Long-Circulating Liposomes: Development and Perspectives

Abstract

Long-circulating liposomes can be prepared by coating liposome surface with a hydrophilic layer of oligosaccharides, glycoproteins, polysaccharides and synthetic polymers in order to make liposomes “invisible” for scavenger cells of the mononuclear phagocyte system. Incorporation of lipid-anchored poly(ethylene glycol) in liposome bilayer allows to prolong its circulation at least tenfold. Various designs of glycolipid- and polymer-based liposomes are presented, possible mechanisms of action are discussed; potential of these liposomes for drug targeting is presented.     

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.     

Enhanced Gene Delivery and Expression in Human Hepatocellular Carcinoma Cells by Cationic Immunoliposomes

Abstract

A targeted vector allowing enhanced gene transfer to human hepatocellular carcinoma (HCC¹) cells in vitro was developed using cationic liposomes covalently conjugated with the mAb AF-20. This high affinity antibody recognizes a rapidly internalized 180 kDa cell surface glycoprotein which is abundantly expressed on the surface of human HCC and other cancer cells. Quantitative binding analysis of liposomes with target cells by flow cytometry showed specific association of mAb-targeted liposomes with human HCC cells. Using mAb-targeted cationic liposomes containing 20% DOTAP, in the presence or absence of serum, gene expression in HuH-7 cells was enhanced up to 40-fold as compared to liposomes conjugated with an isotype-matched non-relevant control antibody. Transfection specificity was not observed in a control cell line that does not express the antigen recognized by mAb AF-20. This study demonstrates that cationic liposome formulations can be targeted with monoclonal antibodies (mAbs) to enhance specific in vitro gene delivery and expression in the presence or absence of serum.     

Formulation of liposomes functionalized with Lotus lectin and effective in targeting highly proliferative cells

BackgroundLiposomes, used to improve the therapeutic index of new and established drugs, have advanced with the insertion of active targeting. The lectin from Lotus tetragonolobus (LTL), which binds glycans containing alpha-1,2-linked fucose, reveals surface regionalized glycoepitopes in highly proliferative cells not detectable in normally growing cells. In contrast, other lectins localize the corresponding glycoepitopes all over the cell surface. LTL also proved able to penetrate the cells by an unconventional uptake mechanism.MethodsWe used confocal laser microscopy to detect and localize LTL-positive glycoepitopes and lectin uptake in two cancer cell lines. We then constructed doxorubicin-loaded liposomes functionalized with LTL. Intracellular delivery of the drug was determined in vitro and in vivo by confocal and electron microscopy.ResultsWe confirmed the specific localization of Lotus binding sites and the lectin uptake mechanism in the two cell lines and determined that LTL-functionalized liposomes loaded with doxorubicin greatly increased intracellular delivery of the drug, compared to unmodified doxorubicin-loaded liposomes. The LTL-Dox-L mechanism of entry and drug delivery was different to that of Dox-L and other liposomal preparations. LTL-Dox-L entered the cells one by one in tiny tubules that never fused with lysosomes. LTL-Dox-L injected in mice with melanoma specifically delivered loaded Dox to the cytoplasm of tumor cells.ConclusionsLiposome functionalization with LTL promises to broaden the therapeutic potential of liposomal doxorubicin treatment, decreasing non-specific toxicity.General significanceDoxorubicin-LTL functionalized liposomes promise to be useful in the development of new cancer chemotherapy protocols.     

New approaches in the chemical design of gd-containing liposomes for use in magnetic resonance imaging of lymph nodes

Abstract

Several approaches to Improve Gd-containing liposomes as magnetic resonance contrast medium for the visualization of lymph nodes are discussed. The modification of the liposome surface with a polymer was chosen as a chemical solution to control the contrast enhancement properties of the medium. It was found that liposome modification with Gd-diethylenetriaminepentaacetic acid (DTPA)-polylysine-based chelating polymer can increase several fold the metal load per vesicle, while surface modification with polyethylene glycol (PEG) might lead to the increased relaxivity of paramagnetic vesicles. Examples are given on how chemical modification of the liposome surface can improve the performance of Gd-containing liposomes in the visualization of lymph nodes.     

Targeting of Liposomes Within Cardiovascular System

Abstract

Our studies on the targeting of liposomes and liposome-associated pharmaceuticals within the cardiovascular system are reviewed. The delivery of diagnostic and therapeutic agents in plain liposomes, immunoliposomes, long-circulating liposomes and long-circulating immunoliposomes into the sites of vascular injuries and myocardial infarction is discussed. In vitro, ex vivo, and in vivo experiments present a general view on the advantages and limitations of using liposome-mediated targeting. Liposomes capable of targeting pathological areas of the blood vessel wall both, in vitro and ex vivo are described, as well as liposome able to be internalized by normal endothelial cells. Liposome-mediated drug targeting to compromised myocardium is reviewed with a primary impact on liposomes with anti-cardiac myosin antibodies. Targeted visualization of myocardial infarction with diagnostic liposomes is discussed. Efficient accumulation of long-circulating immunoliposomes in the infarct zone is demonstrated, and a relative importance of different variables, such as liposome size, targetability, and prolonged circulation time, for target accumulation is analyzed. The use of immunoliposomes for targeted sealing of hypoxia-caused damages in plasmic membranes of cardiocytes is considered as a new approach in the therapeutic use of liposomes.     

Glycoprotein Mediated Cell Binding of Lectin Coated Liposomes

Abstract

The malignant transformation of cells may cause changes in the oligosaccharide composition of their surface glycoproteins. These could be utilized for specific targeting of exogenous lectin probes to tumor cells. However, because of the usually low affinity of monovalent carbohydrate/lectin reactions, the formation of multiple cluster bonds would be required for stable binding. Because of the lateral mobility of protein molecules incorporated into lipid bilayers, lectin coated liposomes were supposed to be promising multivalent probes. This hypothesis was tested by determining the binding affinities of lectin coated liposomes toward erythrocytes bearing multiantennary glycophorin molecules as a major glycoprotein on their surface, and lymphoblastoid Croco II cells exhibiting glycoligands of a different nature. Actually, the measured affinities provide no or only poor evidence for lectin cluster effects. However, with erythrocytes a complementary affinity enhancing effect was observed that is caused by the multiantennary nature of the glycophorin molecule. Apparently, the presentation of 16 rather equivalent oligosaccharide substituents along the polypeptide backbone of this glycoprotein is cluster-like too, and may cause high affinity cell binding of lectin probes. Lectin coated liposomes might therefore be considered for targeting cells that bear mucin-like glycoligands on their surface.     

Liposome Elimination by Non-Phagocytic Cells of the Liver

Abstract

In this contribution we summarize our observations over a period of nearly two decennia on the role of hepatocytes in the hepatic clearance of intravenously administered liposomes. We demonstrate that, although size is an important parameter, it is not decisive in determining access of liposomes to the hepatocytes. Also lipid composition is an important parameter, including charge, rigidity and headgroup composition. The role of the fenestrated sinusoidal endothelial cells in accessibility is discussed as well as the involvement of opsonizing plasma proteins such as apolipoprotein E. Our observations led us to hypothesize at least four different mechanisms of interaction of liposomes with hepatocytes.     

Intrahepatic Distribution of Long-Circulating Liposomes Containing Polyethylene Glycol) Distearoyl Phosphatidylethanolamine

Abstract

We investigated the intrahepatic distribution in rats of liposomes of 85 or 130 nm diameter, which were sterically stabilized with a polyethylene glycol) derivative of phosphatidylethanolamine (PEG-PE) so as to increase their circulation time in blood. Various times after intravenous injection of radiolabeled ([³H-]cholesterylether) liposomes, parenchymal and non-parenchymal cells of the liver were isolated and their radioactivity content was determined. Control liposomes of 85 nm without PEG-PE distributed in an approximately 80:20 ratio to hepatocytes (H) and macrophages (M), respectively; the 130-nm control liposomes showed a 50:50 H/M distribution. Incorporation of PEG-PE reduced the rate of total liver uptake about 4-fold for liposomes of either size and shifted the H/M ratio to 60:40 for the smaller vesicles and to 40:60 for the larger ones. For both liposome sizes, PEG-PE apparently causes a shift in intrahepatic distribution in favor of the macrophages. It is concluded that PEG-PE has a stronger inhibitory effect on liposome uptake by hepatocytes than on uptake by macrophages. Attempts to shift liposome uptake more in favor of hepatocytes, by incorporation of lactosylceramide, failed. This compound, although causing an increase in hepatic uptake, particularly for the 130-nm liposomes, shifted the H/M ratio further towards the macrophages. We conclude that the galactose moiety of the glycolipid is sufficiently exposed on the surface of (PEG-PE)-containing liposomes to allow interaction with the galactose-binding lectin at the surface of the liver macrophage and that the extent of exposure is dependent on vesicle size.     

Investigation of parameters influencing incorporation,retention and cellular cytotoxicity in liposomal formulations of poorly soluble camptothecin

Abstract

Improving tumor delivery of lipophilic drugs through identifying advanced drug carrier systems with efficient carrier potency is of high importance. We have performed an investigative approach to identify parameters that affect liposomes’ ability to effectively deliver lipophilic camptothecin (CPT) to target cells. CPT is a potent anticancer drug, but its undesired physiological properties are impairing its therapeutic use. In this study, we have identified parameters influencing incorporation and retention of lipophilic CPT in liposomes, evaluating the effect of lipid composition, lipid chemical structure (head and tail group variations, polymer inclusion), zeta potential and anisotropy. Polyethyleneglycol (PEG) surface decoration was included to avoid liposome fusing and increase the potential for prolonged in vivo circulation time. The in vitro effect of the different carrier formulations on cell cytotoxicity was compared and the effect of active targeting of one of the formulations was evaluated. We found that a combination of liposome surface charge, lipid headgroup and carbon chain unsaturation affect CPT incorporation. Retention in liposomes was highly dependent on the liposomal surroundings and liposome zeta potential. Inclusion of lipid tethered PEG provided stability and prevented liposome fusing. PEGylation negatively affected CPT incorporation while improving retention. In vitro cell culture testing demonstrated that all formulations increased CPT potency compared to free CPT, while cationic formulations proved significantly more toxic to cancer cells that healthy cells. Finally, antibody mediated targeting of one liposome formulation further enhanced the selectivity towards targeted cancer cells, rendering normal cells fully viable after 1 hour exposure to targeted liposomes.     

Grafting of different glycosides on the surface of liposomes and its effect on the tissue distribution of 125I-labelled γ-globulin encapsulated in liposomes

Different glycosides were grafted on the surface of liposomes containing ¹²⁵I-labelled γ-globulin by two ways: (1) by using glycolipid and (2) by covalent coupling of $\text{p-}\text{aminophenyl-}\text{d}\text{-glycosides}$ to phosphatidylethanolamine liposomes using glutaraldehyde. The distribution of ¹²⁵I-labelled γ-globulin was determined in mouse tissues from 5–60 min after a single injection of these liposomes. The liver uptake of encapsulated ¹²⁵I-labelled γ-globulin was highest from liposomes having galactose and mannose on the surface. Competition experiments and cross-inhibition studies indicate that this uptake are mediated by specific recognition of the surface galactose and mannose residues of liposomes by the receptors present on the plasma membrane of liver cells. Stearylamine-containing liposomes were found to be more efficient in mediating the uptake of ¹²⁵I-labelled γ-globulin by the lung, whereas in the case of spleen, phosphatidylethanolamine liposomes were more efficient. The extent of uptake of ¹²⁵I-labelled γ-globulin from all types of liposome decreases as the amount of given liposomes increases. The uptake of ¹²⁵I-labelled γ-globulin from liposomes containing asialogangliosides depends upon the phospholipid/ glycolipid ratio. These experiments clearly demonstrate that enhanced liposome uptake by liver cells could be achieved by grafting galactose and mannose on the liposomal surface.     

Liposomes for liver scintigraphy

Abstract

Liposomes were prepared by detergent dialysis method and ^99mTc-GHA, a radiopharmaceutical of choice for kidney imaging was entrapped in SUVs of different surface charges. The organ specificity of the agent with and without encapsulation in liposomes was studied in experimental animals. Results obtained show that ^99mTc-GHA entrapped SUV can be targeted to liver. These results were corroborated by scintigraphy studies. The effect of entrapping the agent in SUV on its half-life is discussed.     

Interaction of pH-Sensitive Liposomes With Blood Components

Abstract

Avoidance of lysosomal degradation of drugs entrapped in liposomes has been one of the major efforts in liposome research. The achievement of high drug deliver}' efficiency using pH-sensitive liposomes over the pH-insensitive liposomes has greatly influenced our strategies in liposome drug delivery. The success of pH-sensitive liposomes in delivering compounds such as fluorescence dye, anti-cancer reagents, toxins and DNA to target cells with high efficiency in vitro shows a great potential to apply the same strategy to in vivo systems. Using human plasma as a simplified model for blood, we have systematically examined the interaction of pH-sensitive liposomes composed of dioleoylphosphatidyl-ethanolamine (DOPE) and oleic acid (OA) with plasma components. Our results show that the bilayer structure of liposomes in plasma depends on their sizes. Small liposomes (d<200nm) were stabilized by plasma components while the larger ones (d>600nm) were rapidly lysed upon the exposure to plasma. Such differences in their stability in plasma may derive from their differences in lipid packing which determines the surface pressure of the membrane. Using purified serum proteins, we found that albumin such as bovine serum albumin (BSA) lyse liposomes by extracting OA from the bilayer. However, BSA induced lysis could be blocked by lipoproteins including HDL, LDL and VLDL, but not by immunoglobulins. Further studies with purified components of HDL demonstrated that apoAl, not the lipids of the HDL, contains the stabilization activity. The extraction of OA from liposomes and the insertion of plasma components into the bilayer modified the bilayer properties such that plasma stabilized liposomes were no longer pH sensitive. Using dipalmitoylsuccinylglycerol (DPSG), a double-chain pH senser for DOPE liposomes, we could preserve 50% pH sensitivity after plasma treatment. The potential application of such liposomes and other essential properties of pH-sensitive liposomes for drug delivery in vivo are also discussed.     

Influence of the Main Phospholipid (MPL) from Thermoplasma Acidophilum and of Liposomes from MPL on Living Cells: Cytotoxicity and Mutagenicity

Abstract

Liposomes of the main phospholipid (MPL) from the archaebacterium Thermplasma acidophilum were investigated for their interference with living cells. Growth of mouse lymphoma cells L5178Y, permanent hamster fibroblasts V79, Ehrlich-mouse-ascites tumor (EMAT) cells and a variety of other celltypes was not influenced by these liposomes. Mutagenicity and antimutagenic efficacy were tested with Salmonella typhimurium TA100 in the “Ames plate-incorporation test”. No cytotoxicity and mutagenicity of liposomes from MPL was detected. The influence of MPL liposomes on ion transport, intracellular pH, electrolytes, membrane potential, energy metabolism, and the biosynthesis of proteins and nucleic acids in EMAT cells is demonstrated in detail.     

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.     

透明质酸修饰的绿原酸脂质体对U14宫颈癌荷瘤小鼠的抑制作用

目的:对透明质酸(HA)靶向绿原酸(CA)脂质体(HA-CA脂质体)进行处方筛选,以及对U14宫颈癌小鼠的抑制作用实验。方法:筛选制备HA-CA脂质体的方法,并以磷脂比、药脂比、PBS的p H为单因素考察指标通过正交实验筛选最优处方;采用透析袋法考察HA-CA的体外释放;Bal b/c小鼠右腋皮下接种U14宫颈癌瘤株,连续尾静脉注射给药14 d后,摘取瘤体称重,并计算肿瘤生长抑制。结果:采用薄膜分散法制备脂质体,最优处方为磷脂比为4:1,药脂比为1:30,PBS的p H为7.4。HA-CA脂质体与CA脂质体释放曲线基本一致,都具有一定的缓释效果。48 h时,HA-CA脂质体和CA脂质体的累计释放度分别为78.39%、83.01%。HA-CA脂质体对U14宫颈癌小鼠的抑瘤率为60.39%,与阳性对照组环磷酰胺相当,高于CA和CA脂质体。结论:HA-CA脂质体由于其具有主动靶向配体HA的修饰,使其抑制U14宫颈癌裸鼠的效果明显高于CA和CA脂质体。     

Biological Action of Liposomes on Main Pathogenetic Mechanisms of Inflammation

Abstract

Small neutral liposomes from phosphatidylholine and cholesterol (7:5 molar ratio) were used to test their antiexudative and antialterative action on rats with acute foot oedema and chemical necrosis of soft hip tissues. The action of liposomes on erythrocytes and liver cells homogenates depending on the peroxidation level of liposomal lipids was studied in vitro. Bactericidal action of liposomes was tested on main types of conditionally pathogenic microorganisms. The interaction of liposomes with blood serum components was studied in vitro. Showed that liposomes reduced the degree of tissue hyperhydratation and shortened time of healing chemically induced necrosis. Rate of liposomal lipids oxidation had a great influence on erythrocytes resistance and lipids oxidation in liver cells homogenates. Showed that liposomes had an evident bactericidal action and could adsorbe toxic components (middle mass peptides) of blood serum. The results showed that liposomes could be used as independent drug to influence on main pathogenetic mechanisms of local and general inflammatory disease.     

Functionalization of liposomes: microscopical methods for preformulative screening

Abstract

The development of smart delivery systems able to deliver and target a drug to the site of action is one of the major challenges in the field of pharmaceutical technology. The surface modification of nanocarriers, such as liposomes, is widely investigated either for increasing the blood circulation time (by pegylation) or for interacting with specific tissues or cells (by conjugation of a selective ligand as a monoclonal antibody, mAb). Microscopical analysis thereby is a useful approach to evaluate the morphology and the size owing to resolution and versatility in defining either surface modification or the architecture and the internal structure of liposomes. This contribution aims to connect the outputs obtained by transmission electron (TEM) and atomic force (AFM) microscopical techniques for identifying the modifications on the liposomal surface. To reach this objective, we prepared liposomes applying two different pegylation technologies and further modifying the surface by mAb conjugation. This work demonstrates the feasibility to apply the combined approach (TEM and AFM analysis) in the evaluation of the efficacy of a surface engineering process.     

Targeted Delivery of Drugs and DNA with Liposomes

Abstract

This presentation is divided into three parts: long-circulating liposomes, immunoliposomes and gene transfer with liposomes. The mechanism of action for the poly(ethylene glycol)-phospholipid conjugates to prolong the circulation time of liposomes can be understood on the basis of steric barrier activity imposed by the flexible PEG chains on the liposome surface. The action of ganglioside GM₁, on the other hand, probably involves specific interactions with serum protein(s). Immunoliposomes can efficiently bind with the target only if the target is readily accessible and the liposomes stay in the circulation for a relatively long period of time. Coating the liposome surface with PEG chains or GM₁ enhances the target binding of immunoliposomes, except when PEG of greater than 5000 dalton is used. In this case, immunoliposome binding to the target is sterically hindered by the long PEG chains. To overcome the problem, antibody molecule is conjugated to the distal end of the PEG chain. This approach works well except that the liver uptake of immunoliposomes is somewhat enhanced. For the delivery of DNA into cells, a novel cationic amphiphile (DC-chol) is synthesized and is now used in clinical trials of gene therapy for melanoma. Current effort is concentrated on the means to enhance the level and duration of transgene expression.     

UPTAKE AND INTRACELLULAR PROCESSING OF PEG-LIPOSOMES AND PEG-IMMUNOLIPOSOMES BY KUPFFER CELLS IN VITRO*

Specific targeting of drugs to for instance tumors or sites of inflammation may be achieved by means of immunoliposomes carrying site-specific antibodies on their surface. The presence of these antibodies may adversely affect the circulation kinetics of such liposomes as a result of interactions with cells of the mononuclear phagocyte system (MPS), mainly represented by macrophages in liver and spleen. The additional insertion of poly(ethylene glycol) chains on the surface of the immunoliposomes may, however, attenuate this effect.

We investigated the influence of surface-coupled rat or rabbit antibodies and of PEG on the uptake of liposomes by rat Kupffer cells in culture with ³H-cholesteryloleyl ether as a metabolically stable marker. Additionally, we assessed the effects of surface-bound IgG and PEG on the intracellular processing of the liposomes by the Kupffer cells, based on a double-label assay using the ³H-cholesteryl ether as an absolute measure for liposome uptake and the hydrolysis of the degradable marker cholesteryl-¹⁴C-oleate as relative measure of degradation.

Attachment of both rat and rabbit antibodies to PEG-free liposomes caused a several-fold increase in apparent size. The uptake by Kupffer cells, however, was 3–4 fold higher for the rat than for the rabbit IgG liposomes. The presence of PEG drastically reduced the difference between these liposome types. Uptake of liposomes without antibodies amounted to only about 10% (non-PEGylated) or less (PEGylated) of that of the immunoliposomes.

In contrast to the marked effects of IgG and PEG on Kupffer cell uptake, the rate of intracellular processing of the liposomes remained virtually unaffected by the presence of these substances on the liposomal surface.

These observations are discussed with respect to the design of optimally formulated liposomal drug preparations, combining maximal therapeutic efficacy with minimal toxicity.