RESUMO
Bisensitive interpenetrating polymer network (IPN) hydrogels of temperature sensitive net-poly(N-isopropylacrylamide) and pH sensitive net-poly(acrylic acid-co-acrylamide) for microfluidic applications were prepared via a sequential synthesis using free radical polymerization. The IPN indicated a suitable reversible alteration of swelling in response to the change in pH and temperature. The adequate change of the hydrogel volume is a basic requirement for microfluidic applications. Using the introduced correction factor f, it is possible to determine the cooperative diffusion coefficient (Dcoop) of cylindrical samples at any aspect ratio. The determined cooperative diffusion coefficient allowed the evaluation of varying swelling processes of different network structures. The presence of the second sub-network of the IPN improved the swelling behaviour of the first sub-network compared to the individual networks.
RESUMO
Dendritic cells (DCs) play a crucial role in the development of cell-mediated immunotherapy due to their ability to induce and maintain strong immune responses. In our study, we evaluated a biocompatible Ni(II)-NTA-modified poly(ethylene imine) dendritic glycopolymer (Ni(II)-NTA-DG) as new carrier system to increase the antigen uptake into iDCs for future DC-based immunotherapy. Ni(II)-NTA-DG led to an increase in His6-Gp160 uptake in monocytes and iDCs, where His6-Gp160 is localized in the early endosomal and lysosomal compartments. Ni(II)-NTA-DG and the formed polyplexes induced an activation of iDCs, showing an increasing expression of costimulatory molecules CD86, CD80, and proinflammatory cytokines IL-6 and IL-8. Beside no influencing effect of Ni(II)-NTA-DG and polyplexes on the maturation of antigen-bearing DCs, the mature peptide bearing DCs remained their ability to migrate along a gradient of CCR7 ligands. Thus, Ni(II)-NTA-DG with advancing biological properties is a promising carrier system for the future application in DC-based immunotherapy.
Assuntos
Células Dendríticas/citologia , Iminas/química , Imunoterapia , Polietilenos/química , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Dendríticas/química , Células Dendríticas/imunologia , Citometria de Fluxo , Humanos , Iminas/imunologia , Interleucina-6/química , Interleucina-6/metabolismo , Interleucina-8/química , Interleucina-8/metabolismo , Lipopolissacarídeos/metabolismo , Monócitos/citologiaRESUMO
Poly(propylene imine) (PPI) glycodendrimers are promising candidates as drug carriers and antiamyloidogenic and antiprionic agents. In this study the anti-ß-amyloid capacity of PPI glycodendrimers of the fourth and fifth generations was investigated in vitro and in vivo. We assessed distinct PPI glycodendrimers including G4mDS and G5mDS, with electroneutral maltose shell, and G4mOS and G4m-IIIOS, with cationic maltose or maltotriose shell. Our results show that in vitro PPI maltose dendrimers reduce the toxicity of Aß(1-42). However, only the electroneutral maltose dendrimers G4mDS and G5mDS reduce the toxicity of Alzheimer's disease brain extracts in SH-SY5Y neuroblastoma cells. PPI maltose dendrimers with electroneutral or cationic surface penetrate the cytoplasm of cultured cells, and they reach the brain when administered intranasally. Both cationic G4mOS and electroneutral G4mDS are able to modify the total burden of ß-amyloid in APP/PS1 mice. The studied dendrimers did not reverse memory impairment in APP/PS1 mice following chronic administration; moreover, cationic G4mOS caused cognitive decline in nontransgenic mice. In spite of the capacity of G4mDS and G4mOS to cross the blood-brain barrier and modulate Aß aggregation in APP/PS1 mice, further studies are needed to learn how to reduce the harmful effects of maltose dendrimers in vivo.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Precursor de Proteína beta-Amiloide/metabolismo , Dendrímeros/farmacologia , Glicoconjugados/farmacologia , Polipropilenos/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Administração Intranasal , Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Sobrevivência Celular/efeitos dos fármacos , Dendrímeros/administração & dosagem , Dendrímeros/química , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Glicoconjugados/administração & dosagem , Glicoconjugados/química , Humanos , Masculino , Maltose/química , Camundongos , Camundongos Transgênicos , Tamanho da Partícula , Polipropilenos/administração & dosagem , Polipropilenos/química , Proteínas Serina-Treonina Quinases/genética , Relação Estrutura-Atividade , Propriedades de Superfície , Células Tumorais CultivadasRESUMO
The aim of this study was to investigate the interactions between poly(propylene imine) (PPI) dendrimers and 2'-/3'-O-(N'-methylanthraniloyl)-ATP (Mant-ATP). Mant-ATP was used as a model molecule. Purine and pyrimidine nucleoside analogues are antimetabolites commonly used in therapy for cancer. Drug molecules can complex with dendrimers in two ways: therapeutic agents may be attached in dendrimer interior or bind to functional groups on the surface. Drugs attached to nanoparticles are characterized by improved solubility, pharmacokinetics and stability. Here, we have used poly(propylene imine) dendrimers of the 4th and 5th generations (PPI G4 and PPI G5) with primary amino surface groups partially modified with maltose (PPI-m) or without modification (PPI). We assessed the efficiency of complex formation in relation to dendrimer generation, pH of solution and the type of dendrimer used. A double fluorimetric titration method was used to estimate the binding constant (K b ) and the number of binding centers per molecule of the binding agent (n).
Assuntos
Trifosfato de Adenosina/análogos & derivados , Dendrímeros/química , Polipropilenos/química , ortoaminobenzoatos/química , Trifosfato de Adenosina/química , Espectrometria de FluorescênciaRESUMO
Dendritic cells (DC), which play a major role in development of cell-mediated immunity, represent opportunities to develop novel anti-HIV vaccines. Dendrimers have been proposed as new carriers to ameliorate DC antigen loading and in this way, we have determined the potential use of maltose decorated neutrally and positively charged G4 glycodendrimers. Thus, immunostimulatory properties of these glycodendrimers on human DC were evaluated in the context of HIV infection. We have demonstrated that DC treated with glycodendrimers were fully functional with respect to viability, maturation and HIV-derived antigens uptake. Nevertheless, iDC and mDC phenotypes as well as mDC functions such as migration ability and cytokines profile production were changed. Our results showed the potential carrier properties of glycodendrimers to activate the immune system by the way of DC stimulation. This is the first study for exploring the use of maltose-functionalized dendrimers-peptides complexes as a potential DC-based vaccine candidate. FROM THE CLINICAL EDITOR: In this paper, maltose-functionalized dendrimer-peptide complexes are demonstrated to activate the immune system by way of dendritic cell (DC) stimulation. DC vaccination using this methodology may be applicable to a variety of conditions, including infections and potentially cancer.
Assuntos
Dendrímeros/química , Células Dendríticas/imunologia , Infecções por HIV/imunologia , Infecções por HIV/terapia , HIV/imunologia , Imunoterapia , Biomarcadores/metabolismo , Morte Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Citocinas/metabolismo , Dendrímeros/farmacologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , HIV/efeitos dos fármacos , Humanos , Maltose/química , Peptídeos/imunologia , Fenótipo , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacosRESUMO
Glycopolymers are promising materials in the field of biomedical applications and in the fabrication of supramolecular structures with specific functions. For tunable design of supramolecular structures, glycopolymer architectures with specific properties (e.g., controlled self-assembly) are needed. Using the concept of dendronized polymers, a series of H-bond active giant glycomacromolecules with maleimide backbone and lysine dendrons of different generations were synthesized. They possess different macromolecular size and functionality along the backbone. Their peripheral maltose units lead to solubility under physiological conditions and controlled aggregation behavior. The aggregation behavior was investigated depending on generation number, pH value, and concentration. A portfolio of complementary analytical tools give an insight into the influence of the different parameters in shaping a rod-, coil-, and worm-like molecular structure and their controlled aggregate formation. MD simulation helped us to understand the complex aggregation behavior of the linear polymer chain without dendritic units.
Assuntos
Dendrímeros/química , Lisina/química , Maleimidas/química , Maltose/química , Polímeros/química , Materiais Biocompatíveis/química , Cromatografia em Gel , Fracionamento por Campo e Fluxo , Concentração de Íons de Hidrogênio , Substâncias Macromoleculares , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Simulação de Dinâmica Molecular , Estrutura MolecularRESUMO
Photodynamic therapy is one of the best alternatives to chemo-, radio- or surgical therapy, as it is noninvasive and causes no severe side effects. The mechanism of photodynamic therapy involves activation of the drug (photosensitizer) with light of appropriate wavelength, which combined with molecular oxygen, leads to production of reactive oxygen species. This starts a cascade of reactions leading to cell death. Thus, the efficiency of this therapy is based mainly on the properties of a photosensitizer, including singlet oxygen yield and accumulation in the tumor area. Current research is aimed at applying nanosystems for the improvement of availability and photodynamic properties of photosensitizers. In order to improve the activity and increase photodynamic potential of rose bengal, one of the most promising drugs in anticancer photodynamic therapy, several drug delivery systems were developed. Among them, polymersomes represent a group of innovative polymeric vesicles mimicking membranous cell structures. Polymersomes are nanosystems made of amphiphilic block copolymers, possessing a spherical, liposome-like architecture. Within this study we present biophysical and in vitro biological characterization of this novel pH-stable nanosystem, which due to the improvement of singlet oxygen and reactive oxygen species (ROS) production by rose bengal is a good candidate for nanocarrier in photodynamic therapy.
Assuntos
Fotoquimioterapia , Linhagem Celular Tumoral , Concentração de Íons de Hidrogênio , Fármacos Fotossensibilizantes/química , Polímeros/química , Espécies Reativas de Oxigênio/metabolismo , Rosa Bengala/química , Oxigênio SingleteRESUMO
A major challenge in designing cancer therapies is the induction of cancer cell apoptosis, although activation of intrinsic apoptotic pathways by targeting gold nanoparticles to mitochondria is promising. We report an in vitro procedure targeting mitochondria with conjugated gold nanoparticles and investigating effects on apoptosis induction in the human breast cancer cell line Jimt-1. Gold nanoparticles were conjugated to a variant of turbo green fluorescent protein (mitoTGFP) harbouring an amino-terminal mitochondrial localization signal. Au nanoparticle conjugates were further complexed with cationic maltotriose-modified poly(propylene imine) third generation dendrimers. Fluorescence and transmission electron microscopy revealed that Au nanoparticle conjugates were directed to mitochondria upon transfection, causing partial rupture of the outer mitochondrial membrane, triggering cell death. The ability to target Au nanoparticles into mitochondria of breast cancer cells and induce apoptosis reveals an alternative application of Au nanoparticles in photothermal therapy of cancer.
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Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Ouro/uso terapêutico , Nanopartículas Metálicas/uso terapêutico , Mitocôndrias/efeitos dos fármacos , Fotoquimioterapia/métodos , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Linhagem Celular Tumoral , Ouro/química , Proteínas de Fluorescência Verde , Humanos , Nanopartículas Metálicas/química , Mitocôndrias/química , Mitocôndrias/efeitos da radiação , Terapia de Alvo Molecular/métodos , Radiossensibilizantes/química , Radiossensibilizantes/uso terapêutico , Resultado do TratamentoRESUMO
This paper examines the formation and stability of nano-complexes that could provide a new therapeutic approach against HIV-1 infection. Poly(propylene imine) glycodendrimers decorated with 2(nd) generation cationic carbosilane dendrons were generated and their use in polyplex formation checked. Owing to their positively-charged terminal amino groups the hybrid glycodendrimers can bind anionic peptides. It was shown that they form nano-complexes with the HIV-derived peptides P24, Gp160 and Nef. Complexes 130-190 nm in size were formed in molar ratios (dendrimer/ peptide) of (3-4):1. These were sufficiently stable over time and at different pHs. The results obtained suggest that the hybrid dendrimers studied can be considered as alternative carriers for delivering HIV peptides to dendritic cells.
Assuntos
Dendrímeros/química , Portadores de Fármacos/química , HIV-1 , Nanopartículas/química , Peptídeos/química , Proteína do Núcleo p24 do HIV/química , Proteína gp160 do Envelope de HIV/química , Polipropilenos/química , Silanos/química , Produtos do Gene nef do Vírus da Imunodeficiência Humana/químicaRESUMO
The diagnosis and treatment of malignant melanoma by means of the formulation of active principles with dendrimeric nanoparticles is an area of great current interest. The identification and understanding of molecular mechanisms which ensure the integration of particular dendrimeric nanostructures in tumor cellular environment can provide valuable guidance in their coupling strategies with antitumor or diagnostic agents. Two structurally distinct maltose-shell modified 5th generation (G5) poly(propylene imine) (PPI) glycodendrimers fluorescently labeled, (a) with open maltose shell, cationic charged G5-PPI-OS and (b) with dense maltose shell and nearly neutral G5-PPI-DS, were tested in relation with several melanoma cell lines. We found that three melanoma cell lines internalize G5-PPI-DS structure more efficiently than non tumoral HEK297T cells. Furthermore, the internalization pathways of G5-PPI-OS and G5-PPI-DS are characteristic for each tumor cell phenotype and include more than one mechanism. As a general trend, large amounts of both G5-PPI-OS and G5-PPI-DS are internalized on cholesterol-dependent pathway in MJS primary melanoma cells and on non conventional pathways in SK28 metastatic melanoma cells. G5-PPI-OS, temporarily retained at plasma membrane in both cell lines, is internalized slower in metastatic than in primary phenotype. Unlike G5-PPI-OS, G5-PPI-DS is immediately endocytosed in both cell lines. The unconventional internalization pathway and trafficking, exclusively used by G5-PPI-DS in metastatic cells, is described at molecular level. The decay kinetics of fluorescent labeled G5-PPI-OS and G5-PPI-DS is distinct in the two cellular phenotypes. Both cationic and neutral maltose G5-PPI glycodendrimeric structures represent molecules based on which designing of new formulations for therapy or/and diagnosis of melanoma can be further developed.
Assuntos
Dendrímeros/farmacocinética , Maltose/farmacocinética , Melanoma/metabolismo , Polipropilenos/farmacocinética , Animais , Linhagem Celular Tumoral , Células Cultivadas , Dendrímeros/química , Humanos , Maltose/química , Camundongos , Polipropilenos/químicaRESUMO
Dendrimers are artificial polymeric macromolecules which are widely considered to be a promising tool for future gene therapy applications. They have been used as efficient delivery vehicles for antisense oligonucleotides targeting the interior of cells. We demonstrate that dendriplexes formed from anti-HIV oligodeoxynucleotides ANTI-TAR, GEM91, and SREV in complex with generation 4 maltose (PPI-Mal G4) and maltotriose (PPI-Mal-III G4) modified poly(propylene imine) dendrimers are able to self-assemble into highly organized 1D and 3D nanostructures. The resulting nanostructures were characterized by fluorescence methods, laser Doppler electrophoresis, dynamic light scattering (DLS), atomic force microscopy (AFM) and molecular modeling. The results show that ANTI-TAR and GEM 91 dendriplexes self-assemble into fibrils with length scales up to several hundreds of nm. SREV, on the contrary, forms quadrilateral- like 3D nanostructures. A good correlation between the various experimental methods and molecular modeling indicates the formation of those nanostructures in solution. Space symmetry of the oligonucleotides and the resulting dendriplex monomeric units are probably the most important factors which influence the way of self-assembling.
Assuntos
Fármacos Anti-HIV/química , Dendrímeros/química , Oligonucleotídeos Antissenso/química , Polipropilenos/química , Fármacos Anti-HIV/administração & dosagem , Polarização de Fluorescência , Infecções por HIV/tratamento farmacológico , Humanos , Luz , Maltose/química , Modelos Moleculares , Nanoestruturas/química , Oligonucleotídeos Antissenso/administração & dosagem , Espalhamento de Radiação , Tionucleotídeos/químicaRESUMO
The aggregation behavior of the amyloid peptide Aß(1-28) and the prion peptide PrP(185-208) - both responsible for neurodegenerative disorders - was analyzed in the absence and in the presence of poly(propylene imine) (PPI) dendrimers at generation 5 (G5) with a dense shell of maltose and maltotriose units. Thioflavin T (ThT) fluorescence assay and circular dichroism (CD) experiments indicated that fibril formation is enhanced at low dendrimer concentration, while it is prevented at relatively high dendrimer concentrations. Computer aided EPR analysis by means of the selected spin probe 4-octyl-dimethylammonium,2,2,6,6-tetramethyl-piperidine-1-oxyl bromide (CAT8) further demonstrated this behavior, but also provided detailed information on the mechanism of fibril formation and on the different behavior of the differently decorated dendrimers. The CAT8 radicals were progressively trapped at the peptide interphase when peptide aggregates were formed, also monitoring pre-fibrillar structures. At later time, a phase separation of the CAT8 radicals monitors the formation of further supramolecular structures where the probes become squeezed among fibrillar aggregates. The addition of small amounts of dendrimers promotes the formation of peptide fibrils breaking them and providing a larger amount of ends that serve as sites of replications. Conversely, a high amount of dendrimers allows the peptides to well separate from each other such preventing their aggregation. EPR results also indicate that the perturbation played by PPI(G5)-Maltose are more effective onto PrP(185-208) than onto Aß(1-28), while PPI(G5)-Maltotriose is less effective towards PrP(185-208) in both promoting aggregation and preventing it by changing the dendrimer concentration. These results provide useful information about the mechanism and interactions which regulate the ability of macromolecules like the dendrimers to favor, prevent or cure neurodegenerative diseases.