Kai Schulze-Forster

Cytotoxicity of Solid Lipid Nanoparticles as a Function of the Lipid Matrix and the Surfactant

AbstractPurpose. Assessment of the in vitro cytotoxicity of solid lipid nanoparticles (SLNs) as a function of lipid matrix (Dynasan 114, Compritol ATO 888), and stabilizing surfactant (poloxamers, Tween 80, soya lecithin, and sodium dodecyl sulphate). Comparison with other colloidal carriers should determine their potential use in the clinic. Methods. SLNs were produced by high pressure homogenisation. Cytotoxicity was assessed by measuring the viability of HL60 cells and human granulocytes after incubation with SLNs. Particle internalisation was quantified by chemiluminescence measurements. Results. The nature of the lipid had no effect on viability; distinct differences were found for the surfactants. Binding to the SLN surface reduced markedly the cytotoxic effect of the surfactants, e.g., up to a factor of 65 for poloxamer 184. The permanent HL60 cell line— differentiated from cells with granulocyte characteristics by retinoic acid treatment—yielded results identical to freshly isolated human granulocytes. In general, the SLNs showed a lower cytotoxicity compared to polyalkylcyanoacrylate and polylactic/glycolic acid (PLA/ GA) nanoparticles. Conclusions. Because the results are identical when using human granulocytes, differentiated HL60 cells can be used as an easily accessible in vitro test system for i.v. injectable SLN formulations. The SLNs appear suitable as a drug carrier system for potential intravenous use due to their very low cytotoxicityin vitro.

Isolation and partial characterization of basic proteinases from stem bromelain.

Crude bromelain extracts from pineapple stems (Ananas comosus) were fractionated by two-step FPLC-cation-exchange chromatography. At least eight basic proteolytically active components were detected. The two main components F4 and F5 together with the most active proteinase fraction F9 were characterized by SDS-PAGE, mass spectroscopy, multizonal cathodal electrophoresis, partial amino acid sequence, and monosaccharide composition analysis. F9 amounts to about 2% of the total protein and has a 15 times higher specific activity against the substratel-pyroglutamyl-l-phenylanalyl-l-leucine-p-nitroanilide (PFLNA) than the main component F4. The molecular masses of F4, F5, and F9 were determined to 24,397, 24,472, and 23,427, respectively, by mass spectroscopy. Partial N-terminal amino acid sequence analysis (20 amino acids) revealed that F9 differs from the determined sequence of F4 and F5 by an exchange at position 10 (tyrosine→serine) and position 20 (asparagine→ glycine). F4 and F5 contained fucose, N-acetylglucosamine, xylose, and mannose in ratio of 1.0∶2.0∶1.0∶2.0, but only 50% of the proteins seem to be glycosylated, whereas F9 was found to be unglycosylated. Polyclonal antibodies (IgG) against F9 detected F4 and F5 with tenfold reduced reactivity. ThepH optimum of F4 and F5 was betweenpH4.0 and 4.5 and for F9 close to neutralpH. The kinetic parameters for PFLNA hydrolysis were similar for F4 (Km 2.30 mM,kcat 0.87 sec−1 and F5 (Km 2.42 mM,kcat 0.68 sec−1), and differed greatly from F9 (Km 0.40 mM,kcat 3.94 sec−1).

Prothymosin αl effects in vitro on chemotaxis, cytotoxicity and oxidative response of neutrophils from melanoma, colorectal and breast tumor patients

Immunoregulatory effects of thymic peptides on functions of polymorphonuclear leukocytes (PMNs) are poorly investigated. We studied the effects of prothymosin alpha 1 (Pro alpha 1) on PMNs from patients with colorectal tumors, breast tumors and melanoma (total n = 37) in comparison with healthy donors (n = 18), with respect to chemotaxis, cytotoxicity against HCT-116 colon tumor cells, oxidative response (chemiluminescence reaction) as well as expression of surface marker molecules. We found that Pro alpha 1 was equally effective in stimulating the chemotactic activity of PMNs from tumor patients and healthy donors (43% increase). PMNs from tumor patients, especially with breast tumor, showed a significant enhancement of cytotoxicity against the tumor target cells in comparison with healthy donors. With respect to the PMNs cytotoxicity, only about 50% of the colorectal tumor patients and healthy donors responded to Pro alpha 1 and FMLP. As to the oxidative response of PMNs, elevated levels were found only among colorectal tumor patients. Pro alpha 1 significantly increased the oxidative response in breast and colorectal tumor patients by 55% and 25%, respectively. Pro alpha 1 decreased the expression of CD16 on PMNs of healthy donors, but not that of CD11a, CD11b, CD11c, CD13, CD14, CD15 and CD32. Therefore, we suggest, that Pro alpha 1 may improve some PMN functions of tumor patients, associated with the proposed role in host-tumor interaction.

Interaction of differentiated HL60 cells with poloxamer and poloxamine surface modified model drug carriers

Abstract The human promyelotic cell line HL60 can be differentiated with retinoic acid (RA) along the granulocyte pathway. RA-treated cells were used as an alternative in vitro test system for colloidal drug carriers compared to human granulocytes. The test system should allow identification of surface-modified particles possessing the potential to avoid uptake by the mononuclear phagocytic system (MPS) after intravenous injection. Differently sized model drug carriers (polystyrene particles) with known behaviour in the human granulocytes test system were investigated and the uptake data compared. The particles were surface-modified by the adsorption of poloxamer 188, 338 and 407 and poloxamine 908, the uptake was studied in the presence and absence of human and bovine sera and the albumins, respectively. The extent and kinetics of uptake were found to be comparable therefore the HL60 cells can be used as an in vitro test system with the advantages of a permanent cell line in contrast to the variability of isolated human primary cells.

Effect of selenite combined with chemotherapeutic agents on the proliferation of human carcinoma cell lines

Selenite is frequently used in combination with cancer chemotherapeutic agents to reduce side effects. However, the cytoprotective activity of selenite may also reduce the efficacy of chemotherapeutic drugs on tumor cells. This study was designed to examine the effects of selenite combined with cytotoxic agents used in clinical protocols [e.g., doxorubicine, docetaxel, 5-fluorouracil (5-FU), methotrexate (MTX), mafosphamide, mitomycin C, gemcitabine, etoposide, cisplatin, irinotecan, and oxaliplatin] on the proliferation of various carcinoma cell types. The data demonstrated that selenite had no marked effects on the antiproliferative activity of docetaxel, doxorubicine, 5-FU, MTX, and mafosphamide in MDA-MB-231 breast cancer cells. Likewise, no consistent changes were observed in A549 lung cancer cell proliferation when selenite was combined with cisplatin, etoposide, gemcitabine, or mitomycin C. On the other hand, selenite potentiated the cytotoxicity of 5-FU, oxaliplatin, and irinotecan in HCT116 colon cancer cells by approx 1.1-fold, 2.7-fold, and 2.6-fold, respectively. In SW620 colon cancer cells, selenite induced a 1.5-fold and 4.3-fold increase of the antiproliferative activity of 5-FU and oxaliplatin, respectively. Whereas irinotecan showed no effects on SW620 cell growth, a combination with selenite resulted in 23% inhibition. Our results indicate that selenite did not reduce the antiproliferative activity of chemotherapeutic agents in vitro. In addition, selenite was able to increase the inhibitory activity of docetaxel in A549 lung cancer cells, and of 5-FU, oxaliplatin, and irinotecan in HCT116 and SW620 colon cancer cells implying selenite is potentially useful as an adjuvant chemotherapeutic agent.

The thymus extract Thymex-L potentiates the retinoic acid-induced differentiation of the human myeloid leukemia cell line HL-60.

The human promyelocytic cell line HL-60 can be differentiated with retinoic acid (RA) along the granulocytic pathway. Numerous studies have identified many synergistic combinations of RA with cytostatics, cytokines and other inducers. A combination of RA with the crude thymus extract Thymex-L increased differentiation of HL-60 cells as confirmed by two functional assays and morphology, whereas the extract itself did not show any effect. The functional markers phagocytosis-associated chemiluminescence and nitroblue tetrazolium reduction were more enhanced (up to 4-fold with 1000 micrograms/ml Thymex-L) than morphology. The effect was found over a wide RA concentration range (10(-11) - 10(-6) M) and was dependent on extract concentration. The half-maximal induction of both functional markers was reached at 400 micrograms/ml. To achieve the same effect with the combination in comparison with RA alone, an RA dose reduction of about 100-fold was estimated. The effect was also seen when the cells were pretreated with the thymus extract for two days. The enhancement of RA action by Thymex-L was not correlated with an increase of extracellular or intracellular RA concentration. The active compound in Thymex-L is heat stable and bigger than 5 kDa as confirmed by gelfiltration. The defined thymus peptides thymosin alpha 1, prothymosin alpha 1 and thymopentin were unable to synergistically enhance HL-60 differentiation. These data suggest that the treatment with a thymus extract can increase the sensitivity of HL-60 cells for RA. This may have clinical implications.

Antibodies to Signaling Molecules and Receptors in Alzheimer’s Disease are Associated with Psychomotor Slowing, Depression, and Poor Visuospatial Function

BACKGROUND Alzheimers disease (AD) is associated with several antibodies as well as signaling molecules and receptors. These may be detrimental in the presence of a disrupted blood-brain barrier (BBB). OBJECTIVE To investigate whether the levels of antibodies toward 33 signaling molecules involved in neurotransmitter, vascular, and immune functions were associated with AD and, within the AD group; cognitive function and mood. METHODS Antibodies in sera from patients with mild AD [(n = 91) defined as a Mini-Mental State Examination ≥ 20 or a Clinical Dementia Rating Scale≤1] and healthy controls (n = 102) were measured with enzyme-linked immunosorbent assays. Levels in AD and controls were compared by Mann-Whitney test. In the AD group, associations between antibodies and psychometric test scores were analyzed by robust regression. The false discovery threshold was set to 0.05. RESULTS Antibodies to serotonin receptors [5-HT2AR (effect size (r) = 0.21, p = 0.004), 5-HT2CR (r = 0.25, p = 0.0005) and 5-HT7R (r = 0.21, p = 0.003)], vascular endothelial growth factor receptor 1 [VEGFR1 (r = 0.29, p < 0.001)] and immune-receptors (Stabilin-1 (r = 0.23, p = 0.001) and C5aR1 (r = 0.21, p = 0.004) were higher in AD. Psychomotor speed was associated with D1R-abs (β 0.49, p < 0.001), depression with ETAR-abs (β 0.31, p < 0.001), and visuospatial function with 5-HT1AR-abs (β 0.27, p = 0.004) despite similar antibody levels compared to controls. CONCLUSIONS Antibody levels to VEGFR1, serotonergic receptors, and receptors in the immune system were increased in AD. Antibodies at similar levels as in controls were associated cognitive dysfunction and depression in AD.