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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).

Polysaccharides are indispensable macromolecules widely used in pharmaceutical industry. Hyaluronic acid, mannans, cellulose derivatives, alginates, and dextrans are applied for the preparation of polysaccharide-modified nanoparticles to develop targeted delivery systems for drugs, theranostics, vaccines, and immunotherapeutics. Liposomes represent the most versatile nanoparticles and liposomal products have already found clinical applications, due to their biocompatibility, biodegradability, low toxicity, and ability to internalize substances with different physical-chemical properties. Modification of liposomal surface with polysaccharides is a special part of bioconjugation chemistry and application of click chemistry for orthogonal binding of polysaccharides onto liposomal surface is a new challenging approach for the development of drug and vaccine targeting nanosystems. New technologies for laboratory and industrial production of polysaccharide-modified liposomes can be developed by combination of microfluidics technology integrated together with other processes (dialysis, click chemistry, photochemistry, and drug loading) into a single device by utilizing so-called Lab-on-Chip technology enabling automated high-throughput processes for the production of liposome-based products.

Fluorescent diamond nanocrystals are attracting increasing interest for a broad range of applications, from biolabeling and single particle tracking to nanoscale magnetic field sensing. Their fluorescence stems from nitrogen-vacancy color centers created within synthetic diamond nanoparticles by high-temperature annealing, which results in the association of pre-existing nitrogen impurities and vacancies generated by high-energy particle (electron, proton, or helium ion) beam irradiation. Up to now, diamond nanocrystals have been irradiated as dry powder in a container or deposited as a thin layer on a flat substrate, depending on the type and energy of the irradiating particles. However, these techniques suffer from intrinsic inhomogeneities: the fluence of particles may vary over the whole sample area, as well as the thickness and density of the nanodiamond layer. Here, we present an approach based on direct large-scale irradiation of nanodiamonds in aqueous colloidal solution by high-energy protons. This approach results in a larger fraction of fluorescent particles, with a more homogenous distribution of nitrogen-vacancy centers per particle and less severe lattice damages compared to dry powder irradiation.

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.

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.

In this paper, new steps toward a better understanding and utilization of high-pressure high-temperature nanodiamonds (NDs) containing nitrogen-vacancy (NV) centers have been taken. NV–-related long-term luminescence of oxygenated particles increased in comparison to plasma hydrogenated NDs’ NV– luminescence. The optically detected NV– electron spin resonance process can be also significantly affected by ND termination. For H-terminated ND particles the NV– to NV0 conversion energy is lower than the NV– excitation energy, so that the delocalized triplet electrons can be more easily released from the original positions and drawn to the electron-attracting localities in the material. The final result of this study was application of luminescent NDs in cells, showing the detectability of luminescent NDs in a standard confocal microscope and ND subcellular distribution in the cells by TEM.

Nanodiamonds (NDs) are versatile nanoparticles that are currently being investigated for a variety of applications in drug delivery, biomedical imaging and nanoscale sensing. Although initial studies indicate that these small gems are biocompatible, there is a great deal of variability in synthesis methods and surface functionalization that has yet to be evaluated. Here we present a comprehensive analysis of the cellular compatibility of an array of nanodiamond subtypes and surface functionalization strategies. These results demonstrate that NDs are well tolerated by multiple cell types at both functional and gene expression levels. In addition, ND-mediated delivery of daunorubicin is less toxic to multiple cell types than treatment with daunorubicin alone, thus demonstrating the ability of the ND agent to improve drug tolerance and decrease therapeutic toxicity. Overall, the results here indicate that ND biocompatibility serves as a promising foundation for continued preclinical investigation.

The novel synthesis of a polymeric interface grown from the surface of bright fluorescent nanodiamonds is reported. The polymer enables bioorthogonal attachment of various molecules by click chemistry; the particles are resistant to nonspecific protein adsorption and show outstanding colloidal stability in buffers and biological media. The coating fully preserves the unique optical properties of the nitrogen-vacancy centers that are crucial for bioimaging and sensoric applications. Abstract: /irua/be5622/6542.pdf

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.

Dendritic poly(L-lysines) (DGL) constitute promising nanomaterials applicable as a nonviral gene-delivery vector. In this study, we evaluate the transfection abilities of four DGL generations with special emphasis on the systematic description of the relationship of how generation (i.e., molecule size) affects the transfection efficacy. Using Hep2 cells, we demonstrated that the capability of unmodified DGL to deliver plasmid is of a magnitude lower than that of jetPEI. On the other hand, employing the Hep2 cell line stably transduced with eGFP, we observed that DGL G5 delivers the siRNA oligonucleotide with the same efficiency as Lipofectamine 2000. In further experiments, it was shown that DGL affords excellent ability to bind DNA, protect it against DNase I attack, and internalize it into cells.

High biocompatibility, variable size ranging from ≈5 nm, stable luminescence from its color centers, and simple carbon chemistry for biomolecule grafting make nanodiamond (ND) particles an attractive alternative to molecular dyes for drug-delivery. A novel method is presented that can be used for remote monitoring of chemical processes in biological environments based on color changes from photoluminescent (PL) nitrogen-vacancy (NV) centers in ND. The NV luminescence is driven chemically by alternating the surface chemical potential by interacting atoms and molecules with the diamond surface. Due to the small ND size, the changes of the surface chemical potential modify the electric field profile at the diamond surfaces (i.e., band bending) and intermingle with the electronic NV states. This leads to changes in NV−/NV° PL ratio and allows construction of optical chemo-biosensors operating in cells, with PL visible in classical confocal microscopes. This phenomenon is demonstrated on single crystal diamond containing engineered NV centers and on oxidized and hydrogenated ND in liquid physiological buffers for variously sized ND particles. Hydrogenation of NDs leads to quenching of luminescence related to negatively charged (NV−) centers and as a result produces color shifts from NV− (638 nm) to neutral NV° (575 nm) luminescence. How the reduction of diamond size increases the magnitude of the NV color shift phenomena is modeled.

A plausible strategy to reduce tumor progress is the inhibition of angiogenesis. Therefore, agents that efficiently suppress angiogenesis can be used for tumor suppression. We tested the antiangiogenic potential of a mitochondrially targeted analog of α-tocopheryl succinate (MitoVES), a compound with high propensity to induce apoptosis.MitoVES was found to efficiently kill proliferating endothelial cells (ECs) but not contact-arrested ECs or ECs deficient in mitochondrial DNA, and suppressed angiogenesis in vitro by inducing accumulation of reactive oxygen species and induction of apoptosis in proliferating/angiogenic ECs. Resistance of arrested ECs was ascribed, at least in part, to the lower mitochondrial inner transmembrane potential compared with the proliferating ECs, thus resulting in the lower level of mitochondrial uptake of MitoVES. Shorter-chain homologs of MitoVES were less efficient in angiogenesis inhibition, thus suggesting a molecular mechanism of its activity. Finally, MitoVES was found to suppress HER2-positive breast carcinomas in a transgenic mouse as well as inhibit tumor angiogenesis. The antiangiogenic efficacy of MitoVES was corroborated by its inhibitory activity on wound healing in vivo.We conclude that MitoVES, a mitochondrially targeted analog of α-tocopheryl succinate, is an efficient antiangiogenic agent of potential clinical relevance, exerting considerably higher activity than its untargeted counterpart. MitoVES may be helpful against cancer but may compromise wound healing.

Fluorescent cellular biomarkers play an essential role in biology and medicine for in vitro and in vivo imaging in living cells. Recently, we have demonstrated that photoluminescence (PL) can be driven chemically by changing the occupation of NV− and NV0 states by H- or O-termination. In the presented work we study, how the luminescence of NV centers at controlled depth from the surface changes upon different surface treatment. We compare NV luminescence of hydrogen (H) and oxygen (O) terminated surfaces in single crystal diamond (SCD) containing shallow-implanted NV centers (3–10 nm) with similarly treated nanodiamond particles (ND) of various size distributions. The H-termination leads to reduction of NV− related luminescence in both, ND and SCD. The effect is much stronger in ND in comparison with SCD. We mathematically model both situations and discuss parameters influencing the effect, including implantation energy and different contents of nitrogen in the diamond crystal lattice.

Mitochondrial complex II (CII) has been recently identified as a novel target for anti-cancer drugs. Mitochondrially targeted vitamin E succinate (MitoVES) is modified so that it is preferentially localized to mitochondria, greatly enhancing its pro-apoptotic and anti-cancer activity. Using genetically manipulated cells, MitoVES caused apoptosis and generation of reactive oxygen species (ROS) in CII-proficient malignant cells but not their CII-dysfunctional counterparts. MitoVES inhibited the succinate dehydrogenase (SDH) activity of CII with IC(50) of 80 μM, whereas the electron transfer from CII to CIII was inhibited with IC(50) of 1.5 μM. The agent had no effect either on the enzymatic activity of CI or on electron transfer from CI to CIII. Over 24 h, MitoVES caused stabilization of the oxygen-dependent destruction domain of HIF1α fused to GFP, indicating promotion of the state of pseudohypoxia. Molecular modeling predicted the succinyl group anchored into the proximal CII ubiquinone (UbQ)-binding site and successively reduced interaction energies for serially shorter phytyl chain homologs of MitoVES correlated with their lower effects on apoptosis induction, ROS generation, and SDH activity. Mutation of the UbQ-binding Ser(68) within the proximal site of the CII SDHC subunit (S68A or S68L) suppressed both ROS generation and apoptosis induction by MitoVES. In vivo studies indicated that MitoVES also acts by causing pseudohypoxia in the context of tumor suppression. We propose that mitochondrial targeting of VES with an 11-carbon chain localizes the agent into an ideal position across the interface of the mitochondrial inner membrane and matrix, optimizing its biological effects as an anti-cancer drug.

Liposomes represent a biocompatible platform for the construction of self-assembling proteoliposomes using nickel or zinc metallochelation. Potential applications of such structures consist in the development of new biocompatible vaccination nanoparticles and drug delivery nanoparticle systems. Here, we describe the design and construction of a flow-through ultrafiltration cell suitable for the preparation of monodisperse liposomes enabled for metallochelation and, hence, the formation of proteoliposomes. The linkage of the cell with a fast protein liquid chromatography system facilitates automation of the procedure, which fits the criteria for upscaling. Proof-of-concept experiments are performed using a mixture of egg phosphatidyl choline and nickel-chelating lipid DOGS–NTA–Ni (1,2-dioleoyl- sn -glycero-3-{[ N (5-amino-1-carboxypentyl)iminodiacetic acid]succinyl}(nickel salt)) to formulate proteoliposomes with proteins attached by metallochelation, including histidine (His)-tagged recombinant green fluorescent protein and rgp120 (derived from HIV-1 Env). These model proteoliposomes are characterized by gel permeation chromatography and by dynamic light scattering. Transmission electron microscopy and immunogold staining are used to characterize surface-bound proteins, revealing the tendency of rgp120 to form microdomains on liposome surfaces. These microdomains possess a two-dimensional crystal-like structure that is seen more precisely by atomic force microscopy.

Nanodiamond is a novel and promising material for in vitro and in vivo imaging in living cells. In this work, we studied (using quantum chemical calculation methods) how the various surface terminations affect the conditions for the fluorescence of nitrogen-vacancy (NV) centers in nanodiamond particles. We worked with clusters containing between 35 and 86 atoms of carbon containing NV centers with different charge states of their vacancies (NV ― and NV 0 ) and with different terminations: OH, H, NH 2 , carbonyl, carboxyl, and hydroxyl groups. The systems under study were modeled by DFT-based calculations using the Gaussian 09 and Turbomole-5.10 program packages.

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.

Herein, we present the synthesis of three new 2-azido-2-deoxy-D-mannopyranose building blocks that are useful in the preparation ofoligosaccharides consisting ofa(1→4)-linked 2-azido-2-deoxy-D-mannopyranose units. The successful utilization of these intermediates in the stepwise synthesis of the corresponding trisaccharide is also described in detail.

Overexpression of erbB2 is associated with resistance to apoptosis. We explored whether high level of erbB2 expression by cancer cells allows their targeting using an erbB2-binding peptide (LTVSPWY) attached to the proapoptotic α-tocopheryl succinate (α-TOS). Treating erbB2-low or erbB2-high cells with α-TOS induced similar levels of apoptosis, whereas α-TOS-LTVSPWY induced greater levels of apoptosis in erbB2-high cells. α-TOS rapidly accumulated in erbB2-high cells exposed to α-TOS-LTVSPWY. The extent of apoptosis induced in erbB2-high cells by α-TOS-LTVSPWY was suppressed by erbB2 RNA interference as well as by inhibition of either endocytotic or lysosomal function. α-TOS-LTVSPWY reduced erbB2-high breast carcinomas in FVB/N c-neu transgenic mice. We conclude that a conjugate of a peptide targeting α-TOS to erbB2-overexpressing cancer cells induces rapid apoptosis and efficiently suppresses erbB2-positive breast tumors. [Cancer Res 2007;67(7):3337–44]

Biosensors based on nanodiamonds are able to penetrate through the cell membrane in a targeted manner and probe changes in real-time in the inner cellular space. In this work we performed exclusive theoretical and experimental study of nanodiamond particles adjusted for application in optically-traceable intracellular nanodiamond sensors. Theoretical and experimental study of specific optical properties of high-pressure high-temperature nanodiamonds containing NV- and NV0 centres were performed. The results are supported by theoretical modeling. The final result of this study was detection of luminescence ND in living cells and in vivo application od luminiscence NDs in chicken embryo, showing the detectability of luminescence ND using a standard confocal microscope. On the level of in cells selectivity numerous clusters of ND particles were present within the cytoplasm and at the same time no particles were absent in the nucleus-ND particles can be used as imaging or delivery system for specific cell parts targeting. From our study we can say that biosensors based on nanodiamonds (NDs) are able to penetrate through the cell membrane in a targeted manner and probe changes in the inner cellular space.

The synthesis of ethyl 2-azido-2-deoxy-1-thio-β-D-mannopyranosides 7-11, starting from appropriate synthons with gluco configuration, via S N 2 substitution at C(2), and their rearrangement to 2-S-ethyl-2-thio-β-D-mannopyranosylamines 12-14 are presented.

The effects of a liposomal preparation of lipophilic immunomodulator β-D-GlcNstearoyl-(1-4)-norMurNAc-L-Abu-D-isoGln (N-L18-norAbu-GMDP) were investigated on resistance toCryptosporidium parvuminfection in neonatal kids. The liposomal preparation was administered subcutaneously or intranasally/orally (i.n./p.o.) twice at doses of 100 μg, 200 μg, or 1000 μg per kid pre-infection challenge. The treatment schemes were (i) 72 and 24 h pre-infection challenge, (ii) 24 h pre-infection challenge and 24 h post-infection challenge (oral inoculation with 1×107oocysts ofC. parvumin 5 ml of PBS). Administration of liposomal N-L18-norAbu-GMDP by i.n./p.o. route at the cumulative dose of 2000 μg per kid 72 and 24 h pre-infection challenge, lead to substantially increased clearance of coccidian parasites from various parts of the intestine. On the basis of histological examination, the distribution of cryptosporidia in the intestine and the severity of the infection, treated kids were classified on day 5 as having a strong reduction in infection in comparison to the control group (P

A systematic study is presented of the efficiency of the most common glycosylation methods using standard 2-deoxy-2-phthalimidogalactopyranosyl donors ethyl 4-O-acetyl-3,6-di-O- benzyl-2-deoxy-2-phthalimido-1-thio-β-D-galactopyranoside (3a), 4-O-Acetyl-3,6-di-O-benzyl- 2-deoxy-2-phthalimido-β-D-galactopyranosyl bromide (4), 4-O-acetyl-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-galactopyranosyl fluoride (5b), O-(4-O-acetyl-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-galactopyranosyl) trichloroacetimidate (7) and ethyl 3,6-di-O-benzyl-2-deoxy-2-phthalimido-1-thio-β-D-galactopyranoside (8), pent-4-enyl 3,6-di-O-benzyl- and 3-O-allyl-6-O-benzyl-2-deoxy-2-phthalimido-β-D-galactopyranoside (10a) and (10b) and pent-4-enyl 3,6-di-O-benzyl-2-deoxy-2-phthalimido-4-O-(trimethylsilyl)-β-D-galactopyranoside (11) as glycosyl acceptors in the synthesis of 2-amino-2-deoxy-β-D-galactopyranosyl-(1→4)-2-amino-2-deoxy-β-D-galactopyranosides 12, 16a and 17a. It was found that due to a low reactivity of the axial OH(4) group of glycosyl acceptors, disaccharides 16b and 17b with α(1→4) bond were also formed. The unexpected intermolecular migration of ethylsufanyl group from the reducing end of glycosyl acceptor 8 the reducing end of the activated form of glycosyl donor 4 in the glycosylation step to give ethylsulfanyl derivative 3a was proved. For preparation of the glycosyl donors and glycosyl acceptors with galacto configuration an approach based on epimerization of 4-O-mesyl derivatives of appropriate synthons with gluco configuration 2a and 2b was employed.

Regioisomer of chitobiose13with β(1→3) glycosidic bond and branched analog of chitotriose25having β(1→4) and β(1→3) glycosidic bonds, were prepared and tested as potential mimetics of natural oligosaccharide ligands for activating lectin receptors NKR-P1A and CD69 of natural killer (NK) cells. The structural requirements of NKR-P1 lectin receptor on effective mimetics of its natural ligands has been discussed. A significant binding activity of the branched trisaccharide25to the receptor CD69 was observed.

High pressure high temperature (HPHT) nanodiamonds (NDs) represent extremely promising materials for construction of fluorescent nanoprobes and nanosensors. However, some properties of bare NDs limit their direct use in these applications: they precipitate in biological solutions, only a limited set of bio-orthogonal conjugation techniques is available and the accessible material is greatly polydisperse in shape. In this work, we encapsulate bright 30-nm fluorescent nanodiamonds (FNDs) in 1020-nm thick translucent (i.e., not altering FND fluorescence) silica shells, yielding monodisperse near-spherical particles of mean diameter 66 nm. High yield modification of the shells with PEG chains stabilizes the particles in ionic solutions, making them applicable in biological environments. We further modify the opposite ends of PEG chains with fluorescent dyes or vectoring peptide using click chemistry. High conversion of this bio-orthogonal coupling yielded circa 2000 dye or peptide molecules on a single FND. We demonstrate the superior properties of these particles by in vitro interaction with human prostate cancer cells: while bare nanodiamonds strongly aggregate in the buffer and adsorb onto the cell membrane, the shell encapsulated NDs do not adsorb nonspecifically and they penetrate inside the cells.

Carba analogs of normuramic acid, i.e., 3-(benzyl 2-acetamido-2,3-dideoxy-4,6-O-isopropylidene-α-D-glucopyranosid-3-yl)propanoic acid derivatives (nitrile or esters) 3a-3c were prepared by addition of radicals generated from benzyl 2-acetamido-2-deoxy-4,6-O-isopropylidene-3-O-[(methylsulfanyl)thiocarbonyl]- (2a) or -3-O-(phenoxythiocarbonyl)-α-D-glucopyranoside (2b) with Bu3SnH to acrylonitrile or acryl esters. Alkaline hydrolysis of ethyl ester 3c afforded 3-(benzyl 2-acetamido-2,3-dideoxy-4,6-O-isopropylidene-α-D-glucopyranosid-3-yl)propanoic acid (5). Coupling of acid 5 with L-2-aminobutanoyl-D-isoglutamine benzyl ester trifluoroacetate and subsequent deprotection of the intermediate 6 furnished N-[3-(2-acetamido-2,3-dideoxy-α-D-glucopyranosid-3-yl)propanoyl]-L-2-aminobutanoyl-D-isoglutamine (7).

The preparation of 2,3,4,6-tetra-O-benzyl-5-D-xylo-hex-5-ulosonamides 3a and 3b and their cyclization to 5-amino-2,3,4,6-tetra-O-benzyl-5-deoxy-D-glucono-1,5-lactam (4) and 5-amino- 2,3,4,6-tetra-O-benzyl-5-deoxy-D-talono-1,5-lactam (5) or to 2,3,4,6-tetra-O-benzyl-1,5-dideoxy-1,5-imino-D-glucitol (9), and also their direct transformation into 2,3,4,6-tetra-O-benzyl- D-talono-1,5-lactone (6), are described and discussed. The atypical boat conformation of D-talonolactam 5 and D-talonolactone 6 was found.

Multiple antigenic peptides containing dimeric Tn antigen [Ac-(Tn)2-γ-Abu]4-(Lys-X)2-Lys-β-Ala (V: X=0; VIII: X=γ-Abu) and [Ac-(Tn)2-γ-Abu]8-(Lys-X)4-(Lys-X)2-Lys-β-Ala (XI: X=0; XIV: X=γ-Abu), immobilized on biocompatible Tenta Gel S NH2 support were prepared by SPPS. Rosetting tests of V, VIII, XI and XIV showed positive reactions with anti-Tn (DAKO) and Tn+ erythrocytes, with anti-Tn/A (BRIC 66) and Tn+ and A erythrocytes, other combinations were negative. In all the animals immunized with XIV, we found a remarkable increase in the level of anti-Tn (titre 2000–64 000, score 105–167) and no change of anti-A levels (titre 8, score 13–17). Neither non-immune nor immune sera showed any reactivity with T+, Cad+ and blood group O erythrocytes. Immunized mice did not exhibit any sign of adverse reaction to the administered conjugates. Biological activities were correlated with molecular modelling and molecular dynamic calculations. The biological activities of these synthetic Tn antigen conjugates (good availability for the immunological interactions, highly specific immunogenicity, good biological tolerance) together with their precise chemical characterization seem to be a promising approach to preparation of anti-tumour vaccine and affinity purification of anti-Tn antibodies. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.

Ethyl 3,4,6-tri-O-benzyl-2-deoxy-2-phthalimido-1-thio-β-D-glucopyranoside (5), prepared by benzylation of ethyl 2-deoxy-2-phthalimido-1-thio-β-D-glucopyranoside (4), was transformed by reaction with bromine into 3,4,6-tri-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl bromide (6). Thioglycoside 5 in the presence of methyl triflate and glycosylbromide 6 in the presence of silver triflate were used as glycosyl donors for condensation with benzyl 2-acetamido-3-O-allyl-6-O-benzyl-2-deoxy-α-D-glucopyranoside (7), to give benzyl 2-acetamido-3-O-allyl-6-O-benzyl-4-O-(3,4,6-tri-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-2-deoxy-α-D-glucopyranoside (8). Its reductive dephthaloylation with NaBH4/AcOH afforded benzyl 2-acetamido-3-O-allyl-4-O-(2-amino-3,4,6-tri-O-benzyl-2-deoxy-β-D-glucopyranosyl)- 6-O-benzyl-2-deoxy-α-D-glucopyranoside (11). Compound 11 was N-acylated to give benzyl 2-acetamido-4-O-(2-acylamino-3,4,6-tri-O-benzyl-2-deoxy-β-D-glucopyranosyl)-3-O-allyl-6-O-benzyl-2-deoxy-α-D-glucopyranosides (12a) or (12b). These compounds were converted into corresponding benzyl 2-acetamido-4-O-(2-acylamino-3,4,6-tri-O-benzyl-2-deoxy-β-D-glucopyranosyl)-6-O-benzyl-3-O-carboxymethyl-2-deoxy-α-D-glucopyranosides which, by condensation with H-L-Abu-D-isoGln(OBzl) followed by hydrogenolysis of protective benzyl groups, furnished glycopeptides 16a and 16b. Intramolecular O→N migration of the allyl protecting group followed by its reduction to the propyl residue by reaction of compound 8 with hydrazine or hydrazinium acetate, to give benzyl 2-acetamido-4-O-(3,4,6-tri-O-benzyl-2-deoxy-2-propylamino-β-D-glucopyranosyl)-6-O-benzyl-2-deoxy-α-D-glucopyranoside (9), is also described.

N-Acetylnormuramoyl-L-2-aminobutanoyl-D-isoglutamine (7) and its lipophilic 6-O-octadecanoyl (8) and 6-O-(2-tetradecylhexadecanoyl) (9) derivatives were prepared and their immunoadjuvant activity and pyrogenicity were tested. Compounds 8 and 9 are less pyrogenic than muramoyl-dipeptide (MDP) and norMDP analog 7. Both lipophilic derivatives 8 and 9 are better adjuvants than MDP in cell mediated immunity.

Protection from undesirable effects of radiotherapy or chemotherapy, primarily from myelosuppression, remains still a crucial problem to be studied. Attention has been therefore paid to various immunomodulatory agents that through the monocyte/macrophage system induced production of cytokines, which can induce and operate restoration of haemopoiesis and thus act radioprotectively. Some synthetic analogues of MDP free of undesirable side-effects, were synthesized in the Czech Republic. Lipophilic beta-D-GlcNstearoyl-(1- > 4)-norMurNAc-L-Abu-D-isoGln (DDD-St) was designed to be easily entrapped into liposomes and this liposomal DDD-St protected efficiently mice against irradiation, when administered i.p., i.v. or s.c. 24 h prior to lethal irradiation (survival rate in the range of 30-80% compared with 0% in control). Especially the subcutaneous application of liposomal DDD-St was very efficient. The parameters characteristic of recovery of haemopoiesis in bone marrow on day 10 after 6.5 Gy irradiation were significantly improved in comparison with the controls. Very high radioprotective effect of s.c. administered liposomal DDD-St can be explained (together with induction of haemopoiesis) by an effective and long-lasting activation of nonspecific immunity, which is able to withhold an onset of septicemia in early days after irradiation. In conclusion, the liposomal DDD-St should be therapeutically beneficial in moderating the haemopoietic damage, which is an undesirable effect of radiotherapy or chemotherapy.

Lan-Feng Dong , Victoria J.A. Jameson , David Tilly , Lubomir Prochazka , Jakub Rohlena , Karel Valis , Jaroslav Truksa , Renata Zobalova , Elahe Mahdavian , Katarina Kluckova , Marina Stantic , Jan Stursa , Ruth Freeman , Paul K. Witting , Erik Norberg , Jacob Goodwin , Brian A. Salvatore , Jana Novotna , Jaroslav Turanek , Miroslav Ledvina , Pavel Hozak , Boris Zhivotovsky , Mark J. Coster , Stephen J. Ralph , Robin A.J. Smith , Jiri Neuzil a,e,n

A series of 5′-substituted analogs of toyocamycin were prepared by condensation of silylated 4-amino-6-bromo-5-cyanopyrrolo[2,3-d]pyrimidine with protected 5-azido-5-deoxy- or 5-fluoro-5-deoxyribofuranose followed by debromination and deblocking. Alternatively, 5′-azido-5′-deoxytoyocamycin was prepared by azidation of toyocamycin. Conversion of the 5-nitrile function of the toyocamycin derivatives into a carboxamide or a thiocarboxamide gave the corresponding analogs of sangivamycin or thiosangivamycin while reduction of the 5′-azido-5′-deoxy nucleosides provided 5′-amino-5′-deoxy derivatives.

Dendritic poly(L-lysines) (DGL) constitute promising nanomaterials applicable as a nonviral gene-delivery vector. In this study, we evaluate the transfection abilities of four DGL generations with special emphasis on the systematic description of the relationship of how generation (i.e., molecule size) affects the transfection efficacy. Using Hep2 cells, we demonstrated that the capability of unmodified DGL to deliver plasmid is of a magnitude lower than that of jetPEI. On the other hand, employing the Hep2 cell line stably transduced with eGFP, we observed that DGL G5 delivers the siRNA oligonucleotide with the same efficiency as Lipofectamine 2000. In further experiments, it was shown that DGL affords excellent ability to bind DNA, protect it against DNase I attack, and internalize it into cells.

Vaccinology as a scientific field is undergoing a dramatic development. Never before such sophisticated techniques and in-depth knowledge of immunological processes have been at hand to exploit fully the potential of protecting from as well as curing diseases through vaccination. In spite of great successes like eradication of smallpox in the 1970s and poliomyelitis elimination from all but six countries in the world (two important milestones in the medical history), new challenges have arisen to be faced. Rapidly changing ecosystems and human behaviour, an ever-increasing density of human and farmed animal populations, a high degree of mobility resulting in rapid spreading of pathogens in infected people and animals, new contacts between human and animals in endemic areas, poverty, and war conflicts in the third world, and many other factors contribute to the more frequent occurrence and rapid dissemination of new diseases. Three diseases that most heavily afflict global health are AIDS, tuberculosis, and malaria. As an example of new viral pathogens we can mention Ebola virus, SARS-coronavirus, or new strains of influenza virus (Wack & Rappuoli, 2005). Among re-emerging diseases of the past few years, diphteria and cholera should be mentioned. Moreover, multi drug-resistant bacteria frequently occur as a result of overdosing on antibiotics. One of the most important future challenges will be to respond promptly to emerging diseases such as those mentioned above. Rapid sequencing of the genome of the pathogen implicated the speed of the development of diagnostic tools as well as the identification and expression of recombinant targets for vaccines and therapeutic agents development (Stadler et al., 2003). Immunotherapy of cancer represents a special field, where anticancer vaccines could be powerful weapons/tools for long-term effective treatment. The progress in the vaccine development is closely related with the progress in immunology and molecular biology. A new term “Reverse vaccinology” was proposed by Rappuoli (Rappuoli, 2000) to specify a complex genome-based approach in the vaccine development. Unlike the conventional approach that requires a laborious process of a selection of individual components important for the induction of protective immune response, reverse vaccinology offers a possibility to use genomic information derived from in silico analysis of the sequenced organisms. This approach can significantly reduce the time necessary to identify the antigens for the development of a candidate vaccine and enables a systematic identification of all potential antigens of pathogens including those which are difficult or

The preparation of E and Z isomers of 3-ureido-2-cyano-2-propenoates Ia - Id and their base-catalyzed isomerization and cyclization to 5-carboxycytosine derivatives IIa - IIf and 5-cyanouracil derivatives IIIa and IIIb is described. Also described is the preparation of 3-ureido-2-acyl-2-propenoates Va - Vd and their base-catalyzed cyclization to 5-carboxy-2(1H)-pyrimidone derivatives VIa - VIc and 5-acyluracils VIIa - VIIc.

We designed and synthesised a series of new cationic lipids based on spermine linked to various hydrophobic anchors. These lipids could be potentially useful for the preparation of stable cationic liposomes intended for the construction of drug targeting systems applicable in the field of anticancer/antiviral therapy, vaccine carriers, and vectors for the gene therapy. Low in vitro toxicity was found for these compounds, especially for LD1, in several cell lines. The delivery of both a fluorescence marker (calcein) and antiviral drugs into cells has been achieved owing to a large extent of internalization of cationic liposomes (labelled by Lyssamine-Rhodamine PE or fluorescein-PE) as demonstrated by fluorescent microscopy and quantified by flow cytometry. The bovine herpes virus type 1 (BHV-1) virus infection in vitro model using MDBK cells was employed to study the effect of the established antiviral drug HPMPC (Cidofovir®) developed by Prof. A. Holý. Inhibition of BHV-1 virus replication was studied by quantitative RT-PCR and confirmed by both Hoffman modulation contrast microscopy and transmission electron microscopy. We found that in vitro antiviral activity of HPMPC was significantly improved by formulation in cationic liposomes, which decreased the viral replication by about 2 orders of magnitude.

Lyme disease caused by spirochete Borrelia burgdorferi sensu lato, is a tick-born illness. If the infection is not eliminated by the host immune system and/or antibiotics, it may further disseminate and cause severe chronic complications. The immune response to Borrelia is mediated by phagocytic cells and by Borrelia-specific complement-activating antibodies associated with Th1 cell activation. A new experimental vaccine was constructed using non-lipidized form of recombinant B. burgdorferi s.s. OspC protein was anchored by metallochelating bond onto the surface of nanoliposomes containing novel nonpyrogenic lipophilized norAbuMDP analogues denoted MT05 and MT06. After i.d. immunization, the experimental vaccines surpassed Alum with respect to OspC-specific titers of IgG2a, IgG2b isotypes when MT06 was used and IgG3, IgM isotypes when MT05 was used. Both adjuvants exerted a high adjuvant effect comparable or better than MDP and proved themselves as nonpyrogenic.

The presented work aims for the development of optically-traceable intracellular nanodiamond sensors, where photoluminescence can be changed by biomolecular attachment/delivery event. High biocompatibility, small size and stable luminescence from its color centers, makes nanodiamond (ND) particles an attractive alternative to molecular dyes for drug-delivery and cell-imaging applications. In our work we study how the surface modification of ND can change ND luminescence spectra. This method can be used as a novel detection tool for remote monitoring of chemical processes in biological systems. We discuss photoluminescence (PL) spectra of oxidized and hydrogenated ND and a single crystal diamond, containing engineered NV centers. The hydrogenation of ND leads to quenching of NV- related luminescence and a PL shift due to changing of occupation from NV- to NV0 states. We model this effect using electrical potential changes at the diamond surface.

With the rise of antibiotic resistance of pathogenic bacteria there is an increased demand for monitoring of the functionality of bacteria membranes, whose disruption can be induced by peptide-lipid interactions. In this work we attempt to monitor formation and subsequent peptide induced disruption of supported lipid membranes (SLBs) on boron-doped nanocrystalline diamond (B-NCD). Electrochemical Impedance Spectroscopy (EIS) was used to study in situ changes related to lipid membrane formation and disruption by peptide-induced interactions. The observed impedance changes were minimal for oxidized B-NCD samples. The sensitivity for the detection of membrane formation and disruption was significantly higher for hydrogenated B-NCD surfaces. Data modelling indicates large differences in the structure of electrical double layer at the B-NCD/SLB interface for hydrogen and oxygen terminated B-NCD surfaces. For oxidized B-NCD surfaces, EIS changes are negligible. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. ispartof: Physica Status Solidi A, Applications and Materials Research vol:208 issue:9 pages:2099-2103 status: published

Mitochondria are emerging as intriguing targets for anti-cancer agents. We tested here a novel approach, whereby the mitochondrially targeted delivery of anti-cancer drugs is enhanced by the addition of a triphenylphosphonium group (TPP(+)). A mitochondrially targeted analog of vitamin E succinate (MitoVES), modified by tagging the parental compound with TPP(+), induced considerably more robust apoptosis in cancer cells with a 1-2 log gain in anti-cancer activity compared to the unmodified counterpart, while maintaining selectivity for malignant cells. This is because MitoVES associates with mitochondria and causes fast generation of reactive oxygen species that then trigger mitochondria-dependent apoptosis, involving transcriptional modulation of the Bcl-2 family proteins. MitoVES proved superior in suppression of experimental tumors compared to the untargeted analog. We propose that mitochondrially targeted delivery of anti-cancer agents offers a new paradigm for increasing the efficacy of compounds with anti-cancer activity.

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.

The preparation of E and Z isomers of 3-ureido-2-cyano-2-propenoates Ia - Id and their base-catalyzed isomerization and cyclization to 5-carboxycytosine derivatives IIa - IIf and 5-cyanouracil derivatives IIIa and IIIb is described. Also described is the preparation of 3-ureido-2-acyl-2-propenoates Va - Vd and their base-catalyzed cyclization to 5-carboxy-2(1 H )-pyrimidone derivatives VIa - VIc and 5-acyluracils VIIa - VIIc .

A series of 5′-substituted analogs of toyocamycin were prepared by condensation of silylated 4-amino-6-bromo-5-cyanopyrrolo[2,3-d]pyrimidine with protected 5-azido-5-deoxy- or 5-fluoro-5-deoxyribofuranose followed by debromination and deblocking. Alternatively, 5′-azido-5′-deoxytoyocamycin was prepared by azidation of toyocamycin. Conversion of the 5-nitrile function of the toyocamycin derivatives into a carboxamide or a thiocarboxamide gave the corresponding analogs of sangivamycin or thiosangivamycin while reduction of the 5′-azido-5′-deoxy nucleosides provided 5′-amino-5′-deoxy derivatives.