| 1. |
- Noiri, Makoto, et al.
(author)
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Exogenous Cell Surface Modification with Cell Penetrating Peptide-Conjugated Lipids Causes Spontaneous Cell Adhesion
- 2021
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In: ACS Applied Bio Materials. - : American Chemical Society (ACS). - 2576-6422. ; 4:5, s. 4598-4606
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Journal article (peer-reviewed)abstract
- 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.
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| 2. |
- Adler, Anna, et al.
(author)
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Synthesis of poly(2-methacryloyloxyethyl phosphorylcholine)-conjugated lipids and their characterization and surface properties of modified liposomes for protein interactions
- 2021
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In: Biomaterials Science. - : Royal Society of Chemistry. - 2047-4830 .- 2047-4849. ; 9:17, s. 5854-5867
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Journal article (peer-reviewed)abstract
- Poly(ethylene glycol) (PEG) is frequently used for liposomal surface modification. However, as PEGylated liposomes are cleared rapidly from circulation upon repeated injections, substitutes of PEG are being sought. We focused on a water-soluble polymer composed of 2-methacryloyloxyethyl phosphorylcholine (MPC) units, and synthesized poly(MPC) (PMPC)-conjugated lipid (PMPC-lipid) with degrees of MPC polymerization ranging from 10 to 100 (calculated molecular weight: 3 to 30 kDa). In addition, lipids with three different alkyl chains, myristoyl, palmitoyl, and stearoyl, were applied for liposomal surface coating. We studied the interactions of PMPC-lipids with plasma albumin, human complement protein C3 and fibrinogen using a quartz crystal microbalance with energy dissipation, and found that adsorption of albumin, C3 and fibrinogen could be suppressed by coating with PMPC-lipids. In particular, the effect was more pronounced for PMPC chains with higher molecular weight. We evaluated the size, polydispersity index, surface charge, and membrane fluidity of the PMPC-lipid-modified liposomes. We found that the effect of the coating on the dispersion stability was maintained over a long period (98 days). Furthermore, we also demonstrated that the anti-PEG antibody did not interact with PMPC-lipids. Thus, our findings suggest that PMPC-lipids can be used for liposomal coating.
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| 3. |
- Fujita, Shohei, et al.
(author)
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Cross-vendor multiparametric mapping of the human brain using 3D-QALAS: A multicenter and multivendor study
- 2024
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In: Magnetic Resonance in Medicine. - : WILEY. - 0740-3194 .- 1522-2594.
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Journal article (peer-reviewed)abstract
- Purpose: To evaluate a vendor-agnostic multiparametric mapping scheme based on 3D quantification using an interleaved Look-Locker acquisition sequence with a T2 preparation pulse (3D-QALAS) for whole-brain T1, T2, and proton density (PD) mapping.Methods: This prospective, multi-institutional study was conducted between September 2021 and February 2022 using five different 3T systems from four prominent MRI vendors. The accuracy of this technique was evaluated using a standardized MRI system phantom. Intra-scanner repeatability and inter-vendor reproducibility of T1, T2, and PD values were evaluated in 10 healthy volunteers (6 men; mean age +/- SD, 28.0 +/- 5.6 y) who underwent scan-rescan sessions on each scanner (total scans = 100). To evaluate the feasibility of 3D-QALAS, nine patients with multiple sclerosis (nine women; mean age +/- SD, 48.2 +/- 11.5 y) underwent imaging examination on two 3T MRI systems from different manufacturers.Results: Quantitative maps obtained with 3D-QALAS showed high linearity (R2 = 0.998 and 0.998 for T1 and T2, respectively) with respect to reference measurements. The mean intra-scanner coefficients of variation for each scanner and structure ranged from 0.4% to 2.6%. The mean structure-wise test-retest repeatabilities were 1.6%, 1.1%, and 0.7% for T1, T2, and PD, respectively. Overall, high inter-vendor reproducibility was observed for all parameter maps and all structure measurements, including white matter lesions in patients with multiple sclerosis.Conclusion: The vendor-agnostic multiparametric mapping technique 3D-QALAS provided reproducible measurements of T1, T2, and PD for human tissues within a typical physiological range using 3T scanners from four different MRI manufacturers.
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| 4. |
- Huang, Tianwei, et al.
(author)
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Surface modulation of extracellular vesicles with cell-penetrating peptide-conjugated lipids for improvement of intracellular delivery to endothelial cells
- 2023
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In: Regenerative Therapy. - : Elsevier. - 2352-3204 .- 2352-3204. ; 22, s. 90-98
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Journal article (peer-reviewed)abstract
- Exosomes (diameter 30-200 nm) are a subtype of extracellular vesicles secreted by cells containing DNA, microRNA (miRNA), and proteins. Exosomes are expected to be valuable as a means of delivering drugs or functional miRNAs in treatment of diseases. However, the delivery of exosomes is not sufficiently effective, even though exosomes have intrinsic delivery functions. Cell-penetrating peptides (CPPs) are short peptide families that facilitate cellular intake of molecules and vesicles. We previously reported that the modification of cells, and liposomes with CPP-conjugated-lipids, CPPs conjugated with poly (ethylene glycol)-conjugated phospholipids (PEG-lipid), that induce adhesion by CPPs, can be useful for cell-based assays and harvesting liposomes. In this study, we aimed to modulate the exosome surface using Tat peptide (YGRKKRRQRRR)-PEG-lipids to improve intracellular delivery to endothelial cells. We isolated and characterized exosomes from the medium of HEK 293 T cell cultures. Tat conjugated PEG -lipids with different spacer molecular weights and lipid types were incorporated into exosomes using fluorescein isothiocyanate labeling to optimize the number of Tat-PEG-lipids immobilized on the exo-some surface. The exosomes modified with Tat-PEG-lipids were incubated with human umbilical vein endothelial cells (HUVECs) to study the interaction. Tat conjugated with 5 kDa PEG and C16 lipids incorporated on the exosome surface were highly detected inside HUVECs by flow cytometry. Fluores-cence was negligible in HUVECs for control groups. Thus, Tat-PEG-lipids can be modified on the exosome surface, improving the intracellular delivery of exosomes.(c) 2022, The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/ 4.0/).
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| 5. |
- Inuzuka, Naoki, et al.
(author)
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Stent coating containing a charged silane coupling agent that regulates protein adsorption to confer antithrombotic and cell-adhesion properties
- 2024
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In: Scientific Reports. - : Springer Nature. - 2045-2322. ; 14:1
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Journal article (peer-reviewed)abstract
- The evolution of endovascular therapies, particularly in the field of intracranial aneurysm treatment, has been truly remarkable and is characterized by the development of various stents. However, ischemic complications related to thrombosis or downstream emboli pose a challenge for the broader clinical application of such stents. Despite advancements in surface modification technologies, an ideal coating that fulfills all the desired requirements, including anti-thrombogenicity and swift endothelialization, has not been available. To address these issues, we investigated a new coating comprising 3-aminopropyltriethoxysilane (APTES) with both anti-thrombogenic and cell-adhesion properties. We assessed the anti-thrombogenic property of the coating using an in vitro blood loop model by evaluating the platelet count and the level of the thrombin-antithrombin (TAT) complex, and investigating thrombus formation on the surface using scanning electron microscopy (SEM). We then assessed endothelial cell adhesion on the metal surfaces. In vitro blood tests revealed that, compared to a bare stent, the coating significantly inhibited platelet reduction and thrombus formation; more human serum albumin spontaneously adhered to the coated surface to block thrombogenic activation in the blood. Cell adhesion tests also indicated a significant increase in the number of cells adhering to the APTES-coated surfaces compared to the numbers adhering to either the bare stent or the stent coated with an anti-fouling phospholipid polymer. Finally, we performed an in vivo safety test by implanting coated stents into the internal thoracic arteries and ascending pharyngeal arteries of minipigs, and subsequently assessing the health status and vessel patency of the arteries by angiography over the course of 1 week. We found that there were no adverse effects on the pigs and the vascular lumens of their vessels were well maintained in the group with APTES-coated stents. Therefore, our new coating exhibited both high anti-thrombogenicity and cell-adhesion properties, which fulfill the requirements of an implantable stent.
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| 6. |
- Sato, Yuya, et al.
(author)
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Extracellular vesicle-liposome hybrids via membrane fusion using cell-penetrating peptide-conjugated lipids
- 2024
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In: Regenerative Therapy. - : Elsevier. - 2352-3204. ; 26, s. 533-540
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Journal article (peer-reviewed)abstract
- Extracellular vesicles (EVs) are natural carriers for intercellular communication within the human body. Mimicking and utilizing EVs by combining them with artificial nanocarriers such as liposomes for drug delivery has garnered considerable attention. However, current technologies for manipulating EVs to facilitate their fusion with liposomes are limited; the existing technique of polyethylene glycol (PEG)induced fusion is highly inefficient for fusion. In our previous study, we demonstrated that membrane fusion could be induced by Tat peptide (YGRKKRRQRRR)-conjugated poly(ethylene glycol)-phospholipids (Tat-PEG-lipids), in which the Tat peptide and lipid domain facilitate membrane attachment and subsequent fusion between cells and liposomes. This approach is promising for forming EV and liposomal hybrids. In this study, we aim to fuse EVs and liposomes using Tat-PEG-lipids. We isolated and characterized EVs derived from HEK293T cell culture medium and treated a mixture of EVs and liposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and cholesterol (1:1, molar ratio), with TatPEG-lipids with different lipid chain lengths. Here, we used nonanoyl (C9), dodecanoyl (C12), and myristoyl (C14) groups as lipid anchors with 5 kDa PEG chains. Dynamic light scattering analysis revealed a large increase in the apparent size of mixture of EVs and liposomes by adding Tat-PEG-lipids (especially C14, C12, followed by C9). Fluorescence resonance energy transfer, confocal laser scanning microscopy, and transmission electron microscopy, used to analyze the reaction process, revealed that the membrane fusion occurred between EVs and liposomes but not their aggregates. The short lipid domain of Tat-PEGlipids effectively induced membrane fusion and the formation of hybrid EVs and liposomes. Thus, TatPEG-lipids (C9 and C12) could be promising candidates for inducing membrane fusion to fabricate EVliposome hybrids.
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| 7. |
- Sato, Yuya, et al.
(author)
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Induction of Spontaneous Liposome Adsorption by Exogenous Surface Modification with Cell-Penetrating Peptide-Conjugated Lipids
- 2021
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In: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 37:32, s. 9711-9723
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Journal article (peer-reviewed)abstract
- 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.
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| 8. |
- Suzuki, Haruna, et al.
(author)
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Impact of spontaneous liposome modification with phospholipid polymer-lipid conjugates on protein interactions
- 2022
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In: Science and Technology of Advanced Materials. - : Taylor & Francis Group. - 1468-6996 .- 1878-5514. ; 23:1, s. 845-857
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Journal article (peer-reviewed)abstract
- Liposome surface coating has been studied to avoid the immunological responses caused by the complement system, and alternative materials to poly(ethylene glycol) (PEG) have been explored recently since the production of anti-PEG IgM antibodies has been found in humans. We previously reported a liposome coating with poly(2-methacryloyloxyethyl phosphorylcholine) (poly(MPC))-conjugated lipids (PMPC-lipids) and demonstrated its protective effect on blood protein interactions. Here, we attempted to modify the liposome surface by exogenously adding PMPC-lipids, which were spontaneously incorporated into the outer membrane via hydrophobic interactions. The polymerization degree of the PMPC segment was regulated from 10 to 100. The incorporated ratio of PMPC-lipid increased with a decrease in the degree of PMPC polymerization. Due to surface modification with PMPC-lipids, increase in the length of the PMPC-chain increased the size of the liposomes. The modified liposomes were kept stable for 14 d in terms of their size, polydispersity, and surface properties, where approximately 70% of PMPC-lipids were incorporated into the liposome surface. We demonstrated that liposome surface modification with PMPC-lipids can inhibit protein adsorption when exposed to serum, regardless of the degree of polymerization of PMPC. In addition, the PMPC-lipid modified surface was not recognized by the anti-PEG IgM antibody, whereas PEG-lipid was recognized by the antibody. Thus, we successfully fabricated an inert liposome surface via spontaneous modification with PMPC-lipids, where only the outer bilayer surface was modified. This technique can be available for full loading of water-soluble active pharmaceutical ingredient inside the modified liposome.
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