Trends and developments in liposome drug delivery systems

Since the discovery of liposomes or lipid vesicles derived from self-forming enclosed lipid bilayers upon hydration, liposome drug delivery systems have played a significant role in formulation of potent drugs to improve therapeutics. Currently, most of these liposome formulations are designed to reduce toxicity and to some extent increase accumulation at the target site(s) in a number of clinical applications. The current pharmaceutical preparations of liposome-based therapeutics stem from our understanding of lipid-drug interactions and liposome disposition mechanisms including the inhibition of rapid clearance of liposomes by controlling size, charge, and surface hydration. The insight gained from clinical use of liposome drug delivery systems can now be integrated to design liposomes targeted to tissues and cells with or without expression of target recognition molecules on liposome membranes. Enhanced safety and heightened efficacy have been achieved for a wide range of drug classes, including antitumor agents, antivirals, antifungals, antimicrobials, vaccines, and gene therapeutics. Additional refinements of biomembrane sensors and liposome delivery systems that are effective in the presence of other membrane-bound proteins in vivo may permit selective delivery of therapeutic compounds to selected intracellular target areas.     

Targeted liposomal drug delivery in cancer

Liposomes, which are biodegradable and essentially non-toxic vehicles, can encapsulate both hydrophilic and hydrophobic materials, and are utilized as drug carriers in drug delivery systems. In addition, liposomes can be used to carry radioactive compounds as radiotracers can be linked to multiple locations in liposomes. One option is the hydrated compartment inside the liposome, another the lipid core into which especially hydrophobic conjugates can be attached, and the third option is the outer lipid leaflet where molecules can be bound by covalent linkage. Delivery of agents to the reticuloendothelial system (RES) is easily achieved, since most conventional liposomes are trapped by the RES. For the purpose of delivery of agents to target organs other than RES, long-circulating liposomes have been developed by modifying the liposomal surface. Understanding of the in vivo dynamics of liposome-carried agents is required for the evaluation of the bioavailability of drugs encapsulated in liposomes. In this review, we focus on the in vivo trafficking of liposomes visualized by positron emission tomography (PET) and discuss the characteristics of liposomes that affect the targeting of drugs in vivo.     

Molecular targeting of liposomal nano-particles to lymphatic system

Conventional liposomal drug delivery has been associated with obvious limitations, such as a rapid absorption by the recticulo-endothelial system in the liver and spleen, a short circulation time and a low therapeutic efficacy. Various modifications of liposomal drugs have been developed to prolong the duration of actions of the drugs at target sites, reduce its adverse effects and increase therapeutic index of drugs such as polymeric conjugation and polymeric fixation on the surface of a liposome. The lymphatic system is an important highway to spread the metastasis of most human cancers including breast, colon, and lung, ovarian and prostate. To eradicate those metastatic cancer cells from the lymphatic system, several efforts have been made to develop new and efficient lymphatic targeting drug delivery systems in order to achieve a high initial lymphatic uptake and lymph node localization. Recently, molecule targeting of liposome to lymphatic system may enhance therapeutic efficacy by improving the initial lymphatic uptake and the lymph nodal retention of liposomes such as the ligand-receptor and antibodies binding on the surface of liposome. This article aims to review the emerging liposomal drug, which is targeting the lymphatic system. The significant factors associated with targeting liposomal drugs will also be discussed in more detail in this review.     

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

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.     

Challenges in Development of Targeted Liposomal Therapeutics

Liposomes, phospholipid vesicles with a bilayered membrane structure, have been widely used as pharmaceutical carriers for drugs and genes, in particular for treatment of cancer. To enhance the efficacy of the liposomal drugs, drug-loaded liposomes are targeted to the tumors by means of passive (enhanced permeability and retention mediated) targeting, based on the longevity of liposomes in blood and its accumulation in pathological sites with compromised vasculature, and active targeting, based on the attachment of specific ligands to the liposomal surface to bind certain antigens on the target cells. Antibody-targeted liposomes loaded with anticancer drugs demonstrate high potential for clinical applications. This review highlights evolution of liposomes for both passive and active targeting and challenges in development of targeted liposomal therapeutics specifically antibody-targeted liposomes.     

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.     

Liposomes as drug carrier systems. Preparation, classification and therapeutic advantages of liposomes]

Bangham et al. (1965) created first the concept of the liposome as a microparticulate lipoidal vesicle separated from its aqueous environment by one or more lipid bilayers. Later Gregoriadis and Ryman (1972) suggested to use liposomes as drug carrier systems. Nowadays liposomes are under extensive investigation for improving the delivery of therapeutic agents, enzymes, vaccines and genetic materials. Liposomes offer an excellent opportunity to selective targeting of drugs which is expected to optimize the pharmacokinetical parameters, the pharmacological effect and to reduce the toxicity of the encapsulated drugs. To understand the system it is important to know the basic properties of these lipoidal vesicles. Our aim was to focus on the lipid composition and the method of liposome preparation what determine the liposomal membrane fluidity, permeability, vesicle size, charge density, steric hindrance and stability of the liposomes as principle factors those influence the fate of liposomes, their interactions with the blood components and other tissues after systemic administration or local use.     

Enhanced Anticancer Therapy Mediated by Specialized Liposomes

It has been a central aim of experimental and clinical therapeutics to deliver therapeutic agents as close as possible to, or if possible within, a diseased cell. Such targeting achieves two major aims of drug delivery, the maximum dose of therapeutic agent to the diseased cell and avoidance of uptake by and, usually, accompanying side-effects to normal, healthy cells. Conventional liposomes, originally used for studies in membrane biophysics and biochemistry, have been used in therapy for the past two decades. However, when applied to deliver drugs into cells, conventional liposomes proved inefficient and so novel unconventional or specialized liposomes are constantly being prepared to enhance cell-specific delivery in-vivo. One possible way of achieving better targeting is combination of the positive attributes of more than one specialized type of liposome into one vesicle. Although a limited number of studies has examined the combined effect of such dual-speciality liposomes, more studies are warranted using appropriate models. Liposomes are composed of one, a few, or many concentric bilayer membranes which alternate with aqueous spaces. The drugs are encapsulated within the aqueous internal volume if they are hydrophilic or in the lipid bilayers if they are hydrophobic (Kim 1993). Liposomes range in size from 25 nm to more than 20 μm (Sugarman & Perez-Soler 1992). Depending on their solubility and method of formulation antimicrobial, cytotoxic and other conventional drugs, hormones, antigens, enzymes, genetic material, viruses and bacteria can be incorporated in either the aqueous or hydrophobic phase. This review discusses the types and characteristics of non-conventional liposomes used in various modes of cancer therapy, mainly chemotherapy and gene therapy. It concludes with suggestions on improving these novel liposomal to effect better targeting to cancer cells.     

Liposomal antibiotics for the treatment of infectious diseases

Introduction: Liposomal delivery systems have been utilized in developing effective therapeutics against cancer and targeting microorganisms in and out of host cells and within biofilm community. The most attractive feature of liposome-based drugs are enhancing therapeutic index of the new or existing drugs while minimizing their adverse effects.

Areas covered: This communication provides an overview on several aspects of liposomal antibiotics including the most widely used preparation techniques for encapsulating different agents and the most important characteristic parameters applied for examining shape, size and stability of the spherical vesicles. In addition, the routes of administration, liposome–cell interactions and host parameters affecting the biodistribution of liposomes are highlighted.

Expert opinion: Liposomes are safe and suitable for delivery of variety of molecules and drugs in biomedical research and medicine. They are known to improve the therapeutic index of encapsulated agents and reduce drug toxicity. Recent studies on liposomal formulation of chemotherapeutic and bioactive agents and their targeted delivery show liposomal antibiotics potential in the treatment of microbial infections.     

A targeted liposome delivery system for combretastatin A4: formulation optimization through drug loading and in vitro release studies

Efficient liposomal therapeutics require high drug loading and low leakage. The objective of this study is to develop a targeted liposome delivery system for combretastatin A4 (CA4), a novel antivascular agent, with high loading and stable drug encapsulation. Liposomes composed of hydrogenated soybean phosphatidylcholine (HSPC), cholesterol, and distearoyl phosphoethanolamine-PEG-2000 conjugate (DSPE-PEG) were prepared by the lipid film hydration and extrusion process. Cyclic arginine-glycine-aspartic acid (RGD) peptides with affinity for alphav beta3-integrins overexpressed on tumor vascular endothelial cells were coupled to the distal end of polyethylene glycol (PEG) on the liposomes sterically stabilized with PEG (non-targeted liposomes; LCLs). Effect of lipid concentration, drug-to-lipid ratio, cholesterol, and DSPE-PEG content in the formulation on CA4 loading and its release from the liposomes was studied. Total liposomal CA4 levels obtained increased with increasing lipid concentration in the formulation. As the drug-to-lipid ratio increased from 10:100 to 20:100, total drug in the liposome formulation increased from 1.05+/-0.11 mg/mL to 1.55+/-0.13 mg/mL, respectively. When the drug-to-lipid ratio was further raised to 40:100, the total drug in liposome formulation did not increase, but the amount of free drug increased significantly, thereby decreasing the percent of entrapped drug. Increasing cholesterol content in the formulation decreased drug loading. In vitro drug leakage from the liposomes increased with increase in drug-to-lipid ratio or DSPE-PEG content in the formulation; whereas increasing cholesterol content of the formulation up to 30 mol-percent, decreased CA4 leakage from the liposomes. Ligand coupling to the liposome surface increased drug leakage as a function of ligand density. Optimized liposome formulation with 100 mM lipid concentration, 20:100 drug-to-lipid ratio, 30 mol-percent cholesterol, 4 mol-percent DSPE-PEG, and 1 mol-percent DSPE-PEG-maleimide content yielded 1.77+/-0.14 mg/mL liposomal CA4 with 85.70+/-1.71% of this being entrapped in the liposomes. These liposomes, with measured size of 123.84+/-41.23 nm, released no significant amount of the encapsulated drug over 48 h at 37 degrees C.     

In vitro optimization of liposomal nanocarriers prepared from breast tumor cell specific phage fusion protein

Fusion proteins created by phage display peptides with tumor cell specificity and the pVIII major coat protein of filamentous phages have been explored recently as a simple and cost-effective means for preparing tumor-targeted liposomes that improve the cytotoxicity of anticancer drugs in vitro. The next step in the development of this approach is the optimization of the liposome composition for the maximum targeting activity and subsequent testing in vivo. This study aimed to investigate the impact of preparation protocols, lipid composition and phage protein content on the targeting efficiency of phage protein-modified liposomes. Analysis of size, zeta potential and morphology was used to investigate the effect of preparation protocols on the stability and homogeneity of the phage liposomes. A previously developed coculture targeting assay and a factorial design approach were used to determine the role of lipid composition of the liposomal membrane on the target cell specificity of the phage liposomes. Western blot combined with proteinase K treatment detected the orientation of targeted phage protein in liposomal membrane. Phage protein, DPPG and PEG_2k-PE showed positive effects on target specificity of phage liposomes. The results served to identify optimal formulation that offer an improved liposomal affinity for target tumor cells over the non-optimized formulation.     

An update on liposomes in drug delivery: a patent review (2014-2018)

ABSTRACT

Introduction: Pharmacotherapy is limited by the inefficient drug targeting of non-healthy cells/tissues. In this pharmacological landscape, liposomes are contributing to the impulse given by Nanotechnology to optimize drug therapy.

Areas covered: The analysis of the state-of-the-art in liposomal formulations for drug delivery purposes have underlined that lately published patents (since 2014) are exploring alternative compositions and ways to optimize the stability and drug loading content/release profile. These improvements are complemented by improved long-circulating structures and further liposome functionalizations, which have definitively opened the road for the (co-)delivery of therapeutics to the site of action. Liposomes are also contributing to new drug delivery approaches involving the generation of extracellular vesicles by targeted cells, while opening new ways to combine disease diagnosis and therapy (theranosis).

Expert opinion: Patent publications on liposomal formulations have expanded new ways in drug delivery. New lipid compositions and strategies to optimize stability and drug vehiculization capabilities have settle solid pillars in liposome fabrication. Despite, their architecture has been satisfactorily adapted for combining passive and active drug targeting concepts, new inputs of liposomes into the disease arena should answer for: a simple/scalable/cost-effective formulation; a safe/stable/controllable formulation meeting quality control regulations; and, a confirmed therapeutic efficiency in clinical investigations.     

Targeting of liposome-associated calcipotriol to the skin: effect of liposomal membrane fluidity and skin barrier integrity

Many dermal diseases like psoriasis are characterized by major changes in skin barrier function, which challenge the reproducible delivery of drugs into specific layers of diseased skin. The purpose of this study was to elucidate how liposomal bilayer fluidity and barrier integrity affected the delivery of liposome-associated calcipotriol to the skin. Calcipotriol-containing gel state and liquid state dipalmitoylphosphatidyl-choline:dilauroylphosphatidylcholine liposomes were prepared by extrusion. Using Langmuir monolayers, calcipotriol was shown to affect the packing of the lipid membrane. The penetration of radioactively labeled lipid and calcipotriol into pig skin was examined using the Franz diffusion cell model, and tape stripping was applied to impose an impaired barrier. Distorting the skin barrier resulted in an enhanced penetration of lipid from both gel and liquid state liposomes. In addition, increased penetration of lipid from liquid state liposomes was observed compared to gel state liposomes into barrier-impaired skin. For barrier-impaired skin, an elevated calcipotriol-to-lipid ratio was found in the receptor fluid for both liposome compositions indicating that calcipotriol is released from the vesicles. This suggests that the liposome-mediated delivery of calcipotriol to the epidermis of diseased skin is affected by the fluidity of the liposomal membrane.     

Recent advances in liposome technologies and their applications for systemic gene delivery

The recent clinical successes experienced by liposomal drug delivery systems stem from the ability to produce well-defined liposomes that can be composed of a wide variety of lipids, have high drug-trapping efficiencies and have a narrow size distribution, averaging less than 100 nm in diameter. Agents that prolong the circulation lifetime of liposomes, enhance the delivery of liposomal drugs to specific target cells, or enhance the ability of liposomes to deliver drugs intracellularly can be incorporated to further increase the therapeutic activity. The physical and chemical requirements for optimum liposome drug delivery systems will likely apply to lipid-based gene delivery systems. As a result, the development of liposomal delivery systems for systemic gene delivery should follow similar strategies.     

Mechanisms and kinetics of liposome-cell interactions

Although the possibility of targeting drugs to specific tissues and cells, as well as facilitating their uptake and cytoplasmic delivery has rendered liposomes a versatile drug carrier system with numerous potential applications in medicine, the molecular mechanisms of liposome-cell interactions are not understood well. Here we have reviewed the early and current concepts of liposome-cell interactions, including possible liposome receptors. Uptake of liposomes by cells can be modified by the lipid composition, particularly by the inclusion of steric stabilizers such as PEG-conjugated lipids. Such modifications also alter the circulation time and biodistribution of liposomes, which can thus be tailored for particular applications. The intracellular fate of encapsulated molecules can be modified by the use of pH-sensitive liposomes which can also be sterically stabilized. Cationic liposomes that can undergo lipid mixing with cellular membranes can deliver complexed DNA to cells, but most likely via an endocytotic process. Kinetic analysis of liposome-cell interactions can elucidate the numbers of liposome receptors of several types and the corresponding binding constants. It is likely that liposomes bind to different cell surface receptors on different cells, and that they utilize more than one type of receptor on a particular cell. The kinetic analysis also provides the rate constants of endocytosis and the percentages of liposomes that are bound or endocytosed.     

Liposome-based depot injection technologies

Liposomes have proven to be versatile carriers for the delivery of drugs. These carriers are biocompatible, since they are generally made from lipids commonly found in biologic systems and are biodegradable by the usual metabolic pathways. A sustained drug delivery system is useful when the efficacy of drugs is limited by the inability to maintain therapeutic concentrations. Furthermore, a depot delivery system can offer important advantages in the clinic, such as significantly reducing dose frequency and providing efficacy without toxicity. Because of their small size (<5μ.m), conventional liposomes (unilamellar and multilamellar) are limited in their ability to provide depot delivery of drugs when administered subcutaneously or intramuscularly. The small size of these liposomes results in relatively fast clearance from the injection site and a short duration of delivery, typically 1–4 days. Multivesicular liposomes (MVLs) are distinct from conventional liposomes in composition, structure, and size and are the only class of commercial liposomes that have demonstrated depot delivery of both small molecule and protein/peptide drugs. These MVLs are characterized by the presence of a continuous bilayer membrane, with numerous internal aqueous compartments that are contiguous and separated by bilayer septums. As a result of their larger size (median diameter typically 10–30μ.m), these MVLs are not rapidly cleared by tissue macrophages and can act as a drug depot providing slow release of drugs delivered through different routes of administration. Moreover, the biocompatibility and biodegradability of the MVL lipid matrix allows for the sustained delivery of drugs to sensitive areas. The unique architecture of MVLs provides high drug loading of water-soluble drugs, reasonable stability during storage, and control over the drug-release rate. Furthermore, the lipid composition of MVLs can be altered to deliver therapeutics over periods ranging from a few days to a month, in order to meet specific therapeutic needs. The capability of altering the rate of drug release from MVLs by varying the lipid composition provides a great deal of versatility for controlled delivery of a wide variety of therapeutics. This article reviews depot delivery with conventional liposomes, demonstrates through several examples the sustained depot delivery of small and macromolecular drugs using MVLs, and summarizes some novel delivery systems that combine liposomes with polymeric matrices and have the potential to expand the platform of liposomal depot delivery.     

Triggered activation and release of liposomal prodrugs and drugs in cancer tissue by secretory phospholipase A2

The selectivity of anticancer drugs in targeting the tumour tissue presents a major problem in cancer treatment. In this article we review a new generation of smart liposomal nanocarriers that can be used for enhanced anticancer drug and prodrug delivery to tumours. The liposomes are engineered to be particularly degradable to secretory phospholipase A2 (sPLA2), which is a lipid hydrolyzing enzyme that is significantly upregulated in the extracellular microenvironment of cancer tumours. Thus, when the long circulatory liposomal nanocarriers extravasate and accumulate in the interstitial tumour space, sPLA2 will act as an active trigger resulting in the release of cytotoxic drugs in close vicinity of the target cancer cells. The sPLA2 generated lysolipid and fatty acid hydrolysis products will furthermore be locally released and function as membrane permeability promoters facilitating the intracellular drug uptake. In addition, the liposomal membrane can be composed of a novel class of prodrug lipids that can be converted selectively to active anticancer agents by sPLA2 in the tumour. The integrated drug discovery and delivery technology offers a promising way to rationally design novel tumour activated liposomal nanocarriers for better cancer treatment.     

Liposomes in the treatment of infectious diseases

Phospholipids and other polar lipids can form liposomes and similar colloidal particles that can be used as drug carrier systems. The potential of liposomal delivery systems to increase the therapeutic index (efficacy to safety ratio) of clinically important drugs has been realised with the recent approval of liposomal oncologic and antifungal drugs. The application of liposomes to the treatment of infectious diseases initially focused on intracellular pathogens, based on the natural targeting of liposomes to phagocytic cells and on the antifungal drug amphotericin B, based on its unique affinity for lipids. Recent studies with small, low-clearance liposomes have shown that more specialised formulations may provide benefits over simpler ‘first generation’ liposomes for the treatment of infectious diseases, including prolonged residence in plasma, increased tissue exposure and targeting to sites of infection. These improved biopharmaceutical properties have been associated with both curative and prophylactic activity against a range of non-intracellular pathogens, including Staphylococcus and Klebsiella. These and other highly engineered liposome formulations may provide effective delivery systems for specific antibacterial, antifungal and antiviral indications in the future. Adequate patent protection will be crucial in fully exploiting these advanced liposome technologies and in maintaining market share for liposomal products. This review discusses some of the patent issues related to liposomes and their use in the treatment of infectious diseases.     

New generation of liposomal drugs for cancer

This review is focused on liposomes as a delivery system for anticancer agents and more specifically on the advantages of using liposomes as drug nanocarrier in cancer chemotherapy. The main advantages of liposomal drugs over the non-encapsulated drugs include: (1) improved pharmacokinetics and drug release, (2) enhanced intracellular penetration, (3) tumor targeting and preventing adverse side effects and (4) ability to include several active ingredients in one complex liposomal drug delivery system (DDS). The review also includes our recent data on advanced liposomal anticancer drug delivery systems. As a conclusion we propose a novel liposomal DDS which includes inhibitors of pump resistance combined in one liposomal drug delivery system with an inhibitor of antiapoptotic cellular defense, an apoptosis inducer (a traditional anticancer drug) and a targeting moiety. The proposed drug delivery system utilizes a novel three tier approach, simultaneously targeting three molecular targets: (1) extracellular receptors or antigen expressed on the surface of plasma membrane of cancer cells in order to direct the whole system specifically to the tumor, preventing adverse side effects on healthy tissues; (2) drug efflux pumps in order to inhibit them and enhance drug retention by cancer cells, increasing intracellular drug accumulation and thereby limiting the need for prescribed high drug doses that cause adverse drug side effects; and (3) intracellular controlling mechanisms of apoptosis in order to suppress cellular antiapoptotic defense.