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INTRODUCTION: Fibrotic lung diseases represent a large subset of diseases with an unmet clinical need. Oligonucleotide therapies (ONT) are a promising therapeutic approach for the treatment of pulmonary disease as they can inhibit pathways that are otherwise difficult to target. Additionally, targeting the lung specifically with ONT is advantageous because it reduces the possibilities of systemic side effects and tolerability concerns. AREAS COVERED: This review presents the chemical basis of designing various ONTs currently known to treat fibrotic lung diseases. Further, the authors have also discussed the delivery vehicle, routes of administration, physiological barriers of the lung, and toxicity concerns with ONTs. EXPERT OPINION: ONTs provide a promising therapeutic approach for the treatment of fibrotic diseases of the lung, particularly because ONTs directly delivered to the lung show little systemic side effects compared to current therapeutic strategies. Dry powder aerosolized inhalers may be a good strategy for getting ONTs into the lung in humans. However, as of now, no dry powder ONTs have been approved for use in the clinical setting, and this challenge must be overcome for future therapies. Various delivery methods that can aid in direct targeting may also improve the use of ONTs for lung fibrotic diseases.
Assuntos
Pneumopatias , Oligonucleotídeos , Humanos , Oligonucleotídeos/efeitos adversos , Pós/metabolismo , Pós/farmacologia , Pulmão/metabolismo , FibroseRESUMO
mRNA lipid nanoparticles (LNPs) are at the forefront of nucleic acid intracellular delivery, as exemplified by the recent emergency approval of two mRNA LNP-based COVID-19 vaccines. The success of an LNP product largely depends on the systematic optimisation of the four lipidic components, namely the ionisable lipid, PEG lipid, structural and helper lipids. However, the in vitro screening of novel lipidic components and LNP compositions is limited by the low-throughput of LNP preparation. To address these issues, we herein present an automated high-throughput screening platform to select novel ionisable lipids and corresponding LNPs encapsulating mRNA in vitro. This high-throughput platform employs a lab-based automated liquid handling system, amenable to high-throughput (up to 384 formulations per plate and several plates per run) and allows precise mixing and reproducible mRNA LNP preparation which ensures a direct head-to-head comparison of hundreds and even thousands of novel LNPs. Most importantly, the robotic process has been successfully applied to the screening of novel LNPs encapsulating mRNA and has identified the same novel mRNA LNP leads as those from microfluidics-mixing technology, with a correlation coefficient of 0.8751. This high-throughput platform can facilitate to narrow down the number of novel ionisable lipids to be evaluated in vivo. Moreover, this platform has been integrated into a fully-automated workflow for LNP property control, physicochemical characterisation and biological evaluation. The high-throughput platform may accelerate proprietary lipid development, mRNA LNP lead optimisation and candidate selection to advance preclinical mRNA LNP development to meet urgent global needs.
Assuntos
Vacinas contra COVID-19/administração & dosagem , COVID-19 , Nanopartículas , Vacinas Sintéticas/administração & dosagem , Vacinas de mRNA/administração & dosagem , COVID-19/prevenção & controle , Humanos , Lipossomos , RNA Interferente PequenoRESUMO
Layer-by-layer assembly of polyelectrolyte multilayer (PEM) films represents a bottom-up approach for re-engineering the molecular landscape of cell surfaces with spatially continuous and molecularly uniform ultrathin films. However, fabricating PEMs on viable cells has proven challenging owing to the high cytotoxicity of polycations. Here, we report the rational engineering of a new class of PEMs with modular biological functionality and tunable physicochemical properties which have been engineered to abrogate cytotoxicity. Specifically, we have discovered a subset of cationic copolymers that undergoes a conformational change, which mitigates membrane disruption and facilitates the deposition of PEMs on cell surfaces that are tailorable in composition, reactivity, thickness, and mechanical properties. Furthermore, we demonstrate the first successful in vivo application of PEM-engineered cells, which maintained viability and function upon transplantation and were used as carriers for in vivo delivery of PEMs containing biomolecular payloads. This new class of polymeric film and the design strategies developed herein establish an enabling technology for cell transplantation and other therapies based on engineered cells.
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Materiais Biocompatíveis/química , Bioengenharia , Membrana Celular/química , Transplante das Ilhotas Pancreáticas , Polímeros/química , Animais , Materiais Biocompatíveis/síntese química , Materiais Biocompatíveis/toxicidade , Eletrólitos/síntese química , Eletrólitos/química , Eletrólitos/toxicidade , Ilhotas Pancreáticas/química , Camundongos , Poliaminas/síntese química , Poliaminas/química , Poliaminas/toxicidade , Polieletrólitos , Polietilenoglicóis/síntese química , Polietilenoglicóis/química , Polietilenoglicóis/toxicidade , Polilisina/análogos & derivados , Polilisina/síntese química , Polilisina/química , Polilisina/toxicidade , Polímeros/síntese química , Polímeros/toxicidadeRESUMO
Resolvins are family of lipid mediators derived from omega-3 polyunsaturated fatty acids, which are generated during the resolution phase of acute inflammation. Resolvin E1 is biosynthesized from eicosapentaenoic acid via 18(R)-hydroxyeicosapentaenoic acid (18R-HEPE) in the Cox-2 and lipoxygenase mediated pathway and has proven to exhibit potent anti-inflammatory activity. We report herein the first total chemical synthesis of 18R-HEPE and demonstrate that this compound displays in vivo bioactivity by blocking neutrophil infiltration in a murine model of zymosan-induced peritonitis.
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Anti-Inflamatórios/síntese química , Anti-Inflamatórios/farmacologia , Ácido Eicosapentaenoico/análogos & derivados , Peritonite/tratamento farmacológico , Animais , Anti-Inflamatórios/química , Modelos Animais de Doenças , Ácido Eicosapentaenoico/síntese química , Ácido Eicosapentaenoico/química , Ácido Eicosapentaenoico/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Infiltração de Neutrófilos/efeitos dos fármacos , Peritonite/induzido quimicamente , Estereoisomerismo , ZimosanRESUMO
In recent years, there has been an increasing interest in designing delivery systems to enhance the efficacy of RNA-based therapeutics. Here, we have synthesized copolymers comprised of dimethylaminoethyl methacrylate (DMAEMA) or diethylaminoethyl methacrylate (DEAEMA) copolymerized with alkyl methacrylate monomers ranging from 2 to 12 carbons, and developed a high throughput workflow for rapid investigation of their applicability for mRNA delivery. The structure activity relationship revealed that the mRNA encapsulation efficiency is improved by increasing the cationic density and use of shorter alkyl side chains (2-6 carbons). Minimal cytotoxicity was observed when using DEAEMA-co-BMA (EB) polyplexes up to 18 h after dosing, independent of a poly(ethylene glycol) (PEG) first block. The lowest molecular weight polymer (EB10,250) performed best, exhibiting greater transfection than polyethyenimine (PEI) based upon the number of cells transfected and mean intensity. Conventional investigations into the performance of polymeric materials for mRNA delivery is quite tedious, consequently limiting the number of materials and formulation conditions that can be studied. The high throughput approach presented here can accelerate the screening of polymeric systems and paves the way for expanding this generalizable approach to assess various materials for mRNA delivery.
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Técnicas de Transferência de Genes/normas , Terapia Genética/métodos , Polímeros/química , RNA Mensageiro/metabolismoRESUMO
Emerging therapeutic treatments based on the production of proteins by delivering mRNA have become increasingly important in recent times. While lipid nanoparticles (LNPs) are approved vehicles for small interfering RNA delivery, there are still challenges to use this formulation for mRNA delivery. LNPs are typically a mixture of a cationic lipid, distearoylphosphatidylcholine (DSPC), cholesterol, and a PEG-lipid. The structural characterization of mRNA-containing LNPs (mRNA-LNPs) is crucial for a full understanding of the way in which they function, but this information alone is not enough to predict their fate upon entering the bloodstream. The biodistribution and cellular uptake of LNPs are affected by their surface composition as well as by the extracellular proteins present at the site of LNP administration, e.g., apolipoproteinE (ApoE). ApoE, being responsible for fat transport in the body, plays a key role in the LNP's plasma circulation time. In this work, we use small-angle neutron scattering, together with selective lipid, cholesterol, and solvent deuteration, to elucidate the structure of the LNP and the distribution of the lipid components in the absence and the presence of ApoE. While DSPC and cholesterol are found to be enriched at the surface of the LNPs in buffer, binding of ApoE induces a redistribution of the lipids at the shell and the core, which also impacts the LNP internal structure, causing release of mRNA. The rearrangement of LNP components upon ApoE incubation is discussed in terms of potential relevance to LNP endosomal escape.
Assuntos
Nanopartículas , Apolipoproteínas E/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/metabolismo , Distribuição TecidualRESUMO
We report herein a new and enabling approach for decorating both abiotic and cell surfaces with the extracellular matrix IKVAV peptide in a site-specific manner using strain promoted azide-alkyne cycloaddition. A cyclooctyne-derivatized IKVAV peptide was synthesized and immobilized on the surface of pancreatic islets through strain-promoted azide-alkyne cycloaddition with cell surface azides generated by the electrostatic adsorption of a cytocompatible poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) copolymer bearing azido groups (PP-N(3)). Both "one-pot" and sequential addition of PP-N(3) and a cyclooctyne-derivatized IKVAV peptide conjugate enabled efficient modification of the pancreatic islet surface in less than 60 min. The ability to bind peptides at controlled surface densities was demonstrated in a quantitative manner using microarrays. Additionally, the technique is remarkably rapid and highly efficient, opening new avenues for the molecular engineering of cellular interfaces and protein and peptide microarrays.
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Ilhotas Pancreáticas/química , Peptídeos/química , Membrana Celular/química , Espectrometria de FluorescênciaRESUMO
The druggability and developability space is rapidly evolving in the post-genomic era. In the past, Lipinski's rule-of-five (Ro5) emerged and served as a guide for drug-like molecule design for oral delivery in the traditional druggable target space. In contrast, in this new era, a transition is occurring in drug discovery towards novel approaches to bind and modulate challenging biological targets that have led to transformative treatments for patients. Consequently, drugging novel targets using a variety of emerging molecular modalities, namely beyond the Ro5 (bRo5) small molecules (such as protein-protein interaction modulators, protein-targeted chimeras, or PROTACs), peptide/peptidomimetics, and nucleic acid-based modalities, have become a key focus in drug discovery. Herein, the emerging druggability and developability space is discussed side by side to build a general understanding of the potential development challenges of these novel modalities. An overview is provided on the evolving novel targets and molecular modalities, followed by a detailed analysis of the druggability aspects as well as the strategies used to progress drug candidate, and the trending chemistry and formulation strategies used to assess developability.
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Desenvolvimento de Medicamentos , Descoberta de Drogas , Terapia de Alvo Molecular , Preparações Farmacêuticas/química , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Estrutura Molecular , Mapas de Interação de Proteínas/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Chemical approaches to cell surface engineering have emerged as powerful tools for resurfacing the molecular landscape of cells and tissues. Here we report a new strategy for re-engineering cell surfaces through electrostatic adsorption of appropriately structured and functionalized poly(l-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) copolymers to cellular interfaces. Grafting of methoxy terminated tetra(ethylene glycol) chains to PLL abrogated polycation cytotoxicity in a charge density and PEG dependent manner, and copolymers structured with a unique balance of grafted PEG chains and free lysine monomers adsorbed to cell surfaces without compromising viability. Structurally analogous PLL-g-PEG copolymers bearing terminally functionalized PEG grafts were used as 'cell surface active' polymeric carriers for biotin, hydrazide, and azide moieties, which selectively captured streptavidin-, aldehyde-, and cyclooctyne-labeled probes, respectively, on cell surfaces. This strategy opens new opportunities in cell surface engineering, including generation of unique cell surface motifs, rapid and combinatorial surface modification, and use of biologically complex solvents. Tailored PLL-g-PEG copolymers offer a promising and enabling tool for bio/chemically remodeling cells and tissues with broad potential in biomedical and biotechnological applications.
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Membrana Celular/química , Polímeros/química , Adsorção , Cátions , Portadores de Fármacos/química , Polietilenoglicóis/química , Polilisina/química , Eletricidade EstáticaRESUMO
Introduction: The delivery of nucleic acid therapeutics through non-viral carriers face multiple biological barriers that reduce their therapeutic efficiency. Despite great progress, there remains a significant technological gap that continues to limit clinical translation of these nanocarriers. A number of polymeric materials are being exploited to efficiently deliver nucleic acids and achieve therapeutic effects. Areas covered: We discuss the recent advances in the polymeric materials for the delivery of nucleic acid therapeutics. We examine the use of common polymer architectures and highlight the challenges that exist for their development from bench side to clinic. We also provide an overview of the most notable improvements made to circumvent such challenges, including structural modification and stimuli-responsive approaches, for safe and effective nucleic acid delivery. Expert opinion: It has become apparent that a universal carrier that follows 'one-size' fits all model cannot be expected for delivery of all nucleic acid therapeutics. Carriers need to be designed to exhibit sensitivity and specificity toward individual targets diseases/indications, and relevant subcellular compartments, each of which possess their own unique challenges. The ability to devise synthetic methods that control the molecular architecture enables the future development that allow for the construction of 'intelligent' designs.
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Técnicas de Transferência de Genes , Polímeros/administração & dosagem , RNA/administração & dosagem , Animais , Humanos , Proteínas/administração & dosagemRESUMO
LewisX (LeX) is a branched trisaccharide Galß1â4(Fucα1â3)GlcNAc that is expressed on many cell surface glycoproteins and plays critical roles in innate and adaptive immune responses. However, efficient synthesis of glycopeptides bearing LeX remains a major limitation for structure-function studies of the LeX determinant. Here we report a total synthesis of a LeX pentasaccharide 1 using a regioselective 1-benzenesulfinyl piperidine/triflic anhydride promoted [3 + 2] glycosylation. The presence of an Fmoc-threonine amino acid facilitates incorporation of the pentasaccharide in solid phase peptide synthesis, providing a route to diverse O-linked LeX glycopeptides. The described approach is broadly applicable to the synthesis of a variety of complex glycopeptides containing O-linked LeX or sialyl LewisX (sLeX).
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Glicopeptídeos/química , Glicopeptídeos/síntese química , Oligossacarídeos/química , Treonina/química , Aminoácidos/química , Glicosilação , Técnicas de Síntese em Fase Sólida/métodosRESUMO
Blockade of P-selectin (P-sel)/PSGL-1 interactions holds significant potential for treatment of disorders of innate immunity, thrombosis and cancer. Current inhibitors remain limited due to low binding affinity or by the recognized disadvantages inherent to chronic administration of antibody therapeutics. Here we report an efficient approach for generating glycosulfopeptide mimics of N-terminal PSGL-1 through development of a stereoselective route for multi-gram scale synthesis of the C2 O-glycan building block and replacement of hydrolytically labile tyrosine sulfates with isosteric sulfonate analogues. Library screening afforded a compound of exceptional stability, GSnP-6, that binds to human P-sel with nanomolar affinity (Kd~22 nM). Molecular dynamics simulation defines the origin of this affinity in terms of a number of critical structural contributions. GSnP-6 potently blocks P-sel/PSGL-1 interactions in vitro and in vivo and represents a promising candidate for the treatment of diseases driven by acute and chronic inflammation.
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Adesão Celular/efeitos dos fármacos , Glicopeptídeos/farmacologia , Glicoproteínas de Membrana/farmacologia , Monócitos/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Selectina-P/antagonistas & inibidores , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Agregação Celular/efeitos dos fármacos , Linhagem Celular , Selectina E/metabolismo , Citometria de Fluxo , Humanos , Técnicas In Vitro , Selectina L/metabolismo , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Masculino , Camundongos , Simulação de Dinâmica Molecular , Monócitos/metabolismo , Músculo Esquelético/metabolismo , Neutrófilos/metabolismo , Selectina-P/metabolismo , Ligação ProteicaRESUMO
Recombinant elastin-like protein (ELP) polymers display several favorable characteristics for tissue repair and replacement as well as drug delivery applications. However, these materials are derived from peptide sequences that do not lend themselves to cell adhesion, migration, or proliferation. This report describes the chemoselective ligation of peptide linkers bearing the bioactive RGD sequence to the surface of ELP hydrogels. Initially, cystamine is conjugated to ELP, followed by the temperature-driven formation of elastomeric ELP hydrogels. Cystamine reduction produces reactive thiols that are coupled to the RGD peptide linker via a terminal maleimide group. Investigations into the behavior of endothelial cells and mesenchymal stem cells on the RGD-modified ELP hydrogel surface reveal significantly enhanced attachment, spreading, migration and proliferation. Attached endothelial cells display a quiescent phenotype.
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Materiais Biocompatíveis/metabolismo , Elastina/metabolismo , Maleimidas/metabolismo , Peptídeos/metabolismo , Compostos de Sulfidrila/metabolismo , Sequência de Aminoácidos , Animais , Materiais Biocompatíveis/farmacologia , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/farmacologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Microscopia Confocal , Dados de Sequência Molecular , Oligopeptídeos/farmacologia , Peptídeos/química , Peptídeos/farmacologia , Sus scrofaRESUMO
Selectins (L, E, and P) are vascular endothelial molecules that play an important role in the recruitment of leukocytes to inflamed tissue. In this regard, P-Selectin glycoprotein-1 (PSGL-1) has been identified as a ligand for P-Selectin. PSGL-1 binds to P-Selectin through the interaction of core-2 O-glycan expressing sialyl Lewis(x) oligosaccharide and the three tyrosine sulfate residues. Herein, we report the synthesis of threonine-linked core-2 O-glycan as an amino acid building block for the synthesis of PSGL-1. This building block was further incorporated in the Fmoc-assisted solid-phase peptide synthesis to provide a portion of the PSGL-1 glycopeptide.