多肽、蛋白类药物脂质体的研究进展

脂质体做为多肽、蛋白类药物一种新型给药载体有控制药物释放、降低药物的毒性、提高药物的靶向性等突出优点,具有广阔的应用前景。本文通过查阅近10年来多肽和蛋白类药物脂质体研究的相关资料,总结论述了脂质体作为多肽、蛋白类药物载体在新的制备方法、新型脂质体、给药途径、产业化进展四个方面的最新研究动向,指出了多肽、蛋白类药物脂质体在研究应用中存在的不足,并展望了多肽、蛋白类药物脂质体未来发展的方向。     

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

Abstract

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

“Smart” liposomal nanocontainers in biology and medicine

The perspectives of using liposomes for delivery of drugs to desired parts of the human body have been intensively investigated for more than 30 years. During this time many inventions have been suggested and different kinds of liposomal devices developed, and a number of them have reached the stages of preclinical or clinical trials. The latest techniques can be used to develop biocompatible nano-sized liposomal containers having some abilities of artificial intellect, such as the presence of sensory and responsive units. However, only a few have been clinically approved. Further improvements in this area depend on our knowledge of the interactions of drugs with the lipid bilayer of liposomes. Further studies on liposomal transport through the human body, their targeting of cells requiring therapeutic treatment, and finally, the development of techniques for controlled drug delivery to desired acceptors on cell surfaces or in cytoplasm are still required.     

Detection of liposomal cholesterol and monophosphoryl lipid A by QS-21 saponin and Limulus polyphemus amebocyte lysate

Liposomes containing cholesterol (Chol) have long been used as an important membrane system for modeling the complex interactions of Chol with adjacent phospholipids or other lipids in a membrane environment. In this study we utilize a probe composed of QS-21, a saponin molecule that recognizes liposomal Chol and causes hemolysis of erythrocytes. The interaction of QS-21 with liposomal Chol results in a stable formulation which, after injection into the tissues of an animal, lacks toxic effects of QS-21 on neighboring cells that contain Chol, such as erythrocytes. Here we have used liposomes containing different saturated phospholipid fatty acyl groups and Chol, with or without monophosphoryl lipid A (MPLA), as model membranes. QS-21 is then employed as a probe to study the interactions of liposomal lipids on the visibility of membrane Chol. We demonstrate that changes either in the mole fraction of Chol in liposomes, or with different chain lengths of phospholipid fatty acyl groups, can have a substantial impact on the detection of Chol by the QS-21. We further show that liposomal MPLA can partially inhibit detection of the liposomal Chol by QS-21. The Limulus amebocyte lysate assay is used for binding to and detection of MPLA. Previous work has demonstrated that sequestration of MPLA into the liposomal lipid bilayer can block detection by the Limulus assay, but the binding site on the MPLA to which the Limulus protein binds is unknown. Changes in liposomal Chol concentration and phospholipid fatty acyl chain length influenced the detection of the liposome-embedded MPLA.     

Liposomes containing chelating agents. Cellular penetration and a possible mechanism of metal removal

Electron microscope studies were done on mouse liver, from 5 min to 8 wk after an intravenous injection of liposomes containing ethylenediaminetetraacetic acid (EDTA). Livers of mice receiving an injection of liposomes containing KCL instead of EDTA or an injection of a solution of EDTA were also examined. Liposomes were shown to be phagocytized by hepatocytes as well as by Kupffer cells within minutes after the injection. Initially, there was a close contact between the liposomal membrane and the cellular membrane, followed by an invagination of the latter and the formation of a distinct vesicle surrounding a single liposome or a cluster of several liposomes. No fusion between the liposomal membrane and the cell membrane was observed. Between 15 min and 6 h after liposome injection, the Kupffer cells were found to have an increased number of lysosomes and autophagic vacuoles. Within the latter, morphologically intact liposomes or remnants of liposomes could be seen. At 12 h after injection, a striking increase in macrophages was observed in the liver sinusoids of EDTA-liposome-injected mice, but not in those of KCl- liposome-injected mice. Within the macrophages, remnants of liposomes occasionally could be observed. However, the origin and the physiological role of these cells are unknown. In the hepatocytes, morphological changes were first observed 24 h after injection; there were large numbers of autophagic vacuoles, and some cells showed extensive areas of focal cytoplasmic degeneration. The morphology of the liver cells returned to normal about 7 days after injection. No morphological changes were observed in livers of mice receiving EDTA solution without liposomes. A possible mechanism by which the liposome- encapsulated chelating agents can successfully remove intracellular toxic metals is discussed. The use of liposomes as carriers seems to be a useful tool for intracellular delivery of chelating agents or drugs in general.     

Cytotoxicity to a Tumor Cell Line of Epoxy-Phosphatidylinositol Liposomes

Abstract

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

Pros and Cons of the Liposome Platform in Cancer Drug Targeting

Coating of liposomes with polyethylene-glycol (PEG) by incorporation in the liposome bilayer of PEG-derivatized lipids results in inhibition of liposome uptake by the reticulo-endothelial system and significant prolongation of liposome residence time in the blood stream. Parallel developments in drug loading technology have improved the efficiency and stability of drug entrapment in liposomes, particularly with regard to cationic amphiphiles such as anthracyclines. An example of this new generation of liposomes is a formulation of pegylated liposomal doxorubicin known as Doxil® or Caelyx®, whose clinical pharmacokinetic profile is characterized by slow plasma clearance and small volume of distribution. A hallmark of these long-circulating liposomal drug carriers is their enhanced accumulation in tumors. The mechanism underlying this passive targeting effect is the phenomenon known as enhanced permeability and retention (EPR) which has been described in a broad variety of experimental tumor types. Further to the passive targeting effect, the liposome drug delivery platform offers the possibility of grafting tumor-specific ligands on the liposome membrane for active targeting to tumor cells, and potentially intracellular drug delivery. The pros and cons of the liposome platform in cancer targeting are discussed vis-à-vis nontargeted drugs, using as an example a liposome drug delivery system targeted to the folate receptor.     

Pros and cons of the liposome platform in cancer drug targeting

Coating of liposomes with polyethylene-glycol (PEG) by incorporation in the liposome bilayer of PEG-derivatized lipids results in inhibition of liposome uptake by the reticulo-endothelial system and significant prolongation of liposome residence time in the blood stream. Parallel developments in drug loading technology have improved the efficiency and stability of drug entrapment in liposomes, particularly with regard to cationic amphiphiles such as anthracyclines. An example of this new generation of liposomes is a formulation of pegylated liposomal doxorubicin known as Doxil or Caelyx, whose clinical pharmacokinetic profile is characterized by slow plasma clearance and small volume of distribution. A hallmark of these long-circulating liposomal drug carriers is their enhanced accumulation in tumors. The mechanism underlying this passive targeting effect is the phenomenon known as enhanced permeability and retention (EPR) which has been described in a broad variety of experimental tumor types. Further to the passive targeting effect, the liposome drug delivery platform offers the possibility of grafting tumor-specific ligands on the liposome membrane for active targeting to tumor cells, and potentially intracellular drug delivery. The pros and cons of the liposome platform in cancer targeting are discussed vis-à-vis nontargeted drugs, using as an example a liposome drug delivery system targeted to the folate receptor.     

Oligonucleotide Therapy for Hematological Malignancies

Abstract

In this contribution we describe and discuss (mostly published) experiments providing evidence favoring a decisive role of opsonizing plasma proteins in the removal of liposomes from the vascular compartment. Our conclusion is that cells will only bind and take up liposomes if they are anatomically accessible for the liposomes and if, in addition, they possess (specific) receptors for one or more proteins adsorbing to the liposomal surface. The relative contribution of each cell type fulfilling these criteria to over-all liposome clearance is dictated by the total number of cells in that population, the density of the receptor(s) involved, the affinity of those receptors for their respective ligands and the localization in the vascular system. It is concluded that only a few cell populations meet the criteria. Most are excluded because of inaccessibility while of the accessible ones several lack the proper opsonin receptors for significant liposome uptake. The significance of localization in the vasculature is illustrated by the hepatocytes whose accessibility is limited by the fenestrations in the endothelial lining of the liver sinusoids. The opsonin concept is extrapolated to cells other than macrophages; for example, the existence of hepatocyte-specific opsonins is proposed in order to explain the efficient uptake of small liposomes by this cell population. Because of their virtually complete lack of participation in plasma elimination of liposomes, some readily accessible cell types, such as the circulating blood cells and the vascular endothelial cells, are proposed to lack appropriate receptors. According to the views developed in this contribution the specialty of cells involved in liposome clearance therefore lies in the condition that they possess one or more receptors for plasma-derived proteins that spontaneously adsorb to the liposomal surface. One possible exception to the opsonin-determined concept is the fate of phosphatidylserine-containing liposomes. These may be cleared without or even in spite of involvement of opsonins, by virtue of a PS-specific receptor on macrophages.     

Lyophilisation and sterilisation of liposomal vaccines to produce stable and sterile products

The advantages of liposomes as delivery systems for peptide, protein and DNA vaccines is well-recognised, unfortunately their application has been stinted by their instability during storage and their limited shelf-life. Further, sterilisation of these systems has been problematic, with degradation of the liposomes being reported after sterilisation using the various techniques available. Work form our laboratory has investigated techniques that can be applied to particulate liposomal vaccines such that they can be prepared in a freeze-dried and sterile format. In this article, we describe techniques for the lyophilisation, cryoprotection and sterilisation of liposomal vaccines. Applying these methods allows for the retention of both the chemical integrity of the lipids and the key physico-chemical characteristics of the liposomes (e.g., particle size, zeta potential, and dynamic viscosity), thus supporting the enhanced transition of liposomal vaccines from the bench to the clinic.     

Plasma membrane-mediated leakage of liposomes induced by interaction with murine thymocytic leukemia cells

The interaction of liposomes with BW 5147 murine thymocytic leukemia cells was studied using fluorescent probes (entrapped carboxyfluorescein and fluorescent phosphatidylethanolamine) in conjunction with a Ficoll-Paque discontinous gradient system for rapid separation of liposomes from cells. Reversible liposomal binding to discrete sites on the BW cell surface was found to represent the major form of interaction; uptake of intact liposomal contents by a process such as liposome-BW cell membrane fusion was found to apparently represent a minor pathway of interaction (2%). Liposomal lysis was found to be associated with the process of liposomal binding (perhaps as a result of the binding itself). Lysis was followed by release of the entrapped carboxyfluorescein into the media and its subsequent uptake by the cells. This lysis was shown to be dependent upon discrete membrane-associated sites that have some of the properties of proteins. The results of these studies suggest that liposomal binding to the cells, subsequent lysis of the liposomes and cellular uptake of their contents should be seriously considered in all studies of liposome-cell interactions as an alternate mode of interaction to the four modes (fusion, endocytosis, adsorption and lipid exchange) previously emphasized in the literature.     

Structural changes in the liposomes and the lamellar packing of liposomal lipids exposed to glycerin

Glycerol-induced structural changes in multilamellar liposomes (ML) were studied by light and thin-section electron microscopy. It is shown that high concentrations of glycerol induce non-bilayer rearrangements of liposomal lipids in ML. The data obtained are discussed regarding the possible mechanisms of cryoprotector effects on natural (biological) and artificial (liposomal) membranes.     

Liposomal chemotherapy in visceral leishmaniasis: an ultrastructural study of an intracellular pathway

The intracellular fate of liposomes administered intracardially was examined in the liver and spleen of hamsters experimentally infected with Leishmania donovani. Separate groups of animals were treated with liposomes containing either an antileishmanial agent, a colloidal gold marker, or saline. Ultrastructural examinations of lysosomal interactions with the parasitophorous vacuole and with phagocytized liposomes were made. Lysosomes readily fused with the parasitophorous vacuoles but appeared to have little effect on the parasite, possibly due to the production of enzyme inhibitors. Liposomes rapidly became localized in lysosomes subsequent to endocytosis by macrophages. Morphologic evidence suggested that secondary lysosomes containing liposomal residues then fused with the parasitophorous vacuole. Aspects of one possible pathway are discussed which may account for the greatly enhanced effectiveness of liposomal chemotherapy for experimental visceral leishmaniasis.     

Liposomal modification with uronate, which endows liposomes with long circulation in vivo, reduces the uptake of liposomes by J774 cells in vitro.

For overcoming rapid removal of liposomes from the bloodstream, we developed reticuloendothelial system (RES)-avoiding liposomes modified with a uronic acid derivative, palmityl-D-glucuronide (PGlcUA). In this current study, we examined the in vitro interaction of PGlcUA-liposomes with J774 cells derived from mouse macrophages. Liposomal association with J774 cells at 37 degrees C did not increase compared with the binding at 4 degrees C when liposomes were modified with PGlcUA. RES-avoiding ability was not specifically endowed by glucuronate but by uronates in general, since palmityl-D-galacturonide showed a similar effect on liposomal clearance in vivo and liposomal uptake in vitro. These facts indicate that modification of the liposomal surface with uronic acid derivatives endows liposomes with a long circulation time in the bloodstream by reducing their uptake by macrophages.     

Spin-labelling study of interactions of ovalbumin with multilamellar liposomes and specific anti-ovalbumin antibodies

Ovalbumin (OVA) has been used continuously as the model antigen in numerous studies of immune reactions and antigen processing, very often encapsulated into liposomes. The purpose of this work was to study the possible interactions of spin-labelled OVA and lipids in liposomal membranes using electron spin resonance (ESR) spectroscopy. OVA was covalently spin-labelled with 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO-maleimide), characterized and encapsulated into multilamellar, negatively charged liposomes. ESR spectra of this liposomal preparation gave evidence for the interaction of OVA with the lipid bilayers. Such an interaction was also evidenced by the ESR spectra of liposomal preparation containing OVA, where liposomes were spin-labelled with n-doxyl stearic acids. The spin-labelled OVA retains its property to bind specific anti-OVA antibodies, as shown by ESR spectroscopy, but also in ELISA for specific anti-OVA IgG.     

Spectrophotometric measurement of intraliposomal trehalose

Trehalose, a non-reducing glucose disaccharide found at high concentrations in many species of anhydrobiotic organisms, shows significant promise in protecting cellular viability and structural integrity during freezing and desiccation. As mammalian cell membranes are impermeable to trehalose, extensive efforts have been taken to introduce trehalose into mammalian cells. In this study, we report on the characterization of trehalose-containing liposomes, with focus on the entrapment of trehalose inside liposomes, as the first step in establishing liposomes as a delivery system in the biopreservation field. Liposomes were synthesized by hydrating a phospholipid/cholesterol lipid bilayer with 200-400 mM trehalose buffer and repeatedly extruding the lipid suspension to form unilamellar vesicles. The trehalose content of the liposomal lysate was determined spectrophotometrically using a commercial kit Megazyme and confirmed with HPLC measurements. The number of liposomes was calculated from the phosphate content of the liposomal preparation and an estimated number of lipid molecules in a 401+/-8 nm liposome. Based on an intraliposomal trehalose content, the calculated liposomal encapsulation efficiency of 200 mM trehalose liposomes was of 92+/-0.7%. This value was in agreement with the 300 and 400 mM trehalose liposomes (91.1+/-8.2% and 102.1+/-9.4%, respectively). The Megazyme method for trehalose measurement is an inexpensive and sensitive technique that does not require specialized instrumentation or extensive technical expertise. Therefore, it can be used to enhance current efforts in the development of alternative strategies for the cryo- and lyoprotection of mammalian cells.     

Uptake of liposomes containing phosphatidylserine by liver cells in vivo and by sinusoidal liver cells in primary culture: in vivo-in vitro differences

The interaction with liver cells of liposomes containing different mol fractions of phosphatidylserine was investigated in vivo and in vitro. Increasing the amount of liposomal phosphatidylserine from 10 to 30 mol% leads to a faster blood disappearance of the liposomes. Within the liver, which is mainly responsible for this elimination, these liposomes are only taken up by the hepatocytes and Kupffer cells. By contrast, sinusoidal endothelial cells, in vitro, do bind and internalize liposomes containing >/=30% phosphatidylserine at least as actively as Kupffer cells. The uptake by endothelial and Kupffer cells is inhibited by poly(inosinic acid) and other anionic macromolecules, suggesting the involvement of scavenger receptors. The lack of liposome uptake by endothelial cells under in vivo conditions can be attributed to plasma effects since addition of various sera caused severe reduction of in vitro uptake of liposomes. In vivo the phosphatidylserine head groups may be masked by plasma proteins adsorbed to the liposomal surface, thus preventing recognition by receptors, which are intrinsically able to recognize phosphatidylserine.     

Preparation of liposomal doxorubicin-graphene nanosheet and evaluation of its in vitro anti-cancer effects

In recent years there has been much interest in development of multifunctional drug delivery systems. In this work, liposomes that contain doxorubicin (Dox), a potent anticancer drug, and graphene nanosheets (GNS) were prepared. The GNSs have excellent optical properties, such as photoluminescence which enables tracking of the liposomes, high absorption in ultra violet region of electromagnetic spectrum which can be exploited in photodynamic and photothermal therapy, and low toxicity to mammalian cells. Nanoliposomes were prepared using the thin film hydration method. Dox and GNSs were loaded to the liposomes during the hydration of the lipid film. Liposomes were characterized and the profile of in vitro drug release, cellular uptake, and cytotoxicity of the prepared liposomes on MCF-7 cells were determined. Despite the earlier reports, the liposomes have kept their spherical structures in the presence of GNSs. The cytotoxicity of liposomal Dox and GNSs were shown to be higher than the free forms of them. Novel nanoliposomes that contain GNSs have provided a multi-functional system with the potential of tracking, photodynamic and photothermal therapy. Further improvements of this versatile nanosystem would be promising for treatment of cancer.     

Do folate-receptor targeted liposomal photosensitizers enhance photodynamic therapy selectivity?

One of the current goals in photodynamic therapy research is to enhance the selective targeting of tumor cells in order to minimize the risk and the extension of unwanted side-effects caused by normal cell damage. Special attention is given to receptor mediated delivery systems, in particular, to those targeted to folate receptor. Incorporation of a model photosensitizer (ZnTPP) into a folate-targeted liposomal formulation has been shown to lead an uptake by HeLa cells (folate receptor positive cells) 2-fold higher than the non-targeted formulation. As a result, the photocytotoxicity induced by folate-targeted liposomes was improved. This selectivity was completely inhibited with an excess of folic acid present in the cell culture media. Moreover, A549 cells (folate receptor deficient cells) have not shown variations in the liposomal incorporation. Nevertheless, the differences observed were slighter than expected. Both folate-targeted and non-targeted liposomes localize in acidic lysosomes, which confirms that the non-specific adsorptive pathway is also involved. These results are consistent with the singlet oxygen kinetics measured in living cells treated with both liposomal formulations.     

Liposomal daunorubicin overcomes drug resistance in human breast,ovarian and lung carcinoma cells

Multi-drug resistance due in part to membrane pumps such as P-glycoprotein (Pgp) is a major clinical problem in human cancers. We tested the ability of liposomally-encapsulated daunorubicin (DR) to overcome resistance to this drug. A widely used breast carcinoma cell line originally selected for resistance in doxorubicin (MCF7ADR) was 4-fold resistant to DR compared to the parent MCF7 cells (IC50 79 nM vs. 20 nM). Ovarian carcinoma cells (SKOV3) were made resistant by retroviral transduction of MDR1 cDNA and selection in vinblastine. The resulting SKOV3MGP1 cells were 130-fold resistant to DR compared to parent cells (IC50 5700 nM vs. 44 nM). Small-cell lung carcinoma cells (H69VP) originally selected for resistance to etoposide were 6-fold resistant to DR compared to H69 parent cells (IC50 180 nM vs. 30 nM). In all three cases, encapsulation of DR in liposomes as Daunoxome (Gilead) did not change the IC50 of parent cells relative to free DR. However, liposomal DR overcame resistance in MCF7ADR breast carcinoma cells (IC50 20 nM), SKOV3MGP1 ovarian carcinoma cells (IC50 237 nM) and H69VP small-cell lung carcinoma cells (IC50 27 nM). Empty liposomes did not affect the IC50 for free DR in the three resistant cell lines, nor did empty liposomes affect the IC50 for other drugs that are part of the multi-drug resistance phenotype (etoposide, vincristine) in lung carcinoma cells. These data indicate the possible value of liposomal DR in overcoming Pgp-mediated drug resistance in human cancer.