Placebo Liposomes Research Articles

Can Liposomes Survive Inkjet Printing? The Effect of Jetting on Key Liposome Attributes for Drug Delivery Applications

PurposeInkjet printing has the potential to enable novel personalized and tailored drug therapies based on liposome and lipid nanoparticles. However, due to the significant shear force exerted on the jetted fluids, its suitability for shear-sensitive materials such as liposomes, has not been verified. We have conducted a proof-of-concept study to examine whether the particle concentration and size distribution of placebo liposomes are affected by common inkjet/dispensing technologies.MethodsWe have subjected three types of liposome-containing fluids (“inks”) to two different commercial dispensing/jetting technologies, which are relevant to most drug printing approaches. The liposome jetting processes were observed in real-time using strobographic imaging techniques. The phospholipid concentrations and particle size distributions were determined before and after jetting via enzymatic colorimetric and dynamic light scattering methods, respectively.ResultsOur results have shown that the jetting dynamics of the liposome inks are well predicted by the established inkjet printing regime map based on their physical properties and the jetting conditions. Importantly, although significant shear forces were confirmed during jetting, the liposome concentrations and particle size distributions in the collected samples remain largely unaffected.ConclusionThese findings, we believe, provide the essential proof-of-concept to encourage further development in this highly topical research area.

Liposomal Formulation of a Melphalan Lipophilic Prodrug: Studies of Acute Toxicity, Tolerability, and Antitumor Efficacy.

Recently we developed a scalable scheme of synthesis of melphalan ester conjugate with 1,2-dioleoyl-sn-glycerol (MlphDG) and a protocol for the fabrication of its lyophilized liposomal formulation. Herein we compared this new convenient in use formulation of MlphDG with parent drug Alkeran® in rats concerning several toxicological parameters and evaluated its antitumor efficacy in the model of breast cancer in mice. Liposomes of approximately 100 nm in diameter, consisting of egg phosphatidylcholine, soybean phosphatidylinositol, and MlphDG, or placebo liposomes without the drug were produced by extrusion and lyophilized. Alkeran® or liposomes recovered by the addition of water were injected into the tail vein of animals. Clinical examination of rats consisted of detailed inspection of the behavior, general status, and hematological parameters. Mice with transplanted breast cancer WNT-1 were subjected to multiple treatments with the drugs; tumor growth inhibition was assessed, together with cellular immunity parameters. Liposomes showed approximately two times lower acute toxicity and better tolerability than Alkeran® in terms of behavioral criteria. The toxic effects of liposomes on hemopoiesis were manifested at higher doses than in the case of Alkeran®, proportionally to the difference in LD50 values. The formulation inhibited tumor growth significantly more effectively than Alkeran®, delaying the start of the exponential growth phase and exhibiting no additional toxic effects toward bone marrow. Lower toxicity of the liposomal formulation of MlphDG promises improved quality of life for cancer patients in need of treatment with melphalan. Presumably, the list of indications for melphalan therapy could be extended.

InVivo PET Tracking of 89Zr-Labeled Vγ9Vδ2T Cells to Mouse Xenograft Breast Tumors Activated with Liposomal Alendronate.

Gammadelta T (γδ-T) cells are strong candidates for adoptive immunotherapy in oncology due to their cytotoxicity, ease of expansion, and favorable safety profile. The development of γδ-T cell therapies would benefit from non-invasive cell-tracking methods and increased targeting to tumor sites. Here we report the use of [89Zr]Zr(oxinate)4 to track Vγ9Vδ2 T cells in vivo by positron emission tomography (PET). In vitro, we showed that 89Zr-labeled Vγ9Vδ2 T cells retained their viability, proliferative capacity, and anti-cancer cytotoxicity with minimal DNA damage for amounts of 89Zr ≤20 mBq/cell. Using a mouse xenograft model of human breast cancer, 89Zr-labeled γδ-T cells were tracked by PET imaging over 1 week. To increase tumor antigen expression, the mice were pre-treated with PEGylated liposomal alendronate. Liposomal alendronate, but not placebo liposomes or non-liposomal alendronate, significantly increased the 89Zr signal in the tumors, suggesting increased homing of γδ-T cells to the tumors. γδ-T cell trafficking to tumors occurred within 48 hr of administration. The presence of γδ-T cells in tumors, liver, and spleen was confirmed by histology. Our results demonstrate the suitability of [89Zr]Zr(oxinate)4 as a cell-labeling agent for therapeutic T cells and the potential benefits of liposomal bisphosphonate treatment before γδ-T cell administration.

Interleukin-2 liposomes for primary immune deficiency using the aerosol route

This is the first report of aerosol interleukin 2 (IL-2) liposome administration to individuals with immune deficiency. Parenteral IL-2 therapy has shown beneficial effects in some patients with cancer, common variable immunodeficiency (CVID), and human immunodeficiency virus (HIV) but is problematic because of side effects including fever and malaise as well as local swelling (delayed type hypersensitivity like reaction) after each subcutaneous IL-2 injection. Provision of an IL-2:human albumin liposome formulation via the aerosol route had few side effects in a recent clinical trial in cancer patients. Details of good manufacturing practice (GMP) synthesis and analysis of IL-2 liposomes ( N=6 lots) made without albumin carrier protein and placebo liposomes (three lots) are presented. After centrifugation, IL-2 was closely associated with the liposome pellet (99%). Mean diameter of liposomes was 1.1 μm. Patient acceptance, safety, toxicity, and immune effects of IL-2 liposomes were studied in individuals with primary immune deficiency ( N=15) and subsequently, a larger cohort of patients with hepatitis C. Experience in the immune deficient patients is the subject of this report. Placebo liposomes (12 weeks) and IL-2 liposomes (12 weeks) were provided using a nebulizer. Aerosol placebo liposomes and IL-2 liposomes were well tolerated. No changes in chest X-ray or pulmonary function were seen. Since biologic activity of aerosol IL-2 liposomes has been seen in viral disease (hepatitis C), additional studies of aerosol IL-2 liposomes in individuals with hepatitis C and HIV are planned.

Administration of liposomal agents and the phagocytic function of the mononuclear phagocyte system

Liposomal drugs used in clinical practice are often administered to patients that are immunocompromised and hence, highly susceptible to develop systemic infections. The resident phagocytic cells of the MPS will clear the microorganisms from the blood and thus prevent generalization of the infection as well as mortality. As substantial MPS uptake of liposomes occurs, the question arises whether administration of liposomes, particularly those containing potentially toxic agents such as amphotericin B and doxorubicin, affect the phagocytic capacity of the MPS. In the present study, at first the effect of administration of three types of ‘empty’ liposomes (i.e. devoid of drug), differing in blood residence time, on carbon clearance and bacterial clearance from blood was studied in mice: (1) Classical EPC:PS:Ch (4:1:5) liposomes, 300 nm, (2) placebo liposomes with lipid composition as in AmBisome®, 100 nm, and (3) placebo liposomes with lipid composition as in Doxil®, 100 nm. Liposomes were administered i.v. as a single dose. Secondly, the effect of multiple dose administration of AmBisome® or Doxil® on bacterial clearance from blood was studied in rats. AmBisome® or Doxil® were administered at various dosage schedules. Blood clearance capacity of the MPS was monitored at different time points after the last liposome dose. The data obtained show that carbon blood clearance capacity of the MPS was impaired only at a high lipid dose of empty classical liposomes. Bacterial blood clearance capacity was not impaired, not even after multiple dose treatment with AmBisome® or Doxil® when administered in a clinically relevant regimen.

Comparative Intravenous Toxicity of Cisplatin Solution and Cisplatin Encapsulated in Long-Circulating, Pegylated Liposomes in Cynomolgus Monkeys

The toxicity of cisplatin encapsulated in pegylated, long-circulating liposomes (SPI-077) was compared with nonliposomal cisplatin in male and female cynomolgus monkeys (n = 2-4 per sex per group) treated with intravenous infusions of 2.5 or 25 mg/kg SPI-077, 2.5 mg/kg cisplatin, placebo liposomes, or saline once every 3 weeks for total of five treatments. All animals survived until scheduled necropsy at 3 days after the final treatment or after a treatment-free 4-week recovery period. Emesis occurred after each treatment in all cisplatin-treated monkeys, but only once in one monkey treated with high-dose SPI-077. Dose-related mild decreases in red blood cell (RBC) count, hemoglobin, and hematocrit to or slightly below low normal range occurred in the high-dose SPI-077 and placebo liposome treatment groups after each treatment, with partial to complete recovery between treatments and no signs of correlating bone marrow toxicity. Decreases were similar in cisplatin-treated monkeys, but resolved only slightly between treatments and after the end of treatment (continuing to decrease in females) and were accompanied by bone marrow hypocellularity. Indirect, but not direct, bilirubin levels were cyclically elevated in the high-dose SPI-077 and placebo-treated animals, but not in the other treatment groups. Levels had either fully resolved or were near baseline and/or saline group values prior to the next treatment. Serum cholesterol levels were cyclically increased in SPI-077- and placebo liposome-treated animals, and minimally increased numbers of foam cells were seen in the liver, spleen, kidney, and other organs; both were considered related to the lipid dose administered. Cisplatin-treated monkeys exhibited sensory polyneuropathy and moderate irreversible toxic tubular nephrosis, but no neuropathy or nephrotoxicity was seen in either SPI-077 treatment group. Microscopically, treatment-related cell death was seen in dorsal root ganglia (DRG), affecting 15% of the cells in cisplatin-treated animals, compared to 8 and 12% in the low- and high-dose SPI-077 treatment groups. Neither drug was ototoxic. In summary, repeated administration of SPI-077 produced minimal, reversible effects related to the lipid dose administered, mostly limited to the 25 mg/kg dose group. The most notable effects in this group were cyclical decreases in hematology parameters thought to be related to increased recycling of a small fraction of RBCs and limited cell death in the DRG in the absence of any neurophysiological changes. Animals treated with a 10-fold lower dose of cisplatin (2.5 mg/kg), in contrast, exhibited myelo-, nephro-, and neurotoxicity, including sensory neuropathy, and were emetic after every dose. The SPI-077 liposomal formulation of cisplatin may provide a less toxic alternative to standard cisplatin solution.

Administration of liposomal agents and blood clearance capacity of the mononuclear phagocyte system.

As liposomes are cleared from the circulation to a substantial extent by the phagocytic cells of the mononuclear phagocyte system (MPS), there is a question whether administration of liposome-based therapeutic agents interferes with clearance of infectious organisms by the MPS from blood. In the present study, at first the effect of administration of three types of empty liposomes (devoid of drug), differing in blood residence time, on carbon clearance and bacterial clearance from blood was studied with mice. Classical liposomes (LIP A) and placebo liposomes with lipid composition as in AmBisome (LIP B) or as in Doxil (LIP C) were used. Liposomes were administered intravenously as a single dose. Second, the effect of multiple-dose administration of AmBisome on bacterial blood clearance was studied with rats. AmBisome was administered with two different dosage schedules. The blood clearance capacity of the MPS was monitored at different time points after the last liposome injection. It was shown that the carbon blood clearance capacity of the MPS was impaired only at a high lipid dose of empty classical liposomes. The bacterial blood clearance capacity was never impaired, not even after prolonged treatment with AmBisome administered in a clinically relevant regimen.

Toxicity of doxorubicin entrapped within long-circulating liposomes

We studied the effect of doxorubicin entrapped within long-circulating liposomes (Dox-LCL) on the phagocytic capacity and bacterial blood clearance capacity of rat liver macrophages. Dox-LCL (125 nm in diameter) were composed of egg phosphatidylcholine (PC), cholesterol (CH) and poly(ethyleneglycol)distearoylphosphatidylethanolamine (PEG-PE) (55:45:5 molar ratio; MW PEG 1900), and loaded with doxorubicin by means of a trans-membrane pH gradient. The doxorubicin/lipid ratio was 0.36:1 (mol/mol). At different time-points after one, two or three intravenous injections of Dox-LCL, radiolabeled negatively charged test liposomes (egg PC, CH, and phosphatidylserine in a 4:5:1 molar ratio) were injected. After 2 h, liver macrophages were isolated and the amount of macrophage-associated radioactivity was determined. Twenty-four hours after a single injection of 5 mg/kg Dox-LCL, no significant effect was observed. However, 48 h after injection, phagocytic activity was reduced significantly (49%). Recovery of phagocytic capacity of the liver macrophages took 8 days after two injections of Dox-LCL (2×5 mg/kg). Seventy-two hours after the last of two injections of Dox-LCL, bacterial blood clearance was significantly reduced as compared to clearance in control rats and in rats injected twice with doxorubicin combined with placebo liposomes. When comparing these Dox-LCL data with previous data on the effects of Dox-L, Dox-LCL appears less toxic than Dox-L for the liver macrophage population following i.v. administration both with respect to specific phagocytic activity and cell numbers. Due to the delay in onset of toxic effects and the faster recovery from Dox-LCL treatment as compared to Dox-L treatment, it is conceivable that therapeutic protocols can be designed with Dox-LCL that circumvent long-term impairment of the liver macrophage population.

Reduction of canine infarct size by bolus intravenous administration of liposomal prostaglandin E 1: Comparison with control, placebo liposomes, and continuous intravenous infusion of prostaglandin E 1

Prostaglandin E 1 (PGE 1) reduces experimental infarct size when administered by prolonged low-dose left atrial infusion during coronary occlusion. Liposomal delivery of PGE 1 may enhance biologic activity and limit adverse hemodynamic effects. The purpose of this study was to test the hypothesis that intravenous bolus administration of liposomal PGE 1 (TLC C-53, The Liposome Company, Princeton, N.J.) during coronary occlusion would result in myocardial salvage. We compared TLC C-53 (0.5 μg/kg intravenous bolus at 10 and 100 min of occlusion of the left anterior descending coronary artery [LAD]), free PGE 1 (0.1 μg/kg/min infused 10 min after LAD occlusion until reperfusion), placebo liposomes, and control ( n = 7 for each group) in an open-chest canine model of 2 hours of LAD occlusion and reperfusion. Infarct size as a percentage of risk area (mean ± SD) in the control group (58.4% ± 20.0%) was similar to that in animals given placebo liposomes (53.1% ± 12.6%) but was significantly reduced in the groups given TLC C-53 (33.5% ± 9.2%; p < 0.01) or free PGE 1 (37.2% ± 4.8%; p < 0.05) groups. Infarct salvage was significant ( p < 0.05) for the TLC C-53— and PGE 1-treated dogs compared with the control dogs, independent of collateral blood flow by analysis of covariance. Moreover, the ischemic-zone blood flow during reperfusion was significantly higher in the TLC C-53 group compared with the control group or the group receiving free PGE 1. Neutrophil infiltration of ischemic myocardium was significantly inhibited by TLC C-53 as determined by myeloperoxidase assay. Unlike free PGE 1, TLC C-53 did not cause significant tachycardia or hypotension during therapy. In conclusion, TLC C-53 administered intravenously during coronary occlusion significantly reduced infarct size, limited neutrophil infiltration, and improved myocardial blood flow during reperfusion without adverse hemodynamic consequences.

Liposomal doxorubicin-induced toxicity: depletion and impairment of phagocytic activity of liver macrophages.

Doxorubicin entrapped within conventional liposomes (200 nm in diameter; lip-Dox) has major toxic effects on liver macrophages of the rat for a considerable period of time following i.v. administration, with respect to both specific phagocytic capacity and cell numbers. At different time-points after injection of lip-Dox or free doxorubicin, radiolabeled, negatively charged, "empty" test liposomes were injected. Phagocytic capacity was determined by isolating the liver macrophages and measuring the amount of macrophage-associated radioactivity. Four subfractions of liver macrophages of different cell-size and with intrinsically different phagocytic capacity were isolated. Twenty-four hours after injection of lip-Dox, the phagocytic capacity of the larger-sized liver macrophages was strongly decreased. The relatively low intrinsic phagocytic capacity of the smaller-sized macrophages was only slightly impaired. Phagocytic capacity after injection of lip-Dox was nearly restored to control values after 14 days. Blood clearance of Klebsiella pneumoniae bacteria after pre-treatment with lip-Dox was strongly decreased. Pre-treatment with the free drug and/or placebo liposomes had no effect on phagocytic and bacterial blood-clearance capacity. A major depletion of the liver macrophage population was observed, as revealed by both macrophage isolation and histology. Only 2 weeks after injection of lip-Dox, the number of cells had returned to that seen in control animals. In view of the important host-defense functions of the liver macrophages, especially in the control of tumor growth and infection, the findings reported here should be taken into consideration when lip-Dox is to be administered in anti-tumor therapy.

Inhibition of bacterial translocation in obstructive jaundice by muramyl tripeptide phosphatidylethanolamine in the rat

Obstructive jaundice is frequently associated with septic complications and enteric bacteria have been isolated from both the infectious focus and bile in jaundiced patients. The present study aimed to evaluate bacterial translocation and the influence of a macrophage-stimulant (muramyl tripeptide phosphatidylethanolamine) on bacterial translocation in obstructive jaundice. Male Sprague-Dawley rats were subjected to sham operation (n = 10) or common bile-duct ligation and transection (n = 35). Two weeks later, jaundiced animals received either physiological saline (n = 15), muramyl tripeptide phosphatidylethanolamine liposomes (n = 10) or placebo (empty) liposomes (n = 10) orally, while sham-operated rats received physiological saline, 48 h prior to evaluation of enteric bacterial translocation. Blood, bile and caecal contents were collected and cultured aerobically and anaerobically, as were tissue samples from the liver, spleen and mesenteric lymph nodes. Positive mesenteric lymph node cultures in animals with jaundice + saline (7/15; 47%) and jaundice + placebo liposomes (4/10; 40%) significantly differed (p < 0.05) from sham-operated animals (1/10; 10%) and muramyl tripeptide phosphatidylethanolamine treated animals (0/10). Caecal counts (CFU/g) of Escherichia coli, Lactobacilli and aerobic and microaerobic bacteria did not differ statistically among the groups, although the number of E. coli tended to be higher in jaundiced animals. Thus, liposomal muramyl tripeptide phosphatidylethanolamine inhibits bacterial translocation, probably by activating mucosal macrophages and enhancing reticuloendothelial system function in rats with biliary obstruction.

Modification of Reticuloendothelial Function by Muramyl Dipeptide-Encapsulated Liposomes in Jaundiced Rats Treated with Biliary Decompression

Rats with 2 weeks of biliary obstruction, with and without 1 week of concomitant biliary decompression relieving the jaundice, were treated with physiologic saline, free muramyl dipeptide (MDP), placebo liposomes, or liposome-encapsulated MDP. Reticuloendothelial system (RES) function was evaluated by blood clearance of intravenously injected 125I-labelled Escherichia coli. The corrected phagocytic index (alpha) after 1 week of biliary decompression returned to normal levels in animals treated with MDP liposomes, whereas RES function was impaired (P < 0.05) in all other jaundiced and biliary-decompressed groups. In the biliary-decompressed, MDP-liposome-treated group, hepatic uptake of radiolabelled bacteria was significantly higher (P < 0.05) and renal entrapment of bacteria was significantly lower (P < 0.05) than in all other jaundiced and biliary-decompressed groups. We conclude that treatment with MDP liposomes improves the otherwise impaired RES function in rats with biliary obstruction and biliary decompression.

The Lipophilic Muramyl Peptide MTP-PE Is a Potent Inhibitor of HIV Replication in Macrophages

Cultured monocyte-derived macrophages were productively infected with human immunodeficiency virus in vitro. Treatment of these cells shortly after infection and several times thereafter with the free form of MTP-PE had an inhibitory effect on virus production. When the liposomal formulation of MTP-PE was used, higher levels of protection were achieved. The drug was not only effective when added to cells immediately after infection, but it also reduced virus production by cells with an established infection. When the liposomal formulation of MTP-PE was used only one treatment was required to achieve maximal effects. During these studies it was noted that the placebo liposomes had some effect in reducing the reverse transcriptase levels found in the supernatants of infected cells. This reduction could not be explained by direct cytotoxic effect. Both free and liposomal MTP-PE lipid significantly prevented formation of giant cells during the course of infection as well as reduced the cell associated viral antigen.

Treatment of Disseminated Mycobacterium avium Complex Infection of Beige Mice with Liposome-Encapsulated Aminoglycosides

Free and liposome-encapsulated amikacin are active in vitro against intracellular Mycobacterium avium complex (MAC). To examine whether liposome-encapsulated aminoglycosides might kill intracellular MAC more effectively in vivo, beige mice were infected with MAC strain 101 (serotype 1) and after 1 week were treated intravenously every other day (5 doses total) with amikacin liposomes (0.2, 1, or 4 mg/dose), amikacin solution (0.2, 1, or 2 mg), gentamicin liposomes or gentamicin solution (0.2 or 1 mg), placebo liposomes (without aminoglycosides), or buffer. Amikacin and gentamicin liposomes significantly reduced bacterial counts in blood, liver, and spleen (98.5%, 92.7%, and 92.8%, respectively, for the 1-mg dose of amikacin and 92.8%, 99.7%, and 99.4% for gentamicin; 95.7%, 69.7%, and 89.1%, respectively, for the 0.2-mg dose of amikacin and 49.9%, 76.7%, and 89.1% for gentamicin) compared with placebo liposomes and buffer. Equivalent doses of free drug were not associated with significant decreases in viable bacteria. Thus, aminoglycoside liposomes improved bactericidal effects over conventional treatment in disseminated MAC infection, offering potential application in treating MAC infection in humans.