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.     

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.     

Functional characterization of an endosome-disruptive peptide and its application in cytosolic delivery of immunoliposome-entrapped proteins

Antibody-directed liposomes (immunoliposomes) are frequently used for targeted drug delivery. However, delivery of large biotherapeutic molecules (i.e. peptides, proteins, or nucleic acids) with immunoliposomes is often hampered by an inefficient cytosolic release of entrapped macromolecules after target cell binding and subsequent endocytosis of immunoliposomes. To enhance cytosolic drug delivery from immunoliposomes present inside endosomes, a pH-dependent fusogenic peptide (diINF-7) resembling the NH(2)-terminal domain of influenza virus hemagglutinin HA-2 subunit was used. Functional characterization of this dimeric peptide showed its ability to induce fusion between liposome membranes and leakage of liposome-entrapped compounds when exposed to low pH. In a second series of experiments, diINF-7 peptides were encapsulated in immunoliposomes to enhance the endosomal escape of diphtheria toxin A chain (DTA), which inhibits protein synthesis when delivered into the cytosol of target cells. Immunoliposomes targeted to the internalizing epidermal growth factor receptor on the surface of ovarian carcinoma cells (OVCAR-3) and containing encapsulated DTA did not show any cytotoxicity toward OVCAR-3 cells. Cytotoxicity was only observed when diINF-7 peptides and DTA were co-encapsulated in the immunoliposomes. Thus, diINF-7 peptides entrapped inside liposomes can greatly enhance cytosolic delivery of liposomal macromolecules by pH-dependent destabilization of endosomal membranes after cellular uptake of liposomes.     

Immunoliposomes and PEGylated Immunoliposomes: Possible Use for Targeted Delivery of Imaging Agents

Liposomes can be made target-specific by immobilizing antibodies on their surface against characteristic components of target organ or tissue. Possible schemes of antibody immobilization on liposomes are briefly considered. The use of immunoliposomes for the targeted delivery of diagnostic and therapeutic agents within the cardiovascular system is discussed. Immunoliposomes are shown to be suitable carriers for targeting blood vessel injuries, lung endothelium, and myocardial infarction. The role of polyethylene glycol in the preparation of long-circulation liposomes is investigated, and a hypothesis explaining the mechanism of polymer protective action in terms of physicochemical properties of diluted polymeric solutions is suggested. Polyethylene glycol-coated liposomes are investigated as possible carriers of imaging agents for gamma and MR visualization of different areas of interest in the body, including lymph nodes. The possibility of simultaneous immobilization of protective polymer and antibody on the liposome surface is proved, and the long-circulating targeted immunoliposomes are used for the targeted delivery of radiolabel to necrotic areas in rabbits with experimental myocardial infarction.     

Targeted accumulation of polyethylene glycol-coated immunoliposomes in infarcted rabbit myocardium.

The less than optimal accumulation of immunoliposome-associated reagents at target sites has often been attributed to the rapid in vivo clearance of immunoliposomes from the blood. In an attempt to overcome the drawback of rapid clearance and use the targeting potential of immunoliposomes, we have prepared long-circulating, 111In-labeled immunoliposomes. Targeting properties and enhanced circulation times were demonstrated in a rabbit model of acute experimental myocardial infarct. The specificity of liposomes for newly exposed intracellular cardiac myosin at the necrotic sites was achieved by incorporating monoclonal antimyosin antibody. Extended circulation times were achieved by cocoating the antimyosin-liposomes with polyethylene glycol (PEG). The half-life of the immunoliposomes was 40 min, which increased to 200 min with 4% mol PEG and to approximately 1000 min with 10% mol PEG. The degree of binding of modified immunoliposomes at the target sites was also dependent on the concentration of PEG incorporated at the liposome surface. This study demonstrates the accumulation of long-circulating targeted liposomes at the area of acute rabbit experimental myocardial infarction.     

Interactions of immunoliposomes with target cells

We have covalently attached a monoclonal antibody (11-4.1) against the murine major histocompatibility antigen, H-2Kk, on the surface of liposomes. The interaction of these antibody-coated liposomes (immunoliposomes) with target cells, RDM-4 lymphoma (H-2Kk), was investigated. About 90% of the immunoliposomes taken up by target cells at 4 degrees C could be removed by a mild protease treatment of the cells, whereas only 30% of the uptake at 37 degrees C was labile to the same treatment. Furthermore, the uptake of immunoliposomes at 37 degrees C was inhibitable by cytochalasin B or by a combination of 2-deoxyglucose and NaN3. These results suggest that immunoliposome binding to the target cell surface is the primary uptake event at 4 degrees C and that the surface-bound liposomes are rapidly internalized by the cells at 37 degrees C, probably via an endocytic pathway. Studies with fluorescence microscopy of target cells treated with immunoliposomes containing carboxyfluorescein also supported this conclusion. If endocytosis is the mechanism by which immunoliposomes gain entry into target cells, the efficacy of a cytotoxic drug encapsulated would depend on the resistance of the drug to lysosomal inactivation and its ability to escape from the lysosomal system. Consistent with this notion, we observed that methotrexate encapsulated in liposomes bearing 11-4.1 antibody specifically inhibited deoxy[6-3H]uridine incorporation into DNA in target RDM-4 cells but not in P3-X63-Ag8 myeloma cells (H-2Kd) at the same doses. The observed cytotoxic effect of encapsulated methotrexate could be reversed by the treatment of cells with a lysosomotropic amine, chloroquine, which has been shown to increase the intralysosomal pH of mammalian cells. On the other hand, cytosine-beta-D-arabinofuranoside encapsulated in immunoliposomes showed no target-specific killing, probably because the drug is readily inactivated in the lysosomal system. These results are discussed in terms of the drug carrier potential of immunoliposomes.     

Plasmid DNA adsorbed to pH-sensitive liposomes efficiently transforms the target cells

We have previously reported that plasmid DNA entrapped in the pH-sensitive immunoliposomes effectively transforms the target cells (Proc. Natl. Acad. Sci. USA, in press). In the present study, we demonstrate that DNA adsorbed on the same liposome also transforms the target cells. The transformation activity is antibody dependent, as liposomes containing no targeting antibody had reduced activity. The activity could be significantly inhibited by excess non-specific DNA (salmon sperm). Since some DNA are likely adsorbed to the liposomes during the entrapment process, the activity of the entrapped DNA is partially accounted for by the adsorbed DNA. The possibility of developing a simple DNA-mediated transfection protocol using liposome adsorbed DNA is discussed.     

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.     

Preparation and characterization of heat-sensitive immunoliposomes

Immunoliposomes able to bind specifically to target cells and to release their encapsulated contents upon brief heating were prepared. Monoclonal anti-H2Kk was covalently derivatized with palmitic acid by the method of Huang, A. et al. (Huang, A., Tsao, Y.S., Kennel, S.J. and Huang, L. (1982) Biochim. Biophys. Acta 716, 140-150). The palmitoyl antibody was injected at a controlled rate into a suspension of fused unilamellar dipalmitoylphosphatidylcholine liposomes maintained at a constant temperature. The final protein-to-lipid ratio of the resultant liposomes with incorporated antibody (immunoliposomes) was dependent upon the rate of antibody injection and the lipid concentration. Injection of palmitoyl antibody into a liposome suspension containing 50 mM carboxyfluorescein at 41 degrees C resulted in simultaneous antibody incorporation and entrapment of dye. Immunoliposomes were able to release the entrapped carboxyfluorescein upon heating. The release of dye at temperatures between the pre- and main-transition temperatures of DPPC was abolished by the addition of calf serum (5%). Furthermore, the presence of serum resulted in an increase in the temperature of the maximal release rate and also in the rate of release at that temperature. Retention of antigen-binding capacity was demonstrated by the ability of the immunoliposomes to bind specifically to the target cells. Rapid release of entrapped carboxyfluorescein from immunoliposomes bound to target cells at 4 degrees C was achieved upon brief exposure (less than 3 min) at 41 degrees C. These heat-sensitive immunoliposomes may be useful in enhancing drug delivery to target cells.     

Possibility of the unification of directed drug transport as illustrated by liposome transport to target antigens

A new approach to the targeted drug delivery is described. Unlike previous methods, associated with the necessity of specific immunoglobulin immobilization on the surface of drug-containing microcontainer, the present approach permits targeted transport of standardized container to a set of target antigens, using intermediate molecules-mediators possessing high and specific affinity to both vector antibody and standardized container. It was shown that simultaneous targeting of 14C-labeled liposomes to three target antigens using avidin-biotin system permits the increase in liposome binding to target monolayer by 30-50%, as compared to targeting of the same amount of liposomes to one antigen. The method developed is particularly promising in cases when relative availability of target antigens in the target organ is unknown.     

Development of the novel PEG-PE-based polymer for the reversible attachment of specific ligands to liposomes: synthesis and in vitro characterization

Surface grafting of liposomes with the wide variety of ligands including antibodies and other proteins is a promising approach for targeted delivery of therapeutics. In this paper, we describe a simple method of synthesizing a hydrazine-functionalized poly(ethylene glycol)-phosphatidylethanolamine (PEG-PE)-based amphiphilic polymer which can conjugate a variety of ligands via a reversible, pH-cleavable bond. In this method, the targeting ligand is attached to the distal end of the PEG chain, which facilitates its easy access to the targeted site of interaction. The reversible attachment of targeting ligands is useful especially in multifunctional liposomal systems, whereafter successfully performing the function of targeting to the specific site, the bulky ligands, such as proteins or antibodies, are cleaved off in response to an environmental stimulus to expose some other functionalities such as ligands for intracellular penetration or organelle-specific targeting. To investigate the applicability of the protocol, the model ligands monoclonal antinucleosome antibody 2C5 and antimyosin antibody 2G4, and glycoproteins concanavalin A (Con-A) and avidin were conjugated to the synthesized polymer and incorporated into liposomes. In vitro assays including biochemical, enzyme-linked immunosorbent, fluorescence microscopy, and flow cytometry were used to confirm three key characteristics of the modified and/or liposome-attached proteins: successful conjugation of the targeting ligands to the polymer, preservation of specific activity of the ligands after the conjugation and liposome attachment, and the facile pH-sensitive ligand detachment. Monoclonal antibody 2C5 and 2G4, immobilized on the liposome surface, retained their binding affinity to corresponding antigens as confirmed by ELISA. The Con A-bearing liposomes showed significantly higher agglutination in the presence of its substrate mannan compared to plain liposomes (PL) and avidin-functionalized liposomes bound specifically with biotin-agarose. The study on the pH-dependence showed that almost 80% of the hydrazone bond was cleaved after rather brief preincubation of the immunoliposomes at pH 5 for 0.5 to 1 h. Fluorescence microscopy and flow cytometry analysis of cancer cells (HeLa and MCF-7) treated with cancer cell-specific targeting ligand mAb 2C5-bearing liposomes showed enhanced cellular binding. Studies at low pH clearly confirmed the easy cleavability of the targeting ligand from the liposomes resulting in significantly less or virtually no cellular association.     

Biodistribution and Therapeutic Utility of Liposomal Drug Carrier Systems

Abstract

Delivery of the drug at a specific site (drug targeting) or controlled and prolonged release of the liposome-bound drug are the two major considerations for adding liposomes to the existing arsenal of drug delivery systems. In particular the concept of liposomal drug targeting has been evolving rapidly in the past 10 years with the development of 'second generation' carriers such as immunoliposomes (liposomes bearing covalently coupled antibodies as homing device) and, more recently, the long-circulating liposomes. In this contribution novel approaches in the field of liposomal drug targeting will be briefly described: (1) immunoliposomes for chemotherapy of intraperitoneal malignancies, such as ovarian carcinoma, (2) a new type of immunoliposomes for mediating the targeting of enzymes to be used for site-specific prodrug activation (immuno-enzymosomes), (3) long-circulating liposomes for the targeting of antibiotics to sites of bacterial infection, and (4) polyethyleneglycol (PEG)-modified proteoliposomes with the homing device coupled to the ends of the long PEG chains for achieving effective target binding along with prolonged circulation times.     

Selective transfer of a lipophilic prodrug of 5-fluorodeoxyuridine from immunoliposomes to colon cancer cells.

A monoclonal antibody against the rat colon carcinoma CC531 was covalently coupled to liposomes containing a dipalmitoylated derivative of the anticancer drug FUdR as a prodrug in their bilayers. We investigated the in vitro interaction of these liposomes with CC531 target cells and the mechanism by which they deliver the active drug FUdR intracellularly to the cells by monitoring the fate of the liposomal bilayer markers cholesterol-[(14)C]oleate and [(3)H]cholesteryloleylether as well as the (3)H-labeled prodrug and colloidal gold as an encapsulated liposome marker. After binding of the immunoliposomes to the cell surface, only limited amounts were internalized as demonstrated by a low level of hydrolysis of liposomal cholesterol ester and by morphological studies employing colloidal gold-labeled immunoliposomes. By contrast, already within 24 h immunoliposome-incorporated FUdR-dP was hydrolyzed virtually completely to the parent drug FUdR intracellularly. This process was inhibited by a variety of endocytosis inhibitors, indicating that the prodrug enters and is processed by the cells by a mechanism involving an endocytic process, resulting in intracellular FUdR concentrations up to 3000-fold higher than those in the medium. Immunoliposomes containing poly(ethyleneglycol) (PEG) chains on their surface, with the antibody coupled either directly to the bilayer or at the distal end of the PEG chains were able to deliver the prodrug into the tumor cells at the same rate as immunoliposomes without PEG. Based on these observations, we tentatively conclude that during the interaction of the immunoliposomes with the tumor cells the lipophilic prodrug FUdR-dP is selectively transferred to the cell surface and subsequently internalized by constitutive endocytic or pinocytic invaginations of the plasma membrane, thus ultimately delivering the prodrug to a lysosomal compartment where hydrolysis and release of parent drug takes place. This concept allows for an efficient delivery of a liposome-associated drug without the need for the liposome as such to be internalized by the cells.     

Targeting of drug loaded immunoliposomes to herpes simplex virus infected corneal cells: an effective means of inhibiting virus replication in vitro

The goal of our studies was to develop liposomes containing antiviral drugs and targeted with antiviral antibody (immunoliposomes) that would be effective at inhibiting replication of herpes simplex virus (HSV) in vitro. To achieve this, a monoclonal antibody to glycoprotein D of HSV was derivatized with palmitic acid and was incorporated into the lamellae of dehydration-rehydration vesicles. The gD containing immunoliposomes were shown to bind specifically to HSV-infected rabbit corneal cells in vitro, whereas control immunoliposomes prepared with a monoclonal antibody of the same class as the anti-gD failed to preferentially bind to virus-infected cells. The gD immunoliposome binding was inhibitable by pretreatment with rabbit anti-HSV serum but not by aggregated normal serum. Thus liposome binding was judged to represent an antigen-antibody reaction not binding to Fc receptors expressed by cells infected with HSV. Immunoliposomes loaded with iododeoxyuridine (IUDR) leaked drug rapidly at 37 degrees C, whereas acyclovir (ACV)-loaded liposomes still contained 48% of drug after 24 hr at 37 degrees C. The ACV-liposomes retained 44% of drug after 14 days at 4 degrees C. The ability of immunoliposomes to inhibit virus replication was compared with that of untargeted and empty liposomes by means of virus yield assays in vitro, Immunoliposomes loaded with either IUDR or ACV inhibited virus replication, although ACV-containing immunoliposomes were the most efficacious. The implications of our in vitro results for the development of immunoliposomes suitable for the treatment of ocular herpes infection are briefly discussed.     

ADVENTURES IN TARGETING

ABSTRACT

An overview of our experiences in the field of immunoliposomal anticancer drugs is provided with respect to choice of ligand, and choice of model system, in order to provide some guidance as to the rational use of this new technology. Liposomes targeted by either peptide or monoclonal antibodies showed significantly higher binding to their respective target cells in vitro compared to non-targeted liposomes in all model systems examined. This higher binding led to higher cytotoxicities relative to non-targeted liposomes. For the immunoliposomes to deliver their entrapped drug to target cell in vivo, long circulations half-lives are required. We have evaluated the pharmacokinetics of liposomes prepared by several different coupling techniques, and have found significant differences in the clearance of these immunoliposomes from the circulation. Immunoliposomes prepared with whole anti-CD19 IgG coupled by the Mal-PEG-DSPE method demonstrated a short plasma half-life, which may reflect the random orientation of the MAb on the liposome surface. Coupling methods that mask or eliminate the Fc region result in immunoliposomes that have clearance rates more similar to untargeted liposomes. Insertion of peptides or antibodies into pre-formed liposomes through incubation with ligand-coupled PEG micelles resulted in immunoliposomes, termed post-insertion liposomes, that demonstrated comparable in vitro binding, pharmacokinetic profiles and in vivo therapeutic efficacy to liposomes made by conventional coupling methods. The therapeutic efficacy of liposomes, prepared by various coupling methods and targeted by different ligands, was compared in several different animal models of either haematological malignancies, pseudometastatic disease or solid tumours. In our hands, successful in vivo targeting has been obtained when the target is either small or readily accessible from the vasculature, where the liposomes have longer circulating half-lives and/or where a ligand against an internalizing epitope has been chosen. These results should aid in the rational design of applications for immunoliposomal drugs in the future.     

Adventures in targeting

An overview of our experiences in the field of immunoliposomal anticancer drugs is provided with respect to choice of ligand, and choice of model system, in order to provide some guidance as to the rational use of this new technology. Liposomes targeted by either peptide or monoclonal antibodies showed significantly higher binding to their respective target cells in vitro compared to non-targeted liposomes in all model systems examined. This higher binding led to higher cytotoxicities relative to non-targeted liposomes. For the immunoliposomes to deliver their entrapped drug to target cell in vivo, long circulations half-lives are required. We have evaluated the pharmacokinetics of liposomes prepared by several different coupling techniques, and have found significant differences in the clearance of these immunoliposomes from the circulation. Immunoliposomes prepared with whole anti-CD19 IgG coupled by the Mal-PEG-DSPE method demonstrated a short plasma half-life, which may reflect the random orientation of the MAb on the liposome surface. Coupling methods that mask or eliminate the Fc region result in immunoliposomes that have clearance rates more similar to untargeted liposomes. Insertion of peptides or antibodies into pre-formed liposomes through incubation with ligand-coupled PEG micelles resulted in immunoliposomes, termed post-insertion liposomes, that demonstrated comparable in vitro binding, pharmacokinetic profiles and in vivo therapeutic efficacy to liposomes made by conventional coupling methods. The therapeutic efficacy of liposomes, prepared by various coupling methods and targeted by different ligands, was compared in several different animal models of either haematological malignancies, pseudometastatic disease or solid tumours. In our hands, successful in vivo targeting has been obtained when the target is either small or readily accessible from the vasculature, where the liposomes have longer circulating half-lives and/or where a ligand against an internalizing epitope has been chosen. These results should aid in the rational design of applications for immunoliposomal drugs in the future.     

Stable target-sensitive immunoliposomes.

Interaction of immunoliposomes composed of dioleoylphosphatidylethanolamine (DOPE) (80%), dioleoylphosphatidic acid (DOPA) (20%), and a small amount of specific antibody with Herpes Simplex virus (HSV) were studied by detecting the immune-dependent lysis of liposomes. DOPA was used as the principal stabilizer of the immunoliposomes. Antibodies conjugated with N-glutarylphosphatidylethanolamine or oxidized GM1 served as the target-specific ligands of immunoliposomes. These immunoliposomes (d = 160-180 nm) were stable for at least one month when stored at 4 degrees C. However, they undergo a rapid aggregation and lysis reaction in the presence of a membrane-bound target such as intact HSV virions. We have also employed epitope peptide-containing liposomes (target liposomes) to mimic the virus and showed that the immunoliposomes could be aggregated and lysed by the target liposomes in an antigen-dependent manner. Immunoliposome lysis could be accelerated by increasing the incubation temperature to 60-70 degrees C. No immunoliposome lysis was observed if the target liposomes were absent, indicating the prolonged stability of the immunoliposomes. Liposome lysis was always accompanied by liposome aggregation. However, the aggregation-induced liposome destabilization is unique to the HII phase-forming lipids such as DOPE. DOPC-containing immunoliposomes did not lyse despite the fact that massive liposome aggregation had taken place.     

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。     

Research Advances on Stimuli-responsive Liposomes in the Targeted Drug Delivery System

Liposome, a kind of nanoscale vesicle, is applied in the drug delivery systems (DDS) extensively because of its low toxicity, biodegradability and biocompatibility. However, defects of liposome drugs, such as low rates of drug release, insufficiency in active targeting and inefficient bioavailability still remain to be solved. Therefore, stimuli-responsive liposomes are brought to DDS to improve the efficacy of controlled drug release, assure specific release in targeted sites and alleviate side-effects as much as possible. Stimuli-responsive liposomes could maintain stability in circulation, tissues and cells under physiological conditions. Once delivered, they could be activated by relevant internal or external stimuli to release cargos accurately in target areas. This review highlights the design, functional principles and recent advances on application of pH-sensitive liposomes and thermosensitive liposomes respectively, which are two typical stimuli-responsive liposomes. Common targeting modifications of liposomes are discussed as well. We also summarize recent challenges of stimuli-responsive liposomes and their further applications.     

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.