Comparing therapeutic and prophylactic protection against the lethal effect of paraoxon.

Prophylactic and therapeutic efficacy against organophosphorus (OP) intoxication by pralidoxime (2-PAM) and atropine were studied and compared with sterically stabilized long-circulating liposomes encapsulating recombinant organophosphorus hydrolase (OPH), either alone or in various specific combinations, in paraoxon poisoning. Prophylactic and therapeutic properties of atropine and 2-PAM are diminished when they are used alone. However, their prophylactic effects are enhanced when they are used in combination. Present studies indicate that sterically stabilized liposomes (SL) encapsulating recombinant OPH (SL-OPH) alone can provide much better therapeutic and prophylactic protection than the classic 2-PAM + atropine combination. This protection was even more dramatic when SL-OPH was employed in combination with 2-PAM and/or atropine: the magnitude of prophylactic antidotal protection was an astounding 1022 LD(50) [920 mg/kg (LD(50) of paraoxon with antagonists)/ 0.95 mg/kg (LD(50) of control paraoxon)], and the therapeutic antidotal protection was 156 LD(50) [140 mg/kg (LD(50) of paraoxon with antagonists)/0.9 mg/kg (LD(50) of control paraoxon)]. The current study firmly establishes the value of using liposome encapsulating OPH.

Tumor targeting using anti-her2 immunoliposomes.

We have generated anti-HER2 (ErbB2) immunoliposomes (ILs), consisting of long circulating liposomes linked to anti-HER2 monoclonal antibody (MAb) fragments, to provide targeted drug delivery to HER2-overexpressing cells. Immunoliposomes were constructed using a modular strategy in which components were optimized for internalization and intracellular drug delivery. Parameters included choice of antibody construct, antibody density, antibody conjugation procedure, and choice of liposome construct. Anti-HER2 immunoliposomes bound efficiently to and internalized in HER2-overexpressing cells in vitro as determined by fluorescence microscopy, electron microscopy, and quantitative analysis of fluorescent probe delivery. Delivery via ILs in HER2-overexpressing cells yielded drug uptake that was up to 700-fold greater than with non-targeted sterically stabilized liposomes. In vivo, anti-HER2 ILs showed extremely long circulation as stable constructs in normal adult rats after a single i.v. dose, with pharmacokinetics that were indistinguishable from sterically stabilized liposomes. Repeat administrations revealed no increase in clearance, further confirming that ILs retain the long circulation and non-immunogenicity of sterically stabilized liposomes. In five different HER2-overexpressing xenograft models, anti-HER2 ILs loaded with doxorubicin (dox) showed potent anticancer activity, including tumor inhibition, regressions, and cures (pathologic complete responses). ILs were significantly superior vs. all other treatment conditions tested: free dox, liposomal dox, free MAb (trastuzumab), and combinations of dox+MAb or liposomal dox+MAb. For example, ILs produced significantly superior antitumor effects vs. non-targeted liposomes (P values from <0.0001 to 0.04 in eight separate experiments). In a non-HER2-overexpressing xenograft model (MCF7), ILs and non-targeted liposomal dox produced equivalent antitumor effects. Detailed studies of tumor localization indicated a novel mechanism of drug delivery for anti-HER2 ILs. Immunotargeting did not increase tumor tissue levels of ILs vs. liposomes, as both achieved very high tumor localization (7.0-8.5% of injected dose/g tissue) in xenograft tumors. However, histologic studies using colloidal-gold labeled ILs demonstrated efficient intracellular delivery in tumor cells, while non-targeted liposomes accumulated within stroma, either extracellularly or within macrophages. In the MCF7 xenograft model lacking HER2-overexpression, no difference in tumor cell uptake was seen, with both ILs and non-targeted liposomes accumulating within stroma. Thus, anti-HER2 ILs, but not non-targeted liposomes, achieve intracellular drug delivery via receptor-mediated endocytosis, and this mechanism is associated with superior antitumor activity. Based on these results, anti-HER2 immunoliposomes have been developed toward clinical trials. Reengineering of construct design for clinical use has been achieved, including: new anti-HER2 scFv F5 generated by screening of a phage antibody library for internalizing anti-HER2 phage antibodies; modifications of the scFv expression construct to support large scale production and clinical use; and development of methods for large-scale conjugation of antibody fragments with liposomes. We developed a scalable two-step protocol for linkage of scFv to preformed and drug-loaded liposomes. Our final, optimized anti-HER2 ILs-dox construct consists of F5 conjugated to derivatized PEG-PE linker and incorporated into commercially available liposomal doxorubicin (Doxil). Finally, further studies of the mechanism of action of anti-HER2 ILs-dox suggest that this strategy may provide optimal delivery of anthracycline-based chemotherapy to HER2-overexpressing cancer cells in the clinic, while circumventing the cardiotoxicity associated with trastuzumab+anthracycline. We conclude that anti-HER2 immunoliposomes represent a promising technology for tumor-targeted drug delivery, and that this strategy may also be applicable to other receptor targets and/or using other delivered agents.

Expression of DeltaF508 CFTR in normal mouse lung after site-specific modification of CFTR sequences by SFHR.

The development of gene targeting strategies for specific modification of genomic DNA in human somatic cells has provided a potential gene therapy for the treatment of inherited diseases. One approach, small fragment homologous replacement (SFHR), directly targets and modifies specific genomic sequences with small fragments of exogenous DNA (400-800 bp) that are homologous to genomic sequences except for the desired modification. This approach has been effective for the in vitro modification of exon 10 in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in human airway epithelial cells. As another step in the development of SFHR for gene therapy, studies were carried out to target and modify specific genomic sequences in exon 10 of the mouse CFTR (mCFTR) in vivo. Small DNA fragments (783 bp), homologous to mCFTR except for a 3-bp deletion (DeltaF508) and a silent mutation which introduces a unique restriction site (KpnI), were instilled into the lungs of normal mice using four different DNA vehicles (AVE, LipofectAMINE, DDAB, SuperFect). Successful modification was determined by PCR amplification of DNA or mRNA-derived cDNA followed by KpnI digestion. The results of these studies showed that SFHR can be used as a gene therapy to introduce specific modifications into the cells of clinically affected organs and that the cells will express the new sequence.

Long circulating liposomes encapsulating organophosphorus acid anhydrolase in diisopropylfluorophosphate antagonism.

These studies are focused on antagonizing organophosphorous (OP) intoxications by a new conceptual approach using recombinant enzymes encapsulated within sterically stabilized liposomes to enhance diisopropylfluorophosphate (DFP) degradation. The OP hydrolyzing enzyme, organophosphorous acid anhydrolase (OPAA), encapsulated within the liposomes, was employed either alone or in combination with pralidoxime (2-PAM) and/or atropine. The recombinant OPAA enzyme, from the ALTEROMONAS: strain JD6, has high substrate specificity toward a wide range of OP compounds, e.g., DFP, soman, and sarin. The rate of DFP hydrolysis by liposomes containing OPAA (SL)* was measured by determining the changes in fluoride-ion concentration using a fluoride ion-selective electrode. This enzyme carrier system serves as a biodegradable protective environment for the OP-metabolizing enzyme (OPAA), resulting in an enhanced antidotal protection against the lethal effects of DFP. Free OPAA alone showed some antidotal protection; however, the protection with 2-PAM and/or atropine was greatly enhanced when combined with (SL)*.

In vitro studies on sterically stabilized liposomes (SL) as enzyme carriers in organophosphorus (OP) antagonism.

This study describes a new approach for organophosphorous (OP) antidotal treatment by encapsulating an OP hydrolyzing enzyme, OPA anhydrolase (OPAA), within sterically stabilized liposomes. The recombinant OPAA enzyme was derived from Alteromonas strain JD6. It has broad substrate specificity to a wide range of OP compounds: DFP and the nerve agents, soman and sarin. Liposomes encapsulating OPAA (SL)* were made by mechanical dispersion method. Hydrolysis of DFP by (SL)* was measured by following an increase of fluoride ion concentration using a fluoride ion selective electrode. OPAA entrapped in the carrier liposomes rapidly hydrolyze DFP, with the rate of DFP hydrolysis directly proportional to the amount of (SL)* added to the solution. Liposomal carriers containing no enzyme did not hydrolyze DFP. The reaction was linear and the rate of hydrolysis was first order in the substrate. This enzyme carrier system serves as a biodegradable protective environment for the recombinant OP-metabolizing enzyme, OPAA, resulting in prolongation of enzymatic concentration in the body. These studies suggest that the protection of OP intoxication can be strikingly enhanced by adding OPAA encapsulated within (SL)* to pralidoxime and atropine.

Antagonism of paraoxon intoxication by recombinant phosphotriesterase encapsulated within sterically stabilized liposomes.

This investigation effort is focused on increasing organophosphate (OP) degradation by phosphotriesterase to antagonize OP intoxication. For these studies, sterically stabilized liposomes encapsulating recombinant phosphotriesterase were employed. This enzyme was obtained from Flavobacterium sp. and was expressed in Escherichia coli. It has a broad substrate specificity, which includes parathion, paraoxon, soman, sarin, diisopropylfluorophosphate, and other organophosphorous compounds. Paraoxon is rapidly hydrolyzed by phosphotriesterase to the less toxic 4-nitrophenol and diethylphosphate. This enzyme was isolated and purified over 1600-fold and subsequently encapsulated within sterically stabilized liposomes (SL). The properties of this encapsulated phosphotriesterase were investigated. When these liposomes containing phosphotriesterase were incubated with paraoxon, it readily degraded the paraoxon. Hydrolysis of paraoxon did not occur when these sterically stabilized liposomes contained no phosphotriesterase. These sterically stabilized liposomes (SL) containing phosphotriesterases (SL)* were employed as a carrier model to antagonize the toxic effects of paraoxon by hydrolyzing it to the less toxic 4-nitrophenol and diethylphosphate. This enzyme-SL complex (SL)* was administered intravenously to mice either alone or in combination with pralidoxime (2-PAM) and/or atropine intraperitoneally. These results indicate that this carrier model system provides a striking enhanced protective effects against the lethal effects of paraoxon. Moreover when these carrier liposomes were administered with 2-PAM and/or atropine, a dramatic enhanced protection was observed.

Ultrastructural characterization of cationic liposome-DNA complexes showing enhanced stability in serum and high transfection activity in vivo.

We have investigated the morphology and transfection activity of cationic liposome-DNA complexes (CLDC) under conditions relevant to both in vivo and in vitro studies. Moreover we have attempted to establish structure-function relationships relevant for high transfection activities under both conditions. CLDC were composed of dimethyldioctadecylammonium bromide with either 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or cholesterol (Chol) interacting either with pre-condensed DNA or with uncondensed plasmid DNA. Furthermore for steric stabilization 1% poly(ethylene glycol)-phospholipid conjugate was added to CLDC containing Chol and plasmid DNA. The in vivo studies were carried out in mice following i.v. injection, and the in vitro studies were performed on SK-BR-3 human breast cancer cells in the presence of media with serum. The morphology of the CLDC, monitored by freeze-fracture electron microscopy, was investigated after mixing with mouse serum or the medium where the cells were kept. The substitution of DOPE with Chol, and the addition of N-[omega-methoxypoly(oxyethylene)-alpha-oxycarbonyl-DSPE+ ++ are producing CLDC which are stabilized with respect to time and serum, and are relatively small (100-300 nm). These stabilized complexes show high expression of a marker gene in mouse lungs reaching expression values up to 10 ng luciferase per mg tissue protein, but relatively low expression in SK-BR-3 cells in vitro. Additionally, some of the complexes containing pre-condensed DNA look like 'map-pin' structures showing heads of the size of liposomes and short, stiff and tapering tails. The in vivo transfection activity of these preparations is highest. Similar complexes containing DOPE rather than Chol as helper lipid precipitate in the presence of serum and especially of cell medium and convert into hexagonal lipid (HII) phase. Such complexes, despite their high transfection activity in vitro, show very little transfection activity in vivo. These comparisons may help us to understand the fundamental difference between in vitro and in vivo activity of CLDC: high in vitro transfection activity seems to be associated with hexagonal lipid precipitates whereas high in vivo activity seems to be related with small, stabilized complexes, which in our case also exhibit some protrusions (map-pin structures).

Cationic liposomes coated with polyethylene glycol as carriers for oligonucleotides.

Modification of liposome surface with polyethylene glycol was used to improve oligodeoxyribonucleotide (ODN) loading, stability of the resulting complexes, and specificity of cellular delivery of ODN by cationic liposomes. Liposomes composed of a cationic lipid (DOTAP, DOGS, DDAB), a neutral lipid (DOPE), and a phospholipid derivative of polyethylene glycol (PEG-PE) formed a complex with 18-mer phosphorothioate up to ODN/lipid molar ratio of 0.25. The complexes showed intact vesicular structures similar to original liposomes and their size (100-130 nm) was unchanged after several weeks of storage, whereas complexes lacking PEG-PE showed progressive aggregation and/or precipitation. After exposure to human plasma, PEG-modified cationic liposomes retained over 60% of the originally bound ODN. PEG-coated complexes resulted in 4-13-fold enhancement of the ODN uptake by human breast cancer cells in serum-supplemented growth medium, relative to free ODN. Complexes containing conjugated anti-HER2 F(ab') fragments at the distal termini of PEG chains efficiently delivered ODN primarily into the cytoplasm and nuclei of HER2 overexpressing cancer cells and greatly enhanced the biological activity of antisense ODN. The development of PEG-modified cationic liposomes may lead to improved ODN potency in vivo.

Inhibition of tracheal vascular extravasation by liposome-encapsulated albuterol in rats.

PURPOSE: To develop a liposome-based system for systemic delivery of anti-inflammatory drugs to airways and other inflamed tissues. METHODS: Postcapillary venular gap junctions open during airway inflammation and allow fluid accumulation and permit molecules (e.g. complement, kininogen) to enter tissues, initiating inflammatory cascades. Beta-adrenergic agonists prevent inflammatory plasma extravasation, but because of their deleterious side effects, they are not used intravenously. When sterically stabilized "stealth" liposomes are injected i.v., they remain in the circulation for long periods. Inflammatory mediators [e.g., substance P(SP)] open postcapillary venular gaps and allow liposomes and their contents to be deposited selectively in the inflamed tissue. RESULTS: We hypothesized that liposomes encapsulating a beta-adrenergic agonist, such as albuterol, would deposit selectively in inflamed airway tissue, where the drug would slowly leak out of the liposomes, resulting in closure of the gaps, thus preventing subsequent inflammatory extravasation. To test this hypothesis, we delivered albuterol-loaded liposomes i.v. in rats. Then we injected SP to open the venular gaps and allow accumulation of the drug-loaded liposomes in airway tissue. We examined whether this treatment resulted in inhibition of subsequent plasma extravasation induced by SP. The results indicate that liposome-encapsulated albuterol inhibits subsequent extravasation, presumably by leaking out of liposomes in airway tissue. This inhibition occurs for prolonged periods of time and with limited side effects compared to the effect of free albuterol. CONCLUSIONS: We conclude that liposomes loaded with appropriate drugs, by migrating to inflamed tissue and subsequently inhibiting inflammatory cascades, may be of therapeutic value in inflammatory diseases.

Copolymers of N-isopropylacrylamide can trigger pH sensitivity to stable liposomes.

Stable liposomes were rendered pH-sensitive by complexation to a polymer that undergoes marked temperature- and pH-dependent water solubility changes. The N-isopropylacrylamide-methacrylic acid copolymer was prepared with or without octadecyl acrylate. At pH below the phase transition of the polymer, egg phosphatidylcholine liposomes quickly released a part of their contents only when associated with the octadecyl aliphatic chain grafted polymer at 37 degrees C. Similarly, sterically stabilized liposomes also quickly released a significant part of the entrapped fluorescent markers at pH 5.5-4.9, values corresponding to those of endosomes/lysosomes. This new pH-sensitive liposome-polymer system may further improve the efficiency of liposomal drug delivery.

Simultaneous measurement of liposome extravasation and content release in tumors.

OBJECTIVE: The success of liposome-based drug delivery systems for tumor targeting relies on maximum extravasation of liposomes into tumor interstitium, as well as optimal release of contents from the liposomes once within the tumor Liposome extravasation and content release are two separate processes that can be individually or jointly manipulated so a method is needed to monitor these two processes independently and simultaneously. In this report, we describe a method to measure liposome extravasation and content release in tumor tissues growing in a rat skinfold window chamber preparation. METHODS: Mixtures of liposomes containing either doxorubicin or calcein, both of which are fluorescent, and liposomes surface-labeled with rhodamine were injected intravenously. Fluorescent, light intensities in a tumor region in two fluorescent channels were measured using an image-processing system. Light intensities of plasma from blood samples were also measured using this system. These measurements were used to calculate the amounts of liposomes and released contents in both plasma and tumor interstitium. The calculations were based on the fact that the liposome surface labels and contents emit fluorescent light at different wavelengths and when encapsulated, the contents fluorescence is self-quenched. The model included equations to account for fluorescent light "cross-contamination" by the two fluorochromes as well as equations relating the measured fluorescent light intensities to the amounts of liposomes and released contents. This method was applied to three situations in which liposome extravasation and content release were manipulated in different, predictable ways. RESULTS AND CONCLUSION: Our results indicate that this method can perform simultaneous independent and quantitative measurements of liposome extravasation and content release. This method can potentially be used to study drug delivery of other carrier systems in vivo.

Sterically stabilized anti-HER2 immunoliposomes: design and targeting to human breast cancer cells in vitro.

Liposomes (70-100 nm) of 1-palmitoyl-2-oleoylphosphatidylcholine, cholesterol, and poly(ethylene glycol) (PEG)-modified phosphatidylethanolamine (PEG-DSPE) were conjugated to Fab' fragments of a humanized recombinant MAb against the extracellular domain of HER2/neu to create sterically stabilized immunoliposomes (anti-HER2 SL) as a drug carrier targeting HER2-overexpressing cancers. Conjugation employed maleimide-terminated membrane-anchored spacers of two kinds: a short spacer, providing attachment of Fab' close to the liposome bilayer, or a long spacer, with Fab' attachment at the distal terminus of the PEG chain. Confocal microscopy and spectrofluorometry of HER2-overexpressing breast cancer cells incubated with fluorescently labeled anti-HER2 SL prepared with either spacer showed binding of liposomes (8000-23000 vesicles/cell) followed by endocytosis (rate constant ke = 0.012-0.033 min-1) via the coated-pit pathway, evidenced by intracellular acidification and colocalization with transferrin. Uptake of anti-HER2 immunoliposomes by breast cancer cells with low HER2 expression, or after preincubation of cells with free anti-HER2 Fab', was less than 0.2% and 4.3%, respectively, of the uptake by HER2-overexpressing cells. Increasing PEG-DSPE content (up to 5.7 mol %) in anti-HER2-SL prepared with the short spacer decreased liposome-cell binding affinity 60-100-fold, while ke decreased only 2-fold; however, when Fab' fragments were conjugated via a PEG spacer, both binding affinity and ke were unaffected by PEG-DSPE content. Cell binding and internalization of anti-HER2 immunoliposomes increased at higher surface density of conjugated Fab' fragments, reaching plateaus at approximately 40 Fab'/liposome for binding and approximately 10-15 Fab'/liposome for internalization. Uptake of anti-HER2 immunoliposomes correlated with the cell surface density of HER2 and significantly (p < 0.005) correlated with the antiproliferative effect of the targeting antibody but not with the total level of cellular HER2 expression. The results obtained were used to optimize in vivo preclinical studies of anti-HER2 SL loaded with antineoplastic drugs.

Stabilization of cationic liposome-plasmid DNA complexes by polyamines and poly(ethylene glycol)-phospholipid conjugates for efficient in vivo gene delivery.

Stable complexes of cationic liposomes with plasmid DNA were prepared by (1) including a small amount of poly(ethylene glycol)-phospholipid conjugate or (2) condensing the DNA with polyamines prior to the formation of liposome-plasmid complexes. These preparations were stable for months at 4 degrees C and gave reproducible high transfection activity for in vivo gene delivery after intravenous injection in mice. Under these conditions, the expression of marker gene (luciferase) was primarily in the lungs (reaching values up to 3 ng expression per mg tissue protein), but also in other tissues to a lesser extent. Non-stabilized formulations lost all their transfection activity in 4 days. In these formulations cholesterol, not dioleoylphosphatidylethanolamine, was the helper lipid effective for sustaining high transfection activity in vivo. These new developments in formulation technology should enhance the potential for liposome-mediated gene therapy.

Anti-HER2 immunoliposomes for targeted therapy of human tumors.

Anti-HER2 immunoliposomes (ILs) have been constructed by conjugation of Fab' fragments of recombinant humanized monoclonal antibody rhuMAbHER2 to small sterically stabilized unilamellar liposomes, to create a targeted drug delivery vehicle for the treatment of HER2 (c-erbB-2, neu)-overexpressing cancers. Parameters affecting in vitro binding and internalization of ILs include liposome composition, Fab' linkage site and Fab' density. Anti-HER2 ILs have been constructed to optimize intracellular drug delivery. Doxorubicin (dox)-loaded ILs are highly stable and exhibit prolonged circulation in rats. In nude mice bearing HER2-overexpressing tumor xenografts, anti-HER2 ILs administered i.v. resulted in efficient tumor localization, with penetration of the ILs throughout the tumor mass and accumulation within tumor cells. In contrast, non-targeted liposomes resulted in extracellular tumor accumulation only. In multiple HER2-overexpressing human breast tumor xenograft models, treatment with dox-loaded anti-HER2 ILs produces significantly increased antitumor cytotoxicity as compared to free dox or dox-loaded non-targeted liposomes and significantly less systemic toxicity than free dox. To explore further the intracellular delivery advantages of ILs, anti-HER2 ILs bearing cationic lipids are being developed for nucleic acid delivery. These cationic immunoliposomes mediate efficient and specific transfection of target cells with reporter genes, as well as intracellular delivery of labeled oligonucleotides. Thus, anti-HER2 ILs represent an efficient and feasible strategy to achieve targeted intracellular delivery of therapeutic agents.

Fluctuations in red cell flux in tumor microvessels can lead to transient hypoxia and reoxygenation in tumor parenchyma.

Hypoxia occurs in two forms in tumors. Chronic or diffusion-limited hypoxia is relatively well characterized. In contrast, intermittent or perfusion-limited hypoxia is not well characterized, and it is not known how common it is in tumors. The purpose of this study was to determine whether spontaneous fluctuations in tumor microvessel flow rate can modify vessel oxygen tension (pO2) sufficiently to cause intermittent hypoxia (IH; tissue pO2 < 3 mmHg) in the tumor parenchyma supplied by such vessels. Microvessel red cell flux (RCF) and perivascular pO2 were measured simultaneously and continuously in dorsal flap window chambers of Fischer-344 rats with implanted R3230Ac tumors. In all vessels, RCF was unstable, with apex/nadir ratios ranging from 1.5 to 10. RCF and pO2 were temporally coordinated, and there were linear relationships between the two parameters. Vascular pO2 was less sensitive to changes in RCF in well-vascularized tumor regions compared with poorly vascularized regions. Simulations of oxygen transport in a well-vascularized region of a tumor demonstrated that two-fold variations in RCF can produce IH in 30% of the tissue in that region. In poorly vascularized regions, such fluctuations would lead to an even greater percentage of tissue involved in transient hypoxia. These results suggest that IH is a relatively common phenomenon. It could affect binding of hypoxic cytotoxins to tumor cells, in addition to being an important source of treatment resistance. Intermittent hypoxia also could contribute to tumor progression by providing repeated exposure of tumor cells to hypoxia-reoxygenation injury.

Thermosensitive liposomes: extravasation and release of contents in tumor microvascular networks.

PURPOSE: The purpose of this study was to determine whether hyperthermic exposure would accelerate drug release from thermosensitive sterically stabilized liposomes and enhance their extravasation in tumor tissues. MATERIALS AND METHODS: In vivo fluorescence video microscopy was used to measure the extravasation of liposomes, as well as release of their contents, in a rat skin flap window chamber containing a vascularized mammary adenocarcinoma under defined thermal conditions (34 degrees, 42 degrees, and 45 degrees C). Images of tissue areas containing multiple blood vessels were recorded via a SIT camera immediately before, and for up to 2 h after i.v. injection of two liposome populations with identical lipid composition: one liposome preparation was surface labeled with Rhodamine-PE (Rh-PE) and the other contained either Doxorubicin (Dox) or calcein at self-quenching concentrations. The light intensity of the entire tissue area was measured at 34 degrees C (the physiological temperature of the skin) for 1 h, and at 42 degrees or 45 degrees C for a second hour. These measurements were then used to calculate the fluorescent light intensity arising from each tracer (liposome surface label and the released contents) inside the vessel and in the interstitial region. RESULTS: The calculated intensity of Rh-PE for the thermosensitive liposomes in the interstitial space (which represents the amount of extravasated liposomes) was low during the first hour, while temperature was maintained at 34 degrees C and increased to 47 times its level before heating, when the tumor was heated at 42 degrees or 45 degrees C for 1 h. The calculated intensity of the liposome contents (Dox) in the interstitial space was negligible at 34 degrees C, and increased by 38- and 76-fold, when the tumor was heated at 42 degrees and 45 degrees C for 1 h, respectively. Similar values were obtained when calcein was encapsulated in liposomes instead of Dox. A similar increase in liposome extravasation was seen with nonthermosensitive liposomes, but negligible release of Dox occurred when the window chamber was heated to 45 degrees C for 1 h. Extravasation of liposomes continued after heating was stopped, but content release stopped after removal of heat. Release of Dox from extravasated liposomes was also seen if heating was applied 24 h after liposome administration, but no further enhancement of liposome extravasation occurred in this case. CONCLUSIONS: Our data suggest that hyperthermia can be used to selectively enhance both the delivery and the rate of release of drugs from thermosensitive liposomes to targeted tissues.

Microvascular studies on the origins of perfusion-limited hypoxia.

Two forms of hypoxia are thought to exist in tumours: (1) hypoxia caused by limitations of its diffusion (chronic hypoxia); and (2) hypoxia caused by changes in perfusion (acute hypoxia). Indirect information suggests the existence of perfusion-limited hypoxia, but there is no direct proof that fluctuations in blood flow can lead to hypoxia, nor is there any information regarding potential causes of fluctuant flow. In this study, we have begun to explore these questions using R3230AC tumours transplanted into rat dorsal-flap window chambers. Two types of fluctuant flow have been observed. The first type, usually confined to single vessels, is typified by instability of flow magnitude and direction, and total vascular stasis occurs, but only for a few seconds at a time (4% incidence). The second type of fluctuation occurs in groups of vessels and is cyclic, with cycle times ranging from 20-60 min. Total vascular stasis does not necessarily occur, but the fluctuations in red cell flux are accompanied by changes in vascular oxygen content, as measured by microelectrodes. Another source of chronic hypoxia has also been identified in these experiments. Nine per cent (9%) of vessels examined had plasma flow, but very low or absent red cell flux over periods of many minutes.