Liposomal delivery of biological response modifiers to macrophages

Macrophages activated to the tumoricidal state can recognize and destroy neoplastic cells and leave normal cells unharmed. Systemic activation of macrophages can be achieved by the intravenous administration of liposomes containing various immunomodulators. Much like any particle, liposomes are cleared from the circulation by phagocytic cells. This passive but specific targeting of immunomodulators to macrophages results in their activation forin vitro andin vivo lysis of tumor cells that can be resistant to conventional therapies.     

Uptake of Archaeobacterial Liposomes and Conventional Liposomes by Phagocytic Cells

Abstract

Liposomes in the 200 nm size range were prepared from the ether lipids extracted from various Archaeobacteria (coined archaeosomes), and from conventional lipids. The entrapment of peroxidase or carboxyfluorescein was used to compare the in vitro uptake of various liposomes by murine peritoneal macrophages, J774A.1 macrophages and several non phagocytic cell lines. While liposomes composed of ester lipids dipalmitoylphosphatidylcholine, or dimyristoylphosphatidylcholine: dimyristoylphosphatidylglycerol: cholesterol (1.8:0.2:1.5, molar ratio) were taken up by macrophage species, the uptake of archaeosomes was 3 to 53 times greater. Uptake by non phagocytic HEp-2, HeLa, and EJ/28 cells was considerably less. Evidence from time-course studies using cytochalasins B+D, sub-optimal temperature or formaldehyde treatments of macrophages, indicated that the archaeosomes lost structural integrity following internalization within the J774A. 1 phagocytic cells. No cytotoxicity was observed in viability or growth assays with J774A. 1 cells, using high doses of three representative types of archaeosomes and one type of conventional-liposome. Therefore, archaeosomes may be well suited to applications where phagocytic cells are a target site.     

Pharmacokinetics,Biodistribution and Therapeutic Efficacy of Doxorubicin Encapsulated in Stealth® Liposomes (Doxil®)

Abstract

Doxil® (Stealth® liposomal doxorubicin HCl) Injection is doxorubicin HCl incorporated into long circulating liposomes that contain surface-grafted polyoxyethylene chains. These surface-grafted polymer chains reduce the interaction of the liposomes with the mononuclear phagocytic system, accounting for the long circulation and altered biodistribution of Stealth liposomes. They also reduce adhesion of the liposomes to cells, blood vessel walls and other surfaces and result in increased vascular permeability of Stealth liposomes compared to conventional liposomes of equal size. Efficacy studies in several tumor models, including human xenograft models, have demonstrated that Doxil is more effective than unencapsulated doxorubicin (Adriamycin) or doxorubicin encapsulated in non-coated conventional liposomes. Doxil exhibits altered plasma pharmacokinetics, with a longer plasma half-life, large AUC and markedly smaller volume of distribution than Adriamycin. Tissue levels of doxorubicin are generally lower in Doxil-treated animals than in animals that receive an equivalent dose of Adriamycin, and Doxil is less cardiotoxic, myelotoxic and nephrotoxic than Adriamycin. Phase I and II studies evaluating the efficacy of Doxil in AIDS-related Kaposi sarcoma have been encouraging, with evidence of increased delivery of drug to the lesions and an overall good response to therapy. The increased efficacy of Doxil is believed to be related to its increased extravasation through the leaky tumor vasculature and its accumulation in tumor tissue.     

In situ activation of murine macrophages by liposomes containing lymphokines

Murine alveolar and peritoneal macrophages harvested after injection of lymphokines encapsulated within multilamellar phospholipid vesicles (liposomes) were tumoricidal in vitro. The state and degree of activation depended on the route of liposome administration. Activation of peritoneal macrophages was achieved by intraperitoneal injection of liposomes and alveolar macrophages were activated by injecting liposomes intravenously but not intraperitoneally. The in vivo rendering of macrophages with tumoricidal properties might be useful toward destruction of tumor cells in vivo.     

INTERFERENCE OF MACROPHAGES WITH IMMUNOTARGETING OF LIPOSOMES

ABSTRACT

We investigated the binding, uptake and intracellular degradation of immunoliposomes by isolated rat liver macrophages in vitro. Immunoliposomes were prepared either by coupling a randomly thiolated anti-CC531 rat colon adenocarcinoma monoclonal antibody to bilayer-incorporated MPB-PE by means of a thioether linkage or by attaching it through its F_c moiety to the distal terminus of hydrazide-modified PEG-DSPE. The two immunoliposome preparations clearly differ in their interaction with the tumor target cells, as well as with the macrophages. At comparable antibody densities both cell types show 1.5–2-fold higher levels of association for the Hz-PEG-immunoliposomes than for the MPB-PEG-immunoliposomes. We provide evidence that immunoliposome macrophage-interaction is both F_c-receptor and scavenger receptor mediated to about equal extents. At low antibody density the hydrazide immunoliposomes favor interaction with the tumor cells to that with macrophages. At higher antibody densities, on the other hand, interaction of these liposomes with the macrophages is increasingly favored, mostly due to enhanced scavenger receptor mediated uptake. The rate of intracellular degradation of (immuno)liposomes internalized by liver macrophages is barely influenced by the presence of either PEG or immunoglobulins on the liposomal surface.     

On the Molecular Mechanism of Steric Stabilization of Liposomes in Biological Fluids

Abstract

Liposomes with specific surface modification overcome rapid in vivo uptake by cells of the mononuclear phagocytic system (MPS) resulting in prolonged circulation in the blood. The structure-function relationship of this effect has been examined by measurements both in vitro and in vivo. The results are reviewed and compared with those from liposomes without surface modification. For example, in the best cases with polyethylene glycol-derivatized phosphatidylethanolamine (PEG-PE) up to 35% of the injected dose remains in the blood and less than 10% is taken up by the two major organs of the MPS, liver and spleen, after 24 hr. This compares with less than 1% in the blood and up to 40% uptake for liposomes without PEG-PE. Steric stabilization has been proposed as a theoretical basis for these results, and some initial results testing this basis have been reported. Here, we discuss steric stabilization in terms of the physico-chemical properties of the liposomes.     

Liposome-mediated therapy of human immunodeficiency virus type-1 and mycobacterium infections

Abstract

We review our recent work on the use of liposomes for the delivery of antiviral agents to human immunodeficiency virus type-1 (HIV-1) infected cells, and antimycobactcrial drugs to cells harboring Mycobacterium avium complex or Mycobacterium tuberculosis. Soluble CD4 has been used to target liposomes to HIV-1-infected cells. Antisense oligodeoxynucleotides have been effectively delivered into HIV-1-infected macrophages using pH-sensitive liposomes. pH-sensitive liposomes with serum stability are being developed as in vivo delivery vehicles. Liposomes encapsulating an HIV-1 protease inhibitor were more effective in inhibiting virus production in infected macrophages than the free drug. Anionic liposomes were found to inhibit HIV-1 infectivity, while cationic liposomes had a differential toxicity for HIV-1-infected macrophages. Lipophilic sulfated cyclodextrins have been synthesized as novel antiviral agents. Liposome-encapsulated ciprofloxacin treatment reduced the number of viable M. avium in macrophages more than the free antibiotic. Liposome-encapsulated paromomycin and sparfloxacin were effective against M. tuberculosis inside macrophages, including multi-drug-resistant strains. Streptomycin encapsulated in liposomes and delivered intravenously or subcutaneously reduced the number of viable M. tuberculosis in infected mice and prevented mortality.     

Induction of alloreactive cytotoxic T lymphocytes by intra-splenic immunization with allogeneic class I Major Histocompatibility Complex DNA and DC-chol cationic liposomes

Abstract

A simple strategy for designing a cancer immunotherapeutic system involves modification of tumor cells from tumor-bearing animals in vivo in such a way that the host can evoke a specific immune response against them. We have expressed allogeneic class I major histocompatibility complex (MHC) molecules on tumor cells, through ex vivo DNA-mediated gene transfer. These molecules are potent immuno-modulators for the stimulation of strong immune reactions against certain malignancies. In order to achieve efficient gene delivery to tumor cells in vivo we have compared the efficiencies of gene transfer into mammalian tumor cells by the biolistic particle delivery system and cationic liposomes. In this report, we have demonstrated that cationic liposomes prepared by DC-chol and DOPE gives the best efficiency of transfection for tumor cells in vivo. We also showed that a strong anti-H-2K^b allo-reactive cytotoxic T lymphocyte (CTL) response could be generated following in vivo immunization of AKR/J mouse spleens with the H-2K^b gene and DC-chol cationic liposomes. The direct immunization of mouse spleens to induce cell-mediated immunity against exogenous antigens may allow alternative treatment strategies for cancer immunotherapy.     

Purine Nucleoside 3′,5′-Cyclic Monophosphates as Hormonal Modulators of Cellular Proliferation,Metastases and Lymphocyte Response

Abstract

A review of the potential role of cAMP and cGMP as hormonal regulators of tumor cell proliferation, metastases and lymphocyte activation reveals that several synthetic purine nucleoside 3′,5′-cyclic monophosphates are more potent and more selective in modulating certain specific responses than the parent natural cyclic nucleotides. cAMP derivatives have been prepared which will temporarily restore transformed cells to the normal phenotype. cAMP analogs may well be found which will selectively inhibit tumor metastases. Certain cGMP analogs could selectively stimulate the lymphocyte response toward the destruction of tumor cells. The synthesis of new cyclic nucleotides should provide unique nontoxic agents that could combat neoplasia on a hormonal basis.     

Release of Active Drug from Intracellular Liposomal Prodrug Depots in Macrophages Controlled by Liposome Composition

Abstract

Several drugs have limited potency due to their rapid elimination or inactivation. The anticancer drug 5-fluoro-deoxyuridine (FUdR), which is frequently used in therapeutic treatment of liver metastases from colon tumors, is an example of such drugs. It is rapidly eliminated from circulation and metabolized, mainly by the hepatocytes in the liver. Over the past few years we have investigated the possibility to keep the drug away from the hepatocytes and to save it from rapid inactivation by encapsulating it in liposomes. In this way the liposomal drug is expected to accumulate in the macrophages of the liver (Kupffer cells), which form a major target site for intravenously administered liposomes. There, as the liposomal structure is gradually degraded by lysosomal enzymes, the drug will be released, initially within the lysosomal compartment, while subsequently it will leak out of the lysosomes and eventually out of the cells so as to become available for uptake by intrahepatically situated tumor cells. In this contribution we describe this system for the prodrug dipalmitoyl-FUdR, incorporated in the liposomal bilayer, requiring an additional step for the drug to become available, i.e. the enzymatic deacylation of the prodrug. It is demonstrated that the rate of intralysosomal degradation of liposomes in Kupffer cells varies substantially with liposomal lipid composition and that the rate of release of active drug from the Kupffer cells parallels the rate of liposome degradation. In addition, it is demonstrated that in this way the antitumor activity of the FUdR can be enhanced by more than two orders of magnitude and that the degree of antitumor activity reflects, to a limited extent, the rate at which the liposomes are degraded.     

Activation of local and systemic anti-tumor immune responses by ablation of solid tumors with intratumoral electrochemical or alpha radiation treatments

Cancer, the most devastating chronic disease affecting humankind, is treated primarily by surgery, chemotherapy, and radiation therapy. Surgery and radiotherapy are mainly used for debulking the primary tumor, while chemotherapy is the most efficient anti-metastatic treatment. To control better metastatic cancer, the host immune system should be stimulated. Yet, successful specific stimulation of the immune system against tumors was seldom achieved even in antigenic tumors. Our working hypothesis is that aggressive in situ tumor ablation can release tumor antigens and danger signals, which will enhance anti-tumor T cell responses resulting in the destruction of residual malignant cells in primary tumors and distant metastases. We developed two efficient in situ ablation treatments for solid cancer, which can be used to destroy the primary tumors and stimulate anti-tumor immune responses. The first treatment, electrochemical ablation, is applied through intratumoral electrodes, which deliver unipolar-pulsed electric currents. The second treatment, diffusing alpha-emitters radiation therapy (DaRT), is based on intratumoral ²²⁴Ra-loaded wire(s) that release by recoil its daughter atoms. These short-lived alpha-emitting atoms spread in the tumor and spray it with lethal alpha particles. It was confirmed that these treatments effectively destroy various malignant animal and human primary solid tumors. As a consequence of such tumor ablation, tumor-derived antigenic material was released and provoked systemic T cell-dependent anti-tumor immunological reactions. These reactions conferred protection against a secondary tumor challenge and destroyed remaining malignant cells in the primary tumor as well as in distant metastases. Such anti-tumor immune responses could be further amplified by the immune adjuvant, CpG. Electrochemical ablation or DaRT together with chemotherapy and immunostimulatory agents can serve as treatment protocols for solid metastatic tumors and can be applied instead of or in combination with surgery.     

Impact of Mycobacterium Avium Infection on Macrophage-Liposome Interaction

Abstract

Mycobacterium avium (M. avium) can survive within macrophages, and alters various functions of its cellular host. Liposomes and other particulate drug carriers have been investigated as a means for enhancing drug delivery to the intracellular site of infection via the endocytic pathway. We have investigated the impact of M. avium infection on the endocytosis, retention, and intracellular disposition of liposomes, as these are essential macrophage functions that may determine the activity of liposome-encapsulated antibiotics. J774, a macrophage-like cell line, was infected with M. avium at various multiplicities. Infected cells accumulated and retained liposomes in a manner similar to uninfected cells, regardless of the time after infection. Moreover, the intracellular trafficking and disposition of liposomes was unaltered in J774 cells that contained live or heat-killed M. avium, as evidenced by the vesicular pH which liposomes encountered within cells. These results suggest that specific essential cellular functions involved in processing particulate drug carriers remain intact in macrophages infected with M. avium.     

Repeated Intravenous Administration of Liposome-Encapsulated Muramyl Tripeptide-Phosphatidyl-Ethanolamine Diminishes Cytotoxic Potential of Subpopulations of Rat Liver Macrophages

Abstract

In this study we investigated the effects of repeated intravenous (i.v.) administration of liposome-encapsulated muramyl tripeptide-phosphatidylethanolamine (lipMTP-PE) on the antitumor functions of rat liver macrophages. Liver macrophage subtractions, differing in cell-size, were isolated by elutriation 24h after the final injection of liposomes and tested for cytotoxicity against radio-labeled C26 adenocarcinoma cells. Prolonged multiple i.v. injections of lipMTP-PE resulted in decreased tumoricidal activity in all but the small-sized subtraction, compared to a single injection.

Immunohistochemical staining of liver sections revealed that while the number of liver macrophages increased significantly after multiple lipMTP-PE injections, the expression of the la antigens on these cells decreased. Additionally, multiple lipMTP-PE treatment resulted in a reduced nitric oxide release in response to muramyl dipeptide in vitro, while incubation with another immunomodulator, lipopoly saccharide, resulted in substantial levels of NO secretion in all subtractions.

Taken together, these results demonstrate a diminished antitumor response and activated state of a significant part of the liver macrophage population after prolonged repeated administration of lipMTP-PE, and provide further insight in the behavior of the liver macrophage population during in vivo treatment with liposomal immunomodulators.     

Optimization and limitations of systemic treatment of murine melanoma metastases with liposomes containing muramyl tripeptide phosphatidylethanolamine

Summary The purpose of these studies was to determine the optimal conditions and limitations for the eradication of spontaneous melanoma metastases by the systemic administration of liposomes containing MTP-PE. Mice whose primary melanoma had been excised were given i.v. injections of liposomes at various schedules. Optimal treatment was achieved by twice weekly administration for 4 weeks (eight i.v. injections). Bioassays failed to reveal the presence of melanoma cells in lungs of mice surviving to day 250 of the experiment. The success of liposome treatment of metastases diminished when the first i. v. injection of liposomes-MTP-PE commenced on day 10 after surgical excision of the local melanoma, as compared with day 3 or day 7 after surgery. We conclude that the major limitation for macrophage-mediated destruction of metastases is the number of tumor cells in the lesions. Because of this limitation, it is unlikely that the systemic activation of macrophages could be used as a single modality for treatment of advanced metastases.     

Enzyme-Controlled Release with Liposomes

Abstract

Achievements of avoiding the RES uptake and seccess in tumor targeting of liposomes in vivo have brought a great challenge to improve drug delivery efficiency of liposomes to cytoplasmic targets of a cell. Enzymes could be used as a built-in mechanism to regulate the drug release from liposomes. It involves disruption of the permeability barrier of liposome bilayer and has advantages of high specificity and high efficiency. Comparing to other methods, this system has increased the controllability of drug release from liposomes.     

LIPOSOME RECOGNITION BY RESIDENT AND NEWLY RECRUITED MURINE LIVER MACROPHAGES

ABSTRACT

The evidence in this communication indicate that, unlike resident Kupffer cells, newly recruited liver macrophages (following monocyte migration from the blood to the liver) use complement receptors to recognize and internalize stearylamine-incorporated liposomes. Within two weeks of hepatic residency complement receptors no longer participate in liposome recognition and uptake.     

Cytotoxicity to a Tumor Cell Line of Epoxy-Phosphatidylinositol Liposomes

Abstract

Selective cytotoxicity of tumor cells induced by liposomal plant phosphatidylinositol (Ptdlns) has been studied. We could not always obtain cytotoxic plant Ptdlns liposomes in a series of experiments. Moreover, animal Ptdlns occasionally showed cytotoxicity towards tumor cells. By ¹H nuclear magnetic resonance analysis of non-and cytotoxic Ptdlns, it has been suggested that oxidized acyl residues, such as hydroperoxide or dioxetan, may have been present in the cytotoxic Ptdlns. We have prepared epoxy-Ptdlns, as an analogous compound of the oxidized lipid, from noncytotoxic Ptdlns by chemical synthesis. the epoxy-PtdIns liposomes showed cytotoxicity towards tumor cells. In the presence of 100 µM epoxy-Ptdlns liposomes, normal human peripheral lymphocytes survived for 3 days, but Raji human lymphoblastoid-like cells were almost all killed. However, at higher concentrations, epoxy-PtdIns liposomes were also cytotoxic to normal cells.     

Liposomal muramyl dipeptide therapy of experimental M5076 liver metastases in mice

Summary The effectiveness ofN-acetylmuramyl-l-alanyl-d-isoglutamine (MDP) or of liposomes containing a lipophilic MDP derivative, MDP-glyceroyldipalmitate MDP-GDP in inhibiting the growth of M5076 reticulum cell sarcoma liver metastases in C57BL/6 mice has been determined. MDP (100 µg) or liposomal MDP-GDP (2.5 µmol containing 1 µg) were equally effective in inhibiting liver metastatic growth when given as a single treatment 3 days before tumor cell injection. Therapeutic treatment, initiated 3 days after tumor cell injection and continued for a period of 2 weeks, failed to inhibit metastatic growth. Activation of thioglycollate-elicited peritoneal macrophages or Kupffer cells in vitro with MDP or liposomal MDP-GDP resulted in the expression of tumoricidal activity against M5076 tumor cells. Adoptive cellular therapy with four injections of 2 × 10⁶ macrophages was ineffective: activation of the macrophages with either MDP or liposomal MDP-GDP prior to injection was effective in inhibiting liver metastatic growth. Incorporation of the macrophage toxin dichlorodimethylene diphosphonate within liposomes containing MDP-GDP abolished the ability of such liposomes to induce macrophage or Kupffer cell tumoricidal activity in vitro as well as the antitumor activity when administered 3 days before tumor cell challenge.     

Receptor-Specific Delivery of Liposomes Via Folate-Peg-Chol

Abstract

A novel lipophilic conjugate of folate, folate-PEG-Chol, was synthesized and evaluated for receptor-mediated targeting of liposomes to tumor cells. Liposomes composed of DSPC/Chol/PEG-DSPE/folate-PEG-Chol (60/ 34/5/1, m/m) were taken up by cultured folate receptor-bearing KB cells via a saturable mechanism. Cellular binding of these liposomes could be competitively inhibited by free folic acid with an IC₅₀ of 0.39 mM, indicating an extraordinarily high binding affinity. Fluorescence micrographs of KB cells treated with targeted liposomes encapsulating calcein showed that they were distributed both on the cell surface and in intracellular vesicular compartments. Targeted liposomes carrying doxorubicin were shown to be 38 times more toxic to KB cells than non-targeted control liposomes. A biodistribution study in receptor-positive tumor-bearing C57BL/6 mice showed no significant differences between the tumor uptake of folate-PEG-liposomes and non-targeted control liposomes. This study has demonstrated that cholesterol could be used as an alternative to phospholipids as an effective anchor for incorporation of a targeting ligand into liposomes.     

Destruction of tumor cells by BCG-activated alqolar macrophages.

Alveolar macrophages obtained from Syrian golden hamsters were tested for their ability to destroy tumor cells. Only macrophages obtained from BCG immune animals rechallenged intratracheally with BCG five days before assay exhibited cytotoxic activity. Maximum destruction of tumor cells occurred after 5 days of incubation. Immunologic activation of macrophages was required to attain cytotoxic alveolar macrophages. Induction of inflammatory lung exudates by a variety of nonspecific irritants did not result in tumor cell destruction by macrophages. These observations may prove useful in designing an approach for immunotherapy of lung cancer.