Multifunctional Micromachines Constructed by Combining Multiple Protein-Based Components with Different Functions.

Untethered mobile micromachines have considerable potential to realize more effective and minimally invasive medicine. Although diverse medical micromachines have been reported over the past few decades, these machines were developed for performing only specific tasks and the functions imparted to them were limited to a few. Hence, the methodologies for imparting a wide variety of functions to machines have not been fully explored. In this study, a novel construction strategy for the multifunctional micromachines is presented, where a specific function can be added to the machine in one step by directly combining the protein-based component, possessing the biological function of constituent proteins, to an arbitrary position of the machine by using an inkjet printing technique. As a proof-of-concept demonstration, various types of machines were constructed by combining multiple components with different functions. These constructed machines successfully performed functions as diverse as enzyme-powered self-propulsion, collection of target objects, including the bilirubin and living cells, enzyme-mediated conversion of substrate molecules to different ones, magnetic guidance, and release of anti-inflammatory drug diapocynin. The study's progressive approach as well as multifunctional and biocompatible machines composed of proteins will profoundly impact the development of intelligent machines equipped with multiplex sophisticated functionalities.

Potential of the Coordinated Actions of Multiple Protein-Based Micromachines for Medical Applications.

Untethered mobile micromachines hold great promise in the development of effective and minimally invasive therapies. Although diverse medical micromachines for specific applications have been developed over the past few decades, the coordinated action of multiple machines with different functions remains largely unexplored. In this study, we created three types of biocompatible micromachines using proteins and demonstrated the potential of their coordinated action for medical applications. As a proof of concept, we demonstrated neural replacement therapy, in which neuroblastomas were killed by using an anticancer prodrug and the first machine that contains enzymes, enabling the conversion of the prodrug into a cytotoxic drug. Subsequently, a second machine composed of extracellular matrix was placed on the dead cancer cells to provide a suitable environment for cell adhesion, on which embryonic stem (ES) cells and stromal cells that promote neural differentiation of stem cells were attached by using third machines capable of delivering cells to target positions with desired patterns. As a result, neuroblastomas were replaced with novel healthy neurons derived from ES cells by teaming multiple protein-based machines. We believe that this work highlights the potential of heterogeneous machine groups for medical treatment and the utility of highly biocompatible and functional micromachines made from proteins, representing an important step forward in building more sophisticated micromachine-based therapies.

Superior Adhesion of a Multifunctional Protein-Based Micropatch to Intestinal Tissue by Harnessing the Hydrophobic Effect.

Drug delivery systems comprising drug carriers capable of adhering to intestinal tissue have considerable potential to realize more sophisticated systemic drug delivery and topical drug treatments in the intestinal tract. The development of innovative strategies for improving the adhesion efficiency of carriers is of high importance for the advancement of this field. Herein, a novel approach to achieving high adhesion efficiency of drug carriers is presented, where the accessibility of the carrier to the intestinal surface and its subsequent adhesion to the intestinal tissue are promoted by utilizing the thermodynamic tendency of the hydrophobic carrier and its dispersion solvent, triacetin, to be excluded from the aqueous environment. Drug carriers are fabricated using proteins, imparting multiple functions, including drug release and the removal of reactive oxygen species (ROS). Results of ex vivo studies indicate that this multifunctional protein-based carrier, "protein micropatch," adheres to various mouse intestinal tissues, including the small intestine, colon, and inflamed colon, with high efficiency. Furthermore, protein micropatches, administered to mice via oral or rectal routes, successfully adhere to the intestinal tract. This approach and the highly functionalized carrier described in the study have the potential to significantly contribute to the development of bioadhesive carrier-based drug delivery systems.

Flow analysis on microcasting with degassed polydimethylsiloxane micro-channels for cell patterning with cross-linked albumin.

Patterned cell culturing is one of the most useful techniques for understanding the interaction between geometric conditions surrounding cells and their behaviors. The authors previously proposed a simple method for cell patterning with an agarose gel microstructure fabricated by microcasting with a degassed polydimethylsiloxane (PDMS) mold. Although the vacuum pressure produced from the degassed PDMS can drive a highly viscous agarose solution, the influence of solution viscosity on the casting process is unknown. This study investigated the influences of micro-channel dimensions or solution viscosity on the flow of the solution in a micro-channel of a PDMS mold by both experiments and numerical simulation. It was found experimentally that the degassed PDMS mold was able to drive a solution with a viscosity under 575 mPa·s. A simulation model was developed which can well estimate the flow rate in various dimensions of micro-channels. Cross-linked albumin has low viscosity (1 mPa·s) in aqueous solution and can undergo a one-way dehydration process from solution to solid that produces cellular repellency after dehydration. A microstructure of cross-linked albumin was fabricated on a cell culture dish by the microcasting method. After cells were seeded and cultivated on the cell culture dish with the microstructure for 7 days, the cellular pattern of mouse skeletal myoblast cell line C2C12 was observed. The microcasting with cross-linked albumin solution enables preparation of patterned cell culture systems more quickly in comparison with the previous agarose gel casting, which requires a gelation process before the dehydration process.

Antibody immobilization technique using protein film for high stability and orientation control of the immobilized antibody.

The development of antibody immobilization techniques is essential for creating antibody-based biomaterials. Although numerous methods for antibody immobilization have been demonstrated, low stability and disordered orientation of the immobilized antibody remain an important problem. In this work, an original antibody immobilization technique using a protein film, which achieved a high stability and orientation control of the immobilized antibody, has been described. In this method, an antibody-immobilized albumin film was prepared by adding the cross-linked albumin solution to the substrate, where antibodies were attached in uniform orientation, followed by subsequent drying, and detaching the formed film from the substrate by heating at 120 °C in a dry state. Antibodies in the film showed high antigen-binding capacity, at a level comparable to the oriented immobilized antibody using protein G. The stability of antibodies in the film was found to be significantly high; their antigen-binding capacity was completely retained even after storage at 40 °C in a dry state for one month. Thus, this approach provides useful information to immobilize the antibody on solid surfaces while controlling its orientation and increasing its stability.

Spectroscopic study on the conformation of serum albumin in film state.

Protein is a promising material for fabricating the biocompatible films used in the biomedical fields and food industry. Previously, we successfully prepared a water-insoluble albumin film possessing native albumin properties such as resistance to cell adhesion and drug-binding ability. Here, I quantitatively investigated the conformation of albumin in a film state using circular dichroism (CD) spectroscopy. The albumin film was prepared by crosslinking albumin with ethylene glycol diglycidyl ether (EGDE). CD measurements of albumin films revealed that approximately 70% of the α-helical structure was retained after film formation. Albumin molecules in the films acquired high stability. The conformation of albumin was completely retained even after heating at 100 °C for 1 h. For comparison, crosslinked albumin film was also prepared using glutaraldehyde (GA). Unlike EGDE-crosslinking, GA-crosslinking induced significant conformational changes in albumin; 46% of the α-helical structure was destroyed in GA-crosslinked albumin films. Cell adhesion studies showed that EGDE-crosslinked albumin film maintained the cell-nonadhesive property inherent in native albumin. This property was lost in GA-crosslinked albumin film, and cells adhesion occurred at a level comparable to that of cell culture dishes. These results indicate that EGDE-crosslinking is a useful method for preparing albumin films in which the native albumin structure and property are retained. The approach described here provides valuable information for creating protein films possessing high functionality.

Multifunctional protein microparticles for medical applications.

Micro- and nano-scale intelligent devices can revolutionize the medical field. Although proteins are promising materials for creating biocompatible miniature medical devices with biological functions, construction of complicated solid-state architectures, using inherently vulnerable proteins, remains challenging. Here, I present a sophisticated strategy for constructing a multifunctional microparticle for medical applications using multiple proteins; this strategy achieved the retention of function, increased stability, and orientation control of the proteins in the fabricated particle. As proof-of-concept, the particle, designed to cope with excess reactive oxygen species (ROS) involved in many diseases, was constructed by combining three proteins with different functions. The body of the particle was fabricated using albumin and superoxide dismutase (SOD), and the antibody was incorporated into the surface of the particle in an orientation-controlled manner. The constructed protein microparticle exhibited coordinated activities for coping with ROS, such as capture of the ROS-secreting cells by the incorporated antibody, followed by the elimination of 70% ROS, secreted from the captured cells, by the SOD in the particle. Additionally, diapocynin, loaded to the particle via the drug-binding ability of albumin, was released from the particle, preventing ROS production in the cells. This multifunctional microparticle, constructed from proteins, will profoundly impact the development of intelligent protein-based miniature devices used in medical fields.

Multicomponent Coculture System of Cancer Cells and Two Types of Stromal Cells for In Vitro Evaluation of Anticancer Drugs.

In vitro evaluation of anticancer drugs using cancer cells has long been performed for the development of novel drugs and the selection of effective drugs for different patients. Recent studies have suggested that tumor stromal cells affect the drug sensitivity of cancer cells; however, most conventional culture systems for drug evaluation lack stromal cells. In this study, we fabricated a multicomponent coculture system that takes account of cancer-stroma interactions for drug evaluation. In this system, small-cell and nonsmall-cell lung cancer cells embedded in collagen gel were cocultured with two types of stromal cells, including stromal fibroblasts and proinflammatory cytokine-secreting monocytes, thus recreating the in vivo cancer microenvironment. Cancer drug sensitivity was significantly altered by the presence of stromal cells. Fibroblasts induced resistance of cancer cells to anticancer drugs. Monocytes induced the upregulation of thymidine phosphorylase in cancer cells, promoting the conversion of an anticancer prodrug to a cytotoxic drug, and consequently enhanced the sensitivity of cancer cells to the anticancer prodrug. These results clearly show the importance of incorporating stromal cells into culture systems for drug evaluation. Our system will help to improve the accuracy of in vitro drug evaluation and provide useful information for the in vitro recreation of cancer microenvironments.

Generation of a patterned co-culture system composed of adherent cells and immobilized nonadherent cells.

Patterned co-culture is a promising technique used for fundamental investigation of cell-cell communication and tissue engineering approaches. However, conventional methods are inapplicable to nonadherent cells. In this study, we aimed to establish a patterned co-culture system composed of adherent and nonadherent cells. Nonadherent cells were immobilized on a substrate using a cell membrane anchoring reagent conjugated to a protein, in order to incorporate them into the co-culture system. Cross-linked albumin film, which has unique surface properties capable of regulating protein adsorption, was used to control their spatial localization. The utility of our approach was demonstrated through the fabrication of a patterned co-culture consisting of micropatterned neuroblastoma cells surrounded by immobilized myeloid cells. Furthermore, we also created a co-culture system composed of cancer cells and immobilized monocytes. We observed that monocytes enhanced the drug sensitivity of cancer cells and its influence was limited to cancer cells located near the monocytes. Therefore, the incorporation of nonadherent cells into a patterned co-culture system is useful for creating culture systems containing immune cells, as well as investigating the influence of these immune cells on cancer drug sensitivity. STATEMENT OF SIGNIFICANCE: Various methods have been proposed for creating patterned co-culture systems, in which multiple cell types are attached to a substrate with a desired pattern. However, conventional methods, including our previous report published in Acta Biomaterialia (2010, 6, 526-533), are unsuitable for nonadherent cells. Here, we developed a novel method that incorporates nonadherent cells into the co-culture system, which allows us to precisely manipulate and study microenvironments containing nonadherent and adherent cells. Using this technique, we demonstrated that monocytes (nonadherent cells) could enhance the drug sensitivity of cancer cells and that their influence had a limited effective range. Thus, our technique is useful for recreating complex tissues in order to investigate cellular interactions involving nonadherent cells.

Changes in Cell Adhesiveness and Physicochemical Properties of Cross-Linked Albumin Films after Ultraviolet Irradiation.

We discovered the unique cell adhesive properties of ultraviolet (UV)-irradiated albumin films. Albumin films prepared using a cross-linking reagent with epoxy groups maintained native albumin properties, such as resistance to cell adhesion. Interestingly, the cell adhesive properties of films varied depending upon the UV irradiation time; specifically, cell adhesiveness increased until 2 h of UV irradiation, when the cell number attached to the film was similar to that of culture dishes, and then cell adhesiveness decreased until 20 h of UV irradiation, after which the surface returned to the initial non-adhesive state. To elucidate the molecular mechanisms underlying this phenomenon, we examined the effect of UV irradiation on albumin film properties. The following changes occurred in response to UV irradiation: decreased α-helical structure, cleavage of albumin peptide bonds, and increased hydrophilicity and oxygen content of the albumin film surface. In addition, we found a positive correlation between the degree of cell adhesion and the amount of fibronectin adsorbed on the film. Taken together, UV-induced changes in films highly affect the amount of cell adhesion proteins adsorbed on the films depending upon the irradiation time, which determines cell adhesion behavior.

Facile immunostaining and labeling of nonadherent cells using a microfluidic device to entrap the cells.

We fabricated a microfluidic device for the entrapment of nonadherent cells. Solution exchange was easily performed by introducing the solution into the cell-trapping microchannel. Immunostaining and labeling of the cell membrane of THP-1 cells were demonstrated using this device, which does not require cumbersome repetition of centrifugation and resuspension steps.

Fabrication of protein micropatterns using a functional substrate with convertible protein-adsorption surface properties.

A functional substrate capable of regulating protein adsorption was prepared using a crosslinked albumin (cl-albumin) film for use in the fabrication of protein micropatterns. The adsorption of proteins with different characteristics onto cl-albumin film, including serum proteins, serum albumin, and lysozyme, was investigated using a quartz crystal microbalance. The results showed that surfaces coated with cl-albumin film are highly resistant to protein adsorption, regardless of protein charge and rigidity. In addition, this adsorption-resistance property can be easily converted to promote protein adsorption by exposing the cl-albumin film to a charged polymer solution. By combining the convertible surface property of cl-albumin film and inkjet printing techniques, a precise protein micropattern was successfully fabricated on the substrate. Protein adsorption onto the wall surface of microchannels could also be suppressed or promoted by coating the surface with cl-albumin film. This approach will aid in the development of biomaterials carrying protein micropatterns, such as biosensors, biochips, and cellular scaffolds.

Preparation of arrays of cell spheroids and spheroid-monolayer cocultures within a microfluidic device.

This study describes a novel method for generation of an array of three-dimensional (3D) multicellular spheroids within a microchannel in patterned cultures containing one or multiple cell types. This method uses a unique property of a cross-linked albumin coated surface in which the surface can be switched from non-adhesive to cell adhesive upon electrostatic adsorption of a polycation. Introduction of a solution containing albumin and a cross-linking agent into a microchannel with an array of microwells caused the entire surface, with the exception of the interior of the microwells, to become coated with the cross-linked albumin layer. Cells that were seeded within the microchannel did not adhere to the surface of the microchannel and became entrapped in the microwells. HepG2 cells seeded in the microwells formed 3D spheroids with controlled sizes and shapes depending upon the dimensions of the microwells. When the albumin coated surface was subsequently exposed to an aqueous solution containing poly(ethyleneimine) (PEI), adhesion of secondary cells, fibroblasts, occurred in the regions surrounding the arrayed spheroids. This coculture system can be coupled with spatially controlled fluids such as gradients and focused flow generators for various biological and tissue engineering applications.

Combining the cell-encapsulation technique and axon guidance for cell transplantation therapy.

In cell transplantation therapy for the treatment of neurodegenerative disorders, encapsulation of implanted cells in a semipermeable membrane is a promising approach to protect the implanted cells from host immune rejection and inhibit the invasion of tumor into surrounding tissue if the implanted cells form a tumor after transplantation. However, implanted neurons isolated by capsules could not build connections with host neurons, preventing the implanted neurons from responding to stimuli from host neurons. In the present study, we focused on the passage of neurites and axons navigated by axon guidance molecules through membrane pores to enable encapsulated neurons and host neurons to form connections. The type of matrix coated on membranes and the pore size of the membranes greatly affected the successful passage of PC12 neurites through membrane pores. PC12 neurites preferably passed through collagen-coated membranes with pores greater than 0.8 µm in diameter, but the neurites did not pass through albumin- or fibronectin-coated membranes or membranes with pores less than 0.1 µm in diameter. We could navigate the direction of commissural neural axon extensions by utilizing the axon guidance molecules secreted from floor plate and make guided axons pass through the membrane pores. These results suggest the feasibility of building connections between encapsulated neurons and host neurons by encapsulating the implanted neurons and axon guidance molecules, which attract the axons of host neurons into the capsule, in the porous membranes with suitable pore size and matrix coating.

Cell micropatterning inside a microchannel and assays under a stable concentration gradient.

We describe the use of a microfluidic device to micropattern cells in a microchannel and investigated the behavior of these cells under a concentration gradient. The microfluidic device consisted of 3 parts: a branched channel for generating a stable concentration gradient, a main channel for culturing cells, and 2 side channels that flowed into the main channel. The main channel was coated with a cross-linked albumin that was initially cell-repellent but that could become cell-adherent by electrostatic adsorption of a polycation. A sheath flow stream, which was generated by introducing a polycation solution from the branched channel and a buffer solution from the 2 side channels, was used to change a specific region in the main channel from cell-repellent to cell-adhesive. In this way, cells attached to the central region along the main channel. The remaining surface was subsequently changed to cell-adhesive, thereby facilitating cell migration from a fixed location under a concentration gradient. We demonstrated that with this device, the gradient generator could be used to conduct simultaneous cytotoxic assays with anticancer agents; further, by combining this device with cell micropatterning, migration assays under a concentration gradient of biological factors could be conducted.

Drug-carrying albumin film for blood-contacting biomaterials.

Surface-induced thrombosis is a major complication in the development of blood-contacting medical devices. Serum albumin has the ability to bind to a wide variety of compounds, including drugs, and neither cells nor proteins adsorb to an albumin-coated surface. These properties of albumin are useful for improving the blood compatibility of biomaterial surfaces. In the present study, we prepared a water-insoluble film by cross-linking pharmaceutical grade recombinant human serum albumin aiming to the clinical applications, and loaded the film with a synthetic antiplatelet drug, cilostazol. The resultant film possessed native albumin characteristics such as drug binding ability and resistance to cell adhesion. Mouse fibroblast L929 cells did not adhere on the albumin film, just as they did not adhere on native albumin-coated surfaces. Furthermore, when the albumin film carrying cilostazol was placed in PBS containing Tween-80, the release of cilostazol was sustained over 144 h. The results indicate that the surface coating with thus prepared albumin film can confer the biomaterials with antithrombogenic surface by virtue of its non-adhesiveness to cells and its release of cilostazol.

Preparation of coculture system with three extracellular matrices using capillary force lithography and layer-by-layer deposition.

Micropatterned cocultures were fabricated with 3 extracellular matrices, hyaluronic acid (HA), fibronectin, and collagen. The feature of the fabrication processes is to avoid the use of potentially cytotoxic materials and utilize capillary force of the solution and interactions between the extracellular matrix components. The coculture system can be used to investigate the effects of heterocellular interactions on cellular fate. Direct heterocellular connections between hepatocytes and fibroblasts were visualized by the transcellular diffusion of fluorescein in this coculture system. The interactions between hepatocytes and fibroblasts were crucial for the maintenance of albumin synthesis by hepatocytes. The coculture system was also beneficial for investigating the effects of cell-cell interactions on the induction of embryonic stem (ES) cell differentiation. In cocultures grown in a sea-island pattern, ES cells formed isolated colonies surrounded by PA6 cells and differentiated into neurons with branched neurites that extended from the colonies. This versatile and biocompatible coculture system could potentially be a powerful tool for investigating cell-cell interaction and for tissue engineering applications.

Cell micropatterning on an albumin-based substrate using an inkjet printing technique.

Positioning of cells in a desired pattern on a substrate is an important technique for cell-based technologies, including the fundamental investigation of cell functions, tissue-engineering applications, and the fabrication of cell-based biosensors and cell arrays. Recently, the inkjet printing technique was recognized as a promising approach to the creation of cellular patterns on substrates, and it has been achieved by the printing of living cells or cell adhesive proteins. In this article, we created complex cellular patterns by using an albumin-based substrate and inkjet printing technique. Albumin was cross-linked using ethylene glycol diglycidyl ether. Subsequent casting of the cross-linked albumin solution onto glass plates prevented cells from adhering to their surfaces. Through screening various chemical reagents, we found that these cross-linked albumin surfaces dramatically changed into cell adhesive surfaces after immersion in cationic polymer solutions. Based on this finding, cell adhesive regions were prepared with a desired pattern by printing the polyethyleneimine (PEI) solution onto a cross-linked albumin substrate using a modified commercial inkjet printer. Various cellular patterns including figures, letters, and gradients could be fabricated by seeding mouse L929 fibroblast cells or mouse Neuro-2a neuroblastoma cells onto the printed PEI-patterned substrate. Compared with the printing of fragile living cells or proteins, printing of stable PEI circumvents clogging of printer head nozzles and enables reproducible printing. Therefore, the present method will allow the creation of complex cell patterns.

Facile cell patterning on an albumin-coated surface.

Fabrication of micropatterned surfaces to organize and control cell adhesion and proliferation is an indispensable technique for cell-based technologies. Although several successful strategies for creating cellular micropatterns on substrates have been demonstrated, a complex multistep process and requirements for special and expensive equipment or materials limit their prevalence as a general experimental tool. To circumvent these problems, we describe here a novel facile fabrication method for a micropatterned surface for cell patterning by utilizing the UV-induced conversion of the cell adhesive property of albumin, which is the most abundant protein in blood plasma. An albumin-coated surface was prepared by cross-linking albumin with ethylene glycol diglycidyl ether and subsequent casting of the cross-linked albumin solution on the cell culture dish. While cells did not attach to the albumin surface prepared in this way, UV exposure renders the surface cell-adhesive. Thus, surface micropatterning was achieved simply by exposing the albumin-coated surface to UV light through a mask with the desired pattern. Mouse fibroblast L929 cells were inoculated on the patterned albumin substrates, and cells attached and spread in a highly selective manner according to the UV-irradiated pattern. Although detailed investigation of the molecular-level mechanism concerning the change in cell adhesiveness of the albumin-coated surface is required, the present results would give a novel facile method for the fabrication of cell micropatterned surfaces.

Preparation of water-insoluble albumin film possessing nonadherent surface for cells and ligand binding ability.

Serum albumin is the most abundant protein in blood plasma. Albumin has the ability to bind to a wide variety of compounds including drugs, and cells as well as proteins do not attach to an albumin-coated surface. These properties of albumin are attractive for biomaterials utilized in biomedical fields. In the present study, we aimed to prepare a water-insoluble albumin film possessing suitable flexibility and native albumin characteristics, such as drug binding ability and resistance to cell adhesion. To confer the film with both water insolubility and flexibility without losing albumin characteristics, we searched a suitable condition for the cross-linking of albumin. As a result, we found that a film having aimed properties could be obtained by conducting the cross-linking reaction at room temperature for 24 h using 215 mM ethylene glycol diglycidyl ether. Mouse fibroblast L929 cells did not adhere on thus obtained film in a similar manner to a native albumin-coated surface. In addition, the film could bind 2-(4'-hydroxyphenylazo)-benzoic acid, a representative albumin binding dye, and gradually release it in a pH-dependent manner.