Engineering Swollen Cubosomes Using Cholesterol and Anionic Lipids

Dispersions of nonlamellar lipid membrane assemblies are gaining increasing interest for drug delivery and protein therapeutic application. A key bottleneck has been the lack of rational design rules for these systems linking different lipid species and conditions to defined lattice parameters and structures. We have developed robust methods to form cubosomes (nanoparticles with porous internal structures) with water channel diameters of up to 171 Å, which are over 4 times larger than archetypal cubosome structures. The water channel diameter can be tuned via the incorporation of cholesterol and the charged lipid DOPA, DOPG, or DOPS. We have found that large molecules can be incorporated into the porous cubosome structure and that these molecules can interact with the internal cubosome membrane. This offers huge potential for accessible encapsulation and protection of biomolecules and development of confined interfacial reaction environments

Peptide-Folding Triggered Phase Separation and Lipid Membrane Destabilization in Cholesterol-Rich Lipid Vesicles

Liposome-based drug delivery systems are widely used to improve drug pharmacokinetics but can suffer from slow and unspecific release of encapsulated drugs. Membrane-active peptides, based on sequences derived or inspired from antimicrobial peptides (AMPs), could offer means to trigger and control the release. Cholesterol is used in most liposomal drug delivery systems (DDS) to improve the stability of the formulation, but the activity of AMPs on cholesterol-rich membranes tends to be very low, complicating peptide-triggered release strategies. Here, we show a de novo designed AMP-mimetic peptide that efficiently triggers content release from cholesterol-containing lipid vesicles when covalently conjugated to headgroup-functionalized lipids. Binding to vesicles induces peptide folding and triggers a lipid phase separation, which in the presence of cholesterol results in high local peptide concentrations at the lipid bilayer surface and rapid content release. We anticipate that these results will facilitate the development of peptide-based strategies for controlling and triggering drug release from liposomal drug delivery systems

Additional file 1 of RegiSTORM: channel registration for multi-color stochastic optical reconstruction microscopy

Additional file 1. Supplementary methods, supplementary figures S1-S4, supplementary tables S1-S5

Design of Lipid-Based Nanocarriers via Cation Modulation of Ethanol-Interdigitated Lipid Membranes

Short-chain alcohols (i.e., ethanol) can induce membrane interdigitation in saturated-chain phosphatidylcholines (PCs). In this process, alcohol molecules intercalate between phosphate heads, increasing lateral separation and favoring hydrophobic interactions between opposing acyl chains, which interpenetrate forming an interdigitated phase. Unraveling mechanisms underlying the interactions between ethanol and model lipid membranes has implications for cell biology, biochemistry, and for the formulation of lipid-based nanocarriers. However, investigations of ethanol–lipid membrane systems have been carried out in deionized water, which limits their applicability. Here, using a combination of small- and wide-angle X-ray scattering, small-angle neutron scattering, and all-atom molecular dynamics simulations, we analyzed the effect of varying CaCl2 and NaCl concentrations on ethanol-induced interdigitation. We observed that while ethanol addition leads to the interdigitation of bulk phase 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers in the presence of CaCl2 and NaCl regardless of the salt concentration, the ethanol-induced interdigitation of vesicular DPPC depends on the choice of cation and its concentration. These findings unravel a key role for cations in the ethanol-induced interdigitation of lipid membranes in either bulk phase or vesicular form

3,4-Ethylenedioxythiophene Hydrogels: Relating Structure and Charge Transport in Supramolecular Gels

Ionic charge transport is a ubiquitous language of communication in biological systems. As such, bioengineering is in constant need of innovative, soft, and biocompatible materials that facilitate ionic conduction. Low molecular weight gelators (LMWGs) are complex self-assembled materials that have received increasing attention in recent years. Beyond their biocompatible, self-healing, and stimuli responsive facets, LMWGs can be viewed as a “solid” electrolyte solution. In this work, we investigate 3,4-ethylenedioxythiophene (EDOT) as a capping group for a small peptide library, which we use as a system to understand the relationship between modes of assembly and charge transport in supramolecular gels. Through a combination of techniques including small-angle neutron scattering (SANS), NMR-based Van’t Hoff analysis, atomic force microscopy (AFM), rheology, four-point probe, and electrochemical impedance spectroscopy (EIS), we found that modifications to the peptide sequence result in distinct assembly pathways, thermodynamic parameters, mechanical properties, and ionic conductivities. Four-point probe conductivity measurements and electrochemical impedance spectroscopy suggest that ionic conductivity is approximately doubled by programmable gel assemblies with hollow cylinder morphologies relative to gels containing solid fibers or a control electrolyte. More broadly, it is hoped this work will serve as a platform for those working on charge transport of aqueous soft materials in general

Revealing Population Heterogeneity in Vesicle-Based Nanomedicines Using Automated, Single Particle Raman Analysis

The intrinsic heterogeneity of many nanoformulations is currently challenging to characterize on both the single particle and population level. Therefore, there is great opportunity to develop advanced techniques to describe and understand nanomedicine heterogeneity, which will aid translation to the clinic by informing manufacturing quality control, characterization for regulatory bodies, and connecting nanoformulation properties to clinical outcomes to enable rational design. Here, we present an analytical technique to provide such information, while measuring the nanocarrier and cargo simultaneously with label-free, nondestructive single particle automated Raman trapping analysis (SPARTA). We first synthesized a library of model compounds covering a range of hydrophilicities and providing distinct Raman signals. These compounds were then loaded into model nanovesicles (polymersomes) that can load both hydrophobic and hydrophilic cargo into the membrane or core regions, respectively. Using our analytical framework, we characterized the heterogeneity of the population by correlating the signal per particle from the membrane and cargo. We found that core and membrane loading can be distinguished, and we detected subpopulations of highly loaded particles in certain cases. We then confirmed the suitability of our technique in liposomes, another nanovesicle class, including the commercial formulation Doxil. Our label-free analytical technique precisely determines cargo location alongside loading and release heterogeneity in nanomedicines, which could be instrumental for future quality control, regulatory body protocols, and development of structure–function relationships to bring more nanomedicines to the clinic

Fate of Liposomes in the Presence of Phospholipase C and D: From Atomic to Supramolecular Lipid Arrangement

Understanding the origins of lipid membrane bilayer rearrangement in response to external stimuli is an essential component of cell biology and the bottom-up design of liposomes for biomedical applications. The enzymes phospholipase C and D (PLC and PLD) both cleave the phosphorus–oxygen bonds of phosphate esters in phosphatidylcholine (PC) lipids. The atomic position of this hydrolysis reaction has huge implications for the stability of PC-containing self-assembled structures, such as the cell wall and lipid-based vesicle drug delivery vectors. While PLC converts PC to diacylglycerol (DAG), the interaction of PC with PLD produces phosphatidic acid (PA). Here we present a combination of small-angle scattering data and all-atom molecular dynamics simulations, providing insights into the effects of atomic-scale reorganization on the supramolecular assembly of PC membrane bilayers upon enzyme-mediated incorporation of DAG or PA. We observed that PC liposomes completely disintegrate in the presence of PLC, as conversion of PC to DAG progresses. At lower concentrations, DAG molecules within fluid PC bilayers form hydrogen bonds with backbone carbonyl oxygens in neighboring PC molecules and burrow into the hydrophobic region. This leads initially to membrane thinning followed by a swelling of the lamellar phase with increased DAG. At higher DAG concentrations, localized membrane tension causes a change in lipid phase from lamellar to the hexagonal and micellar cubic phases. Molecular dynamics simulations show that this destabilization is also caused in part by the decreased ability of DAG-containing PC membranes to coordinate sodium ions. Conversely, PLD-treated PC liposomes remain stable up to extremely high conversions to PA. Here, the negatively charged PA headgroup attracts significant amounts of sodium ions from the bulk solution to the membrane surface, leading to a swelling of the coordinated water layer. These findings are a vital step toward a fundamental understanding of the degradation behavior of PC lipid membranes in the presence of these clinically relevant enzymes, and toward the rational design of diagnostic and drug delivery technologies for phospholipase-dysregulation-based diseases

Fate of Liposomes in the Presence of Phospholipase C and D: From Atomic to Supramolecular Lipid Arrangement

Understanding the origins of lipid membrane bilayer rearrangement in response to external stimuli is an essential component of cell biology and the bottom-up design of liposomes for biomedical applications. The enzymes phospholipase C and D (PLC and PLD) both cleave the phosphorus–oxygen bonds of phosphate esters in phosphatidylcholine (PC) lipids. The atomic position of this hydrolysis reaction has huge implications for the stability of PC-containing self-assembled structures, such as the cell wall and lipid-based vesicle drug delivery vectors. While PLC converts PC to diacylglycerol (DAG), the interaction of PC with PLD produces phosphatidic acid (PA). Here we present a combination of small-angle scattering data and all-atom molecular dynamics simulations, providing insights into the effects of atomic-scale reorganization on the supramolecular assembly of PC membrane bilayers upon enzyme-mediated incorporation of DAG or PA. We observed that PC liposomes completely disintegrate in the presence of PLC, as conversion of PC to DAG progresses. At lower concentrations, DAG molecules within fluid PC bilayers form hydrogen bonds with backbone carbonyl oxygens in neighboring PC molecules and burrow into the hydrophobic region. This leads initially to membrane thinning followed by a swelling of the lamellar phase with increased DAG. At higher DAG concentrations, localized membrane tension causes a change in lipid phase from lamellar to the hexagonal and micellar cubic phases. Molecular dynamics simulations show that this destabilization is also caused in part by the decreased ability of DAG-containing PC membranes to coordinate sodium ions. Conversely, PLD-treated PC liposomes remain stable up to extremely high conversions to PA. Here, the negatively charged PA headgroup attracts significant amounts of sodium ions from the bulk solution to the membrane surface, leading to a swelling of the coordinated water layer. These findings are a vital step toward a fundamental understanding of the degradation behavior of PC lipid membranes in the presence of these clinically relevant enzymes, and toward the rational design of diagnostic and drug delivery technologies for phospholipase-dysregulation-based diseases

Potent Virustatic Polymer–Lipid Nanomimics Block Viral Entry and Inhibit Malaria Parasites In Vivo

Infectious diseases continue to pose a substantial burden on global populations, requiring innovative broad-spectrum prophylactic and treatment alternatives. Here, we have designed modular synthetic polymer nanoparticles that mimic functional components of host cell membranes, yielding multivalent nanomimics that act by directly binding to varied pathogens. Nanomimic blood circulation time was prolonged by reformulating polymer–lipid hybrids. Femtomolar concentrations of the polymer nanomimics were sufficient to inhibit herpes simplex virus type 2 (HSV-2) entry into epithelial cells, while higher doses were needed against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Given their observed virustatic mode of action, the nanomimics were also tested with malaria parasite blood-stage merozoites, which lose their invasive capacity after a few minutes. Efficient inhibition of merozoite invasion of red blood cells was demonstrated both in vitro and in vivo using a preclinical rodent malaria model. We envision these nanomimics forming an adaptable platform for developing pathogen entry inhibitors and as immunomodulators, wherein nanomimic-inhibited pathogens can be secondarily targeted to sites of immune recognition

Potent Virustatic Polymer–Lipid Nanomimics Block Viral Entry and Inhibit Malaria Parasites In Vivo

Infectious diseases continue to pose a substantial burden on global populations, requiring innovative broad-spectrum prophylactic and treatment alternatives. Here, we have designed modular synthetic polymer nanoparticles that mimic functional components of host cell membranes, yielding multivalent nanomimics that act by directly binding to varied pathogens. Nanomimic blood circulation time was prolonged by reformulating polymer–lipid hybrids. Femtomolar concentrations of the polymer nanomimics were sufficient to inhibit herpes simplex virus type 2 (HSV-2) entry into epithelial cells, while higher doses were needed against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Given their observed virustatic mode of action, the nanomimics were also tested with malaria parasite blood-stage merozoites, which lose their invasive capacity after a few minutes. Efficient inhibition of merozoite invasion of red blood cells was demonstrated both in vitro and in vivo using a preclinical rodent malaria model. We envision these nanomimics forming an adaptable platform for developing pathogen entry inhibitors and as immunomodulators, wherein nanomimic-inhibited pathogens can be secondarily targeted to sites of immune recognition