Purpose miR-29b directly or indirectly targets genes involved in acute myeloid leukemia (AML) i.e., DNMTs, CDK6, SP1, KIT and FLT3. Higher miR-29b pretreatment expression is associated with improved response to decitabine and better outcome in AML. Thus designing a strategy to increase miR-29b levels in AML blasts may be of therapeutic value. However, free synthetic miRs are easily degraded in bio-fluids and have limited cellular uptake. To overcome these limitations, we developed a novel transferrin-conjugated nanoparticle delivery system for synthetic miR-29b (Tf-NP-miR-29b). Experiment Design Delivery efficiency was investigated by flow-cytometry, confocal microscopy and quantitative-PCR. The expression of miR-29b targets was measured by immunoblotting. The anti-leukemic activity of Tf-NP-miR-29b was evaluated by measuring cell proliferation and colony formation ability and in a leukemia mouse model. Results Tf-NP-miR-29b treatment resulted in >200-fold increase of mature miR-29b compared to free miR-29b and was about twice as efficient as treatment with non-Tf-conjugated NP-miR-29b. Tf-NP-miR-29b treatment significantly downregulated DNMTs, CDK6, SP1, KIT and FLT3 and decreased AML cell growth by 30–50% and impaired colony formation by approximately 50%. Mice engrafted with AML cells and then treated with Tf-NP-miR-29b had significantly longer survival compared to Tf-NP-scramble (P=0.015) or free miR-29b (P=0.003). Furthermore, priming AML cell with Tf-NP-miR-29b before decitabine resulted in strong cell viability decrease in vitro and showed improved anti-leukemic activity compared with decitabine alone (P=0.001) in vivo. Conclusion Tf-NP effectively delivered functional miR-29b, resulting in target downregulation and anti-leukemic activity, and warrants further investigation as a novel therapeutic approach in AML.

Electroporation has been one of the most popular non-viral technologies for cell transfection. However, conventional bulk electroporation (BEP) shows significant limitations in efficiency, cell viability and transfection uniformity. Recent advances in microscale-electroporation (MEP) resulted in improved cell viability. Further miniaturization of the electroporation system (i.e., nanoscale) has brought up many unique advantages, including negligible cell damage and dosage control capabilities with single-cell resolution, which has enabled more translational applications. In this review, we give an insight into the fundamental and technical aspects of micro- and nanoscale/nanochannel electroporation (NEP) and go over several examples of MEP/NEP-based cutting-edge research, including gene editing, adoptive immunotherapy, and cellular reprogramming. The challenges and opportunities of advanced electroporation technologies are also discussed.

While electroporation has been widely used as a physical method for gene transfection in vitro and in vivo, its application in gene therapy of cardiovascular cells remains challenging. Due to the high concentration of ion-transport proteins in the sarcolemma, conventional electroporation of primary cardiomyocytes tends to cause ion-channel activation and abnormal ion flux, resulting in low transfection efficiency and high mortality. In this work, we report a high-throughput nano-electroporation technique based on a nanochannel array platform, which enables massively parallel delivery of genetic cargo (microRNA, plasmids) into mouse primary cardiomyocytes in a controllable, highly efficient and benign manner. A simple ‘dewetting’ approach was implemented to precisely position a large number of cells on the nano-electroporation platform. With dosage control, our device precisely titrated the level of miR-29, a potential therapeutic agent for cardiac fibrosis, and determined the minimum concentration of miR-29 causing side effects in mouse primary cardiomyocytes. Moreover, the dose-dependent effect of miR-29 on mitochondrial potential and homeostasis was monitored. Altogether, our nanochannel array platform provides efficient trapping and transfection of primary mouse cardiomyocyte, which could improve the quality control for future microRNA therapy in heart diseases.

A living cell interrogation platform based on nanochannel electroporation is demonstrated with analysis of RNAs in single cells. This minimally invasive process is based on individual cells and allows both multi‐target analysis and stimulus‐response analysis by sequential deliveries. The unique platform possesses a great potential to the comprehensive and lysis‐free nucleic acid analysis on rare or hard‐to‐transfect cells.

Low-temperature assembly techniques are desirable for further evolving polymer-based microelectromechanical systems (MEMS) and nanoelectromechanical systems (NEMS). Especially for systems containing biomolecules and cells, biologically benign processing techniques that meet strict design constraints (e.g., non-contaminating, non-deforming, low temperature) are necessary. Traditional solvent-based and thermal polymer processing methods have unacceptable drawbacks for these applications. Here, we propose a new and universal method for assembling polymer nanostructures. This method is based on using a low carbon dioxide (CO 2 ) pressure to significantly reduce the glass-transition temperature (T g ) of the polymer surface, thus allowing fusion of nanoscale structures at low temperatures. As a benign volatile solvent, CO 2 is a completely unregulated, non-contaminating processing aid.It has been reported that polymer properties at the free surface and near the polymer/substrate interface are different from those in the bulk, [1] as extensively demonstrated by the T g of ultrathin polymer films that are supported on a substrate [2][3][4] or freely standing. [5][6][7] For example, the T g could be significantly lowered at the surface when the substrate effect was negligible. [2,8] Based on these results, interfacial fusion of polymers at temperatures below their bulk T g has been achieved. [9,10] However, these studies showed that the adhesive strength was very low and developed very slowly below the T g , for example, 0.08 MPa after 4 h at 62°C for polystyrene (PS), making it unsuitable for practical applications. Unlike the fusion of polymers by using supercritical CO 2 in previous studies, [11,12] we have successfully demonstrated that CO 2 can enhance interfacial fusion of microstructures at low temperatures by applying a low CO 2 pressure to the polymer surface. By selecting proper processing conditions, microscale features (as small as 3.9 lm) on the polymer surface were well-preserved. The adhesive strength of poly(D,L-lactic-coglycolic acid) (PLGA) copolymer approached 1 MPa at 35°C and a CO 2 pressure of 0.79 MPa.[13]Most thin-film T g studies only record the global behavior of thin films, except for Ellison and Torkelson who measured the local T g distribution at the surface.[8] Moreover, the enhancement of the surface-chain mobility by CO 2 also needs to be explored. Here, we use atomic force microscopy (AFM) with gold nanoparticles as probes [14,15] to investigate the surface T g of polymers in a CO 2 environment. A polymer film is spincoated onto a silicon wafer with a surface roughness in the range of 1-2 nm. Gold nanoparticles are then placed onto the polymer surface as the probes. After annealing at a prespecified temperature and applying CO 2 pressure for 4 h, the system reaches an equilibrium state, [15] and the apparent height of the nanoparticles embedded into the surface is measured using AFM. From this data, the mobile surface layer at the annealing temperature can be probed. In the case of PS (M ...