Targeted drug delivery: a two-compartment growth inhibition assay demonstrates that fluorodeoxyuridine and fluorodeoxyuridine monophosphate are liposome-independent drugs

The targeted delivery to cells by liposomes and leakage under delivery conditions of fluorodeoxyuridine (FdUR) and fluorodeoxyuridine monophosphate (FdUMP) have been evaluated using a two-compartment growth inhibition assay. Under cell culture conditions, FdUR leaks 100% from all liposomes regardless of charge or phase transition temperature. Under the same conditions, FdUMP leaks 100% from egg yolk phosphatidylglycerol liposomes, 47% from distearoylphosphatidylglycerol liposomes, 44% from egg yolk phosphatidylcholine liposomes, and 10% from distearoylphosphatidylcholine liposomes. All liposomes were prepared from a 67:33 mixture of phospholipid and cholesterol. The two-compartment assay demonstrates directly that neither of these drugs is delivered selectively to the target cells by the liposomes, suggesting that they are liposome independent drugs.     

Thermosensitive liposomes: Extravasation and release of contents in tumor microvascular networks

: 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.

: 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°, 42°, and 45°C). Images of tissue areas containing multiple blood vessels were recorded via a SIT camera immediately before, and for upto 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°C (the physiological temperature of the skin) for 1 h, and at 42° or 45°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.

: The calculated intensity of Rh-PE for the thermosensitive liposomes in the interstitial space (which represents the amout of extravasated liposomes) was low during the first hour, while temperature was maintained at 34°C and increased to 47 times its level before heating, when the tumor was heated at 42° or 45° C for 1 h. The calculated intensity of the liposome contents (Dox) in the interstitial space was negligible at 34°C, and increased by 38- and 76-fold, when the tumor was heated at 42° and 45° 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°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.

: 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.     

Enhancement of hyperthermic damage on M14 melanoma cells by liposome pretreatment

Exponentially growing human melanoma cells (M14 cell line) were pretreated with various amounts of dipalmitoylphosphatidylcholine-containing multilamellar liposomes and then exposed to heat treatment (42.5 degrees C). Cell damage produced by the treatments, given separately or in combination, was evaluated in terms of cell survival. Our results demonstrate that the cell survival at 37 degrees C was not affected by liposome concentrations up to 1000 nmol of phospholipid/2.5 x 10(6) cells, while liposome treatment of cells before heat exposure determined a marked damaging effect even at 100 nmol of phospholipid/2.5 x 10(6) cells. The mechanisms of liposome-cell interaction have been investigated by electron microscopy or by electron spin resonance measurements of spin-labeled membranes of intact cells. Evidence has been obtained that liposomal lipids are either taken up by M14 cells or become incorporated in the cell membrane. The present data suggest the possibility that liposome treatments per se could be of potential value as a therapeutic approach, by increasing the effect of heat therapy.     

Effect of liposome composition and other factors on the targeting of liposomes to experimental tumors: biodistribution and imaging studies.

We have examined the distribution of radiolabeled liposomes in tumor-bearing mice after i.v. injection. Two mouse tumors (B16 melanoma, J6456 lymphoma) and a human tumor (LS174T colon carcinoma) inoculated i.m., s.c., or in the hind footpad were used in these studies. When various liposome compositions with a mean vesicle diameter of approximately 100 nm were compared using a radiolabel of gallium-67-deferoxamine, optimal tumor localization was obtained with liposomes containing a phosphatidylcholine of high phase-transition temperature and a small molar fraction of monosialoganglioside or hydrogenated phosphatidylinositol (HPI). At 24 h after injection, average values of tumor uptake higher than 10% of the injected dose per g and liver-to-tumor ratios close to 1 were reproducibly obtained. Increasing the molar fraction of HPI from 9% to 41% of the total phospholipid resulted in enhancement of liver uptake and decrease of tumor uptake. Methodological aspects that influence vesicle size appear to affect significantly liposome localization in the tumor. However, varying the phospholipid dose within a 10-fold range caused only minor changes in the percent of injected dose recovered in the tumor. A high uptake by tumors was also observed using other radiolabels [[3H]inulin and indium-111-labeled bleomycin (111In-Bleo)] in monosialoganglioside- and HPI-containing liposomes. In the case of 111In-Bleo, encapsulation in liposomes resulted in approximately 20- to 40-fold increase in tumor accumulation of the radiolabel at 24 h after injection. The marked localization of liposomes in the mouse footpad inoculated with tumor as opposed to the contralateral mock-injected footpad was also documented by imaging experiments with gallium-67-deferoxamine and 111In-Bleo-labeled liposomes. These results support the contention that some glycolipid-containing liposomes previously shown to have long circulating half-lives accumulate significantly in a variety of tumors and are promising tools for the delivery of anti-tumor agents.     

Effect of vesicle size and lipid composition on the in vivo tumor selectivity and toxicity of the non-cross-resistant anthracycline annamycin incorporated in liposomes

Annamycin (Ann) is a non-cross-resistant lipophilic anthracy-cline antibiotic optimally suited for liposome delivery. We studied how vesicle size and presence of phospholipids with a high phase transition temperature and monosialoganglioside (GM1) in the liposome bilayers affect the pharmacokinetics, tumor selectivity and toxicity of Ann. Entrapment of Ann in multilamellar vesicles (L-Ann) resulted in a 20% lower heart AUC and a 30–40% higher tumor and liver AUC. Reduction of the liposome size from 1.6 to 0.03 μm increased Ann plasma circulation time and tumor AUC by 2-fold, enhanced Ann tumor selectivity and decreased Ann subacute toxicity by 2-fold. The presence of phospholipids with a high phase transition temperature and GM1 in the liposome bilayers further prolonged Ann plasma circulation time by 2- to 4-fold, did not increase Ann tumor AUC and moderately increased Ann subacute toxicity. The anti-tumor activity of Ann correlated with the tumor AUC achieved with each particular formulation. Our results strongly suggest that vesicle size may be an important determinant of the therapeutic index of liposomal Ann, but they fail to demonstrate a beneficial tumor-targeting effect of liposomes composed of GM1 and phospholipids with a high phase transition temperature, as has been reported for the hydrophilic parent compound doxorubicin. © 1995 Wiley-Liss, Inc.     

Influence of lipid composition on the antitumor activity exerted by doxorubicin-containing liposomes in a rat solid tumor model

The effect of changes in lipid composition on the antitumor activity of doxorubicin (DXR)-containing liposomes was studied in immunoglobulin solid immunocytoma-bearing Lou/M Wsl rats. Rats bearing a tumor with a diameter between 20 and 30 mm were treated i.v. with 2 mg/kg free DXR or different DXR-containing liposome types for 5 consecutive days followed by one injection more at day 11 after start of therapy. A similar pattern of tumor regression was observed for free DXR and DXR entrapped in "fluid" liposome types. However, DXR entrapped in "solid" liposome types expressed an antitumor activity which was significantly delayed; during the first 3 days after start of therapy solid DXR-containing liposomes were less effective in inducing antitumor activity than fluid DXR-containing liposomes. In order to gain more insight into the mode of action of DXR-containing liposomes, one of the solid liposome types [composed of distearoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, and cholesterol (chol)] was compared with one of the fluid liposome types [composed of egg phosphatidylcholine, phosphatidylserine, and chol] with respect to distribution and integrity in vivo. Results obtained after i.v. administration of [3H]inulin-labeled vesicles to tumor-bearing animals suggested that a differential liposome uptake by the tumor was not relevant for the explanation of the delayed antitumor effect. To monitor the structural integrity of liposomes after i.v. injection, the liposomes were double radiolabeled with [3H]inulin as a marker of the aqueous phase and cholesteryl [14C]oleate as a marker of the lipid phase. The bilayer structure of both liposome types remained intact during their presence in the blood compartment. Intact liposomes were taken up primarily by liver and spleen with subsequent degradation of the liposome structure. The degradation rate appeared to be dependent on the lipid composition of the liposomal membranes; phosphatidylcholine/phosphatidylserine/chol liposomes were degraded much faster than distearoylphosphatidylcholine/dipalmitoylphosphatidylglycerol/chol liposomes. The difference in degradation rate was manifested more clearly in the spleen than in the liver. In vitro investigations on uptake and processing of liposomes by liver macrophages indicated that the difference in degradation rate between liver and spleen was caused by intrahepatic reutilization of [14C]oleate liberated from the liposome structures.(ABSTRACT TRUNCATED AT 400 WORDS)     

Efficacy of liposomes and hyperthermia in a human tumor xenograft model: importance of triggered drug release

The tumor drug concentrations, drug distributions, and therapeutic efficacies achieved by three fundamentally different liposomes, nonthermosensitive liposome (NTSL), traditional thermosensitive liposome (TTSL), and low temperature sensitive liposome (LTSL); free doxorubicin (DOX); and saline in combination with hyperthermia (HT) were directly compared in a human tumor xenograft model. NTSL is a nonthermosensitive liposome in the physiological temperature range, TTSL is a traditional thermosensitive liposome that triggers in the range of approximately 42-45 degrees C and releases drug over approximately 30 min, and LTSL is a new low temperature sensitive liposome that triggers in the range of approximately 39-40 degrees C and releases drug in a matter of seconds. Because of the different attributes of the liposomes, it was possible to delineate the relative importance of liposome drug encapsulation, HT cytotoxicity, HT-drug interaction, HT-induced liposomal delivery, and HT-triggered liposomal drug release in achieving antitumor activity. Athymic nude mice bearing the FaDu human tumor xenograft were given a single i.v. dose of 5 mg/kg of DOX (free drug or liposome encapsulated), and the tumors were then heated to either 34 degrees C or 42 degrees C for 1 h at 34 degrees C. All treatment groups were similar, achieving low concentrations of DOX (0-4.5 ng/mg). At 42 degrees C, the LTSL (25.6 ng/mg) achieved the highest DOX concentration (P < 0.04), but all three liposomal formulations (7.3-25.6 ng/mg) were higher than saline or DOX (0-0.7 ng/mg; P < 0.02). LTSL + HT was also the only group that resulted in significant amounts of DNA-bound DOX (silver nitrate-extractable fraction; P < 0.02). Tumor tissue sections were visualized for DOX fluorescence to investigate the local distribution of the drug in the tumor and confirm the relative drug concentrations based on fluorescence intensity. There was relatively little fluorescence seen with treatment groups at 34 degrees C. At 42 degrees C, the LTSL showed the most DOX fluorescence (P < 0.01), and the fluorescence, although not homogeneous, was pervasive throughout the tumor sections. Therapeutic efficacy of treatments was determined from tumor growth time. At 34 degrees C, the only treatment group significantly better than the saline group (9.8 days) was the NTSL group, with a growth time of 20.9 days (P < 0.02). At 42 degrees C, all three liposomal formulations were more efficacious than DOX. LTSL + HT had the longest growth time (51.4 days) and the most number of local controls at 60 days (six of nine tumors). With HT, the DOX concentrations and fluorescence were tightly correlated with tumor growth delay, indicating that adequate (increased) drug delivery can be predictive of therapeutic effect. Overall, the LTSL + HT group showed the largest DOX concentration, the highest and most pervasive DOX fluorescence, and the most antitumor effect. Thus, HT-triggered liposomal drug release may account for the largest differential therapeutic effect and demonstrates the importance of rapid drug release from the drug carriers at the tumor site.     

Targeting of stealth liposomes to erbB-2 (Her/2) receptor: in vitro and in vivo studies.

Long-circulating (stealth) liposomes coated with polyethylene glycol (PEG), which show reduced uptake by the reticuloendothelial system (RES) and enhanced accumulation in tumours, were used for conjugation to monoclonal antibodies (MAbs) as a drug-targeting device. A MAb (N-12A5) directed against erbB-2 oncoprotein, a functional surface antigen, was used. Amplification and overexpression of the erbB-2 gene product, being unique to malignancy, confer onto this antibody-mediated therapy high tumour specificity. In vitro binding of [3H]cholesteryl ether ([3H]Chol ether) labelled anti-erbB-2 conjugated liposomes to N-87 cells (erbB-2-positive human gastric carcinoma) was compared with the binding of non-targeted liposomes and indicated a 16-fold increase in binding for the targeted liposomes. No difference in binding to OV1063 cells (erbB-2-negative human ovary carcinoma) was observed. These results indicate highly selective binding of antibody-targeted liposomes to erbB-2-overexpressing cells. Despite increased cell binding, doxorubicin (DOX) loaded in anti-erbB-2-conjugated liposomes did not cause increased in vitro cytotoxicity against N-87 cells, suggesting lack of liposome internalisation. In vivo, the critical factor needed to decrease the non-specific RES uptake and prolong the circulation time of antibody-conjugated liposomes is a low protein to phospholipid ratio ( < 60 micrograms mumol-1). Using these optimised liposome preparations loaded with DOX and by monitoring the drug levels and the [3H]Chol ether label, biodistribution studies in nude mice bearing subcutaneous implants of N-87 tumours were carried out. No significant differences in liver and spleen uptake between antibody-conjugated and plain liposomes were observed. Nevertheless, there was no enhancement of tumour liposome levels over plain liposomes. Both liposome preparations considerably enhanced DOX concentration in the tumour compared with free drug administration. Therapeutic experiments with N-87 tumour-bearing nude mice indicated that anti-tumour activity of targeted and non-targeted liposomes was similar, although both preparations had an increased therapeutic efficacy compared with the free drug. These studies suggest that efficacy is dependent on drug delivery to the tumour and that the rate-limiting factor of liposome accumulation in tumours is the liposome extravasation process, irrespective of liposome affinity or targeting to tumour cells.     

Hyperthermia increases accumulation of technetium-99m-labeled liposomes in feline sarcomas.

The effect of hyperthermia on the accumulation of technetium-99m-labeled liposomes was studied in feline sarcomas. Each cat received two separate injections of liposomes. The first was used to quantify the amount of technetium-99m-labeled liposomes within the tumor under normothermic conditions. The second injection was made at the beginning of a 60-min hyperthermia procedure. Planar scintigraphy was used to measure the activity of technetium-99m-labeled liposomes within the tumor at predetermined times up to 18 h after injection. Regions of interest were drawn for the tumor, lungs, liver, kidney, and aorta. Counts in the regions of interest were decay corrected. Counts/pixel in the tumor under normothermic and hyperthermic conditions were normalized to aorta counts/pixel. A total of 16 cats were eligible for the study. In two of the 16 cats, incomplete count data precluded analysis. In the remaining 14 cats, hyperthermia resulted in a significant increase in liposome accumulation in the tumor (P = 0.001). Tumor volume ranged from 1.2 to 236.2 cm3, and thermal dose ranged from 2.0 to 243.3 CEM43CT90 (equivalent time that the 10th percentile temperature was equal to 43 degrees C). There was not a relationship between either tumor volume or hyperthermia dose on the magnitude of increased liposome accumulation, suggesting that this method has application across a range of tumor volumes and degrees of heatibility.     

Intravenous infusion of high doses of liposomes containing NSC 251635, a water-insoluble cytostatic agent. A pilot study with pharmacokinetic data

In patients with resistant malignant tumors, we performed a pilot trial of intravenous infusion of a water-insoluble cytostatic agent, NSC 251635, entrapped in large volumes of liposomes made of egg yolk lecithin, cholesterol, and stearylamine (4:3:1). Forty liposome infusions were given to 14 patients in 38 courses. The volume of liposomes (20 mg of lipids/mL) varied from 205 to 1,000 mL or 124 to 617 mL/m2 of body surface, and amounts of NSC 251635 varied from 82 to 456 mg/m2. Three patients received repeated single courses. Liposomal therapy was very well tolerated. Side effects observed during some infusions were mild sedation, fever, chills, lumbar pain, urticarial rash, and bronchospasm. In all patients investigated, an important activation of the complement system was observed. No objective regression of the tumors was observed. The limiting factor in the phase I study was not toxicity but the volume of liposomes that could be prepared at once because of the long time required for its preparation. Pharmacokinetic data showed that maximal serum phospholipid and NSC 251635 concentrations were obtained at the end of the liposome infusion. The drug's peak was followed by a decreasing phase leading to a kind of plateau and a prolonged presence of the drug in the blood until 120 hours after its administration. Comparison of the pharmacokinetics of phospholipids and NSC 251635 suggests a rather rapid dissociation of the drug from the liposome.     

Application of novel drug delivery system, fusogenic liposome, for cancer therapy

Over the past decade, many studies concerning novel anti-cancer therapies have been reported. It has been occasionally noted that a powerful anti-cancer drug, especially one whose target is the cytoplasm or cell nucleus, does not work due to the low permeability across a plasma membrane, degradation by lysosomal enzymes through an endocytosis-dependent pathway, and other reasons. Thus, several approaches using drug delivery systems (DDS) are focused on overcoming these difficulties, eventually leading to the induction of maximal ability of anti-cancer drug. In this respect, we have developed a new paradigm for cancer therapy using a novel drug delivery system, fusogenic liposome. Fusogenic liposomes are composed of the ultraviolet-inactivated Sendai virus and conventional liposomes. Fusogenic liposomes effectively and directly deliver their encapsulated contents into the cytoplasm using a fusion mechanism of the Sendai virus, whereas conventional liposomes are taken up by endocytosis. Thus, fusogenic liposome is a good candidate as a vehicle to deliver drugs into the cytoplasm in an endocytosis-independent manner. In this report, we show the feasibility of fusogenic liposome as a delivery vehicle for anti-cancer drugs using a fragment A of diphtheria toxin as an anti-cancer reagent. We also demonstrate the application of fusogenic liposome for cancer gene therapy and cancer vaccines using a TNF-alpha-expression plasmid and a chicken egg ovalbumin, respectively.     

From conventional to stealth liposomes: a new frontier in cancer chemotherapy

Many attempts have been made to achieve good selectivity to targeted tumor cells by preparing specialized carrier agents that are therapeutically profitable for anticancer therapy. Among these, liposomes are the most studied colloidal particles thus far applied in medicine and in particular in antitumor therapy. Although they were first described in the 1960s, only at the beginning of 1990s did the first therapeutic liposomes appear on the market. The first-generation liposomes (conventional liposomes) comprised a liposome-containing amphotericin B, Ambisome (Nexstar, Boulder, CO, USA), used as an antifungal drug, and Myocet (Elan Pharma Int, Princeton, NJ, USA), a doxorubicin-containing liposome, used in clinical trials to treat metastatic breast cancer. The second-generation liposomes ("pure lipid approach") were long-circulating liposomes, such as Daunoxome, a daunorubicin-containing liposome approved in the US and Europe to treat AIDS-related Kaposi's sarcoma. The third-generation liposomes were surface-modified liposomes with gangliosides or sialic acid, which can evade the immune system responsible for removing liposomes from circulation. The fourth-generation liposomes, pegylated liposomal doxorubicin, were called "stealth liposomes" because of their ability to evade interception by the immune system, in the same way as the stealth bomber was able to evade radar. Actually, the only stealth liposome on the market is Caelyx/Doxil (Schering-Plough, Madison NJ, USA), used to cure AIDS-related Kaposi's sarcoma, resistant ovarian cancer and metastatic breast cancer. Pegylated liposomal doxorubicin is characterized by a very long-circulation half-life, favorable pharmacokinetic behavior and specific accumulation in tumor tissues. These features account for the much lower toxicity shown by Caelyx in comparison to free doxorubicin, in terms of cardiotoxicity, vesicant effects, nausea, vomiting and alopecia. Pegylated liposomal doxorubicin also appeared to be less myelotoxic than doxorubicin. Typical forms of toxicity associated to it are acute infusion reaction, mucositis and palmar plantar erythrodysesthesia, which occur especially at high doses or short dosing intervals. Active and cell targeted liposomes can be obtained by attaching some antigen-directed monoclonal antibodies (Moab or Moab fragments) or small proteins and molecules (folate, epidermal growth factor, transferrin) to the distal end of polyethylene glycol in pegylated liposomal doxorubicin. The most promising therapeutic application of liposomes is as non-viral vector agents in gene therapy, characterized by the use of cationic phospholipids complexed with the negatively charged DNA plasmid. The use of liposome formulations in local-regional anticancer therapy is also discussed. Finally, pegylated liposomal doxorubicin containing radionuclides are used in clinical trials as tumor-imaging agents or in positron emission tomography.     

Stealth liposomes: an improved sustained release system for 1-beta-D-arabinofuranosylcytosine.

Newly developed liposomes with prolonged circulation half-lives and dose-independent pharmacokinetics (Stealth liposomes) have been tested for their efficacy as a slow release system for the rapidly degraded, schedule-dependent, antineoplastic drug 1-beta-D-arabinofuranosylcytosine (ara-C) in the treatment of murine L1210/C2 leukemia. Mice were given injections of either 10(5) cells or 10(6) cells by either the i.v. or the i.p. routes. Leukemia-bearing mice were treated with either i.v. or i.p. injections of free drug, i.v. or i.p. injections of liposome-entrapped drug, or 24-h i.v. infusions of free drug. Long-circulating liposomes contained, as the stealth component, either monosialoganglioside or polyethylene glycol-distearoylphosphatidylethanolamine. Liposomes lacking the stealth components (non-stealth liposomes) were also injected for comparison. At lower dose ranges, stealth liposomes were superior to non-stealth liposomes in prolonging mean survival times of the mice, and all liposome preparations were superior to injections of the free drug. Drug entrapped in stealth liposomes, when administered at or near the maximum tolerated dose of 100 mg/kg ara-C were considerably superior to 24-h free drug infusions given at the same total drug dose. Therapeutic effect was related to the half-life of leakage of ara-C from the liposome formulations, as well as to circulation half-life, with maximum therapeutic effect achieved with long circulation half-lives and more rapid leakage rates. The therapeutic efficacy of non-stealth liposomes increased with increasing liposome (and drug) dose as a result of saturation of liposome uptake by the mononuclear phagocyte system, which resulted in longer circulation half-lives for these liposomes at higher doses (Michaelis-Menten pharmacokinetics). Liposome entrapment can protect rapidly degraded drugs from breakdown in vivo, with release of the drugs in a therapeutically active form over periods of up to several days. The dose-independent pharmacokinetics and reduced mononuclear phagocyte system uptake of stealth liposomes gives them distinct advantages over non-stealth liposomes.     

Reduced thermal sensitivity of the vasculature in a slowly growing tumour

The thermal sensitivity of a slowly growing murine mammary carcinoma has been studied, and correlated with several vascular parameters. This tumour, CaRH, had previously been shown to be particularly resistant to hyperthermia when applied as 1 h immersion in a 42.8°C water bath, with or without added X-rays. In the present study water bath temperatures of 43, 44 and 45°C were used as well as a more complex system of water bath plus radiofrequency currents which produced a uniform intratumour temperature of 43 °C. Following treatment the tumour blood volume and relative capillary perfusion were estimated using radioactive tracer techniques. Only the treatments which gave at least 60 per cent reduction in blood perfusion yielded significant growth delays or thermal radiosensitization. These data have been compared with values for six other murine tumours. If, instead of comparing the exposure temperature, we compare the blood flow reduction, it is seen that all of the tumours are similar in their thermosensitivity. A higher temperature may be needed to cause vascular shutdown in more slowly growing tumours, but it is achieved with an intratumour temperature of 43 °C for 1 h. This may correlate with endothelial cell proliferation rates, which are similar in CaRH to the values measured in human tumours. The more rapid vascular expansion of the fast-growing tumours may result in a more fragile and thermosensitive capillary network. The hypoxic fraction, which is a measure of vascular inadequacy, also correlates with thermal sensitivity.     

Reduced thermal sensitivity of the vasculature in a slowly growing tumour

The thermal sensitivity of a slowly growing murine mammary carcinoma has been studied, and correlated with several vascular parameters. This tumour, CaRH, had previously been shown to be particularly resistant to hyperthermia when applied as 1 h immersion in a 42.8 degrees C water bath, with or without added X-rays. In the present study water bath temperatures of 43, 44 and 45 degrees C were used as well as a more complex system of water bath plus radiofrequency currents which produced a uniform intratumour temperature of 43 degrees C. Following treatment the tumour blood volume and relative capillary perfusion were estimated using radioactive tracer techniques. Only the treatments which gave at least 60 per cent reduction in blood perfusion yielded significant growth delays or thermal radiosensitization. These data have been compared with values for six other murine tumours. If, instead of comparing the exposure temperature, we compare the blood flow reduction, it is seen that all of the tumours are similar in their thermosensitivity. A higher temperature may be needed to cause vascular shutdown in more slowly growing tumours, but it is achieved with an intratumour temperature of 43 degrees C for 1 h. This may correlate with endothelial cell proliferation rates, which are similar in CaRH to the values measured in human tumours. The more rapid vascular expansion of the fast-growing tumours may result in a more fragile and thermosensitive capillary network. The hypoxic fraction, which is a measure of vascular inadequacy, also correlates with thermal sensitivity.     

Targeting Chemotherapy to Solid Tumors with Long-circulating Thermosensitive Liposomes and Local Hyperthermia

The effectiveness of the combination of long-circulating, thermosensitive liposomes and hyperthermia is described. Small-sized, thermosensitive liposomes that encapsulate doxorubicin (DXR-PEGTSL (SUV)) have a prolonged circulation time and are extravasated to targeted solid tumors in vivo , where they preferentially release the agent in an anatomical site subjected to local hyperthermia. Liposomes were prepared by the incorporation of amphipathic polyethyleneglycol (PEG) to prolong their circulation time. DXR-PEG-TSL (SUV) was retained longest and was accumulated most efficiently in solid tumors in Balb/c mice. The combination of DXR-PEG-TSL (SUV) and hyperthermia at the tumor sites 3 h after injection, gave high concentrations of doxorubicin in tumor tissue and resulted in more effective tumor retardation and increased survival time. A large amount of DXR-PEG-TSL (SUV) was extravasated into the tumors during circulation for 3 h after injection, suggesting that the encapsulated drug was released into the interstitial spaces of the lesions by local hyperthermia. This system is expected to be clinically valuable for the delivery of a wide range of chemotherapeutic agents in the treatment of solid tumors.     

Liposome induction or enhancement of macrophage-mediated cancer cell lysis

Liposomes of different composition have been used to modify macrophage-mediated destruction of syngeneic cancer cells through a modulation of membrane lipid content of macrophages and/or tumor cells. Dipalmitoylphos-phatidylcholine (DPPC)¹ liposomes induce cancer cell lysis by normal, non-tumoricidal, peritoneal macrophages and enhance tumor cell destruction by BCG-activated macrophages. This effect was produced by large and small unilamellar liposomes, which are in the 25,000 g supernatant of sonicated preparations. Addition of cholesterol or negative charges carried by dicetylphosphate supressed the effect of DPPC liposomes on macrophage-mediated cytolysis. Enhancement of macrophage-mediated tumor cell lysis was observed when both cancer cells and macrophages were incubated with DPPC liposomes, but not when macrophages and/or tumor cells were preincubated with liposomes prior to their coincubation. Liposomes did not promote the binding of the cancer cells to the macrophages. Liposomes could promote formation of phospholipid domains within the plasma membrane of both tumor cells and macrophages leading to the destruction of cancer cells through a temporary fusion with the macrophages.     

Introductory experiments on ligand liposomes as delivery agents for boron neutron capture therapy

Liposomes are, when coupled to receptor ligands, candidates for receptor mediated delivery of boron for tumour therapy since they have capacity to deliver large amounts of boron per receptor interaction. With EGF-liposomes we present a pegylated ligand liposome delivery vehicle, containing water soluble boronated phenanthridine, WSP1, or water soluble boronated acridine, WSA1, for EGFR targeting. In the case of WSA1 a ligand dependent uptake was obtained and the boron uptake was as good as if free WSA1 was given. No ligand dependent boron uptake was seen for WSP1 containing liposomes. Thus, WSA1 is a candidate for further studies. Approximately 10(5) boron atoms were in each liposome. A critical assessment indicates that after optimization up to 10(6) boron atoms can be loaded. Since it is known that, for therapeutic effect, approximately 10(8)-10(9) boron atoms are needed in a single tumour cell it is realized that 10(2)-10(3) receptor interactions are needed to meet the demand. Tests applying cultured glioma cells indicate, without optimization of the delivery conditions, a boron uptake in the ppm range, which is necessary for successful BNCT. Thus, it seems possible to kill micro-invasive tumour cells with targeted liposomes if the delivery conditions are optimal.     

Targeting chemotherapy to solid tumors with long-circulating thermosensitive liposomes and local hyperthermia.

The effectiveness of the combination of long-circulating, thermosensitive liposomes and hyperthermia is described. Small-sized, thermosensitive liposomes that encapsulate doxorubicin (DXR-PEG-TSL (SUV)) have a prolonged circulation time and are extravasated to targeted solid tumors in vivo, where they preferentially release the agent in an anatomical site subjected to local hyperthermia. Liposomes were prepared by the incorporation of amphipathic polyethyleneglycol (PEG) to prolong their circulation time. DXR-PEG-TSL (SUV) was retained longest and was accumulated most efficiently in solid tumors in Balb/c mice. The combination of DXR-PEG-TSL (SUV) and hyperthermia at the tumor sites 3 h after injection, gave high concentrations of doxorubicin in tumor tissue and resulted in more effective tumor retardation and increased survival time. A large amount of DXR-PEG-TSL (SUV) was extravasated into the tumors during circulation for 3 h after injection, suggesting that the encapsulated drug was released into the interstitial spaces of the lesions by local hyperthermia. This system is expected to be clinically valuable for the delivery of a wide range of chemotherapeutic agents in the treatment of solid tumors.     

Tumor regression in mice by delivery of Bcl-2 small interfering RNA with pegylated cationic liposomes

The pharmacokinetics and antitumor activity of pegylated small interfering RNA (siRNA)/cationic liposome complexes were studied after systemic administration to mice. We designed pegylated-lipid carriers for achieving increased plasma concentrations of RNA and hence improved accumulation of RNA in tumors by the enhanced permeability and retention effect. We compared the pharmacokinetics of siRNA complexed with liposomes incorporating pegylated lipids with longer (C-17 or C-18), shorter (C-12 to C-16), or unsaturated (C-18:1) acyl chains. When longer acyl chains were used, the plasma concentrations of siRNA obtained were dramatically higher than when shorter or unsaturated chains were used. This may be explained by the higher gel-to-liquid-crystalline phase-transition temperature (Tc) of lipids with longer acyl chains, which may form more rigid liposomes with reduced uptake by the liver. We tested a siRNA that is sequence specific for the antiapoptotic bcl-2 mRNA complexed with a pegylated liposome incorporating a C-18 lipid (PEG-LIC) by i.v. administration in a mouse model of human prostate cancer. Three-fold higher accumulation of RNA in the tumors was achieved when PEG-LIC rather than nonpegylated liposomes was used, and sequence-specific antitumor activity was observed. Our siRNA/PEG-LIC complex showed no side effects on repeated administration and the strength of its antitumor activity may be attributed to its high uptake by the tumors. Pegylation of liposomes improved the plasma retention, uptake by s.c. tumors, and antitumor activity of the encapsulated siRNA. PEG-LIC is a promising candidate for siRNA cancer therapy.