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Cytoplasmic Delivery of Liposomal Contents Mediated by an Acid-Labile Cholesterol-Vinyl Ether-PEG Conjugate
Abstract
An acid-cleavable PEG lipid, 1'-(4'-cholesteryloxy-3'-butenyl)-omega-methoxy-polyethylene[112] glycolate (CVEP), has been developed that produces stable liposomes when dispersed as a minor component (0.5-5 mol %) in 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). Cleavage of CVEP at mildly acidic pHs results in dePEGylation of the latently fusogenic DOPE liposomes, thereby triggering the onset of content release. This paper describes the synthesis of CVEP via a six-step sequence starting from the readily available precursors 1,4-butanediol, cholesterol, and mPEG acid. The hydrolysis rates and release kinetics from CVEP/DOPE liposome dispersions as a function of CVEP loading, as well as the cryogenic transmission electron microscopy and pH-dependent monolayer properties of 9:91 CVEP/DOPE mixtures, also are reported. When folate receptor-positive KB cells were exposed to calcein-loaded 5:95 CVEP/DOPE liposomes containing 0.1 mol % folate-modified 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-polyethylene[76] glycolamide (folate-PEG-DSPE), delivery of the calcein cargo to the cytoplasm of the cells was observed as determined by fluorescence microscopy and flow cytometry. Fluorescence resonance energy transfer analysis of lipid mixing in these cells was consistent with membrane-membrane fusion between the liposome and endosomal membranes.
... (Anderson et al. 1999). The fusogenic property of dioleoylphosphatidylethanolamine and transactivator of transcription (TAT) peptide will facilitate liposomes escaping from endosomes/lysosomes and normal cells (Boomer et al. 2009;Hatakeyama et al. 2009). These liposomes will release their cargos when they reach cytosols of ischemic cells that have a pH of around 6.75. ...
... DOPE based nanoliposomes can be optimized to release contents at mild acidic environment that may mimic ischemic penumbra. DOPE also has fusogenic property that facilitates liposomes escaping from endosomes and lysosomes (Boomer et al. 2009;Hatakeyama et al. 2009). The mild acidic activation and fusogenic property of DOPE plus residual blood flow in penumbral region give DOPEbased liposomes a higher chance of drug delivery to ischemic penumbra. ...
... When DSPE-PEG is anchored on to pH-sensitive liposome with a proper molar ratio, it may be randomly extracted and shed from liposome surface allowing uptake by cells when its anchor, the base lipid, becomes unstable in low pH environment. The fusogenic property of DOPE lipid (Boomer et al. 2009;Hatakeyama et al. 2009) may work synergically with TAT peptide to initiate internalization and intracellular delivery. The methodologies for construction and purification of DSPE-PEG-coated nanoliposomes have been well established. ...
... This review focuses on PEGylated and dePEGylated DDS by PEG-Chol conjugates (Beugin et al., 1998b;Boomer et al., 2009;Ishiwata et al., 1997;Zhao et al., 2007), and active targeted DDS based on functionalized-PEG-Chol conjugates He et al., 2010;Pan et al., 2007). In addition, some novel PEG-Chol conjugates (Hofmann et al., 2010;Rao et al., 2011;van de Manakker et al., 2009) have been reviewed. ...
... These studies indicated that this pH-sensitive liposome was acid-triggered, released more of the model drug, exhibited higher cellular uptake of Paclitaxel and eliminated the ABC phenomenon when compared with conventional liposomes alone. Another acid-cleavable PEG-Chol (Fig. 3C) conjugate has been developed and used to prepare stable PEGylated liposomes loaded with calcein in a physiological buffered solution or cell culture medium with serum (Boomer et al., 2009). Cleavage of PEG-Chol at mildly acidic condition (pH 5.0) resulted in dePEGylation of the latently fusogenic liposomes, thereby triggering the onset of release of its contents. ...
... Cleavage of PEG-Chol at mildly acidic condition (pH 5.0) resulted in dePEGylation of the latently fusogenic liposomes, thereby triggering the onset of release of its contents. In uptake experiments, the liposomal calcein cargo was directly delivered to the cytoplasm via an acid-triggered dePEGylation and liposome-endosomal membrane fusion process (Boomer et al., 2009). Therefore, the "dePEGylation triggering strategy" appears to be a very promising biomedical formulation process technique for the site-specific cytoplasmic delivery of liposomal contents (Takae et al., 2008). ...
... Endosomal trapping of liposomes (and their contents within) pose a significant challenge to cytoplasmic delivery of the drug payload. Thus, pH-sensitive functionalities have been incorporated into liposomes to promote the mixing of lipids (between the liposome and the endosomal membrane) under the acidic environment of the endosomes, which in turn facilitates payload release into the cytoplasm [2][3][4][5][6][7][8]. Meanwhile, to mask against opsonization and to increase circulation time in blood, liposomes are usually coated with poly(ethyleneglycol) (PEG) polymers [9][10][11][12]. ...
... Unfortunately, the presence of PEG on the surface of liposomes hinders drug release, even when liposomes have been internalized via receptor mediated endocytosis [13]. One approach to avoid this difficulty is to use pH-sensitive PEG polymers that disassemble in the endosomal low pH environment, thus exposing lipid bilayer membrane of the liposome for direct interaction with the membrane of the endosome [2,6,14]. ...
... The N-capped pHLIP peptide, Acetyl-NH-ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT-CO 2 ...
We develop a method for pH-dependent fusion between liposomes and cellular membranes using pHLIP® (pH Low Insertion Peptide), which inserts into lipid bilayer of membrane only at low pH. Previously we establish the molecular mechanism of peptide action and show that pHLIP can target acidic diseased tissue. Here we investigate how coating of PEGylated liposomes with pHLIP might affect liposomal uptake by cells. The presence of pHLIP on the surface of PEGylated-liposomes enhanced membrane fusion and lipid exchange in a pH dependent fashion, leading to increase of cellular uptake and payload release, and inhibition of cell proliferation by liposomes containing ceramide. A novel type of pH-sensitive, "fusogenic" pHLIP-liposomes was developed, which could be used to selectively deliver various diagnostic and therapeutic agents to acidic diseased cells.
... Such moieties include hydrazone, hydrazide, lysine, imine, histidine, acrylamide, methacrylic acid, maleic acid, acylic acid, etc. [17,79,99,100]. Besides, a few fusogenic lipid and peptide were also used as components of pHsensitive carriers [96,[101][102][103][104]. ...
... Thompson et al. used PEG-cleavable lipid, via an acid-labile vinyl ether-linker, for PEGylation of (1,2-dioleoylsnglycero-3-phosphoethanolamine) (DOPE) liposomes. At acidic lysosomal pH, the vinyl ether linker hydrolyzed and the PEG layer was removed from the DOPE liposomes, enabling DOPE, which has excellent fusogenic capacity, to fuse with the lysosomal membrane for escape [104]. In the case of fusogenic peptide, artificial amphiphatic peptides GALA and KALA, as well as several peptides derived from pathogen toxins, such as hemagglutinin, anthrax toxin, and diphtheria toxin, can disrupt endosomal membranes upon endocytosis within cells, supporting escape into the cytosol [116,117]. ...
In cancer therapy, drug delivery is a complex process that aims to transit the cargo to the destination with as little damage to the normal tissue as possible. In the last decade, tremendous development and research on nanomedicine have been exploring an ideal system with efficient drug transportation and release property. For this end, series of barriers need to be circumvented by nanomedicine, including systemic barriers, such as biosurface adsorption, phagocytic clearance, blood stream washing, interstitial pressure, degradation, as well as intracellular barriers, such as cell membrane reorganization and internalization, endo/lysosomal escape, cytosolic or subcellular localization. Rather than being random, these barriers follow a specific spatial-temporal sequence. Therefore, the nanocarriers have to be endowed with characteristics that are adaptive to particular biological milieu on systemic and intracellular levels. To this end, we reviewed the correlations between the spatial-temporal sequences of drug delivery and nanocarrier characteristics in cancer therapy, as well as strategies to achieve efficient drug delivery upon both systemic and intracellular levels.
... Liposomes can interact with cells by five different mechanisms [24]. ...
Fungal meningitis is an infection which is caused by fungus which spreads through the blood to the spinal cord. People with weakened immunity get this disease easily like persons with AIDs, etc. To make sure the disease is fungal meningitis, a sample is taken from the cerebrospinal fluid and it is sent to the laboratory. Usually, fungal meningitis is not mediated from person to person, but it is caused when a fungi are inhaled from the surrounding and spread into the blood to the central nervous system. Normally medications such as vaccines, IV, and oral suspensions are given to the people for curing fungal meningitis. Commonly used drugs are Amphotericin B and fluconazole oral suspension. Amphotericin B is an antifungal, antiprotozoal, and hydrophobic drug. However, these drugs cannot give a directly as medication therapy for the patients, because it offers toxic effect and side effects, absorption rate is slower, and crossing the blood–brain barrier (BBB) is getting difficult. Adverse effects can be minimized with the application of nanotechnology. Therefore, in human medical services, the availability of molecular nanotechnology will provide rapid progress. Nanoparticle (NP) systems help to improve the solubility of poorly water-soluble drugs which has been explained using Noyes–Whitney equations. Nanoparticles offers several advantages as a drug delivery system, such as better drug bioavailability, reduction of dosing frequency enables them for the betterment of diseases, can cross the BBB, and it is very cost-effective. Types of NP include polymeric NP, carbon nanotubes, metallic structures, nanocrystals, and fusogenic liposomes. Fusogenic liposomes are a peculiar class of phospholipid vesicles. The fusogenic liposomes can deliver encapsulated NP into the targeted sites and also can cross the BBB. On comparing with cationic liposomes, fusogenic liposomes are more effective as well as rapid in the drug delivery.
... 62 PEG has a wide range of bioengineering applications ranging from a membrane patch to 3D scaffolds and drug delivery vehicles. [9][10][11][12][13]49,63 In this section, we will discuss specific applications of PEG in SCI in greater depth (Table 1). ...
Polyethylene glycol (PEG) is a synthetic biocompatible polymer with many useful properties for developing therapeutics to treat spinal cord injury. Direct application of PEG as a fusogen to the injury site can repair cell membranes, mitigate oxidative stress, and promote axonal regeneration to restore motor function. PEG can be covalently or noncovalently conjugated to proteins, peptides, and nanoparticles to limit their clearance by the reticuloendothelial system, reduce their immunogenicity, and facilitate crossing the blood–brain barrier. Cross-linking PEG produces hydrogels that can act as delivery vehicles for bioactive molecules including growth factors and cells such as bone marrow stromal cells, which can modulate the inflammatory response and support neural tissue regeneration. PEG hydrogels can be cross-linked in vitro or delivered as an injectable formulation that can gel in situ at the site of injury. Chemical and mechanical properties of PEG hydrogels are tunable and must be optimized for creating the most favorable delivery environment. Peptides mimicking extracellular matrix protein such as laminin and n-cadherin can be incorporated into PEG hydrogels to promote neural differentiation and axonal extensions. Different hydrogel cross-linking densities and stiffness will also affect the differentiation process. PEG hydrogels with a gradient of peptide concentrations or Young’s modulus have been developed to systematically study these factors. This review will describe these and other recent advancements of PEG in the field of spinal cord injury in greater detail.
... As shown in Figs. 2 and 3, substantial AS014 was efficiently internalized into the cells via the interaction between SPG and Dectin-1. Multiple complexes would be contained in an endosomal vesicle and account for a considerable portion of endosomal vesicles, leading to a change in osmotic pressure by a depletion effect [41]. Subsequently, the collapse of endosomal vesicles would be induced, resulting in an influx of most AS014 into the cytoplasmic compartment. ...
Antisense oligonucleotides (AS-ODNs) hybridize with specific mRNAs, resulting in interference with the splicing mechanism or the regulation of protein translation. We previously demonstrated that the β-glucan schizophyllan (SPG) can form a complex with AS-ODNs with attached dA40 (AS-ODNs/SPG), and this complex can be incorporated into cells, such as macrophages and dendritic cells, expressing the β-glucan receptor Dectin-1. We have achieved efficient gene silencing in animal models, but the uptake mechanism and intracellular distribution are unclear. In this study, we prepared the complex consisting of SPG and AS-ODNs (AS014) for Y-box binding protein-1 (YB-1). After treatment with endocytosis inhibitor Pitstop 2 and small interfering RNA targeting Dectin-1, we found that AS014/SPG complexes are incorporated into cells by Dectin-1-mediated endocytosis and inhibit cell growth in a Dectin-1 expression level-dependent manner. After treatment with AS014/SPG complexes, we separated the cell lysate into endosomal and cytoplasmic components by ultracentrifugation and directly determined the distribution of AS014 by reverse transcription PCR using AS014 ODNs as a template or a reverse transcription primer. In the cytoplasm, AS014 clearly hybridized with YB-1 mRNAs. This is the first demonstration of the distinct distribution of the complex in cells. These results could facilitate the clinical application of the complex.
... Several strategies that improve the subcellular delivery of the nanocarrier and its payload have been identified and reviewed (Rajendran et al. 2010). These include, among others, grafting of ligands that are internalised via receptor-mediated endocytosis and thus targeted to the endosomes (as discussed above) (Rajendran et al. 2010;Duncan and Gaspar 2011;Ritchie et al. 2013;Kong et al. 2013), adding of peptidic sequences (Li et al. 2004;Leopold and Crystal 2007;Mastrobattista et al. 2007), pH-sensitive polymers (Yessine et al. 2003;Yessine and Leroux 2004;Richardson et al. 2010;Cheng et al. 2013) or fusogenic lipids (Boomer et al. 2009) thus enhancing the fusogenic ability of the nanocarrier in a pH-sensitive manner and mediate its release or of its content from the endolysosomal compartments to the cytosol (Rajendran et al. 2010;Duncan and Gaspar 2011;Kong et al. 2013). ...
Extensive research has demonstrated promising results for particulate vaccine delivery systems. This emerging field, and considerable advances in the identification of the most immunogenic epitopes, has promoted the formulation of a next generation of vaccines able to induce potent antigen-specific humoral, cellular, and local immune responses. These delivery systems can combine multiple antigens and adjuvants in one vaccine system, which may overcome the need for the numerous inoculations required to address important childhood immunization programs. However, essential technical and regulatory limitations are hurdles to the translation of particulate vaccines to clinics. These include the study of validation methods used for antigen selection, formulation stability, and antigen release rates as well as difficulties inherent to the characterization of carrier pharmacokinetics and biodistribution. This review addresses the current translational requirements for vaccines and debates the main aspects under discussion for nanoparticulate-based systems in immunomodulation.
... 6 An ideal nanocarrier should have the following features: (a) biocompatibility, (b) long circulation time in vivo, (c) sufficiently high stability, (d) high amount of drug-loading capacity (DLC), and (e) no premature release of the drug molecules. There are a number of conventional nanocarriers like liposomes, 7,8 polymersomes, 9,10 dendrimers, 11 micelles, 12,13 vesicles, 14 microgels, 15 and nanogels, 16−18 these have been applied for advanced drug delivery. Among them, nanogels are the most appropriate for approaching the critical therapeutic level for cancer. ...
A smart ultra-fast light responsive nanogel is a potential carrier for on-demand and immediate delivery of the therapeutic agents. Here, a novel branched pentaerythritol poly(caprolactone)-b-poly(acrylic acid) (PE-PCL-b-PAA) based smart light responsive nanogel has been fabricated by using ferric ion (Fe3+) as a crosslinker. The mentioned block copolymer has been synthesized by combining both the ring opening and atom transfer radical polymerization techniques. Branched structure of the polymer offers a minute amount (1.5 mole%) of Fe3+ sufficient for nanogel formation. The nanogels look like a spherically shaped human brain holding the water molecule as like cerebrospinal fluid in the brain. The particles size of the nanogel has been tailored (in between 30 to 450 nm) by varying both the molar concentration of Fe3+ and polymer chain length, separately. The highly negative zeta potential (-46 mV) of the nanogel promotes their impressive colloidal stability and prolong circulation time (in vivo). Nanogels securely hold the DOX molecules (DLC; 26.2%). On exposure of light onto the nanogel (in presence of lactic acid) produces immediate initiation of de-cross-linking followed by the release of DOX molecules (85.2 % at 120 min.). The nanogel shows significantly high uptake and acute toxicity against cancerous cell line (C6 glioma, in vitro). Administration of the DOX-loaded nanogel on C6 glioma rat model (in vivo) offered a tremendous inhibition (about 91%) of the tumor growth without any toxic side effects (confirmed by histopathology).
... b action is proposed in an increasing number of publications in this field. Several examples have been reported for controlled release including acetals, 18,19 vinyl ethers, [20][21][22] (di-)ortho esters, [23][24][25] hydrazones, [26][27][28] and esters (e.g. succinates). ...
Hyperbranched polyether-based lipids with cleavable acetal units were obtained via copolymerization of the epoxide inimer 1-(glycidyloxy)ethyl ethylene glycol ether (GEGE) and glycidol, using anionic ring-opening polymerization. Cholesterol-linear polyglycerol (Ch-linPG) was used as a macroinitiator, resulting in branched polyethers with an adjustable amount of acid-cleavable units. Random copolymerization led to Ch-P(GEGEx-co-Gy) copolymers, whereas sequential copolymerization provided access to Ch-P(GEGEx-b-Gy) amphiphiles. The amount of GEGE was varied between 8-49 mol% of the total amount of monomer units. In addition, hyperbranched polyethers with a single acetal unit were prepared using glycol-1-(cholesteryloxy)ethylether as an initiator for the polymerization of allyl glycidyl ether (AGE) in bulk. Subsequent thiol-ene coupling of mercaptoethanol resulted in the hydroxyl functional macroinitiator used for the polymerization of glycidol. The novel polyether-based lipids were characterized in detail by ¹H NMR spectroscopy and size exclusion chromatography, revealing narrow to moderate molecular weight distributions. Degradation was achieved at pH 2 in a proof-of-principle experiment. Acid-triggered shedding of liposomes was proven using the linear analogue α-(1-(cholesteryloxy)ethoxy)-ω-hydro-PEG-CH2-CCH with one cleavable group and a fluorescence label, Atto 488 azide. Investigation of the acetal-cleavage under neutral and acidic pH (7.4-2.0) via fluorescence spectroscopy was carried out.
... Studies suggested that phenyl VE linkers were readily and reliably incorporated into a variety of compounds to enable more control of acid triggered intracellular drug delivery [141,142]. Cytoplasmic delivery has also been mediated by an acid-labile cholesterol-VE-PEG modified DOPE liposomes via dePEGylation of the latent fusogenic DOPE liposomes [189]. ...
... The pressure-area isotherms shown in Figure S1 for 1:99 and 5:95 NTA-PEG2000-DTPE:mPEG350-DTPE lipid mixtures displayed gradually increasing surface pressures upon film compression, except that the onset of surface pressure occurred at larger molecular areas as the NTA-PEG2000-DTPE composition in the film increased. We attribute this observation to the displacement of surface-adsorbed NTA-PEG2000 from the air-water interface as previously described 25 for mixed mPEG2000-lipid monolayers. As this desorption process progresses, the majority component, mPEG350, undergoes a mushroom-brush regime transition upon compression to 30 mN/m while the NTA-PEG2000 fraction remains in the mushroom configuration. ...
We report the preparation and performance of TEM grids bearing stabilized nonfouling lipid monolayer coatings. These films contain NTA capture ligands of controllable areal density at the distal end of a flexible poly(ethylene glycol) 2000 (PEG2000) spacer to avoid preferred orientation of surface-bound histidine-tagged (His-tag) protein targets. Langmuir-Schaefer deposition at 30 mN/m of mixed monolayers containing two novel synthetic lipids-1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(5-amido-1-carboxypentyl)iminodiacetic acid]polyethylene glycolamide 2000) (NTA-PEG2000-DSPE) and 1,2-(tricosa-10',12'-diynoyl)-sn-glycero-3-phosphoethanolamine-N-(methoxypolyethylene glycolamide 350) (mPEG350-DTPE)-in 1:99 and 5:95 molar ratios prior to treatment with a 5 min, 254 nm light exposure was used for grid fabrication. These conditions were designed to limit nonspecific protein adsorption onto the stabilized lipid coating by favoring the formation of a mPEG350 brush layer below a flexible, mushroom conformation of NTA-PEG2000 at low surface density to enable specific immobilization and random orientation of the protein target on the EM grid. These grids were then used to capture His6-T7 bacteriophage and RplL from cell lysates, as well as purified His8-green fluorescent protein (GFP) and nanodisc solubilized maltose transporter, His6-MalFGK2. Our findings indicate that TEM grid supported, polymerized NTA lipid monolayers are capable of capturing His-tag protein targets in a manner that controls their areal densities, while efficiently blocking nonspecific adsorption and limiting film degradation, even upon prolonged detergent exposure.
... virsomes comprising liposomes modified with a CPP fusogenic viral envelope protein; diINF-7), (c) undergo conformational changes in response to pH changes, which then leads to fusion of the carrier with the endosomal membrane (e.g., hemagglutinin) [152][153][154][155][156][157][158][159][160][161][162][163][164][165][166], (d) dioleoylphosphatidylethanolamine or DOPE that fuses with endosomal membrane, and (e) pH-sensitive lipids that are degraded under the acidic conditions in late endosomes, e.g., citraconyl-DOPE, promote the unpackaging and release of cargo within the endosome [92]. For pegylated NP, PEG-lipids that are cleaved in the acidic environment of endosomes are used to promote fusion between liposomes with endosomal membrane [152,[167][168][169][170]. ...
... PEG-cholesteryl conjugates are often used as drug carrier owing to their excellent biocompatibility with host molecules, low toxicity, and high solution stability at their CMC [18]. Although pH-sensitive PEG-cholesteryl conjugates have been studied to determine if they exhibit complete drug release inside cancer cells [25,26], there are no previous reports of PEG-cholesteryl conjugates with cleavable linkage in response to thiols. Therefore, we investigated the disintegration or destabilization of Chol-ss-PEG-ss-Chol micelles following cleavage of disulfide in the presence of DTT using NMR, gel permeation chromatography (GPC), and dynamic light scattering (DLS). ...
The ability of polymeric micelles to self-assemble into nanosized particles has created interest in their application as potential anticancer drug delivery systems. A poly(ethylene glycol)-cholesteryl conjugate (Chol-ss-PEG-ss-Chol) connected by cleavable disulfide linkages was synthesized and used as a nanocarrier for in vitro release of doxorubicin (DOX). Owing to its amphiphilic structure, Chol-ss-PEG-ss-Chol was able to self-assemble into micelles with an average diameter 18.6 nm in aqueous solution. The micelles formed large aggregates due to the shedding of the PEG shell through cleavage of disulfide bonds in a reductive environment. The in vitro release studies revealed that Chol-ss-PEG-ss-Chol micelles released 80% and approximately 9% of the encapsulated DOX within 6 h under reductive and non-reductive conditions, respectively. The glutathione (GSH)-mediated intracellular drug delivery was investigated in a KB cell line. The cytotoxicity of DOX-loaded micelles indicated a higher cellular anti-proliferative effect against GSH-pretreated than untreated KB cells. Furthermore, confocal laser scanning microscopy (CLSM) measurement demonstrated that Chol-ss-PEG-ss-Chol micelles exhibited faster drug release in GSH-pretreated KB cells than untreated KB cells. These results suggest the potential usefulness of disulfide-based polymeric micelles as controlled drug delivery carriers.
... Acid-labile functional groups, which rely upon the pH gradient present in the endosome/lysosome pathway, have been incorporated as triggers to regulate the membrane lytic activity of delivery vehicles. Labile bonds such as acetals [1][2][3], hydrazone [4], ortho esters [5][6][7][8], vinyl ethers [9] and maleamates [10][11][12] have been incorporated into liposomes and amphipathic polymers to control interactions with membrane s and provide a trigger for cytoplasmic delivery. ...
The safe and efficacious delivery of membrane impermeable therapeutics requires cytoplasmic access without the toxicity of nonspecific cytoplasmic membrane lysis. We have developed a mechanism for control of cytoplasmic release which utilizes endogenous proteases as a trigger and results in functional delivery of small interfering RNA (siRNA). The delivery approach is based on reversible inhibition of membrane disruptive polymers with protease-sensitive substrates. Proteolytic hydrolysis upon endocytosis restores the membrane destabilizing activity of the polymers thereby allowing cytoplasmic access of the co-delivered siRNA. Protease-sensitive polymer masking reagents derived from polyethylene glycol (PEG), which inhibit membrane interactions, and N-acetylgalactosamine, which targets asialoglycoprotein receptors on hepatocytes, were synthesized and used to formulate masked polymer-siRNA delivery vehicles. The size, charge and stability of the vehicles enable functional delivery of siRNA after subcutaneous administration and, with modification of the targeting ligand, have the potential for extrahepatic targeting.
Copyright © 2015. Published by Elsevier B.V.
... Different types of FLs have been proposed over the last few years, comprising either viral material or natural or synthetic lipids to achieve the required fusogenic properties [5][6][7][8]. Among the various studied systems, FLs based on 1,2-dioleoylphosphatidylethanolamine (DOPE) and cholesteryl hemisuccinate (CHEMS) have shown a high degree of cell association. ...
Many antibacterial drugs have some difficulty passing through the bacterial cell membrane, especially if they have a high molecular weight or large spatial structure. Consequently, intrinsic resistance is shown by some bacterial strains. Reduced cell membrane permeability is one of the mechanisms of resistance known for fusidic acid (FUS), a bacteriostatic steroidal compound with activity limited to Gram-positive bacteria. Moreover, the lipophilic character of FUS has been shown to cause drug retention inside the bilayers of cell membranes, preventing its diffusion towards target sites inside the cytoplasm. Targeting antimicrobial agents by means of liposomes may be a valid strategy in the treatment of infections refractory to conventional routes of antimicrobial treatment. On this basis, loading of FUS in fusogenic liposomes (FLs) was planned in this study. Fusogenic small unilamellar vesicles loaded with FUS were produced to evaluate their influence on improving the cell penetration and antibacterial activity of the antibiotic. The produced carriers were technologically characterised and were subjected to an in vitro microbiological assay against several strains of Gram-negative and Gram-positive bacteria. The experimental results showed that encapsulating FUS in a liposomal carrier can improve antimicrobial efficacy and reduce the effective concentration required, probably through putative mechanisms of increased diffusion through the bacterial cell membrane. In fact, whilst free FUS was active only on the tested Gram-positive strains, incubation of FUS-loaded FLs exhibited growth inhibitory activity both against Gram-positive and Gram-negative strains. The lowest MICs were obtained against Staphylococcus epidermidis (≤0.15μg/mL) and Acinetobacter baumannii (37.5μg/mL) clinical strains.
Copyright © 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
... In general, their bilayers show an enhanced ability of interacting in their liquid crystalline phase with cell membranes, favouring the reciprocal lipid mixing and thus the release of the vesicle content inside the cytoplasm. Fusogenic liposomes can be produced by incorporating either special lipids, that make the vesicles more fluid and able to promote the destabilization of biological membranes [25][26][27], or inactivated Sendai virus envelope components [28,29] as well as other fusogenic peptides, which can achieve the same goal [30]. The possible mechanisms by which fusogenic liposomes interact with cells, as a function of their composition and properties, have been extensively reviewed by Simões et al [31]. ...
Gram-negative bacteria often show a resistance to many antibiotics because of the inability of the latter to cross the outer membrane present in these bacterial cells and surrounding the cell wall. Different chemical and technological strategies have been tried to overcome this problem. We explored the possibility of using fusogenic liposomes, up to now used to transfer drugs inside eukaryotic cells, for localizing glycopeptide antibiotics in the bacterial cell perisplamic space, thus allowing them to exert their activity. Small unilamellar liposomes were prepared using an extrusion procedure (SUVETs) from special phospholipid-cholesterol hemisuccinate mixtures and efficiently loaded with vancomycin (VAN). The in vitro microbiological experiments showed that the fusogenic vesicles can inhibit to a different extent the growth of wild and standard Gram-negative bacterial strains, against which the parent drug was ineffective, as well as 'classical' (non fusogenic) VAN-loaded liposomes.
... For instance, PEG-cholesteryl methyl carbamate (PEG-CHMC) and PEG-cholesteryl hemisuccinate (PEG-CHEMS, 5) are susceptible to esterase-mediated cleavage; the liposomes carrying these two lipopolymers are destabilized in the presence of esterase, enabling the release of encapsulated contents [76]. Similarly, liposomes containing ethers linked Chol-PEG,1'-(4'-cholesteryloxy-3'-butenyl)-ω-PEG 5000 are susceptible to acid-induced content release [77]. Yet another modification is the liposomes containing photo-cleavable Chol-PEG (6) [67], which potentially appears to be more controllable than the acid/esterase-mediated cleavage. ...
Liposomes are used as a delivery vehicle for drug molecules and imaging agents. The major impetus in their biomedical applications comes from the ability to prolong their circulation half-life after administration. Conventional liposomes are easily recognized by the mononuclear phagocyte system and are rapidly cleared from the blood stream. Modification of the liposomal surface with hydrophilic polymers delays the elimination process by endowing them with stealth properties. In recent times, the development of various materials for surface engineering of liposomes and other nanomaterials has made remarkable progress. Poly(ethylene glycol)-linked phospholipids (PEG-PLs) are the best representatives of such materials. Although PEG-PLs have served the formulation scientists amazingly well, closer scrutiny has uncovered a few shortcomings, especially pertaining to immunogenicity and pharmaceutical characteristics (drug loading, targeting, etc.) of PEG. On the other hand, researchers have also begun questioning the biological behavior of the phospholipid portion in PEG-PLs. Consequently, stealth lipopolymers consisting of non-phospholipids and PEG-alternatives are being developed. These novel lipopolymers offer the potential advantages of structural versatility, reduced complement activation, greater stability, flexible handling and storage procedures and low cost. In this article, we review the materials available as alternatives to PEG and PEG-lipopolymers for effective surface modification of liposomes.
... One possibility to achieve this goal is through the construction of a TDS which is triggered through hydrolysis of pH-sensitive linkages. Subsequently, various pH-sensitive cross-linkages have been developed and the most common are ortho esters, [17][18][19] cisaconityl, 20,21 hydrazones, 22,23 acetals, 24,25 silyl ether, 26 and vinyl ethers 27,28 Liposomes are currently the most established drug delivery vehicles and bilayer membranebased nanocarriers where the membrane is typically composed of phospholipids. Liposomes can be used for the delivery of both water-soluble and water-insoluble active pharmaceutical agents. ...
Novel pH-responsive assemblies (PEG-lipid:DOPE liposomes) containing tunable and bifunctional phenyl-substituted vinyl ether (PIVE) cross-linkers were prepared. The assemblies consisted of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), acid-cleavable poly(ethylene glycol) (PEG)-conjugated lipids, pDNA, and protamine sulfate (PS). The PIVE linkage was designed to hydrolyze under acidic conditions, and the hydrolysis studies of PEG-lipid compounds containing PIVE at pH 4.2, 5.4, and 7.4 indicated that the hydrolysis rates of PIVE linker were influenced by the substitution of electron withdrawing or electron donating groups on the phenyl ring. Acid-catalyzed hydrolysis of PIVE leads to destabilization of the acid labile PEG-PIVE-lipid:DOPE liposomes via dePEGylation, thereby triggering content release. Content release assays showed that dePEGylation was highly pH-dependent and correlated with the PIVE proton affinity of the phenyl group. These results indicated that the dePEGylative triggering based on a new pH-sensitive PIVE linkage can be controlled. In vitro transfection studies on the pH-responsive assemblies containing mPEG-(MeO-PIVE)-conjugated 1,3-dioctadecyl-rac-glycerol lipids (mPEG-(MeO-PIVE])-DOG) showed higher transfection efficiency compared to that of polyethylenimine (PEI), a positive control, on HEK 293 and COS-7 cells. In addition, lower cytotoxicity of PEG-PIVE-lipid:DOPE liposomes/PS/DNA was observed in comparison to PEI. These results suggest that PEG-PIVE-lipid:DOPE liposomes can be considered as nonviral vehicles for drug and gene delivery applications.
Liposomes have achieved remarkable distinction in nanocarriers systems owing to the efficient delivery of both hydrophilic and lipophilic therapeutics at targeted sites. Several merits, including high drug payload, biocompatibility, and aptitude for self-assembly along with feasibility for physicochemical modification enabled liposomes as suitable candidates for site-specific drug delivery systems. However, inadequate encapsulation, partly controlled vesicle size, and improper cellular internalization at the target site encouraged modification of the surface chemistry of conventional liposomes. The chapter focuses on various functionalization approaches of conventional liposomes through diverse ligands, including peptides, antibodies (immunoliposomes), affibody (affisomes), aptamers (aptamosomes), carbohydrates (glycoliposomes), vitamins, polyethylene glycol (PEGylated), etc. Amended second-generation or functionalized liposomes portrayed higher solubility, stability, and prolonged circulation time with precise targeting for the management of cancerous diseases and other ailments.
The domain of nanomedicine owns a wide-ranging variety of lipid-based drug carriers, and novel nanostructured drug carriersthat are further added to this range every year. The primary goal behind the exploration of any new lipid-based nanoformulation is the improvement of the therapeutic index of the concerned drug molecule along with minimization in the associated side-effects. However, for maintaining a sustained delivery of these intravenously injected lipoidal nanomedicines to the targeted tissues and organ systems in the body, longer circulation in the bloodstream, as well as their stability, are important. After administration, upon recognition as foreign entities in the body, these systems are rapidly cleared by the cells associated with the mononuclear phagocyte system. In order to provide these lipid-based systems with long circulation characteristics,techniques such as coating of the lipoidal surface with an inert polymeric material like polyethylene glycol (PEG) assists in imparting ‘stealth properties’ to thesenanoformulations for avoiding recognition by the macrophages of the immune system. In this review, detailed importance is given to the hydrophilic PEG polymer and the role played by PEG-linked lipid polymers in the field of nanomedicine-based drug carriers. The typical structure and classification of stealth lipids, clinical utility, assemblage techniques, physicochemical characterization, and factors governing the in-vivo performance of the PEG-linked lipids containing formulations will be discussed.Eventually, the novel concept of accelerated blood clearance (ABC) phenomenon associated with the use of PEGylated therapeuticswill bedeliberated.
PEGylation is a well-established strategy for improving the target specificity, circulation time and stability of liposomes, thereby improving their stealth properties. This brief review provides an insight on the composition of PEGylated liposomes and the characteristics that dictate the functionality of PEGylated liposomes such as surface density, molecular weight, presence of linkers and acyl groups. Physicochemical techniques used to characterize the PEG liposomes and test their stability are also discussed along with their clinical implications. This review provides the readers with a broad range of understanding of various PEGylated lipids, techniques to access their stability in liposomal formulations and state-of-the-art development of PEGylated liposomal formulations.
Interest in the application of nanotechnology to medicine has surged in recent years and could transform the way we diagnose, treat and prevent diseases such as cancer. However, the clinical success of nanomedicine is limited because of problems with toxicity and therapeutic efficacy. To overcome this it is essential to produce new nanosystems with specific functions, which can be achieved by designing new polymers with particular properties that can be used for nanomedicine. Functional Polymers for Nanomedicine provides a complete overview of the different strategies for designing polymers for nanomedicine applications. The first part of the book looks at the current problems and direction in nanomedicine including a review of current design and targeting of nanocarriers. The second part explores the design of polymers with different functions including hyperbranched polymers, polymersomes, polysaccharides, polymeric micelles and zwitterionic polymers and their applications in gene therapy and drug delivery. This timely book is edited by a leading scientist in nanomedicine and provides a suitable introduction and reference source for advanced undergraduates, postgraduates and academic and industrial researchers in polymer science, nanotechnology and pharmacy interested in materials for medical applications.
To maximize drug targeting to solid tumors, cancer nanomedicines with prolonged circulation times are required. To this end, poly(ethylene glycol) (PEG) has been widely used as a steric shield of nanomedicine surfaces to minimize serum protein absorption (opsonisation) and subsequent recognition and clearance by cells of the mononuclear phagocyte system (MPS). However, PEG also inhibits interactions of nanomedicines with target cancer cells, limiting the effective drug dose that can be reached within the target tumor. To overcome this dilemma, nanomedicines with stimuli-responsive cleavable PEG functionality have been developed. These benefit from both long circulation lifetimes en route to the targeted tumor as well as efficient drug delivery to target cancer cells. In this review, various stimuli-responsive strategies to dePEGylate nanomedicines within the tumor microenvironment will be critically reviewed.
The recent expansion of pharmaceutical nanotechnology (nanomedicines) and targeting strategies for antimicrobial delivery are highlighted and utilized in the applied fields of biomedicine, cosmetology, pharmaceutical, and food technologies. Nanoliposomes are artificially prepared membranous vesicles often composed of natural phospholipids and cholesterol. Their structure is similar to the cell membrane, and they can trap and release compounds with different properties, including both hydrophobic and hydrophilic drugs. Nanoliposomes can be used to achieve a slow release of drugs; the reduced particle size enhances the surface area, improving bioavailability and solubility, and thereby reducing its toxicity. As pharmaceutical nanocarriers, liposomes have been extensively studied for topical/dermal use against fungal diseases and as alternative therapy for diseases such as tuberculosis, due to the increase of antimicrobial efficacy, improving the interactions between pathogen and encapsulated drugs. Conventional liposomes have some limitations, such as low stability for long periods of storage and rapid uptake. Actually, a new generation of liposomes is developed with modifications in their surface, optimized size, high loading efficiency, ease of interaction with the cell membrane, and increased target specificity, gaining novel applications and increasing attention in the medicine area.
A variety of active pharmaceutical ingredients (APIs) currently used for cancer treatment are cytotoxic, and show nonspecific distribution when administered systemically resulting in toxicity to normal tissues, hence limiting their clinical application. To overcome these challenges, nanocarriers such as liposomes and micelles have been widely used to deliver APIs for cancer chemotherapy. Delivery of nanocarriers is achieved either via “passive targeting” owing to the enhanced permeability and retention (EPR) effect or via “active targeting” due to the presence of various ligands on the surface of nanocarriers, such as antibodies, peptides, etc. Numerous factors are involved in successful delivery of chemotherapeutic agents; these depend on the tumor microenvironment, formulation factors such as choice of ligand use, physiochemical properties of the nanocarriers, and the choice of target. In this chapter, we discuss the fundamentals of EPR effect, factors affecting passive and active targeting, and current clinical update of various actively and passively targeted liposomes and micelles.
Current cancer nanomedicines can only mitigate adverse effects but fail to enhance therapeutic efficacies of anticancer drugs. Rational design of next-generation cancer nanomedicines should aim to enhance their therapeutic efficacies. Taking this into account, this review first analyzes the typical cancer-drug-delivery process of an intravenously administered nanomedicine and concludes that the delivery involves a five-step CAPIR cascade and that high efficiency at every step is critical to guarantee high overall therapeutic efficiency. Further analysis shows that the nanoproperties needed in each step for a nanomedicine to maximize its efficiency are different and even opposing in different steps, particularly what the authors call the PEG, surface-charge, size and stability dilemmas. To resolve those dilemmas in order to integrate all needed nanoproperties into one nanomedicine, stability, surface and size nanoproperty transitions (3S transitions for short) are proposed and the reported strategies to realize these transitions are comprehensively summarized. Examples of nanomedicines capable of the 3S transitions are discussed, as are future research directions to design high-performance cancer nanomedicines and their clinical translations.
The drug delivery field using liposome or liposome complexes is extremely active with a large number of publications and reports appearing each year. This chapter reviews the current status of liposome-based drug delivery vehicles. It examines several triggered release mechanisms from the perspective of the lipid bilayers' physical and chemical properties. Before discussing various types of triggered release systems, it is necessary to review the basic biophysical properties of lipids and their assemblies. Targeted drug deliveries to specific disease sites and then releasing the drugs at desirable rates at the intended sites have been the goals for drug delivery studies. Liposomes as drug delivery vehicles will find broader applications with the study of tailor-designed lipids in conjunction with the therapeutic agents to be delivered. It is important that for certain types of drugs, the liposomes used should also be tailored to match the physical and chemical properties of the agents.
pH-Sensitive doxorubicin conjugated polymeric micelles entrapped with near infrared (NIR) photosensitizer BODIPY (which works as an imaging agent at the same time) were designed and synthesized by ring opening polymerization of N-carboxyanhydride with mPEG-NH2 as the initiator, following reaction with doxorubicin to form the hydrazone-bond linker for pH responsiveness. Then the NIR dye (BODIPY) was loaded in the micelles for both bioimaging and photodynamic therapy (PDT). A significant cytotoxicity of NIR imaging-guided combined PDT and chemotherapy could be found by MTT assays, which was also confirmed with a fluorescence microscope, indicating a new kind of polymeric nanoparticle for potential theranostic treatment of cancers. In addition, the energy density of the laser for the PDT is extremely low.
A novel branched amphiphilic macromolecule on the basis of a sensitive Si-O-C structure, was synthesized through a one-pot procedure. The self-assembly behavior of the molecule was tunable in non-aqueous media and the aggregates exhibited water stability thanks to the formation of vesicles with multi-chamber structures, which protected the Si-O-C bonds from hydrolysis. The relation between the chemical bond nature and the self-assembly morphology was highlighted. The prepared aggregates were pH and temperature responsive.
This work describes the synthesis of a hexadecyl-modified anthracene-based photoacid generator for use as alight-activated trigger, which will induce contents release from phospholipid1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE):lysoplasmenylcholine liposomes. The liposomes containing photosensitizers such as bacteriochlorophylla or aluminum phthalocyaninetetrasulfonate are also described. Finally, biodistribution of aluminum phthalocyaninetetrasulfonate loaded liposomes in a murine tumor xenograft model is discussed.
Gene therapy shows promise as a potentially revolutionizing strategy for treatment of many genetically-related diseases, such as cancer. However, the lack of safe and effective gene delivery carriers (or vectors) has become a bottleneck in its basic research and clinical application. Generally, gene delivery carriers can be divided into viral and non-viral ones. Although non-viral gene delivery carriers can offer some advantages such as safety and facile fabrication, they don't possess the same high gene delivery efficiency as viral gene delivery carriers do, due to lack of functionality to overcome many intracellular gene-delivery obstacles. Currently, many kinds of "smart" non-viral gene-delivery carriers have been developed in order to realize efficient gene-delivery, since such carriers can undergo physical or chemical reactions in response to changes in pH, oxidative state, or enzymatic activity. As these stimuli or cues may be specific to a biological site, tissue, or condition, it may facilitate the release of the nucleic acid cargo at the desired site in an efficient manner. Among all these stimuli- responsive carriers, pH-responsive one has attracted major attention and great impetus has been directed towards utilizing the subtle yet significant change in pH value within the cellular compartments. In this review, we give an overview of pH-sensitive lipids and polymers which have been designed and developed in recent years, with focus on their structural features and consequent functional attributes to achieve efficient transfection. The underlying modes of actions relating to structure and differential pH environment have also been discussed. It is worthy to note that despite many pH-sensitive carriers have shown success in vitro and a few in vivo, none have entered clinical phase for their transfection activity is still insufficient. To develop more efficient gene delivery carriers, the exact mechanisms of how these pH-sensitive carriers overcome each intracellular obstacle, as well as some concepts such as "proton sponge", have to be maken more clear or verified further. © 2013 Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences.
Poly(ethylene glycol) (PEG) is the gold standard polymer for biomedical applications. PEG is known for its biocompatibility and antifouling properties and is widely used for bioconjugation. However, like other synthetic polymers in the field, PEG is not biodegradable, limiting its use for parenteral formulations and protein conjugation to a molecular weight range with a specific upper limit (commonly 40–60 kDa) to avoid polyether accumulation in human tissue. For these biomedical applications, but also for other purposes such as cleavable hydrogels and templates for porous membranes, several routes for the insertion of in-chain biocleavable moieties, such as acetals or disulfides, into PEG have been developed. Recently, the synthetic strategies have been extended from step-growth polymerizations of commercially available, telechelic PEGs to more sophisticated routes based on ethylene oxide (co)polymerizations, permitting the incorporation of predetermined breaking points at any position in the PEG chains.
The objective of this Feature Article is to reflect on the importance of established and emerging principles of supramolecular organic chemistry to address one of the most persistent problems in life sciences. The main topic is dynamic covalent chemistry on cell surfaces, particularly disulfide exchange for thiol-mediated uptake. Examples of boronate and hydrazone exchange are added for contrast, comparison and completion. Of equal importance are the discussions of proximity effects in polyions and counterion hopping, and more recent highlights on ring tension and ion pair–π interactions. These lessons from supramolecular organic chemistry apply to cell-penetrating peptides, particularly the origin of “arginine magic” and the “pyrenebutyrate trick,” and the currently emerging complementary “disulfide magic” with cell-penetrating poly(disulfide)s. They further extend to the voltage gating of neuronal potassium channels, gene transfection, and the delivery of siRNA. The collected examples illustrate that the input from conceptually innovative chemistry is essential to address the true challenges in biology beyond incremental progress and random screening.
We describe the synthesis and self-assembly of an asparagine-derived amphiphile. The self-assembled systems formulated with the inclusion of cholesterol (0-50 mol%) show encapsulation for a hydrophobic model drug and rapidly disintegrate in response to mild acidic conditions.
The design and development of engineered nanosystems for disease diagnosis, prevention and treatment has been boosted by significant advances observed in distinct areas. This has been combined in order to attain efficient and safe products. Even if some of those nanoproducts have successfully reached the market, the consensus underlying the nanomedicines-related regulatory requirements has still to be fully acquired by part of the scientific community and thus academia and pharmaceutical companies could be facing considerable obstacles during the research and development life cycle of these medicinal products. However, considerable progress has been made in the last years, reflecting the recognition by Regulatory Authorities of specific features associated to the nanosystems-based medicines. As a consequence, the regulatory environment for those innovative medicinal nanosystems has been increasingly challenged by key issues, which has been an opportunity to provide clearer guidance for their development.
The current chapter aims at providing an overview of the regulatory requirements required for nanomedicine-related products, highlighting some differences among those established by different government agencies worldwide. In addition, we will also emphasise the translational aspects underlying the nanotechnology-based products in clinical use or in the pipelines of pharmaceutical companies.
Abstract
Liposomes are artificially-prepared bilayered phospholipid vesicles. Since their
discovery, they have emerged as a promising vehicle for the efficient delivery
of active biological compounds. They can be loaded with hydrophilic drugs in
their aqueous interior and/or hydrophobic drugs in the lipid membrane while
simultaneously protecting the incorporated drug from degradation or inactivation
and changing drug pharmacokinetics and bio-distribution in a favorable
fashion.
The coating of the liposome surface with polyethylene glycol (PEG) gave rise
to long-circulating liposomes. This coating helps prevent liposomal uptake by the
mononuclear phagocyte system. Longer circulation times allow for an increased
concentration of the liposomes in the blood and when combined with active targeting
through the attachment of targeting ligands, lead to efficient liposomal
delivery to the target sites increasing thus drug concentration in the required zone
and decreasing toxicity.
This chapter aims to provide the reader with a clear understanding of the
advantages and limitations of different types of liposomes and bring to light the
recent advances in the use of liposomes in the field of drug delivery.
A new type of acid-labile cationic copolymer consisting of a hydrophilic poly(ethylene glycol) (PEG) block and a polymethacrylamide block bearing tertiary amines linked by acid-labile ortho ester rings in side chains (PAOE), with defined chain length, had been synthesized via RAFT polymerization. The copolymers could efficiently bind and condense plasmid DNA at neutral pH into narrowly dispersed nano-scale polyplexes. The hydrolysis of ortho ester group in the side-chains of PAOE followed a distinct exocyclic mechanism and the rate of hydrolysis was much accelerated at mildly acidic pH, resulting in the accelerated disruption of polyplexes and the release of intact plasmid DNA. The three polymers were not toxic to cultured COS-7 cells as measured by MIT assay. As expected, PEG segments of the PEG-b-PAOE copolymers prevented nonspecific transfection of COS-7 cells. Once conjugated to a targeting ligand to enhance cell-specific entry, PEG-b-PAOE with its pH-triggered DNA release properties may achieve efficient intracellular delivery of DNA or other nucleic acid therapeutics.
Inflammatory pathologies are a growing burden for developed societies, and the tools offered by medicinal chemistry/biotechnology have provided us with an armamentarium of active principles. Unfortunately, most of them are marred by very significant side effects that can be summarized by two words: poor targeting (of cells, of tissues, etc.). This review provides a broad overview of the approaches and of the materials used to obtain an inflammation-responsive or inflammation-targeted behavior, which can be used to improve anti-inflammatory therapies. Here, we first define the biochemical factors that may allow both to recognize and to target an inflamed environment, for example, the leakiness of capillaries, the presence of oxidants (reactive oxygen species), or of upregulated receptors/enzymes. We then review the systems that have shown the possibility to accumulate in inflamed tissues, respond to its stimuli, or bind to activated inflammatory cells, separately discussing payload/carrier (guest/host) systems and macromolecular prodrugs/conjugates. Copyright © 2014 John Wiley & Sons, Ltd.
A new type of homopolymers, PMAOE, bearing acid-cleavable cationic side-chains is synthesized and characterized. PMAOE is obtained via free radical polymerization, and they could efficiently bind and condense plasmid DNA at neutral pH. The strength of DNA binding is dependent on the length of PMAOE chains. NMR analysis reveals that hydrolysis of the ortho ester group of PMAOE follows an exocyclic mechanism and the rate of hydrolysis is much accelerated at mildly acidic pH, leading to accelerated disruption of polyplexes and release of DNA in mildly acidic environment. PMAOE is not toxic to cultured NIH 3T3 and COS-7 cells measured by MTT. This study demonstrates a unique mechanism of achieving pH-modulated binding and release of DNA from polymers with potential applications for gene delivery.
Gene carriers made from synthetic materials are of interest in relation to gene therapy but suffer from lack of transfection efficiency upon systemic delivery. To address this problem, a novel lipo-peptide-PEG conjugate constituted by a lipid-anchor, a peptide sensitive to proteases and a poly (ethylene glycol) (PEG) chain is investigated. Utilizing ethanol-mediated nucleic acid encapsulation to prepare lipo-nanoparticles (LNPs), LNPs that are stable in serum are obtained. The LNPs constitute a highly effective gene delivery systems in vitro and possess the right features for further investigation in vivo including a PEG layer and a net negative charge that should ensure long-circulating properties before being activated by proteases in diseased tissue. Protease activation leads to detachment of PEG and a charge switching where the LNPs become positive due to the presence of glutamates in the cleaved peptide moiety. The cationic lipid DOTAP is used mainly to complex DNA and proton titratable DODAP is used to increase endosomal escape and enhance transfection efficiency. The idea of using a mixture of permanently charged and titratable cationic lipids shielded by a protease sensitive negatively charged lipo-peptide-PEG coat appears to be a highly efficient solution for achieving effective non-viral gene delivery and the results warrant further investigations.
Neurodegenerative diseases represent a major public health problem, but beneficial clinical treatment with neurotrophic factors has not been established yet. The therapeutic use of neurotrophins has been restrained by their instability and rapid degradation in biological medium. A variety of strategies has been proposed for the administration of these leading therapeutic candidates, which are essential for the development, survival and function of human neurons. In this review, we describe the existing approaches for delivery of brain-derived neurotrophic factor (BDNF), which is the most abundant neurotrophin in the mammalian central nervous system (CNS). Biomimetic peptides of BDNF have emerged as a promising therapy against neurodegenerative disorders. Polymer-based carriers have provided sustained neurotrophin delivery, whereas lipid-based particles have contributed also to potentiation of the BDNF action. Nanotechnology offers new possibilities for the design of vehicles for neuroprotection and neuroregeneration. Recent developments in nanoscale carriers for encapsulation and transport of BDNF are highlighted.
The p53 protein mediated anti-tumor strategy is limited due to the lack of suitable delivery agent with insignificant immunogenic response, serum compatibility, and early and easy detection of the transfected cell population. To overcome these problems, we generated a p53-EGFP-C3 fusion construct which expressed easily detectable green fluorescence protein (GFP) and allowed an estimation of p53 mediated anti-tumor activity. A mixture of cationic cholesterol gemini (Chol-5L) with natural lipid, DOPE (molar ratio 1:4), acronymed as Chol-5LD, formed a nano-liposome as characterized by various physical methods. The prepared clone was evaluated for the expression of GFP and functional p53 in HeLa and two additional cell lines with varied p53 status namely, H1299 (p53(-/-)) and HEK293T (p53(+/+)). Transfected cells were screened using RT-PCR, Western blotting, FACS analysis, MTT, Trypan blue assay and visualized under a fluorescence microscope. The p53-EGFP-C3 fusion protein induced apoptosis in cancer cells as evident from DNA fragmentation, cell cycle analysis, Annexin-V staining and PARP cleavage assays. The transfection and apoptosis induction efficiency of Chol-5LD was significantly higher than commercial reagents Lipofectamine2000 and Effectene irrespective of the cell lines examined. Further it significantly decreases the xenograft tumor volume in nude mice tumors via apoptosis as observed in H&E staining.
Liposomes, vesicles consisting of phospholipids, are well known drug carriers, especially in anti-cancer treatment. Due to their improved pharmacokinetics, "stealth" liposomes, which are polymer coated vesicles, are being used in clinical applications with good results. One of the drawbacks of poly(ethylene glycol) (PEG) that is preferentially incorporated, is its lack of functional groups and its non-biodegradability. In this article new polyether-based lipids are presented that can be synthesized from an epoxide monomer library, resulting in tailored multivalent architectures. The cholesterol-based lipid-like structures offer further possibilities for functionalization, which is important for active targeting. Furthermore, a rather simple synthetic route has been developed, which leads to acid-cleavable cholesteryl PEG, thus leading to possible controlled destabilization of the liposome formulation. This process is crucial for drug release in vivo.
Since the discovery of liposomes, these phospholipid ‘bubbles’ have received enormous attention to be recognized as ‘smart’ pharmaceutical nanocarriers. Recently, much effort has been directed to the development of so-called ‘smart’ stimuli-sensitive liposomes that will respond to certain internal or external stimuli, such as, pH, temperature, redox potential or magnetic field. These programmable delivery systems can also be made ‘multifunctional’ so as to expose certain functions in an orchestrated manner which can be readily modulated by the stimulus. In this article, the evolution of liposomes with emphasis on the recent advances in stimuli-sensitive liposomes has been reviewed.
The pathway for content release from reduction-sensitive liposomes based on a quinone-dioleoylphosphatidylethanolamine lipid conjugate (Q-DOPE) is outlined using results from fluorescent dye content release assays as well as single- and multiple-angle light scattering. Experimental observations are consistent with a shape/size change of the reduced liposomes prior to their aggregation, with subsequent near-quantitative content release achieved only when the lipid membrane experiences conditions favorable to a lamellar to an inverted hexagonal phase transition. Addition of poly(ethyleneglycol)-modified DOPE (PEG-DOPE) to the Q-DOPE liposomal formulation results in stabilization of the lipid bilayer, whereas incorporation of DOPE yields faster content release. At high DOPE concentrations, DOPE/PEG-DOPE/Q-DOPE liposomes exhibit larger content release, indicating a change in pathway for content release. The outcomes here provide a better understanding of the underlying principles of triggered liposomal content release and the potential utility of specific lipid properties for the rational design of drug delivery systems based on the novel Q-DOPE lipid.
Nanotechnology has the potential to revolutionize cancer diagnosis and therapy. Advances in protein engineering and materials science have contributed to novel nanoscale targeting approaches that may bring new hope to cancer patients. Several therapeutic nanocarriers have been approved for clinical use. However, to date, there are only a few clinically approved nanocarriers that incorporate molecules to selectively bind and target cancer cells. This review examines some of the approved formulations and discusses the challenges in translating basic research to the clinic. We detail the arsenal of nanocarriers and molecules available for selective tumour targeting, and emphasize the challenges in cancer treatment.
The tumor drug concentrations, drug distributions, and therapeutic efficacies achieved by three fundamentally different liposomes, nonthermosensitive liposome (NTSL), traditional thermosensitive liposome (TTSL), and low temperature sensitive liposome (LTSL); free doxorubicin (DOX); and saline in combination with hyperthermia (HT) were directly compared in a human tumor xenograft model. NTSL is a nonthermosensitive liposome in the physiological temperature range, TTSL is a traditional thermosensitive liposome that triggers in the range of approximately 42-45 degrees C and releases drug over approximately 30 min, and LTSL is a new low temperature sensitive liposome that triggers in the range of approximately 39-40 degrees C and releases drug in a matter of seconds. Because of the different attributes of the liposomes, it was possible to delineate the relative importance of liposome drug encapsulation, HT cytotoxicity, HT-drug interaction, HT-induced liposomal delivery, and HT-triggered liposomal drug release in achieving antitumor activity. Athymic nude mice bearing the FaDu human tumor xenograft were given a single i.v. dose of 5 mg/kg of DOX (free drug or liposome encapsulated), and the tumors were then heated to either 34 degrees C or 42 degrees C for 1 h at 34 degrees C. All treatment groups were similar, achieving low concentrations of DOX (0-4.5 ng/mg). At 42 degrees C, the LTSL (25.6 ng/mg) achieved the highest DOX concentration (P < 0.04), but all three liposomal formulations (7.3-25.6 ng/mg) were higher than saline or DOX (0-0.7 ng/mg; P < 0.02). LTSL + HT was also the only group that resulted in significant amounts of DNA-bound DOX (silver nitrate-extractable fraction; P < 0.02). Tumor tissue sections were visualized for DOX fluorescence to investigate the local distribution of the drug in the tumor and confirm the relative drug concentrations based on fluorescence intensity. There was relatively little fluorescence seen with treatment groups at 34 degrees C. At 42 degrees C, the LTSL showed the most DOX fluorescence (P < 0.01), and the fluorescence, although not homogeneous, was pervasive throughout the tumor sections. Therapeutic efficacy of treatments was determined from tumor growth time. At 34 degrees C, the only treatment group significantly better than the saline group (9.8 days) was the NTSL group, with a growth time of 20.9 days (P < 0.02). At 42 degrees C, all three liposomal formulations were more efficacious than DOX. LTSL + HT had the longest growth time (51.4 days) and the most number of local controls at 60 days (six of nine tumors). With HT, the DOX concentrations and fluorescence were tightly correlated with tumor growth delay, indicating that adequate (increased) drug delivery can be predictive of therapeutic effect. Overall, the LTSL + HT group showed the largest DOX concentration, the highest and most pervasive DOX fluorescence, and the most antitumor effect. Thus, HT-triggered liposomal drug release may account for the largest differential therapeutic effect and demonstrates the importance of rapid drug release from the drug carriers at the tumor site.
One of the main challenges of gene therapy remains the increase of gene delivery into eukaryotic cells. We tested whether intracellular DNA release, an essential step for gene transfer, could be facilitated by using reducible cationic DNA-delivery vectors. For this purpose, plasmid DNA was complexed with cationic lipids bearing a disulphide bond. This reduction-sensitive linker is expected to be reduced and cleaved in the reducing milieu of the cytoplasm, thus potentially improving DNA release and consequently transfection. The DNA--disulphide-lipid complexation was monitored by ethidium bromide exclusion, and the size of complexes was determined by dynamic light scattering. It was found that the reduction kinetics of disulphide groups in DNA--lipid complexes depended on the position of the disulphide linker within the lipid molecule. Furthermore, the internal structure of DNA--lipid particles was examined by small-angle X-ray scattering before and after lipid reduction. DNA release from lipid complexes was observed after the reduction of disulphide bonds of several lipids. Cell-transfection experiments suggested that complexes formed with selected reducible lipids resulted in up to 1000-fold higher reporter-gene activity, when compared with their analogues without disulphide bonds. In conclusion, reduction-sensitive groups introduced into cationic lipid backbones potentially allow enhanced DNA release from DNA--lipid complexes after intracellular reduction and represent a tool for improved vectorization.
We measured the nonradiative fluorescence resonance energy transfer between 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD) labeled lipids (amine labeled phosphatidylethanolamine or acyl chain labeled phosphatidylcholine) and rhodamine labeled lipids in large unilamellar dioleoylphosphatidylcholine vesicles. Two new rhodamine labeled lipid analogues, one a derivative of monolauroylphosphatidylethanolamine and the other of sphingosylphosphorylcholine, were found to exchange through the aqueous phase between vesicle populations but not to be capable of rapid transbilayer movement between leaflets. Energy transfer from NBD to rhodamine was measured using liposomes with symmetric or asymmetric distributions of these new rhodamine labeled lipid analogues to determine the relative contributions of energy transfer between donor and acceptor fluorophores in the same (cis) and opposite (trans) leaflets. Since the characteristic R0 values for energy transfer ranged from 47 to 73 angstrom in all cases, significant contributions from both cis and trans energy transfer were observed. Therefore, neither of these probes acts strictly as a half-bilayer quencher of NBD lipid fluorescence. The dependence of transfer efficiency on acceptor density was fitted to a theoretical treatment of energy transfer to determine the distances of closest approach for cis and trans transfer. These parameters set limits on the positions of the fluorescent groups relative to the bilayer center, 20-31 angstrom for NBD and 31-55 angstrom for rhodamine, and provide a basis for future use of these analogues in measurements of transbilayer distribution and transport.
Abstract Current fusogenic liposomal delivery systems have limited applicability in vivo due to poor stability in the blood and rapid clearance from the circulation. This is particularly true for liposomes composed of dioleoylphosphatidylethanolamine (DOPE) and cationic lipids, currently being developed for systemic delivery of gene-based drugs. This paper describes a potential strategy to overcome these problems, involving the incorporation of exchangeable amphipathic polyethyleneglycol (PEG) derivatives to transiently stabilize fusogenic liposomes while in the circulation, but where the PEG coating dissipates to reveal fusogenic character at later times after arrival at target sites. It is shown here that large unilamellar vesicles (LUVs) containing Dope and the cationic lipid, N,N-dioleoyl-N, N-dimethylammonium chloride (DODAC) can be stabilized against serum-induced aggregation and fusion by inclusion of at least 2 mol% of PEG coupled to phosphatidylethanolamine (PEG-PE) or ceramide (PEG-Cer). However, low in vitro recovery of fusogenic activity was obtained for the PEG-PE-containing system, presumably due to electrostatic interactions between the negatively charged PEG-PE and the cationic lipid which prevent PEG-PE dissociation from the LUV. Improved recovery of fusogenic activity was achieved for LUVs stabilized by the neutral PEG-Cer derivatives, with shorter chain ceramides exhibiting more rapid recovery rates. Biodistribution studies showed that DOPE/DODAC (85:15, mol/mol) LUVs were rapidly cleared from the circulation, whereas inclusion of 10 mol% PEG-Cer(C20) resulted in significantly prolonged circulation time. Inclusion of shorter ceramide chain lengths resulted in decreased circulation times, consistent with increased exchangeability. These findings demonstrate the feasibility of developing a cationic liposome that is stable in the circulation, but retains its ability to fuse with membranes. This work represents the first step toward the rationale design of fusogenic cationic liposomes for the systemic delivery of gene-based drugs to target tissues, such as tumors.
Although polyethylene glycol (PEG) is widely used for aggregating or fusing cells, the forces responsible for these interactions have remained elusive. Through a variety of techniques including quasi-elastic light scattering, surface force measurements, and 31P-NMR, we have established that while PEG of molecular weight 8000−10000 is effective in causing the aggregation of vesicles, PEG of lower or higher molecular weight (1000 and 18 500, respectively) is ineffective. For the first time, direct force measurements between lipid bilayers in solutions of 8000−10000 molecular weight reveal the existence of an attractive osmotic force due to a polymer depleted layer near the bilayer surface. Lower molecular weight PEG does not have a large enough size (Flory radius, RF) to generate a significant depletion force, while higher molecular weight PEG adsorbs sufficiently on the bilayer surfaces to eliminate the depletion attraction and produces a repulsive steric barrier to aggregation. The measured forces can be quantitatively described in terms of current theories of colloidal and polymer interactions. These findings suggest that the differential osmotic pressure produced by the depletion layer is responsible for vesicle aggregation and that fusion is promoted when the depletion pressure is strong enough to locally destabilize two membranes by possibly thinning them at their point of closest approach. The results provide a physicochemical basis for using PEG of certain molecular weights as fusogens for cells, liposomes, and vesicles.
A novel acid-labile poly(ethylene glycol) (PEG)-conjugated lipid, (R)-1,2-di-O-(1‘Z,9‘Z-octadecadienyl)-glyceryl-3-(ω-methoxy-poly(ethylene glycolate, MW5000) (BVEP), a neutral PEG-derivatized analogue of diplasmenylcholine, has been used at low molar ratios to disperse the nonlamellar, fusogenic lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as unilamellar liposomes. It was anticipated that acid-catalyzed hydrolysis of the vinyl ether linkages would destabilize BVEP/DOPE liposomes by removal of the water-soluble, sterically stabilizing PEG layer, thereby promoting contents release and membrane−membrane fusion. This paper describes the hydrolysis rates, contents release rates, and fusion kinetics of BVEP-stabilized DOPE liposomes at 1:99, 3:97, and 5:95 molar ratios of BVEP/DOPE. Calcein leakage kinetics indicate that 3:97 BVEP/DOPE liposomes offer the best stability at pH 7.4 while retaining favorable leakage properties at pH 4.5 (t50%release ≈ 4 h). N-Rhodamine phosphatidylethanolamine/N-nitrobenzoxadiazole phosphatidylethanolamine lipid mixing assays show that membrane fusion occurs on a much slower time scale than leakage in these systems, with 12% lipid mixing occurring over a 24 h time period at pH 2.0. No appreciable membrane fusion occurred in these liposomes at either pH 7.4 or 4.5 when monitored for up to 3 days. 31P NMR spectra at pH 7.4 contain a single isotropic line shape, consistent with the presence of large liposomes. The 31P NMR line shape did not change significantly even after long exposure times at pH 4.0; however, Mn2+ addition experiments with acid-treated samples produced line-broadened spectra, indicating that all the phosphorus sites were continuous with the bulk water phase. Time-dependent cryogenic transmission electron microscopy experiments indicate that extensive liposome collapse to give small dense aggregates occurs over a 1−4 h period when 3:97 BVEP/DOPE liposomes are acidified to pH 4.5. Taken together, these results suggest that acid-catalyzed hydrolysis of BVEP/DOPE liposomes does result in dePEGylative triggering; however, the primary outcome of this cleavage process is contents leakage and liposome collapse to give <100 nm particles that are presumed to be inverted hexagonal phase structures, with membrane lipid mixing occurring on a kinetically slower time scale.
Most pharmaceutical and gene therapy applications of targeted liposomes presently suffer from inefficient contents delivery to the cytoplasm of target cells. We report a plasma-stable liposome, composed of synthetic, naturally occurring diplasmenylcholine (1,2-di-O-(Z-1‘-hexadecenyl)-sn-glycero-3-phosphocholine; DPPlsC), that rapidly and efficiently releases its contents at endosomal pHs. Acid-catalyzed hydrolysis of these liposomes produces glycerophosphocholine and fatty aldehydes, leading to greatly enhanced liposome permeability (t50% release 1−4 h between pH 4.5−5.5) when >20% of the vinyl ether lipid has been hydrolyzed. Plasma stability of nonhydrolyzed 9:1 DPPlsC/dihydrocholesterol liposomes exceeds 48 h at 37 °C, pH 7.4 in 50% serum; pure DPPlsC liposomes remain stable in 10% serum under the same conditions. Fluorescence assays of KB cells treated with 99.5:0.5 DPPlsC/DSPE−PEG3350−folate liposomes containing encapsulated propidium iodide (PI) indicate that 83% of the PI escapes the endosomal compartment within 8 h to produce intensely stained nucleii. The IC50 value of 1-β-arabinofuranosylcytosine (Ara-C) encapsulated in DPPlsC/DSPE−PEG3350−folate liposomes is 0.49 μM in KB cell cultures, a 6000-fold enhancement in cytotoxicity compared with free drug (2.8 mM). Empty DPPlsC/DSPE−PEG3350−folate liposomes had no effect on DNA synthesis, indicating that DPPlsC and its degradation products are benign to cell function at these lipid concentrations. Our results suggest that concurrent application of selective targeting and membrane translocation mechanisms in drug carriers can significantly increase their efficacy.
The first practical total chemical synthesis of a plasmenylcholine (1–O-1'-(Z)-hexadecenyl-2-hexadecanoyl-sn-glycero-3-phosphocholine) with pure (Z) olefin stereochemistry is reported. Monopalmitin was doubly protected as the 3-TBDPS-2-TBDMS ethers (tert-butyldiphenylsilyl-, tert-butyldimethylsilyl-) and converted to the corresponding 1-O-1′-(Z)-hexadecenyl-2-TBDMS-3-TBDPS-glyceryl ether (by the method of ref. [43]). Clean deprotection with tetra-butylammonium fluoride in the presence of imidazole gave 1-O-1′-(Z)-hexadecenylglycerol in >90% yield. Resilylation with TBDPSCl followed by acylation of the sn-2 alcohol with palmitoyl chloride and deprotection of the resulting 3-TBDPS-2-hexadecanoyl-1–O-1′-(Z)-hexadecenylglycerol at –20°C with Bu4NF gave 2-hexadecanoyl-1–O-1′-(Z)-hexadecenylglycerol in 86% yield. The 3-phosphocholine group was attached by phosphorylating the free hydroxyl with 2-chloro-2-oxo-1,3,2-dioxaphospholane in the presence of pyridine, instead of Et3N, as base to avoid acyl migration; the dioxaphospholane triester intermediate was subsequently cleaved with Me3N to give 1-O-1′-(Z)-hexadecenyl-2-hexadecanoyl-sn-glycero-3-phosphocholine in 18% overall yield from monopalmitin. The efficiency and flexibility of this route makes it well-suited to the preparation of a wide variety of 1-, 2-, and 3-substituted as well as isotopically labeled plasmenylcholines for biophysical and biochemical studies.
We examined the effect of a cholesterol derivative, poly (ethylene glycol) cholesteryl ether on the structure/function of clathrin-coated pits and caveolae. Addition of the compound to cultured cells induced progressive smoothening of the surface. Markedly, when the incorporated amount exceeded 10% equivalent of the surface area, fluid pinocytosis, but not endocytosis of transferrin, became inhibited in K562 cells. In A431 cells, both clathrin-independent fluid phase uptake and the internalization of fluorescent cholera-toxin B through caveolae were inhibited with concomitant flattening of caveolae. In contrast, clathrin-mediated internalization of transferrin was not affected until the incorporated poly (ethylene glycol) cholesteryl ether exceeded 20% equivalent of the plasma membrane surface area, at which point opened clathrin-coated pits accumulated. The cells were ruptured upon further addition of poly (ethylene glycol) cholesteryl ether. We propose that the primary reason for the differential effect of poly (ethylene glycol) cholesteryl ether is that the bulk membrane phase and caveolae are both more elastic than the rigid clathrin-coated pits. We analyzed the results with the current mechanical model (Rauch and Farge, Biophys J 2000;78:3036–3047) and suggest here that the functional clathrin-lattice is much stiffer than typical phospholipid bilayers.
Background and ScopeMethods of Studying EnolizationMethods of Studying KetonizationReaction MechanismsResultsReferences
Free folic acid is believed to enter some cells by folate receptor-mediated endocytosis at membrane invaginations termed caveolae. Folate conjugated macromolecules also enter cells by folate receptor-mediated endocytosis, but their site of entry has never been conclusively identified. In this paper, we show that internalization of folate-macromolecule conjugates by receptor-bearing KB cells can be blocked by agents that specifically inhibit caveolae assembly or internalization such as nystatin and phorbol-12-myristate acetate (PMA). To characterize the intracellular conditions to which the macromolecule-folate conjugates are subsequently exposed, we have measured the pH of the major compartments of the folate endocytosis pathway. pH values of individual endosomal compartments in KB cells were determined by dual-excitation laser-scanning confocal microscopy, where the fluorescence ratio of folate-DM-NERF-dextran (pH-sensitive) and Texas Red-dextran (pH-insensitive) was used to calculate pH. These studies revealed that the pH of folate conjugate-containing endosomes commonly varies between 4.7 and 5.8, with the pH in some endosomes as low as 4.3. The most frequent pH value in these compartments was ≈ 5.0.
Gradients of ammonium sulfate in liposomes [(NH4)2SO4]lip.>[(NH4)2SO4]med. were used to obtain ‘active’ loading of amphipathic weak bases into the aqueous compartment of liposomes. The loading is a result of the base exchange with the ammonium ions. This approach was applied to encapsulate anthracyclines and acridine orange inside the liposomes at very high efficiency (>90%). Doxorubicin was accumulated in the aqueous phase of the liposomes where it reached a level as high as 100-fold the doxorubicin concentration in the remote loading medium. Most of the intraliposomal doxorubicin was present in an aggregated state. The active entrapment and loading stability were dependent on liposome lipid composition, lipid quality, medium composition and temperature, as well as on the pKa and hydrophobicity of the base. The ammonium sulfate gradient approach differs from most other chemical approaches used for remote loading of liposomes, since it neither requires preparation of the liposomes in acidic pH, nor to alkalinize the extraliposomal aqueous phase. The stability of the ammonium ion gradient is related to the low permeability of its counterion, the sulfate, which also stabilizes anthracycline accumulation for prolonged storage periods (>6 months) due to the aggregation and gelation of anthracycline sulfate salt.
The efficiency of nucleic acid-based drugs is usually hampered by the fact that, following their uptake by the cell, these drugs end up in acidic organelles (i.e., endosomes/lysosomes) from which they barely escape. This work relates to the preparation and characterization of polyion complex micelles (PICM) formed by the self-assembly of three polyelectrolytes: a diblock cationic copolymer; a membranolytic, methacrylic acid copolymer; and an oligonucleotide. It is demonstrated that a synthetic membrane-active polyanion can be successfully integrated within the structure of PICM to yield well-defined, narrowly distributed micelles (30 nm) with a core/shell architecture. Besides their ability to protect the oligonucleotide against nuclease degradation, PICM partly dissociate under mildly acidic conditions, releasing chain clusters that destabilize bilayer membranes. This association/dissociation behavior illustrates the potential of these pH-sensitive PICM for the transport and efficient delivery of polyionic drugs.
Dioleoyl-N-(monomethoxy polyethyleneglycol succinyl)-phosphatidylethanolamine (PEG-PE) (mol. wt. of PEG = 5000), an amphipathic polymer, can be incorporated into the liposome membrane and significantly prolong the blood circulation time of the liposome. As little as 3.7 mol% of PEG-PE in liposome resulted in maximal enhancement of liposome circulation time. However, this activity of PEG-PE was only seen with relatively small liposomes (d less than or equal to 200 nm); larger liposomes containing PEG-PE showed an unusually high level (approx. 35% injected dose) of accumulation in the spleen. We have tested whether the small, PEG-PE containing liposomes are suitable for immuno targeting by incorporating a lung-specific monoclonal antibody on the liposome surface. While another amphiphile, ganglioside GM1, which is well known for its activity to prolong the liposome circulation time, significantly enhanced the lung binding of the immunoliposomes, PEG-PE incorporation of immunoliposomes resulted in a low level of target binding. To test if the reduced target binding is due to a steric barrier effect of the surface PEG polymer, we have incorporated a small amount of N-biotinaminocaproylphosphatidylethanolamine into the PEG-PE containing liposomes and examined the liposome agglutination induced by the addition of streptavidin. As little as 0.72 mol% PEG-PE in these liposomes completely abolished agglutination. In contrast, incorporation of GM1 in liposomes only reduced the rate, but not the extent, of liposome agglutination. These results strongly support the hypothesis that PEG-PE prolongs liposome circulation time by providing a strong steric barrier which prevents close contact with another liposome or cell. Since GM1 provides only a weak steric barrier effect, its activity to prolong the liposome circulation time must involve another yet unknown mechanism.
Novel synthetic lipid derivatives of poly(ethylene glycol) (PEG) have been synthesized and tested for their ability to decrease uptake of liposomes into the mononuclear phagocyte system (MPS, reticuloendothelial system) in mice and to prolong circulation half-lives of liposomes. A carbamate derivative of PEG-1900 with distearoylphosphatidylethanolamine (PEG-DSPE) had the greatest ability to decrease MPS uptake of liposomes, at optimum concentrations of 5-7 mol% in liposomes composed of sphingomyelin/egg phosphatidylcholine/cholesterol (SM/PC/Chol, 1:1:1, molar ratio). Results obtained with this compound were equivalent to results previously obtained with 10 mol% monosialoganglioside GM1 in liposomes of similar compositions (Allen, T.M. and Chonn, A. (1987) FEBS Lett. 223, 42-46). Non-derivatized methyl PEG or PEG-stearic acid (PEG-SA) were incapable of decreasing MPS uptake of liposomes. PEG-Chol and PEG-dipalmitoylglycerol (PEG-DPG) were intermediate in their effects on MPS uptake. Altering liposome size for liposomes containing PEG-DSPE resulted in only minor changes in blood levels of liposomes. Half-lives of 0.1 microns liposomes of SM/PC/Chol/PEG-DSPE (1:1:1:0.2, molar ratio) in circulation was in excess of 20 h following either i.v. or i.p. injection. Liver plus spleen liposome levels for these liposomes was below 15% of injected label at 48 h following i.v. liposome injection and below 10% following i.p. injection. The major site of liposome uptake was in carcass tissues, with over 50% of label remaining in vivo at 48 h post-injections, either i.v. or i.p., in the carcass.
Liposomes containing phospholipids with covalently attached poly(ethylene glycol) (PEG-lipids) are being developed for in vivo drug delivery. In this paper we determine the structure and phase behavior of fully hydrated distearoylphosphatidylcholine (DSPC) suspensions containing PEG-lipids composed of distearoylphosphatidylethanolamine with attached PEGs of molecular weights ranging from 350 to 5000. For DSPC:PEG-lipid suspensions containing 0-60 mol % PEG-lipid, differential scanning calorimetry shows main endothermic transitions ranging from 55 to 64 degrees C, depending on the size of the PEG and concentration of PEG-lipid. The enthalpy of this main transition remains constant for all PEG-350 concentrations but decreases with increasing amounts of PEG-750, PEG-2000, or PEG-5000, ultimately disappearing at PEG-lipid concentrations greater than about 60 mol %. Low-angle and wide-angle x-ray diffraction show that tilted gel (L beta') phase bilayers are formed for all PEG-lipid molecular weights at concentrations of about 10 mol % or less, with the distance between bilayers depending on PEG molecular weight and PEG-lipid concentration. At PEG-lipid concentrations greater than 10 mol %, the lipid structure depends on the size of the PEG moiety. X-ray diffraction analysis shows that untilted interdigitated (L beta I) gel phase bilayers form with the incorporation of 40-100 mol % PEG-350 or 20-70 mol % PEG-750, and untilted gel (L beta) phase bilayers are formed in the presence of about 20-60 mol % PEG-2000 and PEG-5000. Light microscopy, turbidity measurements, x-ray diffraction, and 1H-NMR indicate that a pure micellar phase forms in the presence of greater than about 60% PEG-750, PEG-2000, or PEG-5000.
A series of conjugates has been prepared by linking various hydrophilic macromolecules [poly-(ethylene glycols), polylysine, aminodextrans, or apotransferrin] to synthetic phosphatidylethanolamines via linker moieties incorporating a fluorescent bimane group. Using a fluorescence energy transfer-based assay, the rate of transfer of these species between phospholipid vesicles has been monitored as a function of the nature and size of the coupled macromolecule and of the acyl chain composition of the lipid anchor. Conjugates in which the phospholipid anchor is linked to a small hydrophilic terminal residue (e.g., ethanolamine or ethylenediamine) transfer between large unilamellar vesicles of egg phosphatidylcholine with half-times ranging from tens of minutes (for dimyristoyl lipid conjugates) to a few tens of hours (for dipalmitoyl and 1-palmitoyl-2-oleoyl lipid conjugates), in agreement with previous results for unlabeled phospholipids. Conjugation of these same lipid anchors to larger hydrophilic molecules markedly accelerates their rates of intermembrane transfer, by factors ranging from 5-7-fold (for conjugates with apotransferrin and aminodextrans of molecular weight 10,000-70,000) to over 25-fold [for conjugates with poly(ethylene glycol)-5000]. In all cases the observed transfer appears to reflect the diffusion of lipid monomers through the aqueous phase. Our results suggest that substantial intermembrane transfer can occur, on a time scale of several hours or less, for hydrophilic macromolecules conjugated to diacyl(/alkyl) lipids with 14- to 18-carbon chains unless portions of the conjugate other than the lipid anchor also interact strongly with the membrane.
The uptake of the anticancer agent doxorubicin into large unilamellar vesicles (LUVs) exhibiting a transmembrane pH gradient (inside acidic) has been investigated using both kinetic and equilibrium approaches. It is shown that doxorubicin uptake into the vesicles proceeds via permeation of the neutral form and that uptake of the drug into LUVs with an acidic interior is associated with high activation energies (Ea) which are markedly sensitive to lipid composition. Doxorubicin uptake into egg-yolk phosphatidylcholine (EPC) LUVs exhibited an activation energy of 28 kcal/mol, whereas for uptake into EPC/cholesterol (55:45, mol/mol) LUVs Ea = 38 kcal/mol. The equilibrium uptake results obtained are analyzed in terms of a model which includes the buffering capacity of the interior medium and the effects of drug partitioning into the interior monolayer. From the equilibrium uptake behaviour, a doxorubicin partition coefficient of 70 can be estimated for EPC/cholesterol bilayers. For a 100 nm diameter LUV, this indicates that more than 95% of encapsulated doxorubicin is partitioned into the inner monolayer, presumably located at the lipid/water interface. This is consistent with 13C-NMR behaviour as a large proportion of the drug appears membrane associated after accumulation as reflected by a broadening beyond detection of the 13C-NMR spectrum. The equilibrium accumulation behaviour of a variety of other lipophilic amines is also examined in terms of the partitioning model.
We have characterized the surface activity of different-sized poly(ethylene-glycols) (PEG; M(r) 200-100,000 Da) in the presence or absence of lipid monolayers and over a wide range of bulk PEG concentrations (10-8-10% w/v). Measurements of the surface potential and surface pressure demonstrate that PEGs interact with the air-water and lipid-water interfaces. Without lipid, PEG added either to the subphase or to the air-water interface forms relatively stable monolayers. Except for very low molecular weight polymers (PEGs < 1000 Da), low concentrations of PEG in the subphase (between 10-5 and 10-4% w/v) increase the surface potential from zero (with respect to the potential of a pure air-water interface) to a plateau value of ~440 mV. At much higher polymer concentrations, >10-1% (w/v), depending on the molecular weight of the PEG and corresponding to the concentration at which the polymers in solution are likely to overlap, the surface potential decreases. High concentrations of PEG in the subphase cause a similar decrease in the surface potential of densely packed lipid monolayers spread from either diphytanoyl phosphatidylcholine (DPhPC), dipalmitoyl phosphatidylcholine (DPPC), or dioleoyl phosphatidylserine (DOPS). Adding PEG as a monolayer at the air-water interface also affects the surface activity of DPhPC or DPPC monolayers. At low lipid concentration, the surface pressure and potential are determined by the polymer. For intermediate lipid concentrations, the surface pressure-area and surface potential-area isotherms show that the effects due to lipid and PEG are not always additive and that the polymer's effect is distinct for the two lipids. When PEG- lipid-mixed monolayers are compressed to surface pressures greater than the collapse pressure for a PEG monolayer, the surface pressure-area and surface potential-area isotherms approach that of the lipid alone, suggesting that for this experimental condition PEG is expelled from the interface.
The effect of poly(ethylene glycol)--lipid (PEG--lipid) conjugates on liposomal fusion was investigated. Incorporation of PEG--lipids into large unilamellar vesicles (LUVs) composed of equimolar phosphatidylethanolamine (PE) and phosphatidylserine (PS) inhibited calcium-induced fusion. The degree of inhibition increased with increasing molar ratio of the PEG conjugate and with increasing size of the PEG moiety. Inhibition appeared to result from the steric barrier on the surface of the liposomes which opposed apposition of bilayers and interbilayer contact. In the presence of a large excess of neutral acceptor liposomes, however, fusogenic activity was restored. The rate of fusion under these conditions depended on the initial molar ratio of the PEG conjugate in the PE:PS vesicles and the length and degree of saturation of the acyl chains which composed the lipid anchor. These results are consistent with spontaneous transfer of the PEG--lipid from PE:PS LUVs to the neutral lipid sink reducing the steric barrier and allowing fusion of the PE:PS LUVs. The primary determinant of the rate of fusion was the rate of transfer of the PEG--lipid, indicating that liposomal fusion could be programmed by incorporation of appropriate PEG--lipid conjugates. Interestingly, increasing the size of the PEG group did not appear to affect the rate of fusion. The implications of these results with respect to the design of fusogenic liposomal drug delivery systems are discussed.
The influence of poly(ethylene glycol)-lipid conjugates on phospholipid polymorphism has been examined using 31P-NMR and freeze--fracture electron microscopy. An equimolar mixture of dioleoylphosphatidylethanolamine (DOPE) and cholesterol adopts the hexagonal (HII) phase when hydrated under physiological conditions but can be stabilized in a bilayer conformation when a variety of PEG-lipid conjugates are included in the lipid mixture. These PEG conjugates produced an increase in the bilayer to hexagonal (HII) phase transition temperature and a broadening of the temperature range over which both phases coexisted. Further, the fraction of phospholipid adopting the bilayer phase increased with increasing mole fraction of PEG-lipid such that at 20 mole % DOPE--PEG2000 no HII phase phospholipid was observed up to a least 60 degrees C. Increasing the size of the PEG moiety from 2000 to 5000 Da (while maintaining the PEG--lipid molar ratio constant) increased the proportion of lipid in the bilayer phase. In contrast, varying the acyl chains of the PE anchor had no effect on polymorphic behavior. PEG--lipid conjugates in which ceramide provides the hydrophobic anchor also promoted bilayer formation in DOPE:cholesterol mixtures but at somewhat higher molar ratios compared to the corresponding PEG--PE species. The slightly greater effectiveness of the PE conjugates may result from the fact that these derivatives also possess a net negative charge. Phosphorus NMR spectroscopy indicated that a proportion of the phospholipid in DOPE:cholesterol:PEG--PE mixtures experienced isotropic motional averaging with this proportion being sensitive to both temperature and PEG molecular weight. Surprisingly, little if any isotropic signal was observed when PEG--ceramide was used in place of PEG--PE. Consistent with the 31P-NMR spectra, freeze-fracture electron microscopy showed the presence of small vesicles (diameter <200 nm) and lipidic particles in DOPE:cholesterol mixtures containing PEG--PE. We conclude that the effects of PEG--lipid conjugates on DOPE:cholesterol mixtures are 2-fold. First, the complementary "inverted cone" shape of the conjugate helps to accommodate the "cone-shaped" lipids, DOPE and cholesterol, in the bilayer phase. Second, the steric hindrance caused by the PEG group inhibits close apposition of bilayers, which is a prerequisite for the bilayer to HII phase transition.
Plasma-stable liposomes (100 nm) were prepared from dioleoylphosphatidylethanolamine (DOPE) and 3-6 mol% of a new disulfide-linked poly(ethylene glycol)-phospholipid conjugate (mPEG-DTP-DSPE). In contrast to similar preparations containing non-cleavable PEG-phospholipid conjugate, thiolytic cleavage of the grafted polymer chains facilitated rapid and complete release of the liposome contents. Furthermore, the detachment of PEG from DOPE liposomes resulted in liposomal fusion. Finally, while formulation of pH-sensitive DOPE/cholesterol hemisuccinate liposomes with mPEG-DTP-DSPE abolished the pH sensitivity, cleavage of the PEG chains completely restored this property. These are the first examples of new useful properties of liposomes grafted with cleavable polymer.
This laboratory has previously shown (Anderson, V.C. and Thompson, D.H. (1992) Biochim. Biophys. Acta 1109, 33-42; Thompson, D.H., Gerasimov, O.V., Wheeler, J.J., Rui, Y. and Anderson, V.C. (1996) Biochim. Biophys. Acta 1279, 25-34), that plasmenylcholine (1-alk-1'-enyl-2-palmitoyl-sn-glycero-3-phosphocholine; PlsPamCho) liposomes release hydrophilic contents upon photooxidation or acid-catalyzed hydrolysis. We now report the kinetics and chemical mechanism of the acid-catalyzed reaction and its effect on calcein leakage rates. Hydrolysis of the plasmenylcholine vinyl ether linkage generates fatty aldehydes and 1-hydroxy-2-palmitoyl-sn-glycero-3-phosphocholine (lysolipid); HPLC and 1H-NMR experiments establish that the former is readily air-oxidized to fatty acids, while the latter undergoes rapid acid-catalyzed rearrangement to 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine. Lysolipid formation obeys first order kinetics, yielding observed pseudo-first order rate constants that are pH-dependent. Bimolecular hydrolysis rate constants, k(bi), have also been determined. Calcein release rates from plasmenylcholine liposomes are strongly dependent on both the dihydrocholesterol (DHC) content and the extent of PlsPamCho hydrolysis within the bilayer. DHC-free plasmenylcholine liposomes (38 degrees C, pH 2.5) require < 5% PlsPamCho hydrolysis to effect > 50% calcein release within 10 min. The presence of > or = 25 mol% DHC, however, greatly reduces the observed calcein release rate; nearly 30% PlsPamCho hydrolysis is required to effect 50% calcein release over a 70-min period in 6:4 PlsPamCho/DHC liposomes. Bacteriochlorophyll a-sensitized photooxidation of plasmenylcholine liposomes also produces fatty aldehyde and another intermediate, tentatively described as 1-formyl-2-palmitoyl-sn-glycero-3-phosphocholine, that hydrolyzes to form the 1-hydroxy lysolipid. These results have important implications for the quantitative description of lysolipid effects on membrane permeability and on the design of triggerable liposomes for drug delivery.
The addition of polyethylene glycol (PEG), of various molecular weights, to solutions bathing yeast hexokinase increases the affinity of the enzyme for its substrate glucose. The results can be interpreted on the basis that PEG acts directly on the protein or indirectly through water activity. The nature of the effects suggests to us that PEG's action is indirect. Interpretation of the results as an osmotic effect yields a decrease in the number of water molecules, delta Nw, associated with the glucose binding reaction. delta Nw is the difference in the number of PEG-inaccessible water molecules between the glucose-bound and glucose-free conformations of hexokinase. At low PEG concentrations, delta Nw increases from 50 to 326 with increasing MW of the PEG from 300 to 1000, and then remains constant for MW-PEG up to 10,000. This suggests that up to MW 1000, solutes of increasing size are excluded from ever larger aqueous compartments around the protein. Three hundred and twenty-six waters is larger than is estimated from modeling solvent volumes around the crystal structures of the two hexokinase conformations. For PEGs of MW > 1000, delta Nw falls from 326 to about 25 waters with increasing PEG concentration, i.e., PEG alone appears to "dehydrate" the unbound conformation of hexokinase in solution. Remarkably, the osmotic work of this dehydration would be on the order of only one k T per hexokinase molecule. We conclude that under thermal fluctuations, hexokinase in solution has a conformational flexibility that explores a wide range of hydration states not seen in the crystal structure.
The structure of polymer-decorated phospholipid monolayers at the solid-solution interface was investigated using neutron reflectometry. The monolayers were composed of distearoylphosphatidylethanolamine (DSPE) matrixed with varying amounts of DSPE-PEG (DSPE with polyethylene glycol covalently grafted to its headgroup). Mixed lipid monolayers were Langmuir-Blodgett deposited onto hydrophobic quartz or silicon substrates, previously hydrophobized by chemically grafting a robust monolayer of octadecyltrichlorosilane (OTS). We show that this method results in homogeneous and continuous phospholipid monolayers on the silanated substrates and determine that the grafted PEG chains extend away from the monolayers into the solvent phase as a function of their density, as expected from scaling theories. In addition, ligands were coupled to the end of the PEG chains and selective binding was demonstrated using fluorescence microscopy. Our results demonstrate that these constructs are ideal for further characterization and studies with well-defined monomolecular films.
The low pH environments characteristic of endosomal compartments and ischemic tissues provide an intrinsic pathway for triggering site-specific contents release from appropriately designed delivery vehicles. Accordingly, research in this group has focused on the design, synthesis and application of novel acid-sensitive lipids that will undergo facile lamellar (L alpha) to hexagonal (HII) phase transitions within these acidic sites. Previously, it has been demonstrated that plasmenylcholine-type lipids have excellent acid hydrolysis and contents release kinetics (Gerasimov et al., Biochim. Biophys. Acta. 1324 (1997) 200-214; Rui et al., J. Am. Chem. Soc. 120 (1998) 11213-11218). This paper describes the synthesis of three new acid sensitive lipids, based on a chiral 1,2-di-O-(1Z',9Z'-octadecadienyl)-sn-glycerol (6) platform, displaying phosphocholine (7), poly(ethyleneoxide) (8), and O-carbamoyl-N-diethylen-etriamine (10) headgroups. Intermediate 6 was obtained in 28% overall yield via a six step synthesis from (S)-(+)-2,2-dimethyl-1,2-dioxolane-4-methanol. Subsequent conversion to the final products was acheived in moderate (7 and 10) to excellent yields (8).
Programmable fusogenic vesicles (PFVs) are lipid-based drug-delivery systems that exhibit time-dependent destabilization. The rate at which this destabilization occurs is determined by the exchange rate of a bilayer-stabilizing component, polyethylene glycol-phosphatidylethanolamine (PEG-PE) from the vesicle surface. This exchange rate is controlled, in turn, by the acyl chain composition of the PEG-PE. We describe in vitro and in vivo studies using PFVs as delivery vehicles for the anticancer drug mitoxantrone. We demonstrate that the PEG-PE acyl composition determined the rate at which PFVs are eliminated from plasma after intravenous administration, and the rate of mitoxantrone leakage from PFV. The nature of the PEG-PE component also determined the antitumor efficacy of mitoxantrone-loaded PFV in murine and human in murine and human xenograft tumor models. Increased circulation time and improved activity were obtained for PFV containing PEG-PE with an 18-carbon acyl chain length, as a result of slower vesicle destabilization.
After a roller coaster ride of successes and failures, new discovery technologies
and advances in manufacturing promise a brighter future for peptides in human
therapy.
A new strategy for the reversible attachment of methoxypoly(ethylene glycol) (mPEG) to an amino-containing substrate is described. The strategy is based on formation of a benzyl carbamate linkage substituted with a disulfide in the para or ortho position. While being stable under nonreducing conditions, the dithiobenzyl (DTB) urethane linkage is susceptible to cleavage by mild thiolysis with cysteine resulting in release of the parent amino component of the conjugate in its original form. The method is exemplified by preparation of mPEG-DTB-alcohol, its activation and attachment to distearoylphosphatidylethanolamine (DSPE). The resulting lipopolymer incorporates into liposomes, which are capable of losing their polymer coating under conditions approximating those existing in vivo. Implications for drug delivery are briefly discussed.
A series of novel cationic detergents that contain cleavable hydrophilic isothiuronium headgroups was synthesized, and their utility in controlled assembly of plasmid DNA into small stable particles with high DNA concentration investigated. The detergents have alkyl chains of C(8)-C(12) and contain hydrophilic isothiuronium headgroups that give relatively high critical micelle concentration (CMC) to the detergents (>10 mM). The isothiuronium group masks a sulfhydryl group on the detergent and can be cleaved in a controlled manner under basic conditions to generate a reactive thiol group. The thiol group can undergo a further reaction after the detergents have accumulated on a DNA template to form a disulfide-linked lipid containing two alkyl chains. The pH-dependent kinetics of cleavage of the isothiuronium group, the CMC of the surfactants, the formation of the complexes, and the transfection efficiency of the DNA complexes have been investigated. Using the C(12) detergent, a approximately 6 kilo-basepair plasmid DNA was compacted into a small particle with an average diameter of around 40 nm with a approximately -13 mV zeta-potential at high DNA concentration (up to 0.3 mg/mL). The compounds were well tolerated in cell culture and showed no cytotoxicity under their CMCs. Under appropriate conditions, the small particle retained transfection activity.
Poly(ethylene glycol) (PEG)-stabilized liposomes were recently shown to exhibit differences in cell uptake that were linked to the liposome charge. To determine the differences and similarities between charged and uncharged PEG-decorated liposomes, we directly measured the forces between two supported, neutral bilayers with terminally grafted PEG chains. The measurements were performed with the surface force apparatus. The force profiles were similar to those measured with negatively charged PEG conjugates of 1, 2-distearoyl-sn-glycero-3-phosphatidyl ethanolamine (DSPE), except that they lacked the longer ranged electrostatic repulsion observed with the charged compound. Theories for simple polymers describe the forces between end-grafted polymer chains on neutral bilayers. The force measurements were complemented by surface plasmon resonance studies of protein adsorption onto these layers. The lack of electrostatic forces reduced the adsorption of positively charged proteins and enhanced the adsorption of negatively charged ones. The absence of charge also allowed us to determine how membrane charge and the polymer grafting density independently affect protein adsorption on the coated membranes. Such studies suggest the physical basis of the different interactions of charged and uncharged liposomes with proteins and cells.
Folate-diplasmenylcholine (1,2-di-O-(Z-l′-hexadecenyl)-sn-glycero-3-phosphocholine; DPPsC) liposomes have been shown to greatly enhance the potency of water-soluble anti-tumor agents via a selective folate-mediated uptake and acid-catalyzed endosomal escape mechanism (Rui et al. J. Am. Chem. Soc., 1998; 120:11213-18). This study describes an adaptation of this strategy for the delivery of chloroaluminum phthalocyanine tetrasulfonate (AIPcS44-), a water-soluble sensitizer used in photodynamic therapy, in a binary targeting scheme designed to enhance both its tumor selectivity and phototoxicity. AIPcS44-/DPPIsC:folate liposomes (9.8 μM bulk concentration, 2.5 mM intraliposomal concentration) were substantially more phototoxic to folate-deficient KB cells than 12.5 μM free AIPcS44- after a 30 min irradiation (630-910 nm). Considerable differences in phototoxicity were observed, however, between the commercially-available AIPcS44- and an HPLC purified sample of AIPcS44- due to an increased tendency for the latter to aggregate. Experiments with AIPcS44-/DPPC:folate and folate-free Al PcSa4-/DPPIsC liposomes (acid-insensitive and non-targeted controls, respectively) showed significantly reduced phototoicities under the same illumination conditions. Our results imply that higher concentrations of water-soluble sensitizers can be delivered to target cells using the folate receptor-mediated pathway, which can change both the biodistribution and intracellular localization of the sensitizer when acid-labile DPPIsC liposomes are used as the delivery vehicle. Potential advantages of this approach include the use of lower bulk AIPcS44- concentrations, rapid plasma clearance of free Alp and better phototoxic responses, due to higher intracellular AIPcS44- concentrations combined with reduced collateral photodamage arising from misguided sensitizer accumulation, thereby enhancing the selective phototoxicity of PDT treatments.
In order to explore the use of exchangeable poly(ethylene glycol) (PEG)-modified diacylphosphatidylethanolamines (PE) to temporarily shield binding ligands attached to the surface of liposomes, a model reaction based on inhibition and subsequent recovery of biotinylated liposome binding to streptavidin immobilized on superparamagnetic iron oxide particles (SA magnetic particles) was developed. PEG-lipid incorporation into biotinylated liposomes decreased liposome binding to SA magnetic particles in a non-linear fashion, where as little as 0.1 mol% PEG-PE resulted in a 20% decrease in binding. Using an assay based on inhibition of binding, PEG(2000)-PE transfer from donor liposomes to biotinylated acceptor liposomes could be measured. The influence of temperature and acyl chain composition on the transfer of PEG-diacyl PEs from donor liposomes to acceptor liposomes, consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine, cholesterol and N-((6-biotinoyl)amino)hexanoyl)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (54.9:45:0.1 mole ratio), was measured. Donor liposomes were prepared using 1,2-distearoyl-sn-glycero-3-phosphocholine (50 mol%), cholesterol (45 mol%) and 5 mol% of either PEG-derivatized 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG(2000)), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG(2000)), or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG(2000)). Transfer of DSPE-PEG(2000) to the donor liposomes was not detected under the conditions employed. In contrast, DMPE-PEG(2000) was transferred efficiently even at 4 degrees C. Using an acceptor to donor liposome ratio of 1:4, the time required for DMPE-PEG(2000) to become evenly distributed between the two liposome populations (T(EQ)) at 4 degrees C and 37 degrees C was approx. 2 and <0.5 h, respectively. An increase in acyl chain length from C14:0 to C16:0 of the PEG-lipid resulted in a significant reduction in the rate of transfer as measured by this assay. The transfer of PEG-lipid out of biotinylated liposomes was also studied in mice following intravenous administration. The relative rates of transfer for the various PEG-lipids were found to be comparable under in vivo and in vitro conditions. These results suggest that it is possible to design targeted liposomes with the targeting ligand protected while in the circulation through the use of PEG-lipids that are selected on the basis of exchange characteristics which result in exposure of the shielded ligand following localization within a target tissue.
Extraordinary advances in molecular medicine and biotechnology have led to the development of a vast number of anti-cancer therapeutic agents. To reach cancer cells in a tumor, a blood-borne therapeutic molecule, particle or cell must make its way into the blood vessels of the tumor and across the vessel wall into the interstitium and finally migrate through the interstitium. Unfortunately, tumors often develop in ways that hinder each of these steps. Our research goals are to analyze each of these steps experimentally and theoretically and then integrate the resulting information in a unified theoretical framework. This paradigm of analysis and synthesis has allowed us to obtain a better understanding of microcirculatory barriers in solid tumors and to develop novel strategies to exploit and/or to overcome these barriers for improved cancer detection and treatment.
We have generated anti-HER2 (ErbB2) immunoliposomes (ILs), consisting of long circulating liposomes linked to anti-HER2 monoclonal antibody (MAb) fragments, to provide targeted drug delivery to HER2-overexpressing cells. Immunoliposomes were constructed using a modular strategy in which components were optimized for internalization and intracellular drug delivery. Parameters included choice of antibody construct, antibody density, antibody conjugation procedure, and choice of liposome construct. Anti-HER2 immunoliposomes bound efficiently to and internalized in HER2-overexpressing cells in vitro as determined by fluorescence microscopy, electron microscopy, and quantitative analysis of fluorescent probe delivery. Delivery via ILs in HER2-overexpressing cells yielded drug uptake that was up to 700-fold greater than with non-targeted sterically stabilized liposomes. In vivo, anti-HER2 ILs showed extremely long circulation as stable constructs in normal adult rats after a single i.v. dose, with pharmacokinetics that were indistinguishable from sterically stabilized liposomes. Repeat administrations revealed no increase in clearance, further confirming that ILs retain the long circulation and non-immunogenicity of sterically stabilized liposomes. In five different HER2-overexpressing xenograft models, anti-HER2 ILs loaded with doxorubicin (dox) showed potent anticancer activity, including tumor inhibition, regressions, and cures (pathologic complete responses). ILs were significantly superior vs. all other treatment conditions tested: free dox, liposomal dox, free MAb (trastuzumab), and combinations of dox+MAb or liposomal dox+MAb. For example, ILs produced significantly superior antitumor effects vs. non-targeted liposomes (P values from <0.0001 to 0.04 in eight separate experiments). In a non-HER2-overexpressing xenograft model (MCF7), ILs and non-targeted liposomal dox produced equivalent antitumor effects. Detailed studies of tumor localization indicated a novel mechanism of drug delivery for anti-HER2 ILs. Immunotargeting did not increase tumor tissue levels of ILs vs. liposomes, as both achieved very high tumor localization (7.0-8.5% of injected dose/g tissue) in xenograft tumors. However, histologic studies using colloidal-gold labeled ILs demonstrated efficient intracellular delivery in tumor cells, while non-targeted liposomes accumulated within stroma, either extracellularly or within macrophages. In the MCF7 xenograft model lacking HER2-overexpression, no difference in tumor cell uptake was seen, with both ILs and non-targeted liposomes accumulating within stroma. Thus, anti-HER2 ILs, but not non-targeted liposomes, achieve intracellular drug delivery via receptor-mediated endocytosis, and this mechanism is associated with superior antitumor activity. Based on these results, anti-HER2 immunoliposomes have been developed toward clinical trials. Reengineering of construct design for clinical use has been achieved, including: new anti-HER2 scFv F5 generated by screening of a phage antibody library for internalizing anti-HER2 phage antibodies; modifications of the scFv expression construct to support large scale production and clinical use; and development of methods for large-scale conjugation of antibody fragments with liposomes. We developed a scalable two-step protocol for linkage of scFv to preformed and drug-loaded liposomes. Our final, optimized anti-HER2 ILs-dox construct consists of F5 conjugated to derivatized PEG-PE linker and incorporated into commercially available liposomal doxorubicin (Doxil). Finally, further studies of the mechanism of action of anti-HER2 ILs-dox suggest that this strategy may provide optimal delivery of anthracycline-based chemotherapy to HER2-overexpressing cancer cells in the clinic, while circumventing the cardiotoxicity associated with trastuzumab+anthracycline. We conclude that anti-HER2 immunoliposomes represent a promising technology for tumor-targeted drug delivery, and that this strategy may also be applicable to other receptor targets and/or using other delivered agents.
Dioleoylphosphatidylethanolamine (DOPE)-containing liposomes that demonstrated pH-dependent release of their contents were stabilized in the bilayer form through the addition of a cleavable lipid derivative of polyethylene glycol (PEG) in which the PEG was attached to a lipid anchor via a disulfide linkage (mPEG-S-S-DSPE). Liposomes stabilized with either a non-cleavable PEG (mPEG-DSPE) or mPEG-S-S-DSPE retained an encapsulated dye at pH 5.5, but treatment at pH 5.5 of liposomes stabilized with mPEG-S-S-DSPE with either dithiothreitol or cell-free extracts caused contents release due to cleavage of the PEG chains and concomitant destabilization of the DOPE liposomes. While formulations loaded with doxorubicin (DXR) were stable in culture media, DXR was rapidly released in human plasma. pH-Sensitive liposomes, targeted to the CD19 epitope on B-lymphoma cells, showed enhanced DXR delivery into the nuclei of the target cells and increased cytotoxicity compared to non-pH-sensitive liposomes. Pharmacokinetic studies suggested that mPEG-S-S-DSPE was rapidly cleaved in circulation. In a murine model of B-cell lymphoma, the therapeutic efficacy of an anti-CD19-targeted pH-sensitive formulation was superior to that of a stable long-circulating formulation of targeted liposomes despite the more rapid drug release and clearance of the pH-sensitive formulation. These results suggest that targeted pH-sensitive formulations of drugs may be able to increase the therapeutic efficacy of entrapped drugs.
Temperature-sensitive liposomes are considered to be a promising tool to achieve site-specific delivery of drugs. These liposomes have been prepared using lipids whose membranes undergo a gel-to-liquid crystalline phase transition a few degrees above physiological temperature. However, recently, temperature-sensitization of liposomes has been attempted using thermosensitive polymers. So far, functional liposomes whose contents release behavior, surface properties, and affinity to cell surface can be controlled in a temperature-dependent manner, have been developed according to this strategy. The design and function of these thermosensitive polymer-modified liposomes have been outlined in this review.
Over the past several years, photodynamic therapy (PDT) has been approved for the treatment of various cancers. Additional applications of photochemical processes for triggering site-specific drug delivery are in early stages of development at this time. This review focuses on the literature appearing between January 1996-June 2001 that describe new and ongoing studies of phototriggering mechanisms that may ultimately find utility in drug delivery applications. (C) 2001 Published by Elsevier Science B.V.
Poly (ethylene glycol) (PEG) in the external environment of membrane vesicles creates osmotic imbalance that leads to mechanical stress in membranes and may induce local membrane curvature. To determine the relative importance of membrane stress and curvature in promoting fusion, we monitored contents mixing (CM) and lipid mixing (LM) between different sized vesicles under a variety of osmotic conditions. CM between highly curved vesicles (SUV, 26 nm diameter) was up to 10 times greater than between less curved vesicles (LUV, 120 nm diameter) after 5 min incubation at a low PEG concentration (<10 wt%), whereas LM was only approximately 30% higher. Cryo-electron microscopy showed that PEG at 10 wt% did not create high curvature contacts between membranes in LUV aggregates. A negative osmotic gradient (-300 mOs/kg, hypotonic inside) increased CM two- to threefold for both types of vesicles, but did not affect LM. A positive gradient (+220 mOs/kg, hypertonic inside) nearly eliminated CM and had no effect on LM. Hexadecane added to vesicles had no effect on LM but enhanced CM and reduced the inhibitory effect on CM of a positive osmotic gradient, but had little influence on results obtained under a negative osmotic gradient. We conclude that the ability of closely juxtaposed bilayers to form an initial intermediate ("stalk") as soon as they come into close contact was not influenced by osmotic stress or membrane curvature, although pore formation was critically dependent on these stresses. The results also suggest that hexadecane affects the same part of the fusion process as osmotic stress. We interpret this result to suggest that both a negative osmotic gradient and hexadecane reduce the unfavorable free energy of hydrophobic interstices associated with the intermediates of the fusion process.
Anti-HER2 immunoliposomes combine the tumor-targeting of certain anti-HER2 monoclonal antibodies (MAbs) with the pharmacokinetic and drug delivery capabilities of sterically stabilized liposomes. We previously showed that anti-HER2 immunoliposomes bind efficiently to and internalize in HER2-overexpressing cells in vitro, resulting in intracellular drug delivery.
Here we describe the pharmacokinetics and therapeutic efficacy of anti-HER2 immunoliposomes containing doxorubicin (dox) in a series of animal models.
Immunoliposomes displayed long circulation that was identical to that of sterically stabilized liposomes in single- and multiple-dose studies in normal rats. Anti-HER2 immunoliposome-dox produced marked therapeutic results in four different HER2-overexpressing tumor xenograft models, including growth inhibition, regression, and cures. These results demonstrated that encapsulation of dox in anti-HER2 immunoliposomes greatly increased its therapeutic index, both by increasing antitumor efficacy and by reducing systemic toxicity. Immunoliposome-dox was significantly superior to all other treatment conditions tested, including free dox, liposomal dox, and anti-HER2 MAb (trastuzumab). When compared with liposomal dox in eight separate therapy studies in HER2-overexpressing models, immunoliposome delivery produced significantly superior antitumor efficacy in each study (P < 0.0001 to 0.04). Anti-HER2 immunoliposome-dox containing either recombinant human MAb HER2-Fab' or scFv C6.5 yielded comparable therapeutic efficacy. Cure rates for immunoliposome-dox reached 50% (11 of 21) with optimized immunoliposomes and Matrigel-free tumors and overall was 16% (18 of 115) versus no cures (0 of 124) with free dox or liposomal dox. Finally, anti-HER2 immunoliposome-dox was also superior to combinations consisting of free MAb plus free dox or free MAb plus liposomal dox.
Anti-HER2 immunoliposomes produced enhanced antitumor efficacy via targeted delivery.
We report the triggered release of Ca2+ from liposomal compartments to induce rapid gelation of protein-based hydrogels. Phototriggerable liposomes were designed by entrapping CaCl(2) within liposomes composed of 38:57:5 diplasmenylcholine (DPPlsC):disteroylphosphatidylcholine (DSPC):bacteriochlorophyll (Bchl). These liposomes release >80% of their entrapped Ca2+ within 15 min when irradiated at 800 nm (800 mW/cm2). A precursor solution, containing liposomes suspended in aqueous human fibrinogen and transglutaminase (TGase), remained fluid for several hours in the dark, but gelled rapidly when exposed to 800 nm excitation, as a result of photosensitized Ca2+ release and TG-induced fibrinogen cross-linking. TGase and hrFXIII activities, determined using a fluorimetric dansylcadaverine assay, were found to depend strongly on irradiation time and reaction temperature. SDS-PAGE of the photolyzed reaction mixture revealed that gelation arises from enzyme-catalyzed cross-linking of predominately the alpha and gamma chains of fibrinogen. This approach to the phototriggered formation of protein hydrogels creates new opportunities for biomaterials applications in drug delivery, tissue engineering, and wound healing.
The size of condensed DNA particles is a key determinant for both diffusion to target cells in vivo and intracellular trafficking. The smallest complexes are obtained when each DNA molecule collapses individually. This was achieved using a designed cationic thiol-detergent, tetradecyl-cysteinyl-ornithine (C(14)COrn). The resulting particles were subsequently stabilized by air-induced dimerization of the detergent into a disulfide lipid on the DNA template. Particles are anionic (zeta potential = -45 mV), and their size (30 nm) corresponds to the volume of a single plasmid DNA molecule. The electrophoretic mobility of the condensed DNA, though quasi-neutralized, was found higher than that of the extended DNA. Moreover, the dimerized (C(14)COrn)(2) lipid was found to be an efficient transfection reagent for various cell lines. In an attempt to achieve extended circulation times and to target tumors by systemic delivery, we have coated the particles with PEG-folate residues. Plasmid DNA was condensed into monomolecular particles as described above and coated by simple mixing with DPPE-PEG-folate. Physicochemical measurements showed particles coated with 2% of DPPE-PEG(3400)-folate remain monomolecular and are stable in the cell-culture medium. Caveolae-mediated cell entry was demonstrated by ligand-dependence, by competition with excess folic acid as well as by confocal microscopy.
From glycosylated cell surfaces to sterically stabilized liposomes, polymers attached to membranes attract biological and therapeutic interest. Can the scaling laws of polymer "brushes" describe the physical properties of these coats? We delineate conditions where the Alexander-de Gennes theory of polymer brushes successfully fits the intermembrane distance versus applied osmotic stress data of Kenworthy et al. for poly(ethylene glycol)-grafted multilamellar liposomes. We establish that the polymer density and size in the brush must be high enough that, in a bulk solution of equivalent monomer density, the polymer osmotic pressure is independent of polymer molecular weight (the des Cloizeaux semidilute regime of bulk polymer solutions). The condition that attached polymers behave as semidilute bulk solutions offers a rigorous criterion for brush scaling-law behavior. There is a deep connection between the behaviors of semidilute polymer solutions in bulk and polymers grafted to a surface at a density such that neighbors pack to form a uniform brush. In this regime, two-parameter unconstrained fits of the Alexander-de Gennes brush scaling laws to the Kenworthy et al. data yield effective monomer lengths of 3.3-3.6 A, which agree with structural predictions. The fitted distances between grafting sites are larger than expected from the nominal mole fraction of poly(ethylene glycol)-lipids; the chains apparently saturate the surface. Osmotic stress measurements can be used to estimate the actual densities of membrane-grafted polymers.
The mechanism of pH-triggered destabilization of liposomes composed of a polyethyleneglycol-orthoester-distearoylglycerol lipid (POD) and phosphatidyl ethanolamine (PE) has been studied using an ANTS/DPX leakage and a lipid-mixing assay. We developed a kinetic model that relates POD hydrolysis to liposome collapse. This minimum-surface-shielding model describes the kinetics of the pH-triggered release of POD/PE liposomes. In the model, when acid-catalyzed hydrolysis lowers the mole percentage of POD on the liposome surface to a critical level, intervesicular lipid mixing is initiated, resulting in a burst of contents release. Two phases of content leakage are observed: a lag phase and a burst phase. During the lag phase, less than 20% of liposomal contents are released and the leakage begins to accelerate when approaching to the transition point. During the burst phase, the leakage rate is dependent on interbilayer contact. The burst phase occurs when the surface density of the PEG lipid is 2.3 +/- 0.6 mol%, regardless of the pH. Vesicles containing 4 mol% of a pH-insensitive PEG-lipid conjugate and 10% POD did not leak contents or collapse at any pH. These data are consistent with the stalk theory to describe the lamellar-to-inverted hexagonal phase transition and set a lower bound of approximately 16 PE lipids on the external monolayer as the contact site required for lipid mixing between two bilayers.
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.
A series of 1-(acyloxyalkyl)imidazoles (AAI) were synthesized by nucleophilic substitution of chloroalkyl esters of fatty acids with imidazole. The former was prepared from fatty acid chloride and an aldehyde. When incorporated into liposomes, these lipids show an apparent pK(a) value ranging from 5.12 for 1-(palmitoyloxymethyl)imidazole (PMI) to 5.29 for 1-[(alpha-myristoyloxy)ethyl]imidazole (alpha-MEI) as determined by a fluorescence assay. When the imidazole moiety was protonated, the lipids were surface-active, as demonstrated by hemolytic activity towards red blood cells. As expected, AAI were hydrolyzed in serum as well as in cell homogenate. They were significantly less toxic than biochemically stable N-dodecylimidazole (NDI) towards Chinese hamster ovary (CHO) and RAW 264.7 (RAW) cells as determined by MTT assay. When fed to RAW cells, fluorescein-labeled oligonucleotides encapsulated in liposomes containing 20 mol% 1-(stearoyloxymethyl)imidazole (SMI) resulted in punctate as well as partially diffuse fluorescence. In a functional assay involving down-regulation of luciferase in CV-1 cells, neutral liposomes containing imidazole lipids showed suboptimal delivery of antisense phosphorothioate oligomers. Taken together, the results suggest that AAI are of potential use in developing nontoxic, pH-sensitive liposomes. However, these liposomal formulations need to be optimized to achieve higher concentrations of pH-sensitive detergents within the endosome to facilitate efficient cytosolic release of liposome-entrapped contents.