[Study on third-type immunoliposomes loaded drugs and the targeting in vitro and in vivo].

AIM: To study the preparation, targeting and pharmacodynamics of third-type immunoliposome loaded anticancer drugs. METHODS: The monoclonal antibody of human bladder cancer was combined with the terminal of PEG-COOH (polyethyleneglycol carboxylic acid) that make the liposomes not only prolong circulation by the membrane protection of PEG, but also target by spreading the antibody on the liposomes surface. That was the third type immunoliposomes. According to this scheme, the IML-ADM (immunoliposome carried adriamycin) wes prepared in which ADM entrapment was efficient and stability was high and the antibody activity was kept. RESULTS: The % survival of the targeting EJ cells treated with IML-ADM (ADM = 45.45 micrograms.mL-1) was 4.3% +/- 1.0%, but 72% +/- 6% for non-targeting LOVO cells in vitro; the tumor weight in nude mice which were implanted by EJ cells after 27 days were (39 +/- 25) mg, (135 +/- 32) mg, (598 +/- 240) mg treated by IML-ADM, SSL-ADM (steric stable lipsomes carried Adriamycin) and normal saline, respectively, in vivo. CONCLUSION: The results confirmed that the immunoliposme-mediated targeting anticancer drug is a feasible way.

[Effects of different phospholipids on the stabilities of doxorubicin liposomes in vitro and in vivo].

AIM: To study the effects of egg phosphatidylcholine (EPC) and hydrogenated egg phosphatidylcholine (HEPC) on the leakage of doxorubicin liposome in vitro and the resident time in blood. METHODS: By the means of dialysis, the leakage of EPC (EPC-NL) and HEPC normal liposomes (HEPC-NL) in fetal cattle serum (FCS) was determined at 37 degrees C and in phosphate buffer solution (PBS) at 37 degrees C, 20 degrees C and 4 degrees C. Furthermore, the pharmacokinetics of EPC and HEPC sterically stabilized liposomes (EPC-SSL and HEPC-SSL) were studied by HPLC. RESULTS: The leakage of doxorubicin from HEPC-NL is slower than that of EPC-NL in FCS at 37 degrees C. But in PBS at all of three different temperatures, the results are completely reversed, i.e., the leakage of EPC-NL is slower than that of HEPC-NL. Further studies on pharmacokinetics showed that the mean residence time of HEPC-SSL in blood is 23.3 h, while that of EPC-SSL is 12.0 h, and the area under the curve (AUC) of concentration of HEPC-SSL is larger than that of EPC-SSL. CONCLUSION: HEPC-SSL is a better carrier in delivering the drugs to the extravascular sites than EPC-SSL.

[Study on preparation and biodistribution of PEG-immunoliposomes with active carboxylic terminals].

AIM: In order to accumulate into its target specifically, the immunoliposomes must possess two characteristics: specific target efficiency to its target cells and prolonged circulation in blood. A new type of polyethylene glycol (PEG)-immunoliposomes carrying monoclonal antibodies at the distal end of PEG chains should be developed. METHODS: A dipalmitoylphosphatidylethanolamine (DPPE) derivative of PEG with carboxyl group (DPPE-PEG3000-COOH) was newly synthesized. Small unilamellar liposomes were prepared from egg phosphatidyl choline and cholesterol (5:4, mol/mol) containing 6 mol% DPPE-PEG3000-COOH using reverse-phase evaporation method followed with bath sonication. Monoclonal antibody of human bladder cancer cell (BDI-1), which is highly specific to human bladder cancer cell, was conjugated to PEG-liposomes as well as mouse IgG at the distal end of polyethylene glycol chain. Doxorubicin was entrapped into these immunoliposomes by remote (NH4)2SO4 gradient loading method. The specific targeting efficiency of these immnoliposomes was tested by cytotoxicity test in vitro, enzyme-linked immune sorbent assay (ELISA) and indirect fluorescent immunoassay. Its biodistribution was carried out in mice. RESULTS: The specific targeting efficiency of BDI-1 immunoliposomes (BDI-1-IML) to EJ cells has been demonstrated, in contrast to the nonspecific human colon carcinoma cells (LOVO). PEG-liposomes linked with mouse IgG (mouse-IgG-immunoliposomes, IgG-IML) displayed lower reticulo-endothelial systems (RES) uptake and longer circulation time than liposomes without PEG after intravenous injection. CONCLUSION: The long circulation of these PEG-immunoliposomes in vivo, combined with its specific targeting efficiency demonstrated in vitro, guarantees the positive targeting efficiency of these immunoliposomes to its target carcinoma in vivo.

[Interactions of insulin with dipalmitoylphosphatidylcholine liposomes].

AIM: To study the interactions of insulin with dipalmitoylphosphatidylcholine liposomes. METHODS: The liposomes were prepared by reverse-phase evaporation vesicle method. The entrapped efficiency, size and distribution of the liposomes were determined, and the influences of insulin on entrapped efficiency, size and distribution of the liposomes were investigated. The influences of liposomes on the fluorescence emission spectra of insulin and the calcein leakage from the liposomes entrapped calcein induced by insulin were measured. RESULTS: Insulin has little influence on the size and distribution of the liposomes while the sizes of the liposomes were about 170-190 nm. The insertion of tyrosine of insulin into dipalmitoylphosphatidylcholine liposomes membrane was not deep. The insulin disturbed the liposomes membrane, induced the calcein leakage from the calcein-loaded liposomes. CONCLUSION: Amphiphilic, example insulin, may disturb the intact membrane of liposome through the interaction either hydrophobic or hydrophilic. The attention should be paid to the entrapment process of peptides into liposomes.

Determination of eight synthetic food colorants in drinks by high-performance ion chromatography.

Eight synthetic food colorants (Amaranth, Brilliant Blue, Indigo Carmine, New Red, Ponceau 4R, Sunset Yellow, Tartrazine, Allura Red) were determined by high-performance ion chromatography on an anion-exchange analytical column with very low hydrophobicity and visible absorbance detection. Gradient elution with hydrochloric acid-acetonitrile effected both the chromatographic separation of these colorants and the on-line clean-up of the analytical column, which was very advantageous for routine analysis. High-performance ion chromatography may be a solution to the chromatographic analysis for some water-soluble, organic analytes with strong hydrophobicity. The method has been applied to the determination of colorants in drinks and in instant drink powder. No time-consuming pretreatment, as used in conventional liquid chromatography, was needed.

[Effect of membrane modification by PEG on prolongation of circulation time of liposomes in blood in vivo].

PEG-PE (polyethylene glycol-phosphatidyl ethanolamine) of different molecular weight (2000 and 5000) were used to modify the membrane of liposomes. Large unilamellar liposomes containing PEG-PE were prepared by reversed phase evaporation. Fluorescent label-calcein was encapsulated at the internal water phase. To compare the differance between the modified and unmodified membrane, the stability in vitro and distribution in vivo were investigated. The results indicated that the circulation half-life for liposomes unmodified, modified by PEG (2000)-PE and modified by PEG (5000)-PE were 13, 21 and 75 (min) respectively. At 6 h after injection, the ratio b/R (b: distribution in blood, R: distribution in liver and spleen) were 0, 0.8 and 1.4, respectively. The results mean that the stability increased and circulation time was prolonged by the PEG-PE modified membrane. The effect of PEG-PE on membrane was found to be directly proportional to the chain length of the polymer.

Neuro-glial neurotrophic interaction in the S-100 beta retarded mutant mouse (Polydactyly Nagoya). II. Co-cultures study.

The homozygote of a mouse strain with genetic polydactyly (Polydactyly Nagoya, Pdn) shows several brain abnormalities, and significant decrease of S-100 beta in the brain [17]. An accompanying paper [18] demonstrates that the hippocampus and caudo-dorsal cortex of homozygote (Pdn/Pdn) mouse were markedly reduced in S-100 beta positive astrocytes and serotonergic fibers, and the content of 5-HT and 5-HIAA of hippocampus and cortex of Pdn/Pdn mouse was lower than those of heterozygote (Pdn/+) or wild type (+/+) mice. To further clarify the effects of target tissues from different type brains on the development of serotonergic neurons, raphe neurons from Pdn/Pdn or +/+ newborn mice were co-cultured with hippocampus or cortex of +/+ or Pdn/Pdn newborn mice. The growth of the serotonergic neurons in the mesencephalic raphe tissue dissociated cultures was estimated by measuring the specific uptake of [3H]5-HT. The development of both genotypes (Pdn/Pdn and +/+) of serotonergic neurons was enhanced by co-cultures with target tissues (hippocampus and cortex) of +/+ brain. This effect was not observed in the co-cultures with Pdn/Pdn brain as a source of target tissue. The present results support the idea that the developmental defect of serotonergic fibers in the Pdn mutant mouse is caused by the deficiency of S-100 beta in the astrocyte of this mutant, and suggest that S-100 beta is a serotonergic growth factor. This mutant mouse is a useful in vivo model to study neural-glial neurotrophic interactions.

Preparation and in vitro antiviral activity of liposomes of lipophilic esters of acyclovir.

The long chain acyclovir such as the acyclovir laurate and acyclovir palmitate were prepared directly from acyclovir by application of the usual esterification methods with appropriate acyl chlorides. The lipophilic prodrugs were found to be retained easier by liposomes whereas acyclovir escaped readily from liposomes. When assayed in African green monkey cell cultures against herpes simplex virus type I strain, the acyclovir palmitate liposomes proved to be more active compared with the parent drug and its liposome, suggesting an enhanced compatibility between the ester and liposomal lipids and an increased uptake of encapsulated prodrug by infected cells.

[Hydrophobic modification of water soluble drug and its reconstitutable liposomes].

Water soluble drugs carried in liposomes have rather low encapsulation percentage (EP%) and poor stability. In this paper metronidazole (I) was chosen as a model of water soluble drugs which was modified by means of esterification. Its myristic ester (II) was synthesized. On studying the liposomes of I and II, the results have shown that EP% and stability of II were more than ten times as high as that of I. Furthermore, the reconstitutable liposomes of II were prepared successfully. The amoebacide activity of II liposomes was increased also. Therefore, the hydrophobic modification of water soluble drugs is a good way to improve the drug entrapped in liposomes.

[Surface charged liposomes as drug carriers and in vivo tests of mice infected with toxoplasma].

The surface charge is one of the most important properties of liposomes. In this paper surface potential of liposomes was measured by fluorimetry using 1.8-ANS as a probe and by microelectrophoresis. The encapsulation efficiency and leakage rate of ionic drugs were influenced by the surface charge of liposomes. Higher encapsulation efficiency and lower leakage rate were resulted if the surface charge of liposomes was opposite to the ionic charge of encapsulated drug. Mice infected with toxoplasma were used as a model. In in vivo test, liposome-drug was compared with the free drug. The average survival days of groups of liposome-drugs were much longer than that of the group of free drugs. Mouse infected with toxoplasma is a simple, convenient and accurate model for measuring the biological activation of liposome-drugs.

Effects of rat platelet extracts and human platelet-derived growth factor on megakaryocytopoiesis in vitro.

An improved plasma clot culture system was developed for the culture of rat megakaryocyte colonies in vitro. Greater stimulation of megakaryocyte colony growth was provided when both serum and plasma were added to the culture medium than when plasma was added alone. Freeze-thawed extracts prepared from 900 x 10(6) platelets/ml saline and also 9 ng/ml of human platelet-derived growth factor (PDGF) caused maximum increases over the controls of 71% and 42%, respectively, in the numbers of megakaryocyte colonies. The increase in the numbers of megakaryocyte colonies induced by platelet extracts and by PDGF implied that substance(s) released by platelets might play an important role in the control of megakaryocytopoiesis.

The effects of prostaglandin E1 on megakaryocyte proliferation in vitro.

A concentration of 560 nM Prostaglandin E1 (PGE1), in a medium containing either 10% serum and 10% plasma, or 20% plasma and a platelet extract prepared from 24 x 10(6) platelets/ml, caused after four days of incubation in a plasma clot culture system, highly significant increases over the controls in both the number of megakaryocyte colonies/clot and megakaryocytes/clot. A linear dose-response relationship over the entire 14-560 nM range was also demonstrated in cultures containing serum, between the concentration of PGE1 in each culture, and the number of megakaryocyte colonies/clot and megakaryocytes/clot. However, PGE1 did not appear to stimulate megakaryocytes in cultures containing only 20% rat plasma.

Decomposition of aspirin in the solid state in the presence of limited amounts of moisture.

When limited amounts of moisture are added to aspirin in a closed system, the model for decomposition differs from that experienced in an open system, i.e., a system at constant relative humidity. The composition of the liquid decomposition layer on the surface of the aspirin crystals will constantly change, and a model for accounting for both the change in volume and composition of the decomposition layer as a function of time has been developed. Solid-state decomposition data, when plotted as a function of time, exhibit a profile similar to the one predicted, but the position of the decomposition curve observed differs from that of the predicted. The predicted curve indicates better stability than the observed data.

Compressibility of anhydrous tricalcium phosphate.

It is shown that tricalcium phosphate follows a simple Heckel relationship in pressures of usual tablet compression, only if a density of 1.92 g/mL is used. This coincides with the density obtained by wet pycnometry, whereas the crystallographical density is 3.1 g/mL. The interpretation is that some of the pore space is occluded and does not constitute a part of the Heckel pore space.