Different strategies for formation of pegylated EGF-conjugated PEI/DNA complexes for targeted gene delivery.

With the aim of generating gene delivery systems for tumor targeting, we have synthesized a conjugate consisting of polyethylenimine (PEI) covalently modified with epidermal growth factor (EGF) peptides. Transfection efficiency of the conjugate was evaluated and compared to native PEI in three tumor cell lines: KB epidermoid carcinoma cells, CMT-93 rectum carcinoma cells, and Renca-EGFR renal carcinoma cells. Depending on the tumor cell line, incorporation of EGF resulted in an up to 300-fold increased transfection efficiency. This ligand-mediated enhancement and competition with free EGF strongly suggested uptake of the complexes through the EGF receptor-mediated endocytosis pathway. Shielded particles being crucial for systemic gene delivery, we studied the effect of covalent surface modification of EGF-PEI/DNA complexes with a poly(ethylene glycol) (PEG) derivative. An alternative way for the formation of PEGylated EGF-containing complexes was also evaluated where EGF was projected away from PEI/DNA core complexes through a PEG linker. Both strategies led to shielded particles still able to efficiently transfect tumor cells in a receptor-dependent fashion. These PEGylated EGF-containing complexes were 10- to 100-fold more efficient than PEGylated complexes without EGF.

[Change in acylneuraminic acid content of T-lymphocytes and in plasma in breast cancer]. / Veränderung der Acylneuraminsäuregehalte auf T-Lymphozyten und im Plasma bei Erkrankung an Mamma-Karzinom.

Increased sialic acid levels reflecting tumor burden are found on the surface of T-lymphocytes and in the plasma of patients with carcinoma of the mammary gland. The data of the determinations of sialic acid content and distribution on T-cells, using microanalytical methods such as HPLC and a colorimetric test, show that the total sialic acid content is increased by about 60% and that nearly 80-90% of the sialic acids consist of N-acetyl-9-O-acetyl-neuraminic acid, in comparison to the healthy controls (not containing O-acetylated neuraminic acid). Investigations on lymphocytes of malignant melanoma patients show similar changes of sialic acid content and distribution on the cell surface. Increased sialic acid levels are also found in the plasma of patients with cancer but no O-acetylated derivative can be found. Furthermore the examinations show that the separation of the T-lymphocytes from the total lymphocyte fraction is not required. Determination of sialic acids in the total lymphocyte fraction can be a simplification in carrying out further diagnostic investigations. A high level of sialic acids as "antirecognition factor" seems to be not only a marker of tumor cells but also an attribute of T-lymphocytes, involved in the defence against the malignoma (malignant melanoma, breast cancer). Considering the possible contribution of sialic acid to the immunoregulatory protective mechanism during the first stage of pregnancy, sialic acid content and distribution on T-cells of pregnant women are investigated. Both an increase and a change in the distribution of sialic acids can be excluded.

Sialic acids in human lymphocytes. Qualitative and quantitative alterations in cancer cases.

Sialic acids, hydrolyzed from human lymphocytes, were determined, in the nanomole range, with a modified form of the periodic acid-thiobarbituric acid assay and liquid chromatography. The l.c. separations were carried out with two different systems, firstly an Aminex HPX-72 S anion-exchange resin and a 0.15M ammonium sulfate mobile phase, and secondly an amine phase (5 microns) and an acetonitrile-phosphate buffer as mobile phase. The lymphocytes of cancer-stricken persons showed an evident rise of the sialic acid content, combined with a shift of the sialic acid distribution to higher O-acetylated derivatives, as compared to the controls.

Alterations of acyl-neuraminic acids on T-lymphocytes in cases of melanoma.

Content and distribution of the different sialic acids on human lymphocytes, isolated from 7-10 ml of fresh human blood, were determined using microanalytical methods, such as HPLC and a colorimetric test. Comparison of the data of patients with melanoma with those of healthy persons shows an evident increase of the sialic acid content combined with a shift of the sialic acid distribution to higher O-acetylated derivatives.