Matching Together Living Cells and Prototissues: Will There Be Chemistry?

Scientific advancements in bottom-up synthetic biology have led to the development of numerous models of synthetic cells, or protocells. To date, research has mainly focused on increasing the (bio)chemical complexity of these bioinspired micro-compartmentalized systems, yet the successful integration of protocells with living cells remains one of the major challenges in bottom-up synthetic biology. In this review, we aim to summarize the current state of the art in hybrid protocell/living cell and prototissue/living cell systems.

Properties and Curing Kinetics of a Processable Binary Benzoxazine Blend.

A benzoxazine system is presented combining liquid cardanol-based benzoxazine (CA-a) and an effective initiator (3,3'-thiodipropionic acid, TDA) to bisphenol A-based benzoxazine (BA-a). The resultant mixture of monomeric precursors shows excellent fluidity and a relatively low peak polymerization temperature of around 200 °C. Moreover, the cured polybenzoxazine displays a high thermal decomposition temperature ( > 330 °C), a moderately high glass transition temperature (∼148 °C), and robust mechanical strength (storage modulus ∼ 2.

A Photo-degradable Crosslinker for the Development of Light-responsive Protocell Membranes.

The achievement of light-responsive behaviours is an important target for protocell engineering to allow control of fundamental protocellular processes such as communication via diffusible chemical signals, shape changes or even motility at the flick of a switch. As a step towards this ambitious goal, here we describe the synthesis of a novel poly(ethylene glycol)-based crosslinker, reactive towards nucleophiles, that effectively degrades with UV light (405 nm). We demonstrate its utility for the fabrication of the first protocell membranes capable of light-induced disassembly, for the photo-generation of patterns of protocells, and for the modulation of protocell membrane permeability.

Towards the design of self-sorting nanomaterials through kinetically directed chemoselective control over interfacial surface chemistry.

A gold nanoparticle platform is described in which post-synthesis surface modifications can be conducted using kinetically-tunable strain-promoted cycloaddition chemistry, which is dependent on the electronic properties of the complementary dipolar species. This permits chemoselective reactivity with one reactive dipole over another less reactive dipole, providing exciting opportinities for kinetically-directed self-sorting strategies.

Orthogonal Light-Dependent Membrane Adhesion Induces Social Self-Sorting and Member-Specific DNA Communication in Synthetic Cell Communities.

Developing orthogonal chemical communication pathways in diverse synthetic cell communities is a considerable challenge due to the increased crosstalk and interference associated with large numbers of different types of sender-receiver pairs. Herein, the authors control which sender-receiver pairs communicate in a three-membered community of synthetic cells through red and blue light illumination. Semipermeable protein-polymer-based synthetic cells (proteinosomes) with complementary membrane-attached protein adhesion communicate through single-stranded DNA oligomers and synergistically process biochemical information within a community consisting of one sender and two different receiver populations.

Automated analysis of soft material microindentation.

An understanding of the mechanical properties of soft hydrogel materials over multiple length scales is important for their application in many fields. Typical measurement methods provide either bulk mechanical properties (compression, tensile, rheology) or probing of nano or microscale properties and heterogeneity (nanoindentation, AFM). In this work we demonstrate the complementarity of instrumented microindentation to these techniques, as it provides representative Young's moduli for soft materials with minimal influence of the experimental parameters chosen, and allows mechanical property mapping across macroscopic areas.

(Hydroxypropyl)methyl Cellulose-Chitosan Film as a Matrix for Lipase Immobilization-Part ΙΙ: Structural Studies.

The present work reports on the structural study of a film made of a hybrid blend of biopolymers used as an enzyme carrier. A cellulose derivative (HPMC) and chitosan (CS) were combined in order to formulate a film on which lipase was immobilized. The film was successfully used as a biocatalyst; however, little is known about the structure of the system.

Bioinspired Networks of Communicating Synthetic Protocells.

The bottom-up synthesis of cell-like entities or from inanimate molecules and materials is one of the grand challenges of our time. In the past decade, researchers in the emerging field of have developed different protocell models and engineered them to mimic one or more abilities of biological cells, such as information transcription and translation, adhesion, and enzyme-mediated metabolism. Whilst thus far efforts have focused on increasing the biochemical complexity of individual protocells, an emerging challenge in bottom-up synthetic biology is the development of networks of communicating synthetic protocells.

Programmed assembly of bespoke prototissues on a microfluidic platform.

The precise assembly of protocell building blocks into prototissues that are stable in water, capable of sensing the external environment and which display collective behaviours remains a considerable challenge in prototissue engineering. We have designed a microfluidic platform that enables us to build bespoke prototissues from predetermined compositions of two types of protein-polymer protocells. We can accurately control their size, composition and create unique Janus configurations in a way that is not possible with traditional methods.

A Floating Mold Technique for the Programmed Assembly of Protocells into Protocellular Materials Capable of Non-Equilibrium Biochemical Sensing.

Despite important breakthroughs in bottom-up synthetic biology, a major challenge still remains the construction of free-standing, macroscopic, and robust materials from protocell building blocks that are stable in water and capable of emergent behaviors. Herein, a new floating mold technique for the fabrication of millimeter- to centimeter-sized protocellular materials (PCMs) of any shape that overcomes most of the current challenges in prototissue engineering is reported. Significantly, this technique also allows for the generation of 2D periodic arrays of PCMs that display an emergent non-equilibrium spatiotemporal sensing behavior.

A Novel Acid-Degradable PEG Crosslinker for the Fabrication of pH-Responsive Soft Materials.

The design and synthesis of a novel acid-degradable polyethylene glycol-based N-hydroxysuccinimide (NHS) ester-activated crosslinker is reported. The crosslinker is reactive towards nucleophiles and features a central ketal functional group that is stable at pH > 7.5 and rapidly hydrolyses at pH > 6.

From protocells to prototissues: a materials chemistry approach.

Prototissues comprise free-standing 3D networks of interconnected protocell consortia that communicate and display synergistic functions. Significantly, they can be constructed from functional molecules and materials, providing unprecedented opportunities to design tissue-like architectures that can do more than simply mimic living tissues. They could function under extreme conditions and exhibit a wide range of mechanical properties and bio-inspired metabolic functions.

Spontaneous membrane-less multi-compartmentalization aqueous two-phase separation in complex coacervate micro-droplets.

Polyelectrolyte/nucleotide multiphase complex coacervate droplets are produced by internalized aqueous two-phase separation and used for the spatially dependent chemical transfer of sugar molecules, providing a step towards the development of membrane-free "organelles" within coacervate-based protocells.

Catalytic processing in ruthenium-based polyoxometalate coacervate protocells.

The development of programmable microscale materials with cell-like functions, dynamics and collective behaviour is an important milestone in systems chemistry, soft matter bioengineering and synthetic protobiology. Here, polymer/nucleotide coacervate micro-droplets are reconfigured into membrane-bounded polyoxometalate coacervate vesicles (PCVs) in the presence of a bio-inspired Ru-based polyoxometalate catalyst to produce synzyme protocells (RuPCVs) with catalase-like activity. We exploit the synthetic protocells for the implementation of multi-compartmentalized cell-like models capable of collective synzyme-mediated buoyancy, parallel catalytic processing in individual horseradish peroxidase-containing RuPCVs, and chemical signalling in distributed or encapsulated multi-catalytic protocell communities.

Nitrone-Modified Gold Nanoparticles: Synthesis, Characterization, and Their Potential as F-Labeled Positron Emission Tomography Probes via I-SPANC.

A novel bioorthogonal gold nanoparticle (AuNP) template displaying interfacial nitrone functional groups for bioorthogonal interfacial strain-promoted alkyne-nitrone cycloaddition reactions has been synthesized. These nitrone-AuNPs were characterized in detail using H nuclear magnetic resonance spectroscopy, transmission electron microscopy, thermogravimetric analysis, and X-ray photoelectron spectroscopy, and a nanoparticle raw formula was calculated. The ability to control the conjugation of molecules of interest at the molecular level onto the nitrone-AuNP template allowed us to create a novel methodology for the synthesis of AuNP-based radiolabeled probes.

Programmed assembly of synthetic protocells into thermoresponsive prototissues.

Although several new types of synthetic cell-like entities are now available, their structural integration into spatially interlinked prototissues that communicate and display coordinated functions remains a considerable challenge. Here we describe the programmed assembly of synthetic prototissue constructs based on the bio-orthogonal adhesion of a spatially confined binary community of protein-polymer protocells, termed proteinosomes. The thermoresponsive properties of the interlinked proteinosomes are used collectively to generate prototissue spheroids capable of reversible contractions that can be enzymatically modulated and exploited for mechanochemical transduction.

Fluorogenic Gold Nanoparticle (AuNP) Substrate: A Model for the Controlled Release of Molecules from AuNP Nanocarriers via Interfacial Staudinger-Bertozzi Ligation.

The ability to regulate small-molecule release from metallic nanoparticle substrates offers unprecedented opportunities for nanocarrier-based imaging, sensing, and drug-delivery applications. Herein we report a novel and highly specific release methodology off gold nanoparticle (AuNP) surfaces based on the bioorthogonal Staudinger-Bertozzi ligation. A thiol ligand bearing the molecular cargo, a Rhodamine B dye derivative, was synthesized and used to modify small water-soluble 5 nm AuNPs.

"Shine & Click" Photo-Induced Interfacial Unmasking of Strained Alkynes on Small Water-Soluble Gold Nanoparticles.

In this study, we report the design, synthesis, and characterization of small 3 nm water soluble gold nanoparticles (AuNPs) that feature cyclopropenone-masked strained alkyne moieties capable of undergoing interfacial strain-promoted cycloaddition (i-SPAAC) with azides after exposure to UV-A light. A strained alkyne precursor was incorporated onto AuNPs by direct ligand exchange of a thiol-modified cyclopropenone-masked dibenzocyclooctyne (photoDIBO) ligand. These photoDIBO-AuNPs were characterized by H NMR, IR, and UV/Vis spectroscopy, as well as transmission electron microscopy (TEM) and thermogravimetric analysis (TGA), and the extent of modification was quantified.

Insights on the Application of the Retro Michael-Type Addition on Maleimide-Functionalized Gold Nanoparticles in Biology and Nanomedicine.

The glutathione-mediated retro Michael-type addition reaction is demonstrated to take place at the interface of small water-soluble maleimide-functionalized gold nanoparticles (Maleimide-AuNP). The retro Michael-type addition reaction can be blocked by hydrolyzing the Michael addition thioether adduct at the nanoparticle's interface under reaction conditions that do not cause AuNP decomposition. This procedure "locks" the molecule of interest onto the Maleimide-AuNP template for potential uses in medical imaging and bioconjugation, ensuring no loss of the molecular cargo from the nanocarrier.

Gold nanosponges (AuNS): a versatile nanostructure for surface-enhanced Raman spectroscopic detection of small molecules and biomolecules.

Prepared by simple pour and mix chemistry, gold nanosponges (AuNS) are versatile structures for surface-enhanced Raman spectroscopy (SERS). An investigation into the enhancement is performed by relating the nanostructure's morphology to the SERS signal. The potential of the AuNS in SERS-based molecular and biomolecular detection is introduced.

Small gold nanoparticles for interfacial Staudinger-Bertozzi ligation.

Small gold nanoparticles (AuNPs) that possess interfacial methyl-2-(diphenylphosphino)benzoate moieties have been successfully synthesized (Staudinger-AuNPs) and characterized by multi-nuclear MR spectroscopy, transmission electron microscopy (TEM), UV-Vis spectroscopy, thermogravimetric analysis, and X-ray photoelectron spectroscopy (XPS). In particular, XPS was remarkably sensitive for characterization of the novel nanomaterial, and in furnishing proof of its interfacial reactivity. These Staudinger-AuNPs were found to be stable to the oxidation of the phosphine center.

Expanding the scope of strained-alkyne chemistry: a protection-deprotection strategy via the formation of a dicobalt-hexacarbonyl complex.

A protection-deprotection strategy for strained alkynes used for bioorthogonal chemistry is reported. A strained alkyne can be protected with dicobalt-octacarbonyl and we demonstrate for the first time that a strained alkyne can be re-formed and isolated under mild reaction conditions for further bioorthogonal reactivity. The protection-deprotection strategy herein reported will expand the versatility of strained alkynes for the preparation of substrates in chemical biology and materials applications.

Versatile strained alkyne modified water-soluble AuNPs for interfacial strain promoted azide-alkyne cycloaddition (I-SPAAC).

Versatile water- and organic solvent-soluble AuNPs that incorporate an interfacial strained alkyne capable of efficient pour and mix strain promoted interfacial cycloadditions with azide partners have been synthesized and carefully characterized for the first time. The use of XPS to quantitate the loading of the strained alkyne on the AuNPs is noteworthy. The reactivity towards the interfacial strain promoted azide-alkyne cycloaddition reaction was demonstrated by using azide-decorated polymersomes as bioorthogonal reaction partners.

Water-soluble gold nanoparticles (AuNP) functionalized with a gadolinium(iii) chelate via Michael addition for use as a MRI contrast agent.

A contrast agent suitable for magnetic resonance imaging based on small, water soluble gold nanoparticles (AuNP) conjugated to over 50 Gd chelators has been prepared by using an interfacial Michael addition in aqueous media. The resultant chelator-AuNP conjugates have been successfully characterised by H NMR spectroscopy, IR spectroscopy, ICP-OES, ζ-potential analysis, TEM and MRI. T-weighted in vivo images of mouse kidney were obtained using the agent at 9.