Autonomic Integration in Nested Protocell Communities.

The self-driven organization of model protocells into higher-order nested cytomimetic systems with coordinated structural and functional relationships offers a step toward the autonomic implementation of artificial multicellularity. Here, we describe an endosymbiotic-like pathway in which proteinosomes are captured within membranized alginate/silk fibroin coacervate vesicles by guest-mediated reconfiguration of the host protocells. We demonstrate that interchange of coacervate vesicle and droplet morphologies through proteinosome-mediated urease/glucose oxidase activity produces discrete nested communities capable of integrated catalytic activity and selective disintegration. The self-driving capacity is modulated by an internalized fuel-driven process using starch hydrolases sequestered within the host coacervate phase, and structural stabilization of the integrated protocell populations can be achieved by on-site enzyme-mediated matrix reinforcement involving dipeptide supramolecular assembly or tyramine-alginate covalent cross-linking. Our work highlights a semi-autonomous mechanism for constructing symbiotic cell-like nested communities and provides opportunities for the development of reconfigurable cytomimetic materials with structural, functional, and organizational complexity.

Osmotic-Induced Reconfiguration and Activation in Membranized Coacervate-Based Protocells.

The design and construction of synthetic protocells capable of stimuli response and homeostatic regulation is an important challenge for synthetic protobiology. Here, we develop a step toward the construction of model protocells capable of a hypotonic stress-induced volume response that facilitates an increase in membrane permeability and the triggering of endogenous enzyme reactions. We describe a facile self-transformation process for constructing single- or multichambered molecularly crowded protocells based on the osmotic reconfiguration of lipid-coated coacervate droplets into multicompartmentalized coacervate vesicles. Hypotonic swelling broadens membrane permeability and increases transmembrane transport such that protease-based hydrolysis and enzyme cascades can be triggered and enhanced within the protocells by osmotically induced expansion. Specifically, we demonstrate how the enhanced production of nitric oxide (NO) within the swollen coacervate vesicles can be used to induce in vitro blood vessel vasodilation in thoracic artery rings. Our approach provides opportunities for designing reconfigurable model protocells capable of homeostatic volume regulation, dynamic structural reorganization, and adaptive functionality in response to changes in environment osmolarity, and could find applications in biomedicine, cellular diagnostics, and bioengineering.

Spontaneous Membranization in a Silk-Based Coacervate Protocell Model.

Molecularly crowded coacervate micro-droplets are useful protocell constructs but the absence of a physical membrane limits their application as cytomimetic models. Auxiliary surface-active agents have been harnessed to stabilize the coacervate droplets by irreversible shell formation but endogenous processes of reversible membranization have received minimal attention. Herein, we describe a dynamic alginate/silk coacervate-based protocell model in which membrane-less droplets are reversibly reconfigured and inflated into semipermeable coacervate vesicles by spontaneous self-organization of amphiphilic silk polymers at the droplet surface under non-neutral charge conditions in the absence of auxiliary agents. We show that membranization can be reversibly controlled endogenously by programming the pH within the protocells using an antagonistic enzyme system such that structural reconfigurations in the protocell microstructure are coupled to the trafficking of water-soluble solutes. Our results open new perspectives in the design of hybrid protocell models with dynamical structural properties.

Triggerable Protocell Capture in Nanoparticle-Caged Coacervate Microdroplets.

Controlling the dynamics of mixed communities of cell-like entities (protocells) provides a step toward the development of higher-order cytomimetic behaviors in artificial cell consortia. In this paper, we develop a caged protocell model with a molecularly crowded coacervate interior surrounded by a non-cross-linked gold (Au)/poly(ethylene glycol) (PEG) nanoparticle-jammed stimuli-responsive membrane. The jammed membrane is unlocked by either exogenous light-mediated Au/PEG dissociation at the Au surface or endogenous enzyme-mediated cleavage of a ketal linkage on the PEG backbone. The membrane assembly/disassembly process is used for the controlled and selective uptake of guest protocells into the caged coacervate microdroplets as a path toward an all-water model of triggerable transmembrane uptake in synthetic protocell communities. Active capture of the guest protocells stems from the high sequestration potential of the coacervate interior such that tailoring the surface properties of the guest protocells provides a rudimentary system of protocell sorting. Our results highlight the potential for programming surface-contact interactions between artificial membrane-bounded compartments and could have implications for the development of protocell networks, storage and delivery microsystems, and microreactor technologies.

Swellable silk fibroin microneedles for transdermal drug delivery.

In this paper, a swelling-modified silk fibroin (SF) microneedle for transdermal drug delivery is presented. The microneedles undergo a phase transition from a dried and rigid state to a semi-solid, acerose hydrogel state with a controlled 3-dimensional (3D) porous network structure. Different micromolecular reagents have been studied for mixing with aqueous silk fibroin to endow a swellable and insoluble capacity to the SF. The aqueous SF composite is poured on a polydimethylsiloxane (PDMS) mold with arranged micropores on its surface to fabricate SF microneedles with high fidelity and mechanical robustness. The results demonstrate that 2-ethoxyethanol (ECS) modified SF microneedles can easily pierce porcine skin with a depth of ∼200µm in vitro, and transform into semi-solid hydrogels with 50-700nm porous network inside. These swelling-modified microneedles can accomplish a significantly enhanced transdermal drug release capacity in proportion to their swelling characteristics. The better swelling capacity of the microneedles produces larger pores, resulting in higher transdermal drug release kinetics. There is also a relationship between swollen pore dimensions and the molecular weights of encapsulated therapeutics. The controllable properties of these SF microneedles coupled with their high biocompatibility, render swell-to-release ECS/SF composites as viable transdermal delivery devices.

Silk Fibroin/Polyvinyl Pyrrolidone Interpenetrating Polymer Network Hydrogels.

Silk fibroin hydrogel is an ideal model as biomaterial matrix due to its excellent biocompatibility and used in the field of medical polymer materials. Nevertheless, native fibroin hydrogels show poor transparency and resilience. To settle these drawbacks, an interpenetrating network (IPN) of hydrogels are synthesized with changing ratios of silk fibroin/N-Vinyl-2-pyrrolidonemixtures that crosslink by H2O2 and horseradish peroxidase. Interpenetrating polymer network structure can shorten the gel time and the pure fibroin solution gel time for more than a week. This is mainly due to conformation from the random coil to the ß-sheet structure changes of fibroin. Moreover, the light transmittance of IPN hydrogel can be as high as more than 97% and maintain a level of 90% within a week. The hydrogel, which mainly consists of random coil, the apertures inside can be up to 200 µm. Elastic modulus increases during the process of gelation. The gel has nearly 95% resilience under the compression of 70% eventually, which is much higher than native fibroin gel. The results suggest that the present IPN hydrogels have excellent mechanical properties and excellent transparency.

Self-Assembling Silk-Based Nanofibers with Hierarchical Structures.

Self-assembling fibrous supramolecular assemblies with sophisticated hierarchical structures at the mesoscale are of interest from both fundamental and applied engineering. In this paper, the relatively hydrophilic domains of silk fibroin (HSF) were extracted and used in studies of self-assembly. The HSF fraction spontaneously self-assembled into nanofibers, 10 to 100 µm long and 50 to 250 nm in diameter, within 2 to 8 h in aqueous conditions. Interestingly, these HSF nanofibers consisted of dozens of nanofibrils arranged in a parallel organization with assembled diameters of ∼30 nm, similar to the sophisticated hierarchical structure observed in native silk fibers. Dynamic morphology and conformation studies were carried out to determine the mechanisms underlying the HSF self-assembly process at both the nanoscale and mesoscale. The HSF self-assembled into nanofibers in a bottom-to-up manner, from "sticky" colloid particles to cylindrical globules, to form nanofibrous networks. Because of the enhanced HSF self-assembling kinetics and the hierarchical structure of HSF nanofibers, this hydrophilicity-driven approach provides further insight into silk fibroin (SF) self-assembly in vivo and also offers new tools for the recapitulation of high-performance materials for engineering applications.

Natural Non-Mulberry Silk Nanoparticles for Potential-Controlled Drug Release.

Natural silk protein nanoparticles are a promising biomaterial for drug delivery due to their pleiotropic properties, including biocompatibility, high bioavailability, and biodegradability. Chinese oak tasar Antheraea pernyi silk fibroin (ApF) nanoparticles are easily obtained using cations as reagents under mild conditions. The mild conditions are potentially advantageous for the encapsulation of sensitive drugs and therapeutic molecules. In the present study, silk fibroin protein nanoparticles are loaded with differently-charged small-molecule drugs, such as doxorubicin hydrochloride, ibuprofen, and ibuprofen-Na, by simple absorption based on electrostatic interactions. The structure, morphology and biocompatibility of the silk nanoparticles in vitro are investigated. In vitro release of the drugs from the nanoparticles depends on charge-charge interactions between the drugs and the nanoparticles. The release behavior of the compounds from the nanoparticles demonstrates that positively-charged molecules are released in a more prolonged or sustained manner. Cell viability studies with L929 demonstrated that the ApF nanoparticles significantly promoted cell growth. The results suggest that Chinese oak tasar Antheraea pernyi silk fibroin nanoparticles can be used as an alternative matrix for drug carrying and controlled release in diverse biomedical applications.

The influence of the hydrophilic-lipophilic environment on the structure of silk fibroin protein.

The present study examines the influence of the hydrophilic-lipophilic environment, mediated by small molecules, on the structural changes in silk protein fibroin. Small molecules mediate the various hydrophilic-lipophilic balances (HLBs) that impact the organisation of silk protein chains. Changes in the silk fibroin structure due to additives are related to the HLB value. At HLB > 10, silk fibroin primarily forms Silk I crystalline structures. Small molecules with HLB < 8.9 primarily induce the formation of Silk II crystalline structures. When 8.9 < HLB < 10, the crystalline structure of silk is related to the content of small molecules. The Silk I structure is primarily formed when the content of small molecules is low, whereas the Silk II structure is formed when the small molecule content is high. The structure of silk fibroin is maintained by regulating the HLB in the fibroin environment. This type of control for the functional design of materials may play a role in fine-tuning the biomaterial properties of silk fibroin protein.