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, 110 (6), 2082-7

A Vector-Free Microfluidic Platform for Intracellular Delivery

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A Vector-Free Microfluidic Platform for Intracellular Delivery

Armon Sharei et al. Proc Natl Acad Sci U S A.

Abstract

Intracellular delivery of macromolecules is a challenge in research and therapeutic applications. Existing vector-based and physical methods have limitations, including their reliance on exogenous materials or electrical fields, which can lead to toxicity or off-target effects. We describe a microfluidic approach to delivery in which cells are mechanically deformed as they pass through a constriction 30-80% smaller than the cell diameter. The resulting controlled application of compression and shear forces results in the formation of transient holes that enable the diffusion of material from the surrounding buffer into the cytosol. The method has demonstrated the ability to deliver a range of material, such as carbon nanotubes, proteins, and siRNA, to 11 cell types, including embryonic stem cells and immune cells. When used for the delivery of transcription factors, the microfluidic devices produced a 10-fold improvement in colony formation relative to electroporation and cell-penetrating peptides. Indeed, its ability to deliver structurally diverse materials and its applicability to difficult-to-transfect primary cells indicate that this method could potentially enable many research and clinical applications.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Delivery mechanism and system design. (A) Illustration of delivery hypothesis whereby the rapid deformation of a cell, as it passes through a microfluidic constriction, generates transient membrane holes. Includes an electron micrograph of current parallel channel design with blue cells as an illustration. (B) Image of a finished device consisting of Pyrex bound to silicon for sealing. (Scale bar: 2 mm.) (C) Illustration of the delivery procedure in which cells and delivery material are mixed in the inlet reservoir, run through the chip, and collected in the outlet reservoir. The mounting system consists of stainless-steel and aluminum parts interfaced to the chip by inert O rings. (Scale bar: 10 mm.)
Fig. 2.
Fig. 2.
Delivery performance depends on cell speed and constriction design. Constriction dimensions (Fig. S1) are denoted by numbers (e.g., 10 μm − 6 μm × 5) such that the first number corresponds to constriction length, the second to constriction width and the third (if present) to the number of constrictions in series per channel. (A) Delivery efficiency and (B) cell viability 18 h after treatment as a function of cell speed for 40 μm − 6 μm (○), 20 μm − 6 μm (□), and 10 μm − 6 μm × 5 (∆) device designs. Efficiencies and viabilities were measured by flow cytometry after propidium iodide staining. For information regarding cell recovery rates, refer to SI Note S1. All data points were run in triplicate, and error bars represent 2 SDs.
Fig. 3.
Fig. 3.
Diffusive delivery mechanism. (A) Scans of different horizontal planes of a HeLa cell after the delivery of Cascade Blue-conjugated 3-kDa dextran, as measured by confocal microscopy. Note that 3-kDa dextran is small enough to enter the nuclear envelope (43). Scans read from top to bottom, and then left to right, where the top left is at z = 6.98 μm and bottom right is at z = −6.7 μm. (Scale bar: 6 μm.) (B) Live-cell delivery efficiency of 10 μm − 6 μm (□), 20 μm − 6 μm (○), 30 μm − 6 μm (∆), and 40 μm − 6 μm (◇) devices. The time axis indicates the amount of time elapsed from initial treatment of cells before they were exposed to the target delivery solution. All results were measured by flow cytometry 18 h after treatment. (C) Average intensity of the delivered cell population normalized by untreated cells to control for autofluorescence. Fluorescein-conjugated 70-kDa dextran and Cascade Blue-conjugated 3-kDa dextran are delivered to the cell (cycles 1 and 3) and removed from the cell (cycle 2) in consecutive treatment cycles. The control represents cells that were only exposed to the delivery solution and not treated by the device. (D) Gene knockdown, as a function of device type and cell speed, in live destabilized GFP-expressing HeLa cells 18 h after the delivery of anti-eGFP siRNA at a delivery concentration of 5 μM. Lipofectamine 2000 was used as a positive control and scrambled controls were run at 500 mm/s on a 10 μm − 6 μm × 5. All data points were run in triplicate, and error bars represent 2 SDs.
Fig. 4.
Fig. 4.
Applicability across cell types. (A) Delivery efficiency and viability of NuFF cells treated with a 30 μm − 6 μm device to deliver 3- and 70-kDa dextran. (B) Delivery efficiency and viability of spleen-isolated, murine dendritic cells treated with a 10 μm − 4 μm device to deliver 3- and 70-kDa dextran. (C) Delivery efficiency and viability of murine embryonic stem cells treated with a 10 μm − 6 μm device to deliver 3- and 70-kDa dextran. (D) Delivery efficiency of 3-kDa and (E) 70-kDa dextran to B cells (CD19+), T cells (TCR-β+), and macrophages (CD11b+) isolated from whole-mouse blood by centrifugation and treated by 30 μm − 5 μm and 30 μm − 5 μm × 5 devices at 1,000 mm/s. Three- and 70-kDa dextran were labeled with Cascade Blue and fluorescein, respectively. All data points were run in triplicate, and error bars represent 2 SDs.
Fig. 5.
Fig. 5.
Nanomaterial and antibody delivery. (A and B) TEM images of gold nanoparticles (some indicated by arrows) in cells fixed ∼1 s after treatment by a 10 μm − 6 μm × 5 device (SI Note S3). (Scale bars: 500 nm.) (C) Delivery efficiency and viability of HeLa cells treated with a 10 μm − 6 μm × 5 device to deliver Cascade Blue-labeled 3-kDa dextran and Cy5-labeled, DNA-wrapped, carbon nanotubes. (D) Bright-field cell images overlaid with Raman scattering in the G-band (red) to indicate delivery of carbon nanotubes in treated cells (Left) vs. endocytosis (Right). (Scale bars: 2 μm.) (E) Fluorescent micrograph of a HeLa cell 18 h after delivery of Cascade Blue-labeled 3-kDa dextran (Center) and antibodies to tubulin with an Alexa Fluor 488 tag (Right). (Scale bars: 3 μm.) (F) Delivery efficiency and viability of HeLa cells treated with a 10 μm − 6 μm × 5 device, at 500 mm/s, to deliver Alexa Fluor 488-labeled anti-tubulin antibodies. Delivery efficiency at different antibody concentrations is compared with an endocytosis control at 100 μg/mL and untreated cells.
Fig. 6.
Fig. 6.
Altering cell morphology and gene expression by cytosolic delivery of transcription factors. (A) A Western blot analysis of c-Myc, Klf4, Oct4, and Sox2 delivery to NuFF cells by cell-penetrating peptides versus a 10 μm − 6 μm device. Each of the four proteins has an additional nine arginine (9R) groups to facilitate uptake. The lysate (Ly) columns correspond to the protein content of cells that are washed and lysed, whereas the media columns correspond to the protein content of the media environment. (B) Confocal microscopy images of NuFF cells fixed after delivery of the reprogramming factors. The proteins are tagged using an Alexa 488-conjugated anti-FLAG antibody and the nucleus is stained by DAPI. (Scale bar: 15 μm.) (C) A progression of morphological changes from fibroblasts into colonies. The white arrows indicate potentially transformed cells. The red arrow points toward coalescing cells forming a colony. (D–G) Expression of the human embryonic stem cell marker Oct4, SSEA-4, Tra-1-60, and Tra-1-81 in transformed colonies (SI Note S3). Where appropriate, the small box represents a DAPI counterstain. (Scale bars: 100 μm.)

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