RESUMO
Insertion of high molecular weight messenger RNA (mRNA) into lyotropic lipid phases as model systems for controlled release formulations for the mRNA was investigated. Low fractions of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were used as an anchor to load the mRNA into a lamellar lipid matrix. Dispersions of zwitterionic lipid in the aqueous phase in the presence of increasing fractions of mRNA and cationic lipid were prepared, and the molecular organization was investigated as a function of mRNA and cationic lipid fraction. Insertion of both cationic lipid and mRNA was clearly proven from the physicochemical characteristics. The d-spacing of the lipid bilayers, as determined by small-angle X-ray scattering (SAXS) measurements, responded sensitively to the amount of inserted DOTAP and mRNA. A concise model of the insertion of the mRNA in the lipid matrices was derived, indicating that the mRNA was accommodated in the aqueous slab between lipid bilayers. Depending on the DOTAP and mRNA fraction, a different excess of water was present in this slab. Results from further physicochemical characterization, including determination of free and bound mRNA, zeta potential, and calorimetry data, were in line with this assumption. The structure of these concentrated lipid/mRNA preparations was maintained upon dilution. The functionality of the inserted mRNA was proven by cell culture experiments using C2C12 murine myoblast cells with the luciferase-encoding mRNA. The described lipid phases as carriers for the mRNA may be applicable for different routes of local administration, where control of the release kinetics and the form of the released mRNA (bound or free) is required.
Assuntos
Portadores de Fármacos/química , Composição de Medicamentos/métodos , RNA Mensageiro/administração & dosagem , Animais , Cátions/química , Linhagem Celular , Preparações de Ação Retardada/administração & dosagem , Preparações de Ação Retardada/química , Bicamadas Lipídicas/química , Lipossomos , Luciferases/genética , Camundongos , Modelos Moleculares , Mioblastos , RNA Mensageiro/síntese química , Espalhamento a Baixo Ângulo , Transfecção/métodos , Difração de Raios XRESUMO
Targeting mRNA to eukaryotic cells is an emerging technology for basic research and provides broad applications in cancer immunotherapy, vaccine development, protein replacement, and in vivo genome editing. Although a plethora of nanoparticles for efficient mRNA delivery exists, in vivo mRNA targeting to specific organs, tissue compartments, and cells remains a major challenge. For this reason, methods for reporting the in vivo targeting specificity of different mRNA nanoparticle formats will be crucial. Here, we describe a straightforward method for monitoring the in vivo targeting efficiency of mRNA-loaded nanoparticles in mice. To achieve accurate mRNA delivery readouts, we loaded lipoplex nanoparticles with Cre-recombinase-encoding mRNA and injected these into commonly used Cre reporter mouse strains. Our results show that this approach provides readouts that accurately report the targeting efficacy of mRNA into organs, tissue structures, and single cells as a function of the used mRNA delivery system. The method described here establishes a versatile basis for determining in vivo mRNA targeting profiles and can be systematically applied for testing and improving mRNA packaging formats.
Assuntos
Nanopartículas/química , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Animais , Cromatografia Líquida , Lipossomos/química , Espectrometria de Massas , Camundongos , Tamanho da PartículaRESUMO
We present the preclinical pharmacology of BNT142, a lipid nanoparticle (LNP)-formulated RNA (RNA-LNP) encoding a T cell-engaging bispecific antibody that monovalently binds the T cell marker CD3 and bivalently binds claudin 6 (CLDN6), an oncofetal antigen that is absent from normal adult tissue but expressed on various solid tumors. Upon BNT142 RNA-LNP delivery in cell culture, mice, and cynomolgus monkeys, RNA is translated, followed by self-assembly into and secretion of the functional bispecific antibody RiboMab02.1. In vitro, RiboMab02.1 mediated CLDN6 target cell-specific activation and proliferation of T cells, and potent target cell killing. In mice and cynomolgus monkeys, intravenously administered BNT142 RNA-LNP maintained therapeutic serum concentrations of the encoded antibody. Concentrations of RNA-encoded RiboMab02.1 were maintained longer in circulation in mice than concentrations of directly injected, sequence-identical protein. Weekly injections of mice with BNT142 RNA-LNP in the 0.1- to 1-µg dose range were sufficient to eliminate CLDN6-positive subcutaneous human xenograft tumors and increase survival over controls. Tumor regression was associated with an influx of T cells and depletion of CLDN6-positive cells. BNT142 induced only transient and low cytokine production in CLDN6-positive tumor-bearing mice humanized with peripheral blood mononuclear cells (PBMCs). No signs of adverse effects from BNT142 RNA-LNP administration were observed in mice or cynomolgus monkeys. On the basis of these and other findings, a phase 1/2 first-in-human clinical trial has been initiated to assess the safety and preliminary efficacy of BNT142 RNA-LNP in patients with CLDN6-positive advanced solid tumors (NCT05262530).
Assuntos
Anticorpos Biespecíficos , Claudinas , Macaca fascicularis , Linfócitos T , Animais , Humanos , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/farmacocinética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Claudinas/metabolismo , Camundongos , RNA/metabolismo , Feminino , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Lipossomos , NanopartículasRESUMO
mRNA based infectious disease vaccines have opened the venue for development of novel nucleic acids-based therapeutics. For all mRNA therapeutics dedicated delivery systems are required, where different functionalities and targeting abilities need to be optimized for the respective applications. One option for advanced formulations with tailored properties are lipid-polymer hybrid nanoparticles with complex nanostructure, which allow to combine features of several already well described nucleic acid delivery systems. Here, we explored hyaluronic acid (HA) as coating of liposome-mRNA complexes (LRCs) to investigate effects of the coating on surface charge, physicochemical characteristics and biological activity. HA was electrostatically attached to positively charged complexes, forming hybrid LRCs (HLRCs). At different N/P ratios, physico-chemical characterization of the two sets of particles showed similarity in size (around 200 nm) and mRNA binding abilities, while the presence of the HA shell conferred a negative surface charge to otherwise positive complexes. High transfection efficiency of LRCs and HLRCs in vitro has been obtained in THP-1 and human monocytes derived from PBMC, an interesting target cell population for cancer and immune related pathologies. In mice, quantitative biodistribution of radiolabeled LRC and HLRC particles, coupled with bioluminescence studies to detect the protein translation sites, hinted towards both particles' accumulation in the hepatic reticuloendothelial system (RES). mRNA translated proteins though was found mainly in the spleen, a major source for immune cells, with preference for expression in macrophages. The results showed that surface modifications of liposome-mRNA complexes can be used to fine-tune nanoparticle physico-chemical characteristics. This provides a tool for assembly of stable and optimized nanoparticles, which are prerequisite for future therapeutic interventions using mRNA-based nanomedicines.
Assuntos
Nanopartículas , Ácidos Nucleicos , Camundongos , Humanos , Animais , Lipossomos/química , Distribuição Tecidual , Leucócitos Mononucleares , Polímeros/química , Nanopartículas/química , TransfecçãoRESUMO
INTRODUCTION: BNT162b1 is a lipid nanoparticle-formulated, nucleoside-modified mRNA SARS-CoV-2 vaccine. Here, we report safety and immune persistence data following a primary two-dose vaccination schedule administered 21 days apart. METHODS: Immune persistence was determined at month 3 in 72 younger participants (aged 18-55 years) and at month 6 in 70 younger and 69 older participants (aged 65-85 years). RESULTS: In younger participants, neutralizing antibody (nAb) geometric mean titers (GMTs) for the 10 and 30 µg dose levels declined from 233 and 254 (21 days after dose 2) to 55 and 87 at month 3, respectively, and to 16 and 27 at month 6, respectively. In older participants, nAb GMTs declined from 80 and 160 (21 days after dose 2) to 10 and 21 at month 6. Overall, higher antibody titers were observed in younger participants, and the 30 µg dose induced higher levels of nAb, which declined more slowly by month 6. No serious adverse events were reported in the vaccine group. CONCLUSION: This study showed BNT162b1 maintains a favorable safety profile in younger and older participants in the 6 months after vaccination. This study further extends our understanding of immune persistence and the safety of the BNT162b1 vaccine as a candidate vaccine in the BioNTech pipeline. TRIAL REGISTRATION NUMBER: NCT04523571, registered August 21, 2020.
Assuntos
Vacina BNT162 , COVID-19 , Vacinas , Adulto , Idoso , Anticorpos Neutralizantes , Vacina BNT162/efeitos adversos , COVID-19/prevenção & controle , Vacinas contra COVID-19/efeitos adversos , China , Método Duplo-Cego , Humanos , Lipossomos , Nanopartículas , RNA Mensageiro , SARS-CoV-2 , VacinaçãoRESUMO
A study, where uranium was online enriched and laser diode spectrometry was determined, was performed with some bottled mineral water samples. A uranium-imprinted polymer (PMDU) was synthesized and characterized for preparation of a specific adsorbent of UO22+ cations. The homemade system preconcentrated uranium in PMDU resin and determined using an Arsenazo III complex at 650â¯nm in a combined laser diode spectrometer. All analytical parameters of the system were optimized at 5.5 of the retention pH, 6.0â¯N HClO4 of eluent concentration, 0.05% of Arsenazo III complex, and a 40â¯mm coil length. The effect of interferent ions was also investigated and LOD and LOQ values were found to be 0.54 and 1.80â¯ngâ¯mL-1 respectively. Sample throughput was 12â¯h-1, the preconcentration factor was 50, and RSD% value was 1.1. Certified reference materials of TMDA 52.3 and TMDA 62.2 were quantitatively analyzed and the proposed method was successfully applied to the bottled mineral water samples.
Assuntos
Águas Minerais , Urânio , Lasers , Polímeros , EspectrofotometriaRESUMO
Hybrid nanoparticles from lipidic and polymeric components were assembled to serve as vehicles for the transfection of messenger RNA (mRNA) using different portions of the cationic lipid DOTAP (1,2-Dioleoyl-3-trimethylammonium-propane) and the cationic biopolymer protamine as model systems. Two different sequential assembly approaches in comparison with a direct single-step protocol were applied, and molecular organization in correlation with biological activity of the resulting nanoparticle systems was investigated. Differences in the structure of the nanoparticles were revealed by thorough physicochemical characterization including small angle neutron scattering (SANS), small angle X-ray scattering (SAXS), and cryogenic transmission electron microscopy (cryo-TEM). All hybrid systems, combining lipid and polymer, displayed significantly increased transfection in comparison to lipid/mRNA and polymer/mRNA particles alone. For the hybrid nanoparticles, characteristic differences regarding the internal organization, release characteristics, and activity were determined depending on the assembly route. The systems with the highest transfection efficacy were characterized by a heterogenous internal organization, accompanied by facilitated release. Such a system could be best obtained by the single step protocol, starting with a lipid and polymer mixture for nanoparticle formation.
Assuntos
Biopolímeros/química , Lipídeos/química , Nanopartículas/química , RNA Mensageiro/metabolismo , Transfecção/métodos , Animais , Linhagem Celular , Ácidos Graxos Monoinsaturados/química , Feminino , Heparina/química , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Imagem Óptica , Tamanho da Partícula , Compostos de Amônio Quaternário/química , RNA Mensageiro/químicaRESUMO
The development of nucleic acid based vaccines against cancer has gained considerable momentum through the advancement of modern sequencing technologies and on novel RNA-based synthetic drug formats, which can be readily adapted following identification of every patient's tumor-specific mutations. Furthermore, affordable and individual 'on demand' production of molecularly optimized vaccines should allow their application in large groups of patients. This has resulted in the therapeutic concept of an active personalized cancer vaccine, which has been brought into clinical testing. Successful trials have been performed by intranodal administration of sterile isotonic solutions of synthetic RNA vaccines. The second generation of RNA vaccines which is currently being developed encompasses intravenously injectable RNA nanoparticle formulations (lipoplexes), made up from lipid excipients, denoted RNA(LIP). A first product that has made its way from bench to bedside is a therapeutic vaccine for intravenous administration based on a fixed set of four RNA lipoplex drug products, each encoding for one shared tumor antigen (Lipoplex Melanoma RNA Immunotherapy, 'Lipo-MERIT'). This article describes the steps for translating these novel RNA nanomedicines into clinical trials.
Assuntos
Vacinas Anticâncer , Lipossomos/química , Melanoma/terapia , Nanopartículas/química , RNA Mensageiro/química , RNA/química , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Ensaios Clínicos como Assunto , Excipientes , Humanos , Imunoterapia/métodos , Melanoma/imunologia , Nanomedicina , Nanopartículas/uso terapêutico , Medicina de Precisão , RNA/administração & dosagem , RNA/imunologia , RNA Mensageiro/administração & dosagem , RNA Mensageiro/farmacologia , RNA Mensageiro/uso terapêuticoRESUMO
Fundamental development of a biocompatible and degradable nanocarrier platform based on hydroxyethyl starch (HES) is reported. HES is a derivative of starch and possesses both high biocompatibility and improved stability against enzymatic degradation; it is used to prepare nanocapsules via the polyaddition reaction at the interface of water nanodroplets dispersed in an organic miniemulsion. The synthesized hollow nanocapsules can be loaded with hydrophilic guests in its aqueous core, tuned in size, chemically functionalized in various pathways, and show high shelf life stability. The surface of the HES nanocapsules is further functionalized with poly(ethylene glycol) via different chemistries, which substantially enhanced blood half-life time. Importantly, methods for precise and reliable quantification of the degree of functionalization are also introduced, which enable the precise control of the chemistry on the capsules' surface. The stealth properties of these capsules is studied both in-vitro and in-vivo. The functionalized nanocapsules serve as a modular platform for specific cell targeting, as they show no unspecific up-taken by different cell types and show very long circulating time in blood (up to 72 h).
Assuntos
Materiais Biocompatíveis/química , Portadores de Fármacos/química , Teste de Materiais , Nanocápsulas/química , Polissacarídeos/química , Adsorção , Animais , Cicloexanos/química , Feminino , Citometria de Fluxo , Meia-Vida , Humanos , Derivados de Hidroxietil Amido/síntese química , Derivados de Hidroxietil Amido/química , Leucócitos/citologia , Camundongos Endogâmicos BALB C , Nanocápsulas/ultraestrutura , Polietilenoglicóis/química , Distribuição Tecidual , Água/químicaRESUMO
A new on-line separation/preconcentration system was developed for the determination of Cu(II) ions by flame atomic absorption spectrometry in urine samples. A newly synthesized chelating resin, by anchoring eriochrome blue black R reagent to Amberlite XAD-16 resin, was used as a packing material for the selective separation/preconcentration of Cu(II) ions. The influence of the parameters on the determination of Cu(II) ions such as pH of sample solution, amount of the resin, eluent type, interfering ions and flow variables was studied. The detection limit of the method was 1.0 µg L(-1) while precision was 2.3% (n=15) at 50 µg L(-1) Cu(II) level. The adsorption capacity of the resin was 217 µg g(-1) Cu(II). The accuracy of the method was proven using TMDA-64 standard lake water and synthetic urine sample. The developed method has been applied successfully to the determination of copper in urine with satisfactory results.
Assuntos
Cobre/urina , Extração em Fase Sólida , Espectrofotometria Atômica , Adsorção , Compostos Azo/química , Quelantes/química , Água Doce , Humanos , Concentração de Íons de Hidrogênio , Polímeros/química , Água/químicaRESUMO
A new on-line flow injection (FI) procedure for the sequential separation, preconcentration and speciation of Cr(III)/Cr(VI) species in different matrices is described based on the combining of solid phase extraction and flame atomic absorption spectrometry (FAAS). Poly 2-(5-methylisoxazol)methacrylamide-co-2-acrylamido-2-methyl-1-propanesulfonic acid-co-divinyl-benzene and Dowex 21K resins were used as chelating and ion-exchange materials for the separation/preconcentration of Cr(III) and Cr(VI) ions, respectively. Trace amounts of chromium retained on the resins were eluted sequentially with HNO3 and then introduced directly to the nebulizer-burner system of FAAS. The optimum conditions such as pH of the sample solution, amount of the resin, volume of the sample and interfering ions, which are effective on the quantitative recovery of the analytes, were investigated for sequential determination of Cr(III) and Cr(VI) ions. The preconcentration factors were found to be 48 and 30 for Cr(III) and Cr(VI), and the detection limits corresponding to three times the standard deviation of the blank (3s/b) were 0.05 and 0.3 µg L(-1), respectively. The method was verified by analyzing a certified reference material. The proposed method was applied to the determination based on the speciation of chromium in various real samples with satisfactory results.