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 NanoAssemblrTM. 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.
- MeSH
- adjuvancia imunologická farmakologie MeSH
- antigeny povrchové metabolismus MeSH
- Candida glabrata chemie MeSH
- click chemie MeSH
- dendritické buňky imunologie MeSH
- hydroxylaminy chemická syntéza chemie MeSH
- lektiny typu C imunologie MeSH
- lektiny vázající mannosu imunologie MeSH
- lidé MeSH
- lipidy chemická syntéza chemie MeSH
- liposomy chemie imunologie farmakologie MeSH
- mannany chemie imunologie farmakologie MeSH
- mikrofluidika metody MeSH
- myši inbrední BALB C MeSH
- nanočástice chemie MeSH
- receptory buněčného povrchu imunologie MeSH
- velikost částic MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
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.
- MeSH
- antigeny CD44 analýza metabolismus MeSH
- buněčné linie MeSH
- endocytóza MeSH
- fluorescenční barviva MeSH
- kyselina hyaluronová chemie metabolismus MeSH
- lékové transportní systémy metody MeSH
- lidé MeSH
- lipidy chemická syntéza MeSH
- liposomy chemie terapeutické užití MeSH
- mikrofluidika MeSH
- nádory farmakoterapie MeSH
- protinádorové látky aplikace a dávkování MeSH
- transmisní elektronová mikroskopie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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).
- MeSH
- acetylmuramyl-alanyl-isoglutamin analogy a deriváty chemie imunologie farmakologie MeSH
- adjuvancia imunologická chemie farmakologie MeSH
- antigeny povrchové chemie imunologie farmakologie MeSH
- bakteriální vakcíny chemie imunologie farmakologie MeSH
- Borrelia burgdorferi imunologie MeSH
- HEK293 buňky MeSH
- imunizace MeSH
- lidé MeSH
- lipoproteiny chemie imunologie farmakologie MeSH
- lymeská nemoc imunologie mikrobiologie MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- protein NLRP3 agonisté imunologie MeSH
- proteiny vnější bakteriální membrány chemie imunologie farmakologie MeSH
- RAW 264.7 buňky MeSH
- tvorba protilátek MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
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.
- MeSH
- adjuvancia imunologická * MeSH
- antigeny bakteriální aplikace a dávkování chemie imunologie MeSH
- Borrelia burgdorferi imunologie MeSH
- ELISA MeSH
- imunizace MeSH
- lymeská nemoc imunologie MeSH
- modely u zvířat MeSH
- myši MeSH
- proteiny vnější bakteriální membrány aplikace a dávkování chemie imunologie MeSH
- proteolipidy MeSH
- protilátky bakteriální imunologie MeSH
- rekombinantní fúzní proteiny aplikace a dávkování chemie imunologie izolace a purifikace MeSH
- sekundární struktura proteinů MeSH
- specificita protilátek imunologie MeSH
- stabilita proteinů MeSH
- tvorba protilátek imunologie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
PURPOSE: The aim of this work was to demonstrate an immunostimulatory and adjuvant effect of new apyrogenic lipophilic derivatives of norAbuMDP and norAbuGMDP formulated in nanoliposomes. METHODS: Nanoliposomes and metallochelating nanoliposomes were prepared by lipid film hydration and extrusion methods. The structure of the liposomal formulation was studied by electron microscopy, AF microscopy, and dynamic light scattering. Sublethal and lethal γ-irradiation mice models were used to demonstrate stimulation of innate immune system. Recombinant Hsp90 antigen (Candida albicans) bound onto metallochelating nanoliposomes was used for immunisation of mice to demonstrate adjuvant activities of tested compounds. RESULTS: Safety and stimulation of innate and adaptive immunity were demonstrated on rabbits and mice. The liposomal formulation of norAbuMDP/GMDP was apyrogenic in rabbit test and lacking any side effect in vivo. Recovery of bone marrow after sublethal γ-irradiation as well as increased survival of mice after lethal irradiation was demonstrated. Enhancement of specific immune response was demonstrated for some derivatives incorporated in metallochelating nanoliposomes with recombinant Hsp90 protein antigen. CONCLUSIONS: Liposomal formulations of new lipophilic derivatives of norAbuMDP/GMDP proved themselves as promising adjuvants for recombinant vaccines as well as immunomodulators for stimulation of innate immunity and bone-marrow recovery after chemo/radio therapy of cancer.
- MeSH
- acetylmuramyl-alanyl-isoglutamin aplikace a dávkování analogy a deriváty chemie farmakologie terapeutické užití MeSH
- adaptivní imunita účinky léků MeSH
- adjuvancia imunologická aplikace a dávkování chemie farmakologie terapeutické užití MeSH
- analýza přežití MeSH
- antigeny fungální imunologie MeSH
- experimentální radiační poranění imunologie prevence a kontrola MeSH
- králíci MeSH
- liposomy MeSH
- mikroskopie atomárních sil MeSH
- mikroskopie elektronová rastrovací MeSH
- molekulární struktura MeSH
- myši inbrední ICR MeSH
- myši MeSH
- nanočástice MeSH
- nosiče léků chemie MeSH
- přirozená imunita účinky léků MeSH
- proteiny tepelného šoku HSP90 imunologie MeSH
- protilátky fungální krev MeSH
- rekombinantní proteiny imunologie MeSH
- transmisní elektronová mikroskopie MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Hsp90-CA is present in cell wall of Candida pseudohyphae or hyphae-typical pathogenic morphotype for both systemic and mucosal Candida infections. Heat shock protein from Candida albicans (hsp90-CA) is an important target for protective antibodies during disseminated candidiasis of experimental mice and human. His-tagged protein rHsp90 was prepared and used as the antigen for preparation of experimental recombinant liposomal vaccine. Nickel-chelating liposomes (the size around 100nm, PDI≤0.1) were prepared from the mixture of egg phosphatidyl choline and nickel-chelating lipid DOGS-NTA-Ni (molar ratio 95:5%) by hydration of lipid film and extrusion methods. New non-pyrogenic hydrophobised derivative of MDP (C18-O-6-norAbuMDP) was incorporated into liposomes as adjuvans. rHsp90 was attached onto the surface of metallochelating liposomes by metallochelating bond and the structure of these proteoliposomes was studied by dynamic light scattering, AF microscopy, TEM and GPC. The liposomes with surface-exposed C18-O-6-norAbuMDP were well recognised and phagocyted by human dendritic cells in vitro. In vivo the immune response towards this experimental vaccine applied in mice (i.d.) demonstrated both TH1 and TH2 response comparable to FCA, but without any side effects. Metallochelating liposomes with lipophilic derivatives of muramyl dipeptide represent a new biocompatible platform for construction of experimental recombinant vaccines and drug-targeting systems.
- MeSH
- antigeny fungální imunologie metabolismus MeSH
- biokompatibilní potahované materiály metabolismus MeSH
- buněčná imunita MeSH
- Candida imunologie MeSH
- chelátory chemie metabolismus MeSH
- dendritické buňky imunologie metabolismus MeSH
- kultivované buňky MeSH
- lidé MeSH
- liposomy MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- nikl imunologie metabolismus MeSH
- proteiny tepelného šoku HSP90 imunologie metabolismus MeSH
- syntetické vakcíny imunologie metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
On the basis of the highly branched ovomucoid-type undecasaccharide that had been shown previously to be an endogenous ligand for CD69 leukocyte receptor, a systematic investigation of smaller oligosaccharide mimetics was performed based on linear and branched N-acetyl-d-hexosamine homooligomers prepared synthetically using hitherto unexplored reaction schemes. The systematic structure-activity studies revealed the tetrasaccharide GlcNAcbeta1-3(GlcNAcbeta1-4)(GlcNAcbeta1-6)GlcNAc (compound 52) and its alpha-benzyl derivative 49 as the best ligand for CD69 with IC(50) as high as 10(-9) M. This compound thus approaches the affinity of the classical high-affinity neoglycoprotein ligand GlcNAc(23)BSA. Compound 68, GlcNAc tetrasaccharide 52 dimerized through a hydrophilic flexible linker, turned out to be effective in activating CD69(+) lymphocytes. It also proved efficient in enhancing natural killing in vitro, decreasing the growth of tumors in vivo, and activating the CD69(+) tumor infiltrating lymphocytes examined ex vivo. This compound is thus a candidate for carbohydrate-based immunomodulators with promising antitumor potential.
- MeSH
- acetylglukosamin analogy a deriváty chemická syntéza chemie farmakologie MeSH
- aktivace lymfocytů MeSH
- buňky NK účinky léků imunologie metabolismus MeSH
- CD antigeny metabolismus MeSH
- diferenciační antigeny T-lymfocytů metabolismus MeSH
- dimerizace MeSH
- imunologické faktory chemická syntéza chemie farmakologie MeSH
- krysa rodu rattus MeSH
- lektinové receptory NK-buněk - podrodina B metabolismus MeSH
- lektiny typu C metabolismus MeSH
- lidé MeSH
- ligandy MeSH
- melanom experimentální imunologie patologie MeSH
- molekulární mimikry MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- oligosacharidy chemická syntéza chemie farmakologie MeSH
- protinádorové látky chemická syntéza chemie farmakologie MeSH
- rekombinantní proteiny chemie MeSH
- sacharidové sekvence MeSH
- screeningové testy protinádorových léčiv MeSH
- techniky in vitro MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- odvolaná publikace MeSH
- práce podpořená grantem MeSH
In this paper, we present the detailed synthetic protocol and characterization of Fmoc-Lys(Pac)-OH, its use for the preparation of octapeptides H-Gly-Phe-Tyr-N-MePhe-Thr-Lys(Pac)-Pro-Thr-OH and H-Gly-Phe-Phe-His-Thr-Pro-Lys(Pac)-Thr-OH by solid-phase synthesis, trypsin-catalyzed condensation of these octapeptides with desoctapeptide(B23-B30)-insulin, and penicillin G acylase catalyzed cleavage of phenylacetyl (Pac) group from Nepsilon-amino group of lysine to give novel insulin analogs [TyrB25, N-MePheB26,LysB28,ProB29]-insulin and [HisB26]-insulin. These new analogs display 4 and 78% binding affinity respectively to insulin receptor in rat adipose membranes. Copyright (c) 2007 European Peptide Society and John Wiley & Sons, Ltd.
- MeSH
- financování organizované MeSH
- inzulin analogy a deriváty farmakologie chemická syntéza metabolismus MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- oligopeptidy farmakologie chemie metabolismus MeSH
- penicilinamidasa chemie MeSH
- potkani Wistar MeSH
- receptor inzulinu metabolismus MeSH
- vazba proteinů fyziologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- mužské pohlaví MeSH
- zvířata MeSH
