Nanoporous electroporation needle for localized intracellular delivery in deep tissues

Abstract

The exogenous control of intracellular drug delivery has been shown to improve the overall efficacy of therapies by reducing nonspecific off-target toxicity. However, achieving a precise on-demand dosage of a drug in deep tissues with minimal damage is still a challenge. In this study, we report an electric-pulse-driven nanopore-electroporation (nEP) system for the localized intracellular delivery of a model agent in deep tissues. Compared with conventional bulk electroporation, in vitro nEP achieved better transfection efficiency (>60%) with a high cell recovery rate (>95%) under a nontoxic low electroporation condition (40 V). Furthermore, in vivo nEP using a nanopore needle electrode with a side drug-releasing compartment offered better control over the dosage release, time, and location of propidium iodide, which was used as a model agent for intracellular delivery. In a pilot study using experimental animals, the nEP system exhibited two times higher transfection efficiency of propidium iodide in the thigh muscle tissue, while minimizing tissue damage (<20%) compared to that of bulk electroporation. This tissue-penetrating nEP platform can provide localized, safe, and effective intracellular delivery of diverse therapeutics into deep tissues in a controlled manner.

Keywords: electric pulse‐driven drug delivery; electroporation; intracellular delivery; nanopore membrane.

FIGURE 1

Schematic illustration of the in vivo nEP system. That consisting of a conventional metal needle and nanopore needle electrode with a side drug‐releasing compartment, applied into the thigh muscle of a mouse. Application of an electric field through the nanopores of the needle electrode generates transient pores on the cell membrane. The charged molecules accelerated through electrical stimulation are released through the nanopores, thereby enabling intracellular delivery of charged target molecules through the temporarily perforated cell membrane.

FIGURE 2

In vitro testing device for nEP. (a) Schematic showing the device structured by sandwiching cells cultured on a nanopore membrane between two Pt plate electrodes. (b) Top‐surface and (c) cross‐sectional SEM images of the nanopore membrane with cylindrical uniform pore structure (scale bar: 5 μm). (d) Fabricated in vitro testing device and cells cultured on the membrane. (Top) Photograph of the multi‐well nEP device. (Middle) Photographs of the hanging insert with the attached nanopore membrane. (Bottom) Fluorescence micrographs showing differences in the adhesion of cells grown on the nanopore membrane depending on the concentration of the cell adhesion molecule, fibronectin. Cells were fluorescently visualized using Calcein AM staining.

FIGURE 3

Simulation results showing the electric field distributions in the bulk EP and nEP. Since the nanopores on the membrane can be considered as parallel resistors in an electric circuit, a single nanopore was used for electric field simulation. (a) The parallel plate electrodes in a homogeneous fluid generate a uniform electric field, while (b) the addition of the nanopore membrane results in electric field getting focused to the membrane. The applied voltages were 30 V and 40 V for bulk EP and nEP, respectively. (c) Electric field strength profiles along a direction away from a certain point in the bulk EP and nanopore entrance in the nEP.

FIGURE 4

In vitro parametric study to determine optimal electroporation conditions. (a) Effects of 16 electric pulse conditions on transfection efficiency, cell recovery, and cell viability. Each column represents a replicate in the experiment. (b) Comparison between bulk EP and nEP under similar electric pulse conditions. The bulk EP and nEP devices have different inter‐electrode distances of 2 mm and 2.7 mm, respectively. Thus, the electric pulse conditions of 20 and 30 V at the 2 mm inter‐electrode distance correspond to 27 and 40.5 V, respectively (n = 3).

FIGURE 5

In vivo nEP system with 2‐needle array electrodes. (a) Schematic showing the 2‐needle array electrodes that can be connected to a power supply. The array consists of a conventional metal (Au‐coated stainless steel) needle electrode and a 3D‐printed hollow needle with a side hole (nanopore electrode). The nanopore membrane was bonded using a photocurable adhesive to seal the side hole of the 3D‐printed needle. (b, c) SEM images of (b) the nanopore electrode (scale bar: 500 μm) and (c) nanopore membrane adhered on the open side hole. (d) Photographs of the 2‐needle array electrodes connected to a commercial electroporator. (e, f) In vitro model agent release using the nEP system with 2‐needle array electrodes. (e) Electric pulse‐driven release of a model agent (PI). (f) Amount of released PI from the nanopore electrode depending on the number of nEP (40 V, 2 ms, 99 pulses). (n = 3)

FIGURE 6

In vivo nEP tests with experimental animals. (a) Photographs showing the in vivo nEP system applied to the thigh muscle of a mouse and schematic illustration showing the nEP‐based intracellular delivery of PI (model agent). (b) Fluorescence micrographs from the sectioned tissue showing tissue damage (TUNEL, green) and transfection efficiency of PI (red). Tissue nuclei were stained with Hoechst (blue). Commercially available two metal needle electrodes were used for bulk EP. The yellow dotted circles indicate the needle insertion sites. (scale bar: 100 μm). (c) Bar graph showing the fluorescent area obtained in (b), representing damaged (TUNEL assay) and transfection (PI) regions. The data are represented as mean ± SD. **p < 0.01, ***p < 0.001 vs. control. (d) Histological images of muscle tissues subjected to electroporation (bulk or nEP) after H&E staining (scale bar: 1 mm).