Effects of alkyl glycosides incorporated into liposomes prepared from synthetic amphiphiles on their tissue distribution in Ehrlich solid tumor-bearing mice.

A study of the effects of alkyl glycosides incorporated into synthetic liposomes with respect to their stability, their in vivo distribution in Ehrlich solid tumor-bearing mice and their in vitro interaction with liver cells was undertaken. The synthetic liposomes were prepared from N,N-didodecyl-N alpha-[6-(trimethylammonio)hexanoyl]-L-alaninamide bromide (N+C5Ala2C12) and labeled with 99mTc. n-Dodecyl glucoside (DG) and n-dodecyl sucrose (DS) were used as alkyl glycosides. The stability was hardly changed by incorporation of alkyl glycosides into the liposomes in saline and serum. The uptake of DG- and DS-modified N+C5Ala2C12 liposomes decreased in liver and spleen compared with that of unmodified N+C5Ala2C12 liposomes, resulting in an increase in blood and other tissues such as tumor, duodenum and kidney, where the DS-modified N+C5Ala2C12 liposomes had a marked tendency. It was observed with electron micrographs that the size of N+C5Ala2C12 liposomes became small by incorporation of alkyl glycoside. The smaller N+C5Ala2C12 liposomes were found to result in the lower uptake in liver. The interaction of the liposomes with liver cells in vitro indicated that both DG- and DS-modified liposomes had a low affinity for liver cells compared with the unmodified liposomes and the extent of interaction of the DS-modified liposomes was weaker than that of the DG-modified liposomes.     

Preferential production of IL-12 by peritoneal macrophages activated by liposomes prepared from neoglycolipids containing oligomannose residues

The present study demonstrates that liposomes coated with synthesized neoglycolipids constructed from mannotriose and dipalmitoylphosphatidylethanolamine (Man3-DPPE) activate peritoneal macrophages (PEMs) to up-regulate expression of co-stimulatory molecules and preferentially secrete IL-12. Injection of Man3-DPPE-coated liposomes (oligomannose-coated liposome, OMLs) into the peritoneal cavity of mice resulted in specific and rapid incorporation of OMLs into PEMs. Upon OML incorporation, expression of co-stimulatory molecules, CD40, CD80, and CD86, and of MHC class II molecules was clearly enhanced on PEMs. In addition, production of IL-12 from PEMs was clearly promoted in response to OML incorporation, while those of IL-1 and IL-6 were suppressed. In contrast, liposomes coated with other carbohydrates and those without a carbohydrate-coating did not induce production of these cytokines. The cytokine profile produced from PEMs in response to OML clearly differed from those in response to ligands for TLRs, in which productions of IL-1 and/or IL-6 were strongly enhanced. Taken together, the results indicate that OMLs activate PEMs through a particular type of signal pathway distinct from those activated with TLR ligands, leading to specific production of IL-12 and consequent maturation of PEMs. Thus, OMLs can be used as a novel adjuvant for efficient activation of specific cellular immunity.     

Analogues of cyclolinopeptide A containing alpha-hydroxymethyl amino acid residues

Linear and cyclic cyclolinopeptide A (CLA) analogues containing alpha-hydroxymethylleucine (HmL) in positions 1, 4, and 1&4, and alpha-hydroxymethylvaline (HmV) in position 5, were synthesized by the solid-phase peptide strategy and cyclized with the 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide/1-hydroxy-7-azabenzotriazole (EDC/HOAt) reagent. The peptides were examined for their immunosuppressive activity in the lymphocyte proliferation assays (LPA). Only HmL-containing peptides demonstrated at about 25% lower immunosuppressive activity, but they are four times more soluble in water solutions than the native CLA. It seems from the LPA results that peptide [(HmL4)CLA] is the most promising for further studies. This peptide was characterized in solution, at room temperature in CDCl3, and the conformation compared with that observed for CLA in the solid state.     

Tissue distribution of EDTA encapsulated within liposomes containing glycolipids or brain phospholipids

Multilamellar liposomes were prepared with various asialoglycolipids, gangliosides, sialic acid, or brain phospholipids in the liposome membrane and with ethylenediaminetetraacetic acid (EDTA) encapsulated in the aqueous compartments. The liposomes containing glycolipids or sialic acid were prepared from a mixture of phosphatidylcholine, cholesterol, and one of the following test substances: galactocerebroside, glucocerebroside, galactocerebroside sulfate, mixed gangliosides, monosialoganglioside G_M1, monosialoganglioside G_M2, monosialoganglioside G_M3, disialoganglioside G_D1a, or sialic acid. The liposomes containing brain phospholipids were mixtures of either sphingomyelin and cholesterol or a brain total phospholipid extract and cholesterol. Distribution of ¹⁴C-labeled EDTA were determined in mouse tissues from 15 min to 6 h or 12 h after a single injection of liposome prepartion. Liver uptake of encapsulated EDTA was lowest from all liposome preparations containing sialic acid or sialogangliosides regardless of the amount of sialic acid moiety present or the identity of the particular ganglioside; highest uptake of encapsulated EDTA by liver was from the liposomes containing galactocerebroside or brain phospholipids. Lungs and brain took up the largest amounts of EDTA from liposomes containing sphingomyelin and lesser amounts from liposomes containing G_D1a. Use of mouse brain phospholipid extract to prepare liposomes did not increase uptake of encapsulated EDTA by the brain. EDTA in liposomes containing monosialogangliosides, brain phospholipids, galactocerebroside, or sialic acid was taken up well by spleen and marrow. Highest thymus uptake of encapsulated EDTA was from liposomes containing G_D1a. These results demonstrate that inclusion of sialogangliosides in liposome membranes decreases uptake of liposomes by liver, thus making direction of encapsulated drugs to other organs more feasible. Liposomes containing glycolipids also have potential uses as probes of cell surface receptors.     

Tissue distribution of EDTA encapsulated within liposomes containing glycolipids or brain phospholipids.

Multilameller liposomes were prepared with various asialoglycolipids, gangliosides, sialic acid, or brain phospholipids in the liposome membrane and with ethylenediaminetetraacetic acid (EDTA) encapsulated in the aqueous compartments. The liposomes containing glycolipids or sialic acid were prepared from a mixture of phosphatidylcholine, cholesterol, and one of the following test substances: galactocerebroside, glucocerebroside, galactocerebroside sulfate, mixed gangliosides, monosialoganglioside GM1, monosialoganglioside GM2, monosialoganglioside GM3, disialoganglioside GD1a, or sialic acid. The liposomes containing brain phospholipids were mixtures of either sphingomyelin and cholesterol or a brain total phospholipid extract and cholesterol. Distributions of 14C-labeled EDTA were determined in mouse tissues from 15 min to 6 h or 12 h after a single injection of liposome preparation. Liver uptake up encapsulated EDTA was lowest from all liposome preparations containing sialic acid or sialogangliosides, regardless of the amount of sialic acid moiety present or the identity of the particular ganglioside; highest uptake of encapsulated EDTA by liver was from liposomes containing galactocerebroside or brain phospholipids. Lungs and brain took up the largest amounts of EDTA from liposomes containing sphingomyelin and lesser amounts from liposomes containing GD1a. Use of mouse brain phospholipid extract to prepare liposomes did not increase uptake of encapsulated EDTA by the brain. EDTA in liposomes containing monosialogangliosides, brain phospholipids, galactocerebroside, or sialic acid was taken up well by spleen and marrow. Highest thymus uptake of encapsulated EDTA was from liposomes containing GD1a. These results demonstrate that inclusion of sialogangliosides in liposome membranes decreases uptake of liposomes by liver, thus making direction of encapsulated drugs to other organs more feasible. Liposomes containing glycolipids also have potential uses as probes of cell surface receptors.     

Synthesis and activity of dermorphin analogues containing unusual amino acid residues

Summary Solid phase syntheses of analogues of the opioid heptapeptide dermorphin (H-Tyr-dAla-Phe-Gly-Tyr-Pro-Ser-NH₂) containing in the first position 3-aminotyrosine, 3-nitrotyrosine, 4-aminophenylalanine, or nucleoamino acids, 3-(uracilyl-1)alanine, 3-(thyminyl-1)alanine and 3-(6-methyluracilyl-1)alanine are described. The receptor binding properties and analgesic activity of the analogues were examined in comparison with dermorphin. All analogues showed low opioid activity in the binding assays with respect to μ- and δ-receptors. The peptide containing 3-(thyminyl-1)alanine demonstrated a high analgesic activity in different tests when administered intracisternally in mice.     

Synthesis and activity of dermorphin analogues containing unusual amino acid residues

Solid phase syntheses of analogues of the opioidheptapeptide dermorphin(H-Tyr-dAla-Phe-Gly-Tyr-Pro-Ser-NH₂) containingin the first position 3-aminotyrosine, 3-nitrotyrosine,4-aminophenylalanine, or nucleoamino acids,3-(uracilyl-1)alanine, 3-(thyminyl-1)alanine and3-(6-methyluracilyl-1)alanine are described. Thereceptor binding properties and analgesic activity ofthe analogues were examined in comparison withdermorphin. All analogues showed low opioid activityin the binding assays with respect to µ- and-receptors. The peptide containing3-(thyminyl-1)alanine demonstrated a high analgesicactivity in different tests when administeredintracisternally in mice.     

SDS-subtilisin catalyzed synthesis of tetra-peptides containing multifunctional amino acid residues in ethanol

The role of acyl donor structure on the course of peptide bond formation catalyzed by SDS-subtilisin in ethanol was investigated. In the reaction Z---Ala---Ala---Leu---OR+H---Phe---pNA→Z---Ala---Ala---Leu---Phe---pNA, nearly quantitative product yields were observed after 2 h, regardless of whether an activated (R=CH₃, p-C₆H₅Cl) or non-activated (R=H) acyl donor was used. It was found that the enzyme can accept as acyl donors N-protected tri-peptides containing basic or acidic amino acid residues in the P₁-position. Tetra-peptides of general formula Z---Ala---Ala---P₁---P₁′---pNA, where P₁=Glu, Asp, Lys, Arg or His and P₁′=Phe, Arg or Glu have been obtained in good yield.     

Redox-responsive vesicles prepared from supramolecular cyclodextrin amphiphiles

Redox-responsive vesicles self-assembled by supramolecular cyclodextrin amphiphiles, consisting of the guest (N-1-decyl-ferrocenylmethylamine, 1) and the host (2-O-carboxymethyl-β-cyclodextrin, CM-β-CD), were prepared. The morphologies and sizes of these novel vesicles in an aqueous solution were observed by transmission electron microscopy (TEM) and were confirmed by atomic force microscopy (AFM) and dynamic light scattering (DLS) measurements. The effects of the host-guest ratio, the concentration and the solvent composition of water and methanol on vesicles were investigated in detail. The interactions between the host and the guest, the complex stoichiometry, the stability constant and conformations of 1·CM-β-CD in aqueous solution were investigated by cyclic voltammetry (CV), UV and nuclear magnetic resonance (NMR) measurements. According to the complex stoichiometry and ‘tadpole-like’ spatial conformations, the supramolecular cyclodextrin amphiphiles made from 1·CM-β-CD were proposed to form the membranes of the vesicles. This kind of vesicle system was responsive to an oxidizing agent, which could pave the way to combine supramolecular host-guest chemistry and membrane chemistry for potentially functional applications.     

Modified in vivo behavior of liposomes containing synthetic glycolipids

Liposomes with synthetic saccharide determinants were prepared from synthetic cholesterol conjugates of D-mannose and 6-amino-6-deoxy-D-mannose and labeled with [⁵¹Cr]chromate. The kinetics and tissue distribution of label in mice were determined after footpad and subcutaneous injection. Liposomes bearing either of these saccharide determinants greatly increased retention of label at the injection sites compared to control liposomes, which contain no glycolipid, and to free [⁵¹Cr]chromate. Draining lymph nodes contained small fractions of the injected radioactivity but in some cases this retention was saccharide-dependent and highly concentrated. These results show that incorporation of synthetic glycolipids can substantially alter the in vivo lifetime and distribution of liposomes outside the bloodstream. Such surface-modified liposomes may be useful for sustained release or selective delivery of therapeutic or diagnostic agents.     

Permeability of liposomes towards amino alcohol and amino acid nitroxides

Electron spin resonance (ESR) allowed the analysis of the encapsulation of amino alcohols and amino acids labeled with nitroxide groups into multilamellar liposomes. The stability and permeability of these phospholipid vesicles were studied in terms of time dependance and structure of these biomolecules.     

Characteristics of distribution of amino acid residues in the primary structure of calmodulin

Systemic analysis of the peculiarities of distribution of di-, tri- and tetrapeptide residues in amino acid sequence of calmodulins of different origin has been carried out. A conclusion is made that all the examined repeated tri- and tetrapeptide residues with comparatively low conformation mobility enter the alpha-helical conformations with low mobility in beta-turns, and tri- and tetrapeptides with intermediate meaning of conformation entropy in beta-sheet conformations of the calmodulin molecule.     

Cationic amphiphiles induce fusion of acidic liposomes

Decylamine, dodecylamine and tetradecylamine induced aggregation and fusion of acidic liposomes at concentrations of about 1 mM, 75 μM and 75 μM, respectively. Aggregation was assayed as increase in turbidity. Fusion was assayed as intermixing of membranes and contents, and was observed in the electron-microscope to form large liposomes. Only at higher concentrations did these amphiphiles induce massive leakage of the liposomes' contents. Similar effects were caused by hexadecylpyridinium bromide (CP) and hexadecyltrimethylammonium bromide (CTAB). The trivalent cation 4-dodecyldiethylenetriamine and the more hydrophobic amphiphile, trioctylmethylammonium chloride, induced fusion at concentrations of about 10–20 μM. Octylamine and heptylamine induced size increase at mM concentrations. They induced membrane intermixing but little or no content intermixing. Thus, these amphiphiles seem to promote size increase either by transfer of lipid or mainly by ‘cracking and annealing’.     

Tissue distribution and cellular distribution of liposomes encapsulating muramyltripeptide phosphatidyl ethanolamide

In previous studies it was shown that administration of liposome-encapsulated MTPPE (LE-MTPPE) led to resistance againstKlebsiella pneumoniae infection. To get more insight in the cell types that are involved in this by LE-MTPPE induced antibacterial resistance, the tissue distribution of liposomes encapsulating MTPPE and the distribution over the cells in the main target organs were investigated. After intravenous injection of the liposomes in mice a substantial amount was recovered from liver and spleen and a smaller amount from the lung. In the liver 83% of the liposomes was taken up by the macrophages. In the spleen also most liposomes were taken up by macrophages of the red and white pulp as well as by dendrocytes. The liver and spleen were also the organs in which, after intravenous inoculation,K. pneumoniae was trapped. It was observed that cells containing LE-MTPPE often had not taken up bacteria. Most bacteria, about 73%, were found in cells not containing liposomes. The capacity of the liposome-containing cells to take up bacteria did not change with time. This suggests that the by LE-MTPPE immunostimulating effect is due to the production of cytokines by the cells that take up LE-MTPPE. These cytokines might stimulate other cells to the killing of bacteria.     

Thiol containing compounds and amino acid hydroxamates as reversible synthetic inhibitors of Astacus protease

Reversible synthetic inhibitors are characterized for Astacus protease, a 22,614-Da zinc containing neutral endopeptidase from the digestive tract of crayfish. Effective inhibition was demonstrated for several simple thiol containing compounds and a series of amino acid hydroxamates. Both classes of inhibitors had ID50 values ranging from 10(-2) to 10(-4) M for inhibition of hydrolysis of succinyl-Ala-Ala-Ala-p-nitroanilide. Tyrosine hydroxamate was found to be the most effective inhibitor with an ID50 of 175 microM and the mode of inhibition by this compound was determined to be of the simple noncompetitive type. In contrast to the other inhibitors tested, cysteine was seen to partially inactivate the enzyme in a time-dependent manner. The kinetics of this process was studied in detail using progress curve analysis. It was determined that cysteine was acting as a weak chelator and slowly establishing an equilibrium between metallo- and apoenzyme. In the presence of the strong zinc scavenger EDTA, cysteine can, in effect, function as a catalyst in transferring the metal from the protein to the secondary chelator at a rate 10,000 times faster than the rate of unassisted zinc dissociation. The series of amino acid hydroxamates served as probes into the microenvironment of the active site. Possible binding modes of the inhibitors are discussed on the basis of the relationship between the chemical nature of the inhibitor side chains and the strength of inhibition.     

Conserved amino acid residues in ribosome-inactivating proteins from plants.

The amino acid sequences of eleven RIPs sequenced to date have been compared in the expectation that this would be useful in the location of functionally and/or structurally important sites of these molecules. In addition to several highly conserved hydrophobic amino acids, thirteen absolutely conserved residues have been found in ricin A-chain: Tyr21, Phe24, Arg29, Tyr80, Tyr123, Gly140, Ala165, Glu177, Ala178, Arg180, Glu208, Asn209 and Trp211. The role of these residues as well as of the C-terminal region have been discussed based on the results of chemical and enzymatic modifications, site-directed mutagenesis, and deletion studies.     

Permeability behavior of liposomes prepared from fatty acids and fatty acid methyl esters

The permeability properties of liposomes prepared at pH 8.7 from a fatty acid and either methyl oleate or methyl elaidate, with or without cholesterol, were investigated. The fatty acids used were oleic acid, elaidic acid, and the selenium-containing fatty acids 9-selenaheptadecanoic acid and 13-selenaheneicosanoic acid. The liposomes trapped sucrose and carboxyfluorescein. Their volume change resulting from osmotic shock was directly proportional to the change in absorbance (light scattering). Liposomes prepared from oleic acid and either methyl oleate or methyl elaidate underwent osmotic swelling much more slowly than liposomes prepared from elaidic acid and either methyl oleate or methyl elaidate. Incorporation of cholesterol decreased the initial rate of erythritol permeation, especially in liposomes containing methyl oleate. The swelling rates of liposomes prepared with the selenium-containing fatty acids indicated that incorporation of methyl elaidate gave more tightly packed bilayers than did incorporation of methyl oleate. The effect of cholesterol on the initial rate of erythritol influx was greater in oleic acid and elaidic acid liposomes than in selenium-containing fatty acid liposomes, indicating that the large bulk of the selenium heteroatom suppresses the ability of cholesterol to interact with the hydrocarbon chain.