Current Drug Delivery - Volume 2, Issue 4, 2005

Editorial: Lipid-Based Nanosystems and Complexes in Experimental and Clinical Therapeutics An intriguing spectrum of novel nanoparticulate systems and complex nanostructures, some with remarkable diagnostic and biomimetic properties and others suitable for site-specific drug delivery and targeting are in existence today. Examples include quantum dots, capable of labelling biological systems for detection by optical or electrical means, tris-malonic acid derivatives of the fullerene C60, which exhibit superoxide dismutase mimetic properties, and porous titanium oxide and silicon nanoparticulate systems with sustained drug release properties. The ability to create these assemblies as well as other unusual nanostructures such as bundles, tubes, and sheets holds promise for new and powerful diagnostic and drug delivery systems, which will surely change the foundations of disease treatment and diagnosis. These technological innovations and nanoscience approaches to particle design are the focus of the US National Institute of Health's Nanomedicine Roadmap Initiative and the National Cancer Institute's Alliance for Nanotechnology in Cancer and several new European Commission-funded initiatives. Indeed, February 2004 saw the birth of Nano2Life, Europe's first Network of Excellence in nanobiotechnology, and in February 2005, the European Science Foundation launched its Scientific Forward Look on Nanomedicine Initiative, with strong emphasis on engineering of smart nanoparticle-based medicines and nanodevices. Although within the remit of the above mentioned nanoscience initiatives, lipid-based nanosystems such as nanoemulsions, lipid-core micelles, small unilamellar vesicles and variations thereof, have long been in existence and some have long been improving patient's lives. Indeed, lipid-based nanoformulations are among the most attractive candidates for improving drug solubility and for site-specific targeting following parenteral administration. This Theme Issue of Current Drug Delivery features contributions focusing on selected molecularbased strategies and nanoscience approaches to design, development, and site-specific targeting of a wide range of lipid-based nanocarriers and complex systems currently at preclinical and clinical stages. Specifically, great strides are being made with such complexes and nanosystems in combating the growth and spread of cancerous tissues (e.g., through exploitation of angiogenic tumour vasculature, combination chemotherapy, and endogenous triggered activation and release of encapsulated lipid pro-drugs), treatment of macrophage infections (through exploitation of macrophage clearance mechanisms), gene transfer (by breaching the endo-lysosomal barrier with cationic lipid vectors) and stimulation of immune responses to antigens (with the aid of vesicular systems and lipid-complexes with self-adjuvanting properties), and are discussed in the first thirteen articles in this Theme Issue. Although, lipidbased nanocarriers may overcome solubility or stability issues for the drug and minimize drug-induced side effects through favourable pharmacokinetic profiles and site-specific targeting, there are significant toxicity issues with carriers themselves that need to be addressed. Selected toxicity issues are dealt with in the last two articles. The first contribution discusses toxicogenomics of the delivery system and the impact of the microarray technology; in assessing cellular toxicity, while in the final article, the reader's attention is drawn towards pseudoallergic reactions, which are believed to be secondary to complement activation. Here, endogenous lipid-based delivery vehicles (lipoproteins) seem to play a complex modulatory role in complement-mediated pseudoallergic responses to nanomedicines. We believe that this Issue has provided a broad sample of the state of the art. We are indebted to all contributors for their attempt to communicate and discuss the most promising ideas, approaches, applications and developments of the most advanced lipid-based nanomedicines.

Nanoemulsions, usually spherical, are a group of dispersed particles used for pharmaceutical and biomedical aids and vehicles that show great promise for the future of cosmetics, diagnostics, drug therapies and biotechnologies. They exist in a wide variety of forms that are dictated by the particle components. Nanoemulsions are generally considered to be in the size range of less than and around 100 nm in diameter. The particles can exist as water-in-oil and oil-inwater forms, where the core of the particle is either water or oil, respectively. More complex variations also exist but these are often larger. The longer-term properties of the particle are dependent on the composition of the adsorbed material lying at the dispersed droplet interface with the dispersion medium. This has an impact on the partitioning and extraction of droplet contents. Thermodynamically stable particles are characterized by having a very low surface tension and this produces a very large surface area. Nanoemulsions can also include small meta-stable very small-scale emulsions; here the surface properties and chemistry can strongly influence behaviour. Processing, storage and formulation composition can also have an impact on the longevity of a pharmaceutical preparation. Some revolutionary new nanoemulsion droplets based on fluorinated compounds are finding a number of widespread biomedical roles and applications. Developments in nanoemulsion technology are likely to lead to a much greater use of this medium in future pharmaceuticals.

Successful homing of drugs to the desired biological compartment of the host usually depends on the intrinsic properties of the drug molecules. However, it can always be manipulated by appropriate designing of the carrier/ delivery system, as little can be done to influence the target and its surroundings. Various carrier systems have emerged to deliver drugs to macrophages, albeit the efficacy, reliability and selectivity of these carriers are still in question. To date, the most extensively studied carriers are liposomes and microspheres. In fact, physicochemical properties of these carriers can alter their efficacy and specificity to a great extent. These properties include hydrophilicity, surface charge, composition, concentration, and presence of various target specific ligands on their surface. Incidentally, the particulate nature of these vehicles may facilitate passive homing of the entrapped drug molecules to the macrophages, which may harbour many of the important pathogens in their intracellular compartments, such as Mycobacterium sps, Leishmania and dengue virus etc., belonging to three different major classes of microbes. Moreover, macrophages upon interaction with particulate drug delivery vehicles may act as secondary drug depot, thus helping in localized delivery of the drug at the infected site. In the present article, a comprehensive review of literature is presented on the suitability of some lipid-based and polymeric materials as vehicles in delivery of drugs to macrophages in parasitic infections.

Micelles, self-assembling nanosized colloidal particles with a hydrophobic core and hydrophilic shell are currently successfully used for the solubilization of various poorly soluble pharmaceuticals and demonstrate a series of attractive properties as drug carriers. Polymeric micelles, i.e. micelles formed by amphiphilic block co-polymers possess high stability both in vitro and in vivo and good biocompatibility. Among those micelles, lipid-core micelles, i.e. micelles formed by conjugates of soluble copolymers with lipids (such as polyethylene glycol-phosphatidyl ethanolamine conjugate, PEG-PE) are of special interest. These micelles can effectively solubilize a broad variety of poorly soluble drugs (anticancer drugs in particular) and diagnostic agents. Drug-loaded lipid-core micelles can spontaneously target body areas with compromised vasculature (tumors, infarcts) via the enhanced permeability and retention (EPR) effect. Lipid-core mixed micelles containing certain specific components (such as positively charged lipids) are capable of escaping endosomes delivering incorporated drugs directly into the cell cytoplasm. Various specific targeting ligand molecules (such as antibodies) can be attached to the surface of the lipid-core micelles and bring drug-loaded micelles to and into target cells. Lipid-core micelles carrying various reporter (contrast) groups may become the imaging agents of choice in different imaging modalities.

Phospholipid nanosomes are small, uniform liposomes manufactured utilizing supercritical fluid technologies. Supercritical fluids are first used to solvate phospholipid raw materials, and then decompressed to form phospholipid nanosomes that can encapsulate hydrophilic molecules such as proteins and nucleic acids. Hydrophobic therapeutics are co-solvated with phospholipid raw materials in supercritical fluids that, when decompressed, form phospholipid nanosomes encapsulating these drugs in their lipid bilayers. Mathematical modeling and semi-empirical experiments indicate that the size and character of phospholipid nanosomes depend on the several process parameters and material properties including the size and design of decompression nozzle, bubble size, pressure and the rate of decompression, interfacial forces, charge distribution and the nature of compound being encapsulated. Examples are presented for the encapsulation of a protein and hydrophobic drugs. In vitro and in vivo data on breast cancer cells and xenografts in nude mice indicate that paclitaxel nanosomes are less toxic and much more effective than paclitaxel in Cremophor EL® (Taxol®). Camptothecin nanosomes demonstrate that the normally very water-insoluble camptothecin can be formulated in a biocompatible aqueous medium while retaining in vivo efficacy against lymphoma xenografts in nude mice. In vitro data for betulinic acid nanosomes demonstrate enhanced efficacy against HIV-1 (EC50 of 1.01 μg/ml versus 6.72 μg/ml for neat betulinic acid). Phospholipid nanosomes may find utility in the enhanced delivery of hydrophilic drugs such as recombinant proteins and nucleic acid as well as hydrophobic anticancer and anti-HIV drugs.

The introduction of combination chemotherapeutic regimens for the treatment of childhood leukaemia in the 1960s provided the proof-of-principle that cytotoxic drugs were capable of curing cancer. However, in the four decades since this discovery, the majority of cancers still cannot be cured by chemotherapy. Clinical evidence supports the hypothesis of Goldie and Coldman that treating cancers with all the available effective agents simultaneously provides the greatest chance of eliciting a cure. Unfortunately, for traditional cytotoxic agents with narrow therapeutic indices, lifethreatening toxicity precludes combination chemotherapy regimens employing multiple agents. This review discusses the concept of fixed dose combination chemotherapy with emphasis on capturing therapeutic efficacy described as synergistic as a basis for improving the effectiveness of combination chemotherapy. The use of lipid-based nanotechnologies, focusing on liposomes, as an enabling technology to facilitate the delivery of cytotoxic agents to the tumour site at concentrations and/or drug ratios judged to be synergistic will be discussed. It is envisaged that the development of this model system will be supported by cell-based screening technologies, pharmacokinetic and pharmacodynamic parameters and mathematical models describing therapeutic drug:drug interactions (the Median Effect Principle of Chou and Talalay). Experiments using preclinical models are presented to support the benefits of drug delivery systems as a foundation for fixed dose anticancer drug combinations. The ultimate goal of this research is to prepare a 'single vial' fixed dose combination product that encompasses both traditional cytotoxic agents and new molecularly targeted modalities with optimum therapeutic effects and acceptable toxicity.

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.

Active targeting of angiogenic vasculature represents a promising therapeutic strategy to fight a variety of diseases. In particular, attention has been focused on inhibition of tumor growth. In this review, the recent progress in targeting liposomes to angiogenic endothelial cells is discussed.

Selective targeting of ligand-targeted liposomes containing anticancer drugs or therapeutic genes to cell surface receptors expressed on cancer cells is a recognized strategy for improving the therapeutic effectiveness of conventional chemotherapeutics or gene therapeutics. Some recent advances in the field of ligand-targeted liposomes for the treatment of cancer are summarized including: selection criteria for the receptors to be targeted, choice of targeting ligands and choice of encapsulated therapeutics. Targeting of liposomes to solid tumors, versus angiogenic endothelial cells versus vascular targets is discussed. Ligand-targeted liposomes have shown considerable promise in preclinical xenograft models and are poised for clinical development.

Lipopeptides incorporating epitopes for CD4+ T cells and either CD8+ T cells or B cells have proven to be immunogenic in animal models and in humans and are well tolerated in these species. This form of vaccine candidate has great benefits over competing technology in terms of providing a totally synthetic and pure product that is effective when administered in the absence of any adjuvant, and is immunogenic when delivered by a variety of routes, including application to mucosal surfaces. The immune response can be focused on critical epitopes of the pathogen or tumour antigen to provide clearing immunity, and responses can also be invoked that modulate hormone activity. This review will cover examples of lipopeptides of different design and their efficacy in different systems as well as challenges for the future. Our recent understanding of how the lipid component confers the "self-adjuvanting" property on these immunogens by targeting the cell at the heart of immune response induction, the dendritic cell, will also be discussed.

Dramatic developments in vaccinology and immunology over the last decade have led to the identification of novel antigens, adjuvants and delivery systems, and combinations thereof, that may prove to be highly efficacious vaccines. Despite this explosion in technological developments, in reality, very few novel vaccines for human use have reached the market. Liposomes are one of the most intensively studied areas of vaccine and delivery system research. Several intrinsic features of liposomes lend themselves to their use in vaccines, particularly in terms of safety, versatility and adjuvant properties. Modifications to liposomal structures in terms of additional adjuvants are also playing a role in potential medical applications. The most significant progress to date has been made with virosomes, a novel antigen delivery system that incorporates the haemagglutinin from influenza virus into liposomes and can induce both humoral- and cell-mediated immunity to antigens. This review discusses the presentation and processing of antigens delivered by virosomes, in light of both licensed vaccines and potential vaccine candidates.

The development of novel 'new generation' vaccine systems that is based on proteins, peptides or DNA is of great current interest. However, due to the lower efficiencies of these new generation vaccines, they are seldomly used alone. Rather, their formulations often contain adjuvants, either to enhance the immune responses or to reduce dosing. The present chapter will provide a brief overview of the recent advances in peptide-based cancer vaccine adjuvants, focusing mainly on Liposome-Protamine-DNA (LPD) nanoparticle-mediated antigen delivery.

Archaeosomes are liposomes made from the polar ether lipids of Archaea. These lipids are unique and distinct in structure from the ester lipids found in Eukarya and Bacteria. The regularly branched and usually fully saturated isopranoid chains of archaeal polar lipids are attached via ether bonds to the sn-2,3 carbons of the glycerol backbone(s). The polar head groups are usually the same as those encountered in the ester lipids from the other two domains, except that phosphatidylcholine is rarely present. These lipid structures provide formulary advantages, and contribute to the excellent physico-chemical stability of the archaeosomes and their efficacy as self-adjuvanting vaccine delivery vesicles. The uptake of archaeosomes by phagocytic cells is several folds greater than that of liposomes made from ester lipids. In addition, archaeosomes enhance the recruitment and activation of professional antigen presenting cells in vivo, and deliver the antigen to both MHC class I and II pathways for antigen presentation, without eliciting overt inflammatory responses. In murine models, systemic administration of archaeosomes containing encapsulated antigen(s) elicits strong and sustained antigen-specific antibody responses which are comparable, in some formulations, to those obtained with Freund's adjuvant. Additionally, archaeosomes promote robust antigen-specific cell-mediated immunity, including CD8+ CTL responses. The immune responses induced by archaeosomes are sustained over long periods and exhibit strong memory responses. More importantly, immunization of mice with archaeosome-based vaccines induces robust protective immunity against intracellular pathogens, and prophylactic and therapeutic efficacies against the development of experimental cancers. Extensive murine model studies suggest that archaeosomes are safe.

A wide variety of lipid molecules used as gene carriers has been reported and compared over the last twenty years. This review highlights a few examples of mechanistic analysis applied to the study of lipid carriers. The modular nature of the lipid structure offers itself up to a controlled, systematic analysis. Key to exploring structural variants is the understanding of the role each component and module plays in the formation of the lipoplex structure itself and their roles in the transfection pathway. Firstly, the lipid carrier must be able to package, and release into the cell its nucleic acid cargo. Uptake of lipoplexes into cells involves endocytic processes that lead inevitably to endosomal/liposomal degradation of the nucleic acid contents, unless lipid structures and designs are optimised to facilitate their release into the cytoplasm. Testing of possible endosomal escape mechanisms has led to improved lipid designs. For example, it was predicted that mixing of cationic lipids with shorter alkyl tails (

Implementation of the high-throughput microarray gene expression profiling technology towards "toxicogenomics" has advanced identification process for safer drugs in the century of 'omics' technology. Applying such technology, in fact, to identify mechanisms for cellular toxicity can provide a means to clarify safety liabilities early in the drug discovery and developments process. The underlying principle in gene therapy is primarily targeting a specific gene (e.g., for silencing). Hence, massive efforts have been devoted to validate the gene-based therapeutics, regardless of toxicogenomics potential of delivery systems. Of the gene delivery systems, viral and non-viral vectors, as two main paradigms, have so far been widely used for delivering of the genome-based therapeutics such as oligonucleotide, small interfering RNA and DNA. However, the use of viral vectors was narrowed due to the safety concerns. Non-viral vectors were utilized as safer alternatives for gene delivery in vitro and ex-vivo; though their success for in vivo gene therapy has been limited due to low efficiency and safety issues. Fundamental principle for gene therapy is to deliver gene-based therapeutics into target cells for specific gene targeting ideally with minimal cellular toxicity. Until now, few works have been conducted about geno-compatibility of delivery systems itself, including cationic lipid-based nanosystems. Inadvertent toxicogenomic impact of gene delivery systems (e.g., cationic lipids) may intrinsically affect the outcome of gene therapy, where often only a single desired genetic change is sought. Further, there exists a possibility that gene changes induced by the lipid delivery system itself could exacerbate, attenuate or even mask the desired effects of the gene-based therapeutics. This review will focus on toxicogenomics impact of the cationic lipid-based formulations for gene therapy.

Self-assembling amphiphilic lipids or polymers have been successfully used in pharmacotherapy as drug solvents or carriers, improving the bioavailability of water-insoluble drugs. This review focuses on an unusual hypersensitivity reaction (HSR) caused by a micellar (Cremophor EL, CrEL) and a monomeric block copolymer (poloxamer 188) representative of these systems. The HSRs, also referred to as anaphylactoid or pseudoallergic, are thought to arise as a consequence of complement (C) activation in blood. However, considering that C activation involves the deposition of multiple C and other (immune) proteins on the activator surface, the mechanism by which small, 8-25 nm CrEL micelles or individual poloxamer 188 molecules activate C is not straightforward. Observations on enlarged lipoproteins and de novo formation of abnormally large lipoprotein-like structures in plasma exposed to CrEL or poloxamer 188 raise the possibility that lipoprotein transformation might play a crucial role in C activation by these amphiphilic emulsifiers. Lipoproteins, furthermore, can also provide a negative feedback control on C activation, as suggested by the inhibition of poloxamer 188-induced C activation in the presence of excess exogenous lipoproteins, and the attenuation of liposome-induced and C activation-related hypotension in pigs by precoating the vesicles with lipoproteins. Thus, lipoproteins may be essential in the induction, and they may also play a complex modulatory role in C activation-related pseudoallergy caused amphiphilic drug solvents and carriers.