A Photo-Switchable Peptide Fibril Esterase.

Recent attempts to mimic enzyme catalysis using simple, short peptides have been successful in enhancing various reactions, but the on-demand, temporal or spatial regulation of such processes by external triggers remains a great challenge. Light irradiation is an ideal trigger for regulating molecular functionality, since it can be precisely manipulated in time and space, and because most reaction mediums do not react to light. We herein report the selection of a photo-switchable amphiphilic peptide catalyst from a small library of isomeric peptides, each containing an azobenzene-based light responsive group and a catalytic histidine residue. In its native fibrillar form, the selected peptide is efficiently and enantio-selectively active for ester hydrolysis, but after irradiation by UV light inducing trans-to-cis azobenzene isomerization, the fibrils disassemble to amorphous aggregates that are much less catalytically active. Significantly, this esterase-like activity can be manipulated multiple times, as the fibrillar peptide assembly is reversibly reduced and restored upon alternate irradiation by UV and visible light, respectively. We propose that this research may shine light on the origin of complex functions in early chemical evolution. Furthermore, it paves the way to regulate additional functions for peptide nanotechnology, such as replication, charge transfer, and delivery.

Structure-function relationship of phase-separated liposomes containing diacylglycerol analogues.

The composition and morphology of lipid-based nanoparticles can influence their overall in vivo behavior. Previously, we demonstrated that phase separation in liposomes composed of DSPC and a diacylglycerol lipid analogue (DOaG) drives the in vivo biodistribution towards a specific subset of endothelial cells in zebrafish embryos. In the absence of traditional targeting functionalities (e.g., antibodies, ligands), this selectivity is mediated solely by the unique liposome morphology and composition, characterized by a DOaG-rich lipid droplet within the DSPC-rich phospholipid bilayer. The phase separation is induced due to the geometry of DOaG lipid and its ability to create non-bilayer phases in lipid membranes. To investigate the underlying principles of phase separation and to optimize the liposome colloidal stability, we performed a structure-function relationship study by synthesizing a library of DOaG analogues with varying molecular properties, such as the number, length and sn-position of the acyl chains, as well as the degree of saturation or carbonyl substituents. We assessed the ability of these lipid analogues to assemble into phase-separated liposomes and studied their morphology, colloidal stability, and in vivo biodistribution in zebrafish embryos. We found that analogues containing unsaturated, medium length (C16-C18) fatty acids were required to obtain colloidally stable, phase-separated liposomes with cell-specific biodistribution patterns. Moreover, we observed that using the pure DOaG isomer, with acyl chains at the sn-1,3 positions, leads to more colloidally stable liposomes than when a mixture of sn-1,2 and sn-1,3 isomers is used. Similarly, we observed that incorporating a DOaG analogue with fatty tails shorter than DSPC, as well as PEGylation, endows liposomes with long term stability while retaining cell-selective biodistribution. Diacylglycerols are known to promote fusion, lipid polymorphism, signaling and protein recruitment on lipid membranes. In this study, we showed that diacylglycerol derivatives can induce phase separation in liposomes, unlocking the potential for cell-specific targeting in vivo. We believe that these findings can be the foundation for future use of diacylglycerols in lipid-based nanomedicines and could lead to the development of novel targeted delivery strategies.

Macrophage-mediated myelin recycling fuels brain cancer malignancy.

Tumors growing in metabolically challenged environments, such as glioblastoma in the brain, are particularly reliant on crosstalk with their tumor microenvironment (TME) to satisfy their high energetic needs. To study the intricacies of this metabolic interplay, we interrogated the heterogeneity of the glioblastoma TME using single-cell and multi-omics analyses and identified metabolically rewired tumor-associated macrophage (TAM) subpopulations with pro-tumorigenic properties. These TAM subsets, termed lipid-laden macrophages (LLMs) to reflect their cholesterol accumulation, are epigenetically rewired, display immunosuppressive features, and are enriched in the aggressive mesenchymal glioblastoma subtype. Engulfment of cholesterol-rich myelin debris endows subsets of TAMs to acquire an LLM phenotype. Subsequently, LLMs directly transfer myelin-derived lipids to cancer cells in an LXR/Abca1-dependent manner, thereby fueling the heightened metabolic demands of mesenchymal glioblastoma. Our work provides an in-depth understanding of the immune-metabolic interplay during glioblastoma progression, thereby laying a framework to unveil targetable metabolic vulnerabilities in glioblastoma.

Lipid conjugate dissociation analysis improves the in vivo understanding of lipid-based nanomedicine.

Lipid conjugates have advanced the field of lipid-based nanomedicine by promoting active-targeting (ligand, peptide, antibody), stability (PEGylation), controlled release (lipoid prodrug), and probe-based tracking (fluorophore). Recent findings indicate lipid conjugates dissociating from nanomedicine upon encountering a biological environment. Yet, implications for (pre)clinical outcomes remain unclear. In this study, using the zebrafish model (Danio rerio), we investigated the fate of liposome-incorporated lipid fluorophore conjugates (LFCs) after intravenous (IV) administration. LFCs having a bilayer mismatch and relatively polar fluorophore revealed counter-predictive outcomes for Caelyx/Doxil (clearance vs. circulating) and AmBisome-like liposomes (scavenger endothelial cell vs. macrophage uptake). Findings on LFC (mis)match for Caelyx/Doxil-like liposomes were supported by translational intravital imaging studies in mice. Importantly, contradicting observations suggest to originate from LFC dissociation in vivo, which was investigated by Asymmetric Flow Field-Flow Fractionation (AF4) upon liposome-serum incubation in situ. Our data suggests that LFCs matching with the liposome bilayer composition - that did not dissociate upon serum incubation - revealed improved predictive outcomes for liposome biodistribution profiles. Altogether, this study highlights the critical importance of fatty acid tail length and headgroup moiety when selecting lipid conjugates for lipid-based nanomedicine.

CAPN2-responsive mesoporous silica nanoparticles: A promising nanocarrier for targeted therapy of pancreatic cancer.

Pancreatic adenocarcinoma (PDAC) is highly resistant to conventional chemotherapeutic interventions, resulting in exceptionally low survival rates. The limited efficacy can in part be attributed to dose limitations and treatment cessation urged by toxicity of currently used chemotherapy. The advent of targeted delivery strategies has kindled hope for circumventing off-target toxicity. We have previously reported a PDAC-specific mesoporous silica nanoparticle (MSN) containing a protease linker responsive to ADAM9, a PDAC-enriched extracellularly deposited protease. Upon loading with paclitaxel these ADAM9-MSNs reduced side effects both in vitro and in vivo, however, disappointing antitumor efficacy was observed in vivo. Here, we propose that an efficient uptake of MSNs by tumor cells might underlie the lack of antitumor efficacy of MSNs functionalized with linker responsive to extracellular proteases. Harnessing this premise to improve antitumor efficacy, we performed an in silico analysis to identify PDAC-enriched intracellular proteases. We report the identification of BACE2, CAPN2 and DPP3 as PDAC enriched intracellular proteases, and report the synthesis of BACE2-, CAPN2- and DPP3-responsive MSNs. Extensive preclinical assessments revealed that paclitaxel-loaded CAPN2- and DPP3-MSNs exhibit high PDAC specificity in vitro as opposed to free paclitaxel. The administration of paclitaxel-loaded CAPN2- and DPP3-MSNs in vivo confirmed the reduction of leukopenia and induced no organ damage. Promisingly, in two mouse models CAPN2-MSNs reduced tumor growth at least as efficiently as free paclitaxel. Taken together, our results pose CAPN2-MSNs as a promising nanocarrier for the targeted delivery of chemotherapeutics in PDAC.

Lipid-Based Nanoparticle Functionalization with Coiled-Coil Peptides for In Vitro and In Vivo Drug Delivery.

For the delivery of drugs, different nanosized drug carriers (e.g., liposomes, lipid nanoparticles, and micelles) have been developed in order to treat diseases that afflict society. Frequently, these vehicles are formed by the self-assembly of small molecules to encapsulate the therapeutic cargo of interest. Over decades, nanoparticles have been optimized to make them more efficient and specific to fulfill tailor-made tasks, such as specific cell targeting or enhanced cellular uptake. In recent years, lipid-based nanoparticles in particular have taken center stage; however, off-targeting side effects and poor endosomal escape remain major challenges since therapies require high efficacy and acceptable toxicity.To overcome these issues, many different approaches have been explored to make drug delivery more specific, resulting in reduced side effects, to achieve an optimal therapeutic effect and a lower required dose. The fate of nanoparticles is largely dependent on size, shape, and surface charge. A common approach to designing drug carriers with targeting capability is surface modification. Different approaches to functionalize nanoparticles have been investigated since the attachment of targeting moieties plays a significant role in whether they can later interact with surface-exposed receptors of cells. To this end, various strategies have been used involving different classes of biomolecules, such as small molecules, nucleic acids, antibodies, aptamers, and peptides.Peptides in particular are often used since there are many receptors overexpressed in different specific cell types. Furthermore, peptides can be produced and modified at a low cost, enabling high therapeutic screening. Cell-penetrating peptides (CPPs) and cell-targeting peptides (CTPs) are frequently used for this purpose. Less studied in this context are fusogenic coiled-coil peptides. Lipid-based nanoparticles functionalized with these peptides are able to avoid the endolysosomal pathway; instead such particles can be taken up by membrane fusion, resulting in increased delivery of payload. Furthermore, they can be used for targeting cells/organs but are not directed at surface-exposed receptors. Instead, they recognize complementary peptide sequences, facilitating their uptake into cells.In this Account, we will discuss peptides as moieties for enhanced cytosolic delivery, targeted uptake, and how they can be attached to lipid-based nanoparticles to alter their properties. We will discuss the properties imparted to the particles by peptides, surface modification approaches, and recent examples showing the power of peptides for in vitro and in vivo drug delivery. The main focus will be on the functionalization of lipid-based nanoparticles by fusogenic coiled-coil peptides, highlighting the relevance of this concept for the development of future therapeutics.

Deconvolving Passive and Active Targeting of Liposomes Bearing LDL Receptor Binding Peptides Using the Zebrafish Embryo Model.

Improving target versus off-target ratio in nanomedicine remains a major challenge for increasing drug bioavailability and reducing toxicity. Active targeting using ligands on nanoparticle surfaces is a key approach but has limited clinical success. A potential issue is the integration of targeting ligands also changes the physicochemical properties of nanoparticles (passive targeting). Direct studies to understand the mechanisms of active targeting and off-targeting in vivo are limited by the lack of suitable tools. Here, the biodistribution of a representative active targeting liposome is analyzed, modified with an apolipoprotein E (ApoE) peptide that binds to the low-density lipoprotein receptor (LDLR), using zebrafish embryos. The ApoE liposomes demonstrated the expected liver targeting effect but also accumulated in the kidney glomerulus. The ldlra-/- zebrafish is developed to explore the LDLR-specificity of ApoE liposomes. Interestingly, liver targeting depends on the LDLR-specific interaction, while glomerular accumulation is independent of LDLR and peptide sequence. It is found that cationic charges of peptides and the size of liposomes govern glomerular targeting. Increasing the size of ApoE liposomes can avoid this off-targeting. Taken together, the study shows the potential of the zebrafish embryo model for understanding active and passive targeting mechanisms, that can be used to optimize the design of nanoparticles.

Liquid crystalline inverted lipid phases encapsulating siRNA enhance lipid nanoparticle mediated transfection.

Efficient cytosolic delivery of RNA molecules remains a formidable barrier for RNA therapeutic strategies. Lipid nanoparticles (LNPs) serve as state-of-the-art carriers that can deliver RNA molecules intracellularly, as exemplified by the recent implementation of several vaccines against SARS-CoV-2. Using a bottom-up rational design approach, we assemble LNPs that contain programmable lipid phases encapsulating small interfering RNA (siRNA). A combination of cryogenic transmission electron microscopy, cryogenic electron tomography and small-angle X-ray scattering reveals that we can form inverse hexagonal structures, which are present in a liquid crystalline nature within the LNP core. Comparison with lamellar LNPs reveals that the presence of inverse hexagonal phases enhances the intracellular silencing efficiency over lamellar structures. We then demonstrate that lamellar LNPs exhibit an in situ transition from a lamellar to inverse hexagonal phase upon interaction with anionic membranes, whereas LNPs containing pre-programmed liquid crystalline hexagonal phases bypass this transition for a more efficient one-step delivery mechanism, explaining the increased silencing effect. This rational design of LNPs with defined lipid structures aids in the understanding of the nano-bio interface and adds substantial value for LNP design, optimization and use.

Noncovalent Conjugation of OVA323 to ELP Micelles Increases Immune Response.

Subunit vaccines would benefit from a safe particle-based adjuvant. Elastin-like polypeptide (ELP)-based micelles are interesting candidate adjuvants due to their well-defined size and easy modification with protein-based cargo. Coiled coils can facilitate noncovalent modifications, while potentially enhancing antigen delivery through interaction with cell membranes. ELP micelles comprise ELP diblock copolymers that self-assemble above a critical micelle temperature. In this study, an amphiphilic ELP was conjugated to peptide "K", which forms a heterodimeric coiled-coil complex with peptide "E". Self-assembled "covalent" micelles containing ELP-OVA323 (i.e., model antigen OVA323 conjugated to ELP), "coiled-coil" micelles containing ELP-K/E-OVA323 and "hybrid" micelles containing ELP-K and ELP-OVA323 were shown to be monodisperse and spherical. Dendritic cells (DCs) were exposed to all micelle compositions, and T-cell proliferation was investigated. The presence of ELP-K enhanced micelle uptake and subsequent DC maturation, resulting in enhanced CD4+ T-cell proliferation, which makes ELPs with coiled coil-associated antigens a promising vaccine platform.

Phase-Separated Lipid-Based Nanoparticles: Selective Behavior at the Nano-Bio Interface.

The membrane-protein interface on lipid-based nanoparticles influences their in vivo behavior. Better understanding may evolve current drug delivery methods toward effective targeted nanomedicine. Previously, the cell-selective accumulation of a liposome formulation in vivo is demonstrated, through the recognition of lipid phase-separation by triglyceride lipases. This exemplified how liposome morphology and composition can determine nanoparticle-protein interactions. Here, the lipase-induced compositional and morphological changes of phase-separated liposomes-which bear a lipid droplet in their bilayer- are investigated, and the mechanism upon which lipases recognize and bind to the particles is unravelled. The selective lipolytic degradation of the phase-separated lipid droplet is observed, while nanoparticle integrity remains intact. Next, the Tryptophan-rich loop of the lipase is identified as the region with which the enzymes bind to the particles. This preferential binding is due to lipid packing defects induced on the liposome surface by phase separation. In parallel, the existing knowledge that phase separation leads to in vivo selectivity, is utilized to generate phase-separated mRNA-LNPs that target cell-subsets in zebrafish embryos, with subsequent mRNA delivery and protein expression. Together, these findings can expand the current knowledge on selective nanoparticle-protein communications and in vivo behavior, aspects that will assist to gain control of lipid-based nanoparticles.

Biological recognition and cellular trafficking of targeted RNA-lipid nanoparticles.

Lipid nanoparticles (LNPs) have unlocked the potential of ribonucleic acid (RNA) therapeutics and vaccines. Production and large-scale manufacturing methods for RNA-LNPs have been established and rapidly accelerate. Despite this, basic research on LNPs is still required, due to their high assembly complexity and fairly new development, including research on lipid organization, transfection optimization, and in vivo behavior. Understanding fundamental aspects of LNPs that is, how lipid composition and physicochemical properties affect their biodistribution, cell recognition, and transfection, could propel their clinical development and facilitate overcoming current challenges. Herein, we review recent developments in the field of LNP technology and summarize the main findings focusing on nano-bio interactions.

Fusogenic Coiled-Coil Peptides Enhance Lipid Nanoparticle-Mediated mRNA Delivery upon Intramyocardial Administration.

Heart failure is a serious condition that results from the extensive loss of specialized cardiac muscle cells called cardiomyocytes (CMs), typically caused by myocardial infarction (MI). Messenger RNA (mRNA) therapeutics are emerging as a very promising gene medicine for regenerative cardiac therapy. To date, lipid nanoparticles (LNPs) represent the most clinically advanced mRNA delivery platform. Yet, their delivery efficiency has been limited by their endosomal entrapment after endocytosis. Previously, we demonstrated that a pair of complementary coiled-coil peptides (CPE4/CPK4) triggered efficient fusion between liposomes and cells, bypassing endosomal entrapment and resulting in efficient drug delivery. Here, we modified mRNA-LNPs with the fusogenic coiled-coil peptides and demonstrated efficient mRNA delivery to difficult-to-transfect induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs). As proof of in vivo applicability of these fusogenic LNPs, local administration via intramyocardial injection led to significantly enhanced mRNA delivery and concomitant protein expression. This represents the successful application of the fusogenic coiled-coil peptides to improve mRNA-LNPs transfection in the heart and provides the potential for the advanced development of effective regenerative therapies for heart failure.

Efficient mRNA delivery using lipid nanoparticles modified with fusogenic coiled-coil peptides.

Gene delivery has great potential in modulating protein expression in specific cells to treat diseases. Such therapeutic gene delivery demands sufficient cellular internalization and endosomal escape. Of various nonviral nucleic acid delivery systems, lipid nanoparticles (LNPs) are the most advanced, but still, are very inefficient as the majority are unable to escape from endosomes/lysosomes. Here, we develop a highly efficient gene delivery system using fusogenic coiled-coil peptides. We modified LNPs, carrying EGFP-mRNA, and cells with complementary coiled-coil lipopeptides. Coiled-coil formation between these lipopeptides induced fast nucleic acid uptake and enhanced GFP expression. The cellular uptake of coiled-coil modified LNPs is likely driven by membrane fusion thereby omitting typical endocytosis pathways. This direct cytosolic delivery circumvents the problems commonly observed with the limited endosomal escape of mRNA. Therefore fusogenic coiled-coil peptide modification of existing LNP formulations to enhance nucleic acid delivery efficiency could be beneficial for several gene therapy applications.

Preclinical Assessment of ADAM9-Responsive Mesoporous Silica Nanoparticles for the Treatment of Pancreatic Cancer.

Pancreatic adenocarcinoma (PDAC) remains largely refractory to chemotherapeutic treatment regimens and, consequently, has the worst survival rate of all cancers. The low efficacy of current treatments results largely from toxicity-dependent dose limitations and premature cessation of therapy. Recently, targeted delivery approaches that may reduce off-target toxicities have been developed. In this paper, we present a preclinical evaluation of a PDAC-specific drug delivery system based on mesoporous silica nanoparticles (MSNs) functionalized with a protease linker that is specifically cleaved by PDAC cells. Our previous work demonstrated that ADAM9 is a PDAC-enriched protease and that paclitaxel-loaded ADAM9-responsive MSNs effectively kill PDAC cells in vitro. Here, we show that paclitaxel-loaded ADAM9-MSNs result in off-target cytotoxicity in clinically relevant models, which spurred the development of optimized ADAM9-responsive MSNs (OPT-MSNs). We found that these OPT-MSNs still efficiently kill PDAC cells but, as opposed to free paclitaxel, do not induce death in neuronal or bone marrow cells. In line with these in vitro data, paclitaxel-loaded OPT-MSNs showed reduced organ damage and leukopenia in a preclinical PDAC xenograft model. However, no antitumor response was observed upon OPT-MSN administration in vivo. The poor in vivo antitumor activity of OPT-MSNs despite efficient antitumor effects in vitro highlights that although MSN-based tumor-targeting strategies may hold therapeutic potential, clinical translation does not seem as straightforward as anticipated.

In vitro and in vivo evaluation of clinically-approved ionizable cationic lipids shows divergent results between mRNA transfection and vaccine efficacy.

Ionizable cationic lipids (ICLs) play an essential role in the effectiveness of lipid nanoparticles (LNPs) for delivery of mRNA therapeutics and vaccines; therefore, critical evaluations of their biological performance would extend the existing knowledge in the field. In the present study, we examined the effects of the three clinically-approved ICLs, Dlin-MC3-DMA, ALC-0315 and SM-102, as well as DODAP, on the in vitro and in vivo performance of LNPs for mRNA delivery and vaccine efficacy. mRNA-LNPs containing these lipids were successfully prepared, which were all found to be very similar in their physicochemical properties and mRNA encapsulation efficiencies. Furthermore, the results of the in vitro studies indicated that these mRNA-LNPs were efficiently taken up by immortalized and primary immune cells with comparable efficiency; however, SM-102-based LNPs were superior in inducing protein expression and antigen-specific T cell proliferation. In contrast, in vivo studies revealed that LNPs containing ALC-0315 and SM-102 yielded almost identical protein expression levels in zebrafish embryos, which were significantly higher than Dlin-MC3-DMA-based LNPs. Additionally, a mouse immunization study demonstrated that a single-dose subcutaneous administration of the mRNA-LNPs resulted in a high production of intracellular cytokines by antigen-specific T cells, but no significant differences among the three clinically-approved ICLs were observed, suggesting a weak correlation between in vitro and in vivo outcomes. This study provides strong evidence that ICLs modulate the performance of mRNA-LNPs and that in vitro data does not adequately predict their behavior in vivo.

In vivo biodistribution of kinetically stable Pt2L4 nanospheres that show anti-cancer activity.

There is an increasing interest in the application of metal-organic cages (MOCs) in a biomedicinal context, as they can offer non-classical distribution in organisms compared to molecular substrates, while revealing novel cytotoxicity mechanisms. Unfortunately, many MOCs are not sufficiently stable under in vivo conditions, making it difficult to study their structure-activity relationships in living cells. As such, it is currently unclear whether MOC cytotoxicity stems from supramolecular features or their decomposition products. Herein, we describe the toxicity and photophysical properties of highly-stable rhodamine functionalized platinum-based Pt2L4 nanospheres as well as their building blocks under in vitro and in vivo conditions. We show that in both zebrafish and human cancer cell lines, the Pt2L4 nanospheres demonstrate reduced cytotoxicity and altered biodistribution within the body of zebrafish embryos compared to the building blocks. We anticipate that the composition-dependent biodistribution of Pt2L4 spheres together with their cytotoxic and photophysical properties provides the fundament for MOC application in cancer therapy.

In vivo metallophilic self-assembly of a light-activated anticancer drug.

Self-assembling molecular drugs combine the easy preparation typical of small-molecule chemotherapy and the tumour-targeting properties of drug-nanoparticle conjugates. However, they require a supramolecular interaction that survives the complex environment of a living animal. Here we report that the metallophilic interaction between cyclometalated palladium complexes generates supramolecular nanostructures in living mice that have a long circulation time (over 12 h) and efficient tumour accumulation rate (up to 10.2% of the injected dose per gram) in a skin melanoma tumour model. Green light activation leads to efficient tumour destruction due to the type I photodynamic effect generated by the self-assembled palladium complexes, as demonstrated in vitro by an up to 96-fold cytotoxicity increase upon irradiation. This work demonstrates that metallophilic interactions are well suited to generating stable supramolecular nanotherapeutics in vivo with exceptional tumour-targeting properties.

Enhanced Liposomal Drug Delivery Via Membrane Fusion Triggered by Dimeric Coiled-Coil Peptides.

An ideal nanomedicine system improves the therapeutic efficacy of drugs. However, most nanomedicines enter cells via endosomal/lysosomal pathways and only a small fraction of the cargo enters the cytosol inducing therapeutic effects. To circumvent this inefficiency, alternative approaches are desired. Inspired by fusion machinery found in nature, synthetic lipidated peptide pair E4/K4 is used to induce membrane fusion previously. Peptide K4 interacts specifically with E4, and it has a lipid membrane affinity and resulting in membrane remodeling. To design efficient fusogens with multiple interactions, dimeric K4 variants are synthesized to improve fusion with E4-modified liposomes and cells. The secondary structure and self-assembly of dimers are studied; the parallel PK4 dimer forms temperature-dependent higher-order assemblies, while linear K4 dimers form tetramer-like homodimers. The structures and membrane interactions of PK4 are supported by molecular dynamics simulations. Upon addition of E4, PK4 induced the strongest coiled-coil interaction resulting in a higher liposomal delivery compared to linear dimers and monomer. Using a wide spectrum of endocytosis inhibitors, membrane fusion is found to be the main cellular uptake pathway. Doxorubicin delivery results in efficient cellular uptake and concomitant antitumor efficacy. These findings aid the development of efficient delivery systems of drugs into cells using liposome-cell fusion strategies.

SNARE mimic peptide triggered membrane fusion kinetics revealed using single particle techniques.

Membrane fusion is an essential part of the proper functioning of life. As such it is not only important that organisms carefully regulate the process, but also that it is well understood. One way to facilitate and study membrane fusion is to use artificial, minimalist, fusion peptides. In this study the efficiency and kinetics of two fusion peptides, denoted CPE and CPK, were studied using single-particle TIRF microscopy. CPE and CPK are helical peptides which interact with each other, forming a coiled-coil motif. The peptides can be inserted into a lipid membrane using a lipid anchor, and if these peptides are anchored in opposing lipid membranes, then the coiled-coil interaction can provide the mechanical force necessary to overcome the energy barrier to initiate fusion, much in the same way the SNARE complex does. In this study we find that the fusogenic facilitation of CPE and CPK in liposomes is, at least partially, dependent on the size of the particle. In addition, under certain fusogenic conditions such as when using small liposomes of ∼60 nm in diameter, CPK alone is enough to facilitate membrane fusion in both bulk and single-particle studies. We show this using bulk lipid mixing assays utilizing FRET and single-particle TIRF, making use of dequenching fluorophores to indicate fusion. This provides us with new insights into the mechanisms of peptide-mediated membrane fusion and illuminates both challenges as well as opportunities when designing drug delivery systems.

Azobenzene-Based Amino Acids for the Photocontrol of Coiled-Coil Peptides.

Coiled-coil peptides are high-affinity, selective, self-assembling binding motifs, making them attractive components for the preparation of functional biomaterials. Photocontrol of coiled-coil self-assembly allows for the precise localization of their activity. To rationally explore photoactivity in a model coiled coil, three azobenzene-containing amino acids were prepared and substituted into the hydrophobic core of the E3/K3 coiled-coil heterodimer. Two of the non-natural amino acids, APhe1 and APhe2, are based on phenylalanine and differ in the presence of a carboxylic acid group. These have previously been demonstrated to modulate protein activity. When incorporated into peptide K3, coiled-coil binding strength was affected upon isomerization, with the two variants differing in their most folded state. The third azobenzene-containing amino acid, APgly, is based on phenylglycine and was prepared to investigate the effect of amino acid size on photoisomerization. When APgly is incorporated into the coiled coil, a 4.7-fold decrease in folding constant is observed upon trans-to-cis isomerization─the largest difference for all three amino acids. Omitting the methylene group between azobenzene and α-carbon was theorized to both position the diazene of APgly closer to the hydrophobic amino acids and reduce the possible rotations of the amino acid, with molecular dynamics simulations supporting these hypotheses. These results demonstrate the ability of photoswitchable amino acids to control coiled-coil assembly through disruption of the hydrophobic interface, a strategy that should be widely applicable.