Collagenase motors in gelatine-based hydrogels.

Nano/micromotors outperform Brownian motion due to their self-propulsive capabilities and hold promise as carriers for drug delivery across biological barriers such as the extracellular matrix. This study employs poly(2-(diethylamino)ethyl methacrylate) polymer brushes to enhance the collagenase-loading capacity of silica particle-based motors with the aim to systematically investigate the impact of gelatine viscosity, motors' size, and morphology on their propulsion velocity. Notably, 500 nm and 1 µm motors achieve similar speeds as high as ∼15 µm s-1 in stiff gelatine-based hydrogels when triggered with calcium. Taken together, our findings highlight the potential of collagenase-based motors for navigating the extracellular matrix, positioning them as promising candidates for efficient drug delivery.

Antioxidant Microgels Support Peroxide-Challenged Hepatic Cells.

Access to therapeutic strategies that counter cellular stress induced by reactive oxygen species (ROS) is an important, long-standing challenge. Here, the assembly of antioxidant artificial cells is based on alginate hydrogels equipped with non-native catalysts, namely platinum nanoparticles and an EUK compound. These artificial cells are able to preserve the viability and lower the intracellular ROS levels of challenged hepatic cells by removing peroxides from the extracellular environment. Conceptually, this strategy illustrates the potential use of artificial cells with a synthetic catalyst toward long-term support of hepatic cells and potentially other mammalian cells.

Artificial Cells and HepG2 Cells in 3D-Bioprinted Arrangements.

Artificial cells are engineered units with cell-like functions for different purposes including acting as supportive elements for mammalian cells. Artificial cells with minimal liver-like function are made of alginate and equipped with metalloporphyrins that mimic the enzyme activity of a member of the cytochrome P450 family namely CYP1A2. The artificial cells are employed to enhance the dealkylation activity within 3D bioprinted structures composed of HepG2 cells and these artificial cells. This enhancement is monitored through the conversion of resorufin ethyl ether to resorufin. HepG2 cell aggregates are 3D bioprinted using an alginate/gelatin methacryloyl ink, resulting in the successful proliferation of the HepG2 cells. The composite ink made of an alginate/gelatin liquid phase with an increasing amount of artificial cells is characterized. The CYP1A2-like activity of artificial cells is preserved over at least 35 days, where 6 nM resorufin is produced in 8 h. Composite inks made of artificial cells and HepG2 cell aggregates in a liquid phase are used for 3D bioprinting. The HepG2 cells proliferate over 35 days, and the structure has boosted CYP1A2 activity. The integration of artificial cells and their living counterparts into larger 3D semi-synthetic tissues is a step towards exploring bottom-up synthetic biology in tissue engineering.

Magnetic micromotors crossing lipid membranes.

Nano/micromotors are self-propelled particles that show enhanced motion upon being triggered by a stimulus. Their use in nanomedicine has been widely explored, with special focus on imaging or drug delivery. However, a thorough understanding of the requirements for more efficient locomotion is still lacking. In this paper, we assembled magnetically propelled motors of different sizes (i.e., 0.5, 1 and 4 µm) and surface chemistries (positive charge or PEGylated) and assessed their motion in the presence of giant unilamellar lipid vesicles (GUVs) of varying compositions (zwitterionic, negatively charged and saturated lipids). Unexpectedly, the size does not seem to be the dominating characteristics that governs the ability of the motors to cross lipid membranes. Specifically, the 0.5 µm PEGylated motors have very limited ability to cross the lipid membrane of GUVs due to their non-interacting nature compared to their equally sized positively charged counterparts. Furthermore, membranes made of saturated lipids and, in particular, in combination with a weak magnetic field facilitate motors' crossing, regardless of their size. The results were validated by in-house data-driven statistical analysis that employs experimental data to allow for the identification of individual motor motion in the ensemble when meeting the lipid membranes. Altogether, we provide insight into motor locomotion when they interact with a biological barrier considering both the entire ensemble and the individual motors, which has the potential to support considerations of future motor designs.

Artificial cells eavesdropping on HepG2 cells.

Cellular communication is a fundamental feature to ensure the survival of cellular assemblies, such as multicellular tissue, via coordinated adaption to changes in their surroundings. Consequently, the development of integrated semi-synthetic systems consisting of artificial cells (ACs) and mammalian cells requires feedback-based interactions. Here, we illustrate that ACs can eavesdrop on HepG2 cells focusing on the activity of cytochrome P450 1A2 (CYP1A2), an enzyme from the cytochrome P450 enzyme family. Specifically, d-cysteine is sent as a signal from the ACs via the triggered reduction of disulfide bonds. Simultaneously, HepG2 cells enzymatically convert 2-cyano-6-methoxybenzothiazole into 2-cyano-6-hydroxybenzothiazole that is released in the extracellular space. d-Cysteine and 2-cyano-6-hydroxybenzothiazole react to form d-luciferin. The ACs respond to this signal by converting d-luciferin into luminescence due to the presence of encapsulated luciferase in the ACs. As a result, the ACs can eavesdrop on the mammalian cells to evaluate the level of hepatic CYP1A2 function.

Surfaces Coated with Polymer Brushes Work as Carriers for Histidine Ammonia Lyase.

The immobilization of enzymes on solid supports is an important challenge in biotechnology and biomedicine. In contrast to other methods, enzyme deposition in polymer brushes offers the benefit of high protein loading that preserves enzymatic activity in part due to the hydrated 3D environment that is available within the brush structure. The authors equipped planar and colloidal silica surfaces with poly(2-(diethylamino)ethyl methacrylate)-based brushes to immobilize Thermoplasma acidophilum histidine ammonia lyase, and analyzed the amount and activity of the immobilized enzyme. The poly(2-(diethylamino)ethyl methacrylate) brushes are attached to the solid silica supports either via a "grafting-to" or a "grafting-from" method. It is found that the grafting-from method results in higher amounts of deposited polymer and, consequently, higher amounts of Thermoplasma acidophilum histidine ammonia lyase. All polymer brush-modified surfaces show preserved catalytic activity of the deposited Thermoplasma acidophilum histidine ammonia lyase. However, immobilizing the enzyme in polymer brushes using the grafting-from method resulted in twice the enzymatic activity from the grafting-to approach, illustrating a successful enzyme deposition on a solid support.

Micromotor-Assisted Keratinocytes Migration in a Floating Paper Chip.

In vitro epidermis models are important to evaluate and study disease progression and possible dermal drug delivery. An in vitro epidermis model using floating paper chips as a scaffold for proliferation and differentiation of primary human keratinocytes is reported. The formation of the four main layers of the epidermis (i.e., basal, spinosum, granulose, and cornified layers) is confirmed. The development of a cornified layer and the tight junction formation are evaluated as well as the alterations of organelles during the differentiation process. Further, this in vitro model is used to assess keratinocyte migration. Finally, magnetic micromotors are assembled, and their ability to aid cell migration on paper chips is confirmed when a static magnetic field is present. Taken together, this attempt to combine bottom-up synthetic biology with dermatology offers interesting opportunities for studying skin disease pathologies and evaluate possible treatments.

Astrocytes in Paper Chips and Their Interaction with Hybrid Vesicles.

The role of astrocytes in brain function has received increased attention lately due to their critical role in brain development and function under physiological and pathophysiological conditions. However, the biological evaluation of soft material nanoparticles in astrocytes remains unexplored. Here, the interaction of crosslinked hybrid vesicles (HVs) and either C8-D1A astrocytes or primary astrocytes cultured in polystyrene tissue culture or floatable paper-based chips is investigated. The amphiphilic block copolymer poly(cholesteryl methacrylate)-block-poly(2-carboxyethyl acrylate) (P1) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine lipids are used for the assembly of HVs with crosslinked membranes. The assemblies show no short-term toxicity towards the C8-D1A astrocytes and the primary astrocytes, and both cell types internalize the HVs when cultured in 2D cell culture. Further, it is demonstrated that both the C8-D1A astrocytes and the primary astrocytes could mature in paper-based chips with preserved calcium signaling and glial fibrillary acidic protein expression. Last, it is confirmed that both types of astrocytes could internalize the HVs when cultured in paper-based chips. These findings lay out a fundamental understanding of the interaction between soft material nanoparticles and astrocytes, even when primary astrocytes are cultured in paper-based chips offering a 3D environment.

Microreactor equipped with naturally acid-resistant histidine ammonia lyase from an extremophile.

Extremophile enzymes are useful in biotechnology and biomedicine due to their abilities to withstand harsh environments. The abilities of histidine ammonia lyases from different extremophiles to preserve their catalytic activities after exposure to acid were assessed. Thermoplasma acidophilum histidine ammonia lyase was identified as an enzyme with a promising catalytic profile following acid treatment. The fusion of this enzyme with the maltose-binding protein or co-incubation with the chaperone HdeA further helped Thermoplasma acidophilum histidine ammonia lyase to withstand acid treatments down to pH 2.8. The assembly of a microreactor by encapsulation of MBP-Thermoplasma acidophilum histidine ammonia lyase into a photocrosslinked poly(vinyl alcohol) hydrogel allowed the enzyme to recover over 50% of its enzymatic activity following exposure to simulated gastric and intestinal fluids. Our results show that using engineered proteins obtained from extremophiles in combination with polymer-based encapsulation can advance the oral formulations of biologicals.

Manganese dioxide nanosheet-containing reactors as antioxidant support for neuroblastoma cells.

Supporting mammalian cells against reactive oxygen species such as hydrogen peroxide (H2O2) is essential. Bottom-up synthetic biology aims to integrate designed artificial units with mammalian cells. Here, we used manganese dioxide nanosheets (MnO2-NSs) as catalytically active entities that have superoxide dismutase-like and catalase-like activities. The integration of these MnO2-NSs into 7 µm reactors was able to assist SH-SY5Y neuroblastoma cells when stressed with H2O2. Complementary, Janus-shaped 800 nm reactors with one hemisphere coated with MnO2-NSs showed directed locomotion in cell media with top speeds up to 50 µm s-1 when exposed to 300 mM H2O2 as a fuel, while reactors homogeneously coated with MnO2-NSs were not able to outperform Brownian motion. These Janus-shaped reactors were able to remove H2O2 from the media, protecting cells cultured in the proximity. This effort advanced the use of bottom-up synthetic biology concepts in neuroscience.

Polymer Micelles vs Polymer-Lipid Hybrid Vesicles: A Comparison Using RAW 264.7 Cells.

Bottom-up synthetic biology aims to integrate artificial moieties with living cells and tissues. Here, two types of structural scaffolds for artificial organelles were compared in terms of their ability to interact with macrophage-like murine RAW 264.7 cells. The amphiphilic block copolymer poly(cholesteryl methacrylate)-block-poly(2-carboxyethyl acrylate) was used to assemble micelles and polymer-lipid hybrid vesicles together with 1,2-dioleoyl-sn-glycero-3-phosphocholine or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) lipids in the latter case. In addition, the pH-sensitive fusogenic peptide GALA was conjugated to the carriers to improve their lysosomal escape ability. All assemblies had low short-term toxicity toward macrophage-like murine RAW 264.7 cells, and the cells internalized both the micelles and hybrid vesicles within 24 h. Assemblies containing DOPE lipids or GALA in their building blocks could escape the lysosomes. However, the intracellular retention of the building blocks was only a few hours in all the cases. Taken together, the provided comparison between two types of potential scaffolds for artificial organelles lays out the fundamental understanding required to advance soft material-based assemblies as intracellular nanoreactors.

Locomotion of micromotors in paper chips.

Locomotion of nano/micromotors in non-aqueous environments remains a challenging task. We assembled magnetic micromotors with different surface coatings and explored their locomotion in paper chips. Poly(L-lysine) deposition resulted in positively charged micromotors. Immobilized cellulase was used to increase the micromotors' paper penetration depth while a polyethylene glycol (PEG) coating was employed to limit the interaction between the micromotors and the cellulose fibers. All micromotors were able to move in the top layers of the paper chips with velocities dependent on the magnetic forces used to induce their locomotion, their sizes and the types of employed paper chips. Maximum speeds of up to ∼25 µm s-1 were observed for PEGylated micromotors in the fibrous cellulose environment. This type of micromotors has the potential to be considered in the area of paper microfluidics to facilitate distribution, or collection of moieties for biosensing or cell culture.

Evaluation of Hybrid Vesicles in an Intestinal Cell Model Based on Structured Paper Chips.

Cell culture-based intestinal models are important to evaluate nanoformulations intended for oral drug delivery. We report the use of a floating structured paper chip as a scaffold for Caco-2 cells and HT29-MTX-E12 cells that are two established cell types used in intestinal cell models. The formation of cell monolayers for both mono- and cocultures in the paper chip are confirmed and the level of formed cell-cell junctions is evaluated. Further, cocultures show first mucus formation between 6-10 days with the mucus becoming more pronounced after 19 days. Hybrid vesicles (HVs) made from phospholipids and the amphiphilic block copolymer poly(cholesteryl methacrylate)-block-poly(2-carboxyethyl acrylate) in different ratios are used as a representative soft nanoparticle to assess their mucopenetration ability in paper chip-based cell cultures. The HV assembly is characterized, and it is illustrated that these HVs cross the mucus layer and are found intracellularly within 3 h when the cells are grown in the paper chips. Taken together, the moist three-dimensional cellulose environment of structured paper chips offers an interesting cell culture-based intestinal model that can be further integrated with fluidic systems or online read-out opportunities.

Enzyme Mimic Facilitated Artificial Cell to Mammalian Cell Signal Transfer.

Catalyzing biochemical reactions with enzymes and communicating with neighboring cells via chemical signaling are two fundamental cellular features that play a critical role in maintaining the homeostasis of organisms. Herein, we present an artificial enzyme (AE) facilitated signal transfer between artificial cells (ACs) and mammalian HepG2 cells. We synthesize metalloporphyrins (MPs) based AEs that mimic cytochrome P450 enzymes (CYPs) to catalyze a dealkylation and a hydroxylation reaction, exemplified by the conversion of resorufin ethyl ether (REE) to resorufin and coumarin (COU) to 7-hydroxycoumarin (7-HC), respectively. The AEs are immobilized in hydrogels to produce ACs that generate the two diffusive fluorophores, which can diffuse into HepG2 cells and result in dual intracellular emissions. This work highlights the use of AEs to promote AC to mammalian signal transfer, which opens up new opportunities for integrating the synthetic and living world with a bottom-up strategy.

Surface polymerization induced locomotion.

Nano- and micromotors are self-navigating particles that gain locomotion using fuel from the environment or external power sources to outperform Brownian motion. Herein, motors that make use of surface polymerization of hydroxyethylmethylacrylate to gain locomotion are reported, synthetically mimicking microorganisms' way of propulsion. These motors have enhanced Brownian motion with effective diffusion coefficients up to ∼0.5 µm2 s-1 when mesoporous Janus particles are used. Finally, indication of swarming is observed when high numbers of motors homogenously coated with atom-transfer radical polymerization initiators are used, while high-density Janus motors lost their ability to exhibit enhanced Brownian motion. This report illustrates an alternative route to self-propelled particles, employing a polymerization process that has the potential to be applied for various purposes benefiting from the tool box of modern polymer chemistry.

Mitochondria Encapsulation in Hydrogel-Based Artificial Cells as ATP Producing Subunits.

Artificial cells (ACs) aim to mimic selected structural and functional features of mammalian cells. In this context, energy generation is an important challenge to be addressed when self-sustained systems are desired. Here, mitochondria isolated from HepG2 cells are employed as natural subunits that facilitate chemically driven adenosine triphosphate (ATP) synthesis. The successful mitochondria isolation is confirmed by monitoring the preserved inner membrane potential, the respiration, and the ATP production ability. The encapsulation of the isolated mitochondria in gelatin-based hydrogels results in similar initial ATP production compared to mitochondria in solution with a sustained ATP production over 24 h. Furthermore, luciferase is coencapsulated with the mitochondria in gelatin-based particles to create ACs and employ the in situ produced ATP to drive the catalytic conversion of d-luciferin. The coencapsulation of luciferase-loaded liposomes with mitochondria in gelatin-based hydrogels is additionally explored where the encapsulation of mitochondria and liposomes resulted in clustering effects that are likely contributing to the functional performance of the active entities. Taken together, mitochondria show potential in cell mimicry to facilitate energy-dependent processes.

Cell mimicry as a bottom-up strategy for hierarchical engineering of nature-inspired entities.

Artificial biology is an emerging concept that aims to design and engineer the structure and function of natural cells, organelles, or biomolecules with a combination of biological and abiotic building blocks. Cell mimicry focuses on concepts that have the potential to be integrated with mammalian cells and tissue. In this feature article, we will emphasize the advancements in the past 3-4 years (2017-present) that are dedicated to artificial enzymes, artificial organelles, and artificial mammalian cells. Each aspect will be briefly introduced, followed by highlighting efforts that considered key properties of the different mimics. Finally, the current challenges and opportunities will be outlined. This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology.

Cell Membrane Coated Particles.

Nanoformulations are widely considered in the biomedical field for drug delivery, imaging, or detoxification purposes. Cell membrane coatings are a growing concept that aims to camouflage nanomaterials. The cell membranes are the first point of contact for cells to other biological or synthetic materials and nature has established signaling pathways in this context. In contrast to using purified membrane associated proteins, the use of purified cell membranes contains the protein of interest in a very native environment. This report provides an overview over the advances in cell membrane coated (nano)particles from the past 2-3 years. The progress in using cell membranes from mammalian cells without nuclei, i.e., red blood cells and platelets, as well as nucleus-containing cells in particular white blood cell and specific cancer cells is outlined. Additionally, highlights from recent reports considering hybrid cell membrane coating that originate from at least two different cell types are discussed. Finally, a future perspective indicating the challenges and potential of cell membrane coated nanomaterials and biomaterials is provided.