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We have developed a pipeline to express, purify, and characterize HIV envelope protein (Env) gp145 from Chinese hamster ovary cells, to accelerate the production of a promising vaccine candidate. First in shake flasks, then in bioreactors, we optimized the growth conditions. By adjusting the pH to 6.8, we increased expression levels to 101 mg/L in a 50 L bioreactor, nearly twice the previously reported titer value. A battery of analytical methods was developed in accordance with current good manufacturing practices to ensure a quality biopharmaceutical. Imaged capillary isoelectric focusing verified proper glycosylation of gp145; dynamic light scattering confirmed the trimeric arrangement; and bio-layer interferometry and circular dichroism analysis demonstrated native-like properties (i.e., antibody binding and secondary structure). MALDI-TOF mass spectrometry was used as a multi-attribute platform for accurate mass determination, glycans analysis, and protein identification. Our robust analysis demonstrates that our gp145 product is very similar to a reference standard and emphasizes the importance of accurate characterization of a highly heterogeneous immunogen for the development of an effective vaccine. Finally, we present a novel guanosine microparticle with gp145 encapsulated and displayed on its surface. The unique properties of our gp145 microparticle make it amenable to use in future preclinical and clinical trials.
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As the skin is the main protective organ of the body, it is exposed to wounds or injuries which carry out a healing process during a period of approximately 15 days depending on the severity of the injury. In the present research, the development of chitosan-based hydrogels loaded with silver nanoparticles and calendula extract (Ch-AgNPs-Ce) was proposed. This can be used to fulfill the hemostatic, anti-infective, antibacterial, healing and anti-inflammatory functions through controlled release of the nanoparticles and calendula extract in substitution of commonly used drugs. The physical properties of the silver nanoparticles were analyzed by UV-visible spectroscopy, scanning and transmission electron microscopy, showing a size between 50 and 100 nm. The antibacterial properties were evaluated by the agar well diffusion method. Antimicrobial testing of the hydrogels showed that the inclusion of silver nanoparticles provides concentration-dependent antibacterial behavior against E. coli and S. aureus. The healing properties of the system were tested in two diabetic patients to whom said hydrogels were placed, obtaining a positive curative result after a few weeks. Therefore, it can be concluded that Ch-AgNPs-Ce hydrogels can achieve healing in chronic or exposed wounds after a period of time which can be used in alternative treatments in patients with poor healing capacity.
Assuntos
Quitosana , Nanopartículas Metálicas , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Calendula , Quitosana/farmacologia , Preparações de Ação Retardada , Escherichia coli , Humanos , Hidrogéis/farmacologia , Extratos Vegetais , Prata/farmacologia , Staphylococcus aureus , CicatrizaçãoRESUMO
We report the synthesis and self-assembly of two lipophilic 2'-deoxyguanosine (G) derivatives whose fluorescence intensity is modulated by self-assembly into supramolecular G-quadruplexes (SGQs). Whereas both derivatives self-assemble isostructurally, one shows up to 100% emission enhancement while the other shows an initial enhancement, followed by 10% quenching. Thus, the rotational restrictions resulting from self-assembly are enough to induce significant changes in emission, but it is critical to consider the specific interactions between fluorophores since they will determine the ultimate emission signature. These findings could open the door to the development of luminescent supramolecular sensors and probes.
Assuntos
Desoxiguanosina/síntese química , Quadruplex G , Desoxiguanosina/análogos & derivados , Desoxiguanosina/química , Fluorescência , Substâncias Macromoleculares/síntese química , Substâncias Macromoleculares/química , Modelos Moleculares , Estrutura MolecularRESUMO
Nanoflowers (NFs) are flowered-shaped particles with overall sizes or features in the nanoscale. Beyond their pleasing aesthetics, NFs have found a number of applications ranging from catalysis, to sensing, to drug delivery. Compared to inorganic based NFs, their organic and hybrid counterparts are relatively underdeveloped mostly because of the lack of a reliable and versatile method for their construction. We report here a method for constructing NFs from a wide variety of biologically relevant molecules (guests), ranging from small molecules, like doxorubicin, to biomacromolecules, like various proteins and plasmid DNA. The method relies on the encapsulation of the guests within a hierarchically structured particle made from supramolecular G-quadruplexes. The size and overall flexibility of the guests dictate the broad morphological features of the resulting NFs, specifically, small and rigid guests favor the formation of NFs with spiky petals, while large and/or flexible guests promote NFs with wide petals. The results from experiments using confocal fluorescence microscopy, and scanning electron microscopy provides the basis for the proposed mechanism for the NF formation.
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Nanopartículas/química , Técnicas de Química Sintética , Desoxiguanosina/análogos & derivados , Desoxiguanosina/química , Dextranos/química , Doxorrubicina/química , Quadruplex G , Estrutura Molecular , Tamanho da Partícula , Plasmídeos , Poliestirenos/química , Porosidade , Temperatura , Xantenos/químicaRESUMO
Supramolecular G-quadruplexes (SGQs) are formed via the cation promoted self-assembly of guanine derivatives into stacks of planar hydrogen-bonded tetramers. Here, we present results on the formation of SGQs made from the 8-(m-acetylphenyl)-2'-deoxyguanosine (mAGi) derivative in the presence of various mono- and divalent cations. NMR and HR ESI-MS data indicate that varying the cation can efficiently tune the molecularity, the fidelity and stability (thermal and kinetic) of the resulting SGQs. The results show that, parallel to the previously reported potassium-templated hexadecamer (mAGi16·3K+), Na+, Rb+ and [Formula: see text] also promote the formation of similar supramolecules with high fidelity and molecularity. In contrast, the divalent cations Pb2+, Sr2+ and Ba2+ template the formation of octamers (mAGi8), with the latter two inducing higher thermal stabilities. Molecular dynamics simulations for the hexadecamers containing monovalent cations enabled critical insights that help explain the experimental observations.
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We report the metallo-responsive high fidelity switching between hexadecameric and octameric supramolecular G-quadruplexes triggered by a change in the metal cation promoter from potassium to strontium, respectively.
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DNA/química , Quadruplex G/efeitos dos fármacos , Potássio/farmacologia , Estrôncio/farmacologiaRESUMO
We have developed the 8-(m-acetylphenyl)-2'-deoxyguanosine (mAG) scaffold for the self-assembly of supramolecules in water and for the synthesis of self-assembled dendrimers (SADs) in organic media. Previously, reported mAG assemblies showed promising characteristics for the construction of SADs. Yet, none of these SADs had large enough dendrons to reach a fractal geometry characteristic of high-generation dendrimers. Here we present the synthesis as well as the molecular and supramolecular characterization of a fourth-generation hydrophilic self-assembled hexadecameric dendrimer [mAGD(4)(OH)(16)](16)·3KI (3(16)) with a size and shape akin to those of globular proteins. The diameter of 3(16) (5.0 nm) was measured by pulsed field gradient NMR and dynamic light scattering experiments, which enabled the construction of a computer-generated molecular model. This SAD represents an attractive platform for biomedical applications due to its water solubility, discreteness, well-defined structure, thermal stability (T(m) = 68 °C), and functional core.
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Dendrímeros/química , Espectroscopia de Ressonância Magnética , Estrutura MolecularRESUMO
Here we show 2'-deoxyguanosine derivatives that self-assemble in aqueous media into discrete supramolecular hexadecamers and exhibit the lower critical solution temperature (LCST) phenomenon. Spectroscopic, calorimetric, and electron microscopy studies support the fact that above the transition temperature (T(t)) the supramolecules further assemble into nanoscopic spherical globules of low polydispersity. Furthermore, the T(t) can be tuned to higher values by the addition of a more hydrophilic derivative. These findings uncover a new paradigm in the development of smart thermosensitive materials with properties and applications complementary to those of polymers.
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Desoxiguanosina/análogos & derivados , Guanosina/análogos & derivados , Temperatura Alta , Guanosina/química , Polímeros/química , Água/químicaRESUMO
We report the self-assembly of a hydrophilic 8-(m-acetylphenyl)-2'-deoxyguanosine (mAG) derivative into a discrete and thermally stable hexadecameric supramolecule in aqueous media. We demonstrate that this hexadecamer is isostructural to the one formed by a related lipophilic derivative in organic media. This mAG moiety represents a rare example of a small-molecule recognition motif that is capable of assembling isostructurally and with high fidelity in both organic and aqueous media.
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Desoxiguanosina/análogos & derivados , Desoxiguanosina/química , Água/química , Isomerismo , Estrutura Molecular , Solventes/químicaRESUMO
Herein we describe the construction of hexadecameric self-assembled dendrimers (SADs) using a series of dendronized 8-(m-acetylphenyl)-2'-deoxyguanosine (mAG) subunits. The azido-substituted mAG subunits were covalently linked to alkynyl polyester dendrons using a copper-catalyzed 1,3-dipolar cycloaddition reaction. Discrete SADs are formed with high fidelity and thermal stability even with the increased steric hindrance offered by the dendrons.