pH Gradient Method Research Articles

Enhanced Tumor Targeting of Doxorubicin by Ganglioside GMl-bearing Long-circulating Liposomes

Doxorubicin (DXR) was encapsulated in long-circulating liposomes, composed of ganglioside GM1 (GM1)/distearoylphosphatidylcholine (DSPC)/cholesterol (CH) (0.13:1:1 in molar ratio) and sized to approximately 100 nm in mean diameter, with 98% entrapping efficiency by the transmembrane pH gradient method. Free DXR, DXR-DSPC/CH and DXR-GM1/DSPC/CH liposomes were injected intravenously into Colon 26 tumor-bearing Balb/c mice via the tail vein at a dose of 5.0 mg DXR/kg. DXR-GM1/DSPC/CH liposomes gave a higher blood level of the drug than did DXR-DSPC/CH liposomes or free DXR up to 24 hours after injection, and the area under the blood concentration-time curve (AUC) for DXR-GM1/DSPC/CH liposomes was 1.5 or 526 times higher than that for DXR-DSPC/CH liposomes or free DXR, respectively. DXR-GM1/DSPC/CH liposomes gave a decreased DXR concentration in the reticuloendothelial system (RES) of the liver and the spleen. Both liposomal formulations effectively reduced the DXR concentration in the heart as compared with that in the case of free DXR. At 6 hours after i.v. injection, DXR-GM1/DSPC/CH liposomes provided an approximately 3.3- or 9-fold higher peak DXR level in the tumor as compared with DXR-DSPC/CH liposomes or the free drug, respectively. These high tumor levels of DXR appear to reflect the prolonged residence time of the liposomes. The results suggest that encapsulation of DXR in GM1-bearing long-circulating liposomes will be useful for cancer chemotherapy.

ｐＨ勾配法によるドキソルビシン封入温度感受性リポソームの調製と評価

Temperature-sensitive liposomes containing doxorubicin (DXR-TSL (pH)) composed of DPPC (Tc = 41°C)/DSPC (Tc = 54°C) (9 : 1 in molar ratio) were prepared by pH gradient method and evaluated concerning tumor uptake of DXR. Colon 26 tumor cells were inoculated in hind foot of BALB/c mice and local hyperthermia (42°C, 20 min) was done at tumor site. The pH gradient method was employed to raise the encapsulation rate of DXR into temperaturesensitive liposomes. This preparation resulted in the remarkably high encapsulation rate with 98%. The tumor uptake of DXR with the combination of DXR-TSL (pH) and the local hyperthermia were 3 times greater than those of free DXR. The higher accumulation of DXR in liver and spleen was observed. In these accumulation, the distribution of liposome rather than released DXR was contributed mainly. Present data indicated that DXR-TSL (pH) have some advantages for the antitumor effect, since DXR content per liposome is high and the controlled release of DXR from liposomes with hyperthermia is possible.

アドリアマイシン封入ガングリオシドＧＭ１付与リポソームの体内動態と抗腫よう効果

Large unilamellar liposomes (LUV), which have a large entrapped volume and efficient drug capture characteristics, are rapidly and efficiently taken up by the RES cells in liver and spleen. A prolonged residence of the drug-entrapped liposomes in the circulation is important for the sustained release of drug form liposomes. Incorporation of 6 mol% of GM1 into LUV composed of DSPC/CH (GM1/DSPC/CH 0.13 : 1 : 1 m/m, 200 nm in size) showed a significant increase of circulation time in the blood circulation. The GM1/DSPC/CH liposomes have been tested for their utility as a sustained release system for adriamycin (ADM) in vivo. ADM was encapsulated into GM1/DSPC/CH liposome with>95% in trapping efficiency by employing the pH gradient method. Mice were given i.p. injection of 2 million L1210 leukemia cells. Twenty-four hrs later, mice were treated with single shot of ADM-GM1/DSPC/CH liposome, ADM-DSPC/CH liposome or free ADM (5 mg/kg). Treatment with ADM-GM1/DSPC/CH liposome significantly increased mean survival times as compared with two controls. Our data indicate that GM1 can significantly enhance the DSPC/CH liposome residence time in bloodstream and GM1/DSPC/CH liposomes with prolonged circulation time are important for the sustained relase of drug in circulation.

Analysis of the Water-soluble Protein Fraction of Glioma Cells by Two-dimensional Electrophoresis

The authors analyzed water-soluble proteins of culture human and rat glioma cells by two-dimensional polyacrylamide gel electrophoresis methods. Glioma cells were suspended in distilled water and then destroyed by freezing and thawing to obtain the water-soluble protein fraction. A modification of O'Farrell's non-equilibrium pH gradient (NEPHGE) method was used to analyze differences in protein mapping. Manabe's microscale two-dimensional electrophoresis without denaturing agents was used to detect proliferating cell nuclear antigen (PCNA/cyclin) by Western blotting. With O'Farrell's NEPHGE method and silver staining, at least 200 different polypeptides were clearly identified in each cell line. Cytoskeletal proteins, such as actin, were consistently separated in all cell lines. Marked differences in the protein map were observed between human and rat glioma cell lines, and even within the same species. Presumably, these differences are attributable to cell-biological difference in the glioma cells lines. Some proteins that were prominent in proliferating cells were scant in the protein maps of cells cultured for 24 hours in medium not containing calf serum, which suppresses cell growth. PCNA, an acidic nuclear protein that appears only in the late G1-S phase and is believed to be involved in cell proliferation, was detected by Western blotting and indirect immunostaining. Quantitative analysis of PCNA spots on the protein map appears useful in assessment of glioma cell proliferation. These results indicate that two-dimensional polyacrylamide gel electrophoresis can contribute to the understanding of the biological features of glioma cells.

Evaluation of protocols for purification of mouse monoclonal antibodies: Yield and purity in two-dimensional gel electrophoresis

Protocols for purification of mouse monoclonal antibodies (MAbs) from nude mice ascites were investigated in order to assess the yield and to compare the purified products in two-dimensional gel electrophoresis (2DGE). Three Mabs (one IgG2 and two IgG1), selected for their differing behaviours towards protein A, were purified by ammonium sulphate precipitation and/or gel filtration, anion exchange (DEAE), hydroxylapatite and affinity (protein A) chromatography, or by a combination of these methods. Protein A constantly provided the highest purity whatever the IgG subclass. The best results in terms of yields and purity were a function of the optimization of the protein A protocol. In our study, they were obtained in a 3 h protocol (IgG2), a 16 h protocol with discontinuous pH gradient method (IggG1 with sufficiently high affinity for protein A) or a multi-step protocol involving DEAE and protein A (IgG1 with low affinity for protein A). DEAE chromatography alone provided a slightly better yield, but only moderate purity. Hydroxylapatite chromatography appeared to be less potent in terms of yield, purity and day-to-day reproducibility. Salt precipitation and gel filtration enabled only relative enrichment of the MAb solution. Some degradation products of both heavy and light chains clearly appeared in the 2DGE patterns of antibodies purified by different protocols, and seem to be partly related to the elution pH and to the duration of the purification procedure. Finally, this work highlights considerable heterogeneity not only between two different MAbs of the IgG1 subclass but also within a monoclonal population of immunoglobulins.

A convenient pH-gradient method for the determination of the maximum and minimum pH for microbial growth.

Previously existing methods for determining the pH limits for the growth of microorganisms have involved (1), the setting up of individual cultures, each having a specific pH; (2), the pH gradient plate technique devised by Sacks (1956) in which a continuous pH gradient is established in a Petri dish by means of a buffer system; and (3), the pH gradient plate technique of Zak (unpublished), in which a continuous pH gradient is established by means of an electric current. The discontinuous pH gradient technique described here provides a convenient method of determining the maximum and minimum pH at which a microorganism can grow. The technique can be used aerobically or anaerobically, and has a precision of about ± 0.1 pH unit. Data are given for several yeasts and forSerratia marcescens. In all cases, the organisms tested continued to metabolize at pH values beyond those representing the limits for growth, sometimes by as much as 0.5 pH unit. The results suggest that pH limits are unsuitable criteria in microbial classification.