High-affinity targeting of biotin-labeled liposomes to streptavidin-conjugated ligands

Abstract

Cell-specific delivery of drug-loaded liposomal carrier systems can be achieved through the use of liposomes with covalently attached proteins. For such targeting strategies to be successful a number of potential difficulties, related to the preparation of the liposomes as well as optimization of properties that maximize in vivo access and binding to a defined target cell population, must be overcome. The studies summarized here have attempted to identify specific factors that will promote binding of targeted liposomes to defined target surfaces. Liposomes containing biotinylated phospha-tidylethanolamine were used to demonstrate that the avidity of a targeted liposome for streptavidin-coated ELISA plates and cells is influenced by liposome lipid composition, the amount of targeting molecule present per liposome, the nature of the targeting ligand, and the target surface. Specifically, it is demonstrated that the three most important factors (in order of importance) controlling the apparent affinity of targeted liposomes are (1) target ligand concentration in the liposomal membrane; (2) the presence of a spacer grout between the biotin and the phospholipid headgroup; and (3) the addition of cholesterol. Other less important factors that influence target liposome binding include whether the target ligand is attached to a saturated phospholipid compared to an unsaturated lipid and whether the bulk phospholipid species in the liposome is unsaturated versus saturated. These studies suggest that targeted liposomes exhibiting a broad range of binding avidities, as estimated by the concentration of liposomes required to achieve saturation of a target surface, can be prepared by selective design of the liposomal carrier. Advantages of the biotinylated liposome for targeting include the relative ease of preparation the possibility of preparation of large-scale batches suitable for clinical development), the ease of incorporation of the targeting ligand, and, importantly, the ability to alter the apparent affinity of the liposome for the target cell through choice of the biotin-labeled lipid and targeting molecule concentration. The potential for developing a two-step targeting strategy based on the use of biotinylated liposomes is discussed.     

Current methods for attaching targeting ligands to liposomes and nanoparticles

Liposomes and nanoparticles have emerged as versatile carrier systems for delivering active molecules in the organism. These colloidal particles have demonstrated enhanced efficacy compared to conventional drugs. However, the design of liposomes and nanoparticles with a prolonged circulation time and ability to deliver active compounds specifically to target sites remains an ongoing research goal. One interesting way to achieve active targeting is to attach ligands, such as monoclonal antibodies or peptides, to the carrier. These surface-bound ligands recognize and bind specifically to target cells. To this end, various techniques have been described, including covalent and noncovalent approaches. Both in vitro and in vivo studies have proved the efficacy of the concept of active targeting. The present review summarizes the most common coupling techniques developed for binding homing moieties to the surface of liposomes and nanoparticles. Various coupling methods, covalent and noncovalent, will be reviewed, with emphasis on the major differences between the coupling reactions, on their advantages and drawbacks, on the coupling efficiency obtained, and on the importance of combining active targeting with long-circulating particles.     

Immunospecific targeting of liposomes to erythrocytes

Immunoliposomes were made by covalently linking Fab' fragments (from rabbit antimouse erythrocyte IgG) to reverse-phase evaporation vesicles (REV) via maleimido-4-(p-phenylbutyrate) phosphatidylethanolamine (MPB-PE) as anchor molecule. These immunoliposomes were characterized in terms of size, charge, stability and antigen binding capacity. The effect of the liposomal Fab' density on the interaction with the target cell was studied. Two isolation methods were tested to separate free Fab' from liposomally bound Fab'. The necessity of deactivation of remaining reactive sites with dithiothreitol preincubation to increase the specificity of immunoliposome target cell interactions was demonstrated.     

葡萄糖受体靶向的钆喷酸葡胺长循环脂质体的肿瘤细胞靶向研究

目的：制备葡萄糖受体靶向的钆喷酸葡胺（GdDTPA）长循环脂质体，并研究其对高表达葡萄糖受体肿瘤细胞的靶向性。方法：合成新型含葡萄糖表面活性剂（N-棕榈酰葡萄糖胺），利用逆向蒸发法制备Gd—DTPA脂质体、PEG修饰Gd—DTPA脂质体、葡萄糖修饰Gd-DTPA脂质体以及葡萄糖PEG修饰GdDTPA脂质体。将4种脂质体分别与前列腺癌细胞一起培养，用MRI检测前列腺癌细胞摄取的Gd-DTPA浓度。结果：成功合成新型含葡萄糖表面活性荆（N棕榈酰葡萄糖胺），以及制备4种Gd-DTPA脂质体。前列腺癌细胞对4种脂质体都有摄取，每种脂质体MRI检测的SBTIW信号值分别为Gd-DTPA脂质体362、PEG修饰Gd-DTPA脂质体299、葡萄糖修饰GdDTPA脂质体397、葡萄糖PEG修饰GdDTPA脂质体377。结论：葡萄糖的修饰有利于脂质体对肿瘤细胞的靶向。     

Enzyme-activated targeting of liposomes.

It has become increasingly evident that tissues utilize specific localization of enzymes to perform certain tasks, often associated with various types of tissue remodeling. The ubiquitous presence of such enzymes, along with their specific localizations, provides an ideal opportunity to elicit specific delivery via an enzyme-triggered mechanism. A survey of some of the recent progress in enzyme-activated targeting of delivery systems, with a focus on a few liposomal systems, is presented.     

Pharmacokinetics of targeting with liposomes

The optimization of drug disposition in the body leads to an increase in its therapeutic effect and to a decrease in adverse effects. Liposomes can serve as a potential drug carrier for achieving this. However, the behavior of a drug carrier system under in vivo conditions is complex. Therefore, a more complete understanding of the pharmacokinetics of liposomes themselves, as well as that of the encapsulated drug, is required. The optimization of the pharmacokinetics of liposomes can be performed by linking a pharmacodynamic model of the free drugs that are encapsulated into liposomes. Sensitivity analysis was applied to optimize the delivery system to maximize the antitumor effect of liposomal doxorubicin (DOX). Advanced technology for ligand-mediated selective targeting and intracellular targeting is also introduced for antitumor agents and for gene delivery systems.     

High-affinity ligands of Siglec receptors and their therapeutic potentials

Sialic acids are one of the important constituents of glycoconjugates in the deuterostome lineage of animals and microorganisms. Siglecs (Sialic acid-binding immunoglobulin like lectins) are a family of cell-surface receptor proteins that recognize sialylated glycoconjugates as ligands. To date, 15 Siglecs have been described in humans and are mainly known as regulators of the immune system. Several of the Siglecs are emerging as potential targets for the treatment of some inflammatory, autoimmune, allergic, neurodegenerative and infectious diseases. In addition to antibody mediated therapy, high-affinity ligand-based probes of Siglec receptors would represent invaluable tools to effectively address therapeutic opportunities of Sialic acid-mediated Siglec recognition. This review discusses some aspects of structure and function of Siglec receptors, and concisely summarizes up-to-date progress on the identification of sialic acid based high-affinity ligands of certain well explored Siglec receptors.     

Possibility of active targeting to tumor tissues with liposomes

In terms of active targeting by immunoliposomes, two anatomical compartments are considerable for targeting sites. One is located a readily accessible site in intravascular, and another is a much less accessible target site located in the extravascular. However, it was made clear that the active targeting with immunoliposomes is determined by two kinetically competing processes, such as binding to the target site and uptake by the RES. To overcome these contradictions, we have designed a new type of long-circulating immunoliposome, which was PEG-immunoliposome attached antibodies at the distal end of PEG chain, so called the pendant type immunoliposome. The pendant type immunoliposome showed much higher targetability than the ordinary immunoliposomes to both targeting sites of lung endothelial cells and solid tumor tissue. This is due to the free PEG chains (not linked to the antibody) effectively avoiding the RES uptake of liposomes, resulting in elevated the blood concentration and enhanced the target binding of immunoliposomes. The presence of free PEG does not interfere with the binding of the terminally linked antibody to the antigen. For targeting to the vascular endothelial surface in the lung, 34A antibody, which is highly specific to mouse pulmonary endothelial cells, was conjugated to make the pendant type immunoliposomes (34A-PEG-ILP). 34A-PFG-ILP showed significantly higher targeting degree than the ordinary type of immunoliposomes. For targeting to the solid tumor tissue, Fab' fragment of 21B2 antibody which is anti-human CFA and transferrin (TF) were used. Both pendant type immunoliposomes (Fab'-PFG-ILP and TF-PEG-ILP) showed the low RES uptake and the long circulation time, and resulted in enhanced accumulation of the liposomes in the solid tumor. TF-PEG-ILP was internalized into tumor cells with receptor mediated endocytosis, after extravasation into tumor tissue. The pendant type immunoliposome can escape from the gaps between adjacent endothelial cells and openings at the vessel termini during tumor angiogenesis by passive convective transport much rather than ligand directed targeting. Active targeting to tumor tissue with the pendant type immunoliposome is particularly important for many highly toxic anticancer drugs for cancer chemotherapy. An ultimate goal of pendant type immunoliposome is the incorporation of a fusogenic molecule that would induce fusion of liposome following their binding to the target cells or their internalization by endocytosis. Such liposomal formulations should be useful for endocytotic internalization of plasmid DNA and other bioactive materials.     

Mannosylated liposomes for bio-film targeting

Vesicular systems in general are investigated to achieve bacterial bio-film targeting as their architecture mimics bio-membranes in terms of structure and bio-behavior. This paper elaborates upon the role of the inherent characteristics of the carrier system and further envisages the role of anchored ligands in navigating the contents in the vicinity of bio-films. Vesicles in the present study were coated with hydrophobic derivatives of mannan (cholesteryl mannan and sialo-mannan). The prepared vesicles were characterized for size, shape, percentage entrapment and ligand binding specificity and results were compared with the uncoated versions. Using a set of in vitro and in vivo models, the bio-film targeting potential of plain and mannosylated liposomal formulations were compared. Results suggested that mannosylated vesicles could be effectively targeted to the model bacterial bio-films, compared with plain vesicles. Moreover, the sialo-mannan coated liposomes recorded superior targetability as reflected in the significantly higher percentage growth inhibition when compared with cholesteryl mannan coated liposomes. The engineered systems thus have the potential use for the delivery of anti-microbial agents to the bio-films.     

In vivo targeting by liposomes

This review deals with the current status of newly developed pendant-type PEG-immunoliposomes (Type C), carrying monoclonal antibodies or their fragments (Fab') at the distal ends of the PEG chains. In terms of target binding of Type C, two different anatomical compartments are considered. They are mouse lung endothelium as a readily accessible site via the intravascular route and the implanted solid tumor as a much less accessible target site reached via extravasation. Distearoyl phosphatidylethanolamine derivatives of PEG with a carboxyl group (DSPE-PEG-COOH) and dipalmitoyl phosphatidylethanolamine derivatives of PEG with a maleimidyl group (DPPE-PEG-Mal) at the PEG terminus were newly synthesized. Small unilamellar liposomes (90-130 nm in diameter) were prepared from phosphatidylcholine and cholesterol (2 : 1, m/m) containing 6 mol% of DSPE-PEG-COOH or DPPE-PEG-Mal. For targeting to the vascular endothelial surface in the lung, 34A antibody, which is highly specific to mouse pulmonary endothelial cells, was conjugated to PEG-liposomes (34A-Type C). The degree of lung binding of 34A-Type C in BALB/c mouse was significantly higher than that of 34A-Type A, which is an ordinary type of immunoliposome (without PEG derivatives). For targeting to solid tumor tissue, 21B2 antibody (anti-human CEA) and its Fab' fragment were used. The targeting ability of Fab'-Type C was examined by using CEA-positive human gastric cancer strain MKN-45 cells inoculated into BALB/c nu/nu mice. Fab'-Type C showed low RES uptake and a long circulation time, and enhanced accumulation of the liposomes in the solid tumor was seen. The small Fab'-Type C predominantly passed through the leaky tumor endothelium by passive convective transport. These studies offer important insights into the potential of Type C liposomes for target-specific drug delivery.     

Vascular targeting of doxorubicin using cationic liposomes

Tumor vessel has been recognized as an important target for anticancer therapy. Cationic liposomes have been shown to selectively target tumor endothelial cells, thus can potentially be used as a carrier for chemotherapy agents. In this study, cationic liposomes containing 20 mol% cationic lipid dimethyl dioctadecyl ammonium bromide (DDAB) and loaded with doxorubicin (DOX) were prepared and characterized. The cationic liposomal DOX showed 10.8 and 9.1 times greater cytotoxicity than control PEGylated liposomal DOX in KB oral carcinoma and L1210 murine lymphocytic leukemia cells, and 7.7- and 6.8-fold greater cytotoxicity compared to control neutral non-PEGylated liposomal DOX, repectively, in these two cell lines. Although cationic liposomal DOX had higher tumor accumulation at 30 min after intravenous administration compared to control liposomes (p<0.05), DOX uptake of these liposomes at 24h post-injection was similar to that of PEGylated liposomal DOX (p>0.05) and approximately twice the levels of the free drug and non-PEGylated liposomes. In a murine tumor model generated using L1210 cells, increased survival rate was obtained with cationic liposomal DOX treatment compared to free DOX (p<0.01), neutral liposome control (p<0.01), as well as PEGylated liposomes (p<0.05). In conclusion, the cationic liposomal DOX formulation produced superior in vitro cytotoxicity and in vivo antitumor activity, and warrants further investigation.     

Nuclear gene targeting using negatively charged liposomes

Oligonucleotides are a very useful tool to control gene activity. Oligos work by complementary base-pairing with target sequences either in the nucleus or in the cytosol (Zelphati, O., Szoka, F.C., Jr., 1996. Liposomes as a carrier for intracellular delivery of antisense oligonucleotides: a real or magic bullet? J. Contr. Rel. 41, 99-119). In a new approach using chimeric oligonucleotides (Yoon, K., Cole Strauss, A., Kmiec, E.B., 1996. Targeted gene correction of episomal DNA in mammalian cells mediated by a chimeric RNA-DNA oligonucleotide. Proc. Natl. Acad. Sci. USA 93, 2071-2076) conversion of single base mutations with help of intranuclear repair mechanisms maybe an advantageous method to cure genetic diseases which are based on single point mutations. These chimeric oligonucleotides are constructed in a way that they form an intramolecular double strand of DNA and modified RNA-bases. We used a fluorescent labelled pure 68-mer DNA-analogue of a chimeric oligonucleotides to follow the intracellular fate of these kind of genetic material. The oligos were complexed with protamine sulfate and coated with three different liposomal formulations. The AVE-3 formulation shows enhanced properties compared to a classical neutral and negatively charged formulation. Nuclear localisation of oligos could only be observed with the AVE-3 formulation. Furthermore only the negatively charged liposome formulations interact with the protamine-complexed oligonucleotides.     

Mannosylated liposomes as antigen delivery vehicles for targeting to dendritic cells

The immune stimulating ability of mannosylated liposomes containing FITC-ovalbumin as a model antigen and displaying either a branched tri-mannose or a mono-mannose ligand on the liposome surface was investigated in human monocyte-derived dendritic cells (MoDCs) and murine bone-marrow-derived dendritic cells (BMDCs). Uptake of liposomes, dendritic cell activation and proliferation of CD8(+) T cells from OT-I transgenic mice were determined by flow cytometry. Uptake of liposomes displaying the tri-mannose ligand was enhanced in human MoDCs compared with both non-mannosylated liposomes and liposomes displaying mono-mannose ligands. However, this increased uptake did not result in an increase in expression of CD80 or CD86 on the surface of the MoDCs. In contrast, neither tri-mannose- nor mono-mannose-containing liposomes were taken up by murine BMDCs to a greater extent than non-mannose-containing liposomes. The expression of CD86 and CD40 on the surface of BMDCs was not increased after exposure to mannosylated liposomes and BMDCs incubated with mannosylated liposomes were not able to stimulate proliferation of CD8(+) T cells to any greater extent than BMDCs incubated with non-mannosylated liposomes. These findings suggest that while mannose-containing ligands can enhance the uptake of antigen-containing liposomes by some dendritic cells, important differences in the affinity of carbohydrate-binding receptors for mannose-containing ligands do exist between species. In addition, the increase in uptake of antigen by dendritic cells using mannosylated liposomes does not necessarily result in enhanced dendritic cell activation.     

抗肿瘤热敏靶向脂质体的研究进展

脂质体具有靶向传递药物进入肿瘤部位的优势,被广泛用于肿瘤的临床治疗,但是靶向传递并不能保证药物在肿瘤部位被生物利用。为了解决此问题,热敏靶向脂质体(TTL)已经成为目前的研究热点。TTL既能提高药物在肿瘤部位的浓度,又能在加热条件下触发释放所载药物,从而显著改善所载药物的抗肿瘤效果。本文主要综述了近年来该领域的研究进展。     

Fibrin targeting of tissue plasminogen activator-loaded echogenic liposomes

We recently reported entrapment of tissue-plasminogen activator (tPA) into echogenic liposomes (ELIP) with retention of echogenicity and thrombolytic effect. Integral to the potential of this agent for ultrasound-detectable local drug delivery is the specific binding of tPA-ELIP to clots. tPA contains fibrin-binding sites; we hypothesized that tPA when associated with ELIP, will maintain fibrin binding properties, rendering further manipulation for targeting of the tPA-ELIP unnecessary. We demonstrated strong fibrin binding of the ELIP-associated tPA. Fibrin binding for ELIP-associated tPA was twice that of free tPA. This strong affinity for fibrin was confirmed using echogenicity analysis of porcine clots in vitro. Both objective (mean gray scale analysis) and subjective (visual estimation by two experienced echocardiographers) evaluation of the clots showed enhanced highlighting of clots treated with tPA-ELIP when compared to control. The findings in this study represent new approaches for fibrin-targeted, ultrasound-directed and enhanced local delivery of a thrombolytic agent.     

A review of the ligands and related targeting strategies for active targeting of paclitaxel to tumours

It has been 30 years since the discovery of the anti-tumour property of paclitaxel (PTX), which has been successfully applied in clinic for the treatment of carcinomas of the lungs, breast and ovarian. However, PTX is poorly soluble in water and has no targeting and selectivity to tumour tissue. Recent advances in active tumour targeting of PTX delivery vehicles have addressed some of the issues related to lack of solubility in water and non-specific toxicities associated with PTX. These PTX delivery vehicles are designed for active targeting to specific cancer cells by the addition of ligands for recognition by specific receptors/antigens on cancer cells. This article will focus on various ligands and related targeting strategies serving as potential tools for active targeting of PTX to tumour tissues, illustrating their use in different tumour models. This review also highlights the need of further studies on the discovery of receptors in different cells of specific organ and ligands with binding efficiency to these specific receptors.     

Enhancing effect of cetylmannoside on targeting of liposomes to Kupffer cells in rats

In order to evaluate whether surface modification of liposomes by cetylmannoside (Man) could be useful for targeting to Kupffer cells, the effect of Man on disposition of liposomes was examined after intravenous administration to rats. In the case of small unilamellar vesicles (SUV), no difference in disposition was observed between control liposomes (PC-SUV) and modified liposomes (Man-SUV). On the other hand, in the case of multilamellar vesicles (MLV), modified liposomes (Man-MLV) were rapidly eliminated from the circulation, and showed higher accumulation (51.4% of dose) in the liver as compared with control liposomes (PC-MLV, 25.7% of dose). In the spleen, splenic clearance of Man-MLV (0.068 ml/min) was comparable to that of PC-MLV (0.068 ml/min), although Man-MLV showed lower accumulation (5.7% of dose) than PC-MLV (14.7% of dose). This lower accumulation in the spleen of Man-MLV might be due to the low blood concentration caused by the high accumulation in the liver. Thus, it is considered that liposomal size is important in revealing the effects of Man, and Man-MLV is able to enhance only the affinity for the liver. The cellular distribution in the liver of Man-MLV 2 h after intravenous administration to rats gave encouraging evidence that Kupffer cells might be involved in the enhanced hepatic uptake of the liposomes. These results suggest the usefulness of Man-MLV for targeting to Kupffer cells. Furthermore, the involvement of plasma protein(s) in the uptake of Man-MLV is suspected.     

靶向肿瘤新生血管的阿霉素阳离子脂质体的体外研究

本研究采用3β-［N-(N′,N′-二甲基胺乙基)胺基甲酰胺基］胆固醇(DC-Chol)和二棕榈酰磷脂酰胆碱为脂材制备了各种DC-Chol含量不同的阿霉素阳离子脂质体，考察了阿霉素阳离子脂质体的体外性质，同时以大鼠主动脉内皮细胞为模型，考察它们对不同阳离子脂质体的摄取情况，并采用静脉注射FITC-Dextran(M_r 500 000)标记体内肿瘤新生血管，为体内靶向肿瘤血管提供依据。结果表明阿霉素阳离子脂质体包封率均在90%以上，粒径在100~200 nm。随着DC-Chol含量的增加，zeta电位升高，但PEG的加入会降低zeta电位。DC-Chol含量的增加会增大阿霉素的释放量，同时也促进脂质体被内皮细胞的摄取，加快摄取速度。因此在进行体内靶向肿瘤血管考察时应充分关注这些体外实验结果。FITC-Dextran标记法可以显影体内新生血管，为体内肿瘤血管靶向实验提供直观的观察方法。     

Drug targeting to inflammation: studies on antioxidant surface loaded diclofenac liposomes

Inflammation is associated with enhanced vascular permeability, production of inflammatory markers and over production of reactive oxygen species (ROS) with depletion of endogenous antioxidants. Several drug targeting approaches to inflammation taking clues from these events have been evolved. Surprisingly, a drug targeting approach utilizing abundant oxidative stress at inflammatory site has not been followed. Antioxidant surface loaded liposomes might preferentially localize at inflammatory sites via redox interaction where at high level of ROS exist. The present study was focused to investigate the role of antioxidant as a targeting ligand on the surface of liposome employing rat granuloma air pouch model of inflammation. We developed conventional and antioxidant loaded diclofenac (DFS) liposomes (co-enzyme Q10 and ascorbyl palmitate) for i.v. administration and characterized for vesicle size, zeta potential and percent entrapment. In vivo drug targeting studies showed an increase in AUC, therapeutic availability of DFS in air pouch fluid (APF) and APF/serum DFS concentration ratios from antioxidant loaded liposomes compared to conventional liposomes and drug solution. The promising results suggest the role of antioxidant as a possible ligand in drug targeting to a site where at abundant ROS exist.