RESUMO
New synthetic aminooxy lipid was designed and synthesized as a building block for the formulation of functionalised nanoliposomes (presenting onto the outer surface of aminooxy groups) by microfluidic mixing. Orthogonal binding of cellular mannan (Candida glabrata (CCY 26-20-1) onto the outer surface of functionalised nanoliposomes was modified by orthogonal binding of reducing termini of mannans to oxime lipids via a click chemistry reaction based on aminooxy coupling (oxime ligation). The aminooxy lipid was proved as a suitable active component for preparation of functionalised nanoliposomes by the microfluidic mixing method performed with the instrument NanoAssemblr™. This "on-chip technology" can be easily scaled-up. The structure of mannan-liposomes was visualized by transmission and scanning electron microscopy, including immunogold staining of recombinant mannan receptor bound onto mannosylated-liposomes. The observed structures are in a good correlation with data obtained by DLS, NTA, and TPRS methods. In vitro experiments on human and mouse dendritic cells demonstrate selective internalisation of fluorochrome-labelled mannan-liposomes and their ability to stimulate DC comparable to lipopolysaccharide. We describe a potentially new drug delivery platform for mannan receptor-targeted antimicrobial drugs as well as for immunotherapeutics. Furthermore, the platform based on mannans bound orthogonally onto the surface of nanoliposomes represents a self-adjuvanted carrier for construction of liposome-based recombinant vaccines for both systemic and mucosal routes of administration.
Assuntos
Células Dendríticas/imunologia , Lectinas Tipo C/imunologia , Lipossomos/imunologia , Mananas/imunologia , Lectinas de Ligação a Manose/imunologia , Nanopartículas/química , Receptores de Superfície Celular/imunologia , Adjuvantes Imunológicos/farmacologia , Animais , Antígenos de Superfície/metabolismo , Candida glabrata/química , Química Click , Humanos , Hidroxilaminas/síntese química , Hidroxilaminas/química , Lipídeos/síntese química , Lipídeos/química , Lipossomos/química , Lipossomos/farmacologia , Mananas/química , Mananas/farmacologia , Receptor de Manose , Camundongos Endogâmicos BALB C , Microfluídica/métodos , Tamanho da PartículaRESUMO
New synthetic aminoxy lipids are designed and synthesized as building blocks for the formulation of functionalized nanoliposomes by microfluidization using a NanoAssemblr. Orthogonal binding of hyaluronic acid onto the outer surface of functionalized nanoliposomes via aminoxy coupling ( N-oxy ligation) is achieved at hemiacetal function of hyaluronic acid and the structure of hyaluronic acid-liposomes is visualized by transmission electron microscopy and cryotransmission electron microscopy. Observed structures are in a good correlation with data obtained by dynamic light scattering (size and ζ-potential). In vitro experiments on cell lines expressing CD44 receptors demonstrate selective internalization of fluorochrome-labeled hyaluronic acid-liposomes, while cells with down regulated CD44 receptor levels exhibit very low internalization of hyaluronic acid-liposomes. A method based on microfluidization mixing was developed for preparation of monodispersive unilamellar liposomes containing aminoxy lipids and orthogonal binding of hyaluronic acid onto the liposomal surface was demonstrated. These hyaluronic acid-liposomes represent a potentially new drug delivery platform for CD44-targeted anticancer drugs as well as for immunotherapeutics and vaccines.
Assuntos
Antineoplásicos/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/química , Lipídeos/síntese química , Lipossomos/química , Linhagem Celular , Endocitose , Corantes Fluorescentes , Humanos , Receptores de Hialuronatos/análise , Ácido Hialurônico/metabolismo , Lipossomos/uso terapêutico , Microfluídica , Microscopia Eletrônica de Transmissão , Neoplasias/tratamento farmacológicoRESUMO
Fatty acyl analogues of muramyldipeptide (MDP) (abbreviated N-L18 norAbuGMDP, N-B30 norAbuGMDP, norAbuMDP-Lys(L18), norAbuMDP-Lys(B30), norAbuGMDP-Lys(L18), norAbuGMDP-Lys(B30), B30 norAbuMDP, L18 norAbuMDP) are designed and synthesized comprising the normuramyl-l-α-aminobutanoyl (norAbu) structural moiety. All new analogues show depressed pyrogenicity in both free (micellar) state and in liposomal formulations when tested in rabbits in vivo (sc and iv application). New analogues are also shown to be selective activators of NOD2 and NLRP3 (inflammasome) in vitro but not NOD1. Potencies of NOD2 and NLRP3 stimulation are found comparable with free MDP and other positive controls. Analogues are also demonstrated to be effective in stimulating cellular proliferation when the sera from mice are injected sc with individual liposome-loaded analogues, causing proliferation of bone marrow-derived GM-progenitors cells. Importantly, vaccination nanoparticles prepared from metallochelation liposomes, His-tagged antigen rOspA from Borrelia burgdorferi, and lipophilic analogue norAbuMDP-Lys(B30) as adjuvant, are shown to provoke OspA-specific antibody responses with a strong Th1-bias (dominance of IgG2a response). In contrast, the adjuvant effects of Alum or parent MDP show a strong Th2-bias (dominance of IgG1 response).
Assuntos
Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Adjuvantes Imunológicos/farmacologia , Antígenos de Superfície/farmacologia , Proteínas da Membrana Bacteriana Externa/farmacologia , Vacinas Bacterianas/farmacologia , Borrelia burgdorferi/imunologia , Lipoproteínas/farmacologia , Acetilmuramil-Alanil-Isoglutamina/química , Acetilmuramil-Alanil-Isoglutamina/imunologia , Adjuvantes Imunológicos/química , Animais , Formação de Anticorpos , Antígenos de Superfície/química , Antígenos de Superfície/imunologia , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/química , Vacinas Bacterianas/imunologia , Feminino , Células HEK293 , Humanos , Imunização , Lipoproteínas/química , Lipoproteínas/imunologia , Doença de Lyme/imunologia , Doença de Lyme/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Proteína 3 que Contém Domínio de Pirina da Família NLR/agonistas , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Células RAW 264.7RESUMO
Like thapsigargin, which is undergoing clinical trials, trilobolide is a natural product with promising anticancer and anti-inflammatory properties. Similar to thapsigargin, it has limited aqueous solubility that strongly reduces its potential medicinal applications. The targeted delivery of hydrophobic drugs can be achieved using liposome-based carriers. Therefore, we designed a traceable liposomal drug delivery system for trilobolide. The fluorescent green-emitting dye BODIPY, cholesterol and trilobolide were used to create construct 6. The liposomes were composed of dipalmitoyl-3-trimethylammoniumpropane and phosphatidylethanolamine. The whole system was characterized by atomic force microscopy, the average size of the liposomes was 150 nm in width and 30 nm in height. We evaluated the biological activity of construct 6 and its liposomal formulation, both of which showed immunomodulatory properties in primary rat macrophages. The uptake and intracellular distribution of construct 6 and its liposomal formulation was monitored by means of live-cell fluorescence microscopy in two cancer cell lines. The encapsulation of construct 6 into the liposomes improved the drug distribution in cancer cells and was followed by cell death. This new liposomal trilobolide derivative not only retains the biological properties of pure trilobolide, but also enhances the bioavailability, and thus has potential for the use in theranostic applications.
RESUMO
Lyme disease, Borrelia burgdorferi-caused infection, if not recognized and appropriately treated by antibiotics, may lead to chronic complications, thus stressing the need for protective vaccine development. The immune protection is mediated by phagocytic cells and by Borrelia-specific complement-activating antibodies, associated with the Th1 immune response. Surface antigen OspC is involved in Borrelia spreading through the host body. Previously we reported that recombinant histidine tagged (His-tag) OspC (rOspC) could be attached onto liposome surfaces by metallochelation. Here we report that levels of OspC-specific antibodies vary substantially depending upon whether rOspC possesses an N' or C' terminal His-tag. This is the case in mice immunized: (a) with rOspC proteoliposomes containing adjuvants MPLA or non-pyrogenic MDP analogue MT06; (b) with free rOspC and Montanide PET GEL A; (c) with free rOspC and alum; or (d) with adjuvant-free rOspC. Stronger responses are noted with all N'-terminal His-tag rOspC formulations. OspC-specific Th1-type antibodies predominate post-immunization with rOspC proteoliposomes formulated with MPLA or MT06 adjuvants. Further analyses confirmed that the structural features of soluble N' and C' terminal His-tag rOspC and respective rOspC proteoliposomes are similar including their thermal stabilities at physiological temperatures. On the other hand, a change in the position of the rOspC His-tag from N' to C' terminal appears to affect substantially the immunogenicity of rOspC arguably due to steric hindrance of OspC epitopes by the C' terminal His-tag itself and not due to differences in overall conformations induced by changes in the His-tag position in rOspC variants.