Gene transfection efficacies of novel cationic gemini lipids possessing aromatic backbone and oxyethylene spacers

Six novel gemini cationic lipids based on aromatic backbone, bearing n-C14H 29 or n-C16H33 hydrocarbon chains, differing in the length of oxyethylene type spacers -CH2-(CH2-O-CH2)m-CH2- between each ammonium headgroups have been synthesized, where m varies from 1 to 3. Each of these lipids formed stable suspensions in aqueous media. Cationic liposomes were prepared from each of these lipids individually and as mixtures of each cationic lipid and DOPE. These were used as nonviral gene delivery agents. Transfection studies showed that among lipids bearing n-C14H29 chains, the transfection efficacies decreased with the increase in the length of the spacer, whereas in case of lipids bearing n-C 16H33 chains, the transfection efficacies increased with the increase in the length of the spacer. Lipid bearing n-C16H33 hydrocarbon chains with a [-(CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2)-] spacer was found to be a potent gene transfer agent and its transfection was highly serum compatible even in the presence of 50% serum conditions.     

Synthesis and gene transfer activities of novel serum compatible cholesterol-based gemini lipids possessing oxyethylene-type spacers

Four novel cholesterol-based gemini cationic lipids differing in the length of oxyethylene-type spacers [-CH2-(CH2-O-CH2)n-CH2-] between each ammonium headgroup have been synthesized. These formed stable suspensions in aqueous media. Cationic liposomes were prepared from each of these lipids individually and as mixtures of cationic lipid and DOPE. These were used as nonviral gene delivery agents. All the cholesterol-based gemini lipids induced better transfection activity than their monomeric counterpart. Inclusion of DOPE in co-liposomal formulation of the cationic gemini lipid potentiates their gene transfer activity significantly. A major characteristic feature of these oxyethylene spacer based cholesterol gemini lipids was that serum does not inhibit the transfection activity of these gemini lipids, whereas the transfection activity of their monomeric counterpart decreased drastically in the presence of serum. One of the cholesterol-based gemini lipids 2a possessing a -CH2-CH2-O-CH2-CH2- spacer showed the highest transfection activity.     

Conjugation of lipid and CpG-containing oligonucleotide yields an efficient method for liposome incorporation

For optimal stimulation of T cells, protein-based vaccines must deliver protein antigens to antigen-presenting cells while simultaneously providing immunostimulatory signals. Listeriolysin O (LLO)-containing liposomes have been utilized to efficiently deliver protein antigens to the cytosolic pathway for antigen processing and major histocompatibility complex class I-dependent presentation while codelivering immunostimulatory CpG-oligodeoxyribonuceotides (ODNs). In this report, we describe the synthesis of lipid-CpG-ODN conjugates utilizing maleimide-phosphatidylethanolamine (PE) lipids and 5'-sulfhdryl-containing CpG-ODNs as a method for facile incorporation of CpG-ODNs in liposomal vaccine carriers, an alternative to co-encapsulation inside liposomes and as a means to enhance delivery of CpG-ODNs to their major receptor, Toll-like receptor 9 (TLR9), in the endosome. The characterization and biological evaluation of the vaccine delivery system made of liposomes, which contain the lipid-CpG-ODN conjugates inserted in the liposomal membrane, is described. We demonstrate in vitro in bone marrow derived macrophages that the lipid-CpG-ODN conjugates incorporated onto the liposome bilayers interact with their receptor TLR9 as readily as liposome-encapsulated ODNs and exert their immunostimulatory capabilities. The liposomal vaccine delivery systems were evaluated in mice using ovalbumin (OVA) as a model antigen, and the results indicate equally robust OVA-specific cytotoxic T lymphocyte responses and similar Th1 immune skewing capabilities between liposomes containing lipid-conjugated or encapsulated CpG-ODNs. Overall, this work indicates that conjugating PE lipids and CpG-ODNs results in an efficient method that allows facile incorporation of CpG-ODNs into a liposome-based delivery platform while retaining the immune-stimulating capabilities of CpG-ODNs.     

Light-sensitive lipid-based nanoparticles for drug delivery: design principles and future considerations for biological applications

Radiation-based therapies aided by nanoparticles have been developed for decades, and can be primarily categorized into two main platforms. First, delivery of payload of photo-reactive drugs (photosensitizers) using the conventional nanoparticles, and second, design and development of photo-triggerable nanoparticles (primarily liposomes) to attain light-assisted on-demand drug delivery. The main focus of this review is to provide an update of the history, current status and future applications of photo-triggerable lipid-based nanoparticles (light-sensitive liposomes). We will begin with a brief overview on the applications of liposomes for delivery of photosensitizers, including the choice of photosensitizers for photodynamic therapy, as well as the currently available light sources (lasers) used for these applications. The main segment of this review will encompass the details of strategies used to develop photo-triggerable liposomes for their drug delivery function. The principles underlying the assembly of photoreactive lipids into nanoparticles (liposomes) and photo-triggering mechanisms will be presented. We will also discuss factors that limit the applications of these liposomes for in vivo triggered drug delivery and emerging concepts that may lead to the biologically viable photo-activation strategies. We will conclude with our view point on the future perspectives of light-sensitive liposomes in the clinic.     

Archaeobacterial ether lipid liposomes (archaeosomes) as novel vaccine and drug delivery systems

Liposomes are artificial, spherical, closed vesicles consisting of one or more lipid bilayer(s). Liposomes made from ester phospholipids have been studied extensively over the last 3 decades as artificial membrane models. Considerable interest has been generated for applications of liposomes in medicine, including their use as diagnostic reagents, as carrier vehicles in vaccine formulations, or as delivery systems for drugs, genes, or cancer imaging agents. The objective of this article is to review the properties and potential applications of novel liposomes made from the membrane lipids of Archaeobacteria (Archaea). These lipids are unique and distinct from those encountered in Eukarya and Bacteria. Polar glycerolipids make up the bulk of the membrane lipids, with the remaining neutral lipids being primarily squalenes and other hydrocarbons. The polar lipids consist of regularly branched, and usually fully saturated, phytanyl chains of 20, 25, or 40 carbon length, with the 20 and 40 being most common. The phytanyl chains are attached via ether bonds to the sn-2,3 carbons of the glycerol backbone(s). It has been shown only recently that total polar lipids of archaeobacteria, and purified lipid fractions therefrom, can form liposomes. We refer to liposomes made with any lipid composition that includes ether lipids characteristic of Archaeobacteria as archaeosomes to distinguish them from vesicles made from the conventional lipids obtained from eukaryotic or eubacterial sources or their synthetic analogs. In general, archaeosomes demonstrate relatively higher stabilities to oxidative stress, high temperature, alkaline pH, action of phospholipases, bile salts, and serum proteins. Some archaeosome formulations can be sterilized by autoclaving, without problems such as fusion or aggregation of the vesicles. The uptake of archaeosomes by phagocytic cells can be up to 50-fold greater than that of conventional liposome formulations. Studies in mice have indicated that systemic administration of several test antigens entrapped within certain archaeosome compositions give humoral immune responses that are comparable to those obtained with the potent but toxic Freund's adjuvant. Archaeosome compositions can be selected to give a prolonged, sustained immune response, and the generation of a memory response. Tissue distribution studies of archaeosomes administered via various systemic and peroral routes indicate potential for targeting to specific organs. All in vitro and in vivo studies performed to date indicate that archaeosomes are safe and do not invoke any noticeable toxicity in mice. The stability, tissue distribution profiles, and adjuvant activity of archaeosome formulations indicate that they may offer a superior alternative to the use of conventional liposomes, at least for some biotechnology applications.     

Light-sensitive lipid-based nanoparticles for drug delivery: design principles and future considerations for biological applications

Abstract

Radiation-based therapies aided by nanoparticles have been developed for decades, and can be primarily categorized into two main platforms. First, delivery of payload of photo-reactive drugs (photosensitizers) using the conventional nanoparticles, and second, design and development of photo-triggerable nanoparticles (primarily liposomes) to attain light-assisted on-demand drug delivery. The main focus of this review is to provide an update of the history, current status and future applications of photo-triggerable lipid-based nanoparticles (light-sensitive liposomes). We will begin with a brief overview on the applications of liposomes for delivery of photosensitizers, including the choice of photosensitizers for photodynamic therapy, as well as the currently available light sources (lasers) used for these applications. The main segment of this review will encompass the details of strategies used to develop photo-triggerable liposomes for their drug delivery function. The principles underlying the assembly of photoreactive lipids into nanoparticles (liposomes) and photo-triggering mechanisms will be presented. We will also discuss factors that limit the applications of these liposomes for in vivo triggered drug delivery and emerging concepts that may lead to the biologically viable photo-activation strategies. We will conclude with our view point on the future perspectives of light-sensitive liposomes in the clinic.     

Cytosolic delivery of macromolecules. II. Mechanistic studies with pH-sensitive morpholine lipids

Drug carriers containing weak acids or bases can promote cytosolic delivery of macromolecules by exploiting the acidic pH of the endosome. We have prepared two pH-sensitive mono-stearoyl derivatives of morpholine, one with a (2-hydroxy) propylene (ML1) linker and the other, an ethylene (ML2) linker. The pK(a) values of lipids ML1 and ML2, when incorporated into liposomes, are 6.12 and 5.91, respectively. Both lipids disrupt human erythrocytes at pH equal to or below their pK(a) but show no such activity at pH 7.4. Confocal microscopy studies suggest partial endosome-to-cytosol transfer of fluorescent dextran (MW 10 kDa) encapsulated in liposomes that contained 20 mol% of morpholine lipids. Interestingly, co-incubation of morpholine lipids in free or micellar form (without liposomal incorporation) with dextran resulted in efficient cytosolic delivery. Upon acidification to the endosomal pH, liposomes containing ML1 revealed: (a). leakage of entrapped solute that is independent of solute size; (b). lack of liposomal collapse into micelles as evidenced by photon correlation spectroscopy and UV light scattering; and (c). minimal inter-bilayer interactions as shown in a fluorescence resonance energy transfer assay. These observations are consistent with progressive intravesicular reorganization of lipids into stable liposomes of smaller size, but of more homogeneous distribution, upon acidification. The results emphasize a need to manipulate liposomal formulations containing ML1 such that ML1 will promote catastrophic collapse of liposomes to mixed micelles upon exposure to acidic pH. It is only then that micelle-mediated permeabilization of the endosomal membrane will lead to efficient cytosolic delivery of macromolecules originally loaded in liposomes.     

Assessment of liposome delivery using scintigraphic imaging

Scintigraphic imaging is a valuable tool for the development of liposome-based therapeutic agents. It provides the ability to non-invasively track and quantitate the distribution of liposomes in the body. Liposomes labeled with technetium-99 m (99mTc) are particularly advantageous for imaging studies because of their favorable physical characteristics. Examples of how scintigraphic imaging studies have contributed to the evaluation and development of a variety of liposome formulations will be presented. These include liposomes for targeting processes with inflammation associated increased vascular permeability such as healing bone fractures and viral infections; liposomes for intraarticular delivery; and liposomes for delivery of agents to lymph nodes located in the extremities, the mediastinum and the peritoneum. Scintigraphic studies of liposome distribution are very informational and often suggest new drug delivery applications for liposomes.     

Interactions of polymerizable phosphatidylcholine vesicles with blood components: relevance to biocompatibility

We have studied the biocompatibility properties of polymerizable phosphatidylcholine bilayer membranes, in the form of liposomes, with a view toward the eventual utilization of such polymerized lipid assemblies in drug carrier systems or as surface coatings for biomaterials. The SH-based polymerizable lipid 1,2-bis[1,2-(lipoyl)dodecanoyl]-sn-glycero-3-phosphocholine (dilipoyl lipid, DLL) and the methacryl-based lipid 1,2-bis[(methacryloyloxy)dodecanoyl]-sn-glycero-3-phosphocholine (dipolymerizable lipid, DPL) were studied in comparison to ‘conventional’ zwitterionic or charged phospholipids. We examined binding of serum proteins to liposomes and effects of liposomes on fibrin clot formation and on platelet aggregation. All types of liposomes tested bound complex mixtures of serum proteins with IgG being the most abundant bound component. DPL vesicles and anionic vesicles bound substantially more protein than other vesicle types. Polymerized DPL vesicles uniquely bound a protein of about 53 kDa which was not bound to other types of phosphatidylcholine liposomes. Likewise polymerized DPL vesicles, but not other types of phosphatidylcholine vesicles, caused a marked alteration in coagulation as measured by activated partial thromboplastin time (APTT) and prothrombin time (PT) tests; this effect was shown to be due to binding and depletion of clothing factor V by the DPL polymerized vesicles. Polymerized DPL liposomes and DLL liposomes in polymerized or nonpolymerized form, were without substantial effect on platelet aggregation. However, DPL nonpolymerized vesicles, while not causing aggregation, did impair ADP-induced aggregation of platelets. These studies suggest that SH based polymerizable lipids of the DLL type may be very suitable for in vivo use in the contexts of drug delivery systems or biomaterials development. Methacryloyl-based lipids of the DPL type seem to display interactions with the hemostatic process which militate against their in vivo utilization.     

One Step Membrane Incorporation of Viral Antigens as a Vaccine Candidate Against HIV

Liposomes can been used as potential immunoadjuvants, because they have the ability to elicit both a cellular mediated immune response and a humoral immune response. Studies have shown liposomes to be effective immunopotentiators in hepatitis A and influenza vaccines. For all these purposes, liposomes can be prepared by different methods. After disperging suitable membrane lipids in an aqueous phase and spontaneous formation of multilamellar large vesicles (MLV), mechanical procedures such as ultrasonication, homogenization by a French press or by other high pressure devices and, or extrusion through polycarbonate membranes with defined pore sizes lead to a reduction in size and number of lamellae of the vesicles. A second group of preparation procedures uses suitable detergents, e.g., bile salts or alkylglycosides. A third group of procedures starts with dissolving the lipids in an organic solvent and mixing it with an aqueous phase. The concentration of the organic solvent is then reduced by suitable procedures.

Here we present a new technique for the preparation of liposomes with associated membrane proteins, where lipid vesicles are formed immediately after injection into a micellar protein solution. The model membrane protein used for these studies is a truncated recombinant gp41 produced in E. coli. This viral membrane antigen is a possible candidate protein for the establishment of HIV-vaccines.

The data presented here, show an efficient and reproducible one step membrane protein encapsulation procedure into liposomes in a closed and sterile containment. We examined encapsulation efficiency, membrane protein conformation and immunogenicity of this possible liposomal vaccine candidate, which can be produced in GMP-compliant quality with the described technique.     

One step membrane incorporation of viral antigens as a vaccine candidate against HIV

Liposomes can been used as potential immunoadjuvants, because they have the ability to elicit both a cellular mediated immune response and a humoral immune response. Studies have shown liposomes to be effective immunopotentiators in hepatitis A and influenza vaccines. For all these purposes, liposomes can be prepared by different methods. After disperging suitable membrane lipids in an aqueous phase and spontaneous formation of multilamellar large vesicles (MLV), mechanical procedures such as ultrasonication, homogenization by a French press or by other high pressure devices and, or extrusion through polycarbonate membranes with defined pore sizes lead to a reduction in size and number of lamellae of the vesicles. A second group of preparation procedures uses suitable detergents, e.g., bile salts or alkylglycosides. A third group of procedures starts with dissolving the lipids in an organic solvent and mixing it with an aqueous phase. The concentration of the organic solvent is then reduced by suitable procedures. Here we present a new technique for the preparation of liposomes with associated membrane proteins, where lipid vesicles are formed immediately after injection into a micellar protein solution. The model membrane protein used for these studies is a truncated recombinant gp41 produced in E. coli. This viral membrane antigen is a possible candidate protein for the establishment of HIV-vaccines. The data presented here, show an efficient and reproducible one step membrane protein encapsulation procedure into liposomes in a closed and sterile containment. We examined encapsulation efficiency, membrane protein conformation and immunogenicity of this possible liposomal vaccine candidate, which can be produced in GMP-compliant quality with the described technique.     

Interaction of pH-Sensitive Liposomes With Blood Components

Abstract

Avoidance of lysosomal degradation of drugs entrapped in liposomes has been one of the major efforts in liposome research. The achievement of high drug deliver}' efficiency using pH-sensitive liposomes over the pH-insensitive liposomes has greatly influenced our strategies in liposome drug delivery. The success of pH-sensitive liposomes in delivering compounds such as fluorescence dye, anti-cancer reagents, toxins and DNA to target cells with high efficiency in vitro shows a great potential to apply the same strategy to in vivo systems. Using human plasma as a simplified model for blood, we have systematically examined the interaction of pH-sensitive liposomes composed of dioleoylphosphatidyl-ethanolamine (DOPE) and oleic acid (OA) with plasma components. Our results show that the bilayer structure of liposomes in plasma depends on their sizes. Small liposomes (d<200nm) were stabilized by plasma components while the larger ones (d>600nm) were rapidly lysed upon the exposure to plasma. Such differences in their stability in plasma may derive from their differences in lipid packing which determines the surface pressure of the membrane. Using purified serum proteins, we found that albumin such as bovine serum albumin (BSA) lyse liposomes by extracting OA from the bilayer. However, BSA induced lysis could be blocked by lipoproteins including HDL, LDL and VLDL, but not by immunoglobulins. Further studies with purified components of HDL demonstrated that apoAl, not the lipids of the HDL, contains the stabilization activity. The extraction of OA from liposomes and the insertion of plasma components into the bilayer modified the bilayer properties such that plasma stabilized liposomes were no longer pH sensitive. Using dipalmitoylsuccinylglycerol (DPSG), a double-chain pH senser for DOPE liposomes, we could preserve 50% pH sensitivity after plasma treatment. The potential application of such liposomes and other essential properties of pH-sensitive liposomes for drug delivery in vivo are also discussed.     

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。     

ASSESSMENT OF LIPOSOME DELIVERY USING SCINTIGRAPHIC IMAGING

ABSTRACT

Scintigraphic imaging is a valuable tool for the development of liposome-based therapeutic agents. It provides the ability to non-invasively track and quantitate the distribution of liposomes in the body. Liposomes labeled with technetium-99 m (^99mTc) are particularly advantageous for imaging studies because of their favorable physical characteristics. Examples of how scintigraphic imaging studies have contributed to the evaluation and development of a variety of liposome formulations will be presented. These include liposomes for targeting processes with inflammation associated increased vascular permeability such as healing bone fractures and viral infections; liposomes for intraarticular delivery; and liposomes for delivery of agents to lymph nodes located in the extremities, the mediastinum and the peritoneum. Scintigraphic studies of liposome distribution are very informational and often suggest new drug delivery applications for liposomes.     

Use of X-ray scattering to aid the design and delivery of membrane-active drugs

Biological membranes can be targets for compounds that either disrupt their barrier function or affect protein function via membrane-mediated processes. Biophysical studies on membrane-mimetic systems composed of membrane lipids have contributed substantially to our knowledge on the pertaining membrane physics and aid the development of membrane-specific drugs. Moreover, lipid membranes and, in particular, liposomes are convenient systems for drug delivery. We review some of our recent work that demonstrates the applicability of X-ray scattering to understanding the molecular mechanisms of drug–membrane interactions. In particular we focus on effects of anesthetics, sphingomyelinase, and antimicrobial peptides. We further discuss X-ray scattering as a quality-control tool for liposomal drug-delivery systems.     

Les nanoparticules

Nanoparticles (NP) are structures comprising from some hundred to some thousand atoms. Owing to their size (1 to 100 nanometres), the physical and chemical properties of these nano-objects differ from those of classical materials. They cover a wide development area, which includes medical research: they can be classified into two major groups, organic NP (liposomes, polymers NP, carbon nanotubes, fullerenes) and inorganic NP (quantum dots, magnetic NP, Raman probes). NP can be conceived to act as a drug delivery system (therapeutic), imaging probe (diagnostic) or both (theranostic). We report recent data from scientific literature and describe main NP within medical area, their state of development, and the limited knowledge of their toxicity in human being.     

Optical characterization of liposomes by right angle light scattering and turbidity measurement

Liposomes have frequently been used as models of biomembranes or vehicles for drug delivery. However, the systematic characterization of lipid vesicles by right angle light scattering and turbidity has not been carried out despite the usefulness of such studies for size estimation. In this study, liposomes of various sizes were prepared by sonication and extrusion. The mean cumulant radii of the vesicles were determined by dynamic light scattering. The lamellarities were estimated based on fluorescence quenching of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)dipalmitoyl-L-alpha-phosph ati dylethanolamine by sodium dithionite. Right angle light scattering intensity and optical density at 436 nm per unit lipid concentration were measured as a function of vesicle radius. With a vesicle radius < or =100 nm, the optical parameters could be well explained by the Rayleigh-Gans-Debye theory in which the liposomes were modeled as homogeneous spheres with mean refractive indices determined by the volume fractions of lipids in vesicles.     

Simultaneous Determination of Polyethylene Glycol-Conjugated Liposome Components by Using Reversed-Phase High-Performance Liquid Chromatography with UV and Evaporative Light Scattering Detection

Liposomes incorporating polyethylene glycol (PEG)-conjugated lipids (PEGylated liposomes) have attracted attention as drug delivery carriers because they show good in vivo stability. The lipid component of PEGylated liposomal formulations needs to be quantified for quality control. In this study, a simple reversed-phase high-performance liquid chromatography (HPLC) method with an evaporative light-scattering detector (ELSD) was established for simultaneous determination of hydrogenated soy phosphatidylcholine, cholesterol, PEG-conjugated lipid, and hydrolysis products of phospholipid in PEGylated liposomal formulations. These lipids were separated using a C18 column with a gradient mobile phase consisting of ammonium acetate buffer and ammonium acetate in methanol at a flow rate of 1.0 ml/min. This method provided sufficient repeatability, linearity, and recovery rate for all lipids. However, the linearity and recovery rates of cholesterol achieved using a ultraviolet (UV) detector were better than those achieved using an ELSD. This validated method can be applied to assess the composition change during the preparation process of liposomes and to quantify lipid components and hydrolysis products contained in a commercially available liposomal formulation DOXIL®. Taken together, this reversed-phase HPLC-UV/ELSD method may be useful for the rapid or routine analysis of liposomal lipid components in process development and quality control.     

Advanced strategies in liposomal cancer therapy: problems and prospects of active and tumor specific drug release

Tumor specific drug delivery has become increasingly interesting in cancer therapy, as the use of chemotherapeutics is often limited due to severe side effects. Conventional drug delivery systems have shown low efficiency and a continuous search for more advanced drug delivery principles is therefore of great importance. In the first part of this review, we present current strategies in the drug delivery field, focusing on site-specific triggered drug release from liposomes in cancerous tissue. Currently marketed drug delivery systems lack the ability to actively release the carried drug and rely on passive diffusion or slow non-specific degradation of the liposomal carrier. To obtain elevated tumor-to-normal tissue drug ratios, it is important to develop drug delivery strategies where the liposomal carriers are actively degraded specifically in the tumor tissue. Many promising strategies have emerged ranging from externally triggered light- and thermosensitive liposomes to receptor targeted, pH- and enzymatically triggered liposomes relying on an endogenous trigger mechanism in the cancerous tissue. However, even though several of these strategies were introduced three decades ago, none of them have yet led to marketed drugs and are still far from achieving this goal. The most advanced and prospective technologies are probably the prodrug strategies where non-toxic drugs are carried and activated specifically in the malignant tissue by overexpressed enzymes. In the second part of this paper, we review our own work, exploiting secretory phospholipase A2 as a site-specific trigger and prodrug activator in cancer therapy. We present novel prodrug lipids together with biophysical investigations of liposome systems, constituted by these new lipids and demonstrate their degradability by secretory phospholipase A2. We furthermore give examples of the biological performance of the enzymatically degradable liposomes as advanced drug delivery systems.