Closing the Gap between Experiment and Simulation─A Holistic Study on the Complexation of Small Interfering RNAs with Polyethylenimine.

Rational design is pivotal in the modern development of nucleic acid nanocarrier systems. With the rising prominence of polymeric materials as alternatives to lipid-based carriers, understanding their structure-function relationships becomes paramount. Here, we introduce a newly developed coarse-grained model of polyethylenimine (PEI) based on the Martini 3 force field. This model facilitates molecular dynamics simulations of true-sized PEI molecules, exemplified by molecules with molecular weights of 1.3, 5, 10, and 25 kDa, with degrees of branching between 50.0 and 61.5%. We employed this model to investigate the thermodynamics of small interfering RNA (siRNA) complexation with PEI. Our simulations underscore the pivotal role of electrostatic interactions in the complexation process. Thermodynamic analyses revealed a stronger binding affinity with increased protonation, notably in acidic (endosomal) pH, compared to neutral conditions. Furthermore, the molecular weight of PEI was found to be a critical determinant of binding dynamics: smaller PEI molecules closely enveloped the siRNA, whereas larger ones extended outward, facilitating the formation of complexes with multiple RNA molecules. Experimental validations, encompassing isothermal titration calorimetry and single-molecule fluorescence spectroscopy, aligned well with our computational predictions. Our findings not only validate the fidelity of our PEI model but also accentuate the importance of in silico data in the rational design of polymeric drug carriers. The synergy between computational predictions and experimental validations, as showcased here, signals a refined and precise approach to drug carrier design.

Regulating Chemically Fueled Peptide Assemblies by Molecular Design.

In living systems, fuel-driven assembly is ubiquitous, and examples include the formation of microtubules or actin bundles. These structures have inspired researchers to develop synthetic counterparts, leading to exciting new behaviors in man-made structures. However, most of these examples are serendipitous discoveries because clear design rules do not yet exist. In this work, we show design rules to drive peptide self-assembly regulated by a fuel-driven reaction cycle. We demonstrate that, by altering the ratio of attractive to repulsive interactions between peptides, the behavior can be toggled between no assembly, fuel-driven dissipative self-assembly, and a state in which the system is permanently assembled. These rules can be generalized for other peptide sequences. In addition, our finding is explained in the context of the energy landscapes of self-assembly. We anticipate that our design rules can further aid the field and help the development of autonomous materials with life-like properties.

Reversible Condensation of Mucins into Nanoparticles.

Mucins are high molar mass glycoproteins that assume an extended conformation and can assemble into mucus hydrogels that protect our mucosal epithelium. In nature, the challenging task of generating a mucus layer, several hundreds of micrometers in thickness, from micrometer-sized cells is elegantly solved by the condensation of mucins inside vesicles and their on-demand release from the cells where they suddenly expand to form the extracellular mucus hydrogel. We aimed to recreate and control the process of compaction for mucins, the first step toward a better understanding of the process and creating biomimetic in vivo delivery strategies of macromolecules. We found that by adding glycerol to the aqueous solvent, we could induce drastic condensation of purified mucin molecules, reducing their size by an order of magnitude down to tens of nanometers in diameter. The condensation effect of glycerol was fully reversible and could be further enhanced and partially stabilized by cationic cross-linkers such as calcium and polylysine. The change of structure of mucins from extended molecules to nano-sized particles in the presence of glycerol translated into macroscopic rheological changes, as illustrated by a dampened shear-thinning effect with increasing glycerol concentration. This work provides new insight into mucin condensation, which could lead to new delivery strategies mimicking cell release of macromolecules condensed in vesicles such as mucins and heparin.

Efficient and Targeted siRNA Delivery to M2 Macrophages by Smart Polymer Blends for M1 Macrophage Repolarization as a Promising Strategy for Future Cancer Treatment.

Cancer remains an issue on a global scale. It is estimated that nearly 10 million people succumbed to cancer worldwide in 2020. New treatment options are urgently needed. A promising approach is a conversion of tumor-promoting M2 tumor-associated macrophages (TAMs) as part of the tumor microenvironment to tumor-suppressive M1 TAMs by small interfering RNA (siRNA). In this work, we present a well-characterized polymeric nanocarrier system capable of targeting M2 TAMs by a ligand-receptor interaction. Therefore, we developed a blended PEI-based polymeric nanoparticle system conjugated with mannose, which is internalized after interaction with macrophage mannose receptors (MMRs), showing low cytotoxicity and negligible IL-6 activation. The PEI-PCL-PEI (5 kDa-5 kDa-5 kDa) and Man-PEG-PCL (2 kDa-2 kDa) blended siRNA delivery system was optimized for maximum targeting capability and efficient endosomal escape by evaluation of different polymer and N/P ratios. The nanoparticles were formulated by surface acoustic wave-assisted microfluidics, achieving a size of ∼80 nm and a zeta potential of approximately +10 mV. Special attention was given to the endosomal escape as the so-called bottleneck of RNA drug delivery. To estimate the endosomal escape capability of the nanocarrier system, we developed a prediction method by evaluating the particle stability via the inflection temperature. Our predictions were then verified in an in vitro setting by applying confocal microscopy. For cellular experiments, however, human THP-1 cells were polarized to M2 macrophages by cytokine treatment and validated through MMR expression. To show the efficiency of the nanoparticle system, GAPDH and IκBα knockdown was performed in the presence or absence of an MMR blocking excess of mannan. Cellular uptake, GAPDH knockdown, and NF-κB western blot confirmed efficient mannose targeting. Herein, we presented a well-characterized nanoparticle delivery system and a promising approach for targeting M2 macrophages by a mannose-MMR interaction.

Engineering poly- and micelleplexes for nucleic acid delivery - A reflection on their endosomal escape.

For the longest time, the field of nucleic acid delivery has remained skeptical whether or not polycationic drug carrier systems would ever make it into clinical practice. Yet, with the disclosure of patents on polyethyleneimine-based RNA carriers through leading companies in the field of nucleic acid therapeutics such as BioNTech SE and the progress in clinical studies beyond phase I trials, this aloofness seems to regress. As one of the most striking characteristics of polymer-based vectors, the extraordinary tunability can be both a blessing and a curse. Yet, knowing about the adjustment screws and how they impact the performance of the drug carrier provides the formulation scientist committed to its development with a head start. Here, we equip the reader with a toolbox - a toolbox that should advise and support the developer to conceptualize a cutting-edge poly- or micelleplex system for the delivery of therapeutic nucleic acids; to be specific, to engineer the vector towards maximum endosomal escape performance at minimum toxicity. Therefore, after briefly sketching the boundary conditions of polymeric vector design, we will dive into the topic of endosomal trafficking. We will not only discuss the most recent knowledge of the endo-lysosomal compartment but further depict different hypotheses and mechanisms that facilitate the endosomal escape of polyplex systems. Finally, we will combine the different facets introduced in the previous chapters with the fundamental building blocks of polymer vector design and evaluate the advantages and drawbacks. Throughout the article, a particular focus will be placed on cellular peculiarities, not only as an additional barrier, but also to give inspiration to how such cell-specific traits might be capitalized on.

Recent advances in superlubricity of liposomes for biomedical applications.

Achieving superlubricity, a state of lubrication where friction nearly vanishes, has become one of the most promising approaches to combat friction-induced energy dissipation and medical device failure. Phospholipids are amphiphilic molecules comprising highly hydrophilic phosphatidylcholine head groups as well as hydrophobic hydrocarbon chains, When solubilized, phospholipids can readily self-assemble to form different structures such as bilayers and vesicles (liposomes). Recently, liposomes have been identified as excellent lubricants, especially in the boundary lubrication regime the most common lubrication status in the field of biotribology. In this review, we summarize recent progress in employing liposomes as key players for employing superlubricity in biomedical applications. The relationship between lipids and liposomes, manufacturing approaches, lubrication regimes, and regulation mechanisms of liposomes are discussed. Finally, we indicate possible future directions for the use of liposome-mediated superlubricity in biomedical applications.

Biopolymer-based nanoparticles with tunable mucoadhesivity efficiently deliver therapeutics across the corneal barrier.

To overcome the natural barriers of the ocular system that limit the topical delivery of therapeutically active molecules to the posterior eye, nanoscale drug carriers can be used to improve transcorneal drug transport. So far, using mucoadhesive drug carriers has been put forward as the most promising strategy to optimize drug transport. However, if the mucoadhesivity of a drug carrier is too high, this might limit the diffusive entry of molecules/drug carriers into the vitreous. In this study, we show how modulating the net charge of biopolymer-based drug carrier particles alters not only their mucoadhesivity but also other important properties, e.g., their stability, drug loading capacity and drug release profiles. Compared to simple aqueous solutions of free drug molecules as used in current treatments, nanoparticulate drug carriers with intermediate mucoadhesivity show improved drug transport across the corneal barrier. Therefore, our study shows that mucoadhesion of drug carrier particles is a feature that needs to be considered with great care - not only for ocular delivery attempts but for all drug delivery approaches dealing with mucosal barriers.

Synthesis and characterization of chemically fueled supramolecular materials driven by carbodiimide-based fuels.

Many supramolecular materials in biological systems are driven to a nonequilibrium state by the irreversible consumption of high-energy molecules such as ATP or GTP. As a result, they exhibit unique dynamic properties such as a tunable lifetime, adaptivity or the ability to self-heal. In contrast, synthetic counterparts that exist in or close to equilibrium are controlled by thermodynamic parameters and therefore lack these dynamic properties. To mimic biological materials more closely, synthetic self-assembling systems have been developed that are driven out of equilibrium by chemical reactions. This protocol describes the synthesis and characterization of such an assembly, which is driven by carbodiimide fuels. Depending on the amount of chemical fuel added to the material, its lifetime can be tuned. In the first step, the protocol details the synthesis and purification of the peptide-based precursors for the fuel-driven assemblies by solid-phase peptide synthesis. Then, we explain how to analyze the kinetic response of the precursors to a carbodiimide-based chemical fuel by HPLC and kinetic models. Finally, we detail how to study the emerging assembly's macro- and microscopic properties by time-lapse photography, UV-visible spectroscopy, shear rheology, confocal laser scanning microscopy and electron microscopy. The procedure is described using the example of a colloid-forming precursor Fmoc-E-OH and a fiber-forming precursor Fmoc-AAD-OH to emphasize the differences in characterization depending on the type of assembly. The characterization of a precursor's transient assembly can be done within 5 d. The synthesis and purification of a peptide precursor requires 2 d of work.

Glyco-Modification of Mucin Hydrogels to Investigate Their Immune Activity.

Mucins are multifunctional glycosylated proteins that are increasingly investigated as building blocks of novel biomaterials. An attractive feature is their ability to modulate the immune response, in part by engaging with sialic acid binding receptors on immune cells. Once assembled into hydrogels, bovine submaxillary mucins (Muc gels) were shown to modulate the recruitment and activation of immune cells and avoid fibrous encapsulation in vivo. However, nothing is known about the early immune response to Muc gels. This study characterizes the response of macrophages, important orchestrators of the material-mediated immune response, over the first 7 days in contact with Muc gels. The role of mucin-bound sialic acid sugar residues was investigated by first enzymatically cleaving the sugar and then assembling the mucin variants into covalently cross-linked hydrogels with rheological and surface nanomechanical properties similar to nonmodified Muc gels. Results with THP-1 and human primary peripheral blood monocytes derived macrophages showed that Muc gels transiently activate the expression of both pro-inflammatory and anti-inflammatory cytokines and cell surface markers, for most makers with a maximum on the first day and loss of the effect after 7 days. The activation was sialic acid-dependent for a majority of the markers followed. The pattern of gene expression, protein expression, and functional measurements did not strictly correspond to M1 or M2 macrophage phenotypes. This study highlights the complex early events in macrophage activation in contact with mucin materials and the importance of sialic acid residues in such a response. The enzymatic glyco-modulation of Muc gels appears as a useful tool to help understand the biological functions of specific glycans on mucins which can further inform on their use in various biomedical applications.

The Lubricity of Mucin Solutions Is Robust toward Changes in Physiological Conditions.

Solutions of manually purified gastric mucins have been shown to be promising lubricants for biomedical purposes, where they can efficiently reduce friction and wear. However, so far, such mucin solutions have been mostly tested in specific tribological settings, i.e., in combination with different material pairings; variations in the composition of the lubricating fluid have not been systematically explored yet. We here fill this gap and determine the viscosity, adsorption behavior, and lubricity of porcine gastric mucin solutions on hydrophobic surfaces at different pH levels, mucin and salt concentrations, and in the presence of other proteins. We demonstrate that mucin solutions provide excellent lubricity even at very low concentrations of 0.01% (w/v), over a broad range of pH levels and even at an elevated ionic strength. Furthermore, we provide mechanistic insights into mucin lubricity, which help explain how certain variations in physiologically relevant parameters can limit the lubricating potential of mucin solutions. Our results show that solutions of manually purified mucin solutions can be powerful biomedical lubricants, e.g., serving as artificial joint fluids for viscosupplementation, as eye drops or mouth spray, or as a personal lubricant for intercourse.

Relation between structural, mechanical and sensory properties of gluten-free bread as affected by modified dietary fibers.

Gluten-free bread was fortified with modified dietary fibers (wheat bran, resistant starch and inulin) and their effects on water mobility, friction coefficient, thermal behavior, crystalline pattern and textural properties were evaluated. Moreover, time-intensity evaluation was used to study temporal dynamics of sensory attributes of fortified-breads. Dietary fibers increased gelatinization temperature while decreasing gelatinization enthalpy, more notably when inulin was used. X-ray diffraction patterns of bread showed the appearance of new peaks after addition of resistant starch and wheat bran, coinciding with an increase in crumb hardness. In contrast, inulin considerably decreased starch crystallinity in the bread, resulting in a softer crumb. Faster decay and shifting of protons to shorter times were found with incorporation of dietary fibers. Friction coefficient determined by tribology measurement was higher in the breads containing resistant starch and wheat bran compared to other samples. Pearson's correlation analysis indicated the sensory attributes of firmness, chewiness and dryness were positively correlated with instrumental findings. Time-intensity evaluation revealed inulin-fortified bread had the lowest firmness and chewiness with less dryness, whereas resistant starch-fortified bread showed the highest intensity of these descriptors.

Macromolecular Coating Enables Tunable Selectivity in a Porous PDMS Matrix.

Whether for laboratory use or clinical practice, many fields in Life Sciences require selective filtering. However, most existing filter systems lack the ability to easily tune their filtration behavior. Two key elements for efficient filtering are a high surface-to-volume ratio and the presence of suitable chemical groups which establish selectivity. In this study, an artificial PDMS-based capillary system with highly tunable selectivity properties is presented. The high surface-to-volume ratio of this filter system is generated by first embedding sugar fibers into a synthetic polymer matrix and then dissolving these fibers from the cured polymer. To functionalize this filter, the inner surface of the capillaries is coated with purified or synthetic macromolecules. Depending on the type of macromolecule used for filter functionalization, selective sieving is observed based on steric hindrance, electrostatic binding, electrostatic repulsion, or specific binding interactions. Furthermore, it is demonstrated that enzymes can be immobilized in the capillary system which allows for performing multiple cycles of enzymatic reactions with the same batch of enzymes and without the need to separate the enzymes from their reaction products. In addition to lab-scale filtration and enzyme immobilization applications demonstrated here, the functionalized porous PDMS matrix may also be used to test binding interactions between different molecules.