Chloroquine delivery to erythrocytes inPlasmodium berghei-infected mice using antibody-bearing liposomes as drug vehicles

Suitability of anti-erythrocyte F_(ab’)2-bearing liposomes as vehicles for chloroquine in the treatment of chloroquine resistantPlasmodium berghei infections in mice has been examined. Free chloroquine or chloroquine encapsulated in antibody-free liposomes failed to show much effect on the resistant infections, but the same doses of this drug after being encapsulated in antibody-bearing liposomes exhibited a significant inhibitory effect on this infection. These results indicate that chloroquine delivery in antibody targeted liposomes may help in the successful treatment of the chloroquine resistant malarial infections. CDRI Communication No. 4705.     

Drug targeting in Leishmania donovani infections using tuftsin-bearing liposomes as drug vehicles

The efficacy of sodium stibogluconate against Leishmania donovani infections was markedly enhanced by encapsulating this drug in tuftsin-bearing liposomes. Also, pretreatment of the animals with these liposomes (free of drug) rendered them resistant to this infection, possibly by activating the host's macrophages. These results demonstrate that tuftsin-bearing liposomes besides delivering the drug to the target cells could also enhance the nonspecific resistance against infections, thus offering an additional advantage over the use of tuftsin-free liposomes as drug carriers in leishmania therapy.     

Antibody-mediated targeting of liposomes to erythrocytes in the whole blood

F(ab′)₂ fragments derived from anti-rat erythrocyte antibody or normal rabbit serum IgG were covalently attached to the surface of liposomes consisting of equimolar amounts of egg phosphatidylcholine and cholesterol. These liposomes were interacted with rat, monkey or mouse blood, and their binding to both red and white blood cells was determined. Results of these studies show that coupling of liposomes to anti-rat erythrocyte F(ab′)₂ considerably enhances their binding to erythrocytes in rat blood. However, no such increase in the binding was observed with rat leukocytes or monkey and mouse erythrocytes. Besides, the interactions between the liposomes and target cells did not affect the permeability properties of the liposome bilayer. These observations indicate that liposomes coupled to cell-specific antibodies may serve as highly useful carriers for homing of drugs/enzymes to specific cells in biophase.     

Biophysical aspects of using liposomes as delivery vehicles

Liposomes are used as biocompatible carriers of drugs, peptides, proteins, plasmic DNA, antisense oligonucleotides or ribozymes, for pharmaceutical, cosmetic, and biochemical purposes. The enormous versatility in particle size and in the physical parameters of the lipids affords an attractive potential for constructing tailor-made vehicles for a wide range of applications. Some of the recent literature will be reviewed here and presented from a biophysical point of view, thus providing a background for the more specialized articles in this special issue on liposome technology. Different properties (size, colloidal behavior, phase transitions, and polymorphism) of diverse lipid formulations (liposomes, lipoplexes, cubic phases, emulsions, and solid lipid nanoparticles) for distinct applications (parenteral, transdermal, pulmonary, and oral administration) will be rationalized in terms of common structural, thermodynamic and kinetic parameters of the lipids. This general biophysical basis helps to understand pharmaceutically relevant aspects such as liposome stability during storage and towards serum, the biodistribution and specific targeting of cargo, and how to trigger drug release and membrane fusion. Methods for the preparation and characterization of liposomal formulations in vitro will be outlined, too.     

CD4 and CD7 molecules as targets for drug delivery from antibody bearing liposomes.

We have examined two T lymphocyte cell surface molecules, CD4 and CD7, as targets for specific delivery of drugs from antibody-directed liposomes. The efficiency of uptake by peripheral lymphocytes, thymocytes, and two CEM sublines (CEM.MRS and CEM-T4) of anti-CD4 and anti-CD7 liposomes containing methotrexate was evaluated by the methotrexate-mediated inhibition of the incorporation of d-[3H]Urd into DNA. This was compared with similar liposomes targeted to MHC-encoded HLA class I molecules, which are known to be efficiently taken up by T cells. Despite the lower expression of CD7 molecules relative to HLA class I on most cell lines, CD7 was shown to be a good target for drug delivery. The results of an internalization study using radiolabeled Protein A showed that a higher proportion of CD7 molecules was internalized than HLA class I molecules. CD4-targeted liposomes, in contrast, were relatively ineffective for drug delivery for lymphoid cells, and only partially inhibited CEM-T4 cells. The lack of toxicity correlated with poor internalization of the target molecule on most cell lines. The drug effect of anti-CD4 liposomes was more pronounced on HeLa-T4, which is an epithelial cell line transfected with the CD4 gene. In contrast to lymphoid cells, these cells efficiently internalized CD4 molecules. PMA is known to down-regulate surface expression of CD4 molecules on various T cells. Internalization of CD4 was induced by PMA, but PMA failed to induce cytotoxicity of CD4-targeted liposomes for CEM.MRS. The internalized drug was probably degraded rapidly because internalized anti-CD4 antibody-bound Protein A was degraded very rapidly.     

Antibody-mediated targeting of liposomes to red cells in vivo

Covalent attachment of anti-rat erythrocyte F(ab')2 to liposomes specifically enhanced their binding to rat erythrocytes in vivo and reduced their uptake by the liver. Furthermore, at least 20-30% of the cell-bound liposomes delivered their contents to the cells. Besides, the liposome binding did not affect the survival time of the target cells at least up to 3 h in the blood circulation. These results demonstrate for the first time that liposomes can be successfully targeted to cells other than liver cells in vivo.     

Affinity targeting of Sendai virions to desialized human erythrocytes using hybrid antibody molecules

F(ab') fragments obtained from anti-Sendai virus antibodies were chemically coupled to F(ab') fragments obtained from anti-human red blood cell antibodies (anti-hRBC-Ab). This led to the formation of hybrid antibody molecules (anti-SV-anti-hRBC(F(ab')2) each of whose F(ab') fragment possessed different binding specificity. The anti-SV(F(ab'] part of the hybrid molecule interacted specifically with Sendai virus particles, while the anti-hRBC(F(ab'] part interacted with the surface of hRBC. These hybrid antibodies were able to mediate binding and fusion of SV to hRBC, from which the virus receptors were removed by treatment with neuraminidase (desialized hRBC). Neither anti-SV-anti-SV(F(ab')2) nor anti-hRBC-anti-hRBC(F(ab')2) possessed the same ability. Thus, it is shown that soluble, hybrid antibody molecules can effectively mediate functional binding of Sendai virus to virus-receptor-depleted cells.     

Use of liposomes as drug delivery vehicles for treatment of melanoma

Melanoma is a progressive disease that claims many lives each year due to lack of therapeutics effective for the long‐term treatment of patients. Currently, the best treatment option is early detection followed by surgical removal. Better melanoma therapies that are effectively delivered to tumors with minimal toxicity for patients are urgently needed. Nanotechnologies provide one approach to encapsulate therapeutic agents leading to improvements in circulation time, enhanced tumor uptake, avoidance of the reticulo‐endothelial system, and minimization of toxicity. Liposomes in particular are a promising nanotechnology that can be used for more effective delivery of therapeutic agents to treat melanoma. Liposomes delivering chemotherapies, siRNA, asODNs, DNA, and radioactive particles are just some of the promising new nanotechnology based therapies under development for the treatment of melanoma that are discussed in this review.     

Monoclonal antibody covalently coupled to liposomes: Specific targeting to cells

We have evaluated optimal conditions for coupling monoclonal antibody to small unilamellar lipisomes. Coupling of an IgG_2a monoclonal anti-β₂-microglobulin antibody, which reacts with human cells, was examined in detail. Liposomes were composed of dipalmitoyl lecithin and cholesterol, and variable quantities of phosphatidylethanolamine substituted with the heterobifunctional cross-linking reagent N-hydroxysuccinimidyl 3-(2-pyridyldithio) propionate (SPDP). They were reacted with antibody derivatized with the same reagent at a 5- to 20-fold molar excess, and activated by mild reduction. This degree of SPDP modification had no effect on the capacity of the antibody to bind to its target antigen. More than 40% of antibody could be reproducibly bound to liposomes, resulting in the coupling of from 1 to 10 antibody molecules per liposome (mean diameter.580 Å). The coupling reaction did not lead to loss of carboxyfluorescein encapsulated within liposomes. At least 80% of liposomes carried nondenatured antibody, as confirmed by precipitation of liposomes and encapsulated carboxyfluorescein by Staphylococcus aureus, strain Cowan I. The liposome-coupled antibody retained its immunological specificity: only cells expressing human β₂-microglobulin bound liposomes in vitro, and the binding was inhibited by the free antibody in solution. Results with antibodies of different antigenic specificity confirm that the technique can be generally applied.     

Development of amphotericin B liposomes bearing antibody specific to Candida albicans

Summary Liposomes expressing external antibody specific for Candida albicans and encapsulating amphotericin B were developed and characterized in this study. Antibody was first modified by the covalent attachment of palmitic acid residues. Liposomes were produced by reverse-phase evaporation and modified antibody was incorporated into these liposomes via the hydrophobic interaction between the palmitic acid and the phospholipids composing the liposomes. The liposomes were characterized as to the amount of amphotericin B by spectroscopy and for the presence of antibody by protein analysis and secondary immunolabeling by fluorescent and electron microscopic methods. Immunogold labeling showed that the antibody was being expressed externally on the liposomes in the electron microscopic studies and the specificity of these liposomes for C. albicans was observed by secondary immunofluorescence.     

Long-living liposomes as potential drug carriers

Neutral, unilamellar liposomes (vesicles) composed of a dialkyl analog of phosphatidylcholine and cholesterol, and containing ¹⁴C-maltose as entrapped marker, were administered intravenously to mice. After one and two days, radioactivity in blood and liver remained 3–4 times higher than after administration of liposomes of egg (diester) phosphatidylcholine and cholesterol. It appears that the vesicles were taken into liver cells by endocytosis, and that phospholipases are involved in the capture as well as in the breakdown of conventional liposomes. Liposomes that are semi-resistant to catabolic enzymes may become useful in the manipulation of drug delivery.     

Rifampicin-loaded liposomes for the passive targeting to alveolar macrophages: in vitro and in vivo evaluation

Mycobacterium avium complex (MAC), the most frequent cause of opportunistic nontuberculous pulmonary infection, is made up of a group of intracellular pathogens that are able to survive and multiply inside lung alveolar macrophages. As nebulized liposomes are reported to be effective to target antibacterial agents to macrophages, in this work we have prepared and characterized re-dispersible freeze-dried rifampicin (RFP)-loaded vesicles by using soy lecithin (SL) and a commercial, enriched mixture of soy phosphatidylcholine (Phospholipon 90, P90) with or without cholesterol. The obtained results showed that RFP could be loaded stably in SL vesicles only when cholesterol was not present in the film preparation, whereas with P90 vesicles, the highest stability was obtained with formulations prepared with P90/cholesterol 7:1 or 4:1 molar ratios. RFP-liposome aerosols were generated using an efficient high-output continuous-flow nebulizer, driven by a compressor. After the experiments, nebulization efficiency (NE%) and nebulization efficiency of the encapsulated drug (NEED%) were evaluated. The results of our study indicated that nebulization properties and viscosity of formulations prepared with the low-transition-temperature phospholipids, SL and P90, are affected by vesicle composition. However, all formulations showed a good stability during nebulization and they were able to retain more than 65% of the incorporated drug. The effect of liposome encapsulation on lung levels of RFP following aerosol inhalation was determined in rats. The in vitro intracellular activity of RFP-loaded liposomes against MAC residing in macrophage-like J774 cells was also evaluated. Results indicated that liposomes are able to inhibit the growth of MAC in infected macrophages and to reach the lower airways in rats.     

Curdlan gels as protein drug delivery vehicles

The use of curdlan, a natural -1,3-glucan, in protein drug delivery vehicles was studied by carrying out in vitro release studies with curdlan gels containing bovine serum albumin (BSA) as a model protein. Addition of urea (8 M) decreased the gel formation temperature to 37°C. Curdlan was hydroxyethylated in order to form gels under mild conditions such as physiological temperature and pH. In gels formed in 8 M urea solution, urea was almost released after 2 h while BSA was completely released after 45–100 h. The total time for complete release of BSA increased with curdlan concentration within gels. The strength of hydroxyethylated curdlan gels (385.7 dyne cm^–2) was weaker than that of curdlan gels formed in 8 M urea solution (6277 dyne cm^–2).     

In vivo liver and lung targeting of adriamycin encapsulated in glutaraldehyde-treated murine erythrocytes

Treatment of adriamycin-loaded erythrocytes from B6D2F1 mice with 0.1% glutaraldehyde produced the following effects: a considerable decrease in the in vitro leakage of the unmodified drug and a selective liver (and, to a lesser extent, lung) uptake of the encapsulated drug (70% of the injected dose) compared to drug leakage from, and tissue distribution of, carrier erythrocytes not treated with glutaraldehyde. The liver vascular bed was not saturated by five daily intravenous injections of 20 microliters of glutaraldehyde-treated erythrocytes, which allows a total dosage of 200 micrograms of the drug (half the LD50 value) to be administered. No appreciable liver damage results from extensive and prolonged uptake of glutaraldehyde-treated carrier erythrocytes. Entrapment of adriamycin within erythrocytes along with glutaraldehyde treatment of the carrier cells seems to be a promising therapeutic strategy against liver (and lung) tumors.     

In vitro targeting of erythrocytes to cytotoxic T-cells by coupling of Thy-1.2 monoclonal antibody.

A method was explored to develop a general means to target erythrocytes to T-cells in vitro. Mouse erythrocytes were coupled with an anti-Thy-1.2 monoclonal antibody by two methods. Chromic chloride coupling of antibody was preferred to biotinylation. The morphology, osmotic fragility, and the hematological values of treated cells were normal compared with those of control erythrocytes. Antibody-coupled erythrocytes were incubated with cytotoxic T-lymphocytes (CTLL) in vitro at a 20:1 ratio. Approximately 60-70% of the CTLL formed rosettes. The cell mixture was subjected to gradient centrifugation and separated into four fractions. THe rosettes were clearly identified only in the treated group containing anti-Thy-1.2-coupled erythrocytes. No rosettes were found when aspecific monoclonal antibody was coupled to erythrocytes. Examination by scanning and transmission electron microscopy revealed CTLL with 4-5 erythrocytes attached to them but did not show any evidence of membrane fusion. Rosettes of CTLL incubated in vitro proliferated as well as CTLL alone and maintained their dependency on interleukin-2. Targeting of erythrocyte carriers to lymphocytes offers the potential for delivery of molecules directly to the target cell.     

Magnetic targeting of rhodamine-labeled superparamagnetic liposomes to solid tumors: in vivo tracking by fibered confocal fluorescence microscopy

Polyethylene glycol (PEG)ylated and rhodamine-labeled liposomes loaded with maghemite nanocrystals provide a novel nanoscaled hybrid system for magnetic targeting to solid tumors in possible combination with double in vivo imaging by fluorescence microscopy and magnetic resonance imaging (MRI). Human prostate adenocarcinoma tumors implanted in mice were used as a system model. A magnetic field gradient was produced at the tumor level by external apposition of a magnet. Noninvasive fibered confocal fluorescence microscopy was successfully used to track the liposomes in vivo within organs and tumor blood vessels. Active targeting to the magnet-exposed tumors was clearly shown, in agreement with previous MRI studies. The liposomes were driven and accumulated within the microvasculature through a process that preserved vesicle structure and content.     

Sterically stabilized liposomes bearing anti-HLA-DR antibodies for targeting the primary cellular reservoirs of HIV-1

The ability of liposomes bearing anti-HLA-DR Fab' fragments at the end termini of polyethyleneglycol chains (sterically stabilized immunoliposomes) to target HLA-DR expressing cells and increase the accumulation of liposomes into lymphoid organs has been evaluated and compared to that of conventional liposomes, sterically stabilized liposomes and conventional immunoliposomes after a single subcutaneous injection to mice. The accumulation of sterically stabilized liposomes in lymph nodes was higher than that of conventional liposomes. Sterically stabilized immunoliposomes accumulated much better than conventional immunoliposomes in all tissues indicating that the presence of PEG has an important effect on the uptake of immunoliposomes by the lymphatic system. Fluorescence microscopy studies showed that sterically stabilized liposomes are mainly localized in macrophage-rich areas such as the subcapsular region of lymph nodes and in the red pulp and marginal zone of the spleen. In contrast, sterically stabilized immunoliposomes mostly accumulated in the cortex in which follicles are located and in the white pulp of the spleen. As the human HLA-DR determinant of the major histocompatibility complex class II is expressed on activated CD4+ T lymphocytes and antigen presenting cells such as monocyte/macrophages and dendritic cells, known as the cellular reservoirs of HIV-1, liposomes bearing anti-HLA-DR antibodies constitute an attractive approach to concentrate drugs in HIV-1 reservoirs and improve their therapeutic effect.     

Characterization of antibody covalently coupled to liposomes

We have explored the covalent coupling of fatty acids to immunoglobulin G(IgG). N-hydroxysuccinimide ester of palmitic acid (NHSP) was used to couple palmitic acid to either a mouse monoclonal antibody to the major histocompatibility antigen, H-2^k, or goat antibody to the major glycoprotein of the Molony Leukemia Virus, gp-70. The reaction was characterized in terms of the time course, input ratio of NHSP to IgG, stoichiometry of the coupling, distribution of palmitic acid in the IgG subunits, and the antigen binding capacity of the coupled antibody. Incorporation of the fatty acid modified IgG into liposomal membranes using a detergent-dialysis method was studied as a function of extent of fatty acid coupling. Finally, the binding of IgG-coated liposomes with cells expressing proper antigens was characterized. The major conclusions were: (1) the optimal molar ratio of NHSP to IgG in the reaction was between 10 and 20, which yields about 4–5 palmitoyl chains per IgG molecule; (2) at this level of coupling, the antigen binding capacity of the IgG antibody decreased about 3–4-fold; (3) incorporation of the coupled antibody into unilamellar liposomes (about 1000 Å diameter) can be achieved with a deoxycholate-dialysis method with an optimal lipid-to-protein ratio of 10:1 (w/w); (4) there were about 48 IgG molecules incorporated per liposome under these conditions; (5) the apparent dissociation constant of the liposome-bound antibody under the optimal condition was about 6–7-fold higher than that of the native antibody; (6) binding of antibody to the target cells was accompanied by binding of liposomal lipids; both bindings could be blocked by pretreatment of cell with unmodified antibody.