Lingqian Chang

Magnetic Tweezers‐Based 3D Microchannel Electroporation for High‐Throughput Gene Transfection in Living Cells

A novel high-throughput magnetic tweezers-based 3D microchannel electroporation system capable of transfecting 40 000 cells/cm(2) on a single chip for gene therapy, regenerative medicine, and intracellular detection of target mRNA for screening cellular heterogeneity is reported. A single cell or an ordered array of individual cells are remotely guided by programmable magnetic fields to poration sites with high (>90%) cell alignment efficiency to enable various transfection reagents to be delivered simultaneously into the cells. The present technique, in contrast to the conventional vacuum-based approach, is significantly gentler on the cellular membrane yielding >90% cell viability and, moreover, allows transfected cells to be transported for further analysis. Illustrating the versatility of the system, the GATA2 molecular beacon is delivered into leukemia cells to detect the regulation level of the GATA2 gene that is associated with the initiation of leukemia. The uniform delivery and a sharp contrast of fluorescence intensity between GATA2 positive and negative cells demonstrate key aspects of the platform for gene transfer, screening and detection of targeted intracellular markers in living cells.

Design of a Microchannel-Nanochannel-Microchannel Array Based Nanoelectroporation System for Precise Gene Transfection

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.

Polyelectrolyte/mesoporous silica hybrid materials for the high performance multiple-detection of pH value and temperature

Mesoporous silica nanoparticles have been widely adopted in energy, biology and medicine due to their well-ordered and stable structures. Nevertheless, few attempts have been made to study these materials as a sensing tool. Herein, we report a “smart” sensor for the dual-detection of the pH value and temperature, which was implemented with environmentally responsive polyelectrolyte/mesoporous silica electrodes. Using SBA-15 silica as the framework, we functionalized the internal mesopores with DMAMEA monomer via surface-initiated RAFT polymerization (“grafting-from” method). By controlling the degree of polymerization, the pore size and the specific surface area can be precisely controlled. When the degree of polymerization was optimized to 75, the hybrid material showed significant sensitivity in response to the pH value in the range of 4–10 and optimally responded to the temperature at 39 °C, setting a pH value of 10. The ionic conductivities of the template Fe(CN)64−/3− and Ru(NH3)62+/3+ ions were switchable in different conditions. These results suggest that the polyelectrolyte/mesoporous silica hybrid materials could have potential for application in dual-functional sensors in environmental detection.

Micro-/nano-electroporation for active gene delivery.

Gene delivery, a process of introducing foreign functional nucleic acids into target cells, has proven to be a very promising tool for inducing specific gene expression in host cells. Many different technologies have been developed for efficient gene delivery. Among them, electroporation has been adopted in gene delivery for decades, and it is currently widely used for transfection of different types of cells. Despite of the success achieved by bulk electroporation (BEP) for gene delivery in vitro and in vivo, it has significant drawbacks such as unstable transfection efficacy and low cell viability. In recent years, there is an emerging interest in understanding how individual cell accepts and responds to exogenous gene materials using single cell based micro-/nano-electroporation (MEP/NEP) technologies. In this review, the authors provide an overview of the recent development of MEP/NEP and their advantages in gene delivery. Additionally, the future perspectives of gene delivery with the application of electroporation are discussed.

Effect of nanoporous structure and polymer brushes on the ionic conductivity of poly(methacrylic acid)/anode aluminum oxide hybrid membranes

Anode aluminum oxide (AAO) porous materials have been widely used in ionic translocation for many biological and chemical studies. However, the lack of stimuli-response of this material limits its applications for the precise control of ionic transportation by the external environment. In this study, we functionalized the internal nanopores of AAO membranes to generate polyelectrolyte-filled pH-responsive membranes whose ionic conductivity could be readily controlled by changing the pH value. AAO membranes with different pore sizes (25, 75 and 100 nm) were modified with poly(methacrylic acid) (PMAA) by a “grafting-to” approach. Thermogravimetric and SEM analysis revealed that the extent of PMAA infiltration strongly depends upon the relative sizes of the nanopores and the PMAA concentration. Increasing the size of the nanopores enables the infiltration of PAA solution with a higher concentration. Electrochemical impedance spectroscopy demonstrated that the membrane conductivity decreases from 7.87 × 10−4 S cm−1 at pH 1 to 5.72 × 10−5 S cm−1 at pH 7. The functionalized AAO nanopores showed significant sensitivity to pH value, whereas a valve effect was observed in the pH range between 4 and 5. Our fabricated PMAA-AAO membranes show promising potential to be used as pH sensors and smart valves in micro-/nano-total analysis chips for biomedical and chemical applications.

Molecular Beacon Nano-Sensors for Probing Living Cancer Cells.

Heterogeneities and oncogenesis essentially result from proteomic disorders orchestrated by changes in DNA and/or cytoplasmic mRNA. These genetic fluctuations, however, cannot be decoded through conventional label-free methods (e.g., patch clamps, electrochemical cellular biosensors, etc.) or morphological characterization. Molecular beacons (MBs) have recently emerged as efficient probes for interrogating biomarkers in live cancer cells. MBs hybridize with their intracellular targets (e.g., mRNAs, DNAs, or proteins), emitting a fluorescent signal that can be quantified and correlated with the expression levels of their targets. In this review we discuss MB probes with different delivery platforms for intracellular probing as well as novel MB designs for detecting a variety of targets in living cancer cells. Finally, we describe current trends in MB-based intracellular biosensors.

On-Chip Clonal Analysis of Glioma-Stem-Cell Motility and Therapy Resistance

Enhanced glioma-stem-cell (GSC) motility and therapy resistance are considered to play key roles in tumor cell dissemination and recurrence. As such, a better understanding of the mechanisms by which these cells disseminate and withstand therapy could lead to more efficacious treatments. Here, we introduce a novel micro-/nanotechnology-enabled chip platform for performing live-cell interrogation of patient-derived GSCs with single-clone resolution. On-chip analysis revealed marked intertumoral differences (>10-fold) in single-clone motility profiles between two populations of GSCs, which correlated well with results from tumor-xenograft experiments and gene-expression analyses. Further chip-based examination of the more-aggressive GSC population revealed pronounced interclonal variations in motility capabilities (up to ∼4-fold) as well as gene-expression profiles at the single-cell level. Chip-supported therapy resistance studies with a chemotherapeutic agent (i.e., temozolomide) and an oligo RNA (anti-miR363) revealed a subpopulation of CD44-high GSCs with strong antiapoptotic behavior as well as enhanced motility capabilities. The living-cell-interrogation chip platform described herein enables thorough and large-scale live monitoring of heterogeneous cancer-cell populations with single-cell resolution, which is not achievable by any other existing technology and thus has the potential to provide new insights into the cellular and molecular mechanisms modulating glioma-stem-cell dissemination and therapy resistance.

Micro-/nanoscale electroporation

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.

Topical tissue nano-transfection mediates non-viral stroma reprogramming and rescue

Although cellular therapies represent a promising strategy for a number of conditions, current approaches face major translational hurdles, including limited cell sources and the need for cumbersome pre-processing steps (for example, isolation, induced pluripotency). In vivo cell reprogramming has the potential to enable more-effective cell-based therapies by using readily available cell sources (for example, fibroblasts) and circumventing the need for ex vivo pre-processing. Existing reprogramming methodologies, however, are fraught with caveats, including a heavy reliance on viral transfection. Moreover, capsid size constraints and/or the stochastic nature of status quo approaches (viral and non-viral) pose additional limitations, thus highlighting the need for safer and more deterministic in vivo reprogramming methods. Here, we report a novel yet simple-to-implement non-viral approach to topically reprogram tissues through a nanochannelled device validated with well-established and newly developed reprogramming models of induced neurons and endothelium, respectively. We demonstrate the simplicity and utility of this approach by rescuing necrotizing tissues and whole limbs using two murine models of injury-induced ischaemia.

PEG/heparin-decorated lipid–polymer hybrid nanoparticles for long-circulating drug delivery

The clinical success of lipid–polymer hybrid nanoparticles (LPHNPs) for effective targeted drug delivery is still hindered by their rapid clearance from the bloodstream. In this work, a novel strategy for surface modification of LPHNPs with combined polyethylene glycol (PEG) and heparin (HEP) was developed to achieve a significant prolongation in blood circulation. All the LPHNPs formulated with a diameter of 100–200 nm were prepared by a modified w/o/w solvent diffusion/evaporation method and physicochemically characterized. The synergistic action of PEG and HEP was observed, as combinatorial modification significantly improved the surface hydrophilicity as well as the suspension stability of nanoparticles and tailored the surface charge close to neutrality, in comparison to LPHNPs surface-treated with PEG or HEP alone. In vitro and in vivo studies showed that the PEG/HEP coating significantly prohibited the macrophage uptake and extended the blood circulation of LPHNPs with concomitant reduced liver sequestration. The in vitro phagocytosis results using murine peritoneal macrophages showed 8.2-fold reduction compared to the control LPHNP group. The in vivo study in ICR mice showed PEG/HEP coating increased the blood circulation half-life of LPHNPs from 0.3 h to 72.6 h. Moreover, PEG/HEP LPHNPs exhibited dramatically reduced liver accumulation when compared to LPHNPs. These results demonstrated that PEG/HEP LPHNPs with optimized particle size, surface hydrophilicity and surface charge, have a promising potential as long-circulating drug delivery systems.