TAT-liposomes: a novel intracellular drug carrier

TAT peptide was attached to the surface of plain and PEGylated liposomes. These TAT peptide-modified liposomes have been shown to translocate into a variety of normal and cancer cells if a non-hindered interaction between the cell surface and liposome-attached TAT peptide was made possible. TAT peptide-liposomes translocated into cells remain intact within first few hours as proved by a co-localization of fluorescent markers entrapped inside liposomes and incorporated into the liposomal membrane. After 2 hours liposomes had slowly migrating towards cell nuclei. Liposomes had completely disintegrated with their inner marker released by approximately 9 hours. TAT peptide-liposomes were made slightly cationic by adding up to 10 mol %. of a cationic lipid (DOTAP). These slightly cationic liposomes were non-toxic towards cells, formed firm complexes with DNA (plasmid encoding for the formation of the Green Fluorescent Protein), and efficiently transfected a variety of cells. TAT peptide-liposomes can be considered as promising carriers for the non-endocytotic intracellular delivery of drugs and DNA.     

PEGylated liposomal doxorubicin targeted to α5β1-expressing MDA-MB-231 breast cancer cells

Targeting drugs selectively to cancer cells can potentially benefit cancer patients by avoiding side effects generally associated with several cancer therapies. One of the attractive approaches to direct the drug cargo to specific sites is to incorporate ligands at the surface of the delivery systems. Integrin α(5)β(1) is overexpressed in tumor vasculature and cancer cells, thus making it an attractive target for use in drug delivery. Our group has developed a fibronectin-mimetic peptide, PR_b, which has been shown to bind specifically to integrin α(5)β(1), thereby providing a tool to target α(5)β(1)-expressing cancer cells in vitro as well as in vivo. Our current work focuses on designing modified stealth liposomes (liposomes functionalized with polyethylene glycol, PEG) for combining the benefits associated with PEGylation, as well as imparting specific targeting properties to the liposomes. We have designed PEGylated liposomes that incorporate in their bilayer the fibronectin-mimetic peptide-amphiphile PR_b that can target several cancer cells that overexpress α(5)β(1), including the MDA-MB-231 breast cancer cells used in this study. We have encapsulated doxorubicin inside the liposomes to enhance its therapeutic potential via PEGylation as well as active targeting to the cancer cells. Our results show that PR_b-functionalized stealth liposomes were able to specifically bind to MDA-MB-231 cells, and the binding could be controlled by varying the peptide concentration. The intracellular trafficking of the doxorubicin liposomes was examined, and within minutes after delivery the majority of them were found to be in the early endosomes, whereas after a longer period of time they had accumulated in the late endosomes and lysosomes. The functionalized liposomes were found to be equally cytotoxic as the free doxorubicin, especially at higher doxorubicin concentrations, and provided higher cytotoxicity than the nontargeted and GRGDSP-functionalized stealth liposomes. Thus, the PR_b-functionalized PEGylated nanoparticles examined in this study offer a promising strategy to deliver their therapeutic payload directly to the breast cancer cells, in an efficient and specific manner.     

Clickable functionalization of liposomes with the gH625 peptide from Herpes simplex virus type I for intracellular drug delivery

Liposomes externally modified with the nineteen residues gH625 peptide, previously identified as a membrane‐perturbing domain in the gH glycoprotein of Herpes simplex virus type I, have been prepared in order to improve the intracellular uptake of an encapsulated drug. An easy and versatile synthetic strategy, based on click chemistry, has been used to bind, in a controlled way, several copies of the hydrophobic gH625 peptide on the external surface of 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPG)‐based liposomes. Electron paramagnetic resonance studies, on liposomes derivatized with gH625 peptides, which are modified with the 2,2,6,6‐tetramethylpiperidine‐1‐oxyl‐4‐amino‐4‐carboxylic acid (TOAC) spin label in several peptide positions, confirm the positioning of the coupled peptides on the liposome external surface, whereas dynamic light scattering measurements indicate an increase of the diameter of the liposomes of approximately 30 % after peptide introduction. Liposomes have been loaded with the cytotoxic drug doxorubicin and their ability to penetrate inside cells has been evaluated by confocal microscopy experiments. Results suggest that liposomes functionalized with gH625 may act as promising intracellular targeting carriers for efficient delivery of drugs, such as chemotherapeutic agents, into tumor cells.     

RNA Hairpin Folding in the Crowded Cell

Precise secondary and tertiary structure formation is critically important for the cellular functionality of ribonucleic acids (RNAs). RNA folding studies were mainly conducted in vitro, without the possibility of validating these experiments inside cells. Here, we directly resolve the folding stability of a hairpin‐structured RNA inside live mammalian cells. We find that the stability inside the cell is comparable to that in dilute physiological buffer. On the contrary, the addition of in vitro artificial crowding agents, with the exception of high‐molecular‐weight PEG, leads to a destabilization of the hairpin structure through surface interactions and reduction in water activity. We further show that RNA stability is highly variable within cell populations as well as within subcellular regions of the cytosol and nucleus. We conclude that inside cells the RNA is subject to (localized) stabilizing and destabilizing effects that lead to an on average only marginal modulation compared to diluted buffer.     

Remotely triggered liposome release by near-infrared light absorption via hollow gold nanoshells

An elusive goal for systemic drug delivery is to provide both spatial and temporal control of drug release. Liposomes have been evaluated as drug delivery vehicles for decades, but their clinical significance has been limited by slow release or poor availability of the encapsulated drug. Here we show that near-complete liposome release can be initiated within seconds by irradiating hollow gold nanoshells (HGNs) with a near-infrared (NIR) pulsed laser. Our findings reveal that different coupling methods such as having the HGNs tethered to, encapsulated within, or suspended freely outside the liposomes, all triggered liposome release but with different levels of efficiency. For the underlying content release mechanism, our experiments suggest that the microbubble formation and collapse due to the rapid temperature increase of the HGN is responsible for liposome disruption, as evidenced by the formation of solid gold particles after the NIR irradiation and the coincidence of a laser power threshold for both triggered release and pressure fluctuations in the solution associated with cavitation. These effects are similar to those induced by ultrasound and our approach is conceptually analogous to the use of optically triggered nano-"sonicators" deep inside the body for drug delivery. We expect HGNs can be coupled with any nanocarriers to promote spatially and temporally controlled drug release. In addition, the capability of external HGNs to permeabilize lipid membranes can facilitate the cellular uptake of macromolecules including proteins and DNA and allow for promising applications in gene therapy.     

On the Origin of Primitive Cells: From Nutrient Intake to Elongation of Encapsulated Nucleotides

Recent major discoveries in membrane biophysics hold the key to a modern understanding of the origin of life on Earth. Membrane bilayer vesicles have been shown to provide a multifaceted microenvironment in which protometabolic reactions could have developed. Cell‐membrane‐like aggregates of amphiphilic molecules capable of retaining encapsulated oligonucleotides have been successfully created in the laboratory. Sophisticated laboratory studies on the origin of life now show that elongation of the DNA primer takes place inside fatty acid vesicles when activated nucleotide nutrients are added to the external medium. These studies demonstrate that cell‐like vesicles can be sufficiently permeable to allow for the intake of charged molecules such as activated nucleotides, which can then take part in copying templates in the protocell interior. In this Review we summarize recent experiments in this area and describe a possible scenario for the origin of primitive cells, with an emphasis on the elongation of encapsulated nucleotides.     

Real‐Time Intracellular Measurements of ROS and RNS in Living Cells with Single Core–Shell Nanowire Electrodes

Nanoelectrodes allow precise and quantitative measurements of important biological processes at the single living‐cell level in real time. Cylindrical nanowire electrodes (NWEs) required for intracellular measurements create a great challenge for achieving excellent electrochemical and mechanical performances. Herein, we present a facile and robust solution to this problem based on a unique SiC‐core–shell design to produce cylindrical NWEs with superior mechanical toughness provided by the SiC nano‐core and an excellent electrochemical performance provided by the ultrathin carbon shell that can be used as such or platinized. The use of such NWEs for biological applications is illustrated by the first quantitative measurements of ROS/RNS in individual phagolysosomes of living macrophages. As the shell material can be varied to meet any specific detection purpose, this work opens up new opportunities to monitor quantitatively biological functions occurring inside cells and their organelles.     

Construction of Liposomes Mimicking Cell Membrane Structure through Frame‐Guided Assembly

The shape of eukaryotic cells is determined by the cytoskeleton associated with membrane proteins; however, the detailed mechanism of how the integral morphologies with structural stability is generated and maintained is still not fully understood. Here, based on the Frame‐Guided Assembly (FGA) strategy, we successfully prepared hetero‐liposomes with structural composition similar to that of eukaryotic cells by screening a series of transmembrane peptides as the leading hydrophobic groups (LHGs). It was demonstrated that the conformation and transmembrane mode of the LHGs played dominant roles during the FGA process. The FGA liposomes were formed with excellent stability, which may further provide evidence for the cytoskeleton–membrane protein–lipid bilayer model. Taking advantage of the biocompatibility and stability, the FGA liposomes were also applied to prepare novel drug delivery vehicles, which is promising in diagnostic imaging and cancer therapy applications.     

Modifying the 5′‐Cap for Click Reactions of Eukaryotic mRNA and To Tune Translation Efficiency in Living Cells

The 5′‐cap is a hallmark of eukaryotic mRNAs and plays fundamental roles in RNA metabolism, ranging from quality control to export and translation. Modifying the 5′‐cap may thus enable modulation of the underlying processes and investigation or tuning of several biological functions. A straightforward approach is presented for the efficient production of a range of N7‐modified caps based on the highly promiscuous methyltransferase Ecm1. We show that these, as well as N²‐modified 5′‐caps, can be used to tune translation of the respective mRNAs both in vitro and in cells. Appropriate modifications allow subsequent bioorthogonal chemistry, as demonstrated by intracellular live‐cell labeling of a target mRNA. The efficient and versatile N7 manipulation of the mRNA cap makes mRNAs amenable to both modulation of their biological function and intracellular labeling, and represents a valuable addition to the chemical biology toolbox.     

In Situ Spatial Complementation of Aptamer‐Mediated Recognition Enables Live‐Cell Imaging of Native RNA Transcripts in Real Time

Direct cellular imaging of the localization and dynamics of biomolecules helps to understand their function and reveals novel mechanisms at the single‐cell resolution. In contrast to routine fluorescent‐protein‐based protein imaging, technology for RNA imaging remains less well explored because of the lack of enabling technology. Herein, we report the development of an aptamer‐initiated fluorescence complementation (AiFC) method for RNA imaging by engineering a green fluorescence protein (GFP)‐mimicking turn‐on RNA aptamer, Broccoli, into two split fragments that could tandemly bind to target mRNA. When genetically encoded in cells, endogenous mRNA molecules recruited Split‐Broccoli and brought the two fragments into spatial proximity, which formed a fluorophore‐binding site in situ and turned on fluorescence. Significantly, we demonstrated the use of AiFC for high‐contrast and real‐time imaging of endogenous RNA molecules in living mammalian cells. We envision wide application and practical utility of this enabling technology to in vivo single‐cell visualization and mechanistic analysis of macromolecular interactions.     

Single siRNA Nanocapsules for Enhanced RNAi Delivery

Synthetic siRNA has been considered as a highly promising therapeutic agent for human diseases. However, clinical use of siRNA has been hampered by instability in the body and inability to deliver sufficient RNA interference compounds to the tissues or cells. To address this challenge, we present here a single siRNA nanocapsule delivery technology, which is achieved by encapsulating a single siRNA molecule within a degradable polymer nanocapsule with a diameter around 20 nm and positive surface charge. As proof-of-concept, since CCR5 is considered a major silencing target of HIV therapy, CCR5-siRNA nanocapsules were delivered into 293T cells and successfully downregulated the CCR5 RNA fused with mCherry reporter RNA. In the absence of human serum, nanocapsules and lipofectamine silenced expression of CCR5-mCherry expression to 8% and 15%, respectively. Such nanocapsules maintain the integrity of siRNA inside even after incubation with ribonuclease and serum for 1 h; under the same conditions, siRNA is degraded in the native form or when formulated with lipofectamine. In the presence of serum, CCR5-siRNA nanocapsules knocked down CCR5-mCherry expression to less than 15% while siRNAs delivered through lipofectamine slightly knocked down the expression to 55%. In summary, this work provides a novel platform for siRNA delivery that can be developed for therapeutic purposes.     

The origin of the eukaryotic cell

The endosymbiotic hypothesis for the origin of the eukaryotic cell has been applied to the origin of the mitochondria and chloroplasts. However as has been pointed out by Mereschowsky in 1905, it should also be applied to the nucleus as well. If the nucleus, mitochondria and chloroplasts are endosymbionts, then it is likely that the organism that did the engulfing was not a DNA-based organism. In fact, it is useful to postulate that this organism was a primitive RNA-based organism. This hypothesis would explain the preponderance of RNA viruses found in eukaryotic cells. The centriole and basal body do not have a double membrane or DNA. Like all MTOCs (microtubule organising centres), they have a structural or morphic RNA implicated in their formation. This would argue for their origin in the early RNA-based organism rather than in an endosymbiotic event involving bacteria. Finally, the eukaryotic cell uses RNA in ways quite unlike bacteria, thus pointing to a greater emphasis of RNA in both control and structure in the cell. The origin of the eukaryotic cell may tell us why it rather than its prokaryotic relative evolved into the metazoans who are reading this paper.     

Gene Expression Inside Liposomes: From Early Studies to Current Protocols

Synthesizing proteins inside liposomes and other microcompartments is a well-established practice. However, the origin of this research is not from the distant past, dating back to 1999–2004, when the first successful attempts were published. Protein synthesis inside artificial compartments is now under strong expansion in the context of synthetic biology (in bottom-up approaches), and, in particular, it strongly contributes to the construction of artificial cell-like systems. These systems, often called “synthetic cells”, can be used to model cellular processes, including membrane-centered ones. They are very innovative models that complement traditional studies and promise future applications. This review does not discuss all current directions in synthetic cell research; in particular, it does not include all kinds of artificial compartments. Instead, it is uniquely dedicated to the analysis of historical and technical developments of protein synthesis inside liposomes, highlighting a selected list of open questions. One of the goals is to note the importance of mastering liposome technology together with cell-free systems for the successful realization of this specific type of synthetic cell. With this aim, four currently employed protocols are compared and discussed, with a major emphasis on the droplet transfer method, which is attractive due to its simplicity and encapsulation efficiency.     

Artificial cells in medicine and biotechnology

Since the feasibility of artificial cells was first demonstrated in 1957 [Chang (1, 2)], an increasing number of approaches to their preparation and use have become available. Thus artificial cell membranes can now be formed using a variety of synthetic or biological materials to produce desired variations in their permeability, surface properties, and blood compatibility. Almost any material can be included within artificial cells. These include enzyme systems, cell extracts, biological cells, magnetic materials, isotopes, antigens, antibodies, vaccines, hormones, adsorbents, and others. Since cells are the fundamental units of living organisms, it is not surprising that artificial cells can have a number of possible applications. This is especially so since artificial cells can be “tailor-made” to have very specialized functions. A number of potential applications suggested earlier have now reached a developmental stage appropriate for clinical trial or application. These clinical applications include the use of such cells as a red blood cell substitute, in hemoperfusion, in an artifical kidney or artificial liver, as detoxifiers, in an artificial pancreas, and so on. Artificial red blood cells based on lipid-coated fluorocarbon or crosslinked hemoglobin are being investigated in a number of centers. The principle of the artificial cells is also being used in biotechnology to immobilize enzymes and cells. Developments in biotechnology have also resulted in the use of the principle underlying the artificial cell to help produce interferons and monoclonal antibodies; to create immunosorbents; to develop an artificial pancreas; and to bring enzyme technology usefully into biotechnology and biomedical applications. Artificial cells are also being used as drug delivery systems based on slow release, on magnetic target delivery, on biodegradability, on liposomes, or other approaches. The present status and recent advances will be emphasized in this paper.