RESUMEN
BACKGROUND/AIMS: Unrestricted increased table salt (NaCl) intake is associated with oxidative stress and inflammation, leading to endothelial dysfunction and atherosclerosis. However, data on salt-induced immunomodulatory effects in the earliest phase of salt loading are scarce. METHODS: In the present study, an animal model of short-term salt loading was employed, including male Sprague Dawley rats consuming a high-salt diet (HSD; 4% NaCl) or standard laboratory chow (low-salt; LSD; 0.4% NaCl) during a 7-day period. The contribution of angiotensin II (ANGII) suppression was tested by adding a group of rats on a high-salt diet receiving ANGII infusions. Samples of peripheral blood/mesenteric lymph node leukocytes, brain blood vessels, and serum samples were processed for flow cytometry, quantitative real-time PCR, total proteome analysis, and multiplex immunoassay. RESULTS: Data analysis revealed the up-regulation of Il 6 gene in the microcirculation of high-salt-fed rats, accompanied by an increased serum level of TNF-alpha cytokine. The high-salt diet resulted in increased proportion of serum mono-unsaturated fatty acids and saturated fatty acids, reduced levels of linoleic (C18:2 ω-6) and α-linolenic (C18:3 ω-3) acid, and increased levels of palmitoleic acid (C16:1 ω-7). The high-salt diet had distinct, lymphoid compartment-specific effects on leukocyte subpopulations, which could be attributed to the increased expression of salt-sensitive SGK-1 kinase. Complete proteome analysis revealed high-salt-diet-induced vascular tissue remodeling and perturbations in energy metabolism. Interestingly, many of the observed effects were reversed by ANGII supplementation. CONCLUSION: Low-grade systemic inflammation induced by a HSD could be related to suppressed ANGII levels. The effects of HSD involved changes in Th17 and Treg cell distribution, vascular wall remodeling, and a shift in lipid and arachidonic acid metabolism.
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Cloruro de Sodio Dietético , Cloruro de Sodio , Ratas , Masculino , Animales , Cloruro de Sodio/farmacología , Ratas Sprague-Dawley , Linfocitos T Reguladores , Ácidos Grasos , Proteoma , Angiotensina II/farmacología , Inflamación , DietaRESUMEN
Doxorubicin (DOX) is one of the most effective cytotoxic agents against malignant diseases. However, the clinical application of DOX is limited, due to dose-related toxicity. The development of DOX nanoformulations that significantly reduce its toxicity and affect the metabolic pathway of the drug requires improved methods for the quantitative determination of DOX metabolites with high specificity and sensitivity. This study aimed to develop a high-throughput method based on high-performance liquid chromatography with fluorescence detection (HPLC-FD) for the quantification of DOX and its metabolites in the urine of laboratory animals after treatment with different DOX nanoformulations. The developed method was validated by examining its specificity and selectivity, linearity, accuracy, precision, limit of detection, and limit of quantification. The DOX and its metabolites, doxorubicinol (DOXol) and doxorubicinone (DOXon), were successfully separated and quantified using idarubicin (IDA) as an internal standard (IS). The linearity was obtained over a concentration range of 0.05-1.6 µg/mL. The lowest limit of detection and limit of quantitation were obtained for DOXon at 5.0 ng/mL and 15.0 ng/mL, respectively. For each level of quality control (QC) samples, the inter- and intra-assay precision was less than 5%. The accuracy was in the range of 95.08-104.69%, indicating acceptable accuracy and precision of the developed method. The method was applied to the quantitative determination of DOX and its metabolites in the urine of rats treated by novel nanoformulated poly(lactic-co-glycolic acid) (DOX-PLGA), and compared with a commercially available DOX solution for injection (DOX-IN) and liposomal-DOX (DOX-MY).
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Doxorrubicina/análogos & derivados , Naftacenos/orina , Orina/química , Animales , Doxorrubicina/farmacocinética , Doxorrubicina/farmacología , Doxorrubicina/orina , Femenino , Masculino , Polietilenglicoles/farmacocinética , Polietilenglicoles/farmacología , Ratas , Ratas WistarRESUMEN
Acetylcholine-induced vasorelaxation (AChIR) and responses to reduced pO2 (hypoxia-induced relaxation (HIR), 0% O2) were assessed in vitro in aortic rings of healthy male Sprague-Dawley rats (N = 252) under hyperbaric (HBO2) protocols. The studied groups consisted of the CTRL group (untreated); the A-HBO2 group (single HBO2; 120 min of 100% O2 at 2.0 bars); the 24H-HBO2 group (examined 24 h after single exposure) and the 4D-HBO2 group (four consecutive days of single HBO2). AChIR, sensitivity to ACh and iNOS expression were decreased in the A-HBO2 group. HIR was prostanoid- and epoxyeicosatrienoic acid (EET)-mediated. HIF-1α expression was increased in the 24H-HBO2 and 4D-HBO2 groups. LW6 (HIF-1α inhibitor) decreased HIR in the 24H-HBO2 group. HBO2 affected the expression of COX-1 and COX-2. CYP2c11 expression was elevated in the 24H-HBO2 and 4D-HBO2 groups. Concentrations of arachidonic acid (AA) metabolites 14(15)-DiHET, 11(12)-DiHET and 8(9)-DiHET were increased in A-HBO2 and 24H-HBO2. An increased concentration of 8(9)-EET was observed in the A-HBO2 and 24h-HBO2 groups vs. the CTRL and 4D-HBO2 groups, and an increased concentration of 5(6)-DiHET was observed in the 24H-HBO2 group vs. the 4D-HBO2 group. The 20-HETE concentration was increased in the A-HBO2 group. All were determined by LC-MS/MS of the aorta. The results show that AChIR in all groups is mostly NO-dependent. HIR is undoubtedly mediated by the CYP450 enzymes' metabolites of AA, whereas HIF-1α contributes to restored HIR. Vasoconstrictor metabolites of CYP450 enzymes contribute to attenuated AChIR and HIR in A-HBO2.
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Aorta/efectos de los fármacos , Ácidos Araquidónicos/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Endotelio/efectos de los fármacos , Oxigenoterapia Hiperbárica/métodos , Estrés Oxidativo/efectos de los fármacos , Vasodilatación/fisiología , Acetilcolina/farmacología , Animales , Aorta/metabolismo , Endotelio/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Vasodilatación/efectos de los fármacosRESUMEN
Multicomponent self-assembled supramolecular nanovesicles based on an amphiphilic derivative of ß-cyclodextrin and phosphatidylcholine liposomes (PC-liposomes) functionalized with four structurally different adamantyl guanidines were prepared and characterized. Incorporation efficiency of the examined adamantyl guanidines as well as size and surface charge of the prepared supramolecular nanovesicles was determined. Changes in the surface charge of the prepared nanovesicles confirmed that guanidinium groups were exposed on the surface. ITC and 1H NMR spectroscopy complemented by molecular dynamics (MD) simulations were used to elucidate the structural data and stability of the inclusion complexes of ß-cyclodextrin and adamantyl guanidines (AG1-5). The results are consistent and point to a significant contribution of the guanylhydrazone residue to the complexation process for AG1 and AG2 with ß-cyclodextrin. In order to evaluate the potential of the self-assembled supramolecular nanomaterial as a nonviral gene delivery vector, fluorescence correlation spectroscopy was used. It showed that the prepared nanovesicles functionalized with adamantyl guanidines AG1-4 effectively recognize and bind the fluorescently labelled DNA. Furthermore, gel electrophoretic assay confirmed the formation of nanoplexes of functionalized nanovesicles and plasmid DNA. These findings together suggest that the designed supramolecular nanovesicles could be successfully applied as nonviral gene delivery vectors.
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Adamantano/análogos & derivados , Portadores de Fármacos/química , Guanidinas/química , Liposomas/química , beta-Ciclodextrinas/química , Línea Celular Tumoral , ADN/química , ADN/genética , Difusión , Técnicas de Transferencia de Gen , Células HEK293 , Humanos , Simulación de Dinámica Molecular , Fosfatidilcolinas/química , PlásmidosRESUMEN
The adamantane moiety is widely applied in design and synthesis of new drug delivery systems and in surface recognition studies. This review focuses on liposomes, cyclodextrins, and dendrimers based on or incorporating adamantane derivatives. Our recent concept of adamantane as an anchor in the lipid bilayer of liposomes has promising applications in the field of targeted drug delivery and surface recognition. The results reported here encourage the development of novel adamantane-based structures and self-assembled supramolecular systems for basic chemical investigations as well as for biomedical application.
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Adamantano/química , Sistemas de Liberación de Medicamentos , Animales , Ciclodextrinas/química , Dendrímeros/química , Portadores de Fármacos/química , Humanos , Liposomas/química , Estructura Molecular , NanotecnologíaRESUMEN
A series of mono and bis-aminoguanidinium adamantane derivatives has been synthesized and incorporated into liposomes. They combine two biomedically significant molecules, the adamantane moiety and the guanidinium group. The adamantane moiety possesses the membrane compatible features while the cationic guanidinium subunit was recognized as a favourable structural feature for binding to complementary molecules comprising phosphate groups. The liposome formulations of adamantyl aminoguanidines were characterized and it was shown that the entrapment efficiency of the examined compounds is significant. In addition, it was demonstrated that liposomes with incorporated adamantyl aminoguanidines effectively recognized the complementary liposomes via the phosphate group. These results indicate that adamantane derivatives bearing guanidinium groups might be versatile tools for biomedical application, from studies of molecular recognition processes to usage in drug formulation and cell targeting.
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Adamantano/síntesis química , Guanidinas/síntesis química , Liposomas/química , Adamantano/química , Animales , Pollos , Guanidinas/química , Microscopía de Contraste de Fase , Conformación Molecular , Tamaño de la Partícula , Electricidad Estática , Sus scrofa , TermodinámicaRESUMEN
The aim of the present study was to encapsulate mannosylated 1-aminoadamantane and mannosylated adamantyltripeptides, namely [(2R)-N-(adamant-1-yl)-3-(α,ß-d-mannopyranosyloxy)-2-methylpropanamide and (2R)-N-[3-(α-d-mannopyranosyloxy)-2-methylpropanoyl]-d,l-(adamant-2-yl)glycyl-l-alanyl-d-isoglutamine] in liposomes. The characterization of liposomes, size and surface morphology was performed using dynamic light scattering (DLS) and atomic force microscopy (AFM). The results have revealed that the encapsulation of examined compounds changes the size and surface of liposomes. After the concanavalin A (ConA) was added to the liposome preparation, increase in liposome size and their aggregation has been observed. The enlargement of liposomes was ascribed to the specific binding of the ConA to the mannose present on the surface of the prepared liposomes. Thus, it has been shown that the adamantyl moiety from mannosylated 1-aminoadamantane and mannosylated adamantyltripeptides can be used as an anchor in the lipid bilayer for carbohydrate moiety exposed on the liposome surface.
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Membrana Dobles de Lípidos/química , Liposomas/química , Manosa/química , Péptidos/química , Biofisica/métodos , Cromatografía/métodos , Concanavalina A/química , Concentración de Iones de Hidrógeno , Lectinas/química , Luz , Microscopía de Fuerza Atómica/métodos , Modelos Químicos , Conformación Molecular , Dispersión de Radiación , Electricidad Estática , Propiedades de Superficie , UltracentrifugaciónRESUMEN
The aim of this biophysical study is to characterize reconstructed membrane vesicles obtained from microalgae in terms of their morphology, properties, composition, and ability to transport a model drug. The reconstructed vesicles were either emptied or non-emptied and exhibited a non-uniform distribution of spherical surface structures that could be associated with surface coat proteins, while in between there were pore-like structures of up to 10 nm that could contribute to permeability. The reconstructed vesicles were very soft and hydrophilic, which could be attributed to their composition. The vesicles were rich in proteins and were mostly derived from the cytoplasm and chloroplasts. We demonstrated that all lipid classes of D. tertiolecta are involved in the formation of the reconstructed membrane vesicles, where they play fundamental role to maintain the vesicle structure. The vesicles appeared to be permeable to calcein, impermeable to FITC-ovalbumin, and semipermeable to FITC-concanavalin A, which may be due to a specific surface interaction with glucose/mannose units that could serve as a basis for the development of drug carriers. Finally, the reconstructed membrane vesicles could pave a new way as sustainable and environmentally friendly marine bioinspired carriers and serve for studies on microtransport of materials and membrane-related processes contributing to advances in life sciences and biotechnology.
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Microalgas , Fluoresceína-5-Isotiocianato , Sistemas de Liberación de Medicamentos , Portadores de Fármacos/química , Proteínas de la MembranaRESUMEN
General adhesion behavior of phospholipid vesicles was examined in a wide range of potentials at the mercury electrode by recording time-resolved adhesion signals. It was demonstrated that adhesion-based detection is sensitive to polar headgroups in phospholipid vesicles. We identified a narrow potential window around the point of zero charge of the electrode where the interaction of polar headgroups of phosphatidylcholine vesicles with the substrate is manifested in the form of bidirectional signals. The bidirectional signal is composed of the charge flow due to the nonspecific interaction of vesicle adhesion and spreading and of the charge flow due to a specific interaction of the negatively charged electrode and the most exposed positively charged choline headgroups. These signals are expected to appear only when the electrode surface charge density is less than the surface charge density of the choline groups at the contact interface. In comparison, for the negatively charged phosphatidylserine vesicles, we identified the potential window at the mercury electrode where charge compensation takes place, and bidirectional signals were not detected.
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Alcanos/química , Electroporación , Fosfatidilcolinas/química , Fosfatidilserinas/química , Liposomas Unilamelares/química , Adsorción , Algoritmos , Electroquímica , Electrodos , Liposomas/química , Mercurio/química , Fosfolípidos/química , TermodinámicaRESUMEN
The aim of this work was to prepare L- and D-(adamant-1-yl)-Gly-L-Ala-D-isoGln peptides in order to study their adjuvant (immunostimulating) activities. Adjuvant activity of adamant-1-yl tripeptides was tested in the mouse model using ovalbumin as an antigen and in comparison to the peptidoglycan monomer (PGM; ß-D-GlcNAc-(1â4)-D-MurNAc-L-Ala-D-isoGln-mesoDAP(εNH(2) )-D-Ala-D-Ala) and structurally related adamant-2-yl tripeptides.
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Adamantano/análogos & derivados , Adamantano/farmacología , Adyuvantes Inmunológicos/química , Adyuvantes Inmunológicos/farmacología , Oligopéptidos/química , Oligopéptidos/farmacología , Adamantano/síntesis química , Adyuvantes Inmunológicos/síntesis química , Animales , Ratones , Oligopéptidos/síntesis química , Ovalbúmina/inmunología , Peptidoglicano/química , Peptidoglicano/farmacologíaRESUMEN
The mannosylated derivative of adamant-1-yl tripeptide (D-(Ad-1-yl)Gly-L-Ala-D-isoGln) was prepared to study the effects of mannosylation on adjuvant (immunostimulating) activity. Mannosylated adamant-1-yl tripeptide (Man-OCH(2) CH(Me)CO-D-(Ad-1-yl)Gly-L-Ala-D-isoGln) is a non-pyrogenic, H(2) O-soluble, and non-toxic compound. Adjuvant activity of mannosylated adamantyl tripeptide was tested in the mouse model with ovalbumin as an antigen and in comparison to the parent tripeptide and peptidoglycan monomer (PGM, ß-D-GlcNAc-(1â4)-D-MurNAc-L-Ala-D-isoGln-mesoDAP(εNH(2) )-D-Ala-D-Ala), a well-known effective adjuvant. The mannosylation of adamantyl tripeptide caused the amplification of its immunostimulating activity in such a way that it was comparable to that of PGM.
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Adamantano/análogos & derivados , Adyuvantes Inmunológicos/química , Inmunización , Manosa/química , Oligopéptidos/química , Adamantano/química , Animales , Modelos Animales de Enfermedad , Glicosilación , Ratones , Estructura Molecular , Ovalbúmina/inmunologíaRESUMEN
The success of vaccination with subunit vaccines often relies on the careful choice of adjuvants. There is great interest in developing new adjuvants that can elicit a cellular immune response. Here, we address this challenge by taking advantage of the synergistic cross-talk between two pattern recognition receptors: nucleotide-binding oligomerization-domain-containing protein 2 (NOD2) and Toll-like receptor 7 (TLR7). We designed two conjugated NOD2/TLR7 agonists, which showed potent immunostimulatory activities in human primary peripheral blood mononuclear cells and murine bone-marrow-derived dendritic cells. One of these, 4, also generated a strong antigen-specific immune response in vivo, with a Th1-polarized profile. Importantly, our study shows that novel NOD2/TLR7 agonists elicit sophisticated and fine-tuned immune responses that are inaccessible to individual NOD2 and TLR7 agonists.
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Leucocitos Mononucleares , Receptor Toll-Like 7 , Humanos , Ratones , Animales , Receptor Toll-Like 7/agonistas , Adyuvantes Inmunológicos/farmacología , Inmunidad Celular , Inmunización , Proteína Adaptadora de Señalización NOD2RESUMEN
NOD2 is an innate immune receptor that constitutes an important target for the development of small molecule immunopotentiators with great potential to be used as vaccine adjuvants. We report here the results of an in vivo study of the adjuvant properties of a desmuramylpeptide NOD2 agonist SG29 and its lipidated analogs featuring an adamantyl moiety or a stearoyl group. These compounds have been synthesized, incorporated into liposomes, and evaluated for their in vivo adjuvant activity. The characterization of liposome formulations of examined compounds revealed that their size increased in comparison to that of empty liposomes. The introduction of a stearoyl or an adamantane lipophilic anchor into the structure of SG29, to produce SG115 and ZSB63, respectively, substantially improved the in vivo adjuvant activity. Of note, the attachment of the stearoyl moiety produced a Th2-biased immune response, while the incorporation of the adamantyl moiety greatly enhanced the production of total IgG but mostly augmented the production of IgG2a antibodies, which indicated a shift toward a Th1 immune response. The identified bona fide capacity of ZSB63 to initiate a cellular immune response thus highlights its untapped potential as an alternative vaccine adjuvant.
RESUMEN
Nano-drug delivery systems may potentially overcome current challenges in the treatment of Parkinson's disease (PD) by enabling targeted delivery and more efficient blood-brain penetration ability. This study investigates novel gold nanoparticles (AuNPs) to be used as delivery systems for L-DOPA and dopamine by considering their binding capabilities in the presence and absence of a model protein, bovine serum albumin (BSA). Four different AuNPs were prepared by surface functionalization with polyethylene glycol (PEG), 1-adamantylamine (Ad), 1-adamantylglycine (AdGly), and peptidoglycan monomer (PGM). Fluorescence and UV-Vis measurements demonstrated the strongest binding affinity and L-DOPA/dopamine loading efficiency for PGM-functionalized AuNPs with negligible impact of the serum protein presence. Thermodynamic analysis revealed a spontaneous binding process between L-DOPA or dopamine and AuNPs that predominantly occurred through van der Waals interactions/hydrogen bonds or electrostatic interactions. These results represent PGM-functionalized AuNPs as the most efficient at L-DOPA and dopamine binding with a potential to become a drug-delivery system for neurodegenerative diseases. Detailed investigation of L-DOPA/dopamine interactions with different AuNPs was described here for the first time. Moreover, this study highlights a cost- and time-effective methodology for evaluating drug binding to nanomaterials.
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Nanopartículas del Metal , Enfermedad de Parkinson , Dopamina , Sistemas de Liberación de Medicamentos , Oro , Humanos , Levodopa , Enfermedad de Parkinson/tratamiento farmacológicoRESUMEN
We report on the design, synthesis, and biological evaluation of a series of nucleotide-binding oligomerization-domain-containing protein 2 (NOD2) desmuramylpeptide agonists with improved in vitro and in vivo adjuvant properties. We identified two promising compounds: 68, a potent nanomolar in vitro NOD2 agonist, and the more lipophilic 75, which shows superior adjuvant activity in vivo. Both compounds had immunostimulatory effects on peripheral blood mononuclear cells at the protein and transcriptional levels, and augmented dendritic-cell-mediated activation of T cells, while 75 additionally enhanced the cytotoxic activity of peripheral blood mononuclear cells against malignant cells. The C18 lipophilic tail of 75 is identified as a pivotal structural element that confers in vivo adjuvant activity in conjunction with a liposomal delivery system. Accordingly, liposome-encapsulated 75 showed promising adjuvant activity in mice, surpassing that of muramyl dipeptide, while achieving a more balanced Th1/Th2 immune response, thus highlighting its potential as a vaccine adjuvant.
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Acetilmuramil-Alanil-Isoglutamina/química , Adyuvantes Inmunológicos/química , Proteína Adaptadora de Señalización NOD2/agonistas , Acetilmuramil-Alanil-Isoglutamina/metabolismo , Acetilmuramil-Alanil-Isoglutamina/farmacología , Adyuvantes Inmunológicos/metabolismo , Adyuvantes Inmunológicos/farmacología , Animales , Formación de Anticuerpos/efectos de los fármacos , Línea Celular , Diseño de Fármacos , Humanos , Inmunoglobulina G/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Liposomas/química , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Proteína Adaptadora de Señalización NOD2/metabolismo , Ovalbúmina/inmunología , Relación Estructura-Actividad , Células TH1/citología , Células TH1/inmunología , Células TH1/metabolismo , Células Th2/citología , Células Th2/inmunología , Células Th2/metabolismoRESUMEN
Plasma membrane vesicles can be effective, non-toxic carriers for microscale material transport, provide a convenient model for probing membrane-related processes, since intracellular biochemical processes are eliminated. We describe here a fine-tuned protocol for isolating ghost plasma membrane vesicles from the unicellular alga Dunaliella tertiolecta, and preliminary characterization of their structural features and permeability properties, with comparisons to giant unilamellar phospholipid vesicles. The complexity of the algal ghost membrane vesicles reconstructed from the native membrane material released after hypoosmotic stress lies between that of phospholipid vesicles and cells. AFM structural characterization of reconstructed vesicles shows a thick envelope and a nearly empty vesicle interior. The surface of the envelope contains a heterogeneous distribution of densely packed, nanometer-scale globules and pore-like structures which may be derived from surface coat proteins. Confocal fluorescence imaging reveals the highly pigmented photosynthetic apparatus located within the thylakoid membrane and retained in the vesicle membrane. Transport of the fluorescent dye calcein into ghost and giant unilamellar vesicles reveals significant differences in permeability. Expanded knowledge of this unique membrane system will contribute to the design of marine bio-inspired carriers for advanced biotechnological applications.
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Membrana Celular/metabolismo , Chlorophyceae/citología , Fluorescencia , Liposomas Unilamelares/metabolismo , Fraccionamiento Celular , Permeabilidad de la Membrana CelularRESUMEN
Various amounts of Ovalbumin (OVA) were encapsulated into positively and negatively charged multilamellar liposomes, with the aim to investigate the entrapment efficiency in different buffers and to study their effects on the liposome size and zeta potential. Results showed that the entrapment efficiency of OVA in anionic liposomes was the same in 10 mM Phosphate Buffer (PB) as in Phosphate-Buffered Saline (PBS; PB + 0.15 M NaCl). Also, liposome size was approximately 1200 nm for all anionic liposomes incorporating OVA. The entrapment efficiency of OVA in cationic liposomes was highly dependent on ionic strength. The size of cationic liposomes was approximately 1200 nm in PBS, regardless of protein content, but increased with the amount of the incorporated protein in PB. Aggregation of cationic liposomes in PB was observed when the mass of the protein was 2.5 mg or greater. The zeta potential of anionic liposomes was negative and of cationic liposomes positive in the whole range of protein mass tested. These results show how different compositions of lipid and aqueous phases can be used to vary the entrapment efficiency, liposome size, and zeta potential--the factors that are of great importance for the use of liposomes as drug carriers.
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Liposomas/química , Ovalbúmina/química , Aniones/química , Cationes/química , Electroquímica , Liposomas/síntesis química , Nanopartículas/química , Nanotecnología/métodos , Tamaño de la Partícula , Sensibilidad y Especificidad , Propiedades de SuperficieRESUMEN
Muramyl dipeptide (MDP), a fragment of bacterial peptidoglycan, has long been known as the smallest fragment possessing adjuvant activity, on the basis of its agonistic action on the nucleotide-binding oligomerization domain-containing protein 2 (NOD2). There is a pressing need for novel adjuvants, and NOD2 agonists provide an untapped source of potential candidates. Here, we report the design, synthesis, and characterization of a series of novel acyl tripeptides. A pivotal structural element for molecular recognition by NOD2 has been identified, culminating in the discovery of compound 9, the most potent desmuramylpeptide NOD2 agonist to date. Compound 9 augmented pro-inflammatory cytokine release from human peripheral blood mononuclear cells in synergy with lipopolysaccharide. Furthermore, it was able to induce ovalbumin-specific IgG titers in a mouse model of adjuvancy. These findings provide deeper insights into the structural requirements of desmuramylpeptides for NOD2-activation and highlight the potential use of NOD2 agonists as adjuvants for vaccines.
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Acetilmuramil-Alanil-Isoglutamina/agonistas , Adyuvantes Inmunológicos/síntesis química , Adyuvantes Inmunológicos/farmacología , Inmunidad Innata/efectos de los fármacos , Proteína Adaptadora de Señalización NOD2/efectos de los fármacos , Animales , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Línea Celular Tumoral , Citocinas/metabolismo , Diseño de Fármacos , Descubrimiento de Drogas , Femenino , Humanos , Inmunoglobulina G/biosíntesis , Lipopolisacáridos/farmacología , Ratones , Modelos Moleculares , Conformación Molecular , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Proteína Adaptadora de Señalización NOD2/metabolismo , Relación Estructura-ActividadRESUMEN
Ovalbumin (OVA) has been used continuously as the model antigen in numerous studies of immune reactions and antigen processing, very often encapsulated into liposomes. The purpose of this work was to study the possible interactions of spin-labelled OVA and lipids in liposomal membranes using electron spin resonance (ESR) spectroscopy. OVA was covalently spin-labelled with 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO-maleimide), characterized and encapsulated into multilamellar, negatively charged liposomes. ESR spectra of this liposomal preparation gave evidence for the interaction of OVA with the lipid bilayers. Such an interaction was also evidenced by the ESR spectra of liposomal preparation containing OVA, where liposomes were spin-labelled with n-doxyl stearic acids. The spin-labelled OVA retains its property to bind specific anti-OVA antibodies, as shown by ESR spectroscopy, but also in ELISA for specific anti-OVA IgG.
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Anticuerpos/inmunología , Liposomas/química , Liposomas/metabolismo , Ovalbúmina/inmunología , Ovalbúmina/metabolismo , Marcadores de Spin , Animales , Pollos , Espectroscopía de Resonancia por Spin del Electrón , Concentración de Iones de Hidrógeno , Ovalbúmina/análisis , Ovalbúmina/aislamiento & purificación , Peptidoglicano/metabolismo , Porcinos , TemperaturaRESUMEN
Multivalent mannosyl-lipoconjugates may be of interest for glycosylation of liposomes and targeted drug delivery because the mannose specifically binds to C-type lectin receptors on the particular cells. In this paper syntheses of two types of novel O-mannosides are presented. Conjugates 1 and 2 with a COOH- and NH2-functionalized spacer and the connection to a lysine and FmocNH-PEG-COOH, are described. The coupling reactions of prepared intermediates 6 and 4 with a PEGylated-DSPE or palmitic acid, respectively, are presented. Compounds 5, mono-, 8, di- and 12, tetravalent mannosyl-lipoconjugates, were synthesized. The synthesized compounds were incorporated into liposomes and liposomal preparations featuring exposed mannose units were characterized. Carbohydrate liposomal quartz crystal microbalance based assay has been established for studying carbohydrate-lectin binding. It was demonstrated that liposomes with incorporated mannosyl-lipoconjugates were effectively recognized by Con A and have great potential to be used for targeted liposomal drug delivery systems.