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1.
Acta Crystallogr D Struct Biol ; 79(Pt 2): 177-187, 2023 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-36762863

RESUMO

During the initiation step of bacterial genome replication, replicative helicases depend on specialized proteins for their loading onto oriC. DnaC and DnaI were the first loaders to be characterized. However, most bacteria do not contain any of these genes, which are domesticated phage elements that have replaced the ancestral and unrelated loader gene dciA several times during evolution. To understand how DciA assists the loading of DnaB, the crystal structure of the complex from Vibrio cholerae was determined, in which two VcDciA molecules interact with a dimer of VcDnaB without changing its canonical structure. The data showed that the VcDciA binding site on VcDnaB is the conserved module formed by the linker helix LH of one monomer and the determinant helix DH of the second monomer. Interestingly, DnaC from Escherichia coli also targets this module onto EcDnaB. Thanks to their common target site, it was shown that VcDciA and EcDnaC could be functionally interchanged in vitro despite sharing no structural similarity. This represents a milestone in understanding the mechanism employed by phage helicase loaders to hijack bacterial replicative helicases during evolution.


Assuntos
Proteínas de Escherichia coli , Proteínas de Escherichia coli/química , Replicação do DNA , DnaB Helicases/química , DnaB Helicases/genética , DnaB Helicases/metabolismo , DNA Helicases/química , Bactérias/metabolismo , Escherichia coli/genética , Sítios de Ligação , Proteínas de Bactérias/química
2.
Int J Mol Sci ; 24(2)2023 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-36674944

RESUMO

DciA is the ancestral bacterial replicative helicase loader, punctually replaced during evolution by the DnaC/I loaders of phage origin. DnaC helps the helicase to load onto DNA by cracking open the hexameric ring, but the mechanism of loading by DciA remains unknown. We demonstrate by electron microscopy, nuclear magnetic resonance (NMR) spectroscopy, and biochemistry experiments that DciA, which folds into a KH-like domain, interacts with not only single-stranded but also double-stranded DNA, in an atypical mode. Some point mutations of the long α-helix 1 demonstrate its importance in the interaction of DciA for various DNA substrates mimicking single-stranded, double-stranded, and forked DNA. Some of these mutations also affect the loading of the helicase by DciA. We come to the hypothesis that DciA could be a DNA chaperone by intercalating itself between the two DNA strands to stabilize it. This work allows us to propose that the direct interaction of DciA with DNA could play a role in the loading mechanism of the helicase.


Assuntos
Proteínas de Escherichia coli , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , DNA Helicases/metabolismo , DNA , Replicação do DNA , Bactérias/metabolismo , DNA de Cadeia Simples , Proteínas de Bactérias/genética , Proteínas de Bactérias/química
3.
ACS Appl Mater Interfaces ; 14(36): 40501-40512, 2022 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-36044427

RESUMO

We have designed a new Bodipy scaffold for efficient in vivo photoacoustic (PA) imaging of nanoparticles commonly used as drug nanovectors. The new dye has an optimized absorption band in the near-infrared window in biological tissue and a low fluorescence quantum yield that leads to a good photoacoustic generation efficiency. After Bodipy-initiated ring-opening polymerization of lactide, the polylactide-Bodipy was formulated into PEGylated nanoparticles (NPs) by mixing with PLA-PEG at different concentrations. Formulated NPs around 100 nm exhibit excellent PA properties: an absorption band at 760 nm and a molar absorption coefficient in between that of molecular PA absorbers and gold NPs. Highly improved photostability compared to cyanine-labeled PLA NPs as well as innocuity in cultured macrophages were demonstrated. After intravenous injection in healthy animals, NPs were easily detected using a commercial PA imaging system and spectral unmixing, opening the way to their use as theranostic agents.


Assuntos
Nanopartículas , Técnicas Fotoacústicas , Animais , Compostos de Boro , Meios de Contraste , Técnicas Fotoacústicas/métodos , Poliésteres , Polímeros
4.
Int J Pharm ; 626: 122131, 2022 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-36028084

RESUMO

A common approach to tackle the poor intestinal membrane permeability of peptides after oral administration is to formulate them with a permeation enhancer (PE). Increased oral bioavailability for oral peptide candidates has been reported from clinical trials when either salcaprozate sodium (SNAC) or sodium caprate (C10) is incorporated in the formulation. However, little is known about how they physically interact with peptides in solution. Our objective was to compare the biophysical interactions between the GLP-1 analogue exenatide (Byetta®, Lilly), and C10 or SNAC using a variety of advanced analytical techniques. First, critical micelle concentration was measured in different buffers for both PEs. Dynamic light scattering (DLS) measurements revealed specific supramolecular structures arising from exenatide-PE association. Surface plasmon resonance (SPR) indicated the formation of exenatide-PE complexes with a high contribution from non-specific interactions and rapid binding kinetics, resulting in overall low affinities. DLS and isothermal titration calorimetry (ITC) were used to examine the supramolecular organization of the PEs, and revealed thermodynamic signatures characterized by unfavourable enthalpic contributions compensated by favourable entropic ones, but with low-affinity estimates in water (KD in the 10-100 µM range). With affinity capillary electrophoresis (ACE), weak interactions between exenatide and SNAC or C10 were confirmed in saline, with a dissociation constant around 10 µM and 30 µM respectively. In biorelevant intestinal media, the bile salts in FaSSIF and FeSSIF further reduced the binding of both agents to exenatide (KD ≈ 100 µM), indicating that the interaction between the PEs and exenatide might be inhibited by bile salts in the GI lumen. This study suggests that the interactions of both PEs with exenatide follow a similar non-covalent mechanism and are of low affinity.


Assuntos
Absorção Intestinal , Micelas , Ácidos e Sais Biliares , Caprilatos , Ácidos Decanoicos , Exenatida , Peptídeo 1 Semelhante ao Glucagon , Peptídeos , Água
5.
Proc Natl Acad Sci U S A ; 119(19): e2120098119, 2022 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-35507869

RESUMO

Microtubule dynamics is regulated by various cellular proteins and perturbed by small-molecule compounds. To what extent the mechanism of the former resembles that of the latter is an open question. We report here structures of tubulin bound to the PN2-3 domain of CPAP, a protein controlling the length of the centrioles. We show that an α-helix of the PN2-3 N-terminal region binds and caps the longitudinal surface of the tubulin ß subunit. Moreover, a PN2-3 N-terminal stretch lies in a ß-tubulin site also targeted by fungal and bacterial peptide-like inhibitors of the vinca domain, sharing a very similar binding mode with these compounds. Therefore, our results identify several characteristic features of cellular partners that bind to this site and highlight a structural convergence of CPAP with small-molecule inhibitors of microtubule assembly.


Assuntos
Tubulina (Proteína) , Vinca , Microtúbulos/metabolismo , Ligação Proteica , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina , Vinca/metabolismo
6.
Int J Pharm ; 609: 121147, 2021 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-34600059

RESUMO

A better knowledge on influence of nanomedicine characteristics on their biological efficacy and safety is expected to accelerate their clinical translation. This work aimed understanding of the oral fate of polymer-based nanomedicines designed with different characteristics. The influence of nanoparticle characteristics (size, zeta potential, molecular architecture surface design) was explored on biological responses evaluating their retention and absorption by rat jejunum using the Ussing chamber experimental model. Thermodynamic aspects of interactions between nanoparticles and model mucins were elucidated by isothermal titration calorimetry. The retention on mucosa varied between nanoparticles from 18.5 to 97.3 % of the initial amount after a simulation considering the entire jejunum length. Different mechanisms were proposed which promoted mucosal association or oppositely precluded any interactions. Strikingly, mucosal retention was profoundly affected by the size and nature of interactions with the mucus which depended on the nature of the coating material, but not on the zeta potential. The nanoparticle absorption simulated along the whole length of the intestine was low (0.01 to almost 3% of the initial amounts). A saturable mechanism including an upper nanoparticle size limit was evidenced but, needs now to be further elucidated. This work showed that the molecular design and formulation of nanoparticles can guide mechanisms by which nanoparticles interact with the mucosa. The data could be useful to formulators to address different oral drug delivery challenges ranging from the simple increase of residence time and proximity to the absorptive epithelium and systemic delivery using the most absorbed nanoparticles.


Assuntos
Quitosana , Nanopartículas , Animais , Portadores de Fármacos , Mucosa , Tamanho da Partícula , Ratos
7.
Int J Pharm ; 609: 121076, 2021 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-34481886

RESUMO

A simple approach to achieve a lipoprotein (LP)-mediated drug delivery is to trigger the spontaneous drug insertion into endogenous lipoproteins in the bloodstream, by means of its chemical modification. Nanoparticles (NPs) made of the squalene-gemcitabine (SQGem) conjugate were found to have a high affinity for plasma lipoproteins while free gemcitabine did not, suggesting a key role of the lipid moiety in this event. Whether the drug conjugation to cholesterol, one of the major lipoprotein-transported lipids, could also promote an analogous interaction was a matter of question. NPs made of the cholesterol-gemcitabine conjugate (CholGem) have been herein thoroughly investigated for their blood distribution profile both in vitro and in vivo. Unexpectedly, contrarily to SQGem, no trace of the CholGem prodrug could be found in the lipoprotein fractions, nor was it interacting with albumin. The investigation of isolated NPs and NPs/LPs physical mixtures provided a further insight into the lack of interaction of CholGem NPs with LPs. Although essential for allowing the self-assembly of the prodrug into nanoparticles, the lipid moiety may not be sufficient to elicit interaction of the conjugated drug with plasma lipoproteins but the whole NP physicochemical features must be carefully considered.


Assuntos
Desoxicitidina , Sistemas de Liberação de Medicamentos , Nanopartículas , Pró-Fármacos , Animais , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacocinética , Humanos , Lipídeos , Masculino , Ratos Sprague-Dawley , Gencitabina
8.
Toxins (Basel) ; 12(4)2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32316084

RESUMO

Envenoming due to Loxosceles spider bites still remains a neglected disease of particular medical concern in the Americas. To date, there is no consensus for the treatment of envenomed patients, yet horse polyclonal antivenoms are usually infused to patients with identified severe medical conditions. It is widely known that venom proteins in the 30-35 kDa range with sphingomyelinase D (SMasesD) activity, reproduce most of the toxic effects observed in loxoscelism. Hence, we believe that monoclonal antibody fragments targeting such toxins might pose an alternative safe and effective treatment. In the present study, starting from the monoclonal antibody LimAb7, previously shown to target SMasesD from the venom of L. intermedia and neutralize its dermonecrotic activity, we designed humanized antibody V-domains, then produced and purified as recombinant single-chain antibody fragments (scFvs). These molecules were characterized in terms of humanness, structural stability, antigen-binding activity, and venom-neutralizing potential. Throughout this process, we identified some blocking points that can impact the Abs antigen-binding activity and neutralizing capacity. In silico analysis of the antigen/antibody amino acid interactions also contributed to a better understanding of the antibody's neutralization mechanism and led to reformatting the humanized antibody fragment which, ultimately, recovered the functional characteristics for efficient in vitro venom neutralization.


Assuntos
Anticorpos Monoclonais , Antivenenos , Anticorpos de Cadeia Única , Venenos de Aranha/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Antígenos/imunologia , Antivenenos/administração & dosagem , Antivenenos/imunologia , Eritrócitos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Modelos Moleculares , Testes de Neutralização , Anticorpos de Cadeia Única/administração & dosagem , Anticorpos de Cadeia Única/imunologia , Picada de Aranha/terapia , Venenos de Aranha/efeitos adversos , Aranhas/imunologia
9.
Pharm Res ; 37(2): 22, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31897766

RESUMO

PURPOSE: The work aimed to compare quality of a siRNA carrier prepared with chitosan of two different sources having similar degree of deacetylation and molecular weights. Differences were analyzed from thermodynamic characteristics of interactions with siRNA. METHODS: The siRNA carrier (chitosan-coated poly(isobutylcyanoacrylate) nanoparticles) was prepared with home-prepared, CSLab, and commercial, CSCom, chitosans. Chitosan counterion was identified and chitosans CSCommod1 and CSCommod2 were obtained from CSCom exchanging counterion with that found on CSLab. Carrier quality was checked considering the size, zeta potential and siRNA association capacity by gel electrophoresis. Thermodynamic parameters of interactions between siRNA and chitosans in solution or immobilized at the carrier surface were determined by isothermal titration calorimetry (ITC). RESULTS: CSLab and CSCommod2 having a high content of acetate counterion associated better siRNA than CSCom and CSCommod1 which counterion included mainly chloride. ITC measurements indicated that siRNA interactions with chitosan and the siRNA carrier were driven by entropic phenomena including dehydration, but thermodynamic parameters of interactions clearly differed according to the nature of the counterion of chitosan. The influence of chitosan counterions was interpreted considering their different lyotropic character. CONCLUSION: Association of siRNA with our siRNA carrier was influenced by the nature of counterions associated with chitosan. Driven by entropic phenomena including dehydration, interactions were favored by acetate counterion. Although more work would be needed to decipher the influence of the counterion of chitosan during association with siRNA, it was pointed out as a new critical attribute of chitosan to consider while formulating siRNA carrier with this polysaccharide.


Assuntos
Quitosana/química , RNA Interferente Pequeno/química , Calorimetria/métodos , Eletroforese/métodos , Nanopartículas/química , Termodinâmica
10.
Nanomedicine (Lond) ; 14(6): 689-705, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30691340

RESUMO

AIM: To develop a monoclonal antibody against dehydroepiandrosterone (DHEA) and miniaturize it, generating a single-chain antibody variable fragment (scFv) against DHEA as an adrenocortical carcinoma (ACC) marker. MATERIAL & METHODS: DHEA conjugated to keyhole limpet hemocyanin was used as an immunogen to obtain anti-DHEA hybridomas. Variable fragments were cloned from hybridoma 5B7 total RNA, and used to detect DHEA in normal adrenal tissue and ACC cells. RESULTS: IgM monoclonal antibody was highly specific, and the recombinant scFv preserved parental antibody characteristics, allowing tissue localization of DHEA. CONCLUSION: Undefined small lesions are challenges for clinicians and impact clinical adrenocortical tumor management. Generating an anti-DHEA scFv facilitates development of imaging tests for early diagnosis of pediatric ACC.


Assuntos
Neoplasias do Córtex Suprarrenal/diagnóstico , Carcinoma Adrenocortical/diagnóstico , Biomarcadores Tumorais/análise , Desidroepiandrosterona/análise , Anticorpos de Cadeia Única/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Desidroepiandrosterona/metabolismo , Expressão Gênica , Humanos , Camundongos Endogâmicos BALB C , Simulação de Acoplamento Molecular , Conformação Proteica , Engenharia de Proteínas/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Anticorpos de Cadeia Única/genética , Zona Reticular/metabolismo
11.
J Med Chem ; 61(16): 7202-7217, 2018 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-30028133

RESUMO

The nucleoprotein (NP) of influenza A virus (IAV) required for IAV replication is a promising target for new antivirals. We previously identified by in silico screening naproxen being a dual inhibitor of NP and cyclooxygenase COX2, thus combining antiviral and anti-inflammatory effects. However, the recently shown strong COX2 antiviral potential makes COX2 inhibition undesirable. Here we designed and synthesized two new series of naproxen analogues called derivatives 2, 3, and 4 targeting highly conserved residues of the RNA binding groove, stabilizing NP monomer without inhibiting COX2. Derivative 2 presented improved antiviral effects in infected cells compared to that of naproxen and afforded a total protection of mice against a lethal viral challenge. Derivative 4 also protected infected cells challenged with circulating 2009-pandemic and oseltamivir-resistant H1N1 virus. This improved antiviral effect likely results from derivatives 2 and 4 inhibiting NP-RNA and NP-polymerase acidic subunit PA N-terminal interactions.


Assuntos
Antivirais/química , Antivirais/farmacologia , Inibidores de Ciclo-Oxigenase 2/farmacologia , Vírus da Influenza A/efeitos dos fármacos , Naproxeno/análogos & derivados , Células A549 , Animais , Sítios de Ligação , Inibidores de Ciclo-Oxigenase 2/química , Cães , Desenho de Fármacos , Reposicionamento de Medicamentos , Farmacorresistência Viral/efeitos dos fármacos , Feminino , Humanos , Vírus da Influenza A/patogenicidade , Influenza Humana/tratamento farmacológico , Influenza Humana/patologia , Células Madin Darby de Rim Canino , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Naproxeno/farmacologia , Proteínas do Nucleocapsídeo , Oseltamivir/farmacologia , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ressonância de Plasmônio de Superfície , Proteínas do Core Viral/química , Proteínas do Core Viral/metabolismo
12.
Nanoscale ; 10(23): 10952-10962, 2018 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-29850714

RESUMO

In the field of dendrimers targeting small interfering RNA (siRNA) delivery, dendrimer structural properties, such as the flexibility/rigidity ratio, play a crucial role in the efficiency of complexation. However, advances in organic chemistry have enabled the development of dendrimers that differ only by a single atom on their surface terminals. This is the case for cationic phosphorus dendrimers functionalized with either pyrrolidinium (DP) or morpholinium (DM) terminal groups. This small change was shown to strongly affect the dendrimer-siRNA complexation, leading to more efficient anti-inflammatory effects in the case of DP. Reasons for this different behavior can hardly be inferred only by biological in vitro and in vivo experiments due to the high number of variables and complexity of the investigated biological system. However, an understanding of how small chemical surface changes may completely modify the overall dendrimer-siRNA complexation is a significant breakthrough towards the design of efficient dendrimers for nucleic acid delivery. Herein, we present experimental and computational approaches based on isothermal titration calorimetry and molecular dynamics simulations to elucidate the molecular reasons behind different efficiencies and activities of DP and DM. Results of the present research highlight how chemical surface modifications may drive the overall dendrimer-siRNA affinity by influencing enthalpic and entropic contributions of binding free energy. Moreover, this study elucidates molecular reasons related to complexation stoichiometry that may be crucial in determining the dendrimer complexation efficiency.


Assuntos
Dendrímeros/química , Fósforo/química , RNA Interferente Pequeno/química , Cátions , Biologia Computacional , Simulação de Dinâmica Molecular , Termodinâmica
13.
Pharm Res ; 35(7): 129, 2018 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-29700622

RESUMO

PURPOSE: A Surface Plasmon Resonance chip (SPR) was developed to study the activation of complement system triggered by nanomaterials in contact with human serum, which is an important concern today to warrant safety of nanomedicines. METHODS: The developed chip was tested for its specificity in complex medium and its longevity of use. It was then employed to assess the release of complement fragments upon incubation of nanoparticles in serum. A comparison was made with other current methods assessing complement activation (µC-IE, ELISA). RESULTS: The SPR chip was found to give a consistent response for C3a release upon activation by nanoparticles. Results were similar to those obtained by µC-IE. However, ELISA detection of iC3b fragments showed an explained high non-specific background. The impact of sample preparation preceding the analysis was assessed with the newly develop SPR method. The removal of nanoparticles before analysis showed an important modification in the obtained response, possibly leading to false negative results. CONCLUSION: The SPR chip developed in this work allows for an automated assessment of complement activation triggered by nanoparticles with possibility of multiplexed analysis. The design of the chip proved to give consistent results of complement activation by nanoparticles.


Assuntos
Complemento C3/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Nanopartículas/administração & dosagem , Nanopartículas/metabolismo , Ressonância de Plasmônio de Superfície/métodos , Animais , Complemento C3/agonistas , Eletroforese/métodos , Cabras , Humanos , Camundongos
14.
Biomacromolecules ; 18(8): 2379-2388, 2017 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-28639789

RESUMO

Inflammation is an essential component of many lung diseases, including asthma, chronic obstructive pulmonary disease (COPD), or acute lung injury. Our purpose was to design efficient carriers for lung delivery of small interfering RNA (siRNA) targeting tumor necrosis factor (TNF-α) in an acute lung injury model. To achieve this goal, two different types of phosphorus-based dendrimers with either pyrrolidinium or morpholinium as terminal protonated amino groups were selected for their better biocompatibility compared to other dendrimers. Dendriplexes containing pyrrolidinium surface groups demonstrated a stronger siRNA complexation, a higher cellular uptake, and enhanced in vitro silencing efficiency of TNF-α in the lipopolysaccharide (LPS)-activated mouse macrophage cell line RAW264.7, compared to morpholinium-containing dendriplexes. The better performance of the pyrrolidium dendriplexes was attributed to their higher pKa value leading to a stronger siRNA complexation and improved protection against enzymatic degradation resulting in a higher cellular uptake. The superior silencing effect of the pyrrolidinium dendriplexes, compared to noncomplexed siRNA, was confirmed in vivo in an LPS-induced murine model of short-term acute lung injury upon lung delivery via nasal administration. These data suggest that phosphorus dendriplexes have a strong potential in lung delivery of siRNA for treating inflammatory lung diseases.


Assuntos
Lesão Pulmonar Aguda/tratamento farmacológico , Anti-Inflamatórios , Dendrímeros , Inativação Gênica , Morfolinos , RNA Interferente Pequeno , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Lesão Pulmonar Aguda/genética , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/patologia , Administração Intranasal , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Dendrímeros/química , Dendrímeros/farmacologia , Modelos Animais de Doenças , Feminino , Camundongos , Morfolinos/química , Morfolinos/genética , Morfolinos/farmacologia , Células RAW 264.7 , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Fator de Necrose Tumoral alfa/genética
15.
Biochim Biophys Acta Gen Subj ; 1861(6): 1606-1616, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28137620

RESUMO

BACKGROUND: Metal-organic framework nanoparticles (nanoMOFs) are biodegradable highly porous materials with a remarkable ability to load therapeutic agents with a wide range of physico-chemical properties. Engineering the nanoMOFs surface may provide nanoparticles with higher stability, controlled release, and targeting abilities. Designing postsynthetic, non-covalent self-assembling shells for nanoMOFs is especially appealing due to their simplicity, versatility, absence of toxic byproducts and minimum impact on the original host-guest ability. METHODS: In this study, several ß-cyclodextrin-based monomers and polymers appended with mannose or rhodamine were randomly phosphorylated, and tested as self-assembling coating building blocks for iron trimesate MIL-100(Fe) nanoMOFs. The shell formation and stability were studied by isothermal titration calorimetry (ITC), spectrofluorometry and confocal imaging. The effect of the coating on tritium-labeled AZT-PT drug release was estimated by scintillation counting. RESULTS: Shell formation was conveniently achieved by soaking the nanoparticles in self-assembling agent aqueous solutions. The grafted phosphate moieties enabled a firm anchorage of the coating to the nanoMOFs. Coating stability was directly related to the density of grafted phosphate groups, and did not alter nanoMOFs morphology or drug release kinetics. CONCLUSION: An easy, fast and reproducible non-covalent functionalization of MIL-100(Fe) nanoMOFs surface based on the interaction between phosphate groups appended to ß-cyclodextrin derivatives and iron(III) atoms is presented. GENERAL SIGNIFICANCE: This study proved that discrete and polymeric phosphate ß-cyclodextrin derivatives can conform non-covalent shells on iron(III)-based nanoMOFs. The flexibility of the ß-cyclodextrin to be decorated with different motifs open the way towards nanoMOFs modifications for drug delivery, catalysis, separation, imaging and sensing. This article is part of a Special Issue entitled "Recent Advances in Bionanomaterials" Guest Editors: Dr. Marie-Louise Saboungi and Dr. Samuel D. Bader.


Assuntos
Química Click/métodos , Compostos de Ferro/química , Magnetismo/métodos , Manose/química , Nanopartículas Metálicas/química , Nanotecnologia/métodos , Rodaminas/química , Zidovudina/química , beta-Ciclodextrinas/química , Calorimetria/métodos , Liberação Controlada de Fármacos , Cinética , Manose/análogos & derivados , Microscopia Confocal , Fosforilação , Porosidade , Solubilidade , Espectrometria de Fluorescência , Propriedades de Superfície , Termodinâmica
16.
Biochemistry ; 55(31): 4259-62, 2016 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-27431776

RESUMO

The structure of the ribonucleoprotein complexes is crucial to viral transcription and replication of influenza virus, but association of the nucleoprotein (NP) with the polymerase remains to be characterized at the molecular level. Here, we identify a peptide of the polymerase acidic subunit PA(1-27) that associates with NP. Docking and molecular dynamics simulations suggest a similar NP binding site with PA(1-27) and PA(1-186). The PA(1-27)-NP complex is characterized by surface plasmon resonance and fluorescence using recombinant NP proteins and by pull-down assays in infected cells. The PA(1-27)-NP complex may have a role in the final steps of transcription and replication.


Assuntos
Vírus da Influenza A Subtipo H1N1/química , Proteínas de Ligação a RNA/química , RNA Polimerase Dependente de RNA/química , Proteínas do Core Viral/química , Proteínas Virais/química , Animais , Cães , Vírus da Influenza A Subtipo H1N1/fisiologia , Células Madin Darby de Rim Canino , Modelos Moleculares , Simulação de Dinâmica Molecular , Complexos Multiproteicos/química , Proteínas do Nucleocapsídeo , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/química , Proteínas Recombinantes/química , Espectrometria de Fluorescência , Ressonância de Plasmônio de Superfície
17.
Immunol Lett ; 176: 90-6, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27288291

RESUMO

Loxosceles spider bites often lead to serious envenomings and no definite therapy has yet been established. In such a context, it is of interest to consider an antibody-based targeted therapy. We have previously prepared a murine monoclonal IgG (LiMab7) that binds to 32-35kDa components of Loxosceles intermedia venom and neutralizes the dermonecrotic activity of the venom. Here, we re-engineered LiMab7 into a recombinant diabody. The protein was produced in bacteria and then it was functionally characterized. It proved to be efficient at neutralizing sphingomyelinase and hemolytic activities of the crude venom despite the slightly altered binding kinetic constants and the limited stability of the dimeric configuration. This is the first report of a specific recombinant antibody for a next-generation of Loxosceles antivenoms.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Antivenenos/uso terapêutico , Imunoterapia/métodos , Diester Fosfórico Hidrolases/imunologia , Proteínas Recombinantes/uso terapêutico , Anticorpos de Cadeia Única/uso terapêutico , Picada de Aranha/terapia , Venenos de Aranha/imunologia , Animais , Células Cultivadas , Expressão Gênica , Hemólise , Camundongos , Engenharia de Proteínas , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Picada de Aranha/imunologia , Aranhas/imunologia
18.
Langmuir ; 31(41): 11186-94, 2015 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-26375384

RESUMO

The dynamics of the formation of siRNA-lipoplexes coated with hyaluronic acid (HA) and the parameters influencing their supramolecular organization were studied. The insertion of a HA-dioleylphosphatidylethanolamine (DOPE) conjugate in the liposome structure as well as subsequent complexation with siRNA increased the liposome size. Lipoplexes were around 110 nm at high ± charge ratios with a zeta potential around +50 mV and around 230 nm at low ± ratios, with a zeta potential that decreased to negative values, reaching -45 mV. The addition of the conjugate did not compromise siRNA binding to liposomes, although these nucleic acids induced a displacement of part of the HA-DOPE conjugate upon lipoplex formation, as confirmed by capillary electrophoresis. Isothermal titration calorimetry, X-ray diffraction studies, and cryo-TEM microscopy demonstrated that in addition to electrostatic interactions with siRNA a rearrangement of the lipid bilayers takes place, resulting in condensed oligolamellar vesicles. This phenomenon is dependent on the number of siRNA molecules and the degree of modification with HA. Finally, the suitable positioning of HA on the lipoplex surface and its ability to bind specifically to the CD44 receptors in a concentration-dependent manner was demonstrated by surface plasmon resonance analysis.


Assuntos
Sistemas de Liberação de Medicamentos , Receptores de Hialuronatos/química , Ácido Hialurônico/química , Bicamadas Lipídicas/química , RNA Interferente Pequeno/química , Sítios de Ligação , Humanos , Ressonância de Plasmônio de Superfície
19.
Pharm Res ; 32(5): 1794-803, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25504453

RESUMO

PURPOSE: Nanoparticles with prolonged residence time in bone constitute a valuable strategy for bone disease treatments. The aim of this work was to synthesise a simple nanoparticulate system exhibiting both anticancer and hydroxyapatite binding properties for potential bone cancer applications. METHODS: The amphiphilic copolymer poly(γ-benzyl-glutamate)-block-poly(glutamic acid) (PBLG-b-PGlu) was synthetised by ring opening polymerization and nanoparticles were obtained by a simple nanoprecipitation method. Nanoparticles were characterized in terms of cisplatin interaction, association, and release as well as interaction with hydroxyapatite and their cytoxicity was studied in three prostate cancer cell lines. RESULTS: PBLG-b-PGlu nanoparticles of ~50 nm in size were successfully prepared. They could display for the first time dual hydroxyapatite binding and anticancer properties mediated by the PGlu moiety. They could complex cisplatin at a drug loading content of 6.2% (w/w). Cisplatin release was triggered by physiological concentrations of chloride ions according to an almost zero order kinetics during 14 days. Simultaneously, these nanoparticles showed in vitro hydroxyapatite binding. Finally, they were shown to exert a cytotoxic effect in three prostate cancer cell lines that potentially metastasize to bone. CONCLUSIONS: These properties suggest the potential utility of cisplatin-loaded PBLG-b-PGlu nanoparticles as carrier systems for the treatment of bone metastases.


Assuntos
Antineoplásicos/administração & dosagem , Cisplatino/administração & dosagem , Preparações de Ação Retardada/química , Durapatita/química , Nanopartículas/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Cisplatino/farmacologia , Humanos , Masculino , Ácido Poliglutâmico/química , Próstata/efeitos dos fármacos , Próstata/patologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia
20.
Bioconjug Chem ; 25(11): 1971-83, 2014 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-25313527

RESUMO

We report herein a detailed study concerning the impact of different bioconjugation and nanoformulation strategies on the in vitro targeting ability of peptide-decorated squalenoyl gemcitabine (SQdFdC) nanoparticles (NPs). NPs have been functionalized with the CKAAKN peptide, previously identified as an efficient homing device within the pancreatic pathological microenvironment. Two approaches have been followed: (i) either the CKAAKN peptide was directly conjugated at the surface of preformed SQdFdC nanoparticles (conjugation after NP formation) or (ii) it was first reacted with a maleimide squalenoyl derivative before the resulting bioconjugate was co-nanoprecipitated with SQdFdC to form the peptide-decorated NPs (conjugation before NP formation). NPs were characterized with respect to mean diameter, zeta potential, and stability over time. Then, their specific interaction with the sFRP-4 protein was evaluated by surface plasmon resonance. Although both synthetic strategies allowed us to formulate NPs able to interact with the corresponding receptor, enhanced target binding and better specific avidity were observed with CKAAKN-NPs functionalized before NP formation. These NPs displayed the highest cell uptake and cytotoxicity in an in vitro model of human MIA Paca-2 pancreatic cancer cells.


Assuntos
Portadores de Fármacos/química , Nanopartículas/química , Oligopeptídeos/química , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/química , Portadores de Fármacos/metabolismo , Desenho de Fármacos , Humanos , Ligantes , Camundongos , Células NIH 3T3 , Nanopartículas/metabolismo , Tamanho da Partícula , Proteínas Proto-Oncogênicas/metabolismo , Gencitabina
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