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1.
Transl Res ; 224: 40-54, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32522668

RESUMO

The modulation of voltage-gated K+ (Kv) channels, involved in cell proliferation, arises as a potential therapeutic approach for the prevention of intimal hyperplasia present in in-stent restenosis (ISR) and allograft vasculopathy (AV). We studied the effect of PAP-1, a selective blocker of Kv1.3 channels, on development of intimal hyperplasia in vitro and in vivo in 2 porcine models of vascular injury. In vitro phenotypic modulation of VSMCs was associated to an increased functional expression of Kv1.3 channels, and only selective Kv1.3 channel blockers were able to inhibit porcine VSMC proliferation. The therapeutic potential of PAP-1 was then evaluated in vivo in swine models of ISR and AV. At 15-days follow-up, morphometric analysis demonstrated a substantial reduction of luminal stenosis in the allografts treated with PAP-1 (autograft 2.72 ± 1.79 vs allograft 10.32 ± 1.92 vs allograft + polymer 13.54 ± 8.59 vs allograft + polymer + PAP-1 3.06 ± 1.08 % of luminal stenosis; P = 0.006) in the swine model of femoral artery transplant. In the pig model of coronary ISR, using a prototype of PAP-1-eluting stent, no differences were observed regarding % of stenosis compared to control stents (31 ± 13 % vs 37 ± 18%, respectively; P = 0.372) at 28-days follow-up. PAP-1 treatment was safe and did not impair vascular healing in terms of delayed endothelialization, inflammation or thrombosis. However, an incomplete release of PAP-1 from stents was documented. We conclude that the use of selective Kv1.3 blockers represents a promising therapeutic approach for the prevention of intimal hyperplasia in AV, although further studies to improve their delivery method are needed to elucidate its potential in ISR.


Assuntos
Canal de Potássio Kv1.3/antagonistas & inibidores , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Bloqueadores dos Canais de Potássio/farmacologia , Túnica Íntima/patologia , Aloenxertos/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Reestenose Coronária/patologia , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/lesões , Vasos Coronários/patologia , Modelos Animais de Doenças , Feminino , Artéria Femoral/efeitos dos fármacos , Artéria Femoral/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hiperplasia , Canal de Potássio Kv1.3/genética , Canal de Potássio Kv1.3/metabolismo , Canal de Potássio Kv1.5/genética , Canal de Potássio Kv1.5/metabolismo , Modelos Biológicos , Miócitos de Músculo Liso/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Stents , Suínos , Túnica Íntima/efeitos dos fármacos
2.
Adv Healthc Mater ; 7(17): e1800335, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29923337

RESUMO

The encapsulation of mRNA in nanosystems as gene vaccines for immunotherapy purposes has experienced an exponential increase in recent years. Despite the many advantages envisaged within these approaches, their application in clinical treatments is still limited due to safety issues. These issues can be attributed, in part, to liver accumulation of most of the designed nanosystems and to the inability to transfect immune cells after an intravenous administration. In this context, this study takes advantage of the known versatile properties of the oligopeptide end-modified poly (ß-amino esters) (OM-PBAEs) to complex mRNA and form discrete nanoparticles. Importantly, it is demonstrated that the selection of the appropriate end-oligopeptide modifications enables the specific targeting and major transfection of antigen-presenting cells (APC) in vivo, after intravenous administration, thus enabling their use for immunotherapy strategies. Therefore, with this study, it can be confirmed that OM-PBAE are appropriate systems for the design of mRNA-based immunotherapy approaches aimed to in vivo transfect APCs and trigger immune responses to fight either tumors or infectious diseases.


Assuntos
Células Apresentadoras de Antígenos/metabolismo , RNA Mensageiro/administração & dosagem , RNA Mensageiro/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular , Portadores de Fármacos/química , Células HeLa , Humanos , Imunoterapia , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/química , Polímeros/química , Células RAW 264.7
3.
Mater Sci Eng C Mater Biol Appl ; 76: 295-300, 2017 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-28482530

RESUMO

In this paper, we report the modification of polypirrole (PPy) with dopaminated hyaluronic acid (HADA). This design improves PPy adhesion onto stretchable materials such as poly(dimethylsiloxane) (PDMS) allowing the formation of conducting films on this kind of very flexible, hydrophobic materials. The results revealed that described PPy modification allows to obtain stable PPy:HADA nano-suspension able to cast films directly on PDMS. The comparison of PPy:HADA films with conventional PPy and other modified PPy shows that the modification improved the strength of the films under tension stress and their water resistance. Moreover, the modification proposed does not affect significantly the conductivity of the PPy films. The resulting properties of the material make it especially suitable for bio-integrated device applications, where a biocompatible material with stable electrical behaviour under deformation and water media is needed.


Assuntos
Polímeros/química , Pirróis/química , Condutividade Elétrica , Ácido Hialurônico
4.
ACS Appl Mater Interfaces ; 9(13): 11461-11471, 2017 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-28299925

RESUMO

Gene therapy has arisen as a pioneering technique to treat diseases by direct employment of nucleic acids as medicine. The major historical problem is to develop efficient and safe systems for the delivery of therapeutic genes into the target cells. Carbon nanotubes (CNTs) have demonstrated considerable promise as delivery vectors due to their (i) high aspect ratio and (ii) capacity to translocate through plasma membranes, known as the nanoneedle effect. To leverage these advantages, close attention needs to be paid to the physicochemical characteristics of the CNTs used. CNTs with different diameters (thinner and thicker) were treated by chemical oxidation to produce shorter fragments. Rigid (thick) and flexible (thin) CNTs, and their shortened versions, were coated with polyallylamine (ppAA) by plasma-enhanced chemical vapor deposition. The ppAA coating leads to a positively charged CNT surface that is able to electrostatically bind the green fluorescent protein plasmid reporter. This study shows how rigidity and length can affect their (i) behavior in biological media, (ii) ability to transfect in vitro, and (iii) biodistribution in vivo. This study also generates a set of basic design rules for the development of more efficient CNT-based gene-delivery vectors.


Assuntos
Nanotubos de Carbono , Técnicas de Transferência de Genes , Terapia Genética , Plasmídeos , Distribuição Tecidual
5.
Colloids Surf B Biointerfaces ; 133: 339-46, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26125487

RESUMO

Designing nanocarriers for gene delivery is a multidisciplinary challenge that involves not only DNA condensation with biocompatible polymers, but also DNA-release processes. Once the genetic material is introduced into the cell, the rupture of degradable bonds permits the unpacking and release of the load. In this work, a dual-degradable polycation - composed by a linear poly(ß-amino ester) chain in which ester and disulfide bonds coexist - has been used to condense a DNA plasmid. The goal was to reinforce the spontaneous hydrolysis of the ester groups with the intracellular break-up of the disulfide bonds, since these reducible bonds are degraded in the reductive intracellular environment. For a comparative study, two poly(ß-amino ester) molecules differing only in the presence (or absence) of some SS bonds have been tested. DNA condensation, physico-chemical characterization of the polyplexes formed, and degradation studies have been carried out at pH 5 and pH 7. The acidic conditions gave the best nanoparticles, due to a better solubilization of both polymers and to a higher stability of the ester bonds. Despite the synthesis and storage of polyplexes were much more appropriate at pH 5, transfection efficiency in HeLa cells was similar irrespective the original pH used. Only in those polyplexes formed at low polymer:DNA ratios (i.e. 5 and 10 (w/w)) was transfection more effective when the plasmid was condensed at an acidic pH. With regard to the DNA-release efficiency in the intracellular medium, degradation of the polymers was practically governed by the rapid hydrolysis of the ester groups, this spontaneous and rapid process masking, unfortunately, any potential contribution associated with the breakup of the disulfide bonds.


Assuntos
DNA/química , Polímeros/química , Ensaio de Desvio de Mobilidade Eletroforética , Células HeLa , Humanos
6.
Colloids Surf B Biointerfaces ; 126: 374-80, 2015 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-25601090

RESUMO

Two degradable poly(ß-amino ester)s with an average molecular weight of 2kDa, referred to as B1 and B2, have been synthesized to be tested as non-viral gene delivery systems. B2 polymer exhibits two additional non-polar ethyl groups at both ends. This paper describes the influence of that subtle difference on the compaction ability and temporal stability of the complexes formed with plasmid DNA. Our results suggest that the inclusion of those small hydrophobic fragments into the polycation backbone improves its suitability as synthetic DNA carrier. The improvement is related to the formation and physicochemical properties of the complexes. B2 polyplexes were more stable, the polymer hydrolysis was slowed down and plasmid DNA was better protected which was translated into better transfection efficiencies. Although still not totally understood, the role played by hydrophobic forces is ubiquitous in chemical, biological and physical systems, and they must be considered to design future polymers for gene delivery.


Assuntos
DNA/química , Interações Hidrofóbicas e Hidrofílicas , Polímeros/química , Coloides/química , Ensaio de Desvio de Mobilidade Eletroforética , Células HeLa , Humanos , Plasmídeos , Polímeros/síntese química , Células Tumorais Cultivadas
7.
Methods Mol Biol ; 1025: 261-8, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23918344

RESUMO

The surface modification of carbon nanotubes (CNTs) can be tailored to allow the formation of a complex between these potential carriers with DNA. In this chapter, protocols developed in our lab to prepare transfection vectors through the modification of MWCNTs are described. The protocol includes sections focused on the reduction of CNTs length, protocols to increase the dispersability of CNTs and finally, protocols for surface modification to attach through electrostatic interactions DNA to the CNTs.


Assuntos
DNA/química , Técnicas de Transferência de Genes/instrumentação , Vetores Genéticos , Nanotecnologia/instrumentação , Nanotubos de Carbono/química , DNA/genética , Técnicas de Transferência de Genes/tendências , Nanotecnologia/métodos , Plasma/química , Polimerização , Transfecção
8.
J Control Release ; 130(1): 46-56, 2008 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-18571758

RESUMO

Histidine containing reducible polycations based on CH(6)K(3)H(6)C monomers (His6 RPCs), are highly effective DNA transfection agents combining pH buffering endosomal escape mechanisms with rapid unpackaging following reduction in the cytoplasm. We examined their ability to mediate siRNA uptake into cells focusing on hepatocyte delivery. Co-delivery of EGFP siRNA with pEGFP plasmid DNA reduced reporter gene expression by 85%. However while DNA transfection efficiency increased with polymer size, with 162 k His6 RPCs proving the most effective, delivery of siRNA alone to EGFP stably expressing cells was only possible using 36-80 k polymers. Analysis of particle sizes showed that 80 k polymers formed more compact siRNA complexes than 162 k polymers. The reducible nature of the polymer was necessary for siRNA activity, since siRNA combined with non-reducible polylysine showed little activity. Incorporation of a targeting peptide from the Plasmodium falciparum circumsporozoite (CS) protein onto His6 RPCs, significantly improved transfection of plasmid DNA and siRNA activity in hepatocytes, but not in most non-liver cells tested. siRNA targeted to the hepatitis B virus surface antigen delivered by CS-His6 RPC, mediated falls in both mRNA and protein expression, suggesting that this delivery system could be developed for potential therapies for viral hepatitis.


Assuntos
DNA/administração & dosagem , Técnicas de Transferência de Genes , Histidina/química , Poliaminas/química , RNA Interferente Pequeno/administração & dosagem , Animais , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Citomegalovirus/genética , DNA/genética , Portadores de Fármacos/química , Ensaio de Imunoadsorção Enzimática , Genes Reporter , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Antígenos de Superfície da Hepatite B/genética , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Luciferases/genética , Camundongos , Peso Molecular , Plasmodium falciparum , Polieletrólitos , Proteínas de Protozoários/química , Interferência de RNA , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção
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