Single Living Cell Analysis Nanoplatform for High-Throughput Interrogation of Gene Mutation and Cellular Behavior

Dong, Zaizai; Yan, Shi; Liu, Bing; Hao, Yongcun; Lin, Long; Chang, Tianrui; Sun, Hong; Wang, Yusen; Li, Hu; Wu, Han; Hang, Xinxin; He, Shiqi; Hu, Jiaming; Xue, Xinying; Wu, Nan; Chang, Lingqian

doi:10.1021/acs.nanolett.1c00199

Cited by 37 publications

(49 citation statements)

References 54 publications

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“…Identifying the tumor cells from a large number of background cells without cellular structure damage is a powerful strategy to monitor the targeted cells for the study of biological mechanisms. The electroporation-based method generally relies on the complex probe design [ 44 ], and traditional immunofluorescence (IF) staining is impossible to guarantee both high efficiency and high activity of cell labeling.…”

Section: Resultsmentioning

confidence: 99%

In Situ Electroporation on PERFECT Filter for High-Efficiency and High-Viability Tumor Cell Labeling

Hun

Zhang

et al. 2022

Micromachines

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Labeling-assisted visualization is a powerful strategy to track circulating tumor cells (CTCs) for mechanism study (e.g., tumor metastasis). Due to the rarity of CTCs in the whole blood, efficient simultaneous enrichment and labeling of CTCs are needed. Hereby, novel in situ electroporation on a previously-developed micropore-arrayed filter (PERFECT filter) is proposed. Benefiting from the ultra-small-thickness and high-porosity of the filter plus high precision pore diameter, target rare tumor cells were enriched with less damage and uniform size distribution, contributing to enhanced molecular delivery efficiency and cell viability in the downstream electroporation. Various biomolecules (e.g., small molecule dyes, plasmids, and functional proteins) were used to verify this in situ electroporation system. High labeling efficiency (74.08 ± 2.94%) and high viability (81.15 ± 3.04%, verified via live/dead staining) were achieved by optimizing the parameters of electric field strength and pulse number, ensuring the labeled tumor cells can be used for further culture and down-stream analysis. In addition, high specificity (99.03 ± 1.67%) probing of tumor cells was further achieved by introducing fluorescent dye-conjugated antibodies into target cells. The whole procedure, including cell separation and electroporation, can be finished quickly (<10 min). The proposed in situ electroporation on the PERFECT filter system has great potential to track CTCs for tumor metastasis studies.

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Section: Resultsmentioning

confidence: 99%

In Situ Electroporation on PERFECT Filter for High-Efficiency and High-Viability Tumor Cell Labeling

Hun

Zhang

et al. 2022

Micromachines

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“…According to the principle of fluorescence resonance energy transfer, the fluorophore (labeled on the 5′ end of the sequence F) is quenched by the quencher (labeled on the 3′ end of the sequence Q). [30] Each single cell is trapped and settled onto the bottom of the microwell. The F terminal of the tensioner is linked with cholesterol that has the chance to insert in all hydrophobic areas on the cell membrane [31,32] (Figure 1d).…”

Section: Design Of a Dna Tensioner Platformmentioning

confidence: 99%

High‐Throughput DNA Tensioner Platform for Interrogating Mechanical Heterogeneity of Single Living Cells

Hang

Dong

et al. 2022

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The heterogeneities among individual cells lead to different cellular behaviors and responses against the stimulation. [3] In given scenarios, abnormal regulations of mechanical force in specific cell subtypes may introduce disorders to the organism, such as tumorigenesis, [4] drug resistance, [5] and metastasis. [6][7][8] The understanding of the mechanical properties at the single-cell level is an important index to explore the initiation and development of diseases, and further contribute to the advancement of treatment strategies.Recent years have seen emerging technologies for cell mechanical analysis, which can be categorized into two subgroups in terms of their format: i) the first group, including atomic force microscopy (AFM), [9][10][11] optical tweezers, [12] and magnetic tweezers, [13,14] focuses on the measurement of relevant force curves to obtain basic cell mechanical information, such as Young's modulus and stiffness. [15][16][17] These methods generally apply a mechanical stimulus to the cells by manipulating beams Cell mechanical forces play fundamental roles in regulating cellular responses to environmental stimulations. The shortcomings of conventional methods, including force resolution and cellular throughput, make them less accessible to mechanical heterogeneity at the single-cell level. Here, a DNA tensioner platform is introduced with high throughput (>10 000 cells per chip) and pN-level resolution. A microfluidic-based cell array is trapped on "hairpin-structured" DNA tensioners that enable transformation of the mechanical information of living cells into fluorescence signals. By using the platform, one can identify enhanced mechanical forces of drug-resistant cells as compared to their drug-sensitive counterparts, and mechanical differences between metastatic tumor cells in pleural effusion and nonmetastatic histiocytes. Further genetic analysis traces two genes, VEGFA and MINK1, that may play deterministic roles in regulating mechanical heterogeneities. In view of the ubiquity of cells' mechanical forces in the extracellular microenvironment (ECM), this platform shows wide potential to establish links of cellular mechanical heterogeneity to genetic heterogeneity.

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“…Due to the challenge to reflect the heterogeneity of the tumor environment, it is difficult to precisely show the disturbance of the electric field in the tumor tissue at single cell level. However, a number of research be conducted based on this simulation model to illustrate drug delivery process, [29,45,46] suggesting the rationality of our modeling and simulation. We verified the amount of drug that flowed through 4M platforms with microchannels of different diameters (20 µm/30 µm/40 µm/50 µm/6 0 µm/70 µm/80 µm) under the same pressure within the same period of time using PBS and the 3D hydrogel model, and the experimental results are consistent with the simulation results (Figure S5A,B, Supporting Information).…”

Section: Determination Of the Optimum Diameter Of The Microchannelsmentioning

confidence: 99%

“…are based on circulation, which, albeit simple in operation, the essential anticancer effect may be compromised by the cell membrane that serves as the barrier to the internalization of the cargo. [25][26][27] To enhance the cellular permeability and transcellular delivery, [28,29] local ECTs have been recently proposed in vivo, in which needle-like electrodes are inserted into the solid tumor after intratumoral drug injection. [30][31][32] A pulsed electric field applied between two electrodes has proved efficient to perforate cell membrane, establishing the pathway for the entry of drugs that were randomly distributed around the cell/tissue.…”

Section: Introductionmentioning

confidence: 99%

“…To enhance the cellular permeability and transcellular delivery, [ 28,29 ] local ECTs have been recently proposed in vivo, in which needle‐like electrodes are inserted into the solid tumor after intratumoral drug injection. [ 30–32 ] A pulsed electric field applied between two electrodes has proved efficient to perforate cell membrane, establishing the pathway for the entry of drugs that were randomly distributed around the cell/tissue.…”

Section: Introductionmentioning

confidence: 99%

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Multimicrochannel Microneedle Microporation Platform for Enhanced Intracellular Drug Delivery

Lin

Wang

Cai

et al. 2021

Adv Funct Materials

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Common delivery routes for chemotherapeutics are based on circulation, which faces clinical limitations to local delivery efficiency, and the conflict between the dose for anticancer effect and the systemic toxicity. The recent advances in localized delivery strategies aim to improve drug accumulation at the target site or directly transport into cells. However, most are not equipped to provide additional momentum in the process of cargo release, propagation, and intracellular movement, which limit their locomotion that relies on passive diffusion. In this work, a multimicrochannel microneedle microporation (4M) platform that achieves high efficiency, safety, and uniformity for in vivo intracellular delivery is proposed. By high precision 3D printing, internal microchannels are implemented through the microneedle, which offer a concentrated, safe electric field that not only accelerates the movement of cargo into deep tissue under electrophoresis, but also triggers cell electroporation, achieving enhanced transport across cell membrane. The platform proves efficient for the delivery of chemotherapeutics in solid tumors in vitro and in vivo, with significantly enhanced anticancer effect and reduced systemic toxicity. The platform serves as a general-purpose delivery tool to emerging drugs in vivo.

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Single Living Cell Analysis Nanoplatform for High-Throughput Interrogation of Gene Mutation and Cellular Behavior

Cited by 37 publications

References 54 publications

In Situ Electroporation on PERFECT Filter for High-Efficiency and High-Viability Tumor Cell Labeling

In Situ Electroporation on PERFECT Filter for High-Efficiency and High-Viability Tumor Cell Labeling

High‐Throughput DNA Tensioner Platform for Interrogating Mechanical Heterogeneity of Single Living Cells

Multimicrochannel Microneedle Microporation Platform for Enhanced Intracellular Drug Delivery

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