RESUMO
The complex genetic diversity of chronic lymphocytic leukemia (CLL) makes it difficult to determine the effective and durable therapy beneficial to patients. During the several past years' significant insights in the biology of the disease and its treatment have been made, allowing for the identification of promising novel therapeutic agents. The investigation of signaling pathways to understand the biological character of CLL together with the development of molecular profiling is key in personalized approach in therapy for this disease. As it was already proven, maltotriose (M3) modified fourth generation poly(propylene imine) dendrimers (PPI-G4) modulate BCR, TRAIL and WNT signaling pathway gene expression in CLL cells and strongly influence their survival by inducing apoptosis and inhibiting proliferation. The aim of this study was to evaluate the influence of PPI-G4-M3 dendrimers on NFκB pathway gene expression in CLL (MEC-1) cells with 60â¯K microarray, as it is one of the major factors in the pathogenesis of B-cell neoplasms. The findings were compared with those obtained with Fludarabine (FA) and the results indicate that PPI-G4-M3 dendrimers affect the expression of the examined genes and exert comparable effect on the CLL cells to FA. Dendrimers are one of the most potent groups of nanometer-sized macromolecules for closing the gap between the present ineffective treatment and the future effective personalized therapy due to their potential versatile biological properties.
Assuntos
Dendrímeros/química , Leucemia Linfoide/metabolismo , Nanopartículas/química , Nanopartículas/toxicidade , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , NF-kappa B , Análise Serial de Proteínas , Transdução de Sinais/fisiologia , Transcriptoma , Vidarabina/análogos & derivados , Vidarabina/farmacologiaRESUMO
The influence of maltose-modified poly(propylene imine) (PPI) dendrimers on dimyristoylphosphatidylcholine (DMPC) or dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) (3%) liposomes was studied. Fourth generation (G4) PPI dendrimers with primary amino surface groups were partially (open shell glycodendrimers - OS) or completely (dense shell glycodendrimers - DS) modified with maltose residues. As a model membrane, two types of 100nm diameter liposomes were used to observe differences in the interactions between neutral DMPC and negatively charged DMPC/DMPG bilayers. Interactions were studied using fluorescence spectroscopy to evaluate the membrane fluidity of both the hydrophobic and hydrophilic parts of the lipid bilayer and using differential scanning calorimetry to investigate thermodynamic parameter changes. Pulsed-filed gradient NMR experiments were carried out to evaluate common diffusion coefficient of DMPG and DS PPI in D2O when using below critical micelle concentration of DMPG. Both OS and DS PPI G4 dendrimers show interactions with liposomes. Neutral DS dendrimers exhibit stronger changes in membrane fluidity compared to OS dendrimers. The bilayer structure seems more rigid in the case of anionic DMPC/DMPG liposomes in comparison to pure and neutral DMPC liposomes. Generally, interactions of dendrimers with anionic DMPC/DMPG and neutral DMPC liposomes were at the same level. Higher concentrations of positively charged OS dendrimers induced the aggregation process with negatively charged liposomes. For all types of experiments, the presence of NaCl decreased the strength of the interactions between glycodendrimers and liposomes. Based on NMR diffusion experiments we suggest that apart from electrostatic interactions for OS PPI hydrogen bonds play a major role in maltose-modified PPI dendrimer interactions with anionic and neutral model membranes where a contact surface is needed for undergoing multiple H-bond interactions between maltose shell of glycodendrimers and surface membrane of liposome.
Assuntos
Dendrímeros/química , Bicamadas Lipídicas/química , Maltose/química , Lipídeos de Membrana/química , Polipropilenos/química , Varredura Diferencial de Calorimetria , Dendrímeros/metabolismo , Dimiristoilfosfatidilcolina/química , Dimiristoilfosfatidilcolina/metabolismo , Difenilexatrieno/química , Polarização de Fluorescência , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/metabolismo , Lipossomos/química , Lipossomos/metabolismo , Espectroscopia de Ressonância Magnética , Maltose/metabolismo , Fluidez de Membrana , Lipídeos de Membrana/metabolismo , Fosfatidilgliceróis/química , Fosfatidilgliceróis/metabolismo , Polipropilenos/metabolismo , Eletricidade EstáticaRESUMO
The generation of homogeneously glycosylated proteins is essential for defining glycoform-specific activity and improving protein-based therapeutics. We present a novel glycodendron prosthetic which can be site-selectively appended to recombinant proteins to create 'N-glycosylated' glycoprotein mimics. Using computational modeling, we designed the dendrimer scaffold and protein attachment point to resemble the native N-glycan architecture. Three piperidine-melamine glycodendrimers were synthesized via a chemoenzymatic route and attached to human growth hormone and the Fc region of human IgG. These products represent a new class of engineered biosimilars bearing novel glycodendrimer structures.
Assuntos
Dendrímeros/química , Piperidinas/química , Polissacarídeos/química , Proteínas/química , Dendrímeros/síntese química , Humanos , Estrutura Molecular , Triazinas/químicaRESUMO
Mycobacterium tuberculosis mannose-capped lipoarabinomannan inhibits the release of proinflammatory cytokines by LPS-stimulated human dendritic cells (DCs) via targeting the C-type lectin receptor DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN). With the aim of mimicking the bioactive supramolecular structure of mannose-capped lipoarabinomannan, we designed and synthesized a set of poly(phosphorhydrazone) dendrimers grafted with mannose units, called mannodendrimers, that differed by size and the number and length of their (α1â2)-oligommanoside caps. A third-generation dendrimer bearing 48 trimannoside caps (3T) and a fourth-generation dendrimer bearing 96 dimannosides (4D) displayed the highest binding avidity for DC-SIGN. Moreover, these dendrimers inhibited proinflammatory cytokines, including TNF-α, production by LPS-stimulated DCs in a DC-SIGN-dependent fashion. Finally, in a model of acute lung inflammation in which mice were exposed to aerosolized LPS, per os administration of 3T mannodendrimer was found to significantly reduce neutrophil influx via targeting the DC-SIGN murine homolog SIGN-related 1. The 3T mannodendrimer therefore represents an innovative fully synthetic compound for the treatment of lung inflammatory diseases.
Assuntos
Moléculas de Adesão Celular/metabolismo , Dendrímeros/farmacologia , Células Dendríticas/metabolismo , Lectinas Tipo C/metabolismo , Manosídeos/farmacologia , Pneumonia/tratamento farmacológico , Receptores de Superfície Celular/metabolismo , Animais , Citocinas/antagonistas & inibidores , Citocinas/metabolismo , Dendrímeros/química , Citometria de Fluxo , Humanos , Lipopolissacarídeos/química , Espectroscopia de Ressonância Magnética , Manosídeos/química , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Pneumonia/patologia , Ligação ProteicaRESUMO
This review discusses the role of galectin-1 in the tumor microenvironment. First, the structure and function of galectin-1 are discussed. Galectin-1, a member of the galectin family of lectins, is a functionally dimeric galactoside-binding protein. Although galectin-1 has both intracellular and extracellular functions, the defining carbohydrate-binding role occurs extracellularly. In this review, the extracellular roles of galectin-1 in cancer processes are discussed. In particular, the importance of multivalent interactions in galectin-1 mediated cellular processes is reviewed. Multivalent interactions involving galectin-1 in cellular adhesion, mobility and invasion, tumor-induced angiogenesis, and apoptosis are presented. Although the mechanisms of action of galectin-1 in these processes are still not well understood, the overexpression of galectin-1 in cancer progression indicates that the role of galectin-1 is significant. To conclude this review, synthetic frameworks that have been used to modulate galectin-1 processes are reviewed. Small molecule oligomers of carbohydrates, carbohydrate-functionalized pseudopolyrotaxanes, cyclodextrins, calixarenes, and glycodendrimers are presented. These synthetic multivalent systems serve as important tools for studying galectin-1 mediated cancer cellular functions.
Assuntos
Galectina 1/metabolismo , Neoplasias/patologia , Animais , Adesão Celular , Ciclodextrinas/química , Ciclodextrinas/metabolismo , Dendrímeros/química , Dendrímeros/metabolismo , Matriz Extracelular/metabolismo , Galectina 1/genética , Humanos , Invasividade Neoplásica , Neoplasias/metabolismo , Neovascularização Patológica , Poloxâmero/química , Poloxâmero/metabolismo , Rotaxanos/química , Rotaxanos/metabolismo , Microambiente TumoralRESUMO
Glycan recognition by sugar receptors (lectins) is intimately involved in many aspects of cell physiology. However, the factors explaining the exquisite selectivity of their functional pairing are not yet fully understood. Studies toward this aim will also help appraise the potential for lectin-directed drug design. With the network of adhesion/growth-regulatory galectins as therapeutic targets, the strategy to recruit synthetic chemistry to systematically elucidate structure-activity relationships is outlined, from monovalent compounds to glyco-clusters and glycodendrimers to biomimetic surfaces. The versatility of the synthetic procedures enables to take examining structural and spatial parameters, alone and in combination, to its limits, for example with the aim to produce inhibitors for distinct galectin(s) that exhibit minimal reactivity to other members of this group. Shaping spatial architectures similar to glycoconjugate aggregates, microdomains or vesicles provides attractive tools to disclose the often still hidden significance of nanometric aspects of the different modes of lectin design (sequence divergence at the lectin site, differences of spatial type of lectin-site presentation). Of note, testing the effectors alone or in combination simulating (patho)physiological conditions, is sure to bring about new insights into the cooperation between lectins and the regulation of their activity.
Assuntos
Carboidratos/química , Galectinas/química , Glicoproteínas/química , Polissacarídeos/química , Dendrímeros/química , Galectinas/metabolismo , Glicoproteínas/síntese química , Humanos , Modelos Moleculares , Polissacarídeos/metabolismo , Relação Estrutura-AtividadeRESUMO
Most classical dendrimers are frequently built-up from identical repeating units of low valency (usually AB2 monomers). This strategy necessitates several generations to achieve a large number of surface functionalities. In addition, these typical monomers are achiral. We propose herein the use of sugar derivatives consisting of several and varied functionalities with their own individual intrinsic chirality as both scaffolds/core as well as repeating units. This approach allows the construction of chiral, dense dendrimers with a large number of surface groups at low dendrimer generations. Perpropargylated ß-D-glucopyranoside, serving as an A5 core, together with various derivatives, such as 2-azidoethyl tetra-O-allyl-ß-D-glucopyranoside, serving as an AB4 repeating moiety, were utilized to construct chiral dendrimers using "click chemistry" (CuAAC reaction). These were further modified by thiol-ene and thiol-yne click reactions with alcohols to provide dendritic polyols. Molecular dynamic simulation supported the assumption that the resulting polyols have a dense structure.
Assuntos
Química Click , Dendrímeros/química , Compostos de Sulfidrila/química , Álcoois/química , Dendrímeros/síntese química , Estrutura Molecular , Polímeros/química , Propriedades de SuperfícieRESUMO
Four generations of lactose-functionalized polyamidoamine (PAMAM) were employed to further the understanding of multivalent galectin-1 mediated interactions. Dynamic light scattering and fluorescence microscopy were used to study the multivalent interaction of galectin-1 with the glycodendrimers in solution, and glycodendrimers were observed to organize galectin-1 into nanoparticles. In the presence of a large excess of galectin-1, glycodendrimers nucleated galectin-1 into nanoparticles that were remarkably homologous in size (400-500 nm). To understand augmentation of oncologic cellular aggregation by galectin-1, glycodendrimers were used in cell-based assays with human prostate carcinoma cells (DU145). The results revealed that glycodendrimers provided competitive binding sites for galectin-1, which diverted galectin-1 from its typical function in cellular aggregation of DU145 cells.
RESUMO
A novel water-soluble fluorescent glycodendrimer based on perylene bisimides is synthesized, which exhibits high fluorescence quantum yield of 54%. While the binding interactions of PBI-Man with Concanavalin A (Con A) are studied by fluorescence spectra and CD spectra, which show strong binding affinity for Con A with the binding constant of 3.8 × 10(7) m(-1) for monomeric mannose, nearly four orders of magnitude higher affinity than the monovalent mannose ligand. Furthermore, the fluorescence imaging of macrophage cell with PBI-Man is investigated, and shows selectively binding interaction with the mannose receptor-medicated cell entry. Moreover, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) activities of PBI-Man show that PBI-Man as a biocompatible agent is noncytotoxic to living cells.
Assuntos
Materiais Biocompatíveis/síntese química , Técnicas Biossensoriais , Concanavalina A/química , Dendrímeros/síntese química , Fluorescência , Imidas/síntese química , Imagem Molecular , Perileno/análogos & derivados , Animais , Materiais Biocompatíveis/química , Sobrevivência Celular , Dicroísmo Circular , Dendrímeros/química , Imidas/química , Macrófagos/citologia , Camundongos , Conformação Molecular , Estrutura Molecular , Perileno/síntese química , Perileno/química , Espectrometria de FluorescênciaRESUMO
The present study synthesized a new kind of pH-responsive active targeting glycodendrimer (ATGD) for doxorubicin delivery to cancerous cells. First, the glycodendrimer was synthesized based on the cultivation of chitosan dendrons on amine-functionalized, silica-grafted cellulose nanocrystals. Afterward, glycodendrimer was conjugated with folic acid to provide a folate receptor-targeting agent. The response surface method was employed to obtain the optimum conditions for the preparation of doxorubicin-loaded ATGD. The effect of doxorubicin/ATGD ratio, temperature, and pH on doxorubicin loading capacity was evaluated, and high loading capacity was achieved under optimized conditions. After determining doxorubicin release pattern at acidic and physiological pH, ATGD cytotoxicity was surveyed by MTT assay. Based on the results, the loading behavior of doxorubicin onto ATGD was in good agreement with monolayer-physisorption, and drug release was Fickian diffusion-controlled. ATGD could release the doxorubicin much more at acidic pH than physiological pH, corresponding to pH-responsive release behavior. Results of MTT assay confirmed the cytotoxicity of doxorubicin-loaded ATGD in cancer cells, while ATGD (without drug) was biocompatible with no tangible toxicity. These results suggested that ATGD has the potential for the treatment of cancer.
Assuntos
Quitosana , Nanopartículas , Concentração de Íons de Hidrogênio , Doxorrubicina/farmacologia , Doxorrubicina/química , Liberação Controlada de Fármacos , Quitosana/química , Nanopartículas/química , Sistemas de Liberação de Medicamentos , Portadores de Fármacos/químicaRESUMO
Food allergy induced by lipid transfer proteins (LTPs) of Rosacea fruit family, such as peach, is becoming an important health problem in the Mediterranean area. Current treatments, such as allergen specific immunotherapy (AIT) with allergenic extracts show promising, but in many cases, they need an improvement in homogeneity, availability and induction of tolerant responses. Peptide-based vaccines containing adjuvants, such as carbohydrates for C-type lectin receptors (CLRs) are presented as an alternative approach. In this work, we have prepared fucosylated glycodendropeptides (GDPs) functionalized with Pru p 3 peptides via click chemistry. These GDPs, DnFuc9Prup3, induced changes in moDC maturation and lymphocyte proliferation in food allergic patients, indicating specific recognition via DC-SIGN receptor. From these data, D4Fuc9Prup3 can be considered a promising candidate for specific immunotherapy development.
Assuntos
Antígenos de Plantas , Proteínas de Plantas , Alérgenos , Antígenos de Plantas/metabolismo , Moléculas de Adesão Celular , Células Dendríticas/metabolismo , Humanos , Lectinas Tipo C/metabolismo , Proteínas de Plantas/metabolismo , Receptores de Superfície CelularRESUMO
Label-free detection of pathogens is of major concern to the microbiologist community. Most procedures require several steps and amplification techniques. Carbohydrates are well-established receptors for host-pathogen interactions, which can be amplified using glycodendritic architectures on the basis of multivalent binding interactions. Given that uropathogenic Escherichia coli bacterial FimH is based on such mannopyranoside-binding interactions, we demonstrate herein that synthetic monomeric and trimeric thiolated α-d-mannosides can be effectively bound to gold substrate-functionalized self-assembled monolayers (SAMs) preactivated with maleimide functionalities. Mannosides grafted onto SAMs were followed using Quartz Crystal Microbalance with Dissipation (QCM-D). Binding recognition efficiency was first evaluated using the plant lectin from Canavalia ensiformis (ConA) also using QCM-D. We showed a direct correlation between the amount of mannoside bound and the lectin attachment. Even though there was less trimer bound (nM/cm2) to the surface, we observed a 7-fold higher amount of lectin anchoring, thus further demonstrating the value of the multivalent interactions. We next examined the relative fimbriated E. coli selective adhesion/capture to either the monomeric or the trimeric mannoside bound to the surface. Our results established the successful engineering of the surfaces to show E. coli adhesion via specific mannopyranoside binding but unexpectedly, the monomeric derivative was more efficient than the trimeric analog, which could be explained by steric hindrance. This approach strongly suggests that it could be broadly applicable to other Gram-negative bacteria sharing analogous carbohydrate-dependent binding interactions.
Assuntos
Escherichia coli Uropatogênica , Escherichia coli Uropatogênica/metabolismo , Manose/metabolismo , Manosídeos/química , Concanavalina A , LectinasRESUMO
Click chemistry gives access to unlimited set of multivalent glycoconjugates to explore carbohydrate-protein interactions and discover high affinity ligands. In this study, we have created supramolecular systems based on a carrier protein that was grafted by Cu(I)-catalyzed azide-alkyne cycloaddition with tetravalent glycodendrons presenting αGal, ßGal and/or αFuc. Binding studies of the homo- (4 a-c) and heterovalent (5) neoglycoproteins (neoGPs) with the LecA and LecB lectins from P. aeruginosa has first confirmed the interest of the multivalent presentation of glycodendrons by the carrier protein (IC50 up to 2.8â nM). Moreover, these studies have shown that the heterovalent display of glycans (5) allows the interaction with both lectins (IC50 of 10â nM) despite the presence of unspecific moieties, and even with similar efficiency for LecB. These results demonstrate the potential of multivalent and multispecific neoGPs as a promising strategy to fight against resistant pathogens.
RESUMO
Glycodendrimers are receiving considerable attention to mimic a number of imperative features of cell surface glycoconjugate and acquired excellent relevance to a wide domain of investigations including medicine, pharmaceutics, catalysis, nanotechnology, carbohydrate-protein interaction, and moreover in drug delivery systems. Toward this end, an expeditious, modular, and regioselective triazole-forming CuAAC click approach along with double stage convergent synthetic method was chosen to develop a variety of novel chlorine-containing cyclen cored glycodendrimers of high sugar tethers at low generation of promising therapeutic potential. We developed a novel chlorine-containing hypercore unit with 12 alkynyl functionality originated from cyclen scaffold which was confirmed by its single crystal X-ray data analysis. Further, the modular CuAAC technique was utilized to produce a variety of novel 12-sugar coated (G0) glycodendrimers 12-15 adorn with ß-Glc-, ß-Man-, ß-Gal-, ß-Lac, along with 36-galactose coated (G1) glycodendrimer 18 in good-to-high yield. The structures of the developed glycodendrimer architectures have been well elucidated by extensive spectral analysis including NMR (1H & 13CNMR), HRMS, MALDI-TOF MS, UV-Vis, IR, and SEC (Size Exclusion Chromatogram) data.
Assuntos
Ciclamos , Química Click , DendrímerosRESUMO
Three new spherical sulfated cellobiose-polylysine dendrimers of increasing generations bearing negatively charged sulfate groups were prepared by sulfating the corresponding cellobiose-polylysine dendrimers. The first, second, and third-generation derivatives exhibited potent anti-HIV activity with EC50 values of 3.7, 0.6, and 1.5 µg/mL, respectively, in constant to sulfated oligosaccharides with low anti-HIV activity, while the second-generation sulfated dendrimer was the most active. Surface plasmon resonance measurements with poly-l-lysine bearing positively charged amino acids as a model of the HIV surface glycoprotein gp120, indicated that the second-generation dendrimer had the lowest dissociation constant (KD = 1.86 × 10-12 M). Both the particle size and ζ potential increased in the presence of poly-l-lysine. It was proven that the moderate distance between the terminal sulfated cellobiose units in the second-generation dendrimer favored the high anti-HIV activity, owing to the electrostatic interactions developed due to the cluster effect.
Assuntos
Fármacos Anti-HIV/farmacologia , Celobiose/farmacologia , Dendrímeros/farmacologia , HIV-1/efeitos dos fármacos , Polilisina/farmacologia , Sulfatos/farmacologia , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Linhagem Celular Tumoral , Celobiose/química , Dendrímeros/química , Humanos , Testes de Sensibilidade Microbiana , Conformação Molecular , Polilisina/química , Sulfatos/químicaRESUMO
In the present research, we prepared new glycodendrimer containing ß-cyclodextrin (ß-CD) in three steps. At first, graphene quantum dots (GQDs) synthesized through pyrolysis of the citric acid (CA). Then the polyamidoamine (PAMAM) dendrimer was grown from the surface of the modified GQDs (GQDs-PAMAM). Finally, the prepared GQDs-PAMAM was functionalized with ß-CD to obtain the glycodendrimer (GQDs-PAMAM-ß-CD). The synthesized glycodendrimer characterized using several techniques. The phenol-sulfuric acid test obtained the amount of the ß-CD about 30.37 %. 61.2 % of doxorubicin (DOX) was loaded in the prepared glycodendrimer. DOX@GQDs-PAMAM-ß-CD displayed the pH-sensitive drug release profile, which fitted the Higuchi kinetic model. The biological test outcomes showed that the GQDs-PAMAM-ß-CD is a safe carrier with good capability in penetration to the cancer cells. Moreover, DOX@GQDs-PAMAM-ß-CD exhibited more efficiency in the killing of the cancer cells compared to neat DOX. Obtained results suggested that prepared glycodendrimer could be a potential nanosystem for breast cancer treatment.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Dendrímeros/química , Doxorrubicina/farmacologia , Portadores de Fármacos/síntese química , Substâncias Luminescentes/química , beta-Ciclodextrinas/química , Antibióticos Antineoplásicos/metabolismo , Materiais Biocompatíveis , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ácido Cítrico/química , Doxorrubicina/metabolismo , Liberação Controlada de Fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Grafite/química , Humanos , Concentração de Íons de Hidrogênio , Cinética , Pontos Quânticos/química , Pontos Quânticos/ultraestruturaRESUMO
Prion diseases, such as the sporadic Creutzfeldt-Jakob disease (sCJD), are a class of fatal neurodegenerative disorders. Currently, there is no efficient treatment or therapy available. Hence, the search for molecules that may inhibit the conversion of the cellular prion protein (PrPC) into its pathological counterpart PrPScrapie (PrPSc) is of great urgency. Here, we report the generation- and dose-dependent biological action of dense-shell poly(propylene imine) (PPI) glycodendrimers by using scrapie-infected neuroblastoma (ScN2a) cells and the real-time quaking-induced conversion assay (RT-QuIC) for validation of anti-prion efficiencies. Whereas the 2nd and 3rd generation of PPI glycodendrimers exhibited anti-prion conversion efficiency in ScN2a cells validated by RT-QuIC analysis, we observed that the 4th generation of glycodendrimers had shown no significant effect. Translational RT-QuIC studies conducted with human prions derived from sCJD patients indicated an anti-prion conversion effect (not on PrPRes degradation) of PPI glycodendrimers against human prions with the highest inhibitory activity of the 4th generation of PPI glycodendrimers towards prion aggregation compared to the 2nd and 3rd generation. In conclusion, our study highlights the potential of PPI glycodendrimers as therapeutic compounds due to their anti-conversion activity on human prions in a PrPSc strain depending manner.
Assuntos
Dendrímeros/química , Polipropilenos/química , Príons/metabolismo , Encéfalo/patologia , Linhagem Celular Tumoral , Síndrome de Creutzfeldt-Jakob/líquido cefalorraquidiano , Fluoresceína-5-Isotiocianato/metabolismo , Glicosilação , Humanos , Modelos Moleculares , Agregados Proteicos , Reprodutibilidade dos TestesRESUMO
The human skin is an attractive anti-tumor vaccination site due to the vast network of dendritic cell (DC) subsets that carry antigens to the draining lymph nodes and stimulate tumor specific CD4+ and CD8+ T cells in. Specific vaccine delivery to skin DC can be accomplished by targeting glycan coated antigens to C-type lectin receptors (CLRs) such as DC-SIGN expressed by human dermal DCs and Langerin expressed by Langerhans cells (LCs), which facilitate endocytosis and processing for antigen presentation and T cell activation. Although there are multiple human skin DC subsets, targeting individual DC subsets and receptors has been a focus in the past. However, the simultaneous targeting of multiple human skin DC subsets that mobilize the majority of the skin antigen presenting cells (APC) is preferred to accomplish more robust and efficient T cell stimulation. Dual CLR targeting using a single tumor vaccine has been difficult, as we previously showed Langerin to favor binding and uptake of monovalent glyco-peptides whereas DC-SIGN favors binding of larger multivalent glyco-particles such as glyco-liposomes. Methods: We used branched polyamidoamine (PAMAM) dendrimers as scaffold for melanoma specific gp100 synthetic long peptides and the common DC-SIGN and Langerin ligand Lewis Y (LeY), to create multivalent glyco-dendrimers with varying molecular weights for investigating dual DC-SIGN and Langerin targeting. Using DC-SIGN+ monocyte derived DC (moDC) and Langerin+ primary LC we investigated glyco-dendrimer CLR targeting properties and subsequent gp100 specific CD8+ T cell activation in vitro. In situ targeting ability to human dermal DC and LC through intradermal injection in a human skin explant model was elucidated. Results: Dual DC-SIGN and Langerin binding was achieved using glyco-dendrimers of approximately 100kD, thereby fulfilling our criteria to simultaneously target LCs and CD1a+ and CD14+ dermal DC in situ. Both DC-SIGN and Langerin targeting by glyco-dendrimers resulted in enhanced internalization and gp100 specific CD8+ T cell activation. Conclusion: We designed the first glyco-vaccine with dual CLR targeting properties, thereby reaching multiple human skin DC subsets in situ for improved anti-tumor CD8+ T cell responses.
Assuntos
Vacinas Anticâncer/imunologia , Dendrímeros/química , Células Dendríticas/metabolismo , Células de Langerhans/metabolismo , Poliaminas/química , Antígenos CD/metabolismo , Antígenos CD1/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Vacinas Anticâncer/química , Vacinas Anticâncer/uso terapêutico , Linhagem Celular Tumoral , Humanos , Lectinas Tipo C/metabolismo , Lectinas de Ligação a Manose/metabolismoRESUMO
The first example of the self-assembly and lectin binding properties of photoswitchable glycodendrimer micelles is reported. Light-addressable micelles were assembled from a library of 12 amphiphilic Janus glycodendrimers composed of variable carbohydrate head groups and hydrophobic tail groups linked to an azobenzene core. Spontaneous association in water gave cylindrical micelles with uniform size distribution as determined by dynamic light scattering (DLS) and small angle neutron scattering (SANS). Trans-cis photoisomerization of the azobenzene dendrimer core was used to probe the self-assembly behaviour and lectin binding properties of cylindrical micelles, revealing moderate-to-potent inhibition of lectins LecA and LecB from Pseudomonas aeruginosa.
Assuntos
Adesinas Bacterianas/metabolismo , Dendrímeros/química , Lectinas/química , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , Compostos Azo/química , Compostos Azo/farmacologia , Difusão Dinâmica da Luz , Interações Hidrofóbicas e Hidrofílicas , Lectinas/antagonistas & inibidores , Lectinas/metabolismo , Micelas , Espalhamento a Baixo ÂnguloRESUMO
Glyco-PAMAM dendrimers and glyco-agarose beads bearing a core-fucosylated N-glycan trisaccharide GlcNAcß1,4(Fucα1,6)GlcNAc or a non-fucose disaccharide GlcNAcß1,4GlcNAc were successfully synthesized and characterized by monosaccharide analysis with HPAEC-PAD technique. These glycoconjugates as fucose lectin probes were applied in fucose-specific lectin detection and purification. The model fucose lectin AAL indicated binding activity with the FITC-labeled PAMAM carrying core-fucose trisaccharide. An affinity chromatography column stuffed with the agarose beads carrying core-fucosylated trisaccharide exhibited a good specificity in purification of AAL than non-fucose disaccharide agarose beads. These novel glycoconjugates bearing the precise and simplified core-fucose N-glycan structure provided a potential application for core-fucose-specific lectin discovery.