RESUMO
Inhaled delivery of mRNA has the potential to treat a wide variety of diseases. However, nebulized mRNA lipid nanoparticles (LNPs) face several unique challenges including stability during nebulization and penetration through both cellular and extracellular barriers. Here we develop a combinatorial approach addressing these barriers. First, we observe that LNP formulations can be stabilized to resist nebulization-induced aggregation by altering the nebulization buffer to increase the LNP charge during nebulization, and by the addition of a branched polymeric excipient. Next, we synthesize a combinatorial library of ionizable, degradable lipids using reductive amination, and evaluate their delivery potential using fully differentiated air-liquid interface cultured primary lung epithelial cells. The final combination of ionizable lipid, charge-stabilized formulation and stability-enhancing excipient yields a significant improvement in lung mRNA delivery over current state-of-the-art LNPs and polymeric nanoparticles.
Assuntos
Excipientes , Nanopartículas , Diferenciação Celular , Polímeros , RNA Mensageiro/genética , RNA Interferente PequenoRESUMO
Developing safe and effective nanoparticles for the delivery of messenger RNA (mRNA) is slow and expensive, partly due to the lack of predictive power of in vitro screening methods and the low-throughput nature of in vivo screening. While DNA barcoding and batch analysis present methods for increasing in vivo screening throughput, they can also result in incomplete or misleading measures of efficacy. Here, we describe a high-throughput and accurate method for the screening of pooled nanoparticle formulations within the same animal. The method uses liquid chromatography with tandem mass spectrometry to detect peptide barcodes translated from mRNAs in nanoparticle-transfected cells. We show the method's applicability by evaluating a library of over 400 nanoparticle formulations with 384 unique ionizable lipids using only nine mice to optimize the formulation of a biodegradable lipid nanoparticle for mRNA delivery to the liver. Barcoding lipid nanoparticles with peptide-encoding mRNAs may facilitate the rapid development of nanoparticles for mRNA delivery to specific cells and tissues.
Assuntos
Lipossomos , Nanopartículas , Animais , Camundongos , RNA Mensageiro/química , Nanopartículas/química , PeptídeosRESUMO
Recent medical advances have exploited the ability to address a given disease at the underlying level of transcription and translation. These treatment paradigms utilize nucleic acids - including short interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides (ASO), and messenger RNA (mRNA) - to achieve a desired outcome ranging from gene knockdown to induced expression of a selected target protein. Towards this end, numerous strategies for encapsulation or stabilization of various nucleic acid structures have been developed in order to achieve intracellular delivery. In this review, we discuss several therapeutic applications of nucleic acids directed towards specific diseases and tissues of interest, in particular highlighting recent technologies which have reached late-stage clinical trials and received FDA approval.
Assuntos
Sistemas de Liberação de Medicamentos/tendências , Técnicas de Transferência de Genes/tendências , Ácidos Nucleicos/administração & dosagem , Ácidos Nucleicos/genética , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/genética , Animais , COVID-19/genética , COVID-19/metabolismo , COVID-19/terapia , Ensaios Clínicos como Assunto/métodos , Aprovação de Drogas , Sistemas de Liberação de Medicamentos/métodos , Hepatite/genética , Hepatite/metabolismo , Hepatite/terapia , Humanos , MicroRNAs/administração & dosagem , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/terapia , Ácidos Nucleicos/metabolismo , Oligonucleotídeos Antissenso/metabolismo , RNA Mensageiro/administração & dosagem , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
Catalytic enantioselective addition of N-heteroarenes to terminal and internal 1,3-dienes is reported. Reactions are promoted by 5 mol % of Rh catalyst supported by a new chiral pincer carbodicarbene ligand that delivers allylic substituted arenes in up to 95% yield and up to 98:2 er. Mechanistic and X-ray evidence is presented that supports that the reaction proceeds via a Rh(III)-η3-allyl.
RESUMO
Catalytic intermolecular hydroalkylation of dienes with silyloxyfuran nucleophiles is reported. Reactions are catalyzed by 5 mol % of a (CDC)-Rh complex and proceed in up to 87% yield and 6:1 dr (syn/anti) to provide allylic butenolides bearing vicinal stereocenters. Reactions proceed with terminal aryl and alkyl dienes and with modified silyl enol ether nucleophiles including a thiophenone variant. Utility of the products is demonstrated in the synthesis of a polypropionate anti,syn-stereotriad.
RESUMO
Catalytic C-H activation and hydroamination represent two important strategies for eco-friendly chemical synthesis with high atom efficiency and reduced waste production. Combining both C-H activation and hydroamination in a cascade process, preferably with a single catalyst, would allow rapid access to valuable nitrogen-containing molecules from readily available building blocks. Here we report a single metal catalyst-based approach for N-heterocycle construction by tandem C-H functionalization and alkene hydroamination. A simple catalyst system of cationic rhodium(I) precursor and phosphine ligand promotes redox-neutral [4+2] annulation between N-H aromatic ketimines and internal alkynes to form multi-substituted 3,4-dihydroisoquinolines (DHIQs) in high chemoselectivity over competing annulation processes, exclusive cis-diastereoselectivity, and distinct regioselectivity for alkyne addition. This study demonstrates the potential of tandem C-H activation and alkene hydrofunctionalization as a general strategy for modular and atom-efficient assembly of six-membered heterocycles with multiple chirality centres.
RESUMO
A modular and atom-efficient synthesis of 2-aza-1,3-butadiene derivatives has been developed via nickel-catalyzed intermolecular coupling between internal alkynes and aromatic N-H ketimines. This novel alkyne hydroimination process is promoted by a catalyst system of a Ni(0) precursor ([Ni(cod)2]), N-heterocyclic carbene (NHC) ligand (IPr), and Cs2CO3 additive. The exclusive formation of (Z)-enamine stereoisomers is consistent with a proposed anti-iminometalation of alkyne by π-complexation with Ni(0) and subsequent attack by the N-H ketimine nucleophile. An NHC-ligated Ni(0) π-imine complex, [(IPr)Ni(η(1)-HNâCPh2)(η(2)-HNâCPh2)], was independently synthesized and displayed improved reactivity as the catalyst precursor.
RESUMO
Assisted by a partially aqueous media, a catalyst system of [Rh(cod)(OH)](2) and DPPBenzene ligand effectively promotes direct conjugate additions by perfluoroarenes. This formal C-H alkylation process represents a rare example of olefin hydroarylation with electron-deficient arenes. The catalyst system can be modified to selectively form the corresponding olefination products under anhydrous conditions.