Human interleukin 10 gene therapy decreases the severity and mortality of lethal pancreatitis in rats

BACKGROUND: Studies have proven the validity of interleukin-10 (IL-10) in the treatment of experimental pancreatitis. Prophylactic human IL-10 (hIL-10) gene treatment attenuated the severity in cerulein models. Our research aims to study whether the therapeutic hIL-10 gene could decrease both severity and mortality in a lethal pancreatic model. METHODS: Severe acute pancreatitis (SAP) was induced by sodium taurocholate. A plasmid-hIL-10 construct (pcDNA3-hIL-10) complexed with cationic liposomes was administered to SAP rats by a single intraperitoneal injection. Levels of hIL-10 in the pancreas, liver, and lungs were determined by ELISA kits. The severity of pancreatitis was assessed in terms of serum amylase, histology, and tissue tumor necrosis factor alpha (TNF-alpha). Mortality, observed for 7 days, was evaluated for gene therapy or control groups. RESULTS: After hIL-10 gene therapy, hIL-10 levels in the pancreas, liver, and lungs increased significantly and the serum amylase, tissue TNF-alpha, and histological changes in pancreas, liver, and lungs decreased markedly. Therefore, mortality was significantly reduced in the hIL-10 gene therapy group, in which 70% of rats survived in the 7-day observation, while only 10% survived in untreated groups (P < 0.05). CONCLUSION: We found that liposome/hIL-10 gene therapy decreased severity and mortality in SAP, even carried out after SAP establishment, predicting a more convenient shift to clinical applications.     

Promotion of xenogeneic hematopoietic chimerism in rodents by mononuclear phagocyte depletion

Cheng J, Glaser RM, Kruger-Grey H, White-Scharf ME, Cooper DKC, Thall AD. Promotion of xenogeneic hematopoietic chimerism in rodents by mononuclear phagocyte depletion. Xenotransplantation 2002; 9: 402–409. © Blackwell Munksgaard, 2002
The successful establishment of tolerance toward pig tissues in primates through hematopoietic progenitor cell engraftment is restricted by the rapid disappearance of these cells in the recipient following infusion. We developed and tested the hypothesis that phagocytes of the reticuloendothelial system are responsible for the rapid clearance of infused pig hematopoietic cells using a mouse model. Mice received non-myeloablative conditioning and, on various days, were injected with medronate-encapsulated liposomes (M-L) or control blank liposomes, followed by the intravenous infusion of miniature swine hematopoietic cells. M-L were well-tolerated in mice (n=100) at levels that deplete mononuclear phagocytes. Depletion of mononuclear phagocytes in normal Balb/c mice as well as in severe combined immune deficient mice increased the accumulation of pig hematopoietic cells in the bone marrow (BM) by 10-fold when measured 24 h after the infusion of the cells. Colony-forming unit analysis showed an increased accumulation of pig hematopoietic progenitors in the BM of mice that were infused with medronate-liposomes. We conclude that depletion of mononuclear phagocytes by M-L has the potential to lower the barrier to the establishment of mixed chimerism and tolerance induction in xenotransplantation.     

In vivo characteristics of cationic liposomes as delivery vectors for gene therapy

After a decade of clinical trials, gene therapy seems to have found its place between excessive ambitions and feasible aims, with encouraging results obtained in recent years. Intracellular delivery of genetic material is the key step in gene therapy. Optimization of delivery vectors is of major importance for turning gene therapy into a successful therapeutic method. Nonviral gene delivery relies mainly on the complexes formed from cationic liposomes (or cationic polymers) and DNA, i.e., lipoplexes (or polyplexes). Many lipoplex formulations have been studied, but in vivo activity is generally low compared to that of viral systems. This review gives a concise overview of studies on the application of cationic liposomes in vivo in animal models of diseases and in clinical studies. The transfection efficiency, the pharmacokinetic and pharmacodynamic properties of the lipid-DNA complexes, and potentially relevant applications for cationic liposomes are discussed. Furthermore, the toxicity of, and the induction of an inflammatory response in association with the administration of lipoplexes are described. Increasing understanding of lipoplex behavior and gene transfer capacities in vivo offers new possibilities to enhance their efficiency and paves the path to more extensive clinical applications in the future.     

Design and characterization of liposomes containing long-chain N-acylPEs for brain delivery: penetration of liposomes incorporating GM1 into the rat brain

Purpose. To develop a suitable liposomal carrier to encapsulate neu- roactive compounds that are stable enough to carry them to the brain across the blood-brain barrier with the appropriate surface characteristics for an effective targeting and for an active membrane transport. Methods. Liposomes containing glycosides and a fusogenic lipid were prepared by extrusion. Photon correlation spectroscopy, fluorescence spectroscopy, and differential scanning calorimetry were used to characterize liposomal preparations. Tissue distribution was determined by using ³H-cholesterylhexadecylether as a marker. Results. The incorporation of glycoside determinants and N-palmitoylphosphatidylethanolamine gives liposomes with similar initial size, trapped volume, negative surface charge, bilayer fluidity, and melting temperature, except for monosialoganglioside-containing liposomes, which showed less negative surface charge and the highest size, trapped volume and melting temperature. All glycosilated formulations gave liposomes able to retain up to the 95% of encapsulated carboxyfluorescein after 90 min at physiologic temperature even in the presence of serum. Monosialoganglioside liposomes were recovered in the cortex, basal ganglia, and mesencephalon of both brain hemispheres. The liver uptake was higher for sulfatide- and glucose-liposomes, whereas the higher blood levels were observed for glucose- and mannose-liposomes. Conclusions. These results show the suitability of such liposomal formulations to hold encapsulated drugs. Moreover, the brain uptake of monosialoganglioside liposomes makes them good candidates as drug delivery systems to the brain.     

Cetirizine from topical phosphatidylcholine-hydrogenated liposomes: Evaluation of peripheral antihistaminic activity and systemic absorption in a rabbit model

Cetirizine, an effective, minimally sedating, second-generation H1-antihistamine is widely used orally to treat allergic skin disorders. This study was performed to assess the peripheral H1-antihistaminic activity and extent of systemic absorption of cetirizine from liposomes applied to the skin. Cetirizine was incorporated into small unilamellar vesicles (SUV) and multilamellar vesicles (MLV) prepared using L-alpha-phosphatidylcholine hydrogenated (HPC), and into Glaxal Base (GB) as the control. In a randomized, crossover study, each formulation, containing 10 mg of cetirizine, was applied to the depilated backs of 6 rabbits (3.08 +/- 0.05 kg). Histamine-induced wheal tests and blood sampling were performed before cetirizine application and at designated times for up to 24 hours afterwards. Compared with baseline, histamine-induced wheal formation was suppressed by cetirizine in SUV only at 24 hours, in MLV from 0.5 to 24 hours, and in GB from 0.5 to 8 hours (P < or = .05). Wheal suppression by cetirizine in SUV at 24 hours (91.7% +/- 5.2%) and in MLV from 1 to 24 hours (93.8% +/- 2.2% to 76.2% +/- 6.5%) was greater than in GB (36.5% +/- 7.4% to 60.6% +/- 14.2%) from 1 to 24 hours (P < or = .05). Faster onset, as well as greater and more persistent suppression was obtained from cetirizine in MLV. Plasma cetirizine concentrations from MLV (area under the curve [AUC] of 221.2 +/- 42.3 ng x hr/mL) were lower than from GB (AUC of 248.3 +/- 34.6 ng.hr/mL). In this model, cetirizine from MLV had excellent topical H(1)-antihistamine activity, while systemic exposure was reduced, compared with cetirizine from GB.     

Tumor necrosis factor-alpha augmented tumor response in B16BL6 melanoma-bearing mice treated with stealth liposomal doxorubicin (Doxil) correlates with altered Doxil pharmacokinetics

The application of tumor necrosis factor-alpha (TNF) for the treatment of solid tumors is limited by its severe, life-threatening, toxicity. Therefore, only low dosages of this cytokine can be applied systemically, which results in poor tumor response. It has been demonstrated previously that administration of high-dose TNF in a so-called isolated perfusion system markedly improved tumor response when combined with chemotherapy. It appeared that TNF had a major impact specifically on the tumor-associated vasculature. At these high concentrations, endothelial cell death is induced by TNF, resulting in complete collapse of the tumor vascular bed. Strikingly, this effect alone is not enough to induce a tumor response, but addition of a chemotherapeutic drug is mandatory to obtain an anti-tumor effect. We showed that TNF has no anti-tumor effect by itself but augmented drug accumulation mainly in the tumor, most likely by enhancing vascular leakage. It seems that enhanced vascular leakage, but not endothelial cell death, explains the interaction between TNF and the co-administered drug. We hypothesized that in a low-dose setting TNF could induce tumor accumulation of chemotherapeutic drugs and consequently improve tumor response. We demonstrate that free TNF has a strong effect on the pharmacokinetics of co-administered Doxil in B16BL6 melanoma-bearing mice, resulting in strongly augmented drug accumulation in the tumor and improved tumor response. Co-injection of Stealth liposomal TNF with Doxil resulted in comparable or less pronounced tumor responses as compared to free TNF. These results imply that systemic application of clinically tolerable doses of TNF may improve drug distribution and tumor response and could be useful in a number of anti-cancer therapies.     

Neovascular targeting chemotherapy: encapsulation of paclitaxel in cationic liposomes impairs functional tumor microvasculature

Cationic liposomes have been shown to be internalized selectively by angiogenic tumor endothelial cells after intravenous injection. Therefore, encapsulation of cytotoxic substances in cationic liposomes is a new approach to target tumor vasculature. It was the aim of our study to quantify the effects of paclitaxel encapsulated in cationic liposomes (MBT-0206) on tumor microvasculature and growth in vivo. Experiments were performed in the dorsal skinfold chamber preparation of Syrian Golden hamsters bearing syngeneic A-Mel-3 melanomas. Tumors were treated with intravenous infusion of MBT-0206 (20 mM) resulting in an effective paclitaxel dose of 5 mg/kg body weight (b.w.). Control animals received conventional paclitaxel in Cremophor EL (Taxol(R); 5 mg/kg b.w.), unloaded cationic liposomes (20 mM) or the solvent 5% glucose, respectively. Using intravital microscopy, tumor growth and effects on intratumoral microvasculature were analyzed. Tumor growth was significantly retarded after treatment with MBT-0206 compared to the treatment with paclitaxel. Analysis of intratumoral microcirculation revealed a reduced functional vessel density in tumors after application of liposomal paclitaxel. At the end of the observation time, vessel diameters were significantly smaller in animals treated with paclitaxel encapsulated in cationic liposomes while red blood cell velocity was less affected. This resulted in a significantly reduced blood flow in vessel segments and a reduced microcirculatory perfusion index in these animals. Histochemical TUNEL stain was vessel-associated after treatment with liposomal paclitaxel in contrast to few apoptotic tumor cells in the control groups. Our data demonstrate that encapsulation of paclitaxel in cationic liposomes significantly increased the antitumoral efficacy of the drug. Remarkable microcirculatory changes indicate that encapsulation of paclitaxel in cationic liposomes resulted in a mechanistic switch from tumor cell toxicity to an antivascular therapy.     

Stability and transdermal absorption of topical amphotericin B liposome formulations

The aim of this study was to characterize the stability and transdermal absorption of amphotericin B (AmB: 0.05 mg/mg lipid) in hydrogenated soya phosphatidylcholine/cholesterol/charged lipid {dicetyl phosphate (−) or stearylamine (+)} liposomes at molar ratios of 1:1:0, 7:2:0, 7:2:1(−) and 7:2:1(+). The AmB contents in liposomes were determined by HPLC with UV detection at 382 nm. Stabilities of AmB in liposome formulations were compared with those in solution and powder forms, during storage at 4, 30 and 45 °C for 90 days. Absorption studies of AmB across the rat skin were conducted, using vertical Franz diffusion cells at 37 °C for 24 h. The slowest degradation was observed in the positive liposome (7:2:1(+)AmB), with shelf life of 1 year (30 °C). In comparison, the shelf lives of AmB in solution and powder were 4 and 14 days, respectively. AmB in positive liposomes seemed to demonstrate the highest flux in stratum corneum (58 ng/cm²/h), while the highest flux in viable epidermis (23 ng/cm²/h) was observed in negative liposomes. AmB entrapped in charged liposomes showed sustained skin absorption. The positively charged liposome might be the best formulation for AmB, due to its higher stability than other formulations.     

In Vivo ESR Studies on Subcutaneously Injected Multilamellar Liposomes in Living Mice

PURPOSE: An innovative, noninvasive, low-frequency electron spin resonance (ESR) spectroscopy method was applied and adapted to investigate the integrity of multilamellar liposomes from hydrogenated phospholipids after subcutaneous injection in living mice. Moreover, the fate of the injected liposomal preparations was examined, as well as the possibility to achieve a depot effect. METHODS: Highly concentrated solutions of the spin probe 2,2,6,6-tetramethyl-4-trimethylammoniumpiperidine-1-oxyl-iodide (CAT-1; 138 mM) were encapsulated in liposomes. They were characterized by laser diffraction, and the liberation of spin probe was investigated by ESR spectroscopy. RESULTS: Line shape changes allowed the differentiation between encapsulated and released CAT-1 after subcutaneous injection of liposomes. Multilamellar liposomes form a local depot at the site of injection. A sustained release of the spin probe from the depot was monitored by means of ESR. Whereas 40% of the spin probe was released within the first 96 h after administration, 60% remained in intact liposomes under the skin. No depot formation could be observed after injection of CAT-1 solutions, but a fast signal decrease due to systemic distribution and bioreduction of the nitroxide spin probe. CONCLUSIONS: Noninvasive analysis of liposomal integrity in living animals was successfully accomplished using a new L-Band ESR spectroscopy method. The liberation of CAT-1 from liposomes in vitro and in vivo was monitored by changes in the lineshape of ESR spectra and Heisenberg spin exchange. The significance of liposomal integrity for the formation of a localized drug depot effect was proved.     

Preferential Binding of Polyethylene Glycol-Coated Liposomes Containing a Novel Cationic Lipid,TRX-20, to Human Subendthelial Cells via Chondroitin Sulfate

Purpose. To design novel cationic liposomes, polyethylene glycol (PEG)-coated cationic liposomes containing a newly synthesized cationic lipid, 3,5-dipentadecyloxybenzamidine hydrochloride (TRX-20) were formulated and their cellular binding and uptake investigated in vitro in the following cells: human subendothelial cells (aortic smooth muscle cells and mesangial cells) and human endothelial cells. Methods. Three different PEG-coated cationic liposomes were prepared by the extrusion method, and their mean particle size and zeta potential were determined. Rhodamine-labeled PEG-coated cationic liposomes were incubated with smooth muscle cells, mesangial cells, and endothelial cells at 37°C for 24 h. The amounts of cellular binding and uptake of liposomes were estimated by measuring the cell-associated fluorescence intensity of rhodamine. To investigate the binding property of the liposomes, the changes of the binding to the cells pretreated by various kinds of glycosaminoglycan lyases were examined. Fluorescence microscopy is used to seek localization of liposomes in the cells. Results. The cellular binding and uptake of PEG-coated cationic liposomes to smooth muscle cells was depended strongly on the chemical species of cationic lipids in these liposomes. Smooth muscle cells bound higher amount of PEG-coated TRX-20 liposomes than other cationic liposomes containing N-(1-(2,3-dioleoyloxy) propyl)-N, N, N-trimethylammonium salts or N-(-(trimethylammonio)acetyl)-D-glutamate chloride. Despite of the higher affinity of PEG-coated TRX-20 liposomes for subendothelial cells, their binding to endothelial cells was very small. The binding to subendothelial cells was inhibited when cells were pretreated by certain kinds of chondroitinase, but not by heparitinase. These results suggest that PEG-coated TRX-20 liposomes have strong and selective binding property to subendothelial cells by interacting with certain kinds of chondroitin sulfate proteoglycans (not with heparan sulfate proteoglycans) on the cell surface and in the extracellular matrix of the cells. This binding feature was different from that reported for other cationic liposomes. Conclusions. PEG-coated TRX-20 liposomes can strongly and selectively bind to subendothelial cells via certain kinds of chondroitin sulfate proteoglycans and would have an advantage to use as a specific drug delivery system.