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G Gregoriadis

Publications and source records attributed to G Gregoriadis.

At least 37 records · Page 2Linked to original sources
PubMed1997

Polysialylated asparaginase: preparation, activity and pharmacokinetics.

Erwinia carotovora L-asparaginase was coupled covalently to colominic acid, a low molecular mass polysialic acid, by reductive amination. Depending on the molar ratios of colominic acid-asparaginase (50:1, 100:1 and 250:1), polysialylated constructs contained 4.2-8.1 molecules of colominic acid per molecule of enzyme. Such constructs retained most (82-86%) of the initial asparaginase activity and also maintained the Km values of the native enzyme towards the substrate asparagine. On exposure to (mouse) blood plasma at 37 degrees C, polysialylated asparaginase constructs exhibited resistance to proteolysis with 65-83% of the initial enzyme activity still present after 6 h. In contrast, most of the native enzyme was inactivated under the same conditions. In vivo experiments with intravenously injected mice revealed a significant increase in the half-life of the polysialylated asparaginase over that observed with the native enzyme. Such an increase was greatest (250%, about 38 h) for the construct with the highest degree of polysialylation. Results suggest that polysialylation of asparaginase and other proteins may provide an alternative means to improve their effective use in therapeutics.

Animals↗
PubMed1997

Liposome-mediated DNA vaccination.

Numerous reports have indicated that intramuscular injection of antigen-coding naked plasmid DNA can trigger humoral and cell-mediated protective immunity against infection. This follows DNA uptake by muscle fibres, leading to the expression and extracellular release of the antigen. Here it is shown for the first time that intramuscular immunization of mice with pRc/CMV HBS (encoding the S region of hepatitis B antigen; HBsAg) entrapped into positively charged (cationic) liposomes leads to greatly improved humoral and cell-mediated immunity. These cationic liposome-entrapped DNA vaccines generate titres of anti-HBsAg IgG1 antibody isotype in excess of 100-fold higher and increased levels of both IFN-gamma and IL-4 when compared with naked DNA or DNA complexed with preformed similar (cationic) liposomes. It is likely that immunization with liposome-entrapped plasmid DNA involves antigen-presenting cells locally or in the regional draining lymph nodes.

Animals↗
PubMed1997

Interleukin-15 acts as an immunological co-adjuvant for liposomal antigen in vivo.

The recently discovered interleukin-15 (IL-15) is known to bind to the receptor of interleukin-2 (IL-2) and to share several of the latter's immunological properties. In the present study, the first to our knowledge on IL-15 behaviour in vivo, we examined the possibility that IL-15 also shares the ability of IL-2 to enhance the immunological adjuvant property of liposomes by acting as a co-adjuvant. The cytokine and a model antigen (tetanus toxoid) were either co-entrapped by the dehydration rehydration method into, or covalently co-linked by diazotization to the surface of the same liposomes, or entrapped in different liposome populations. Intramuscular immunization of CD-1 mice with a variety of IL-15 and toxoid formulations revealed that IL-15 augments anti-toxoid IgG (IgG1, IgG2, IgG2b) responses well above (up to ten-fold) those achieved with liposomal toxoid alone (or with a mixture of free IL-15 and toxoid) when the cytokine and the antigen are associated with the same vesicles but not when in different vesicle populations that were mixed before injection. Higher responses were observed for all three subclasses studied only with liposomes where IL-15 and antigen were accommodated on their surface.

Adjuvants, Immunologic↗
PubMed1997

Novel non-acidic formulations of haloperidol complexed with beta-cyclodextrin derivatives.

Haloperidol (Hal), a highly hydrophobic drug, was complexed with two beta-cyclodextrin (beta-CD) derivatives. Hal solubility was increased 20-fold in the presence of a 10-fold excess of methyl beta-CD (Me beta-CD) and 12-fold in the presence of a 10-fold excess of 2-hydroxypropyl beta-CD (HP beta-CD). The stoichiometries and stability constants of Hal-Me beta-CD (1:1 and 2345 M-1 at 27 degrees C) and Hal-HP beta-CD (1:1 and 2112 M-1 at 27 degrees C) complexes were calculated by the continuous variation and phase solubility methods respectively. Differential scanning calorimetry and 1H-NMR were used to confirm the formation of inclusion complexes. Moreover, the enthalpy and entropy of the complexation process were calculated for both complexes in order to obtain such information as the main 'driving force' and whether or not complex formation is thermodynamically favoured. This was achieved by monitoring the isothermic solubility lines at various temperatures.

Anti-Dyskinesia Agents↗
PubMed1997

Fluorimetric studies of the formation of riboflavin-beta-cyclodextrin inclusion complex: determination of the stability constant by use of a non-linear least-squares model.

The non-linear least-squares model for calculation of the stability constant (Kst) of a drug-cyclodextrin complex has been used in fluorimetry studies. Complexation of riboflavin with beta-cyclodextrin (beta-CyD) was monitored fluorimetrically by measuring changes in the fluorescence intensity of the vitamin in the presence of various amounts of beta-CyD. Formation of an inclusion complex was confirmed in the solid state by differential scanning calorimetry (DSC) and in aqueous solution by proton nuclear magnetic resonance spectroscopy (1H NMR). The experimental Kst value (2112 M-1) derived from the fluorimetry studies appeared to fit well to a 1:1 drug-to-cyclodextrin molar ratio according to the non-linear mathematical model. The model is particularly suitable for fluorescent compounds of which fluorescence intensity is influenced by the presence of cyclodextrins.

Calorimetry, Differential Scanning↗
PubMed1996

Comparative studies of the fate of free and liposome-entrapped hydroxypropyl-beta-cyclodextrin/drug complexes after intravenous injection into rats: implications in drug delivery.

Inclusion complexes of 14C-labelled hydroxypropyl-beta-cyclodextrin (HP beta-CD) with tritiated dehydroepiandrosterone (DHEA), retinol (RET) and dexamethasone (DEX) were entrapped in the aqueous phase of liposomes composed of distearoyl phosphatidylcholine. Incubation of liposome-entrapped complexes in the presence of rat blood plasma at 37 degrees C resulted in complex dissociation and drug leakage in the media at a rate which was minimal for DEX/HP beta-CD (11%), modest for RET/HP beta-CD (23%) and considerable for DHEA/HP beta-CD (56%; 60 min). In contrast, the HP beta-CD moiety of the complexes was retained by liposomes quantitatively. Free complexes injected intravenously into rats were cleared rapidly from the circulation with most (up to 94%) of the HP beta-CD moiety recovered in 24 urine together with lesser and variable amounts of drug (up to 46% of the dose) in a pattern (DHEA < RET < DEX) that was consistent with the extent of drug association with HP beta-CD in the presence of plasma. A significant proportion of the drugs (up to 25% of the dose)(but very little HP beta-CD) was removed by the liver where drugs were catabolised rapidly, probably following complex dissociation and transport to the tissue via plasma proteins. Injection of entrapped complexes revealed that liposomes alter their pharmacokinetics with only 6-13% of HP beta-CD and a moderate proportion of drugs (up to 26% of the dose) recovered in the urine. Much of the HP beta-CD moiety of the liposomal complexes was recovered in the liver (up to 83%) and spleen (up to 13% of the dose) by 30 min after injection, together with a variable proportion of drugs in a pattern (DHEA < RET < DEX) which reflected that of complex dissociation in vitro. Longer term experiments where animals injected with liposome-entrapped complexes were killed at time intervals (up to 24 days), revealed that HP beta-CD is eliminated from the tissues, albeit at a very slow rate. Moreover, the metabolism of individual drugs in the tissues following vesicle disintegration, appeared to depend on the rate of complex dissociation with DEX, for instance, reaching values of 4-6% (liver) only after 14 days. It is concluded that administration of drug/cyclodextrin inclusion complexes via liposomes could serve as a means to control the action of a wide range of therapeutic agents.

2-Hydroxypropyl-beta-cyclodextrin↗
PubMed1996

The effect of microfluidization of protein-coated liposomes on protein distribution on the surface of generated small vesicles.

Tetanus toxoid and immunoglobulin G (IgG), model proteins for vaccines and targeting ligands respectively, were covalently coupled to preformed dehydration-rehydration vesicles (DRV) to produce vesicles with surface-bound proteins (DRV-protein) or to preformed small unilamellar vesicles (SUV) which were used to generate DRV with bound protein [(SUV-protein)DRV]. Of the amount of protein employed for coupling (1 mg), 13.8-45.1% was recovered with the liposomes, depending on the type of preparations and the proteins used. Microfluidization of similar DRV-protein or (SUV-protein)DRV for up to 10 cycles led to the formation of smaller vesicles (98-136 mm diameter) which, however, had modestly reduced (estimated as 8.8-21.7%) bound proteins, again depending on the type of preparation and protein used. Treatment of DRV-protein and (SUV-protein) DRV with proteinase revealed that 32.9-45.6% of the total bound protein was exposed on the liposomal surface. With microfluidized liposomes, the proportion of surface-exposed protein increased to 63.1-76.2%. Incubation of intact and microfluidized DRV-IgG and (SUV-IgG) DRV with a protein A-Sepharose 4B CL gel confirmed the presence of IgG on the liposomal surface (47.1-68.4 and 80.5-82.1% of total bound protein respectively). These studies were supplemented with freeze-fracture electron microscopy of (SUV-toxoid)DRV which demonstrated the presence of protein particles (up to 3; 12-14 nm diameter) on the surface of both intact and microfluidized individual liposomes.

Desiccation↗
PubMed1996

Synthesis, characterization and properties of sialylated catalase.

Colominic acid (CA), an alpha-(2-->8) N-acetylneuraminic acid (sialic acid) polymer (average molecular weight of 10 kDa) was activated by periodate oxidation of carbon 7 at the non-reducing end of the saccharide. The oxidized CA was then coupled to catalase by reductive amination in the presence of sodium cyanoborohydride. The extent of sialylation of catalase, estimated by ammonium sulfate precipitation as 3.8+/-0.4 (mean+/-S.D.) moles of CA per mole of catalase, did not improve significantly when depolymerized CA was used in the coupling reaction. At the end of the coupling reaction, sialylated catalase exhibited a two-fold (70%) retention of initial activity compared to enzyme controls (29-35%) subjected to the same conditions. Formation of sialylated catalase was confirmed by ammonium sulfate or trichloroacetic acid precipitation, molecular sieve chromatography and SDS-PAGE electrophoresis. Enzyme kinetics studies revealed an increase in the apparent Km of the enzyme from 70.0 (native) to 122.9 mmol l-1 H2O2 (sialylated catalase) indicating a reduction of enzyme affinity for the substrate (hydrogen peroxide) on sialylation. Compared to native enzyme, sialylated catalase was much more stable in the presence of specific proteinases, completely resisting degradation by chymotrypsin and losing only some of its activity in the presence of trypsin. The increased stability conferred to catalase by sialylation agrees with similar observations on enzymes modified by other hydrophilic molecules (e.g., monomethoxypoly(ethyleneglycol)) and suggests that steric stabilization with the biodegradable polysialic acid may prove an alternative means to render therapeutic proteins more effective in vivo.

Animals↗
PubMed1996

Morphological observations on the fate of liposomes in the regional lymph nodes after footpad injection into rats.

Multilamellar liposomes composed of equimolar egg phosphatidylcholine and cholesterol and containing carboxyfluorescein or colloidal gold were injected subcutaneously into the footpad of the hind-leg of rats. The draining popliteal lymph nodes of animals killed at time intervals after injection were then dissected and sections examined by fluorescence microscopy (carboxyfluorescein), light microscopy using an immunogold silver kit to enhance gold particles or by transmission electron microscopy. Morphological observations confirmed that subcutaneously injected liposomes accumulate in large numbers in the draining lymph node. The majority of liposomes arrived at the subcapsular sinuses, probably via afferent lymphatic vessels, as such, i.e., in a non-cell bound form. Subsequently, liposomes were dispersed throughout the lymph node either by permeation as free vesicles along the sinuses or by cells involved in vesicle uptake. The majority of such cells were free macrophages, littoral cells and reticular cells (fixed macrophages). Once within cells, liposomes were seen digested by the lysosomal apparatus with varying loss of their lamellar structure, leaving free gold particles within the lysosomes.

Animals↗
PubMed1996

Photolysis of retinol in liposomes and its protection with tocopherol and oxybenzone.

In this work the stabilities of retinol in methanolic solutions and liposomal suspensions exposed to UV light were compared using absorbance spectroscopy and the ability of alpha-tocopherol and the sunscreen additive, oxybenzone, to reduce the rate of retinol decomposition assessed. Retinol in methanol decolorized almost completely within a few minutes of exposure to a 6 W 350 nm wavelength lamp. From the concentration dependence of the reaction rates it appears that retinol activated by light can decomposed either directly or after collision with a second retinol molecule. Several reaction products are formed. alpha-Tocopherol solutions were unaffected by 350 nm light but they did darken when irradiated with 250 nm wavelength light. Addition of alpha-tocopherol or removal of oxygen from the retinol in methanol solutions reduced only slightly the rates of retinol photolysis. When dispersed in water within liposomes made of equimolar egg phosphatidylcholine (PC) and cholesterol, up to six-fold increases in the decomposition rate of the retinol were observed. The reaction rate could be reduced but only slightly by increasing the ratio of PC to retinol. A mechanism that explains the concentration dependence of the retinol photolysis is that the reduction in reaction rate on diluting the retinol concentration within a given liposome was due to the prevention of the reaction between one light-activated retinol molecule with another within the same liposome. Incorporation of oxybenzone into the liposomes reduced the reaction rates. The results suggested that most of the protection in this case arises through the oxybenzone closet to the light source absorbing the light, thereby preventing it reaching retinol much further into the sample. Incorporation of alpha-tocopherol into the liposomes could also reduce substantially the photolysis rate of co-entrapped retinol. The mechanism of protection in this case appears to be via the tocopherol quenching activated retinol molecules. The close proximity of the tocopherol to the retinol within a single liposome has shown to be important in this case. Only slight protection of retinol in one liposome by tocopherol in another was observed under the conditions studied. This means that the protection by tocopherol will still be observed if the liposome dispersions are diluted considerably or if only thin samples are exposed to light.

Benzophenones↗
PubMed1996

High yield incorporation of plasmid DNA within liposomes: effect on DNA integrity and transfection efficiency.

Effective use of liposomes in gene therapy requires high yield incorporation of nucleic acids within vesicles which protect their content from nuclease attack and facilitate transfection: To that end, pGL2 plasmid DNA (3.99 x 10(6) Daltons) expressing the luciferase reporter gene was incorporated quantitatively (40-92% of the DNA used) by a mild procedure into neutral and negatively or positively charged multilamellar liposomes which offered considerable protection from deoxyribonuclease attack. Smaller vesicles (210-383 nm diameter) produced from such liposomes, retained much of the original content of DNA which was still significantly inaccessible to the enzyme. Liposomal plasmid DNA was found to retain its structural integrity and to transfect cells in vitro in relation to the size and surface charge of the vesicles. Such DNA-incorporating liposome constructs could prove effective for plasmid DNA expression in vivo.

Cell Line↗
PubMed1995

Engineering liposomes for drug delivery: progress and problems.

Liposomes were first proposed and tested as a drug delivery system 25 years ago. Since then, advances in our understanding of the fate and behaviour of liposomes at the cellular and subcellular level in vivo have allowed the rational design of constructs for use in the treatment and prevention of disease, both in experimental animals and clinically. The involvement of several liposome-based biotechnology companies in the early 1980s, paralleled by great leaps in liposome technology, has culminated in the design and licencing of formulations for the treatment of certain microbial infections and cancers, and the first liposome-based vaccine for use in humans.

Drug Delivery Systems↗
PubMed1995

A non-ionic surfactant vesicle-in-water-in-oil (v/w/o) system: potential uses in drug and vaccine delivery.

An aqueous dispersion of niosomes (non-ionic surfactant vesicles) emulsified in an external oil phase forms the vesicle-in-water-in-oil (v/w/o) system described in this paper. The properties of the surfactant used to form the vesicles, the surfactant or surfactant mixture used to stabilize the emulsion and the nature of the oil phase can be changed to provide systems of different capacities for drug or antigen and different release characteristics. The same nonionic surfactant is used as the principle amphipile to form the niosomes and to stabilize the w/o emulsion, thus promoting stability by decreasing transfer of surfactant between the stabilizing monolayers and the vesicle bilayers. The in vitro release of carboxyfluoroscein and 5-fluorouracil encapsulated within the niosomes of the v/w/o system has been investigated, the nature of the oil phase and surfactant-oil interactions being important in determining the rate of solute release. Initial studies of the system in vivo, as an adjuvant for tetanus toxoid, using cottonseed oil as the external oil phase, showed enhanced immunological activity over the free antigen or vesicles.

Adjuvants, Immunologic↗
PubMed1994

Entrapment of haemoglobin into liposomes by the dehydration-rehydration method: vesicle characterization and in vivo behaviour.

Haemoglobin (Hb) was isolated from human erythrocytes under conditions which maintained NADH-cytochrome-b5 reductase activity and suppressed oxidation of Hb during storage at 4 degrees C (methaemoglobin values < 3% after 29 days). Hb was entrapped into liposomes composed of hydrogenated egg phosphatidylcholine and equimolar cholesterol according to the dehydration/rehydration procedure of Kirby and Gregoriadis ((1984) Biotechnology 2, 979). However, encapsulation of Hb in its intact form was poor (< 5%) as a result of its oxidation and denaturation during freeze-drying. The addition of cryoprotectants and the use of both, higher initial concentrations of Hb and very small void vesicles resulted in Hb-rich dehydration/rehydration vesicles (phospholipid/Hb molar ratio of about 200:1) of the preferred size of 110 nm (mean). Highly homogeneous and small void vesicles as starting material were prepared using the one-step method of Brandl et al. ((1990) Drug Dev. Ind. Pharm. 16, 2167). The cryoprotectants were chosen with respect to their sufficient protection of Hb without affecting its loading into vesicles during freeze-drying and rehydration. 51Cr-labelling of Hb was used for the in vivo monitoring of the fate of Hb-containing vesicles rather than 125I-labelling since the latter induced strong interactions of Hb with liposomes. Upon intravenous administration into rats, liposomal 51Cr-Hb showed greater blood levels and prolonged circulation times in the blood compared to free Hb. The present approach provides high yield entrapment of labile molecules into vesicles of small size known to exhibit long circulation time.

Animals↗
PubMed1994

Liposomes as immunoadjuvants and vaccine carriers: antigen entrapment.

Successful use of liposomes as immunological adjuvants in vaccines requires simple, easy to scale up technology capable of high-yield antigen entrapment. Recent work from this laboratory has led to the development of techniques that can generate liposomes of various sizes containing soluble antigens such as proteins or particulate antigens such as whole, live, or attenuated bacteria or viruses. Entrapment of proteins is carried out by the dehydration-rehydration procedure, which entails freeze-drying of a mixture of "empty" small unilamellar vesicles and free antigens. Upon rehydration, the large multilamellar vesicles that are formed incorporate up to 80% of the antigen used. When such liposomes are microfluidized in the presence of nonentrapped material, their size is reduced to about 100 nm in diameter, with much of the originally entrapped antigen still associated with the vesicles. A similar technique applied to the entrapment of particulate antigens (e.g., Bacillus subtilis spores) consists of freeze-drying giant vesicles (4-5 microns in diameter) in the presence of spores. On rehydration and sucrose gradient fractionation of the suspension, up to 27% of the spores used are associated with generated giant liposomes of similar mean size.

Adjuvants, Immunologic↗
PubMed1994

Late postoperative results in males treated for testicular torsion during childhood.

Successful preservation of a testis that has undergone torsion is directly related to the duration and degree of torsion. From 1979 to 1991, 75 cases of testicular torsion were treated. Eleven orchiectomies were performed; operative detorsion was done in the other 64 cases, with concurrent orchiopexy of both testicles. Twenty-five patients were reexamined 1 to 12 years after the surgery. Age of torsion, duration of symptoms, and operative findings were reevaluated. Testicular volume was determined using Prader's orchidometer, a pachymeter, and ultrasonography. In four postpubertal patients, semen analysis was performed. According to clinical data, results of testicular atrophy correlated with duration of symptoms and operative findings. In all cases of surgical detorsion in which torsion lasted more than 24 hours and viability of the testis was questionable, subsequent atrophy was the rule.

Adolescent↗
PubMed1994

IgG subclass response and protection against challenge following immunisation of mice with various influenza A vaccines.

The serum total IgG and IgG subclass and nasal wash IgA and IgG antibody responses of mice to influenza virus A/Hong Kong/68 (H3N2) subunit preparations administered parenterally as a single dose, incorporated either in immune stimulatory compounds (ISCOMs) or liposomes with Freund's Complete Adjuvant, or as an aqueous material, as well as to live, infectious virus were measured by ELISA at 10 days and 3, 5, 7 and 22 weeks after immunisation. The protection of the upper and lower respiratory tracts provided by these preparations against homologous and heterologous challenge infection was assessed. Of the four variously-presented subunit preparations, influenza subunit ISCOMs induced relatively high and persisting levels of each of the different IgG subclasses, particularly IgG2a, throughout the study, and most nearly approached those observed after intranasal infection of mice with infectious virus. Furthermore, nasal wash IgA and IgG antibody levels, particularly at 5 or 7 weeks after immunisation, were also significantly greater in mice given the subunit ISCOM preparation than those induced by other subunit preparations with adjuvant or subunits given alone, and provided protection of both the upper and lower respiratory tracts against challenge as similar to that elicited by infectious virus.

Animals↗