RESUMO
By combined light scattering and circular dichroism measurements (CD), we have investigated the coil-to-globule transition of the thermosensitive polymer poly(N-isopropylacrylamide) (pNIPAAm) copolymerized with a 1/10 fraction of valine- or leucine-derived groups randomly positioned along the chains. The comonomers provide the pNIPAAm chains with chirality, electric charge, and increased hydrophobicity. For valine-derived copolymers, the coil-globule transition is basically unmodified with respect to pNIPAAm, whereas doping with leucine-derived groups significantly lowers the transition temperature and makes the transition discontinuous. We find the CD signal of the chiral comonomers to cleanly depend on the local chain density. We interpret this behavior as an effect of the whole chain conformation on the conformations accessible to the chiral groups.
Assuntos
Resinas Acrílicas/química , Aminoácidos/química , Biofísica/métodos , Físico-Química/métodos , Dicroísmo Circular , Luz , Conformação Molecular , Polímeros/química , Ligação Proteica , Espalhamento de Radiação , Estereoisomerismo , Temperatura , Valina/químicaRESUMO
Materials and components employed in the presence of intense neutron and gamma fields are expected to absorb high dose levels that may induce deep modifications of their physical and mechanical properties, possibly causing loss of their function. A protocol for irradiating elastomeric materials in reactor mixed neutron and gamma fields and for testing the evolution of their main mechanical and physical properties with absorbed dose has been developed. Four elastomeric compounds used for vacuum O-rings, one fluoroelastomer polymer (FPM) based and three ethylene propylene diene monomer rubber (EPDM) based, presently available on the market have been selected for the test. One EPDM is rated as radiation resistant in gamma fields, while the other elastomers are general purpose products. Particular care has been devoted to dosimetry calculations, since absorbed dose in neutron fields, unlike pure gamma fields, is strongly dependent on the material composition and, in particular, on the hydrogen content. The products have been tested up to about 2 MGy absorbed dose. The FPM based elastomer, in spite of its lower dose absorption in fast neutron fields, features the largest variations of properties, with a dramatic increase in stiffness and brittleness. Out of the three EPDM based compounds, one shows large and rapid changes in the main mechanical properties, whereas the other two feature more stable behaviors. The performance of the EPDM rated as radiation resistant in pure gamma fields does not appear significantly better than that of the standard product. The predictive capability of the accelerated irradiation tests performed as well as the applicable concepts of threshold of radiation damage is discussed in view of the use of the examined products in the selective production of exotic species facility, now under construction at the Legnaro National Laboratories of the Italian Istituto Nazionale di Fisica Nucleare. It results that a careful account of dose rate effects and oxygen penetration in the material, both during test irradiations and in operating conditions, is needed to obtain reliable predictions.
RESUMO
We have examined the cytotoxicity of a number of poly(amidoamine) polymers which have been proposed for use as DNA delivery systems and compared them to the charged polyamino acid polylysine. Most of the poly(amidoamine)s tested were shown to be remarkably non-toxic to both HepG2 and HL60 cell lines. However, one of the structures (NG30, co-monomers methylene bisacrylamide, dimethylethylene diamine) did show cytotoxicity similar to that of polylysine. A second PAA structure (NG37, NG38, NG39, co-monomers bisacryloyl piperazine, 2-methyl piperazine) showed mild cytotoxicity towards both cell lines, related to the degree of polymerisation. The results support the idea that the cytotoxicity of polycations has a strong structural basis rather than being an effect due only to charge. As a consequence of their general reduced level of cytotoxicity, poly(amidoamine)s appear to have possible advantages for complexation with DNA over some other cationic polymers as a key component of DNA delivery systems.
Assuntos
DNA/química , Técnicas de Transferência de Genes , Poliaminas/química , Polímeros/química , Resinas de Troca de Cátion , Portadores de Fármacos , Células HL-60 , Humanos , Lipídeos , Luciferases , Plasmídeos , Poliaminas/síntese química , Poliaminas/toxicidade , Polilisina/toxicidade , Polímeros/síntese química , Polímeros/toxicidade , Relação Estrutura-Atividade , Transfecção/métodosRESUMO
Polyplexes are now emerging as potentially useful vectors for gene therapy. To improve our understanding of how the chemical structure of the polymer affects the properties of these systems, a series of structurally related polymers, the linear poly(amidoamine)s (PAAs), have been examined for their abilities to form complexes with DNA. Structure-dependent differences in DNA binding are shown by gel electrophoretic retardation of DNA and thermal transition analyses. Two PAAs, NG28 and NG30, stand out as having high affinity DNA binding characteristics, similar to the model homopolypeptide, poly-L-lysine. In addition, differences in complex formation, particle size and surface charge are displayed for the different polymer-DNA systems. Electron microscopy studies showed that the polymers condensed DNA into similar unit structures but only complexes with NG30 did not undergo agglomeration. This was attributed to an excess of complexed polymer forming a shell of uncomplexed polymer chain segments around a condensed DNA-polymer core. The transfection activities of these polymer complexes differ greatly, and some of these differences can be explained in a multifactorial way by the physicochemical and colloidal properties. It is concluded that polymer chemical structure dictates the apparent affinity of DNA binding, and also several of the important colloidal characteristics of the resulting complexes.
Assuntos
DNA/química , Terapia Genética , Polímeros/química , Cloroquina , Coloides/química , Portadores de Fármacos , Eletroforese em Gel de Ágar , Humanos , Microscopia Eletrônica , Tamanho da Partícula , Plasmídeos , Polilisina/química , Relação Estrutura-Atividade , Propriedades de Superfície , Temperatura , Transfecção , Células Tumorais CultivadasRESUMO
Different families of functionalized polymers with potential as biomaterials, or for biomaterial modification, have been investigated. In particular, degradation studies have been performed on poly(amidoamines), a family of polymers obtained by polyaddition of amines to bisacrylamides, and endowed with heparin-complexing ability. Some new poly(amidoamines) with more resistance towards hydrolytic degradation than traditional ones have been discovered. Other ter-amino polymers deriving from the polyaddition of ter-amino functionalized bis-thiols to bis-acrylic esters, or other activated unsaturated compounds, have been studied. Their quaternarization products have been proven, in a parallel work, to act as powerful antimicrobial agents. By performing in situ the polyaddition reaction, semi-interpenetrated networks based on silicone rubber and the same polymers have been prepared. Finally, end-functionalized amphiphilic oligomers have been prepared by radical polymerization techniques, and their use for enzyme modification considered.
Assuntos
Materiais Biocompatíveis/química , Polímeros/química , Poliaminas/químicaRESUMO
Surface-modified human serum albumin (HSA) nanospheres with a size of around 100 nm in diameter were prepared from poly(amidoamine)-poly(ethylene glycol) copolymer grafted human serum albumin (HSA-PAA-PEG) and poly(thioetheramido acid)-poly(ethylene glycol) copolymer grafted human serum albumin (HSA-PTAAC-PEG). The nanospheres were produced using a pH-coacervation method and cross-linked with glutaraldehyde. The cross-linking efficiency was affected by the type of albumin conjugate used. The zeta potential of the surface-modified nanospheres was significantly lower than that of unmodified particles. The existence of a hydrated steric barrier surrounding the nanospheres was confirmed by electrolyte- and pH-induced flocculation tests. The surface-modified nanospheres showed a reduced plasma protein adsorption on the particle surface compared with unmodified particles.
Assuntos
Portadores de Fármacos , Microesferas , Polietilenoglicóis/química , Albumina Sérica , Albumina Sérica/química , Adsorção , Proteínas Sanguíneas , Eletrólitos , Humanos , Concentração de Íons de Hidrogênio , Microscopia Eletrônica , Polietilenoglicóis/síntese química , Albumina Sérica/síntese química , Albumina Sérica Humana , Propriedades de SuperfícieRESUMO
Poly(amidoamines) are soluble polymers containing tertiary amino and amido groups regularly arranged along the macromolecular chain, and their net average charge alters considerably as pH changes from neutral to acidic leading to a change in conformation. This property provides the possibility to design polymer-drug conjugates that are, following intravenous administration, relatively compacted and thus protect a drug payload in the circulation, but following pinocytic internalisation into acidic intracellular compartments unfold permitting pH-triggered intracellular drug delivery. To study the feasibility of this approach, a covalent conjugate of a poly(amidoamine) (MBI) was prepared to contain the membrane lytic non-ionic detergent Triton X-100 (as a model), and its ability to lyse red blood cells in vitro was used as an indicator of conjugate conformation at at different pHs. Although Triton X-100 was highly lytic at pH 5.5, 7.4 and 8.0, and the parent polymer MBI was not lytic under any conditions, the conjugate only showed concentration-dependent red blood cell lysis at pH 5.5. Moreover, incubation of human leukaemic cells (CCRF) with these substrates showed conjugate to be more toxic than MBI (IC50 values of 100 micrograms/ml and 650 micrograms/ml respectively) and less toxic than Triton X-100 (IC50 of 1 microgram/ml).
Assuntos
Sistemas de Liberação de Medicamentos , Eritrócitos/efeitos dos fármacos , Octoxinol/administração & dosagem , Polímeros/metabolismo , Animais , Sítios de Ligação , Sobrevivência Celular/efeitos dos fármacos , Cromatografia em Camada Fina , Eritrócitos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Injeções Intravenosas , Nylons/metabolismo , Octoxinol/metabolismo , Poliaminas/metabolismo , Ratos , Espectroscopia de Infravermelho com Transformada de Fourier , Células Tumorais CultivadasRESUMO
Polyelectrolyte complexes between cationic polymers and DNA have emerged as potential nonviral vectors for DNA delivery. For successful in vivo delivery, methods for analyzing their ability to prevent digestion of the DNA payload by serum nucleases are essential. We report here a simple assay to determine degradation of DNA in these complexes using standard electrophoretic techniques. The assay is based on a high pH buffer which can dissociate the complexes under standard electrophoretic conditions. This assay can be used qualitatively to determine the time taken for degradation to occur. Alternatively, with a standard gel analysis program it can be used quantitatively to investigate rates of DNA degradation from complexes in the presence of serum nucleases. We have shown that it can distinguish between different formulations with the same polymer, and also to distinguish between the time taken to degradation and the rates of degradation of DNA in complexes formed with two structurally related, linear polyamidoamine polymers. The assay could also distinguish between the time to degradation using poly-l-lysine complexes, although these were less well dissociated by the electrophoresis buffer, and could not be analyzed quantitatively. This assay will be of value in investigating and developing polyelectrolyte formulations for parenteral administration.
Assuntos
DNA/química , DNA/metabolismo , Desoxirribonucleases/sangue , Eletroforese em Gel de Ágar/métodos , Plasmídeos/genética , Poliaminas/química , Soluções Tampão , Fragmentação do DNA , Terapia Genética , Vetores Genéticos/sangue , Vetores Genéticos/química , Concentração de Íons de Hidrogênio , Plasmídeos/sangue , Polieletrólitos , Reprodutibilidade dos TestesRESUMO
The aim of this presentation is to review some of our recent work mostly on poly(amidoamine)s (PAAs) and some other families of polymers structurally related to PAAs of medical interest. PAAs are obtained by stepwise polyaddition of primary monoamines, or bis secondary amines, to bisacrylamides. There are several other ter-amino polymers structurally related to PAAs, such poly(amido phosphine)s (PAPs), poly(ester-amine)s (PEAs), poly(ketone-amine)s (PKAs), poly(amidothioeteramine)s (PATAs) poly(esterthioether amine)s (PTEAs), and poly(sulphone thioetheramine)s (PSTAs). Most of the PAAs exhibit heparin complexing ability. PAAs are also being considered as soluble carriers for delivering anti-cancer drugs. Some of these polymers have been studied as antimicrobial agents. PAAs with different structures degrade at different rates under physiological conditions. The degradation rate is also strongly influenced by pH. The quaternarized PATAs and PTEAs are reasonably stable over a period of some days, but ultimately degrade to oligomeric products, while the quaternized PAAs do rapidly degrade.