Design of programmable post-translational switch control platform for on-demand protein secretion in mammalian cells.

The development of novel strategies to program cellular behaviors is a central goal in synthetic biology, and post-translational control mediated by engineered protein circuits is a particularly attractive approach to achieve rapid protein secretion on demand. We have developed a programmable protease-mediated post-translational switch (POSH) control platform composed of a chimeric protein unit that consists of a protein of interest fused via a transmembrane domain to a cleavable ER-retention signal, together with two cytosolic inducer-sensitive split protease components. The protease components combine in the presence of the specific inducer to generate active protease, which cleaves the ER-retention signal, releasing the transmembrane-domain-linked protein for trafficking to the trans-Golgi region. A furin site placed downstream of the protein ensures cleavage and subsequent secretion of the desired protein. We show that stimuli ranging from plant-derived, clinically compatible chemicals to remotely controllable inducers such as light and electrostimulation can program protein secretion in various POSH-engineered designer mammalian cells. As proof-of-concept, an all-in-one POSH control plasmid encoding insulin and abscisic acid-activatable split protease units was hydrodynamically transfected into the liver of type-1 diabetic mice. Induction with abscisic acid attenuated glycemic excursions in glucose-tolerance tests. Increased blood levels of insulin were maintained for 12 days.

High fibroin-loaded silk-PCL electrospun fiber with core-shell morphology promotes epithelialization with accelerated wound healing.

Silk fibroin (SF) is a widely explored biopolymer for wound-healing applications due to the presence of amino acids in the biodegradable polymer chain with superior mechanical properties. Herein, a high SF-loaded fibrous matrix along with poly(ε-caprolactone) (PCL) was fabricated using electrospinning of emulsion and blend compositions to modulate nanostructure morphology. A comparative study of the physicomechanical properties of electrospun fibers with emulsion (eS7P3) and homogenous blend (bS7P3) was performed as well. In both compositions, SF loading of up to 70% was successfully achieved in the spun fibers while emulsion yielded core-shell morphology, and the blend resulted in monolith fiber architecture as evidenced by TEM microscopy. Further characterization revealed superior mechanical properties in S7P3 fiber with core-shell morphology, as compared to those in the monolith in terms of a higher degree of crystallinity with Young's modulus of 60 MPa under tensile test and nanoindentation modulus of 1.59 ± 0.8 GPa. Further, eS7P3 nanostructure morphology containing silk in the core with a thin outer layer of PCL facilitated relatively faster biodegradation in the lysozyme medium, as compared to that in the monolith. Owing to the presence of a hydrophobic shell, protein adsorption on the fibrous mat presented slow but steady kinetics up to 24 h. When the scaffold was seeded with human placenta-derived mesenchymal stem cells (hPMSCs), in vitro study confirmed that the eS7P3 structure had marginally higher cell proliferation with superior cell infiltration than the monolith. Further, in vivo study involving a rodent model showed the potential of the eS7P3 fiber substrate with a core-shell structure for accelerating full-thickness wound healing by inducing hair follicle and wound closure with less scar formation after 15 days.

Programmable DARPin-based receptors for the detection of thrombotic markers.

Cellular therapies remain constrained by the limited availability of sensors for disease markers. Here we present an integrated target-to-receptor pipeline for constructing a customizable advanced modular bispecific extracellular receptor (AMBER) that combines our generalized extracellular molecule sensor (GEMS) system with a high-throughput platform for generating designed ankyrin repeat proteins (DARPins). For proof of concept, we chose human fibrin degradation products (FDPs) as markers with high clinical relevance and screened a DARPin library for FDP binders. We built AMBERs equipped with 19 different DARPins selected from 160 hits, and found 4 of them to be functional as heterodimers with a known single-chain variable fragments binder. Tandem receptors consisting of combinations of the validated DARPins are also functional. We demonstrate applications of these AMBER receptors in vitro and in vivo by constructing designer cell lines that detect pathological concentrations of FDPs and respond with the production of a reporter and a therapeutic anti-thrombotic protein.

ß-Cyclodextrin-Based Ultrahigh Stretchable, Flexible, Electro- and Pressure-Responsive, Adhesive, Transparent Hydrogel as Motion Sensor.

In the present work, a multiple-stimuli-responsive hydrogel has been synthesized via polymerization of acrylamide (AAm) and N-hydroxy methyl acrylamide (HMAm) on ß-cyclodextrin (ß-CD). The synthesized hydrogel ß-CD-g-(pAAm/pHMAm) exhibited various striking features like ultrahigh stretchability (>6000%), flexibility, stab resistivity, self-recoverability, electroresponsiveness, pressure-responsiveness, adhesiveness, and high transparency (>90%). Besides, the hydrogel has demonstrated enhanced biocompatibility, UV resistance, and thermoresponsive shape memory behaviors. On the basis of these attractive characteristics of the hydrogel, a flexible pressure sensor for the real-time monitoring of human motion with superior biocompatibility and transparency was fabricated. Moreover, due to the nanofibrillar surface morphology of the ß-CD-g-(pAAm/pHMAm) hydrogel, the sensor based on the gel exhibited high sensitivity (0.053 kPa-1 for 0-3.3 kPa). The flexible sensor demonstrates very fast response time (130 ms-210 ms) with adequate stability (5000 cycles). Interestingly, the sensor can rapidly sense both robust (index finger and wrist) motions as well as tiny (swallowing and phonation) physiological actions. In addition, this adhesive hydrogel patch also acts as a potential carrier for the sustained topical release of (∼80.8% in 48 h) the antibiotic drug gentamicin sulfate.

Hyperbranched Copolymers Forming Polymersome-like Structures Used for Encapsulation and Controlled Release of α-Tocopherol Succinate (TOS): Drug Transport Modeling.

Trimesic acid (TMA) and ethyelene diamine (ED) were reacted in various molar proportions to yield several branched/hyperbranched copolymers which formed polymersome-like structures, and they were used for encapsulation and release of a model drug, α-tocopherol succinate (TOS). The branched topology of the copolymers was established from spectroscopy, viscometry, and rheological measurements. Hydrodynamic size and transmission electron microscopy revealed the self-aggregated polymersome-like features of the copolymers with a dense core. Zeta potential studies unveiled pH-sensitive features of the aggregates. Both hydrodynamic size and viscosity were found to decrease with more branching, whereas the encapsulation efficiency displayed a drastic increase from 68 to 89.5% from the least branched (30%) to mostly branched grade (68%). The hydrophobic drug was primarily accommodated within the macromolecular voids inside the core and was released slowly following the diffusion mechanism. An indigenous model was established to explain the release kinetics from such a pH-sensitive and highly branched core-shell matrix which yielded a unique parameter called effective diffusivity or ED that took account of those parameters affecting the release rate. The copolymers were found to be biologically viable through a series of in vitro tests, thus inviting in vivo trials for future experimentation.

Role of nanofibers on MSCs fate: Influence of fiber morphologies, compositions and external stimuli.

In regenerative medicine, self-regulated tissue regeneration is perceived by Mesenchymal Stem Cells (MSCs) fate due to their tissue-specific differentiation, which is an emerging yet promising tool for therapeutics. MSCs with their innate nature like secretion of bioactive molecules, multilineage differentiation and proliferation supported tissue repair. MSCs interact with extracellular matrix (ECM) components like collagen, glycosaminoglycans (GAGs), proteoglycans and various proteins that are present in the form of nanofibers representing variable matrix elasticity along with topographies and bioactive cues. Synthetic nanofibers also showed to mimic native tissue microenvironment and supported regeneration owing to structural resemblance with ECM for anchorage-dependent cells. Different nanofibers generated using various polymer precursors and their resultant scaffolds, architectures, compositions etc. were studied for their influence on MSCs activities to improvise cell-cell and cell-material interactions. Electrospinning, popular nanotechnology for fiber formation based on electrohydrodynamic theory, is widely used for many applications due to its simplicity, efficacy and environmentally friendliness. Electrospun nanofibers were extensively investigated to understand the influence of material towards manipulating stem cells based on regenerative medicine. Subsequently, the influence of different solutions and process parameters were studied for nanofiber structure repeatability and emphasized on fiber properties such as diameter, mechanical properties, degradation rate, and porosity. Recent approaches towards scale-up for nanofiber production by electrospinning and other novel techniques are also presented briefly. The fate of MSCs, while seeded on nanofibers under external stimuli viz. electrical, mechanical, magnetic and electromagnetic field, is reviewed to find the niche for differentiation pathways. Further, several external stimuli presented as important factors motivating cellular differentiation in combination with specific conditions without the use of any chemical cues.

Eggshell membrane: A natural substrate for immobilization and detection of DNA.

Chemically modified eggshell membranes (ESM) have been explored as potentially novel platforms for immobilization of oligonucleotides and subsequent detection of target DNA. The fibrous network of the native ESM as well those functionalized with acetic acid or n-butyl acetate has been examined by field-emission scanning electron microscopy (FESEM). The formation of surface functional moieties has been confirmed by Fourier-transform infrared spectroscopy (FTIR). DNA molecules, with an end terminal -NH2 group (at 5' end) have been immobilized on the chemically modified ESM surface. The effect of surface modification on the DNA immobilization efficiency has been investigated using fluorescence microscopy and atomic force microscopy (AFM). The above studies concurrently suggest that functionalization of ESM with n-butyl acetate causes a better homogeneity of the DNA probes on the membrane surface. On-chip hybridization of the target DNA with the surface bound capture probes has been performed on the functionalized membranes. It is observed that n-butyl acetate modification of ESM pushes the limit of detection (LOD) of the DNA sensors by at least an order of magnitude compared to the other modification method.