Synthetic molecular motor activates drug delivery from polymersomes.

The design of stimuli-responsive systems in nanomedicine arises from the challenges associated with the unsolved needs of current molecular drug delivery. Here, we present a delivery system with high spatiotemporal control and tunable release profiles. The design is based on the combination of an hydrophobic synthetic molecular rotary motor and a PDMS-b-PMOXA diblock copolymer to create a responsive self-assembled system. The successful incorporation and selective activation by low-power visible light (λ = 430 nm, 6.9 mW) allowed to trigger the delivery of a fluorescent dye with high efficiencies (up to 75%). Moreover, we proved the ability to turn on and off the responsive behavior on demand over sequential cycles. Low concentrations of photoresponsive units (down to 1 mol% of molecular motor) are shown to effectively promote release. Our system was also tested under relevant physiological conditions using a lung cancer cell line and the encapsulation of an Food and Drug Administration (FDA)-approved drug. Similar levels of cell viability are observed compared to the free given drug showing the potential of our platform to deliver functional drugs on request with high efficiency. This work provides an important step for the application of synthetic molecular machines in the next generation of smart delivery systems.

Colloidal Structure Dictates Antimicrobial Efficacy in LL-37 Self-Assemblies With Glycerol Monooleate.

The antimicrobial peptide LL-37 is a promising alternative to conventional antibiotics to combat bacteria in suspension and biofilms. Its self-assembly with polar lipids is suggested to improve its potential for therapeutic applications with higher stability against degradation and bioavailability. This study investigates the self-assembly of LL-37 with glyceryl monooleate (GMO), establishing the link between colloidal structure and antimicrobial activity. Small-angle X-ray scattering, dynamic light scattering and cryogenic transmission electron microscopy show structural transformation from dispersions of inverse bicontinuous structure (cubosomes) to multilamellar vesicles and direct rod-like mixed-micelles upon increasing the content of LL-37 in GMO. In vitro assays against planktonic and biofilm cells demonstrate that 128 µg mL-1 of GMO cubosomes have no impact on Pseudomonas aeruginosa. Still, the cubosomes reduce the Staphylococcus aureus planktonic population by ≈ 1-log after 24 h. Cylindrical micelles formed at LL-37/GMO 9/1 and 8/2 with 128 µg mL-1 LL-37 decrease the Pseudomonas aeruginosa population by 6-log. This activity is gradually abolished when LL-37 is encapsulated in vesicles or cubosomes. They also demonstrate low antibiofilm efficacy and promote the biomass of Staphylococcus aureus biofilms. These results highlight the importance of colloidal structure for therapeutic outcomes, providing insights for advanced lipid nanocarrier designs.

Ex Situ Covalent Functionalization of Germanene via 1,3-Dipolar Cycloaddition: A Promising Approach for the Bandgap Engineering of Group-14 Xenes.

Group-14 Xenes beyond graphene such as silicene, germanene, and stanene have recently gained a lot of attention for their peculiar electronic properties, which can be tuned by covalent functionalization. Up until now, reported methods for the top-down synthesis of covalently functionalized silicene and germanene typically yield multilayered flakes with a minimum thickness of 100 nm. Herein, the ex situ covalent functionalization of germanene (fGe) is reported via 1,3-dipolar cycloaddition of the azomethine ylide generated by the decarboxylative condensation of 3,4-dihydroxybenzaldehyde and sarcosine. Amorphous few-layered sheets (average thickness of ≈6 nm) of dipolarophile germanene (GeX) are produced by thermal dehydrogenation of its fully saturated parent precursor, germanane (GeH). Spectroscopic evidence confirmed the emergence of the dipolarophilic sp2 domains due to the dehydrogenation of germanane, and their sp3 hybridization due to the covalent functionalization of germanene. Elemental mapping of the functionalized germanene revealed flakes with a very high abundance of carbon uniformly covering the germanium backbone. The 500 meV increase of the optical bandgap of germanene observed upon functionalization paves the way toward bandgap engineering of other group-14 Xenes, which could potentially be a major turning point in the fields of electronics, electrocatalysis, and photocatalysis.

Lowest gelation concentration in a complex-coacervate-driven self-assembly system, achieved by redox-RAFT synthesis of high molecular weight block polyelectrolytes.

The objective of this work was to synthesize high molecular weight polyelectrolyte complex (PEC) micelles that are effective in controlling the rheology of aqueous solutions at low concentrations, paving the way for industrial applications of thickeners based on the principle of electrostatic self-assembly. Redox-initiated RAFT (reversible addition-fragmentation chain-transfer) polymerization was used to obtain anionic block polyelectrolytes based on poly(sodium 2-acrylamido-2-methylpropane sulfonate) and poly(acrylamide)-poly(AMPS)-block-poly(AM) (di-block) and poly(AMPS)-block-poly(AM)-block-poly(AMPS) (tri-block), with molecular weights of 237 kDa and 289 kDa and polydispersities of 1.29 and 1.34, respectively. A random poly(AMPS)-co-poly(AM) copolymer was also synthesized for comparison. PEC micelles were obtained upon mixing with cationic poly(N-[3-(dimethylamino)propyl]methacrylamide hydrochloride) - poly(DMAPMA), forming viscoelastic gels at unprecedented low concentrations of <3 wt% for the di-block and <1 wt% for the tri-block, which to date is the lowest demonstrated gelation concentration for a synthetic PEC micelle system. Differences between tri-block and di-block architectures are discussed, with the former being more affected by the addition of salt, which is attributed to percolated network breakdown. The random co-polymer was shown not to be an effective thickener but displayed a surprising lack of phase separation upon coacervation. The assemblies were characterized using dynamic light scattering (DLS) and cryo transmission electron microscopy (cryoTEM), revealing spherical micelles with a diameter of approximately 200 nm for the diblock and a mixture of spherical micelles and network particles for the tri-block PEC micelles. The micelles were not affected by dilution down to a polymer concentration of 7.8 × 10-4% (approx. 0.03 µM). Responsiveness to salinity, pH, and temperature was studied using DLS, revealing a critical NaCl concentration of 1.1 M for the block copolymer micelles.

Aqueous Supramolecular Transformations of Motor Bola-Amphiphiles at Multiple Length-Scale.

Molecular motor amphiphiles have already been widely attempted for dynamic nanosystems across multiple length-scale for developments of small functional materials, including controlling macroscopic foam properties, amplifying motion as artificial molecular muscles, and serving as extracellular matrix mimicking cell scaffolds. However, limiting examples of bola-type molecular motor amphiphiles are considered for constructing macroscopic biomaterials. Herein, this work presents the designed two second generation molecular motor amphiphiles, motor bola-amphiphiles (MBAs). Aside from the photoinduced motor rotation of MBAs achieved in both organic and aqueous media, the rate of recovering thermal helix inversion step can be controlled by the rotor part with different steric hindrances. Dynamic assembled structures of MBAs are observed under (cryo)-transmission electron microscopy (TEM). This dynamicity assists MBAs in further assembling as macroscopic soft scaffolds by applying a shear-flow method. Upon photoirradiation, the phototropic bending function of MBA scaffolds is observed, demonstrating the amplification of molecular motion into macroscopic phototropic bending functions at the macroscopic length-scale. Since MBAs are confirmed with low cytotoxicity, human bone marrow-derived mesenchymal stem cells (hBM-MSCs) can grow on the surface of MBA scaffolds. These results clearly demonstrate the concept of designing MBAs for developing photoresponsive dynamic functional materials to create new-generation soft robotic systems and cell-material interfaces.

Light-Triggered Disassembly of Molecular Motor-based Supramolecular Polymers Revealed by High-Speed AFM.

Photoresponsive supramolecular polymers have a major potential for applications in responsive materials that are externally triggered by light with spatio-temporal control of their polymerisation state. While changes in macroscopic properties revealed the adaptive nature of these materials, it remains challenging to capture the dynamic depolymerisation process at the molecular level, which requires fast observation techniques combined with in situ irradiation. By implementing in situ UV illumination into a High-Speed Atomic Force Microscope (HS-AFM) setup, we have been able to capture the disassembly of a light-driven molecular motor-based supramolecular polymer. The real-time visualisation of the light-triggered disassembly process not only reveals cooperative depolymerisation, it also shows that this process continues after illumination is halted. Combining the data with cryo-electron microscopy and spectroscopy approaches, we obtain a molecular-level description of the motor-based polymer dynamics reminiscent of actin chain-end depolymerisation. Our detailed understanding of supramolecular depolymerisation will drive the development of future responsive polymer systems.

Molecular Engineering of the Kinetic Barrier in Seeded Supramolecular Polymerization.

Seeded supramolecular polymerization (SSP) is a method that enables the controlled synthesis of supramolecular structures. SSP often relies on structures that are capable of self-assembly by interconverting between intramolecular and intermolecular modes of hydrogen bonding, characterized by a given kinetic barrier that is typically low. The control of the polymerization process is thus limited by the propensity of the hydrogen bonds to interconvert between the intramolecular and intermolecular modes of binding. Here, we report on an engineering of the polymerization kinetic barriers by sophisticated molecular design of the building blocks involved in such SSP processes. Our designs include two types of intramolecular hydrogen-bonded rings: on one hand, a central triazine tricarboxamide moiety that prevents self-assembly due to its stable intramolecular hydrogen bonds and on the other hand, three peripheral amide groups that promote self-assembly due to their stable intermolecular hydrogen bonds. We report a series of molecules with increasing bulkiness of the peripheral side chains exhibiting increasing kinetic stability in the monomeric form. Owing to the relative height of the barrier, we were able to observe that the rate constant of seeding is not proportional to the concentration of the seeds used. Based on that, we proposed a new kinetic model in which the rate-determining step is the activation of the monomer, and we provide the detailed energy landscape of the supramolecular polymerization process. Finally, we investigated the hetero-seeding of the building blocks that shows either inhibition or triggering of the polymerization.

Role of Curvature-Sensing Proteins in the Uptake of Nanoparticles with Different Mechanical Properties.

Nanoparticles of different properties, such as size, charge, and rigidity, are used for drug delivery. Upon interaction with the cell membrane, because of their curvature, nanoparticles can bend the lipid bilayer. Recent results show that cellular proteins capable of sensing membrane curvature are involved in nanoparticle uptake; however, no information is yet available on whether nanoparticle mechanical properties also affect their activity. Here liposomes and liposome-coated silica are used as a model system to compare uptake and cell behavior of two nanoparticles of similar size and charge, but different mechanical properties. High-sensitivity flow cytometry, cryo-TEM, and fluorescence correlation spectroscopy confirm lipid deposition on the silica. Atomic force microscopy is used to quantify the deformation of individual nanoparticles at increasing imaging forces, confirming that the two nanoparticles display distinct mechanical properties. Uptake studies in HeLa and A549 cells indicate that liposome uptake is higher than for the liposome-coated silica. RNA interference studies to silence their expression show that different curvature-sensing proteins are involved in the uptake of both nanoparticles in both cell types. These results confirm that curvature-sensing proteins have a role in nanoparticle uptake, which is not restricted to harder nanoparticles, but includes softer nanomaterials commonly used for nanomedicine applications.

Motorized Photomodulator: Making A Non-photoresponsive Supramolecular Gel Switchable by Light.

Introducing photo-responsive molecules offers an attractive approach for remote and selective control and dynamic manipulation of material properties. However, it remains highly challenging how to use a minimal amount of photo-responsive units to optically modulate materials that are inherently inert to light irradiation. Here we show the application of a light-driven rotary molecular motor as a "motorized photo-modulator" to endow a typical H-bond-based gel system with the ability to respond to light irradiation and create a reversible sol-gel transition. The key molecular design feature is the introduction of a minimal amount (2 mol %) of molecular motors into the supramolecular network as photo-switchable non-covalent crosslinkers. Advantage is taken of the subtle interplay of the large geometry change during photo-isomerization of the molecular motor guest and the dynamic nature of a supramolecular gel host system. As a result, a tiny amount of molecular motors is enough to switch the mechanical modulus of the entire supramolecular systems. This study proves the concept of designing photo-responsive materials with minimum use of non-covalent light-absorbing units.

Photoactuating Artificial Muscles of Motor Amphiphiles as an Extracellular Matrix Mimetic Scaffold for Mesenchymal Stem Cells.

Mimicking the native extracellular matrix (ECM) as a cell culture scaffold has long attracted scientists from the perspective of supramolecular chemistry for potential application in regenerative medicine. However, the development of the next-generation synthetic materials that mimic key aspects of ECM, with hierarchically oriented supramolecular structures, which are simultaneously highly dynamic and responsive to external stimuli, remains a major challenge. Herein, we present supramolecular assemblies formed by motor amphiphiles (MAs), which mimic the structural features of the hydrogel nature of the ECM and additionally show intrinsic dynamic behavior that allow amplifying molecular motions to macroscopic muscle-like actuating functions induced by light. The supramolecular assembly (named artificial muscle) provides an attractive approach for developing responsive ECM mimetic scaffolds for human bone marrow-derived mesenchymal stem cells (hBM-MSCs). Detailed investigations on the photoisomerization by nuclear magnetic resonance and UV-vis absorption spectroscopy, assembled structures by electron microscopy, the photoactuation process, structural order by X-ray diffraction, and cytotoxicity are presented. Artificial muscles of MAs provide fast photoactuation in water based on the hierarchically anisotropic supramolecular structures and show no cytotoxicity. Particularly important, artificial muscles of MAs with adhered hBM-MSCs still can be actuated by external light stimulation, showing their ability to convert light energy into mechanical signals in biocompatible systems. As a proof-of-concept demonstration, these results provide the potential for building photoactuating ECM mimetic scaffolds by artificial muscle-like supramolecular assemblies based on MAs and offer opportunities for signal transduction in future biohybrid systems of cells and MAs.

Dynamic Control of a Multistate Chiral Supramolecular Polymer in Water.

Natural systems transfer chiral information across multiple length scales through dynamic supramolecular interaction to accomplish various functions. Inspired by nature, many exquisite artificial supramolecular systems have been developed, in which controlling the supramolecular chirality holds the key to completing specific tasks. However, to achieve precise and non-invasive control and modulation of chirality in these systems remains challenging. As a non-invasive stimulus, light can be used to remotely control the chirality with high spatiotemporal precision. In contrast to common molecular switches, a synthetic molecular motor can act as a multistate chiroptical switch with unidirectional rotation, offering major potential to regulate more complex functions. Here, we present a light-driven molecular motor-based supramolecular polymer, in which the intrinsic chirality is transferred to the nanofibers, and the rotation of molecular motors governs the chirality and morphology of the supramolecular polymer. The resulting supramolecular polymer also exhibits light-controlled multistate aggregation-induced emission. These findings present a photochemically tunable multistate dynamic supramolecular system in water and pave the way for developing molecular motor-driven chiroptical materials.

From Photoinduced Supramolecular Polymerization to Responsive Organogels.

Controlling supramolecular polymerization by external stimuli holds great potential toward the development of responsive soft materials and manipulating self-assembly at the nanoscale. Photochemical switching offers the prospect of regulating the structure and properties of systems in a noninvasive and reversible manner with spatial and temporal control. In addition, this approach will enhance our understanding of supramolecular polymerization mechanisms; however, the control of molecular assembly by light remains challenging. Here we present photoresponsive stiff-stilbene-based bis-urea monomers whose trans isomers readily form supramolecular polymers in a wide range of organic solvents, enabling fast light-triggered depolymerization-polymerization and reversible gel formation. Due to the stability of the cis isomers and the high photostationary states (PSS) of the cis-trans isomerization, precise control over supramolecular polymerization and in situ gelation could be achieved with short response times. A detailed study on the temperature-dependent and photoinduced supramolecular polymerization in organic solvents revealed a kinetically controlled nucleation-elongation mechanism. By application of a Volta phase plate to enhance the phase-contrast method in cryo-EM, unprecedented for nonaqueous solutions, uniform nanofibers were observed in organic solvents.

Tuning of Morphology by Chirality in Self-Assembled Structures of Bis(Urea) Amphiphiles in Water.

We present the synthesis and self-assembly of a chiral bis(urea) amphiphile and show that chirality offers a remarkable level of control towards different morphologies. Upon self-assembly in water, the molecular-scale chiral information is translated to the mesoscopic level. Both enantiomers of the amphiphile self-assemble into chiral twisted ribbons with opposite handedness, as supported by Cryo-TEM and circular dichroism (CD) measurements. The system presents thermo-responsive aggregation behavior and combined transmittance measurements, temperature-dependent UV, CD, TEM, and micro-differential scanning calorimetry (DSC) show that a ribbon-to-vesicles transition occurs upon heating. Remarkably, chirality allows easy control of morphology as the self-assembly into distinct aggregates can be tuned by varying the enantiomeric excess of the amphiphile, giving access to flat sheets, helical ribbons, and twisted ribbons.

Dynamic Assemblies of Molecular Motor Amphiphiles Control Macroscopic Foam Properties.

Stimuli-responsive supramolecular assemblies controlling macroscopic transformations with high structural fluidity, i.e., foam properties, have attractive prospects for applications in soft materials ranging from biomedical systems to industrial processes, e.g., textile coloring. However, identifying the key processes for the amplification of molecular motion to a macroscopic level response is of fundamental importance for exerting the full potential of macroscopic structural transformations by external stimuli. Herein, we demonstrate the control of dynamic supramolecular assemblies in aqueous media and as a consequence their macroscopic foam properties, e.g., foamability and foam stability, by large geometrical transformations of dual light/heat stimuli-responsive molecular motor amphiphiles. Detailed insight into the reversible photoisomerization and thermal helix inversion at the molecular level, supramolecular assembly transformations at the microscopic level, and the stimuli-responsive foam properties at the macroscopic level, as determined by UV-vis absorption and NMR spectroscopies, electron microscopy, and foamability and in situ surface tension measurements, is presented. By selective use of external stimuli, e.g., light or heat, multiple states and properties of macroscopic foams can be controlled with very dilute aqueous solutions of the motor amphiphiles (0.2 weight%), demonstrating the potential of multiple stimuli-responsive supramolecular systems based on an identical molecular amphiphile and providing opportunities for future soft materials.

Caught in the Act: Mechanistic Insight into Supramolecular Polymerization-Driven Self-Replication from Real-Time Visualization.

Self-assembly features prominently in fields ranging from materials science to biophysical chemistry. Assembly pathways, often passing through transient intermediates, can control the outcome of assembly processes. Yet, the mechanisms of self-assembly remain largely obscure due to a lack of experimental tools for probing these pathways at the molecular level. Here, the self-assembly of self-replicators into fibers is visualized in real-time by high-speed atomic force microscopy (HS-AFM). Fiber growth requires the conversion of precursor molecules into six-membered macrocycles, which constitute the fibers. HS-AFM experiments, supported by molecular dynamics simulations, revealed that aggregates of precursor molecules accumulate at the sides of the fibers, which then diffuse to the fiber ends where growth takes place. This mechanism of precursor reservoir formation, followed by one-dimensional diffusion, which guides the precursor molecules to the sites of growth, reduces the entropic penalty associated with colocalizing precursors and growth sites and constitutes a new mechanism for supramolecular polymerization.

Spontaneous Emergence of Self-Replicating Molecules Containing Nucleobases and Amino Acids.

The conditions that led to the formation of the first organisms and the ways that life originates from a lifeless chemical soup are poorly understood. The recent hypothesis of "RNA-peptide coevolution" suggests that the current close relationship between amino acids and nucleobases may well have extended to the origin of life. We now show how the interplay between these compound classes can give rise to new self-replicating molecules using a dynamic combinatorial approach. We report two strategies for the fabrication of chimeric amino acid/nucleobase self-replicating macrocycles capable of exponential growth. The first one relies on mixing nucleobase- and peptide-based building blocks, where the ligation of these two gives rise to highly specific chimeric ring structures. The second one starts from peptide nucleic acid (PNA) building blocks in which nucleobases are already linked to amino acids from the start. While previously reported nucleic acid-based self-replicating systems rely on presynthesis of (short) oligonucleotide sequences, self-replication in the present systems start from units containing only a single nucleobase. Self-replication is accompanied by self-assembly, spontaneously giving rise to an ordered one-dimensional arrangement of nucleobase nanostructures.

Structure of a robust bacterial protein cage and its application as a versatile biocatalytic platform through enzyme encapsulation.

Using a newly discovered encapsulin from Mycolicibacterium hassiacum, several biocatalysts were packaged in this robust protein cage. The encapsulin was found to be easy to produce as recombinant protein. Elucidation of its crystal structure revealed that it is a spherical protein cage of 60 protomers (diameter of 23 nm) with narrow pores. By developing an effective coexpression and isolation procedure, the effect of packaging a variety of biocatalysts could be evaluated. It was shown that encapsulation results in a significantly higher stability of the biocatalysts. Most of the targeted cofactor-containing biocatalysts remained active in the encapsulin. Due to the restricted diameters of the encapsulin pores (5-9 Å), the protein cage protects the encapsulated enzymes from bulky compounds. The work shows that encapsulins may be valuable tools to tune the properties of biocatalysts such as stability and substrate specificity.

A Technical Introduction to Transmission Electron Microscopy for Soft-Matter: Imaging, Possibilities, Choices, and Technical Developments.

With a significant role in material sciences, physics, (soft matter) chemistry, and biology, the transmission electron microscope is one of the most widely applied structural analysis tool to date. It has the power to visualize almost everything from the micrometer to the angstrom scale. Technical developments keep opening doors to new fields of research by improving aspects such as sample preservation, detector performance, computational power, and workflow automation. For more than half a century, and continuing into the future, electron microscopy has been, and is, a cornerstone methodology in science. Herein, the technical considerations of imaging with electrons in terms of optics, technology, samples and processing, and targeted soft materials are summarized. Furthermore, recent advances and their potential for application to soft matter chemistry are highlighted.

Molecular photoswitches mediating the strain-driven disassembly of supramolecular tubules.

Chemists have created molecular machines and switches with specific mechanical responses that were typically demonstrated in solution, where mechanically relevant motion is dissipated in the Brownian storm. The next challenge consists of designing specific mechanisms through which the action of individual molecules is transmitted to a supramolecular architecture, with a sense of directionality. Cellular microtubules are capable of meeting such a challenge. While their capacity to generate pushing forces by ratcheting growth is well known, conversely these versatile machines can also pull microscopic objects apart through a burst of their rigid tubular structure. One essential feature of this disassembling mechanism is the accumulation of strain in the tubules, which develops when tubulin dimers change shape, triggered by a hydrolysis event. We envision a strategy toward supramolecular machines generating directional pulling forces by harnessing the mechanically purposeful motion of molecular switches in supramolecular tubules. Here, we report on wholly synthetic, water-soluble, and chiral tubules that incorporate photoswitchable building blocks in their supramolecular architecture. Under illumination, these tubules display a nonlinear operation mode, by which light is transformed into units of strain by the shape changes of individual switches, until a threshold is reached and the tubules unleash the strain energy. The operation of this wholly synthetic and stripped-down system compares to the conformational wave by which cellular microtubules disassemble. Additionally, atomistic simulations provide molecular insight into how strain accumulates to induce destabilization. Our findings pave the way toward supramolecular machines that would photogenerate pulling forces, at the nanoscale and beyond.

Intracellular Activation of a Prostate Specific Antigen-Cleavable Doxorubicin Prodrug: A Key Feature Toward Prodrug-Nanomedicine Design.

L-377,202 prodrug (Dox-PSA) was in phase I clinical trials for patients with metastatic castration-resistant prostate cancer (mCRPC). It consists of doxorubicin (Dox) conjugated to a prostate specific antigen (PSA)-cleavable peptide that can be selectively activated by secreted PSA at the tumor site. However, despite the initial promising results, further clinical testing with Dox-PSA was halted due to toxicity concerns emerging from non-PSA-specific cleavage, following systemic administration. In the present study, we have reported, for the first time, the intracellular activation of Dox-PSA, where Dox nuclear uptake was specific to C4-2B (PSA-expressing) cells, which agreed with the cytotoxicity studies. This finding was confirmed by encapsulating Dox-PSA prodrug into pH-sensitive liposomes to enable prodrug intracellular release, followed by its enzymatic activation. Interestingly, our results demonstrated that Dox-PSA loaded into pH-responsive nanoparticles exhibited cytotoxicity comparable to free prodrug in C4-2B monolayers, with superior activity in tumor spheroids, due to deeper penetration within tumor spheroids. Our approach could open the doors for novel Dox-PSA nanomedicines with higher safety and efficacy to treat advanced and metastatic prostate cancer.