Distearoylphosphatidylethanolamine Research Articles

Assessment of Targeting Potential of Galactosylated and Mannosylated Sterically Stabilized Liposomes to Different Cell Types of Mouse Liver

Galactose and Mannose residues were tagged on the surface of n-glutaryl-phosphatidyl-ethanolamine (NGPE) containing liposomes with and without polyethylene glycol of molecular weight 2000 Da conjugated to distearoyl phosphatidylethanolamine (PEG-2000-DSPE). Biodistribution studies showed that sugar bearing liposomes were cleared more rapidly from circulation than those not bearing the sugar moieties. However, the rate of clearance of glycosylated conventional liposomes was much faster than the sugar bearing sterically stabilized liposomes. Intrahepatic distribution studies showed that a substantial amount of conventional liposomes without sugar residues were taken up by both parenchymal (P) (40%) and non-parenchymal (NP) cells (60%). However, incorporation of PEG-2000-DSPE shifted this uptake slightly in favour of parenchymal cells (47%). While ratio of distribution of galactosylated conventional liposomes to P and NP cells was found to be 74: 26, galactosylation of sterically stabilized liposomes further enhanced the affinity of these vesicles towards P cells (P: NP ratio being 93: 7). Thus, reduced uptake by Kupffer cells was observed with galactosylated sterically stabilized liposomes as compared to conventional liposomes. Whereas, mannosylation of both the liposomes shifted the distribution towards Kupffer cells in an analogous manner. These findings indicate that sterically stabilized liposomes tagged with galactose residues on their surface are more effective in targeting the entrapped material to hepatocytes as compared to conventional liposomes. This approach can therefore be employed for delivering therapeutic agents like drugs, enzymes, genetic materials, anti-sense oligonucleotides selectively to liver P cells for treatment of hepatic disorders.

A Neutron Reflectivity Study of Polymer-Modified Phospholipid Monolayers at the Solid-Solution Interface: Polyethylene Glycol-Lipids on Silane-Modified Substrates

The structure of polymer-decorated phospholipid monolayers at the solid-solution interface was investigated using neutron reflectometry. The monolayers were composed of distearoylphosphatidylethanolamine (DSPE) matrixed with varying amounts of DSPE-PEG (DSPE with polyethylene glycol covalently grafted to its headgroup). Mixed lipid monolayers were Langmuir-Blodgett deposited onto hydrophobic quartz or silicon substrates, previously hydrophobized by chemically grafting a robust monolayer of octadecyltrichlorosilane (OTS). We show that this method results in homogeneous and continuous phospholipid monolayers on the silanated substrates and determine that the grafted PEG chains extend away from the monolayers into the solvent phase as a function of their density, as expected from scaling theories. In addition, ligands were coupled to the end of the PEG chains and selective binding was demonstrated using fluorescence microscopy. Our results demonstrate that these constructs are ideal for further characterization and studies with well-defined monomolecular films.

Comparison of different hydrophobic anchors conjugated to poly(ethylene glycol): effects on the pharmacokinetics of liposomal vincristine

Poly(ethylene glycol) (PEG) conjugated lipids have been used to increase the circulation longevity of liposomal carriers encapsulating therapeutic compounds. PEG is typically conjugated to distearoylphosphatidylethanolamine (DSPE) via a carbamate linkage that results in a net negative charge on the phosphate moiety at physiological pH. It was anticipated that the presence of this negative charge could have deleterious effects on liposome pharmacokinetic characteristics. We describe here the synthesis of a new class of neutrally charged PEG-lipid conjugates in which the PEG moiety was linked to ceramide (CER). These PEG-CER conjugates were compared with PEG-DSPE conjugates for their effects on the pharmacokinetics of liposomal vincristine. PEG-CER (78% palmitic acid, C16) and PEG-DSPE achieved comparable increases in the circulation lifetimes of sphingomyelin/cholesterol (SM/chol) liposomes. However, PEG-DSPE significantly increased the in vitro and in vivo leakage rates of vincristine from SM/chol-based liposomes compared to vincristine leakage observed when PEG-CER was used. The increase in drug leakage observed in vitro that was due to the presence of PEG-DSPE was likely due to the presence of a negative surface charge. Analysis of the electrophoretic mobilities of these formulations suggested that the negative surface charges were shielded by approx. 80% by the PEG layer extending from the membrane surface. In contrast, formulations containing PEG-CER had no surface charge and no electrophoretic mobility. A comparison of the effects of the ceramide acyl chain length (C8 through C24) on the pharmacokinetics of SM/chol/PEG-CER formulations of vincristine demonstrated that longer acyl chains on the PEG-CER were associated with longer circulation lifetimes of the liposomal carriers and, consequently, higher plasma vincristine concentrations. These data suggest that the short chain PEG-ceramides underwent rapid partitioning from the vesicles after i.v. administration, whereas the longer chain PEG-ceramides had stronger anchoring properties in the liposome bilayers and partitioned slowly from the administered vesicles. These data demonstrate the utility of ceramide-based steric stabilizing lipids as well as the potential for developing controlled release formulations by manipulating the retention of the PEG-ceramide conjugate in liposome bilayers.

Lymphatic uptake and biodistribution of liposomes after subcutaneous injection: IV. Fate of liposomes in regional lymph nodes

The ability of clodronate-containing liposomes to deplete lymph nodes of macrophages was used as a tool to investigate the fate of liposomes in regional lymph nodes after subcutaneous (s.c.) administration. Reduced lymph node localization of liposomes in macrophage-depleted lymph nodes confirmed that phagocytosis by macrophages plays an important role in lymph node retention of liposomes. Depletion of macrophages had less effect on lymph node localization of small liposomes than on the lymph node localization of large liposomes. Inclusion of distearoylphosphatidylethanolamine (DSPE)–poly(ethyleneglycol) (PEG–PE) into the liposomes, which is known to oppose macrophage uptake, did not affect lymph node localization in macrophage-depleted or control lymph nodes. We conclude that PEG-liposomes retained by lymph nodes are also taken up by lymph node macrophages. Morphological observations visualizing the uptake of PEG-liposomes by lymph node macrophages support this conclusion.

Accumulation of protein-loaded long-circulating micelles and liposomes in subcutaneous Lewis lung carcinoma in mice.

The purpose of our work was to compare the biodistribution and tumor accumulation of a liposome- or micelle-incorporated protein in mice bearing subcutaneously-established Lewis lung carcinoma. A model protein, soybean trypsin inhibitor (STI) was modified with a hydrophobic residue of N-glutaryl-phosphatidyl-ethanolamine (NGPE) and incorporated into both polyethyleneglycol(MW 5000)-distearoyl phosphatidyl ethanolamine (PEG-DSPE) micelles (< 20 nm) and PEG-DSPE-modified long-circulating liposomes (ca. 100 nm). The protein was labeled with 111In via protein-attached diethylene triamine pentaacetic acid (DTPA), and samples of STI-containing liposomes or micelles were injected via the tail vein into mice bearing subcutaneously-established Lewis lung carcinoma. At appropriate time points, mice were sacrificed and the radioactivity accumulated in the tumor and main organs was determined. STI incorporated into PEG-lipid micelles accumulates in subcutaneously established Lewis lung carcinoma in mice better than the same protein anchored in long-circulating PEG-liposomes. Small-sized long-circulating delivery systems, such as PEG-lipid micelles, are more efficient in the delivery of protein to Lewis lung carcinoma than larger long-circulating liposomes.

An immune response to ovalbumin covalently coupled to liposomes is prevented when the liposomes used contain doxorubicin

It is now well established that liposomes with surface associated proteins are immunogenic. Repeated administration of protein coated liposomes elicits the generation of antibodies and the elimination of proteoliposome increases markedly in animals `immunized' with such liposomes. This immune response compromises the therapeutic potential of liposomal formulations that rely on the use of protein- or peptide-based targeting ligands to enhance cell specificity. Strategies to suppress or inhibit such immune responses must be developed if this technology is going to prove therapeutically viable. This study evaluates whether an immune response to a protein, covalently attached to liposomes by a thioether bond between N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP)-modified-protein and N-(4-( P-maleimidophenyl)butyryl) (MPB)-activated lipids, can be suppressed when the liposomes used contain the anti-cancer drug doxorubicin. To assess this, the highly immunogenic protein ovalbumin was conjugated onto liposomes composed of distearoylphosphatidylcholine/cholesterol (DSPC/Chol) with sufficient poly(ethylene glycol)-modified distearoyl phosphatidylethanolamine (PEG-DSPE) (2 mol%) to prevent liposome aggregation during protein coupling and to engender increased circulation lifetimes. The immune response to these liposomes with and without encapsulated doxorubicin was measured by: (1) monitoring liposome elimination after 3 weekly i.v. injections in C3H/HeJ mice and (2) measuring the anti-ovalbumin antibody levels by an ELISA assay. One week after a single dose of ovalbumin-coated PEG liposomes (50 μg protein/mouse) the immune response resulted in rapid elimination of a second dose of ovalbumin-coated PEG liposomes. Rapid liposome elimination was correlated to generation of high levels (>9 μg/ml plasma) of circulating anti-ovalbumin IgG. In contrast, anti-ovalbumin antibodies were not detected when the liposomes used contained doxorubicin. Plasma elimination of these drug loaded protein coated liposomes decreased following repeated weekly i.v. doses, an effect that is consistent with liposomal doxorubicin mediated suppression of phagocytic cells in the liver.

Nanometer-Scale Surface Properties of Mixed Phospholipid Monolayers and Bilayers

Mixed distearoylphosphatidylethanolamine (DSPE) and dioleoylphosphatidylethanolamine (DOPE) monolayers and bilayers have been deposited on mica using the Langmuir-Blodgett (LB) technique, as a model system for biomembranes. Investigation with atomic force microscopy revealed phase-separation for both monolayers in air and bilayers in water in the form of microscopic DSPE domains embedded in a DOPE matrix. For the monolayers in air, the step height measured between the higher DSPE phase and the lower DOPE phase was larger than expected from the molecular lengths, and a significant contrast in adhesion and friction was observed despite identical lipid end groups. This unexpected behavior resulted primarily from a difference in the film mechanical properties, the DOPE phase being inelastically deformed by the probe. For the bilayers in water, similar trends were found in terms of height, adhesion, and friction, but an additional short-range repulsive hydration/steric force over the DSPE phase contributed to the observed differences.

Immunogenicity and pharmacokinetic attributes of poly(ethylene glycol)-grafted immunoliposomes

Immunoliposomes composed of hydrogenated soy phosphatidylcholine, cholesterol, methoxypoly(ethylene glycol)-distearoyl phosphatidylethanolamine (mPEG-DSPE), and hydrazide-PEG-DSPE (mole ratio, 57:38:3.3:1.7) linked to periodate-oxidized chimerized mouse IgG (C225, anti-human epidermal growth factor receptor) were prepared by an optimized aggregation-free procedure. The antigen-binding activity of the immunoliposomes was well preserved. When injected intravenously into naive rats, the immunoliposomes (∼18 IgG per 100 nm liposome) exhibited long circulation times (MRT=8.5 h, Cl=0.2 ml/h). Subsequent injections of the immunoliposomes into the same animals resulted in rapid clearance (MRT≤0.7 h, Cl≥7 ml/h), which was accompanied by a significant increase in anti-C225 specific titers. Upon repeated injection or coinjection with the parent liposomes free C225 consistently exhibited prolonged circulation without any increase in C225-specific antisera, but was cleared quickly when administered into animals that had been pretreated with the immunoliposomes. Screening of the immunoliposome induced antisera against human polyclonal IgG and C225-derived Fab′ fragment revealed that the immune response was specifically triggered by the constant human region of C225. These results demonstrate that the preparations of PEG-grafted immunoliposomes are more immunogenic than the free IgG component, which is of profound importance to the antibody-mediated liposomal drug delivery effort.

Grafted poly-(ethylene glycol) on lipid surfaces inhibits protein adsorption and cell adhesion

Monolayers of dipalmitoyl-phosphatidylethanolamine (DPPE) mixing with various mole percentages of distearoyl-phosphatidylethanolamine (DSPE)-conjugated poly-(ethylene glycol) (PEG m.w. 750–5000) were deposited on DPPE-coated glass surfaces by the Langmuir-Blodgett method. Increasing percentages of grafted PEG in these supported lipid surfaces increasingly inhibit the adsorption of bovine serum albumin (BSA), laminin, and fibronectin. Increasing percentages of grafted PEG also inhibit the adhesion of erythrocytes, lymphocytes, and macrophages to these supported lipid surfaces. The adsorption of proteins on lipid coated glass surfaces were assayed by the fluorescence of FITC-labelled proteins. Cell adhesion was measured mainly by microscopic counting. The concentration of PEG-grafted lipids required for the inhibition of erythrocyte adhesion decreases with increasing molecular weight of the grafted PEG. The inhibitory effects are strongly dependent on the graft density of PEG at low concentrations, but weakly dependent on graft density at higher concentrations. For DSPE-PEG5000, the change of graft density dependency occurs approximately at the complete coverage of the lipid surface by the grafted polymer in the mushroom conformation (0.7 mol%), and the transition to partial brush conformation. The change-overs become less distinctive for grafted PEG of lower molecular weights, probably due to the failure of strictly mushroom and brush models of the polymer. The relative inhibitory efficiency is protein or cell dependent. The implication on the function of stealth liposomes is discussed.

Thermodynamics of Transfer of Amphiphiles between the Liquid−Air Interface and a Solid SurfaceWetting Tension Study of Langmuir−Blodgett Films

Wetting tension was determined by measuring the meniscus height on a mica surface during spreading, compression, and subsequent Langmuir−Blodgett deposition of an insoluble monolayer of DSPE (distearoylphosphatidylethanolamine) spread at the water−air interface, and on reimmersion of the surface in pure water. While such systems are essentially nonequilibrium and irreversible in various aspects of their behavior, we show that fundamental principles behind Langmuir−Blodgett phenomena can be understood by consideration of the thermodynamic equilibrium at the three-phase line. At large areas per molecule, long before the monolayer at the liquid−vapor interface is condensed, it undergoes spontaneous condensation and compression at the three-phase line. By this thermodynamically driven mechanism the contact angle increases and the layer is transferred onto the surface of the solid. This is a general effect which occurs also for soluble surfactants. Free energies of transfer of a lipid molecule between the inte...

Poly(ethylene glycol)-grafted liposomes with oligopeptide or oligosaccharide ligands appended to the termini of the polymer chains.

Novel conjugates tailor-made for inclusion in liposomal formulations, containing distearoylphosphatidylethanolamine (DSPE) as a lipid anchor, heterobifunctional polyethylene glycol (PEG) with a molecular weight of 2000 as a linking moiety, and a biological cell adhesive ligand [YIGSR peptide or Sialyl Lewis(X) oligosaccharide (SLX)], were synthesized. They were characterized by NMR, chromatography, and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOFMS). Inclusion of either of the ligand-PEG-lipid conjugates (2 mol %) in a lecithin/cholesterol/ methoxy-PEG2000-DSPE (55:40:3 mole ratio) lipid mixture followed by preparation of unilamellar vesicles (100 nm) resulted in positioning of 55% of the YIGSR and 63% of the SLX ligands on the periphery of the outer surface-grafted polymeric "brush", as determined by a combination of specific enzymatic alterations of each ligand and HPLC. Similar densities of ligand-bearing PEG chains were incorporated into liposomes by simply incubating (37 degrees C, 5 h) either one of the ligand-PEG-lipid conjugates with preformed lipid vesicles. This conjugate insertion process was aggregation free. Using enzymatic derivatization-HPLC, it was demonstrated that all the ligands incorporated into lipid membranes by this new approach were positioned exclusively on the outer leaflet of the liposomal bilayers. Since liposomes of this type are intended for in vivo use as long-circulating, ligand-presenting platforms, the insertion approach is preferable because of the more efficient utilization of ligand-PEG-lipid conjugates.

Comparative pharmacokinetics and antitumor efficacy of doxorubicin encapsulated in soybean-derived sterols and poly(ethylene glycol) liposomes in mice

The blood clearance, tissue uptake and antitumor efficacy of doxorubicin (DOX) encapsulated in two different types of liposomes against hepatoma 22 (H22) and sarcoma 180 (S180) tumors were examined in mice. Liposomes were composed of dipalmitoylphosphatidylcholine (DPPC) and soybean-derived sterol mixture (SS) at a 7:4 molar ratio (DPPC/SS-liposomes), a new stabilizer like cholesterol, and 6 mol% distearoylphosphatidylethanolamine (DSPE) derivatized with poly(ethylene glycol) (PEG) (DPPC/SS/PEG-liposomes). Pharmacokinetic analysis of drug disposition was based on the areas under the curve (AUC) for liposome-encapsulated DOX uptake per gram tissue up to 24 h following i.v. injection. The highest tissue AUC values with both liposome types were obtained in spleen and liver. The serum AUC value of DPPC/SS/PEG-liposomes was 1.3 times higher than that of DPPC/SS-liposomes ( P < 0.05). These findings indicate that the encapsulation of DOX in either DPPC/SS- or DPPC/SS/PEG-liposomes markedly prolonged the blood circulation time. The antitumor efficacy of DOX encapsulated in liposomes was compared with that of the free drug at two doses, 5 and 10 mg/kg. The antitumor efficacy of DOX encapsulated in DPPC/SS- and DPPC/SS/PEG-liposomes was different between the H22 and S180 tumor. DPPC/SS-liposomes were significantly more active against the H22 tumor than the free drug and the DPPC/SS/PEG-Iiposomes markedly more active (ILS: 32.1 and 97.7%, respectively, P<0.001), reflecting long circulation. The antitumor efficacy of the DPPC/SS/PEG-liposomes against S180 tumor-bearing mice was significantly high but that of DPPC/SS-liposomes was not, in comparison with free DOX.

Long-Circulating Immunoliposome Targeting in Animal Models

The current status of newly developed PEG-immunoliposomes (Type C), carrying monoclonal antibodies or their fragments (Fab') at the distal ends of the PEG chains, were described in this review. In terms of target binding of Type C, two different anatomical compartments were considered. They are the mouse lung endothelial for a readily accessible site in intravascular and the mouse implanted solid tumor for a much less accessible target site located in extravascular through leaky vascular. Distearoyl phosphatidylethanolamine derivatives of PEG with carboxyl group (DSPE-PEG-COOH) and dipalmitoyl phosphatidylethanolamine derivatives of PEG with maleimidyl group (DPPE-PEG-Mal) at the PEG's terminus were newly synthesized. Small unilamellar liposomes (90 - 130 nm in diameter) were prepared from phosphatidylcholine and cholesterol (2:1, m/m) containing 6 mol% of DSPE-PEG-COOH or DPPE-PEG-Mal. For targeting to the vascular endothelial surface in the lung, 34A antibody, which is highly specific to mouse pulmonary endothelial cells, was conjugated to PEG-liposome (34A-Type C). The degree of lung binding of 34A-Type C in BALB/c mouse was significantly higher than that of 34A-Type A which is an ordinary type of immunoliposome (without PEG derivatives). For targeting to the solid tumor tissue, 21B2 antibody which is anti-human CEA and its Fab' fragment were used. The targeting ability of Fab'-Type C was examined by using CEA-positive human gastric cancer strain MKN-45 cells inoculated into BALB/c nu/nu mice. Fab'-Type C showed the low RES uptake and the long circulation time, and resulted in enhanced accumulation of the liposomes in the solid tumor. The small Fab'-Type C could predominantly pass through the leaky tumor endothelium by passive convective transport. These studies offer some important insights into the potential of target-specific drug delivery.

Lipopeptide conjugates: biomolecular building blocks for receptor activating membrane-mimetic structures

A simple method for the derivatization of phospholipid subunits with short peptide sequences is presented. Amphiphilic conjugates of distearoylphosphatidylethanolamine (DSPE) and the minimal human thrombin-receptor peptide agonist SFLLRN were synthesized via coupling of the bromoacetyl derivative of DSPE (PEBr) with a thiol-terminated decapeptide of SFLLRN. Bromoderivatization of DSPE was performed by condensation of bromoacetic acid and DSPE, with an overall yield of 40%. Coupling of PEBr and the unprotected thiol-terminated decapeptide was performed in chloroform/ methanol/water in the presence of triethylamine and afforded 25.5% of the lipopeptide. The compound was characterized by TLC, 1H-NMR (600 MHz) and mass spectrometry. Lipopeptide bioactivity was confirmed by a platelet aggregation assay. The EC 50 of the all- l amino acid lipopeptide was 38 ± 3 μM. The all- d conjugate was inactive. Model receptor-activating systems, including well-ordered assemblies of amphiphilic phospholipid-peptide conjugates, represent the first step in the construction of bioactive surfaces for tissue engineering based on a membrane-mimetic approach.

Enhanced tumor targeting and improved antitumor activity of doxorubicin by long-circulating liposomes containing amphipathic poly(ethylene glycol)

Enhanced delivery of doxorubicin (DXR) to colon 26 in mice was studied by the long-circulating liposomes (LCL) composed of distearoylphosphatidylcholine cholesterol (DSPC CH) (1 1. m m) and 6 mol° distearoyl phosphatidyl ethanolamine (DSPE) derivatives of poly(ethylene glycol) (PEG) with an average molecular weight of 1000. LCL was approximately 100 nm in mean diameter and encapsulated DXR with > 98% entrapping efficiency by the transmembrane pH gradient method. The control liposomes (LP) which had the same lipid composition, similar size and DXR entrapment as LCL. but did not have amphipathic PEG. were used for comparison. Liposomal DXR and free DXR were injected intravenously at a dose of 5 mg DXR kg to Balb c mice implanted subcutaneously with colon 26 carcinoma. DXR encapsulated in LCL showed high blood levels up to 24 h after injection, compared with the corresponding DXR-LP and free DXR. The value of the area under the curve (AUC) of blood was approximately 2.4-or 810-fold higher than that of DXR-LP or free DXR. respectively. LCL decreased the uptake amount of DXR by the reticuloendothelial system (RES) of liver and spleen. Both liposomal formulations effectively reduced the DXR concentrations in heart compared with free DXR. Compared with DXR-LP or free drug. DXR-LCL produced an approximately 3.6-or 10.5-fold increased DXR level in tumor. respectively, at 6 h after injection. The AUC value of tumor tissue for DXR-LCL was 3.4-or 9.4-fold higher than that of DXR-LP or free DXR. respectively. These high tumor levels of DXR by LCL corresponded to the prolonged residence feature of liposomes. Therapeutic experiments were performed with three different formulations of DXR. Administration of DXR-LCL at a dose of 10 mg DXR kg resulted in effective retardation of tumor growth and 2-fold prolongation of survival times compared with DXR-LP. free drug and saline. Our results indicate that DXR encapsulated in long-circulating liposomes is significantly more active against colon 26 carcinoma than the conventional liposome (DSPC CH. 1:1. m m) formulation of DXR and free drug. Thus long-circulating liposomes should be useful carriers of chemotherapeutic agents for the treatment of solid tumor.

Biodistribution and Antitumor Effect of Adriamycin Encapsulated in Long-Circulating Liposomes Containing Amphipathic Polyethylene Glycol or Ganglioside GM1

AbstractBiodistribution and antitumor effect of adriamycin (ADM) encapsulated in liposomes with reduced uptake by reticuloendothelial system (RES) and prolonged circulation time were investigated in mice. Two different types of long-circulating liposomes, ganglioside GMI (GMl)/distearoyl phosphatidyl choline (DSPC) /cholesterol (CH) (0.13:1:1, m/m) and amphipathic polyethylene glycol (PEG)/DSPC/CH (0.13:1:1, m/m) were used. They were sized to 180-200 nm in mean diameter. In the case of amphipathic PEG, distearoyl phosphatidylethanolamine (DSPE) derivatives of PEG with various molecular weight (1000-12000 in mean molecular weight) were used. ADM was encapsulated by transmembrane pH gradient method. GM~DSPC /CH

Enhanced delivery and antitumor activity of doxorubicin using long-circulating thermosensitive liposomes containing amphipathic polyethylene glycol in combination with local hyperthermia.

Enhanced delivery of doxorubicin (DXR) to a solid tumor subjected to local hyperthermia was achieved by using long-circulating, thermosensitive liposomes (TSL) composed of dipalmitoyl phosphatidylcholine (DPPC)/distearoyl phosphatidylcholine (DSPC) (9:1, m/m) and 3 mol% amphipathic polyethylene glycol (PEG) in colon 26-bearing mice. Inclusion of 3 mol% of distearoyl phosphatidylethanolamine derivatives of PEG (DSPE-PEG, amphipathic PEG) with a mean molecular weight of 1000 or 5000 in DPPC/DSPC liposomes resulted in decreased reticuloendothelial system (RES) uptake and a concomitant prolongation of circulation time, affording sustained increased blood levels of the liposomes. Concomitantly, DXR levels in blood were also kept high over a long period. The presence of amphipathic PEG did not interfere with the encapsulation of DXR by the pH gradient method (> 90% trapping efficiency) or with the temperature-dependent drug release from the liposomes. The optimal size of these liposomes was 180-200 nm in mean diameter for thermosensitive drug release and prolonged circulation time. The DXR levels in the tumor after injection of long-circulating TSL (DXR-PEG1000TSL or DXR-PEG5000TSL, at a dose of 5 mg DXR/kg) with local hyperthermia were much higher than after treatment with DXR-TSL lacking PEG or with free DXR, reaching 7.0-8.5 DXR micrograms/g tumor (approximately 2 times or 6 times higher than that of DXR-TSL or free DXR, respectively). Furthermore, the combination of DXR-PEGTSL and hyperthermia effectively retarded tumor growth and increased survival time.(ABSTRACT TRUNCATED AT 250 WORDS)

The fate of ileorectal anastomosis in Crohn's disease.

An assessment has been made of 25 patients who have undergone ileorectal anastomosis for Crohn's disease. The rate of anastomotic leakage was high (32%) but was fatal in only one patient. The likelihood of leakage was not affected by a safety valve ileostomy, failure to excise all diseased bowel, or steroid therapy. The overall recurrence rate was 60 per cent. This was not affected by the age of the patient, duration of the disease, steroid therapy, or anastomotic leak but was affected adversely by the presence of concurrent small bowel disease and by residual rectal disease at the site of the anastomosis.