Expansion Microscopy with a Thermally Adjustable Expansion Factor Using Thermoresponsive Biospecimen-Hydrogel Hybrids.

Expansion microscopy (ExM) is a technique in which swellable hydrogel-embedded biological samples are physically expanded to effectively increase imaging resolution. Here, we develop thermoresponsive reversible ExM (T-RevExM), in which the expansion factor can be thermally adjusted in a reversible manner. In this method, samples are embedded in thermoresponsive hydrogels and partially digested to allow for reversible swelling of the sample-gel hybrid in a temperature-dependent manner. We first synthesized hydrogels exhibiting lower critical solution temperature (LCST)- and upper critical solution temperature (UCST)-phase transition properties with N-alkyl acrylamide or sulfobetaine monomers, respectively. We then formed covalent hybrids between the LCST or UCST hydrogel and biomolecules across the cultured cells and tissues. The resulting hybrid could be reversibly swelled or deswelled in a temperature-dependent manner, with LCST- and UCST-based hybrids negatively and positively responding to the increase in temperature (termed thermonegative RevExM and thermopositive RevExM, respectively). We further showed reliable imaging of both unexpanded and expanded cells and tissues and demonstrated minimal distortions from the original sample using conventional confocal microscopy. Thus, T-RevExM enables easy adjustment of the size of biological samples and therefore the effective magnification and resolution of the sample, simply by changing the sample temperature.

Development of poly(D,L-lactic-co-glycolic acid) films coated with biomembrane-mimicking polymers for anti-adhesion activity.

A physical barrier is one of the most effective strategies to alleviate excessive postoperative adhesion (POA) between tissues at an injury site. To overcome the limitations of current polymeric film-type physical barriers, we suggest a film of poly(lactic-co-glycolic acid) (PLGA) that is non-covalently coated with poly(2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)) (PMB). While maintaining the degradability and mechanical properties of PLGA, the PMB coating introduces strong anti-adhesive properties to the film by forming a zwitterionic MPC-based surface through the hydrophobic interactions between BMA moieties and PLGA. Compared to SurgiWrap®, the commercially available poly(lactic acid)-based anti-adhesive film against POA, the PMB-coated PLGA film is much more inhibitory against protein adsorption and fibroblast adhesion, processes that are crucial to the POA process. PMB coating also inhibits the expression of fibronectin containing extra domain A (FN-EDA), α-smooth muscle actin (α-SMA), and collagen type IV alpha 2 (COL4A2), which are marker genes and proteins involved in fibroblast activation and excessive fibrosis during POA. Such inhibitory activities are clearly observed in a 3-dimensional culture of fibroblasts within a collagen matrix, which mimics the in vivo environment of an injury site, as well as in a 2-dimensional culture. The kinetics and the stability of the PMB coating suggest potential future clinical use to coat PLGA films to create a film-type anti-adhesion barrier that overcomes the limitations of current products.

Efficient reduction of fibrous capsule formation around silicone breast implants densely grafted with 2-methacryloyloxyethyl phosphorylcholine (MPC) polymers by heat-induced polymerization.

Implants based on silicone elastomers, polydimethylsiloxane (PDMS), have been widely used for breast augmentation and reconstruction, but excessive foreign body reactions around implants often cause serious side effects such as capsular contracture. In our previous study, we covalently grafted 2-methacryloyloxyethyl phosphorylcholine (MPC)-based polymers on the surface of PDMS blocks by UV-induced polymerization and showed effective reduction of capsular formation around the MPC-grafted PDMS in rats. In the present study, we examined the efficacy of heat-induced polymerization of MPC grafting on silicone breast implants intended for humans, and analyzed the in vivo inhibitory effect against capsular formation and inflammation in pigs, which are closely related to humans in terms of epidermal structures and fibrotic processes. The heat-induced polymerization provided a thicker MPC-grafted surface and was more effective than UV-induced polymerization for the grafting of complex shaped non-transparent implants. After 24-week implantation in the submuscular pockets of Yorkshire pigs, the heat-induced MPC-grafted breast implants showed 45% smaller capsular thickness and 20-30% lower levels of inflammatory markers such as myeloperoxidase (MPO), transforming growth factor-ß (TGF-ß), and α-smooth muscle actin (α-SMA) in surrounding tissues compared to non-grafted implants. This study provides important information for future clinical trials of MPC-grafted silicone implants.

De novo formation of citrate-based fluorophores on N-termini of peptides and proteins in cells and tissues.

We developed a new method for the de novo formation of fluorophores based on citrate (DNFC) in biological samples. Use of an amide coupling reagent and microwave irradiation greatly facilitates the fluorophore formation on peptides and proteins with N-terminal cysteine or serine. Since N-terminal cysteine and serine can form thiazolopyridone- or oxazolopyridone-based fluorophores emitting blue and green fluorescence, respectively, by the DNFC staining, each organelle, cell and tissue exhibited a characteristic fluorescence distribution. The DNFC staining is able to provide a new potential protocol for future cell imaging, histology and diagnosis.

Scalable and Isotropic Expansion of Tissues with Simply Tunable Expansion Ratio.

Tissue expansion techniques physically expand swellable gel-embedded biological specimens to overcome the resolution limit of light microscopy. As the benefits of expansion come at the expense of signal concentration, imaging volume and time, and mechanical integrity of the sample, the optimal expansion ratio may widely differ depending on the experiment. However, existing expansion methods offer only fixed expansion ratios that cannot be easily adjusted to balance the gain and loss associated with expansion. Here, a hydrogel conversion-based expansion method is presented, that enables easy adjustment of the expansion ratio for individual needs, simply by changing the duration of a heating step. This method, termed ZOOM, isotropically expands samples up to eightfold in a single expansion process. ZOOM preserves biomolecules for post-processing labelings and supports multi-round expansion for the imaging of a single sample at multiple zoom factors. ZOOM can be flexibly and scalably applied to nanoscale imaging of diverse samples, ranging from cultured cells to thick tissues, as well as bacteria, exoskeletal Caenorhabditis elegans, and human brain samples.

Anti-inflammatory and Antibacterial Effects of Covalently Attached Biomembrane-Mimic Polymer Grafts on Gore-Tex Implants.

Expanded polytetrafluoroethylene (ePTFE), also known as Gore-Tex, is widely used as an implantable biomaterial in biomedical applications because of its favorable mechanical properties and biochemical inertness. However, infection and inflammation are two major complications with ePTFE implantations, because pathogenic bacteria can inhabit the microsized pores, without clearance by host immune cells, and the limited biocompatibility can induce foreign body reactions. To minimize these complications, we covalently grafted a biomembrane-mimic polymer, poly(2-methacryloyloxylethyl phosphorylcholine) (PMPC), by partial defluorination followed by UV-induced polymerization with cross-linkers on the ePTFE surface. PMPC grafting greatly reduced serum protein adsorption as well as fibroblast adhesion on the ePTFE surface. Moreover, the PMPC-grafted ePTFE surface exhibited a dramatic inhibition of the adhesion and growth of Staphylococcus aureus, a typical pathogenic bacterium in ePTFE implants, in the porous network. On the basis of an analysis of immune cells and inflammation-related factors, i.e., transforming growth factor-ß (TGF-ß) and myeloperoxidase (MPO), we confirmed that inflammation was efficiently alleviated in tissues around PMPC-grafted ePTFE plates implanted in the backs of rats. Covalent PMPC may be an effective strategy for promoting anti-inflammatory and antibacterial functions in ePTFE implants and to reduce side effects in biomedical applications of ePTFE.

Suspension arrays of hydrogel microparticles prepared by photopatterning for multiplexed protein-based bioassays.

Suspension arrays for protein-based assays have been developed using shape-coded poly(ethylene glycol) (PEG) hydrogel microparticles to overcome the problems with current systems which use color-coded rigid microparticles as protein supports. Various shapes of hydrogel microparticles were fabricated by a two-step process consisting of photopatterning and flushing using a poly(dimethylsiloxane) (PDMS) channel as a molding insert. Hydrogel microparticles with lateral dimensions ranging from 50 to 300 micrometers were fabricated using different molecular weights of PEG (700, 3,400, and 8,000 Da), by which the water content and swelling behavior of the hydrogel microparticles could be controlled. Protein-entrapped hydrogel microparticles were prepared in a suspension array format, and PEG hydrogel could encapsulate proteins without deactivation for a week due to its high water content and soft nature. The sequential bienzymatic reaction of hydrogel-entrapped glucose oxidase (GOX) and peroxidase (POD) was successfully investigated using fluorescence detection, demonstrating one possible application of suspension arrays. Furthermore, a mixture of two different shapes of hydrogel microparticles containing GOX/POD and alkaline phosphatase (AP), respectively, was prepared and the shape-coded suspension array was used for simultaneous characterization of two different enzyme-catalyzed reactions.

Preparation of poly(ethylene glycol) hydrogels with different network structures for the application of enzyme immobilization.

In this study, poly(ethylene glycol) (PEG)-based hydrogels having different network structures were synthesized by UV-initiated photopolymerization and used for the enzyme immobilization. PEGs with different molecular weight were acrylated by derivatizing both ends with acryloyl chloride and photopolymerization of PEG-diacrylate (PEG-DA) yielded crosslinked hydrogel network within 5 seconds. Attachment of acrylate groups and gelation were confirmed by ATR/FT-IR and FT-Raman spectroscopy. Network structures of hydrogels could be easily controlled by changing the molecular weight (MW) of PEG-DA and characterized by calculating molecular weight between crosslinks and mesh size from the swelling measurement. Synthesis of hydrogels with higher MW of PEG produced less crosslinked hydrogels having higher water content, larger value of Mc and mesh size, which resulted in enhanced mass transfer but loss of mechanical properties. For the enzyme immobilization, glucose oxidase (GOX) was immobilized inside PEG hydrogels by means of physical entrapment and covalent immobilization. Encapsulated GOX were covalently bound to PEG backbone using acryloyl-PEG-N-hydroxysuccinimide and maintained their activity over a week period without leakage. Kinetic study indicated that immobilized enzyme inside hydrogel prepared from higher MW of PEG possessed lower apparent Km (Michaelis-Menten constant) and higher activity.