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1.
Bioorg Med Chem ; 91: 117412, 2023 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-37473615

RESUMO

Semiconducting polymer nanoparticles (SPN), formulated from organic semiconducting polymers and lipids, show promise as exogenous contrast agents for photoacoustic imaging (PAI). To fully realise the potential of this class of nanoparticles for imaging and therapeutic applications, a broad range of active targeting strategies, where ligands specific to receptors on the target cells are displayed on the SPN surface, are urgently needed. In addition, effective strategies for quantifying the level of surface modification are also needed to support development of ligand-targeted SPN. In this paper, we have developed methods to prepare SPN bearing peptides targeted to Epidermal Growth Factor Receptors (EGFR), which are overexpressed at the surface of a wide variety of cancer cell types. In addition to fully characterising these targeted nanoparticles by standard methods (UV-visible, photoacoustic absorption, dynamic light scattering, zeta potential and SEM), we have developed a powerful new NMR method to determine the degree of conjugation and the number of targeting peptides attached to the SPN. Preliminary in vitro experiments with the colorectal cancer cell line LIM1215 indicated that the EGFR-targeting peptide conjugated SPN were either ineffective in delivering the SPN to the cells, or that the targeting peptide itself destabilised the formulation. This in reinforces the need for effective characterisation techniques to measure the surface accessibility of targeting ligands attached to nanoparticles.


Assuntos
Nanopartículas , Técnicas Fotoacústicas , Polímeros/química , Receptores ErbB , Técnicas Fotoacústicas/métodos , Ligantes , Nanopartículas/química
2.
Biomater Sci ; 11(9): 3335-3353, 2023 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-36960608

RESUMO

Lipopolyplexes (LPDs) are of considerable interest for use as gene delivery vehicles. Here LPDs have been prepared from cationic vesicles (composed of a 1 : 1 molar ratio of DOTMA with the neutral helper lipid, DOPE), singly branched cationic peptides and plasmid DNA. All peptides contained a linker sequence (cleaved by endosomal furin) attached to a targeting sequence selected to bind human airway epithelial cells and mediate gene delivery. The current study investigates the effects of novel Arg-containing cationic peptide sequences on the biophysical and transfection properties of LPDs. Mixed His/Arg cationic peptides were of particular interest, as these sequences have not been previously used in LPD formulations. Lengthening the number of cationic residues in a homopolymer from 6 to 12 in each branch reduced transfection using LPDs, most likely due to increased DNA compaction hindering the release of pDNA within the target cell. Furthermore, LPDs containing mixed Arg-containing peptides, particularly an alternating Arg/His sequence exhibited an increase in transfection, probably because of their optimal ability to complex and subsequently release pDNA. To confer stability in serum, LPDs were prepared in 0.12 M sodium chloride solution (as opposed to the more commonly used water) yielding multilamellar LPDs with very high levels of size reproducibility and DNA protection, especially when compared to the (unilamellar) LPDs formed in water. Significantly for the clinical applications of the LPDs, those prepared in the presence of sodium chloride retained high levels of transfection in the presence of media supplemented with fetal bovine serum. This work therefore represents a significant advance for the optimisation of LPD formulation for gene delivery, under physiologically relevant conditions, in vivo.


Assuntos
Peptídeos , Cloreto de Sódio , Humanos , Reprodutibilidade dos Testes , Transfecção , Peptídeos/química , DNA/química , Plasmídeos/genética , Lipossomos/química
3.
Nanoscale ; 13(44): 18520-18535, 2021 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-34730152

RESUMO

In vivo delivery of small molecule therapeutics to cancer cells, assessment of the selectivity of administration, and measuring the efficacity of the drug in question at the molecule level, are important ongoing challenges in developing new classes of cancer chemotherapeutics. One approach that has the potential to provide targeted delivery, tracking of biodistribution and readout of efficacy, is to use multimodal theragnostic nanoparticles to deliver the small molecule therapeutic. In this paper, we report the development of targeted theragnostic lipid/peptide/DNA lipopolyplexes. These simultaneously deliver an inhibitor of the EGFR tyrosine kinase, and plasmid DNA coding for a Crk-based biosensor, Picchu-X, which when expressed in the target cells can be used to quantify the inhibition of EGFR in vivo in a mouse colorectal cancer xenograft model. Reversible bioconjugation of a known analogue of the tyrosine kinase inhibitor Mo-IPQA to a cationic peptide, and co-formulation with peptides containing both EGFR-binding and cationic sequences, allowed for good levels of inhibitor encapsulation with targeted delivery to LIM1215 colon cancer cells. Furthermore, high levels of expression of the Picchu-X biosensor in the LIM1215 cells in vivo allowed us to demonstrate, using fluorescence lifetime microscopy (FLIM)-based biosensing, that EGFR activity can be successfully suppressed by the tyrosine kinase inhibitor, released from the lipopolyplexes. Finally, we measured the biodistribution of lipopolyplexes containing 125I-labelled inhibitors and were able to demonstrate that the lipopolyplexes gave significantly higher drug delivery to the tumors compared with free drug.


Assuntos
Técnicas Biossensoriais , Nanopartículas , Preparações Farmacêuticas , Animais , Linhagem Celular Tumoral , Receptores ErbB/genética , Receptores ErbB/metabolismo , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Distribuição Tecidual
4.
J Pept Sci ; 27(10): e3353, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34142414

RESUMO

Helicobacter pylori (H. pylori) infections have been implicated in the development of gastric ulcers and various cancers: however, the success of current therapies is compromised by rising antibiotic resistance. The virulence and pathogenicity of H. pylori is mediated by the type IV secretion system (T4SS), a multiprotein macromolecular nanomachine that transfers toxic bacterial factors and plasmid DNA between bacterial cells, thus contributing to the spread of antibiotic resistance. A key component of the T4SS is the VirB11 ATPase HP0525, which is a hexameric protein assembly. We have previously reported the design and synthesis of a series of novel 8-amino imidazo[1,2-a]pyrazine derivatives as inhibitors of HP0525. In order to improve their selectivity, and potentially develop these compounds as tools for probing the assembly of the HP0525 hexamer, we have explored the design and synthesis of potential bivalent inhibitors. We used the structural details of the subunit-subunit interactions within the HP0525 hexamer to design peptide recognition moieties of the subunit interface. Different methods (cross metathesis, click chemistry, and cysteine-malemide) for bioconjugation to selected 8-amino imidazo[1,2-a]pyrazines were explored, as well as peptides spanning larger or smaller regions of the interface. The IC50 values of the resulting linker-8-amino imidazo[1,2-a]pyrazine derivatives, and the bivalent inhibitors, were related to docking studies with the HP0525 crystal structure and to molecular dynamics simulations of the peptide recognition moieties.


Assuntos
Adenosina Trifosfatases , Helicobacter pylori , Proteínas de Bactérias , Peptídeos/farmacologia , Pirazinas
5.
Bioorg Med Chem ; 28(22): 115740, 2020 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-33007553

RESUMO

Coenzyme A (CoA) is a highly selective inhibitor of the mitotic regulatory enzyme Aurora A kinase, with a novel mode of action. Herein we report the design and synthesis of analogues of CoA as inhibitors of Aurora A kinase. We have designed and synthesised modified CoA structures as potential inhibitors, combining dicarbonyl mimics of the pyrophosphate group with a conserved adenosine headgroup and different length pantetheine-based tail groups. An analogue with a -SH group at the end of the pantotheinate tail showed the best IC50, probably due to the formation of a covalent bond with Aurora A kinase Cys290.


Assuntos
Aurora Quinase A/antagonistas & inibidores , Coenzima A/farmacologia , Difosfatos/farmacologia , Desenho de Fármacos , Panteteína/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Aurora Quinase A/metabolismo , Coenzima A/síntese química , Coenzima A/química , Difosfatos/química , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Estrutura Molecular , Panteteína/química , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade
6.
Nanoscale ; 12(31): 16570-16585, 2020 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-32749427

RESUMO

Stem cells have been utilised as anti-cancer agents due to their ability to home to and integrate within tumours. Methods to augment stem cell homing to tumours are being investigated with the goal of enhancing treatment efficacy. However, it is currently not possible to evaluate both cell localisation and cell viability after engraftment, hindering optimisation of therapy. In this study, luciferase-expressing human adipocyte-derived stem cells (ADSCs) were incubated with Indium-111 radiolabelled iron oxide nanoparticles to produce cells with tri-modal imaging capabilities. ADSCs were administered intravenously (IV) or intracardially (IC) to mice bearing orthotopic breast tumours. Cell fate was monitored using bioluminescence imaging (BLI) as a measure of cell viability, magnetic resonance imaging (MRI) for cell localisation and single photon emission computer tomography (SPECT) for cell quantification. Serial monitoring with multi-modal imaging showed the presence of viable ADSCs within tumours as early as 1-hour post IC injection and the percentage of ADSCs within tumours to be 2-fold higher after IC than IV. Finally, histological analysis was used to validate engraftment of ADSC within tumour tissue. These findings demonstrate that multi-modal imaging can be used to evaluate the efficiency of stem cell delivery to tumours and that IC cell administration is more effective for tumour targeting.


Assuntos
Neoplasias Mamárias Experimentais/terapia , Transplante de Células-Tronco Mesenquimais/métodos , Imagem Multimodal/métodos , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Proliferação de Células , Sobrevivência Celular , Rastreamento de Células , Sistemas de Liberação de Medicamentos , Feminino , Humanos , Radioisótopos de Índio/administração & dosagem , Radioisótopos de Índio/química , Luciferases/genética , Luciferases/metabolismo , Nanopartículas Magnéticas de Óxido de Ferro/administração & dosagem , Nanopartículas Magnéticas de Óxido de Ferro/química , Neoplasias Mamárias Experimentais/diagnóstico por imagem , Neoplasias Mamárias Experimentais/patologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos
7.
Front Chem ; 8: 228, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32309273

RESUMO

Peptide toxins that adopt the inhibitory cystine knot (ICK) scaffold have very stable three-dimensional structures as a result of the conformational constraints imposed by the configuration of the three disulfide bonds that are the hallmark of this fold. Understanding the oxidative folding pathways of these complex peptides, many of which are important therapeutic leads, is important in order to devise reliable synthetic routes to correctly folded, biologically active peptides. Previous research on the ICK peptide ProTx-II has shown that in the absence of an equilibrating redox buffer, misfolded intermediates form that prevent the formation of the native disulfide bond configuration. In this paper, we used tandem mass spectrometry to examine these misfolded peptides, and identified two non-native singly bridged peptides, one with a Cys(III)-Cys(IV) linkage and one with a Cys(V)-Cys(VI) linkage. Based on these results, we propose that the C-terminus of ProTx-II has an important role in initiating the folding of this peptide. To test this hypothesis, we have also studied the folding pathways of analogs of ProTx-II containing the disulfide-bond directing group penicillamine (Pen) under the same conditions. We find that placing Pen residues at the C-terminus of the ProTx-II analogs directs the folding pathway away from the singly bridged misfolded intermediates that represent a kinetic trap for the native sequence, and allows a fully oxidized final product to be formed with three disulfide bridges. However, multiple two-disulfide peptides were also produced, indicating that further study is required to fully control the folding pathways of this modified scaffold.

8.
Redox Biol ; 28: 101318, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31546169

RESUMO

Aurora A kinase is a master mitotic regulator whose functions are controlled by several regulatory interactions and post-translational modifications. It is frequently dysregulated in cancer, making Aurora A inhibition a very attractive antitumor target. However, recently uncovered links between Aurora A, cellular metabolism and redox regulation are not well understood. In this study, we report a novel mechanism of Aurora A regulation in the cellular response to oxidative stress through CoAlation. A combination of biochemical, biophysical, crystallographic and cell biology approaches revealed a new and, to our knowledge, unique mode of Aurora A inhibition by CoA, involving selective binding of the ADP moiety of CoA to the ATP binding pocket and covalent modification of Cys290 in the activation loop by the thiol group of the pantetheine tail. We provide evidence that covalent CoA modification (CoAlation) of Aurora A is specific, and that it can be induced by oxidative stress in human cells. Oxidising agents, such as diamide, hydrogen peroxide and menadione were found to induce Thr 288 phosphorylation and DTT-dependent dimerization of Aurora A. Moreover, microinjection of CoA into fertilized mouse embryos disrupts bipolar spindle formation and the alignment of chromosomes, consistent with Aurora A inhibition. Altogether, our data reveal CoA as a new, rather selective, inhibitor of Aurora A, which locks this kinase in an inactive state via a "dual anchor" mechanism of inhibition that might also operate in cellular response to oxidative stress. Finally and most importantly, we believe that these novel findings provide a new rationale for developing effective and irreversible inhibitors of Aurora A, and perhaps other protein kinases containing appropriately conserved Cys residues.


Assuntos
Aurora Quinase A/química , Aurora Quinase A/metabolismo , Coenzima A/administração & dosagem , Animais , Coenzima A/química , Coenzima A/farmacologia , Cristalografia por Raios X , Células HEK293 , Células Hep G2 , Humanos , Camundongos , Modelos Moleculares , Estresse Oxidativo , Fosforilação , Conformação Proteica , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/metabolismo
9.
Chemistry ; 25(64): 14572-14582, 2019 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-31599485

RESUMO

Natural products that target lipid II, such as the lantibiotic nisin, are strategically important in the development of new antibacterial agents to combat the rise of antimicrobial resistance. Understanding the structural factors that govern the highly selective molecular recognition of lipid II by the N-terminal region of nisin, nisin(1-12), is a crucial step in exploiting the potential of such compounds. In order to elucidate the relationships between amino acid sequence and conformation of this bicyclic peptide fragment, we have used solid-phase peptide synthesis to prepare two novel analogues of nisin(1-12) in which the dehydro residues have been replaced. We have carried out an NMR ensemble analysis of one of these analogues and of the wild-type nisin(1-12) peptide in order to compare the conformations of these two bicyclic peptides. Our analysis has shown the effects of residue mutation on ring conformation. We have also demonstrated that the individual rings of nisin(1-12) are pre-organised to an extent for binding to the pyrophosphate group of lipid II, with a high degree of flexibility exhibited in the central amide bond joining the two rings.


Assuntos
Nisina/análogos & derivados , Peptídeos/síntese química , Uridina Difosfato Ácido N-Acetilmurâmico/análogos & derivados , Sequência de Aminoácidos , Ligação de Hidrogênio , Nisina/metabolismo , Ressonância Magnética Nuclear Biomolecular , Peptídeos/química , Peptídeos/metabolismo , Conformação Proteica , Uridina Difosfato Ácido N-Acetilmurâmico/química , Uridina Difosfato Ácido N-Acetilmurâmico/metabolismo
10.
J Phys Chem B ; 123(35): 7545-7557, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31449408

RESUMO

Exogenous chemical exchange saturation transfer (CEST) contrast agents such as glucose or 2-deoxy-d-glucose (2-DG) have shown high sensitivities and significant potential for monitoring glucose uptake in tumors with MRI. Here, we show that liposome encapsulation of such agents can be exploited to enhance the CEST signal by reducing the overall apparent exchange rate. We have developed a concise analytical model to describe the liposomal contrast dependence on several parameters such as pH, temperature, irradiation amplitude, and intraliposomal water content. This is the first study in which a model has been constructed to measure the exchange properties of diamagnetic CEST agents encapsulated inside liposomes. Experimentally measured exchange rates of glucose and 2-DG in the liposomal system were found to be reduced due to the intermembrane exchange between the intra- and extraliposomal compartments because of restrictions in water transfer imposed by the lipid membrane. These new theoretical and experimental findings will benefit applications of diamagnetic liposomes to image biological processes. In addition, combining this analytical model with measurements of the CEST signal enhancement using liposomes as a model membrane system is an important new general technique for studying membrane permeability.


Assuntos
Meios de Contraste/química , Desoxiglucose/química , Glucose/química , Lipossomos/química
11.
J Pept Sci ; 24(12): e3131, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30325562

RESUMO

The design, synthesis and formulation of non-viral gene delivery vectors is an area of renewed research interest. Amongst the most efficient non-viral gene delivery systems are lipopolyplexes, in which cationic peptides are co-formulated with plasmid DNA and lipids. One advantage of lipopolyplex vectors is that they have the potential to be targeted to specific cell types by attaching peptide targeting ligands on the surface, thus increasing both the transfection efficiency and selectivity for disease targets such as cancer cells. In this paper, we have investigated two different modes of displaying cell-specific peptide targeting ligands at the surface of lipopolyplexes. Lipopolyplexes formulated with bimodal peptides, with both receptor binding and DNA condensing sequences, were compared with lipopolyplexes with the peptide targeting ligand directly conjugated to one of the lipids. Three EGFR targeting peptide sequences were studied, together with a range of lipid formulations and maleimide lipid structures. The biophysical properties of the lipopolyplexes and their transfection efficiencies in a basal-like breast cancer cell line were investigated using plasmid DNA bearing genes for the expression of firefly luciferase and green fluorescent protein. Fluorescence quenching experiments were also used to probe the macromolecular organisation of the peptide and pDNA components of the lipopolyplexes. We demonstrated that both approaches to lipopolyplex targeting give reasonable transfection efficiencies, and the transfection efficiency of each lipopolyplex formulation is highly dependent on the sequence of the targeting peptide. To achieve maximum therapeutic efficiency, different peptide targeting sequences and lipopolyplex architectures should be investigated for each target cell type.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Neoplasias da Mama/terapia , DNA/química , Técnicas de Transferência de Genes , Lipídeos/química , Peptídeos Catiônicos Antimicrobianos/síntese química , Neoplasias da Mama/metabolismo , DNA/genética , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Feminino , Vetores Genéticos/química , Vetores Genéticos/genética , Humanos , Ligantes , Plasmídeos/química , Conformação Proteica , Propriedades de Superfície , Transfecção
12.
J Am Chem Soc ; 139(37): 13063-13075, 2017 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-28880078

RESUMO

Spider venom toxins, such as Protoxin-II (ProTx-II), have recently received much attention as selective Nav1.7 channel blockers, with potential to be developed as leads for the treatment of chronic nocioceptive pain. ProTx-II is a 30-amino acid peptide with three disulfide bonds that has been reported to adopt a well-defined inhibitory cystine knot (ICK) scaffold structure. Potential drawbacks with such peptides include poor pharmacodynamics and potential scrambling of the disulfide bonds in vivo. In order to address these issues, in the present study we report the solid-phase synthesis of lanthionine-bridged analogues of ProTx-II, in which one of the three disulfide bridges is replaced with a thioether linkage, and evaluate the biological properties of these analogues. We have also investigated the folding and disulfide bridging patterns arising from different methods of oxidation of the linear peptide precursor. Finally, we report the X-ray crystal structure of ProTx-II to atomic resolution; to our knowledge this is the first crystal structure of an ICK spider venom peptide not bound to a substrate.


Assuntos
Dissulfetos/farmacologia , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Peptídeos/farmacologia , Venenos de Aranha/farmacologia , Aranhas/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Animais , Cristalografia por Raios X , Dissulfetos/química , Modelos Moleculares , Conformação Molecular/efeitos dos fármacos , Peptídeos/química , Venenos de Aranha/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/química
13.
Bioconjug Chem ; 28(6): 1734-1740, 2017 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-28561568

RESUMO

Photoacoustic imaging combines both excellent spatial resolution with high contrast and specificity, without the need for patients to be exposed to ionizing radiation. This makes it ideal for the study of physiological changes occurring during tumorigenesis and cardiovascular disease. In order to fully exploit the potential of this technique, new exogenous contrast agents with strong absorbance in the near-infrared range, good stability and biocompatibility, are required. In this paper, we report the formulation and characterization of a novel series of endogenous contrast agents for photoacoustic imaging in vivo. These contrast agents are based on a recently reported series of indigoid π-conjugated organic semiconductors, coformulated with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, to give semiconducting polymer nanoparticles of about 150 nm diameter. These nanoparticles exhibited excellent absorption in the near-infrared region, with good photoacoustic signal generation efficiencies, high photostability, and extinction coefficients of up to three times higher than those previously reported. The absorption maximum is conveniently located in the spectral region of low absorption of chromophores within human tissue. Using the most promising semiconducting polymer nanoparticle, we have demonstrated wavelength-dependent differential contrast between vasculature and the nanoparticles, which can be used to unambiguously discriminate the presence of the contrast agent in vivo.


Assuntos
Meios de Contraste/química , Imagem Molecular/métodos , Nanopartículas/química , Técnicas Fotoacústicas/métodos , Vasos Sanguíneos/diagnóstico por imagem , Humanos , Polímeros/química , Semicondutores , Espectroscopia de Luz Próxima ao Infravermelho
14.
Mol Biosyst ; 12(3): 934-51, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26794416

RESUMO

Ternary nanocomplexes, composed of bifunctional cationic peptides, lipids and siRNA, as delivery vehicles for siRNA have been investigated. The study is the first to determine the optimal sequence and architecture of the bifunctional cationic peptide used for siRNA packaging and delivery using lipopolyplexes. Specifically three series of cationic peptides of differing sequence, degrees of branching and cell-targeting sequences were co-formulated with siRNA and vesicles prepared from a 1 : 1 molar ratio of the cationic lipid DOTMA and the helper lipid, DOPE. The level of siRNA knockdown achieved in the human alveolar cell line, A549-luc cells, in both reduced serum and in serum supplemented media was evaluated, and the results correlated to the nanocomplex structure (established using a range of physico-chemical tools, namely small angle neutron scattering, transmission electron microscopy, dynamic light scattering and zeta potential measurement); the conformational properties of each component (circular dichroism); the degree of protection of the siRNA in the lipopolyplex (using gel shift assays) and to the cellular uptake, localisation and toxicity of the nanocomplexes (confocal microscopy). Although the size, charge, structure and stability of the various lipopolyplexes were broadly similar, it was clear that lipopolyplexes formulated from branched peptides containing His-Lys sequences perform best as siRNA delivery agents in serum, with protection of the siRNA in serum balanced against efficient release of the siRNA into the cytoplasm of the cell.


Assuntos
Técnicas de Transferência de Genes , Peptídeos/química , RNA Interferente Pequeno/metabolismo , Sequência de Aminoácidos , Cátions , Linhagem Celular Tumoral , Dicroísmo Circular , Difusão Dinâmica da Luz , Ensaio de Desvio de Mobilidade Eletroforética , Fluorescência , Técnicas de Silenciamento de Genes , Humanos , Lipossomos/metabolismo , Lipossomos/ultraestrutura , Microscopia Confocal , Dados de Sequência Molecular , Difração de Nêutrons , Peptídeos/metabolismo , Espalhamento a Baixo Ângulo , Soro/metabolismo , Eletricidade Estática
15.
Biomaterials ; 34(36): 9190-200, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23948162

RESUMO

Convection enhanced delivery (CED) is a method of direct injection to the brain that can achieve widespread dispersal of therapeutics, including gene therapies, from a single dose. Non-viral, nanocomplexes are of interest as vectors for gene therapy in the brain, but it is essential that administration should achieve maximal dispersal to minimise the number of injections required. We hypothesised that anionic nanocomplexes administered by CED should disperse more widely in rat brains than cationics of similar size, which bind electrostatically to cell-surface anionic moieties such as proteoglycans, limiting their spread. Anionic, receptor-targeted nanocomplexes (RTN) containing a neurotensin-targeting peptide were prepared with plasmid DNA and compared with cationic RTNs for dispersal and transfection efficiency. Both RTNs were labelled with gadolinium for localisation in the brain by MRI and in brain sections by LA-ICP-MS, as well as with rhodamine fluorophore for detection by fluorescence microscopy. MRI distribution studies confirmed that the anionic RTNs dispersed more widely than cationic RTNs, particularly in the corpus callosum. Gene expression levels from anionic formulations were similar to those of cationic RTNs. Thus, anionic RTN formulations can achieve both widespread dispersal and effective gene expression in brains after administration of a single dose by CED.


Assuntos
Encéfalo/metabolismo , Técnicas de Transferência de Genes , Nanopartículas/química , Ácidos Nucleicos/uso terapêutico , Receptores de Superfície Celular/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Linhagem Celular Tumoral , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Lipossomos/química , Imageamento por Ressonância Magnética , Masculino , Camundongos , Nanosferas , Ácidos Nucleicos/farmacologia , Peptídeos/metabolismo , Plasmídeos/metabolismo , Ratos , Ratos Wistar , Espectrofotometria Atômica , Distribuição Tecidual/efeitos dos fármacos , Transfecção
16.
Biomaterials ; 34(4): 1179-92, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23131536

RESUMO

A series of metal-chelating lipid conjugates has been designed and synthesized. Each member of the series bears a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) macrocycle attached to the lipid head group, using short n-ethylene glycol (n-EG) spacers of varying length. Liposomes incorporating these lipids, chelated to Gd(3+), (64)Cu(2+), or (111)In(3+), and also incorporating fluorescent lipids, have been prepared, and their application in optical, magnetic resonance (MR) and single-photon emission tomography (SPECT) imaging of cellular uptake and distribution investigated in vitro and in vivo. We have shown that these multimodal liposomes can be used as functional MR contrast agents as well as radionuclide tracers for SPECT, and that they can be optimized for each application. When shielded liposomes were formulated incorporating 50% of a lipid with a short n-EG spacer, to give nanoparticles with a shallow but even coverage of n-EG, they showed good cellular internalization in a range of tumour cells, compared to the limited cellular uptake of conventional shielded liposomes formulated with 7% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethyleneglycol)(2000)] (DSPE-PEG2000). Moreover, by matching the depth of n-EG coverage to the length of the n-EG spacers of the DOTA lipids, we have shown that similar distributions and blood half lives to DSPE-PEG2000-stabilized liposomes can be achieved. The ability to tune the imaging properties and distribution of these liposomes allows for the future development of a flexible tri-modal imaging agent.


Assuntos
Meios de Contraste/síntese química , Lipossomos , Imageamento por Ressonância Magnética/métodos , Microscopia de Fluorescência/métodos , Nanocápsulas/química , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Lipossomos/química , Técnica de Subtração
17.
Mol Pharm ; 10(1): 127-41, 2013 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-23210981

RESUMO

Cationic peptide sequences, whether linear, branched, or dendritic, are widely used to condense and protect DNA in both polyplex and lipopolyplex gene delivery vectors. How these peptides behave within these particles and the consequences this has on transfection efficiency remain poorly understood. We have compared, in parallel, a complete series of cationic peptides, both branched and linear, coformulated with plasmid DNA to give polyplexes, or with plasmid DNA and the cationic lipid, DOTMA, mixed with 50% of the neutral helper lipid, DOPE, to give lipopolyplexes, and correlated the transfection efficiencies of these complexes to their biophysical properties. Lipopolyplexes formulated from branched Arg-rich peptides, or linear Lys-rich peptides, show the best transfection efficiencies in an alveolar epithelial cell line, with His-rich peptides being relatively ineffective. The majority of the biophysical studies (circular dichroism, dynamic light scattering, zeta potential, small angle neutron scattering, and gel band shift assay) indicated that all of the formulations were similar in size, surface charge, and lipid bilayer structure, and longer cationic sequences, in general, gave better transfection efficiencies. Whereas lipopolyplexes formulated from branched Arg-containing peptides were more effective than those formulated from linear Arg-containing sequences, the reverse was true for Lys-containing sequences, which may be related to differences in DNA condensation between Arg-rich and Lys-rich peptides observed in the CD studies.


Assuntos
Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Lipídeos/administração & dosagem , Lipídeos/genética , Peptídeos/administração & dosagem , Peptídeos/genética , Cátions/administração & dosagem , Cátions/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Química Farmacêutica/métodos , Dicroísmo Circular/métodos , DNA/administração & dosagem , DNA/química , DNA/genética , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Técnicas de Transferência de Genes , Vetores Genéticos/química , Humanos , Lipídeos/química , Tamanho da Partícula , Peptídeos/química , Plasmídeos/administração & dosagem , Plasmídeos/química , Plasmídeos/genética , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/metabolismo , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/metabolismo , Transfecção/métodos
18.
Biomaterials ; 33(29): 7241-50, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22809644

RESUMO

The efficient targeted delivery of nucleic acids in vivo provides some of the greatest challenges to the development of genetic therapies. We aim to develop nanocomplex formulations that achieve targeted transfection of neuroblastoma tumours that can be monitored simultaneously by MRI. Here, we have compared nanocomplexes comprising self-assembling mixtures of liposomes, plasmid DNA and one of three different peptide ligands derived from ApoE, neurotensin and tetanus toxin for targeted transfection in vitro and in vivo. Neurotensin-targeted nanocomplexes produced the highest levels of transfection and showed a 4.7-fold increase in transfected luciferase expression over non-targeted nanocomplexes in Neuro-2A cells. Transfection of subcutaneous Neuro-2A tumours in vivo with neurotensin-targeted nanocomplexes produced a 9.3-fold increase in gene expression over non-targeted controls. Confocal microscopy analysis elucidated the time course of DNA delivery with fluorescently labelled nanocomplex formulations in cells. It was confirmed that addition of a gadolinium lipid conjugate contrast agent allowed real time in vivo monitoring of nanocomplex localisation in tumours by MRI, which was maintained for at least 24 h. The peptide-targeted nanocomplexes developed here allow for the specific enhancement of targeted gene therapy both in vitro and in vivo, whilst allowing real time monitoring of delivery with MRI.


Assuntos
Técnicas de Transferência de Genes , Imageamento por Ressonância Magnética/métodos , Nanopartículas/química , Neoplasias/terapia , Animais , Linhagem Celular Tumoral , Meios de Contraste/farmacologia , Feminino , Gadolínio/química , Terapia Genética/métodos , Cinética , Ligantes , Lipossomos/química , Luciferases/metabolismo , Camundongos , Modelos Químicos , Nanotecnologia/métodos , Transplante de Neoplasias , Neoplasias/patologia , Neurotensina/química , Peptídeos/química , Toxina Tetânica/química , Transfecção
19.
J Alzheimers Dis ; 32(1): 43-56, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22751177

RESUMO

Enzymatic degradation contributes to the control of intracerebral amyloid-ß (Aß) peptide levels. Previous studies have demonstrated the therapeutic potential of viral vector-mediated neprilysin (NEP) gene therapy in mouse models of Alzheimer's disease (AD). However, clinical translation of NEP gene therapy is limited by ethical and practical considerations. In this study we have assessed the potential of convection-enhanced delivery (CED) as a means of elevating intracerebral NEP level and activity and degrading endogenous Aß. We analyzed the interstitial and perivascular distribution of NEP following CED into rat striatum. We measured NEP protein level, clearance, activity, and toxicity by ELISA for NEP and synaptophysin, NEP-specific activity assay, and immunohistochemistry for NEP, NeuN, glial fibrillary acidic protein and Iba1. We subsequently performed CED of NEP in normal aged rats and measured endogenous Aß by ELISA. CED resulted in widespread distribution of NEP, and a 20-fold elevation of NEP protein level with preservation of enzyme activity and without evidence of toxicity. CED in normal, aged rats resulted in a significant reduction in endogenous Aß(40) (p = 0.04), despite rapid NEP clearance from the brain (half-life ~3 h). CED of NEP has therapeutic potential as a dynamically controllable Aß(40)-degrading therapeutic strategy for AD. Further studies are required to determine the longer term effects on Aß (including Aß(42)) and on cognitive function.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/metabolismo , Neprilisina/administração & dosagem , Neprilisina/uso terapêutico , Envelhecimento/fisiologia , Doença de Alzheimer/metabolismo , Animais , Antígenos Nucleares/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Proteínas de Ligação ao Cálcio/metabolismo , Cateterismo , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos , Ensaio de Imunoadsorção Enzimática , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica , Masculino , Proteínas dos Microfilamentos/metabolismo , Neprilisina/farmacocinética , Proteínas do Tecido Nervoso/metabolismo , Neuroimagem , Nimodipina/farmacologia , Veículos Farmacêuticos , Polietilenoglicóis , Ratos , Ratos Wistar , Sinaptofisina/metabolismo
20.
PLoS One ; 6(10): e26768, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22046351

RESUMO

BACKGROUND: Gene therapy mediated by synthetic vectors may provide opportunities for new treatments for cystic fibrosis (CF) via aerosolisation. Vectors for CF must transfect the airway epithelium efficiently and not cause inflammation so they are suitable for repeated dosing. The inhaled aerosol should be deposited in the airways since the cystic fibrosis transmembrane conductance regulator gene (CFTR) is expressed predominantly in the epithelium of the submucosal glands and in the surface airway epithelium. The aim of this project was to develop an optimised aerosol delivery approach applicable to treatment of CF lung disease by gene therapy. METHODOLOGY: The vector suspension investigated in this study comprises receptor-targeting peptides, cationic liposomes and plasmid DNA that self-assemble by electrostatic interactions to form a receptor-targeted nanocomplex (RTN) of approximately 150 nm with a cationic surface charge of +50 mV. The aerodynamic properties of aerosolised nanocomplexes produced with three different nebulisers were compared by determining aerosol deposition in the different stages of a Next Generation Pharmaceutical Impactor (NGI). We also investigated the yield of intact plasmid DNA by agarose gel electrophoresis and densitometry, and transfection efficacies in vitro and in vivo. RESULTS: RTNs nebulised with the AeroEclipse II BAN were the most effective, compared to other nebulisers tested, for gene delivery both in vitro and in vivo. The biophysical properties of the nanocomplexes were unchanged after nebulisation while the deposition of RTNs suggested a range of aerosol aerodynamic sizes between 5.5 µm-1.4 µm cut off (NGI stages 3-6) compatible with deposition in the central and lower airways. CONCLUSIONS: RTNs showed their ability at delivering genes via nebulisation, thus suggesting their potential applications for therapeutic interventions of cystic fibrosis and other respiratory disorders.


Assuntos
Técnicas de Transferência de Genes , Terapia Genética/métodos , Nebulizadores e Vaporizadores , Mucosa Respiratória/metabolismo , Fibrose Cística/terapia , Epitélio/metabolismo , Terapia de Alvo Molecular , Nanocompostos/administração & dosagem , Plasmídeos/administração & dosagem , Transfecção
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