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Extracellular vesicles nanoarray technology: Immobilization of individual extracellular vesicles on nanopatterned polyethylene glycol-lipid conjugate brushes.

ABSTRACT: Arraying individual extracellular vesicles (EVs) on a chip is expected one of the promising approaches for investigating their inherent properties. In this study, we immobilized individual EVs on a surface using a nanopatterned tethering chip-based versatile platform. A microfluidic device was used to ensure soft, reproducible exposure of the EVs over the whole chip surface. The device is incorporated with a high-density nanoarray chip patterned with 200-nm diameter nanospots composed of polyethylene glycol (PEG)-lipid conjugate brushes. We present a procedure adopted for fabricating high-density PEG-lipid modified nanospots (200 nm?, 5.0 × 105 spots/mm2 in 2 × 2 mm2 area). This procedure involves nanopatterning using electron beam lithography, followed by multistep selective chemical modification. Aqueous treatment of a silane coupling agent, used as a linker between PEG-lipid molecules and the silicon surface, was the key step that enabled surface modification using a nanopatterned resist film as a mask. The nanoarray chip was removed from the device for subsequent measurements such as atomic force microscopy (AFM). We developed a prototype device and individually immobilized EVs derived from different cell lines (Sk-Br-3 and HEK293) on tethering nanospots. We characterized EV's morphology using AFM and showed the possibility of evaluating the deformability of EVs using the aspect ratio as an indicator.

SUBMITTER: Yokota S

PROVIDER: S-EPMC6812765 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Extracellular vesicles nanoarray technology: Immobilization of individual extracellular vesicles on nanopatterned polyethylene glycol-lipid conjugate brushes.

Yokota Shusuke S Kuramochi Hiromi H Okubo Kyohei K Iwaya Akiko A Tsuchiya Shoichi S Ichiki Takanori T

PloS one 20191024 10

Arraying individual extracellular vesicles (EVs) on a chip is expected one of the promising approaches for investigating their inherent properties. In this study, we immobilized individual EVs on a surface using a nanopatterned tethering chip-based versatile platform. A microfluidic device was used to ensure soft, reproducible exposure of the EVs over the whole chip surface. The device is incorporated with a high-density nanoarray chip patterned with 200-nm diameter nanospots composed of polyeth ...[more]

PMID: 31648253

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Project description:Extracellular vesicles (EVs) provide a complex means of intercellular signalling between cells at local and distant sites, both within and between different organs. According to their cell-type specific signatures, EVs can function as a novel class of biomarkers for a variety of diseases, and can be used as drug-delivery vehicles. Furthermore, EVs from certain cell types exert beneficial effects in regenerative medicine and for immune modulation. Several techniques are available to harvest EVs from various body fluids or cell culture supernatants. Classically, differential centrifugation, density gradient centrifugation, size-exclusion chromatography and immunocapturing-based methods are used to harvest EVs from EV-containing liquids. Owing to limitations in the scalability of any of these methods, we designed and optimised a polyethylene glycol (PEG)-based precipitation method to enrich EVs from cell culture supernatants. We demonstrate the reproducibility and scalability of this method and compared its efficacy with more classical EV-harvesting methods. We show that washing of the PEG pellet and the re-precipitation by ultracentrifugation remove a huge proportion of PEG co-precipitated molecules such as bovine serum albumine (BSA). However, supported by the results of the size exclusion chromatography, which revealed a higher purity in terms of particles per milligram protein of the obtained EV samples, PEG-prepared EV samples most likely still contain a certain percentage of other non-EV associated molecules. Since PEG-enriched EVs revealed the same therapeutic activity in an ischemic stroke model than corresponding cells, it is unlikely that such co-purified molecules negatively affect the functional properties of obtained EV samples. In summary, maybe not being the purification method of choice if molecular profiling of pure EV samples is intended, the optimised PEG protocol is a scalable and reproducible method, which can easily be adopted by laboratories equipped with an ultracentrifuge to enrich for functional active EVs.

Project description:BackgroundColorectal cancer is one of the prominent leading causes of fatality worldwide. Despite recent advancements within the field of cancer therapy, the cure rates and long-term survivals of patients suffering from colorectal cancer have changed little. The application of conventional chemotherapeutic agents like doxorubicin is limited by some drawbacks such as cardiotoxicity and hematotoxicity. Therefore, nanotechnology has been exploited as a promising solution to address these problems. In this study, we synthesized and compared the anticancer efficacy of doxorubicin-loaded liposomes that were surface engineered with the 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-matrix metalloproteinase-2 (MMP-2) cleavable peptide-polyethylene glycol (PEG) conjugate. The peptide linker was used to cleave in response to the upregulated MMP-2 in the tumor microenvironment, thus exposing a positive charge via PEG-deshielding and enhancing liposomal uptake by tumor cells/vasculature. Liposomal formulations were characterized in terms of size, surface charge and morphology, drug loading, release properties, cell binding and uptake, and cytotoxicity.ResultsThe formulations had particle sizes of ~ 100-170 nm, narrow distribution (PDI ˂ 0.2), and various surface charges (- 10.2 mV to + 17.6 mV). MMP-2 overexpression was shown in several cancer cell lines (C26, 4T1, and B16F10) as compared to the normal NIH-3T3 fibroblast cells by gelatin zymography and qRT-PCR. In vitro results demonstrated enhanced antitumor efficacy of the PEG-cleavable cationic liposomes (CLs) as compared to the commercial Caelyx® (up to fivefold) and the chick chorioallantoic membrane assay showed their great antiangiogenesis potential to target and suppress tumor neovascularization. The pharmacokinetics and efficacy studies also indicated higher tumor accumulation and extended survival rates in C26 tumor-bearing mice treated with the MMP-2 cleavable CLs as compared to the non-cleavable CLs with no remarkable sign of toxicity in healthy tissues.ConclusionAltogether, the MMP-2-cleavable CLs have great potency to improve tumor-targeted drug delivery and cellular/tumor-vasculature uptake which merits further investigation.Supplementary informationThe online version contains supplementary material available at 10.1186/s12645-023-00169-8.

Dataset Information

Extracellular vesicles nanoarray technology: Immobilization of individual extracellular vesicles on nanopatterned polyethylene glycol-lipid conjugate brushes.

Publications

Extracellular vesicles nanoarray technology: Immobilization of individual extracellular vesicles on nanopatterned polyethylene glycol-lipid conjugate brushes.

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