Many transfection techniques can deliver biomolecules into cells, but the dose cannot be controlled precisely. Delivering well-defined amounts of materials into cells is important for various biological studies and therapeutic applications. Here, we show that nanochannel electroporation can deliver precise amounts of a variety of transfection agents into living cells. The device consists of two microchannels connected by a nanochannel. The cell to be transfected is positioned in one microchannel using optical tweezers, and the transfection agent is located in the second microchannel. Delivering a voltage pulse between the microchannels produces an intense electric field over a very small area on the cell membrane, allowing a precise amount of transfection agent to be electrophoretically driven through the nanochannel, the cell membrane and into the cell cytoplasm, without affecting cell viability. Dose control is achieved by adjusting the duration and number of pulses. The nanochannel electroporation device is expected to have high-throughput delivery applications.

Of great interest to modern medicine and biomedical research is the ability to inject individual target cells with the desired genes or drug molecules. Some advances in cell electroporation allow for high throughput, high cell viability, or excellent dosage control, yet no platform is available for the combination of all three. In an effort to solve this problem, here we show a "3D nano-channel electroporation (NEP) chip" on a silicon platform designed to meet these three criteria. This NEP chip can simultaneously deliver the desired molecules into 40,000 cells per cm(2) on the top surface of the device. Each 650 nm pore aligns to a cell and can be used to deliver extremely small biological elements to very large plasmids (>10 kbp). When compared to conventional bulk electroporation (BEP), the NEP chip shows a 20 fold improvement in dosage control and uniformity, while still maintaining high cell viability (>90%) even in cells such as cardiac cells which are characteristically difficult to transfect. This high-throughput 3D NEP system provides an innovative and medically valuable platform with uniform and reliable cellular transfection, allowing for a steady supply of healthy, engineered cells.

Development of an effective treatment against advanced tumors remains a major challenge for cancer immunotherapy. Approximately 50% of human melanoma is driven by B-Raf proto-oncogene mutation (BRAF mutant). Tumors with such mutation are desmoplastic, highly immunosuppressive, and often resistant to immune checkpoint therapies. We have shown that immunotherapy mediated by low-dose doxorubicin-induced immunogenic cell death was only partially effective for this type of tumor and not effective in long-term inhibition of tumor progression. Wnt family member 5A (Wnt5a), a signaling protein highly produced by BRAF mutant melanoma cells, has been implicated in inducing dendritic cell tolerance and tumor fibrosis, thus hindering effective antigen presentation and T-cell infiltration. We hypothesized that Wnt5a is a key molecule controlling the immunosuppressive tumor microenvironment in metastatic melanoma. Accordingly, we have designed and generated a trimeric trap protein, containing the extracellular domain of Fizzled 7 receptor that binds Wnt5a with a K ∼ 278 nM. Plasmid DNA encoding for the Wnt5a trap was delivered to the tumor by using cationic lipid-protamine-DNA nanoparticles. Expression of Wnt5a trap in the tumor, although transient, was greater than that of any other major organs including liver, resulting in a significant reduction of the Wnt5a level in the tumor microenvironment without systematic toxicity. Significantly, combination of Wnt5a trapping and low-dose doxorubicin showed great tumor growth inhibition and host survival prolongation. Our findings indicated that efficient local Wnt5a trapping significantly remodeled the immunosuppressive tumor microenvironment to facilitate immunogenic cell-death-mediated immunotherapy.

A micro/nano-fabrication process of a nanochannel electroporation (NEP) array and its application for precise delivery of plasmid for non-viral gene transfection is described. A dip-combing device is optimized to produce DNA nanowires across a microridge array patterned on the polydimethylsiloxane (PDMS) surface with a yield up to 95%. Molecular imprinting based on a low viscosity resin, 1,4-butanediol diacrylate (1,4-BDDA), adopted to convert the microridge-nanowire-microridge array into a microchannel-nanochannel-microchannel (MNM) array. Secondary machining by femtosecond laser ablation is applied to shorten one side of microchannels from 3000 to 50 μm to facilitate cell loading and unloading. The biochip is then sealed in a packaging case with reservoirs and microfluidic channels to enable cell and plasmid loading, and to protect the biochip from leakage and contamination. The package case can be opened for cell unloading after NEP to allow for the follow-up cell culture and analysis. These NEP cases can be placed in a spinning disc and up to ten discs can be piled together for spinning. The resulting centrifugal force can simultaneously manipulate hundreds or thousands of cells into microchannels of NEP arrays within 3 minutes. To demonstrate its application, a 13 kbp OSKM plasmid of induced pluripotent stem cell (iPSC) is injected into mouse embryonic fibroblasts cells (MEFCs). Fluorescence detection of transfected cells within the NEP biochips shows that the delivered dosage is high and much more uniform compared with similar gene transfection carried out by the conventional bulk electroporation (BEP) method.

Tumors are inherently heterogeneous in antigen expression, and escape from immune surveillance due to antigen loss remains one of the limitations of targeted immunotherapy. Despite the clinical use of adoptive therapy with chimeric antigen receptor (CAR)-redirected T cells in lymphoblastic leukemia, treatment failure due to epitope loss occurs. Targeting multiple tumor-associated antigens (TAAs) may thus improve the outcome of CAR-T cell therapies. CARs developed to simultaneously target multiple targets are limited by the large size of each single-chain variable fragment and compromised protein folding when several single chains are linearly assembled. Here, we describe single-domain antibody mimics that function within CAR parameters but form a very compact structure. We show that antibody mimics targeting EGFR and HER2 of the ErbB receptor tyrosine kinase family can be assembled into receptor molecules, which we call antibody mimic receptors (amR). These amR can redirect T cells to recognize two different epitopes of the same antigen or two different TAAs in vitro and in vivo.