Liposomes to Induce Potent Humorale Responses to T-Independent Antigens

Abstract

When liposomes are used to present haptens and T-independent antigens to the immune system the response is primarily of the IgM type. This response can be switched to a potent IgG type by the incorporation within the liposomal structure of either exogenous T-epitopes, immunostimulants, or both.

The capability of liposomes to co-present separate B-and T-epitopes and immunostimulants, without the necessity for covalent conjugation, thus makes them ideal candidates as carriers for vaccines where the immune response is limited to recognition of T-epitopes within the antigen.     

Physical properties and biological interactions of liposomes developed as a drug carrier in the field of regenerative medicine

Liposomes are used for encapsulation of the active compounds in different therapies, with the increasing frequency. The important areas of clinical applications of liposomes are cancer targeted treatment, antibiotic delivery or regenerative medicine. The liposomes can transfer both hydrophilic and hydrophobic compounds and have the lipid bilayer which imitates the cell membrane. Liposomes additionally may extend half-live period of drugs and protect them against the elimination in different ways, such as phagocytosis, enzymatic cleavage or exclusion by detoxification. The size and charge of liposomes play an important role in drug distribution and absorption into the cell. Limited data is available on the effects of liposomes on stem cells and progenitor cells. In this article, we examined the effect of charged conventional liposomes on growth of mesenchymal and blood stem cells isolated from umbilical cord. The data suggest a likelihood, that positively charged liposomes could impair stem cell growth and metabolism. Different methodological approaches allowed for the selection of negatively charged liposomes for further experiments, as the only type of liposomes which has the lowest cytotoxicity and does not affect hematopoietic cell proliferation.     

Effect of cisplatin containing liposomes formulated by unsaturated chain-containing lipids on gynecological tumor cells

Gynecological tumors are major therapeutic areas of platinum-based anticancer drugs. Here, we report the characterization and in vitro biological assays of cisplatin-containing Egg L-α-phosphatidylcholine liposomes with different amounts of cholesterol. Dynamic light scattering estimated sizes of all obtained liposomes in the 100?nm range that are suitable for in vivo use. On the basis of these data and of the drug loading values, the best formulation has been selected. Stability and drug release properties of platinum-containing liposomes have been verified in serum. The growth inhibitory effects of both liposomal and free drug in a panel of ovarian and breast human cancer cell lines, characterized by a different drug sensitivity, give comparable or better results with respect to free cisplatin drug.     

Effect of Peg Homopolymer and Grafted Amphiphilic Peg-Palmityl on the Thermotropic Phase Behavior of 1,2-Dipalmitoyl-Sn-Glycero-3-Phosphocholine Bilayer

Abstract

Phospholipids covalently attached to polyethylene glycol (PEG-PE) are routinely used for the preparation of long-circulating liposomes. The common preparation procedure for long-circulating liposomes involves use of organic solvent. Although there is a plethora of studies describing the interaction of PEG-PE with bilayers, little is known about the effects of PEG homopolymers and single chain amphiphilic PEG on liposome structure. In the present investigation the interaction of PEG homopolymer and amphiphilic PEG-palmityl conjugate with large multilamellar liposomes composed of 1,2-dipalmitoyl-sn-glycero-phosphocholine was investigated utilizing differential scanning calorimetry. Vesicle and aggregate sizes were determined by dynamic light scattering. DSC thermograms revealed interaction of PEG homopolymer with DPPC when the two are premixed in organic solvent. The data suggest that PEG interacts with the phospholipid acyl chains deep in the bilayer. Several questions are raised regarding the suitability of the current procedure for preparation of long-circulating liposomes which utilizes organic solvent. Incorporation of only 2 mol% 5 kDa PEG-palmityl conjugate completely solubilized DPPC liposomes. Packing geometry of the lipid anchor, irrespective of the polymer molecular weight, is suggested to be the primary factor for successful grafting of hydrophilic polymers on liposomes. Pure PEG-palmityl formed self-assembled organized structures of potential use in the delivery of poorly soluble drugs.     

The liposomal daunorubicin plus tamoxifen: improving the stability, uptake, and biodistribution of carriers

The synergistic effects of tamoxifen on the sensitivity of MCF-7 cells to daunorubicin have been reported. Whether the effects of daunorubicin on MCF-7/adr cells can be improved by tamoxifen in liposomes and how tamoxifen changes daunorubicin's behavior in vivo remains unclear. The aim of this study was to investigate the effects of tamoxifen on the uptake and biodistribution of daunorubicin liposomes by breast-cancer-resistant MCF-7/adr cells in vitro and in vivo. The uptake of liposomes by MCF-7/adr cells in vitro studies was measured using flow cytometry and laser confocal microscopy. The biodistributions of carriers and free drugs were evaluated by DiR dye using in vivo imaging. Tamoxifen obviously enhanced the cellular uptake of liposomes by MCF-7/adr cells in time-dependent manners. According to the results from in vivo imaging analysis, the mean fluorescence intensity of DiR liposomes with tamoxifen in the tumor regions of MCF-7/adr tumor-bearing nude mice was much stronger than that of DiR liposomes alone (16,450 ± 1,331 versus 3,666 ± 321; n = 3). Pegylated liposomes elongated the existence of daunorubicin in the circulatory system and the enhanced permeability and retention effect enhanced its concentration in local tumor tissues, which may provide the precondition for tamoxifen further promoting the uptake by MCF-7/Adr cells in vivo. Using daunorubicin liposomes and tamoxifen together generates better biodistribution profiles in tumor tissue than using daunorubicin liposomes only, which contributes to improving the therapeutic effect of breast cancer treatment.     

Binding of hydrophobic drugs to lipid bilayers and to the (Ca2+ + Mg2+)-ATPase

Microelectrophoretic studies of the binding of a number of commonly used hydrophobic amine drugs to liposomes demonstrated the existence of relatively large surface potentials associated with binding of the protonated forms of the drugs. A theoretical treatment based on Langmuir adsorption isotherms and the Gouy-Chapman theory of the diffuse double layer allows estimation of drug-binding constants from electrophoretic mobility data. Such constants allow calculation of the charge effects arising from drug binding in more complex membrane systems, and it is shown that shifts in the apparent Ca+ affinity of the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum in the presence of hydrophobic amine drugs are readily explicable in terms of the electrostatic effects of drug binding.     

The effect of nonspecific immune stimulation with Corynebacterium parvum and polidin on the Ehrlich ascites tumor growth

Investigations were performed: a) to compare the effect of two nonspecific immunostimulants, Polidin and Corynebacterium parvum, on the development of Ehrlich ascites carcinoma in mice; b) to determine whether the effects are dependent on the tumor cell dose inoculated into the animals. C. parvum and Polidin administered prior to Ehrlich ascites tumor inoculation have a protective effect evidenced by a delay in tumor development, a retardation in tumor growth and a prolonged survival of the tumor host. The effect of immunostimulants was highly dependent on the tumor cell dose inoculated into mice and was more marked with C. parvum.     

脂质体递药系统的靶向修饰

作为药物递送载体，脂质体（LPs）由于免疫原性低、稳定性好、毒性低和成本低而被认为是有前途的纳米药物递送系统。然而，LPs的靶向递送效果并不理想，往往会对正常的机体细胞造成伤害，因此，如何优化LPs药物，使其具有靶向性仍然是当前研究的重点。本文结合近年来国内外相关研究进展，重点介绍了多肽、抗体、糖类、配体，以及核酸适配体等靶向修饰物对LPs功能的影响，并归纳总结了各种靶向修饰目前存在的优势与挑战，以期对LPs给药系统的进一步研究提供科学参考及新药研发提供理论依据。     

Preparation and characterization of immunoliposomes for targeting of antiviral agents

Antibodies specific to avian myeloblastosis virus envelope glycoprotein gp80 were raised. Immunoliposomes were prepared using anti-avian myeloblastosis virus envelope glycoprotein gp80 antibody. The antibody was palmitoylated to facilitate its incorporation into lipid bilayers of liposomes. The fluorescence emission spectra of palmitoylated IgG have exhibited a shift in emission maximum from 330 to 370 nm when it was incorporated into the liposomes. At least 50% of the incorporated antibody molecules were found to be oriented towards the outside in the liposomes. The average size of the liposome was found to be 300 A, and on an average, 15 antibody molecules were shown to be present in a liposome. When adriamycin encapsulated in immunoliposomes was incubated in a medium containing serum for 72 h, about 75% of the drug was retained in liposomes. In vivo localization studies, revealed an enhanced delivery of drug encapsulated in immunoliposomes to the target tissue, as compared to free drug or drug encapsulated in free liposomes. These data suggest a possible use of the drugs encapsulated in immunoliposomes to deliver the drugs in target areas, thereby reducing side effects caused by antiviral agents.     

Immunostimulants,adjuvants, and vaccine carriers in fish: Applications to aquaculture

Use of immunostimulants, adjuvants, and vaccine carriers in fish culture offers a wide range of attractive methods for inducing and building up protection against diseases. Immunostimulants and adjuvants can be administered before, with, or after vaccines to amplify the specific immune response generating elevations of circulating antibody titers and numbers of plaque-forming cells. Special applications of immunostimulants include assisting shower or other regimens to increase topical uptake of vaccines. In addition, immunostimulants may be used alone, inducing elevated activities in the nonspecific defense mechanisms such as increased oxidative activity of neutrophils, augmented engulfment activity of phagocytic cells, or potentiating cytotoxic cells. In cases where disease outbreaks are cyclical and can be predicted, losses may be reduced by elevating the nonspecific defense mechanisms, and the immunostimulants may be used in anticipation of events to prevent losses from diseases. Complete Freund's adjuvant was one of the first immunostimulants used in animals to elevate the specific immune response, and it has also been successfully used in conjunction with injection of fish bacterins. Other adjuvants, immunostimulants, and biological response modifiers that have been used in fisheries research include levamisole, salt baths, and bacterial lipopolysaccharides. Vaccines have been adsorbed to inert particles, such as bentonite on latex beads, to carry the immunogens to maximize in vivo uptake for bath immunization and to facilitate in vitro phagocytosis. Each substance presents special problems in timing and method of administration (injection, immersion, oral—by feed—or flush treatments), dosage adjustments for size and fish species, storage stability, and cost. An additional consideration is that the nonspecific defense mechanisms and immune responses in fish are highly variable among individuals and statistical validation requires appropriate sample numbers and carefully controlled experiments.This article reviews the literature and present concepts of use of immunostimulants, adjuvants, and vaccine carriers in fish. Cautions for use are noted, as some of these potent substances can suppress or alter biological pathways if used inappropriately. Recent research, defining pathways of the action of immunostimulants, adjuvants, and vaccine carriers, helps explain how these substances activate the protective mechanisms in fish. In addition, immunostimulants used alone hold tremendous potential for use in fish farms, hatcheries, and aquaculture facilities to reduce losses from infectious diseases. Research on the immunostimulant, levamisole, and the light oil adjuvants for use in food fish is in progress. Applications for use of these immunostimulants are proposed.     

Liposome coated stents: a method to deliver drugs to the site of action and improve stent blood-compatibility

A method to correct stent related complications non-invasively, is the local delivery of therapeutic agents. Different drugs have been delivered on stents, after being either dispersed or encapsulated in polymeric materials, and placed on stents to form drug-eluting-stents (DE-stents). Investigation of possibility to cover polymer - coated metallic stents, with liposomal drugs, for preparation of novel DE-liposome-coated-stents, has been initiated few years ago. In this context our research has been focused on answering the following questions: (i) Can liposomes be applied as coatings on polymer covered stents? (ii) Can drug release from liposome coated-stents be controlled? And: (iii) how is haemo-compatibility of stents affected? The results of the experiments carried out demonstrate that liposomal formulations of drugs can be used as coating systems of polymer covered stents for achieving sustained release of drugs at the site of interest. By modifying liposome characteristics, different amounts of drugs may be placed on the stents and their release rates can be adjusted for maximum therapeutic benefit. Finally, haemocompatibility of stents is highly improved (mainly in terms of cell adhesion and activation of coagulation system), when stents are coated with heparin-encapsulating -DRV liposomes.     

The disordering effect of hyoscyamine drugs on phospholipid membranes

The effect of hyoscyamine drugs on the fluidity of dipalmitoylphosphatidylcholine liposomes has been studied by differential scanning calorimetry (DSC), electron spin resonance spectroscopy (ESR), fluorescence polarization and freeze-fracture electron microscopic techniques. DSC results indicate that anisodamine, anisodine, atropine and scopolamine all increase the fluidity of dipalmitoylphosphatidylcholine liposomes but with different degrees of efficiency. The increasing of fluidity of dipalmitoylphosphatidylcholine liposomes by hyoscyamine drugs is in a dose-dependent way. Increase of the fluidity of phosphatidylcholine liposomes by anisodamine was also shown by the other three methods. The possible mechanism of hyoscyamine-membrane interaction is discussed.     

Synergistic Effect of Phospholipid-Based Liposomes and Propylthiouracil on U-937 Cell Growth

Scientific evidence indicates that exogenous phospholipids in the form of liposomes can affect cell growth. Effects of liposomes on cell growth depend on several factors including composition of liposomes, lipid concentration, and type of cells studied. Because phagocytic cells such as monocytes and macrophages are natural targets of liposomes, intracellular delivery of drugs to modulate cellular activity of these cells is of interest. We explored the effects of phospholipid-based liposomes composed of soy bean phosphatidylcholine (PC) as the main lipid component on U-937 cell growth. Effects of charge-imposing lipids and cholesterol were also studied. In addition, we investigated whether phospholipid-based liposomes would exert any interaction on cell growth with propylthiouracil, a drug with known antiproliferative activity. We found that PC in the form of extruded liposomes had intrinsic antiproliferative activity on U‐937 cells at concentrations of 200 μM and up without any appreciable cytotoxiciy. Phosphatidylserine and phosphtidylglycerol, but not dicetlylphosphate, at 10 mol% increased growth retardation activity of PC liposomes. Cholesterol at 30 mol% did not have any effect on cell growth, except for liposomes composed of PC and phosphatidylserine, where growth retardation was negated in the presence of cholesterol. Synergistic effect on cell growth was seen with certain liposome compositions when 5.5 μg/mL of propylthiouracil was coincubated. The results of this study suggest that the effects of exogenous lipids on cell growth should be taken into consideration when PC-based liposomes are to be used as drug delivery systems, especially when the targets are cells with phagocytic activity.     

The disordering effect of hyoscyamine drugs on phospholipid membranes

The effect of hyoscyamine drugs on the fluidity of dipalmitoylphosphatidylcholine liposomes has been studied by differential scanning calorimetry (DSC), electron spin resonance spectroscopy (ESR), fluorescence polarization and freeze-fracture electron microscopic techniques. DSC results indicate that anisodamine, anisodine, atropine and scopolamine all increase the fluidity of dipalmitoylphosphatidylcholine liposomes but with different degrees of efficiency. The increasing of fluidity of dipalmitoylphosphatidylcholine liposomes by hyoscyamine drugs is in a dose-dependent way. Increase of the fluidity of phosphatidylcholine liposomes by anisodamine was also shown by the other three methods. The possible mechanism of hyoscyamine-membrane interaction is discussed.     

Preparation of liposomes using the phase reversal method without sonication

A simple procedure for preparation of liposomes based on the phase reversal method but differing from the original technique by the absence of ultrasonic treatment, has been developed. Using this method, the effects of NaCl concentration used for the preparation of liposomes from a mixture of neutral and charged lipids, on the inner liposome volume were studied. The experimental data suggest that NaCl concentration may be regarded as a basic criterion determining the size of liposomes in many routine techniques used for the preparation of liposomes from a mixture of neutral and charged lipids.     

Possible implication of macrophages in the regulation of cytochrome P450 activities in carp (Cyprinus carpio)

Macrophages play a key role in the regulation of cytochrome P450 activity induced by immunostimulants in mammals. We investigated the effects of immunostimulants (LPS, dextran sulfate and tilorone) on biotransformation and macrophage activities in carp. The major effect of LPS was its capacity to inhibit 3-MC-induced cytochrome P450 activities in the liver and head kidney. Basal phase I activities were reduced by tilorone and dextran sulfate in immune organs. Tilorone and dextran sulfate differently modulated total cytochrome P450 contents and P4501A activities suggesting differential sensitivity for P450 classes. In immune organs, tilorone and dextran sulfate inhibited basal EROD activity. Tilorone inhibited 3-MC-induced EROD activity whereas dextran sulfate enhanced this activity. LPS and dextran sulfate increased ROS production by macrophages and all the immunostimulants induced macrophage activating factor (MAF) production. This study demonstrates for the first time in fish the capacity of CYP-regulated immunostimulants to activate macrophages and provides initial insight into the capacity of macrophages to regulate CYP activity induced by immunostimulants in fish.     

Factors affecting the pharmacokinetics and pharmacodynamics of liposomal drugs

Various attempts to increase the therapeutic index of the drug while minimizing side effects have been made in drug delivery systems. Among several promising strategies, liposomes represent an advanced technology to target active molecules to the site of action. Rapid clearance of circulating liposomal drugs administered intravenously has been a critical issue because circulation time in the blood affects drug exposure at the target site. The clinical use of liposomal drugs is complicated by large intra- and interindividual variability in their pharmacokinetics (PK) and pharmacodynamics (PD). Thus, it is important to understand the factors affecting the PK/PD of the liposomal formulation of drugs and to elucidate the mechanisms underlying the variability in the PK/PD of liposomal drugs. In this review article, we describe the characteristics of liposome formulations and discuss the effects of various factors, including liposome-associated factors, host-associated factors, and treatment on the PK/PD of liposomal agents.     

Factors affecting the pharmacokinetics and pharmacodynamics of liposomal drugs

Various attempts to increase the therapeutic index of the drug while minimizing side effects have been made in drug delivery systems. Among several promising strategies, liposomes represent an advanced technology to target active molecules to the site of action. Rapid clearance of circulating liposomal drugs administered intravenously has been a critical issue because circulation time in the blood affects drug exposure at the target site. The clinical use of liposomal drugs is complicated by large intra- and interindividual variability in their pharmacokinetics (PK) and pharmacodynamics (PD). Thus, it is important to understand the factors affecting the PK/PD of the liposomal formulation of drugs and to elucidate the mechanisms underlying the variability in the PK/PD of liposomal drugs. In this review article, we describe the characteristics of liposome formulations and discuss the effects of various factors, including liposome-associated factors, host-associated factors, and treatment on the PK/PD of liposomal agents.     

Ligand self-association at the surface of liposomes: A complication during equilibrium-binding studies

Daunomycin and carminomycin, two anthracycline antibiotics known to bind phospholipid bilayers, appear to self-associate at the surface of liposomes at high bound drug/lipid ratios (r). Fluorescence intensity, lifetime, and anisotropy measurements have been used to monitor the equilibrium binding of these drugs to small unilamellar solid-phase dipalmitoylphosphatidylcholine vesicles. Association of an anthracycline with excess liposome (low r) resulted in an increase in both the observed intensity and the fluorescence lifetime. At low vesicle concentrations (high r), a decrease in the total emission intensity was observed which was not paralleled by the excited-state lifetime. The data from these experiments are consistent with the formation of nonfluorescent anthracycline complexes at the surface of liposomes. Such ligand self-association is a potential complication in any studies on the interaction of amphipathic molecules with liposomes conducted at high r values. Because ligand self-association limits the collection of binding data over certain concentration ranges, this consequently results in greater uncertainty in the determination of the maximum value of r (n) in equilibrium binding studies.     

The effect of blood on the uptake of liposomal lipid by perfused rat liver

Liposomes have been prepared from dipalmitoylphosphatidylcholine (DPPC) and its mixtures with phosphatidylinositol (PI) and stearylamine. The absorption of the liposomes by perfused rat liver has been studied as a function of blood level (0-7% haematocrit). It has been found that the rate constant for uptake of liposomes (perfusion constant, kp) is markedly reduced by addition of blood to the perfusate particularly in the haematocrit range 0-3%. The perfusion constant is dependent on the liposome composition and decreases with incorporation of PI and increases with incorporation of stearylamine into DPPC liposomes, but is independent of the initial size of the liposomes in the range of weight-average diameter from 40-400 nm. The possible effects of blood components on the liposomes and their subsequent absorption by the liver are discussed.