Oxygenation of malignant tumors after localized microwave hyperthermia

Summary The oxyhemoglobin saturation (HbO₂) of single red blood cells within tumor microvessels (diameter: 3–12 µm) of DS-Carcinosarcoma was studied using a cryophotometric micromethod. In untreated control tumors (mean tissue temperature approx. 35° C) the measured values scattered over the whole saturation range from zero to 100 sat.%, the mean being 51 sat.%. Upon heating at 40° C for 30 min, the oxygenation of the tumor tissue significantly improved as compared with control conditions. After 40° C-hyperthermia a mean oxyhemoglobin saturation of 66 sat.% was obtained. In contradistinction to this, after 43° C-hyperthermia the tumor oxygenation was significantly lower and reached a mean HbO₂ saturation value of 47 sat.%. A further temperature rise to 45° C caused the oxygenation to drop drastically (mean oxyhemoglobin saturation value: 24 sat.%). This is due to a severe restriction of nutritive blood flow.The changes in tumor oxygenation after hyperthermia seem to be predominantly mediated through changes in tumor blood flow, including tumor microcirculation, which showed a similar temperature dependence. Metabolic effects probably play a minor role in the oxyhemoglobin saturation distribution within tumor microvessels.Supported by the Deutsche Forschungsgemeinschaft (Va 57/2-1). Presented in part at the International Symposium on Biomedical Thermology, June 30 to July 4, 1981, Strasbourg, France     

Glass-ceramic-mediated, magnetic-field-induced localized hyperthermia: response of a murine mammary carcinoma

Hyperthermia has been found to be a useful modality for cancer therapy. In this report, a biocompatible, ferrimagnetic glass-ceramic capable of inducing localized hyperthermia by hysteresis heating upon exposure to an alternating magnetic field is presented. When the glass-ceramic was placed in the region of a subcutaneously transplanted, weakly antigenic breast carcinoma and subjected to the magnetic field, sufficient temperature rise was obtained to cause significant (approximately 50%) tumor regrowth delay and a 12% permanent control. The data demonstrate that glass-ceramic-mediated hysteresis heating may be a useful therapeutic approach in the treatment of cancer which offers the advantage of producing a highly localized and predictable tumor volume hyperthermia.     

Study of the pilot production process of long-circulating and pH-sensitive liposomes containing cisplatin

Long-circulating and pH-sensitive liposomes, containing cisplatin (SpHL-CDDP), have been developed as an alternative aimed at avoiding severe side effects as well as the appearance of resistance, which can limit the use of free cisplatin. However, physical (i.e., aggregation/fusion) and chemical instabilities limit the use of these drug carriers as pharmaceutical products. The preparation of freeze-dried pharmaceuticals has proven to be a successful strategy implemented to improve the stability of these formulations. In addition, the development of an economically feasible, reproducible process of liposome production, on a large scale, has also become necessary. A pilot production process, using three stages (i.e., reverse-phase evaporation, homogenization under high pressure, and ultrafiltration), was used to prepare SpHL-CDDP. The optimization of factors related to the homonogenization under high pressure (i.e., pressure and number of cycles), ultrafiltration (i.e., number of cycles), and storage stability at 4°C were assessed by means of particle size, zeta potential, and encapsulation percentage. A 500-bar pressure and 9 cycles were adopted as measures for the production of SpHL-CDDP, which presented a mean diameter of 99.0?±?3.9?nm and an encapsulation percentage of 12.9?±?2.3. The use of trehalose as a cryoprotectant was investigated, regarding its effective ability to control the vesicle diameter and retain encapsulated CDDP after the freeze-drying/rehydration step. After 135 days of storage, freeze-dried or liquid SpHL-CDDP showed no significant change in mean diameter. However, the freeze-dried SpHL-CDDP proved to be more efficient, in terms of CDDP retention, than did the liposomal liquid dispersion.     

In situ activation of murine macrophages by liposomes containing lymphokines

Murine alveolar and peritoneal macrophages harvested after injection of lymphokines encapsulated within multilamellar phospholipid vesicles (liposomes) were tumoricidal in vitro. The state and degree of activation depended on the route of liposome administration. Activation of peritoneal macrophages was achieved by intraperitoneal injection of liposomes and alveolar macrophages were activated by injecting liposomes intravenously but not intraperitoneally. The in vivo rendering of macrophages with tumoricidal properties might be useful toward destruction of tumor cells in vivo.     

Acute toxicity of long-circulating and pH-sensitive liposomes containing cisplatin in mice after intraperitoneal administration

AimsThe objective of this work was to evaluate the acute toxicity of long-circulating and pH-sensitive liposomes containing cisplatin (SpHL-CDDP), after their intraperitoneal administration in male and female mice.Main methodsAfter single administration of free CDDP (5,10,and 20 mg/kg) or SpHL-CDDP (7,12,30,45 and 80 mg/kg), the body weight was recorded and the LD₅₀ was calculated. Blood samples were collected for biochemical and hematological analysis. Kidneys, liver, spleen and bone marrow were removed to histopathological examination.Key findingsMice treated with high doses of free CDDP showed a greater loss of body weight and more delayed recovery time than those treated with SpHL-CDDP. The LD₅₀ values for SpHL-CDDP treatment for male and female mice groups were 2.7 and 3.2 fold higher, respectively, than that obtained for free CDDP. The red and white blood cells counts and quantification of hemoglobin and hematocrit presented no change upon administration of SpHL-CDDP treatment. Free CDDP treatment, however, did lead to an appearance of mild anemia and a reduction in total white blood cell counts. As regards nephrotoxicity, it was observed that free CDDP treatment caused pronounced alterations in the blood urea and creatinine levels of mice. In contrast, these parameters were slightly altered only after SpHL-CDDP treatment at a dose of 30 mg/kg. Microscopic analysis of kidneys from mice treated with SpHL-CDDP showed no morphological alteration. Concerning hepatotoxicity, no histopathological alteration was observed after both treatments.SignificanceThese findings reveal that SpHL-CDDP can eliminate CDDP-induced toxicity and is thus a promising candidate for intraperitoneal chemotherapy.     

The change in hypoxic and chronically hypoxic cell fraction in murine tumors treated with hyperthermia

The effect of hyperthermia on the size of hypoxic and chronically hypoxic cell fractions in murine tumors was studied. The chronically hypoxic cell fraction was defined as a fraction of tumor cells which were not oxygenated under hyperbaric oxygen. Animals were C3Hf/Sed mice derived from our defined flora mouse colony. Tumors were FSa-II and MCa which were early generation isotransplants of a spontaneous fibrosarcoma and a mammary carcinoma, respectively. TCD50 (50% tumor control dose) or the radiation dose which yields a local tumor control in half the treated animals and TG (tumor growth) time or the time required for half the treated tumors to reach 1000 mm3 from the first treatment day were experimental end points. Hyperthermia was given by immersing animal feet into a water bath maintained at 43.5 +/- 0.1 degrees C. Animal tumors were irradiated with a 137Cs unit under hypoxic conditions, in air or under O2 30 psi. The hypoxic cell fraction increased immediately after hyperthermia in both MCa and FSa-II tumors. The chronically hypoxic cell fraction was, on the other hand, decreased following hyperthermia. The decrease was more substantial in the MCa than in FSa-II.     

Optimization and limitations of systemic treatment of murine melanoma metastases with liposomes containing muramyl tripeptide phosphatidylethanolamine

Summary The purpose of these studies was to determine the optimal conditions and limitations for the eradication of spontaneous melanoma metastases by the systemic administration of liposomes containing MTP-PE. Mice whose primary melanoma had been excised were given i.v. injections of liposomes at various schedules. Optimal treatment was achieved by twice weekly administration for 4 weeks (eight i.v. injections). Bioassays failed to reveal the presence of melanoma cells in lungs of mice surviving to day 250 of the experiment. The success of liposome treatment of metastases diminished when the first i. v. injection of liposomes-MTP-PE commenced on day 10 after surgical excision of the local melanoma, as compared with day 3 or day 7 after surgery. We conclude that the major limitation for macrophage-mediated destruction of metastases is the number of tumor cells in the lesions. Because of this limitation, it is unlikely that the systemic activation of macrophages could be used as a single modality for treatment of advanced metastases.     

Lymph flow from murine footpad tumors before and after sublethal hyperthermia.

The effect of local hyperthermia (43.5 degrees C for 1 h) on lymph flow from B16-F10 tumor-bearing foot pads of C57BL/6 mice was measured by monitoring the clearance of 99mTc-labeled human serum albumin. The foot was represented by a single-compartment model enabling a quantitative computation of lymphatic flow from the tumor to regional lymph nodes. Lymphatic flow from untreated tumors was 0.0059 +/- 0.0011 ml/min cm3 compared to 0.0118 +/- 0.0027 ml/min cm3 lymphatic flow from tumors immediately following heating. Morphological alterations in tumor blood vessels result in their high vascular permeability. The increase in lymphatic clearance from tumors after sublethal hyperthermia is compatible with the increase in interstitial fluid formation in tumors based on Starling's Law.     

Effect of cisplatin containing liposomes formulated by unsaturated chain-containing lipids on gynecological tumor cells

Gynecological tumors are major therapeutic areas of platinum-based anticancer drugs. Here, we report the characterization and in vitro biological assays of cisplatin-containing Egg L-α-phosphatidylcholine liposomes with different amounts of cholesterol. Dynamic light scattering estimated sizes of all obtained liposomes in the 100?nm range that are suitable for in vivo use. On the basis of these data and of the drug loading values, the best formulation has been selected. Stability and drug release properties of platinum-containing liposomes have been verified in serum. The growth inhibitory effects of both liposomal and free drug in a panel of ovarian and breast human cancer cell lines, characterized by a different drug sensitivity, give comparable or better results with respect to free cisplatin drug.     

Effects on the murine mononuclear phagocyte system of chronic administration of liposomes containing cytotoxic drug or lipid A compared with empty liposomes

In experiments designed to examine the adverse effects of chronic liposome administration in vivo on the mononuclear phagocyte system (reticuloendothelial system), the presence of drug entrapped in the liposomes may increase the level of reticuloendothelial impairment. We have compared the effects on the mononuclear phagocyte system in mice of chronic administration of empty liposomes with the effects of liposomes containing the anti-leishmanial drug meglumine antimoniate. We have also examined the effect on the mononuclear phagocyte system of continued injections of liposomes containing lipid A, a component of bacterial lipopolysaccharide, which is responsible for macrophage activation. Ten intravenous injections of multilamellar liposomes composed of dipalmitoylphosphatidylcholine and cholesterol (1:0.75 M ratio) were given to ICR mice over a 25-day period. Two individual groups of mice received endotoxin-free liposomes in which meglumine antimoniate was either present or absent. One addition group received liposomes containing lipid A derived from Escherichia coli lipopolysaccharide. A control group received sterile saline injections. In each group, a depression of the phagocytic index, as measured by reduction of uptake of particulate carbon, was observed among some of the individual animals 24 h after the first injection. In many mice a marked splenomegaly was observed. A depressed phagocytic index and splenomegaly were most marked for mice receiving lipid A liposomes. However, there was a large individual variability among mice receiving these preparations and some mice in each group had normal spleen size and a nearly normal phagocytic index. Tissue distribution of liposomes containing [14C]dipalmitoylphosphatidylcholine as a phospholipid marker was examined in all groups in mice 24 h after the last injection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of murine hepatic tumor growth by liposomes containing a lipophilic muramyl dipeptide

Summary We have investigated the ability of liposomes containing a lipophilic muramyl dipeptide, N-acetylmuramyl-l-alanyl-d-isoglutamine glycerol dipalmitate (MDP-GDP) to activate Kupffer cell tumoricidal activity in situ and to inhibit the growth of experimental hepatic micrometastases of tumor cell line H-59, a liver-homing variant of the Lewis lung carcinoma. Liposomes prepared from distearoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DSPC/DMPG) and containing MDP-GDP (1 mol and 2 g, respectively) were efficiently taken up by the liver after i.v. administration. A single i.v. injection of DSPC/DMPG liposomes containing MDP-GDP was capable of inducing Kupffer cell tumoricidal activity against H-59 tumor cells as measured in vitro. Control liposomes or 100 g free MDP were ineffective in inducing Kupffer cell tumoricidal activity in situ. Two treatment regimens were evaluated in vivo: firstly, C57BL/6 mice were injected with tumor cell line H-59 and subsequently treated with multiple injections of liposomal MDP-GDP. Secondly, treatment with liposomal MDP-GDP was initiated prior to tumor cell injection and continued after tumor cell injection. The ability of liposomes containing MDP-GDP to reduce the number of hepatic micrometastases using the first protocol was related to the tumor cell inoculum, significant inhibition being observed at lower liver tumor burdens (<25 tumor nodules). Pretreatment of the mice prior to tumor cell challenge followed by treatment afterwards greatly enhanced the efficacy of liposomal MDP-GDP and brought about a highly significant inhibition of the growth of experimental metastases even at high liver tumor burdens (>50 nodules).     

Antineoplastic effect of mer-trichlorobisdimethylsulphoxideaminorutheniumIII against murine tumors: comparison with cisplatin and with ImH[RuIm2Cl4

An asymmetric rutheniumIII complex containing dimethylsulphoxide ligands, namely mer-trichlorobisdimethylsulphoxideaminorutheniumIII (BBR2382), has been tested in mice bearing solid metastasizing tumors. The effects of i.p. treatment with BBR2382 on primary tumor growth and on the survival time of hosts carrying s.c. or i.m. tumors have been compared to those of cisplatin and of a rutheniumIII complex with imidazole ligands, ImH[RuIm2Cl4], described as a potent antitumor agent in a number of experimental models of murine neoplasms. In mice bearing Lewis lung carcinoma, BBR2382 results as effective as cisplatin on s.c. primary tumor growth and more potent than cisplatin on the prolongation of host survival time. The combined treatment of mice bearing Lewis lung carcinoma with cisplatin and BBR2382 causes a reduction of s.c. tumors higher than that caused by each single agent; the effects on host survival time are similar to those caused by BBR2382 alone but significantly superior to those caused by cisplatin alone. In CBA mice bearing MCa mammary carcinoma, the effects of BBR2382 are slightly lower than those of cisplatin on i.m. tumors but are equivalent on host survival time. The comparison of the antineoplastic action of BBR2382 with that of ImH[RuIm2Cl4] is always in favor of the former, independently of the parameter chosen and of the tumor system used. Qualitatively, the antitumor action of BBR2382 seems different from that of cisplatin and of ImH[RuIm2Cl4]; it is supposed that this agent, like other "rutheniumIII dimethylsulphoxide" complexes, could have a particular efficacy for tumors localized in the lungs.     

Enzyme replacement with liposomes containing beta-galactosidase from Charonia lumpas in murine globoid cell leukodystrophy (twitcher)

Enzyme replacement with liposomes containing beta-galactosidase obtained from charonia lumpas was carried out in murine globoid cell leukodystrophy (GLD). Charonia lumpas beta-galactosidase was able to hydrolyze galactocerebroside trapped into liposomes prepared from lecithin, cholesterol and sulfatide (molar ratio; 7:2:1). Liposomes containing charonia lumpas beta-galactosidase were successfully incorporated into the mouse tissues. 3H-galactocerebroside labeled liposomes were also incorporated into mouse liver, spleen and other tissues. The accumulation rate of 3H-galactocerebroside into twithcer mice liver and spleen was almost 40 to 100 times higher than those of controls and degraded to 70 to 80% of accumulated radioactivity of 3H-galactocerebroside by single injection of liposomes containing charonia lumpas beta-galactosidase. Results suggest that exogeneous enzyme trapped in liposomes can be useful for the correction of accumulated compound.     

Interaction of photosensitizers with liposomes containing unsaturated lipid

Small unilamellar liposomes were made of dipalmitoyl-phosphatidylcholine and dioleoyl-phosphatidylcholine, and photosensitized by a symmetrically or an asymmetrically substituted glycosilated tetraphenyl-porphyrin derivative. As differential scanning calorimetry and electron paramagnetic resonance spectroscopy (EPR) revealed these porphyrin derivatives were localized in different depth within the lipid bilayer. Both porphyrin derivatives were able to induce photoreaction and consequent structural changes in the membrane. 5-, 12-, or 16-doxyl stearic acid labeled lipid bilayers were applied and the efficiency of photoinduced reaction was followed by the decay of their EPR signal amplitude. Light dose-dependent destruction of nitroxide radical proved to be dependent on the position of spin label. In this process the porphyrin localized in closer connection with the double bond of unsaturated fatty acid was more effective. EPR signal decay was also dependent on the unsaturated fatty acid content of the liposome and the oxygen saturation of the solvent.     

Further characterization of a murine temperature-sensitive mutant, tsFT20 strain, containing heat-labile DNA polymerase alpha-activity

tsFT20 cells, which have temperature-sensitive DNA polymerase alpha-activity, were characterized mainly at the cellular level. The cells lost their ability to synthesize DNA immediately after a shift to non-permissive temperature. The extent of decrease in the activity of DNA polymerase alpha in whole-cell extracts was the same as that of the decrease in the DNA replication ability determined by [3H]thymidine incorporation. At 39 degrees C, tsFT20 cells lost most of their colony-forming ability in one doubling time (16 h). The cells could not grow at higher than 38 degrees C, but could grow at 37 degrees C. When tsFT20 cells were synchronized at the G1/S boundary and incubated at 39 degrees C, they could not complete the S phase, ceasing cell cycle progression in mid-S phase. A temperature shift (33 degrees C----39 degrees C) experiment indicated that the whole S phase was temperature-sensitive, whereas the G2 and M phases were not. These results confirmed that DNA polymerase alpha plays a key role in DNA replication in mammalian cells.     

Treatment of superficial cancerous tumors by hyperthermia induced by ultrasonics or microwaves

Results of treating superficial human cancerous tumors by ultrasound (1-3 MHz) or microwave (434 MHz) hyperthermia, alone or combined with radiotherapy or chemotherapy are presented according to the heating method. The comparison of in vivo thermal distributions obtained at depth with these two techniques could partially explain their different therapeutic efficiency.     

Preparation and properties of arsonolipid containing liposomes

Arsonolipids are analogs of phosphonolipids which have a chemically versatile head group. In preliminary cell culture studies, liposomes composed solely of arsonolipids or of phosholipid-arsonolipid mixtures, demonstrate a specific toxicity against cancer cells (Gortzi et al., unpublished results). The possibility of using such formulations as an alternative of arsenic trioxide with or without combination of other cytostatic agents (encapsulated in their aqueous interior) prompted the investigation of their physicochemical characteristics. Herein we compared the characteristics of arsonolipid containing vesicles with different lipid compositions. Experimental results and morphological observations reveal that non-sonicated formulations have different structures and stability (when both membrane integrity and aggregation are taken into account) depending on the acyl chain length of the arsonolipid. When phospholipids and especially cholesterol are included in their membranes almost all arsonolipids studied produce more stable vesicles. An interesting aspect of these arsonolipid containing vesicles is also their negative surface charge, which may be modulated by mixing phospholipids with arsonolipids. Sonicated vesicles have smaller sizes and profoundly higher stability, especially when containing cholesterol and phosphatidylcholine mixed with arsonolipids. The only exception is that of the arsonolipid with the C(12) acyl chain which was observed to produce long tubes which break down to cubes by sonication. In conclusion, these initial studies demonstrate that sonicated vesicles composed of arsonolipid and phospholipid mixtures mixed with cholesterol posses the stability required to be used as an arsonolipid delivery system. In addition, although cryo-electron microscopy demonstrated that the sonicated vesicles are elliptical in shape, their encapsulation efficiency is not significantly lower than sonicated phospholipid liposomes. Thereby, these vesicles may be also used for the delivery of other drug molecules which can be sufficiently retained in their aqueous interior.     

Inhibition of cell proliferation with antibody-targeted liposomes containing methotrexate-gamma-dimyristoylphosphatidylethanolamine

We have prepared liposomes containing methotrexate-gamma-dimyristoylphosphatidylethanolamine (MTX-DMPE liposomes), to which protein A was covalently coupled, permitting specific association of these liposomes in vitro with murine cells preincubated with relevant protein A-binding monoclonal antibodies. In the absence of antibody the presence of externally-oriented methotrexate (MTX) in MTX-DMPE liposomes did not result in greater binding to cells than liposomes made without MTX-gamma-DMPE. Derivation of methotrexate with phospholipid permits enhanced drug-liposome association. These liposomes are more resistant than conventional liposomes to repeated cycles of freezing and thawing. MTX-DMPE liposomes are comparable to antibody-targeted liposomes made with encapsulated water-soluble methotrexate both with respect to specific binding to target cells and drug effect. The inhibitory effects of MTX-liposomes, as well as free MTX, were reversible by either thiamin pyrophosphate (Tpp) or N5-formyltetrahydrofolate (F-THF), while the effects of MTX-DMPE liposomes were reversed only by N5-formyltetrahydrofolate. This suggests that the toxicity of non-targeted MTX-liposomes may be due to leakage of the encapsulated MTX. The absence of an effect of thiamin pyrophosphate on non-targeted MTX-DMPE liposomes indicates that they do not enter into the cell via the normal folate transport system.     

Interaction of spermine with polyphosphoinositides containing liposomes and myo-inositol 1,4,5 triphosphate

The interaction of spermine with liposomes containing 2% phosphatidylinositol, phosphatidylinositol 4 phosphate and phosphatidylinositol 4,5 biphosphate was inferred from the ability of these liposomes to interfere with spermine binding to the resin heparin-Sepharose. The inositol phospholipids tested showed different affinities for spermine: the order of binding strength appear to be phosphatidylinositol phosphatidylinositol 4 phosphate phosphatidylinositol 4,5 biphosphate. The ability of vesicles containing 2% polyphosphoinositides to interact with spermine is comparable to that of either single stranded RNAs or highly negatively charged liposomes. Myo-inositol 1,4,5 triphosphate has a much lower ability to bind spermine.     

A critical appraisal of evidence for localized energy coupling. Kinetic studies on liposomes containing bacteriorhodopsin and ATP synthase.

In intact systems (chloroplasts, mitochondria and bacteria) many experiments have been reported which are indicative of localized coupling between ATP synthase and electron transfer complexes. We have carried out similar experiments with a system in which we may assume that specific interactions between the proton pumps are absent: reconstituted vesicles containing bacteriorhodopsin and yeast mitochondrial ATP synthase. The only experiment that gives results which differ from those previously published for intact systems concerns the effect of uncouplers on the rate of ATP synthesis at different levels of inhibition of the ATP synthase. We propose that this type of experiment may discriminate between localized and delocalized coupling.