10 ( =10100) and AGMA20 ( =20500) were tested as nucleic acid carriers in order to establish the relationship between the molecular weight of the samples and their gene transfer ability. All samples proved able to complex DNA forming stable nanoparticles with positively charged surface and dimensions depending on the weight/weight ratio between polymer and DNA, and pH. The molecular weight of the polymeric samples had a significant influence on their efficiency as DNA carrier. AGMA10 and AGMA20 showed good transfection ability compared to AGMA5 which was almost ineffective even at a 1:100 w/w DNA/polymer ratio. The polyplex nanoparticles proved highly biocompatible and were easily internalized in cells escaping from the endosomal vesicles and mostly localizing in perinuclear region. AGMA10 was chosen for animal experiments and proved effective also in vivo. A 1:30 DNA/AGMA10 polyplex after intravenous administration to mice induced remarkable gene expression in the liver but not in other organs, including lungs without detectable toxic side effects.

NOVEL BIOACTIVE AND BIODEGRADABLE BIOMATERIALS OF POLY(AMIDOMINE) STRUCTURE / F. Fenili ; Coordinator: Dominique Roberto ; Tutor: Elisabetta Ranucci ; Co-tutor: Paolo Ferruti. - : . Universita' degli Studi di Milano, 2010 Dec 15. ((23. ciclo, Anno Accademico 2010. [10.13130/fenili-fabio_phd2010-12-15].

NOVEL BIOACTIVE AND BIODEGRADABLE BIOMATERIALS OF POLY(AMIDOMINE) STRUCTURE

F. Fenili
2010-12-15

Abstract

PhD thesis of Fabio Fenili R07830 Tutor: Prof. Elisabetta Ranucci Coordinator: Prof. Dominique Roberto NOVEL BIOACTIVE AND BIODEGRADABLE BIOMATERIALS OF POLY(AMIDOAMINE)S STRUCTURE Biomaterials are any materials designed to interface with biological system to evaluate, support or replace any tissue, organ or body function. Metals, ceramics, polymers and composites are biomaterials commonly used every day for biomedical applications in the form of sutures, vascular grafts, heart valves, intraocular lenses, dental and orthopaedic implants, pacemakers, biosensors, drug delivery systems, gene carriers, etc. Compared to the other types of biomaterials, polymeric biomaterials offer the advantages that can be prepared in different compositions with a variety of structures and properties. In particular, in the case of biodegradable polymers, their physical, chemical, and biological characteristics as well as degradation rate can be tuned to meet specific applications. Among polymeric biomaterials, poly(amidoamine)s (PAA), a family of biodegradable and biocompatible polymers, can be considered as promising materials for different pharmaceuticals and biotechnological applications. The present PhD project aimed at the design of novel poly(amidoamine)s biomaterials of different architecture for diversified biomedical applications, each representing a distinct research sub-objective. Poly(amidoamine)-gelonin conjugates as potential anti-cancer drugs. Two PAAs bearing pendant 2-ethenyldithiopyridine (ISA1-SSPy and ISA23-SSPy) were used to investigate their ability to mediate intracellular delivery of the type I ribosome-inactivating gelonin. The strategy adopted consists of a three step synthesis. In the first step, a cystamine crosslinked PAA was synthesised generating a hydrophilic tri-dimensional network. In the second step, the hydrogel obtained was de-reticulated by direct disulfide exchange reaction with dipyridyl disulfide. Finally, the linear and soluble PAA-SSPy thus obtained was conjugated via thiol-disulfide exchange reaction to a thiol-containing gelonin, a ribosom inactivating protein. Two different recombinant protein, encoding gelonin with an N-terminal 6xHistidine (H or His) and V5 epitope tag (6H-V5 Gelonin) and gelonin encorporating a C-terminal HA and 6xHis tag flanking a cysteine residue (Gelonin HA-Cys-6H) were prepared introducing the required tag on a gelonin plasmid by polymerase chain reaction (PCR), isolated and purified from E. Coli. In vitro experiments were performed on B16F10 cells using non-toxic concentration of gelonin and polymeric samples up to 2 mg/ml. ISA1-SSPy promoted the intracytoplasmic delivery of gelonin more efficiently than the parent non-functionalized ISA1 with IC50 values of 100 µg/ml. The results obtained for ISA1-SSPy-HA-Cys-6H, designed to have a covalent bound between the polymeric vector and the toxin, and ISA1-SSPy-6H-V5 Gelonin were the same, suggesting a non specific conjugation to the thiol-groups in the Gelonin HA-Cys-6H. This findings could be attributed both to the ability of ISA1-SSPy to react with protein’s disulfide groups and to the interactions between the ethenyl-dithiopyridine pendants and the hydrophobic domains of the protein, giving stable complexes. ISA23-SSPy was unable to mediate toxin delivery. Tricarbonyl-rhenium complexes of a thiol-functionalized amphoteric poly(amidoamine). In this work we investigate the possibility to design new polymeric radiopharmaceuticals using poly(amidoamine)s polymers (PAA)s and [Re(CO)3(H2O)3]+. An amphoteric thiol-functionalized ISA23 copolymer (ISA23SH10%) has been obtained following a two step pathway: the first was the synthesis of polymers containing cystamine-m-Boc pendants, the second one was the reduction of the disulphide bond in the presence of dithiotreitol (DTT) as reducing agent. The copolymer was able to tightly bind up to 0.8 equivalents of Re(CO)3+ fragments, with respect to the thiol groups. The polymeric complexes, containing 0.5 or 0.8 equivalents of rhenium, respectively, were easily obtained by reacting ISA23SH10% with [Re(CO)3(H2O)3](CF3SO3), in aqueous solution, at pH 5.5. The complexes maintained the water solubility of the parent polymer and were stable in physiological conditions and also in the presence of cysteine. The coordination of the Re(CO)3 fragment involves the cysteamine-deriving moiety. A detailed 1H, 13C and 15N NMR characterization of ISA23SH10% and of complex with 0.8 equivalents of Re(CO)3+ provided a clear evidence that the binding of rhenium occurs by chelation through the S and N atoms of the -amino-thiolate fragment. A morphological evaluation by TEM analysis showed that both complexes form nanoparticles with a regular spherical morphology and a narrow size distribution. In vivo toxicological tests showed that ISA23SH10% is highly biocompatible, with a maximum tolerated dose of 500 mg/kg. Preliminary biological studies in vitro and in vivo have been performed also on both complexes. No hemolytic activity was observed, up to a concentration of 5 mg/mL, neither citotoxicity effect was observed on Hela cell after 48 h of incubation. No toxic side effects were observed after the intravenous injection in mice of the two complexes in doses up to 20 mg/kg. Poly(amidoamine)-based hydrogels as scaffold for tissue engineering applications. In this work a new synthetic method has been developed leading to PAA hydrogels with improved mechanical strength and good biological properties for tissue engineering applications. In particular a two-step pathway has been followed: in the first step an acryloyl end-capped linear Agma1 oligomer was synthesised using a controlled excess of the bisacrylamide; in the second step the oligomer was photopolymerized by UV irradiation producing hydrogels with the required mechanical characteristics. Using this new synthetic procedure, PAA-UV made hydrogels with different form and shape were prepared. In particular, tubular scaffolds with 1 mm inner diameter were tested in vivo as conduit for nerve regeneration in a rat sciatic nerve cut model. The implants were analyzed at 30, 90 and 180 days post-surgery and resulted particularly promising in many important respects, such as biodegradability, biocompatibility, lack of inflammatory reaction upon degradation and capability of promoting optimum morphological and functional nerve regeneration. The regenerated nerves showed several interesting signs of morphological improvements even at 30 days post-surgery. At 180 days the scaffold was almost completely reabsorbed and the regenerated nerve morphologically comparable to the control animals. Cationic PAA as non-viral vector for gene therapy. Recently, a new linear and amphoteric but prevailing cationic PAA, nicknamed Agma1 (Scheme 4) has been successfully tested as trasfection promoters in vitro without any signs of toxicity is despite its polycationic behavior. Based on this premise, in this work, three samples of Agma1 having different molecular weights, named AGMA5 ( =5100), AGMA10 ( =10100) and AGMA20 ( =20500) were tested as nucleic acid carriers in order to establish the relationship between the molecular weight of the samples and their gene transfer ability. All samples proved able to complex DNA forming stable nanoparticles with positively charged surface and dimensions depending on the weight/weight ratio between polymer and DNA, and pH. The molecular weight of the polymeric samples had a significant influence on their efficiency as DNA carrier. AGMA10 and AGMA20 showed good transfection ability compared to AGMA5 which was almost ineffective even at a 1:100 w/w DNA/polymer ratio. The polyplex nanoparticles proved highly biocompatible and were easily internalized in cells escaping from the endosomal vesicles and mostly localizing in perinuclear region. AGMA10 was chosen for animal experiments and proved effective also in vivo. A 1:30 DNA/AGMA10 polyplex after intravenous administration to mice induced remarkable gene expression in the liver but not in other organs, including lungs without detectable toxic side effects.
RANUCCI, ELISABETTA
ROBERTO, DOMINIQUE MARIE
Biomaterials ; poly(amidoamine) ; biodegrdable polymers ; tissue enginnering ; polymer therapeutics ; gene therapy
Settore CHIM/04 - Chimica Industriale
NOVEL BIOACTIVE AND BIODEGRADABLE BIOMATERIALS OF POLY(AMIDOMINE) STRUCTURE / F. Fenili ; Coordinator: Dominique Roberto ; Tutor: Elisabetta Ranucci ; Co-tutor: Paolo Ferruti. - : . Universita' degli Studi di Milano, 2010 Dec 15. ((23. ciclo, Anno Accademico 2010. [10.13130/fenili-fabio_phd2010-12-15].
Doctoral Thesis
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