Application: Acoustofluidic Sonoporation Using Chemyx Fusion 4000 Syringe Pump

We developed a gene-delivery platform that utilizes acoustofluidic- mediated sonoporation of target cells to facilitate DNA uptake across plasma membranes. With optimization of our device, we demonstrated plasmid delivery from model cells (Jurkat) to clini- cally relevant cell types (PBMCs, CD34+ HSPCs) with throughputs of 200,000 cells/min and viabilities exceeding 80%. This device employs a facile and cost-effective design, taking advantage of a commercially available square glass capillary as the microfluidic channel, thereby circumventing the need for specialized facilities and complex microfluidic geometries. These data indicate scalable and economical acoustofluidic strategies for applications involving disease treatment. For example, successful eGFP expression in PBMCs suggests a strong potential to manufacture cells expressing chimeric antigen receptors for cancer immunotherapies. Further- more, analyses of intracellular delivery revealed disruption of the cell membrane and the nuclear membranes of Jurkat and mouse embryonic fibroblasts, respectively. Further investigation of mem- brane disruption with our acoustofluidic platform will make it possible to examine membrane rupture, repair, and membrane mechanics in a variety of cell types. These studies, along with pro- spective applications in the delivery of CRISPR-Cas9 and other targeted nuclease systems, are important steps for the clinical ap- plication of the acoustofluidic platform for gene editing.

Materials and Methods
Surface Functionalization of Glass Microcapillaries. Square glass micro- capillaries (Vitrocom) with 5 cm × 80 μm × 80 μm in internal dimensions were cleaned in piranha solution (3:1 concentrated sulfuric acid and 30% hydro- gen peroxide) for 30 min to remove organic molecules while adding hy- droxyl functionalities to the glass surface. Next, the capillaries were rinsed and sonicated in 18-MΩ deionized water (Millipore) for five cycles of 5 min and placed in a drying oven at 110 °C for 6 h. The dried capillaries were then dipped in a 5% (vol/vol) ethanolic solution of APTES (Sigma Aldrich) and placed in an oven at 60 °C for 5 min followed by three cycles of sonication in ethanol for 5 min to remove any passively adsorbed APTES molecules from the channel walls. Clean functionalized capillaries were stored in ethanol until device assembly.

Device Fabrication. The acoustofluidic devices are comprised of a piezoelectric lead zirconate titanate (PZT) transducer (SMPL26W16T07111, StemInc), a functionalized glass microcapillary, and a glass slide that provides a sup- porting substrate. The PZT transducers were mounted onto the glass slide with a thin layer of Devcon 5-min epoxy adhesive (300007-392, VWR) after soldering 30-gauge wire to the front and back electrodes of the PZT trans- ducer. A functionalized glass microcapillary was attached onto the trans- ducer with adhesive and cured for 30 min. Polyethylene tubing (PE-50, Instech) was connected to both ends of the microcapillary and sealed with small drops of epoxy. After curing, the tubing was secured to the glass slide with double-sided tape and tested for leaks. The resonant frequency for each device was determined with a vector network analyzer (VNA-120, Array Solutions).

Operation. Fabricated acoustofluidic devices were vertically aligned in a custom-built stage that aligned the cross-section of the microfluidic channel within the optical path of a Nikon TE300 optical microscope. Tubing was connected to a syringe by inserting a 23-gauge needle adapter, and the flow rate was controlled with a syringe pump (Fusion 4000, Chemyx). The PZT transducers were excited with a sinusoidal wave at the desired frequency and an amplitude of 40 Vp-p with a signal generator (81150A, Agilent) and a broadband amplifier (25A250B, Amplifier Research).

DNA and Plasmid Delivery. The APTES-treated glass capillaries were prerinsed with 5 mL of 70% ethanol, followed by 3 mL of 1× phosphate-buffered