Enhancement of intercellular interaction between iPSC-derived neural progenitor cells and activated endothelial cells using cell surface modification with functional oligopeptides.

Cell-based therapy has been used to treat stroke related disorders, which have no treatment options available 4.5 hours after onset. Although the administration of tissue plasminogen activator and mechanical thrombectomy are potent treatments, their clinical implementation is limited within the available time. Here, we aimed to use induced pluripotent stem cell-derived neural progenitor cells (NPCs) for stroke treatment with higher delivery efficiency in stroke areas, which will improve the therapeutic effect. E-selectin binding oligopeptide (Esbp) was conjugated with poly(ethylene glycol)-conjugated-lipid (Esbp-PEG-lipid) with different molecular weights of PEG (5 and 40 kDa) for cell surface modification. Then, we optimized the cell surface modification of NPCs by studying cell-binding ability onto the model surfaces of stroke areas, such as recombinant E-selectin-immobilized surfaces and TNF-α activated endothelium. As a result, the cell surface modification of NPCs with Esbp-PEG-lipid was found to induce specific intercellular interactions with the activated endothelium through the binding of Esbp with E-selectin. Additionally, the shorter PEG spacer was suitable for intercellular interactions. Thus, our technique shows potential for use in cell therapy with enhanced cell accumulation in infarct areas.

Induction of Spontaneous Liposome Adsorption by Exogenous Surface Modification with Cell-Penetrating Peptide-Conjugated Lipids.

The use of amphiphilic molecules such as poly(ethylene glycol)-conjugated phospholipid (PEG-lipid) enables incorporation into liposome surfaces by exogenous addition as a result of the self-assembly with lipids. This technique can be applicable for manipulation of both liposomes and cells. In this study, we aimed to characterize Tat peptide (YGRKKRRQRRR)-conjugated PEG-lipids when used to exogenously surface modify liposomes (size: ca. 100 nm). We earlier reported that cells, which were surface modified with Tat peptides conjugated to PEG-lipids could attach spontaneously to material surfaces without any chemical modification. Here, we synthesized different types of Tat-PEG-lipids by combining PEG of different molecular weights (5 and 40 kDa) with different lipids with three acyl chains (myristoyl, palmitoyl, and stearoyl, respectively) and then studied the spontaneous adsorption of modified liposomes onto a substrate surface induced by the different Tat-PEG-lipids. The amount of adsorbed liposomes strongly depended on the number of incorporated Tat-PEG-lipid moieties: a decrease in both the PEG and the acyl chain lengths led to adsorption of higher amounts of liposomes. Furthermore, when a collagenase-cleavable amino acid sequence was inserted between the Tat sequence and the PEG segment, adsorbed liposomes could be harvested from the substrate by collagenase treatment with no difference in desorption efficiency between the different Tat-PEG-lipids. Thus, Tat-PEG-lipid can be a suitable tool for the manipulation of liposomes and cells.

Exogenous Cell Surface Modification with Cell Penetrating Peptide-Conjugated Lipids Causes Spontaneous Cell Adhesion.

The technique of cell patterning on a substrate is of great importance for platforms in cell-based assays. Chemical treatment of the substrate is commonly performed for cell patterning using cationic polymers, extracellular matrices, and antibodies. However, cell patterning could be easier if there is an approach to immobilize cells without treating the substrate surface. We previously reported that cell adhesion could be induced by the modification of the cellular surface with a cell-penetrating peptide (CPP)-conjugated poly(ethylene glycol)-phospholipid (CPP-PEG-lipid). This approach does not require chemical modification of the substrate surface, such as polystyrene or glass, and can be used for the cell patterning of floating cells. Here, we aimed to study the mechanism of induced cell adhesion using a representative CPP, Tat peptide (Tat-PEG-lipid). We found that cell adhesion was induced via electrostatic interactions between the Tat peptide and the substrate surface, which could be induced more efficiently by increasing the molecular weight of PEG together with CPPs but not with cationic peptides. The excluded volume effect between neighboring PEG chains could stretch the cell shape better than PEG with lower molecular weight, allowing the cell to spread firmly. In addition, Tat-PEG-lipid did not activate actin filament formation and did not influence the expression of focal adhesion kinase. Thus, the induced cell adhesion by CPP-PEG-lipid did not affect internal cell signaling.

Quantitative evaluation of the impact of artificial cell adhesion via DNA hybridization on E-cadherin-mediated cell adhesion.

Programmable cell adhesion with DNA hybridization is a promising approach for fabricating various tissue architectures without sophisticated instrumentation. However, little is known about how this artificial interaction influences the binding of cell adhesion proteins, E-cadherin. In this work, we designed a planar and fluid lipid membrane displaying E-cadherin and/or single-strand DNA with well-defined densities. Visualization of cells on membranes by fluorescence and interference microscopy revealed cell adhesion to be a two-step process: artificial adhesion by DNA hybridization within a few minutes followed by biological adhesion via cadherin-cadherin binding within hours. Furthermore, we discovered that DNA hybridization can substantially facilitate E-cadherin-mediated cell adhesion. The promotive effect is probably due to the enforced binding between E-cadherin molecules in geometrical confinement between two membranes. Our in vitro model of cell adhesion can potentially be used to design functional synthetic molecules that can regulate cell adhesion via cell adhesion proteins for tissue engineering.

Enhancing Detection Sensitivity of ZnO-Based Infrared Plasmonic Sensors Using Capped Dielectric Ga2O3 Layers for Real-Time Monitoring of Biological Interactions.

Surface plasmon resonances on Ga-doped ZnO (ZnO/Ga) layer surfaces (ZnO-SPRs) have attracted substantial attention as alternative plasmonic materials in the infrared range. We present further enhancement of the detection limits of ZnO-SPRs to monitor biological interactions by introducing thin dielectric layers into ZnO-SPRs, which remarkably modify the electric fields and the corresponding decay lengths on the sensing surfaces. The presence of a high-permittivity dielectric layer of Ga2O3 provides high wavelength sensitivities of the ZnO-SPRs due to the strongly confined electric fields. The superior sensing capabilities of the proposed samples were verified by real-time monitoring of the biological interactions between biotin and streptavidin molecules. Introduction of the high-permittivity dielectric layer into ZnO-SPRs effectively enhances the detection sensitivity and therefore allowed for the observation of biological interactions. This paper provides useful information for the development of optical detection techniques for use in biological fields based on ZnO from the viewpoints of plasmonic applications.

Modification of human MSC surface with oligopeptide-PEG-lipids for selective binding to activated endothelium.

Promising cell therapies using mesenchymal stem cells (MSCs) is proposed for stroke patients. Therefore, we aimed to efficiently accumulate human MSC (hMSC) to damaged brain area to improve the therapeutic effect using poly(ethylene glycol) (PEG)-conjugated phospholipid (PEG-lipid) carrying an oligopeptide as a ligand, specific for E-selectin which is upregulated on activated endothelial cells under hypoxia-like stroke. Here we synthesized E-selectin-binding oligopeptide (ES-bp) conjugated with PEG spacer having different molecular weights from 1 to 40 kDa. We found that ES-bp can be immobilized onto the hMSC surface through PEG-lipid without influence on cell growth and differentiation into adipocytes and osteocytes, respectively. It is also possible to control the immobilization of ES-bp on hMSC surface (<108 ES-bp per cell). Immobilized ES-bp can be continuously immobilized at the outside of cell membrane when PEG-lipids with PEG 5 and 40 kDa were used. In addition, the modified hMSC can specifically attach onto E-selectin-immobilized surface as a model surface of activated endothelium in human blood, indicating the sufficient number of immobilized ES-bp onto hMSC. Thus, this technique is one of the candidates for hMSC accumulation to cerebral infarction area. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1779-1792, 2019.

Influence of cell adhesive molecules attached onto PEG-lipid-modified fluid surfaces on cell adhesion.

The involvement of intercellular interactions in various biological events indicates the importance of studying cell-cell interactions using fluid model surfaces. Here, we propose a fluid surface composed of a self-assembled monolayer (SAM) and poly(ethylene glycol)-conjugated phospholipid (PEG-lipid) derivatives, which can be an alternative to supported lipid membranes. The modification of SAM surfaces with PEG-lipids carrying functional peptides resulted in the formation of the fluid surfaces with different mobility, which was quantitatively determined by quartz crystal microbalance with dissipation (QCM-D) and fluorescence recovery after photobleaching (FRAP). Different types of fluid surfaces with calculated diffusion coefficients between 0.9 ± 0.25 and 0.16 ± 0.03 µm2/sec for PEG-lipids derivatives were fabricated, onto which arginylglycylaspartate (RGD) peptides were immobilized for cell adhesion, and compared to solid surfaces with the same surface density of RGD peptides. The fluid surfaces revealed that cell adhesions of epithelial cells (MCF-10 A) and human umbilical vein endothelial cells (HUVEC) could not be established on the surfaces with higher fluidity, while cells could adhere onto surfaces with lower fluidity, where the lateral diffusion of PEG-lipids was approximately 20 times lower, and solid surfaces. Interestingly, cells that adhered onto the surface with lower fluidity proliferated at a normal rate while maintaining their round morphology, which was a different shape from that observed on solid surfaces. Thus, the scaffold fluidity greatly influenced cell adhesion behaviors, demonstrating that it is an important parameter for designing novel biomimetic scaffolds for biomedical applications.