Circulation lifetime is a crucial parameter for a successful therapy with nanoparticles. Reduction and alteration of opsonization profiles by surface modification of nanoparticles is the main ...strategy to achieve this objective. In clinical settings, PEGylation is the most relevant strategy to enhance blood circulation, yet it has drawbacks, including hypersensitivity reactions in some patients treated with PEGylated nanoparticles, which fuel the search for alternative strategies. In this work, lipopolysarcosine derivatives (BA‐pSar, bisalkyl polysarcosine) with precise chain lengths and low polydispersity indices are synthesized, characterized, and incorporated into the bilayer of preformed liposomes via a post insertion technique. Successful incorporation of BA‐pSar can be realized in a clinically relevant liposomal formulation. Furthermore, BA‐pSar provides excellent surface charge shielding potential for charged liposomes and renders their surface neutral. Pharmacokinetic investigations in a zebrafish model show enhanced circulation properties and reduction in macrophage recognition, matching the behavior of PEGylated liposomes. Moreover, complement activation, which is a key factor in hypersensitivity reactions caused by PEGylated liposomes, can be reduced by modifying the surface of liposomes with an acetylated BA‐pSar derivative. Hence, this study presents an alternative surface modification strategy with similar benefits as the established PEGylation of nanoparticles, but with the potential of reducing its drawbacks.
Amphiphilic pSar derivatives are synthesized and incorporated into the bilayer of liposomes to impart stealth‐like properties. Formulations show similar characteristics as PEGylated liposomes regarding their surface charge shielding potential and reduced clearance by macrophages in zebrafish. Liposomes modified with an acetylated pSar derivative show lower complement activation levels compared to those observed for PEGylated liposomes.
Perfluorocarbon nanoemulsions (PFC-NE) are disperse systems consisting of nanoscale liquid perfluorocarbon droplets stabilized by an emulsifier, usually phospholipids. Perfluorocarbons are chemically ...inert and non-toxic substances that are exhaled after in vivo administration. The manufacture of PFC-NE can be done in large scales by means of high pressure homogenization or microfluidization. Originally investigated as oxygen carriers for cases of severe blood loss, their application nowadays is more focused on using them as marker agents in 19F Magnetic Resonance Imaging (19F MRI). 19F is scarce in organisms and thus PFC-NE are a promising tool for highly specific and non-invasive imaging of inflammation via 19F MRI. Neutrophils, monocytes and macrophages phagocytize PFC-NE and subsequently migrate to inflamed tissues. This technique has proven feasibility in numerous disease models in mice, rabbits and mini pigs. The translation to clinical trials in human needs the development of a stable nanoemulsion whose droplet size is well characterized over a long storage time. Usually dynamic light scattering (DLS) is applied as the standard method for determining particle sizes in the nanometer range. Our study uses a second method, analysis of transmission electron microscopy images of cryo-fixed samples (Cryo-TEM), to evaluate stability of PFC-NE in comparison to DLS. Four nanoemulsions of different composition are observed for one year. The results indicate that DLS alone cannot reveal the changes in particle size, but can even mislead to a positive estimation of stability. The combination with Cryo-TEM images gives more insight in the particulate evolution, both techniques supporting one another. The study is one further step in the development of analytical tools for the evaluation of a clinically applicable perfluorooctylbromide nanoemulsion.
Nanoscale biological materials formed by the assembly of defined block-domain proteins control the formation of cellular compartments such as organelles. Here, we introduce an approach to ...intentionally 'program' the de novo synthesis and self-assembly of genetically encoded amphiphilic proteins to form cellular compartments, or organelles, in Escherichia coli. These proteins serve as building blocks for the formation of artificial compartments in vivo in a similar way to lipid-based organelles. We investigated the formation of these organelles using epifluorescence microscopy, total internal reflection fluorescence microscopy and transmission electron microscopy. The in vivo modification of these protein-based de novo organelles, by means of site-specific incorporation of unnatural amino acids, allows the introduction of artificial chemical functionalities. Co-localization of membrane proteins results in the formation of functionalized artificial organelles combining artificial and natural cellular function. Adding these protein structures to the cellular machinery may have consequences in nanobiotechnology, synthetic biology and materials science, including the constitution of artificial cells and bio-based metamaterials.
Secretion of extracellular vesicles (EVs) is a ubiquitous mechanism of intercellular communication based on the exchange of effector molecules, such as growth factors, cytokines, and nucleic acids. ...Recent studies identified tumor-derived EVs as central players in tumor progression and the establishment of the tumor microenvironment (TME). However, studies on EVs from classical Hodgkin lymphoma (cHL) are limited. The growth of malignant Hodgkin and Reed-Sternberg (HRS) cells depends on the TME, which is actively shaped by a complex interaction of HRS cells and stromal cells, such as fibroblasts and immune cells. HRS cells secrete cytokines and angiogenic factors thus recruiting and inducing the proliferation of surrounding cells to finally deploy an immunosuppressive TME. In this study, we aimed to investigate the role of tumor cell-derived EVs within this complex scenario. We observed that EVs collected from Hodgkin lymphoma (HL) cells were internalized by fibroblasts and triggered their migration capacity. EV-treated fibroblasts were characterized by an inflammatory phenotype and an upregulation of alpha-smooth muscle actin (α-SMA), a marker of cancer-associated fibroblasts. Analysis of the secretome of EV-treated fibroblast revealed an enhanced release of pro-inflammatory cytokines (e.g., IL-1α, IL-6, and TNF-α), growth factors (G-CSF and GM-CSF), and pro-angiogenic factors such as VEGF. These soluble factors are known to promote HL progression. In line, ingenuity pathway analysis identified inflammatory pathways, including TNF-α/NF-κB-signaling, as key factors directing the EV-dependent phenotype changes in fibroblasts. Confirming the
data, we demonstrated that EVs promote α-SMA expression in fibroblasts and the expression of proangiogenic factors using a xenograft HL model. Collectively, we demonstrate that HL EVs alter the phenotype of fibroblasts to support tumor growth, and thus shed light on the role of EVs for the establishment of the tumor-promoting TME in HL.
Liposomes act as efficient drug carriers. Recently, epirubicin (EPI) formulation was developed using a novel EDTA ion gradient method for drug encapsulation. This formulation displayed very good ...stability and drug retention in vitro in a two-year long-term stability experiment. The cryo-TEM images show drug precipitate structures different than ones formed with ammonium sulfate method, which is usually used to encapsulate anthracyclines. Its pharmacokinetic properties and its efficacy in the human breast MDA-MB-231 cancer xenograft model were also determined. The liposomal EPI formulation is eliminated slowly with an AUC of 7.6487, while the free drug has an AUC of only 0.0097. The formulation also had a much higher overall antitumor efficacy than the free drug.
The aim of this study was to investigate the fusogenic properties of poly(ethylene glycol) (PEG)ylated dioleoylphosphatidylethanolamine/cholesteryl hemisuccinate (DOPE/CHEMS) liposomes. These ...pH-sensitive liposomes were prepared by incorporating two different PEG lipids: Distearoylphosphatidylethanolamine (DSPE)-PEG2000 was mixed with the liposomal lipids using the conventional method, whereas sterol-PEG1100 was inserted into the outer monolayer of preformed vesicles. Both types of PEGylated liposomes were characterized and compared for their entrapment efficiency, zeta potential and size, and were tested in vitro for pH sensitivity by means of proton-induced leakage and membrane fusion activity. To mimic the routes of intracellular delivery, fusion between pH-sensitive liposomes and liposomes designed to simulate the endosomal membrane was studied. Our investigations confirmed that DOPE/CHEMS liposomes were capable of rapidly releasing calcein and of fusing upon acidification. However, after incorporation of DSPE-PEG2000 or sterol-PEG1100 into the membrane, pH sensitivity was significantly reduced; as the mol ratio of PEG-lipid was increased, the ability to fuse was decreased. Comparison between two different PEGylated pH-sensitive liposomes showed that only vesicles containing 0.6 mol% sterol-PEG1100 in the outer monolayer were still capable of fusing with the endosome-like liposomes and showing leakage of calcein at pH 5.5.
The emergence of antibiotic-resistant bacteria presents a severe challenge to medicine and public health. While bacteriophage therapy is a promising alternative to traditional antibiotics, the ...general inability of bacteriophages to penetrate eukaryotic cells limits their use against resistant bacteria, causing intracellular diseases like tuberculosis. Bacterial vectors show some promise in carrying therapeutic bacteriophages into cells, but also bring a number of risks like an overload of bacterial antigens or the acquisition of virulence genes from the pathogen.
As a first step in the development of a non-bacterial vector for bacteriophage delivery into pathogen-infected cells, we attempted to encapsulate bacteriophages into liposomes.
Here we report effective encapsulation of the model bacteriophage λeyfp and the mycobacteriophage TM4 into giant liposomes. Furthermore, we show that liposome-associated bacteriophages are taken up into eukaryotic cells more efficiently than free bacteriophages.
These are important milestones in the development of an intracellular bacteriophage therapy that might be useful in the fight against multi-drug-resistant intracellular pathogens like Mycobacterium tuberculosis.
The delivery of nucleic acids such as DNA or siRNA still represents a major hurdle, especially with regard to possible therapeutic applications in vivo. Much attention has been focused on the ...development of non-viral gene delivery vectors, including liposomes or cationic polymers. Among them, polyethylenimines (PEIs) have been widely explored for the delivery of nucleic acids and show promising results. The combination of cationic polymers and liposomes (lipopolyplexes) for gene delivery may further improve their efficacy and biocompatibility, by combining the favourable properties of lipid systems (high stability, efficient cellular uptake, low cytotoxicity) and PEIs (nucleic acid condensation, facilitated endosomal release). In this study, we systematically analyse various conditions for the preparation of liposome–polyethylenimine-based lipopolyplexes with regard to biological activity (DNA transfection efficacy, siRNA knockdown efficacy) and physicochemical properties (size, zeta potential, stability). This includes the exploration of lipopolyplex compositions containing different liposomes and different relevant branched or linear low-molecular-weight PEIs. We establish optimal parameters for lipopolyplex generation, based on various PEIs, N/P ratios, lipids, lipid/PEI ratios and preparation conditions. Importantly, we also demonstrate that certain lipopolyplexes retain their biological activity and physicochemical integrity upon prolonged storage, even at 37°C and/or in the presence of serum, thus providing formulations with considerably higher stability as compared to polyplexes. In conclusion, we establish optimal liposome–polyethylenimine lipopolyplexes that allow storage under ambient conditions. This is the basis and an essential prerequisite for novel, promising and easy-to-handle formulations for possible therapeutic applications.
Physicochemical characterization of different doxorubicincontaining liposomes after ultrasound mediated drug release. Display omitted
► Mechanisms of ultrasound-mediated drug release from liposomes ...were investigated. ► The drug release mechanism was dependent on the liposome membrane composition. ► Liposomes based on the lipid DOPE were irreversible disrupted after US-release. ► Most of the HSPC-liposomes remained intact without entrapped drug after US-release. ► Distinctive release mechanism may explain differences in sonosensitive properties.
Ultrasound is investigated as a novel drug delivery tool within cancer therapy. Non-thermal ultrasound treatment of solid tumours post i.v.-injection of drug-carrying liposomes may induce local drug release from the carrier followed by enhanced intracellular drug uptake. Recently, ultrasound-mediated drug release of liposomes (sonosensitivity) was shown to strongly depend on liposome membrane composition. In the current study the ultrasound-mediated drug release mechanism of liposomes was investigated. The results showed that differences in ultrasound drug release kinetics obtained for different liposomal compositions were caused by distinctive release mechanisms of the carriers. Two types of liposomes composed of 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) and hydrogenated soy l-α-phosphatidylcholine (HSPC) as main lipids, respectively, were recently shown to vary in sonosensitivity. Here, these liposomes were analyzed prior to and after a given ultrasound-exposure for their mean size, size distribution and morphology. Cryo-transmission electron microscopy, dynamic light scattering and asymmetric flow field-flow fractionation in combination with multi-angle light scattering revealed a significant change in mean size, size distribution and morphology of DOPE-based liposomes after ultrasound, pointing to an irreversible disruption of the vesicles and concomitant drug release. In contrast, the HSPC-based liposomes remained unchanged in size and structure after ultrasound application, indicating pore-mediated release mechanisms. The results show that the release mechanisms and interactions between ultrasound and liposomes depend on the liposome membrane-composition, explaining their sonosensitive properties.
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