Robert L. Gutman

Targeted drug delivery for brain cancer treatment.

The blood brain barrier (BBB) and the systemic toxicity of conventional chemotherapy present obstacles to the success of future blood-borne drug therapies of brain tumors. The work with polymer-encapsulated cancer drugs suggests an alternative and more focused treatment approach. Our experimental strategy integrates direct intracerebral drug delivery, sustained drug release from liposomes or polymer implants, and increased targeting of the drug either by chemically modifying the drug or by using tumor-specific carriers. This review will present some of the recent work on targeted drug delivery for brain cancer treatment.

Cellular localization of antiviral polyoxometalates in J774 macrophages

The cellular localization of the polyoxometalates, K12H2[P2W12O48].24H20 (JM 1591), K10[P2W18-Zn4(H2O)2O68].20H2O (JM 1596), and [Me3NH]8[Si2W18Nb6O77] (JM 2820) were examined in cultured J774 cells by inhibition of cellular uptake of acetylated low-density lipoprotein (LDL) and by electron microscopy. All three polyoxometalates inhibited the cellular uptake of acetylated LDL, suggesting that the polyoxometalates block the association of acetylated LDL with cellular scavenger receptors. Fluorescence microscopy showed increased numbers of vacuoles in the presence of polyoxometalates, suggesting their uptake by cells. Using scanning electron microscopy (SEM), no significant cell surface morphological differences were observed between treated and non-treated J774 cells, suggesting that the compounds are not toxic to J774 cells up to a concentration of 200 micrograms/ml. Transmission electron microscopy (TEM) revealed large amounts of high electron dense granules were observed in the ramifying system of tubular cavities and vacuoles. TEM-energy dispersive spectroscopy (EDS) X-ray microanalysis was unable to differentiate the dense particles, most likely because the amount of tungsten in the cells was below the limit of detection. X-ray microanalysis conducted using the SEM-wavelength dispersive spectroscopy (WDS) detected tungsten, averaging 0.45 +/- 0.16% (mean +/- S.D.), in the J774 cells treated with JM 2820, suggesting that this polyoxometalate was taken up by the macrophages or was bound to their surface. Polyoxometalates interact at the cell surface and appear to be taken up by J774 macrophages. The cellular localization of polyoxometalates may be associated with anti-HIV activity.

Association of negatively-charged phospholipids with low-density lipoprotein (LDL) increases its uptake and the deposition of cholesteryl esters by macrophages

LDL, the major carrier of cholesterol in blood, is poorly metabolized by macrophages. In contrast, macrophages can recognize and endocytose anionic phospholipids such as phosphatidylserine, phosphatidylglycerol and cardiolipin. Since macrophages can take up large amounts of these phospholipids, experiments were performed to ascertain whether pre-incubation of native LDL with negatively-charged phospholipids would enhance the metabolism of LDL by macrophages. When 125I-LDL was incubated with cardiolipin liposomes for 18 h at 37 degrees C before addition to macrophages, an approx. 40-fold increase of LDL metabolism by these cells was observed. Similar results were found when LDL was pre-incubated with phosphatidylserine or phosphatidylglycerol; however, pre-incubation of LDL with phosphatidylcholine liposomes did not lead to an increase of LDL metabolism. The macrophage uptake of LDL pre-incubated with cardiolipin was reduced to approx. 40% of control values in the presence of dextran sulfate and fucoidin, inhibitors of anionic phospholipid uptake. Cytochalasin D, an inhibitor of phagocytosis, reduced the lysosomal degradation of LDL pre-incubated with cardiolipin to approx. 10% of control values. When the LDL-cardiolipin mixture was chromatographed on agarose gel, two peaks containing LDL were observed in the elution profile: the first peak appeared at the void volume and the second peak was detected just ahead of native LDL. The LDL in both peaks was much more extensively metabolized by macrophages than was native LDL; the LDL in the first peak was metabolized at a rate that was 8 times the second peak. The results demonstrate that negatively-charged phospholipids can form a complex with LDL which facilitates its phagocytosis by macrophages.

Advances in agarose gel electrophoresis of serum lipoproteins

Agarose gel electrophoresis has been extensively employed by researchers to gain a greater understanding of lipoprotein biology and its relationship to cardiovascular disease. Advances in this technique have been made in the visualization and quantitation of separated lipoproteins, in the use of agarose gel electrophoresis for detection and quantitation of apolipoproteins of the separated lipoproteins, and in the detection of lipoprotein heterogeneity. Agarose gel electrophoresis has been employed for two-dimensional electrophoretic analysis of lipoproteins as well as in several different methods which probe the immunological properties of lipoproteins. Agarose gel electrophoresis has thus become an important tool in the study of serum lipoproteins in both clinical and basic science laboratories.

Endocytosis of sulfatides by macrophages: relationship to the cellular uptake of phosphatidylserine.

These studies initially examined the effect of sulfatides on the endocytosis of phosphatidylserine (PS) liposomes in J774 macrophages employing liposomes composed entirely of PS and the nonexchangeable radiolabel [3H]cholesteryl hexadecyl ether. Bovine brain sulfatides significantly inhibited the uptake of PS liposomes by macrophages to a level of approximately 15% of control values. To examine whether macrophages were also capable of recognizing and internalizing sulfatides, sulfatide‐containing liposomes were prepared using phosphatidylcholine (PC), cholesterol, and sulfatides (6:2:4 molar ratio) incorporating the same radiolabel. The sulfatide‐containing liposomes were found to be avidly endocytosed by macrophages. Uptake of the sulfatide‐containing liposomes by macrophages was significantly greater than the uptake of liposomes made without sulfatides. When the macrophages were incubated with the anionic compounds dextran sulfate and fucoidin, both the binding of the liposomes to the macrophages at 4° and the internalization of the liposomes at 37° were inhibited to approximately 10% of control values. The negatively charged phospholipids phosphatidylglycerol and cardiolipin significantly inhibited the uptake of sulfatide‐containing liposomes, and PS was not effective in reducing the cellular uptake of these liposomes. Both oxidized low‐density lipoprotein (LDL) and acetylated LDL reduced the uptake of the sulfatide‐containing liposomes; the uptake observed in the presence of acetylated LDL and oxidized LDL was approximately 70% and 40%, respectively, of control values. These findings demonstrate that macrophages can efficiently endocytose both sulfatides and negatively charged phospholipids to remove them from the circulation. J. Leukoc. Biol. 55: 99–104; 1994.

Effects of phosphatidylserine on the oxidation of low density lipoprotein.

1. LDL was incubated in the presence of 1 microM CuSO4 for 18 hr at 37 degrees C. The content of lipoperoxides was found to be approx. 40 nmol MDA equivalents/mg LDL protein. The addition of 50 microM phosphatidylserine (PS) reduced the content of lipoperoxides to 15% of control values. 2. The electrophoretic mobility observed for LDL oxidized in the presence of PS approximated the mobility observed for native LDL. 3. The formation of conjugated dienes was strongly inhibited when LDL was oxidized in the presence of PS. 4. The addition of 50 microM phosphatidylcholine, phosphatidylglycerol and cardiolipin did not alter the extent of LDL oxidation. 5. PS did not inhibit the oxidation of LDL mediated by J774 macrophages in the presence of Hams F-10 culture medium. Under these conditions, PS was found to be an excellent substrate for oxidation.

Iron-ascorbate-phospholipid mediated modification of low density lipoprotein

LDL can be oxidized by a variety of agents to form a modified lipoprotein which is capable of being avidly metabolized by macrophages. While previous in vitro studies have focused exclusively on the oxidation of LDL, other lipids found in the atheroma are also subject to oxidation and its lipoperoxide byproducts may contribute to the process of LDL modification. To examine the relationship between the oxidation of phospholipids and the subsequent modification of LDL, we incubated 250 microM phosphatidylcholine with 10 microM ferrous sulfate and 50 microM ascorbic acid in 10 mM Tris (pH 7.0). After 18 h at 37 degrees C, significant amounts of thiobarbituric acid reactive substances (TBARS) were formed. The inclusion of LDL (100 micrograms protein/ml) elevated the TBARS and increased the electrophoretic mobility of the lipoprotein. LDL treated with iron and ascorbate in the absence of phosphatidylcholine did not result in the modification of this lipoprotein. LDL that was incubated with phosphatidylcholine, iron and ascorbate was found to be metabolized by macrophages to a far greater extent than native LDL or LDL treated with phosphatidylcholine alone. Probucol (10 microM) inhibited the LDL modification process. These results demonstrate that while iron and ascorbate cannot oxidize LDL directly, the addition of phosphatidylcholine to these initiators of lipid peroxidation can mediate and lead to the modification of LDL.