High-Throughput and Efficient Intracellular Delivery Method via a Vibration-Assisted Nanoneedle/Microfluidic Composite System

Abstract

Intracellular delivery and genetic modification have brought a significant revolutionary to tumor immunotherapy, yet existing methods are still limited by low delivery efficiency, poor throughput, excessive cell damage, or unsuitability for suspension immune cells, specifically the natural killer cell, which is highly resistant to transfection. Here, we proposed a vibration-assisted nanoneedle/microfluidic composite system that uses large-area nanoneedles to rapidly puncture and detach the fast-moving suspension cells in the microchannel under vibration to achieve continuous high-throughput intracellular delivery. The nanoneedle arrays fabricated based on the large-area self-assembly technique and microchannels can maximize the delivery efficiency. Cas9 ribonucleoprotein complexes (Cas9/RNPs) can be delivered directly into cells due to the sufficient cellular membrane nanoperforation size; for difficult-to-transfect immune cells, the delivery efficiency can be up to 98%, while the cell viability remains at about 80%. Moreover, the throughput is demonstrated to maintain a mL/min level, which is significantly higher than that of conventional delivery techniques. Further, we prepared CD96 knockout NK-92 cells via this platform, and the gene-edited NK-92 cells possessed higher immunity by reversing exhaustion. The high-throughput, high-efficiency, and low-damage performance of our intracellular delivery strategy has great potential for cellular immunotherapy in clinical applications.

Advanced Nanomaterials in Medical 3D Printing

“…[69] The use of nanoneedles in drug delivery enables precise membrane perforation and subsequent intracellular delivery of drug substances by concentrating force on a small area of the cell surface, showing great potential in solving the efficiency and viability of intracellular delivery. [70][71][72] In addition, nanoneedles can serve as a multifunctional tool for probing and manipulating biological processes and biophysical properties in living cells, such as cell mechanics, cell signaling and cell differentiation. [73] Nanoneedles can achieve high spatial and temporal resolution detection and manipulation of biological systems, revealing the mysteries and laws of life sciences.…”

Artificial Breakthrough of Cell Membrane Barrier for Transmembrane Substance Exchange: A Review of Recent Progress

“…[56] This combination enables the achievement of high-efficiency and high-throughput intracellular delivery of drugs or genes of suspended cells and implements massive parallelism. Li et al [18] designed a composite chip consisting of a microchannel chip and a nanoneedle chip. With the aid of vibration, the nanoneedles could rapidly puncture and separate fast-moving cells within microchannels, enabling continuous and high-throughput intracellular delivery (Figure 2e).…”

“…(i) Reproduced with permission. [ 18 ] Copyright 2023, American Chemical Society. (ii) Reproduced with permission.…”

“…a) Cell perforation. (i) Reproduced with permission [18]. Copyright 2023, American Chemical Society.…”

Analysis of the Pattern Shapes Obtained By Micro/Nanospherical Lens Photolithography

“…This is the fundamental concept behind modern nanotechnology. In recent years, materials with micro/nano structures have found diverse applications in various fields such as nanophononics, − surface wettability modification, , mechanical metamaterials, energy storage, flexible sensors bioengineering, and so on. Several fabrication techniques are already available, including electron-beam lithography, focused-ion-beam lithography, X-ray lithography, nanoimprint lithography, probe-based methods, two-photon polymerization, and other so-called “top-down” methods.…”