O2 permeability of lipid bilayers is low, but increases with membrane cholesterol.

Oxygen on its transport route from lung to tissue mitochondria has to cross several cell membranes. The permeability value of membranes for O2 (PO2), although of fundamental importance, is controversial. Previous studies by mostly indirect methods diverge between 0.6 and 125 cm/s. Here, we use a most direct approach by observing transmembrane O2 fluxes out of 100 nm liposomes at defined transmembrane O2 gradients in a stopped-flow system. Due to the small size of the liposomes intra- as well as extraliposomal diffusion processes do not affect the overall kinetics of the O2 release process. We find, for cholesterol-free liposomes, the unexpectedly low PO2 value of 0.03 cm/s at 35 °C. This PO2 would present a serious obstacle to O2 entering or leaving the erythrocyte. Cholesterol turns out to be a novel major modifier of PO2, able to increase PO2 by an order of magnitude. With a membrane cholesterol of 45 mol% as it occurs in erythrocytes, PO2 rises to 0.2 cm/s at 35 °C. This PO2 is just sufficient to ensure complete O2 loading during passage of erythrocytes through the lung's capillary bed under the conditions of rest as well as maximal exercise.

Phospholipid-Block Copolymer Hybrid Vesicles with Lysosomal Escape Ability.

The success of nanoparticulate formulations in drug delivery depends on various aspects including their toxicity, internalization, and intracellular location. Vesicular assemblies consisting of phospholipids and amphiphilic block copolymers are an emerging platform, which combines the benefits from liposomes and polymersomes while overcoming their challenges. We report the synthesis of poly(cholesteryl methacrylate)- block-poly(2-(dimethylamino) ethyl methacrylate) (pCMA- b-pDMAEMA) block copolymers and their assembly with phospholipids into hybrid vesicles. Their geometry, their ζ-potential, and their ability to adsorb onto polymer-coated surfaces were assessed. Giant unilamellar vesicles were employed to confirm the presence of both the phospholipids and the block copolymer in the same membrane. Furthermore, the cytotoxicity of selected hybrid vesicles was determined in RAW 264.7 mouse macrophages, primary rat Kupffer cells, and human macrophages. The internalization and lysosomal escape ability of the hybrid vesicles were confirmed using RAW 264.7 mouse macrophages. Taken together, our findings illustrate that the reported hybrid vesicles are a promising complementary drug delivery platform for existing liposomes and polymersomes.

Multicompartmentalized Microreactors Containing Nuclei and Catalase-Loaded Liposomes.

Multicompartmentalized microreactors are considered as cell mimics with hierarchical structures inspired by mammalian cells. We report the successful assembly and encapsulation of purified nuclei from RAW 264.7 cells (pNuc) into alginate-based microreactors. We demonstrate the preserved function of nuclei within the microreactors for mRNA production. Further, we load catalase-loaded liposomes (Lcat) into the microreactors to break down hydrogen peroxide (H2O2) into oxygen and water. Assemblies containing both natural pNuc and synthetic Lcat show significantly higher mRNA production in the presence of H2O2 compared to microreactors without Lcat or no H2O2 present, suggesting a beneficial effect of the locally enzymatically produced oxygen for transcription. This novel type of microreactors, containing both natural and synthetic compartments, presents a substantial advancement from assemblies equipped with solely synthetic units and offers opportunities as hypoxia models or for cell-free protein synthesis.

Low CO2 permeability of cholesterol-containing liposomes detected by stopped-flow fluorescence spectroscopy.

Here we ask the following: 1) what is the CO2 permeability (Pco2) of unilamellar liposomes composed of l-α-phosphatidylcholine (PC)/l-α-phosphatidylserine (PS) = 4:1 and containing cholesterol (Chol) at levels often occurring in biologic membranes (50 mol%), and 2) does incorporation of the CO2 channel aquaporin (AQP)1 cause a significant increase in membrane Pco2? Presently, a drastic discrepancy exists between the answers to these two questions obtained from mass-spectrometric (18)O-exchange measurements (Chol reduces Pco2 100-fold, AQP1 increases Pco2 10-fold) vs. from stopped-flow approaches observing CO2 uptake (no effects of either Chol or AQP1). A novel theory of CO2 uptake by vesicles predicts that in a stopped-flow apparatus this fast process can only be resolved temporally and interpreted quantitatively, if 1) a very low CO2 partial pressure (pCO2) is used (e.g., 18 mmHg), and 2) intravesicular carbonic anhydrase (CA) activity is precisely known. With these prerequisites fulfilled, we find by stopped-flow that 1) Chol-containing vesicles possess a Pco2 = 0.01cm/s, and Chol-free vesicles exhibit ∼1 cm/s, and 2) the Pco2 of 0.01 cm/s is increased ≥ 10-fold by AQP1. Both results agree with previous mass-spectrometric results and thus resolve the apparent discrepancy between the two techniques. We confirm that biologic membranes have an intrinsically low Pco2 that can be raised when functionally necessary by incorporating protein-gas channels such as AQP1.

Dynamics of Membrane Proteins within Synthetic Polymer Membranes with Large Hydrophobic Mismatch.

The functioning of biological membrane proteins (MPs) within synthetic block copolymer membranes is an intriguing phenomenon that is believed to offer great potential for applications in life and medical sciences and engineering. The question why biological MPs are able to function in this completely artificial environment is still unresolved by any experimental data. Here, we have analyzed the lateral diffusion properties of different sized MPs within poly(dimethylsiloxane) (PDMS)-containing amphiphilic block copolymer membranes of membrane thicknesses between 9 and 13 nm, which results in a hydrophobic mismatch between the membrane thickness and the size of the proteins of 3.3-7.1 nm (3.5-5 times). We show that the high flexibility of PDMS, which provides membrane fluidities similar to phospholipid bilayers, is the key-factor for MP incorporation.

Does Membrane Thickness Affect the Transport of Selective Ions Mediated by Ionophores in Synthetic Membranes?

Biomimetic polymer nanocompartments (polymersomes) with preserved architecture and ion-selective membrane permeability represent cutting-edge mimics of cellular compartmentalization. Here it is studied whether the membrane thickness affects the functionality of ionophores in respect to the transport of Ca2+ ions in synthetic membranes of polymersomes, which are up to 2.6 times thicker than lipid membranes (5 nm). Selective permeability toward calcium ions is achieved by proper insertion of ionomycin, and demonstrated by using specific fluorescence markers encapsulated in their inner cavities. Preservation of polymersome architecture is shown by a combination of light scattering, transmission electron microscopy, and fluorescence spectroscopy. By using a combination of stopped-flow and fluorescence spectroscopy, it is shown that ionomycin can function and transport calcium ions across polymer membranes with thicknesses in the range 10.7-13.4 nm (7.1-8.9 times larger than the size of the ionophore). Thicker membranes induce a decrease in transport, but do not block it due to the intrinsic flexibility of these synthetic membranes. The design of ion selective biomimetic nanocompartments represents a new path toward the development of cellular ion nanosensors and nano-reactors, in which calcium sensitive biomacromolecules can be triggered for specific biological functions.

Selective ion-permeable membranes by insertion of biopores into polymersomes.

In nature there are various specific reactions for which highly selective detection or support is required to preserve their bio-specificity or/and functionality. In this respect, mimics of cell membranes and bio-compartments are essential for developing tailored applications in therapeutic diagnostics. Being inspired by nature, we present here biomimetic nanocompartments with ion-selective membrane permeability engineered by insertion of ionomycin into polymersomes with sizes less than 250 nm. As a marker to assess the proper insertion and functionality of ionomycin inside the synthetic membrane, we used a Ca(2+)-sensitive dye encapsulated inside the polymersome cavity prior to inserting the biopore. The calcium sensitive dye, ionomycin, and Ca(2+) did not influence the architecture and the size of polymersomes. Successful ionomycin functionality inside the synthetic membrane with a thickness of 10.7 nm was established by a combination of fluorescence spectroscopy and stopped-flow spectroscopy. Polymersomes equipped with ion selective membranes are ideal candidates for the development of medical applications, such as cellular ion nanosensors or nanoreactors in which ion exchange is required to support in situ reactions.

Dual Functional Antibacterial-Antioxidant Core/Shell Alginate/Poly(ε-caprolactone) Nanofiber Membrane: A Potential Wound Dressing.

Core/shell nanofibers offer the advantage of encapsulating multiple drugs with different hydrophilicity in the core and shell, thus allowing for the controlled release of pharmaceutic agents. Specifically, the burst release of hydrophilic drugs from such fiber membranes causes an instantaneous high drug concentration, whereas a long and steady release is usually desired. Herein, we tackle the problem of the initial burst release by the generation of core/shell nanofibers with the hydrophilic antibiotic drug gentamycin loaded within a hydrophilic alginate core surrounded by a hydrophobic shell of poly(ε-caprolactone). Emulsion electrospinning was used as the nanofibrous mesh generation procedure. This process also allows for the loading of a hydrophobic compound, where we selected a natural antioxidant molecule, betulin (BTL), to detoxify the radicals. The resulting nanofibers exhibited a cylindrical shape with a core/shell structure. In vitro tests showed a controlled release of gentamicin from nanofibers via diffusion. The drug reached 93% release in an alginate hydrogel film but only 50% release in the nanofibers, suggesting its potential to minimize the initial burst release. Antibacterial tests revealed significant activity against both Gram-negative and Gram-positive bacteria. The antioxidant property of betulin was confirmed through the DPPH assay, where the incorporation of 20% BTL revealed 37.3% DPPH scavenging. The nanofibers also exhibited favorable biocompatibility in cell culture studies, and no harmful effects on cell viability were observed. Overall, this research offers a promising approach to producing core/shell nanofibrous mats with antibacterial and antioxidant properties, which could effectively address the requirements of wound dressings, including infection prevention and wound healing acceleration.

The Effect of Substrate Properties on Cellular Behavior and Nanoparticle Uptake in Human Fibroblasts and Epithelial Cells.

The delivery of nanomedicines into cells holds enormous therapeutic potential; however little is known regarding how the extracellular matrix (ECM) can influence cell-nanoparticle (NP) interactions. Changes in ECM organization and composition occur in several pathophysiological states, including fibrosis and tumorigenesis, and may contribute to disease progression. We show that the physical characteristics of cellular substrates, that more closely resemble the ECM in vivo, can influence cell behavior and the subsequent uptake of NPs. Electrospinning was used to create two different substrates made of soft polyurethane (PU) with aligned and non-aligned nanofibers to recapitulate the ECM in two different states. To investigate the impact of cell-substrate interaction, A549 lung epithelial cells and MRC-5 lung fibroblasts were cultured on soft PU membranes with different alignments and compared against stiff tissue culture plastic (TCP)/glass. Both cell types could attach and grow on both PU membranes with no signs of cytotoxicity but with increased cytokine release compared with cells on the TCP. The uptake of silica NPs increased more than three-fold in fibroblasts but not in epithelial cells cultured on both membranes. This study demonstrates that cell-matrix interaction is substrate and cell-type dependent and highlights the importance of considering the ECM and tissue mechanical properties when designing NPs for effective cell targeting and treatment.

CO2 permeability of cell membranes is regulated by membrane cholesterol and protein gas channels.

Recent observations that some membrane proteins act as gas channels seem surprising in view of the classical concept that membranes generally are highly permeable to gases. Here, we study the gas permeability of membranes for the case of CO(2), using a previously established mass spectrometric technique. We first show that biological membranes lacking protein gas channels but containing normal amounts of cholesterol (30-50 mol% of total lipid), e.g., MDCK and tsA201 cells, in fact possess an unexpectedly low CO(2) permeability (P(CO2)) of ∼0.01 cm/s, which is 2 orders of magnitude lower than the P(CO2) of pure planar phospholipid bilayers (∼1 cm/s). Phospholipid vesicles enriched with similar amounts of cholesterol also exhibit P(CO2) ≈ 0.01 cm/s, identifying cholesterol as the major determinant of membrane P(CO2). This is confirmed by the demonstration that MDCK cells depleted of or enriched with membrane cholesterol show dramatic increases or decreases in P(CO2), respectively. We demonstrate, furthermore, that reconstitution of human AQP-1 into cholesterol-containing vesicles, as well as expression of human AQP-1 in MDCK cells, leads to drastic increases in P(CO2), indicating that gas channels are of high functional significance for gas transfer across membranes of low intrinsic gas permeability.

Protein-polymer nanoreactors for medical applications.

Major challenges that confront nanoscience in medicine today include the development of efficacious therapies with minimum side effects, diagnostic methods featuring significantly higher sensitivities and selectivities, and personalized diagnostics and therapeutics for theragnostic approaches. With these goals in mind, combining biological molecules and synthetic carriers/templates, such as polymer supramolecular assemblies, represents a very promising strategy. In this critical review, we present protein-polymer systems as reaction spaces at the nano-scale in which the enzymatic reactions take place inside polymer supramolecular assembly, at its interface with the environment or in a combination of both. The location of the protein(s) with respect to the polymer assembly generates a diversity of systems ranging from nanoreactors to active enzymatic polymer surfaces. We describe these both in terms of general modelling and addressing the specific conditions and requirements related to the medical domain. We will particularly present protein-polymer nanoreactors that provide protected spaces for enzymatic reactions. We also show how polymer supramolecular structures, such as micelles, capsules, dendrimers and vesicles, can accommodate sensitive biomolecules to mimic natural systems and functions, and to serve as avenues for new medical approaches. Even though not yet on the market, we will emphasize possible applications of protein-polymer systems that generate reaction nanospaces-as novel ways to advanced medicine (264 references).

Functional Fiber Membranes with Antibacterial Properties for Face Masks.

Reusable face masks are an important alternative for minimizing costs of disposable and surgical face masks during pandemics. Often complementary to washing, a prolonged lifetime of face masks relies on the incorporation of self-cleaning materials. The development of self-cleaning face mask materials requires the presence of a durable catalyst to deactivate contaminants and microbes after long-term use without reducing filtration efficiency. Herein, we generate self-cleaning fibers by functionalizing silicone-based (polydimethylsiloxane, PDMS) fibrous membranes with a photocatalyst. Coaxial electrospinning is performed to fabricate fibers with a non-crosslinked silicone core within a supporting shell scaffold, followed by thermal crosslinking and removal of the water-soluble shell. Photocatalytic zinc oxide nanoparticles (ZnO NPs) are immobilized on the PDMS fibers by colloid-electrospinning or post-functionalization procedures. The fibers functionalized with ZnO NPs can degrade a photo-sensitive dye and display antibacterial properties against Gram-positive and Gram-negative bacteria (Escherichia coli and Staphylococcus aureus) due to the generation of reactive oxygen species upon irradiation with UV light. Furthermore, a single layer of functionalized fibrous membrane shows an air permeability in the range of 80-180 L/m2s and 65% filtration efficiency against fine particulate matter with a diameter less than 1.0 µm (PM1.0). Supplementary Information: The online version contains supplementary material available at 10.1007/s42765-023-00291-7.

Emulsion electrospinning of sodium alginate/poly(ε-caprolactone) core/shell nanofibers for biomedical applications.

Electrospun nanofibers have shown great potential as drug vehicles and tissue engineering scaffolds. However, the successful encapsulation of multiple hydrophilic/hydrophobic therapeutic compounds is still challenging. Herein, sodium alginate/poly(ε-caprolactone) core/shell nanofibers were fabricated via water-in-oil emulsion electrospinning. The sodium alginate concentration, water-to-oil ratio, and surfactant concentration were optimized for the maximum stability of the emulsion. The results demonstrated that an increasing water-to-oil ratio results in more deviation from Newtonian fluid and leads to a broader distribution of the fibers' diameters. Moreover, increasing poly(ε-caprolactone) concentration increases loss and storage moduli and increases the diameter of the resulting fibers. The nanofibers' characteristics were investigated by scanning electron microscopy, transmission electron microscopy, confocal laser scanning microscopy, Fourier transform infrared spectroscopy, X-ray diffraction, and water contact angle measurements. It was observed that using an emulsion composition of 10% (w/v) PCL and a water-to-oil ratio of 0.1 results in smooth, cylindrical, and uniform core/shell nanofibers with PCL in the shell and ALG in the core. The in vitro cell culture study demonstrated the favorable biocompatibility of nanofibers. Overall, this study provides a promising and trustworthy material for biomedical applications.

pH-Responsive Chitosan/Alginate Polyelectrolyte Complexes on Electrospun PLGA Nanofibers for Controlled Drug Release.

The surface functionalization of electrospun nanofibers allows for the introduction of additional functionalities while at the same time retaining the membrane properties of high porosity and surface-to-volume ratio. In this work, we sequentially deposited layers of chitosan and alginate to form a polyelectrolyte complex via layer-by-layer assembly on PLGA nanofibers to introduce pH-responsiveness for the controlled release of ibuprofen. The deposition of the polysaccharides on the surface of the fibers was revealed using spectroscopy techniques and ζ-potential measurements. The presence of polycationic chitosan resulted in a positive surface charge (16.2 ± 4.2 mV, pH 3.0) directly regulating the interactions between a model drug (ibuprofen) loaded within the polyelectrolyte complex and the layer-by-layer coating. The release of ibuprofen was slowed down in acidic pH (1.0) compared to neutral pH as a result of the interactions between the drug and the coating. The provided mesh acts as a promising candidate for the design of drug delivery systems required to bypass the acidic environment of the digestive tract.

Tailoring the multiscale architecture of electrospun membranes to promote 3D cellular infiltration.

Controlling the architecture of engineered scaffolds is of outmost importance to induce a targeted cell response and ultimately achieve successful tissue regeneration upon implantation. Robust, reliable and reproducible methods to control scaffold properties at different levels are timely and highly important. However, the multiscale architectural properties of electrospun membranes are very complex, in particular the role of fiber-to-fiber interactions on mechanical properties, and their effect on cell response remain largely unexplored. The work reported here reveals that the macroscopic membrane stiffness, observed by stress-strain curves, cannot be predicted solely based on the Young's moduli of the constituting fibers but is rather influenced by interactions on the microscale, namely the number of fiber-to-fiber bonds. To specifically control the formation of these bonds, solvent systems of the electrospinning solution were fine-tuned, affecting the membrane properties at every length-scale investigated. In contrast to dichloromethane that is characterized by a high vapor pressure, the use of trifluoroacetic acid, a solvent with a lower vapor pressure, favors the generation of fiber-to-fiber bonds. This ultimately led to an overall increased Young's modulus and yield stress of the membrane despite a lower stiffness of the constituting fibers. With respect to tissue engineering applications, an experimental setup was developed to investigate the effect of architectural parameters on the ability of cells to infiltrate and migrate within the scaffold. The results reveal that differences in fiber-to-fiber bonds significantly affect the infiltration of normal human dermal fibroblasts into the membranes. Membranes of loose fibers with low numbers of fiber-to-fiber bonds, as obtained from spinning solutions using dichloromethane, promote cellular infiltration and are thus promising candidates for the formation of a 3D tissue.

Hybrid vesicles as intracellular reactive oxygen species and nitric oxide generators.

Artificial organelles are envisioned as nanosized assemblies with intracellular biocatalytic activity to provide the host cells with non-native or missing/lost function. Hybrid vesicles loaded with glucose oxidase (NRGOx) or ß-galactosidase (NRß-Gal) and equipped with lysosomal escape ability are assembled using phospholipids and the block copolymer poly(cholesteryl methacrylate)-block-poly(2-(dimethylamino)ethyl methacrylate). The co-localization of the building blocks and the catalytic activity of NRGOx and NRß-Gal are illustrated. The intracellular activity of the nanoreactors in RAW 264.7 macrophages is confirmed by an enhanced reduction in viability for cells pre-incubated with NRGOx in the presence of glucose due to the generation of cytotoxic hydrogen peroxide compared to the controls. In addition, RAW 264.7 macrophages and primary human macrophages equipped with NRß-Gal are able to intracellularly convert ß-Gal-NONOate into nitric oxide. The successful use of these hybrid vesicles to equip host macrophages with additional catalytic activity diversifies the available toolbox of nanocarriers with envisioned application in cell mimicry.

Matrix Vesicles-Containing Microreactors as Support for Bonelike Osteoblasts to Enhance Biomineralization.

Therapeutic cell mimicry aims to provide a source of cell-like assemblies, which exhibit the core structural or functional properties of their natural counterparts with broad envisioned applications in biomedicine. Bone tissue engineering (BTE) aims at promoting and inciting the natural healing process of, for instance, critically sized bone defects. Microreactors designed to co-assemble with biological bone-forming osteoblasts like SaOS-2 cells to start biomineralization are reported for the first time. The alginate-based microparticles are equipped with active alkaline phosphatase-loaded artificial liposomes or SaOS-2-derived matrix vesicles (MVs). Spheroids assembled from SaOS-2 cells and microreactors not only exhibit higher cell viability, but also show enhanced biomineralization when MVs are present. The active biomineralization stimulation of the microreactors is illustrated by colorimetric calcium quantification and micro-computed tomography. These findings show the promising potential of applying cell mimicry in BTE.

Planar and Cell Aggregate-Like Assemblies Consisting of Microreactors and HepG2 Cells.

The assembly of microreactors has made considerable progress toward the fabrication of artificial cells. However, their characterization remains largely limited to buffer solution-based assays in the absence of their natural role model-the biological cells. Herein, the combination of microreactors with HepG2 cells either in planar cell cultures or in the form of cell aggregates is reported. Alginate (Alg)-based microreactors loaded with catalase are assembled by droplet microfluidics, and their activity is confirmed. The acceptance of polymer-coated ∼40 µm Alg particles by proliferating HepG2 cells is depending on the terminating polymer layer. When these functional microreactors are cocultured with HepG2 cells, they can be employed for detoxification, that is, hydrogen peroxide removal, and by doing so, they assist the cells to survive. This report is among the first successful combination of microreactors with biological cells, that is, HepG2 cells, contributing to the fundamental understanding of integrating synthetic and biological partners toward the maturation of this semisynthetic concept for biomedical applications.

Enzymes as key features in therapeutic cell mimicry.

Cell mimicry is a nature inspired concept that aims to substitute for missing or lost (sub)cellular function. This review focuses on the latest advancements in the use of enzymes in cell mimicry for encapsulated catalysis and artificial motility in synthetic bottom-up assemblies with emphasis on the biological response in cell culture or more rarely in animal models. Entities across the length scale from nano-sized enzyme mimics, sub-micron sized artificial organelles and self-propelled particles (swimmers) to micron-sized artificial cells are discussed. Although the field remains in its infancy, the primary aim of this review is to illustrate the advent of nature-mimicking artificial molecules and assemblies on their way to become a complementary alternative to their role models for diverse biomedical purposes.