Zicai Liang

Synthetic oligonucleotides (oligos) are important tools in the fields of molecular biology and genetic engineering. For applications requiring a large number of oligos with high concentration, it is critical to perform high throughput oligo synthesis and achieve high yield of each oligo. This study reports a microreactor chip for oligo synthesis. By incorporating silica beads in the microreactors, the surface area of the solid substrate for oligo synthesis increases significantly in each microreactor. These beads are fixed in the microreactors to withstand the flushing step in oligo synthesis. Compared to conventional synthesis methods, this design is able to avoid protocols to hold the beads and integrate more microreactors on a chip. An inkjet printer is utilized to deliver chemical reagents in the microreactors. To evaluate the feasibility of oligo synthesis using this proof-of-concept synthesizer, an oligo with six nucleotide units is successfully synthesized.

Electrochemotherapy (ECT), as one of the very few available treatments for cutaneous and subcutaneous tumors when surgery and radiotherapy are no longer available, requires applying a proper electric field to the tumor to realize electroporation-mediated cytotoxic drug delivery. It is impossible to exhaust all possible electrical parameters on patients to realize the optimal tradeoff between tumor suppression and adverse effects. To address this issue, this study provides a feasible solution by developing a four-leaf micro-electrode chip (F-MEC) in which the electric field was specially designed by linear distribution to cover all possible electric field strengths for ECT.

Hydrophobization of cationic polymers, as an efficient strategy, had been widely developed in the structure of cationic polymer micelles to improve the delivery efficiency of nucleic acids. However, the distribution of hydrophobic segments in the polymer chains is rarely considered. Here, we have elaborated three types of hydrophobized polyethylene glycol (PEG)-blocked cationic polymers with different distributions of the hydrophobic segments in the polymer chains PEG–PAM–PDP (E–A–D), PEG–PDP–PAM (E–D–A), and PEG–P(AM/DP) (E–(A/D)), which were synthesized by reversible addition–fragmentation chain transfer polymerization of methoxy PEG, cationic monomer aminoethyl methacrylate, and pH-sensitive hydrophobic monomer 2-diisopropylaminoethyl methacrylate, respectively. In aqueous solution, all of the three copolymers, E–A–D, E–D–A, and E–(A/D), were able to spontaneously form nanosized micelles (100–150 nm) (M_E–A–D, M_E–D–A, and M_E–(A/D)) and well-incorporated small interfering RNA (siRNA) into complex micelles (CMs). The effect of distributions of the hydrophobic segments on siRNA delivery had been evaluated in vitro and in vivo. Compared with M_E–D–A and M_E–(A/D), M_E–A–D showed the best siRNA binding capacity to form stable M_E–A–D/siRNA CMs less than 100 nm, mediated the best gene-silencing efficiency and inhibition effect of tumor cell growth in vitro, and showed better liver gene-silencing effect in vivo. In the case of M_E–(A/D) with a random distribution of cationic and hydrophobic segments, a gene-silencing efficiency higher than Lipo2000 but lesser than M_E–A–D and M_E–D–A was obtained. As the mole ratio of positive and negative charges increased, M_E–D–A/siRNA and M_E–A–D/siRNA showed similar performances in size, zeta potential, cell uptake, and gene silencing, but M_E–(A/D)/siRNA showed reversed performances. In addition, M_E–A–D as the best siRNA carrier was evaluated in the tumor tissue in the xenograft murine model and showed good anticancer capacity. Obviously, the distribution of the hydrophobic segments in the amphiphilic cationic polymer chains should be seriously considered in the design of siRNA vectors.

Cell electroporation referring to the formation of micro pores on cell membrane caused by externally applied electrical field is a membrane phenomenon which involves fundamental behavior of cell and artificial bilayer membrane, and increasingly attracts consideration in biology, biotechnology and medicine. To improve cell viability and transfection rate, we systemically studied on electroporation under different conditions: (1) ultrasound with different power and frequency, (2) magnetic fields in electroporation, (3) value of pH during electroporation.

A hybrid cationic bicellar nanodisc was prepared by a conventional Bangham method in combination with a sol–gel reaction and self-assembly process. The relatively small size and disc-like shape was very stable because the incorporation of the organic–inorganic hybrid lipid had formed a crosslinked siloxane net structure on the surface. Physicochemical properties, biocompatibility, intracellular trafficking behavior, in vitro transfection efficiency and in vivo distribution of the bicellar nanodiscs were investigated. The unique characteristics of the cationic PEGylated hybrid bicellar nanodiscs address many of the deficiencies relating to current liposome technology. By optimizing the doping ratio of PEG-containing lipid, we found that a doping ratio of 1 mol% is enough to confer an excellent in vivo delivery performance while not compromising the transfection efficacy in vitro. The distinct disc-like shape, high stability conferred by the hybrid lipid and modest siRNA delivery performance make this platform promising as a siRNA vehicle for efficient siRNA delivery both in vitro and in vivo.

RNA activation (RNAa) is the upregulation of gene expression by small activating RNAs (saRNAs). In order to investigate the mechanism by which saRNAs act in RNAa, we used the progesterone receptor (PR) gene as a model, established a panel of effective saRNAs and assessed the involvement of the sense and antisense strands of saRNA in RNAa. All active saRNAs had their antisense strand effectively incorporated into Ago2, whereas such consistency did not occur for the sense strand. Using a distal hotspot for saRNA targeting at 1.6-kb upstream from the PR transcription start site, we further established that gene activation mediated by saRNA depended on the complementarity of the 5′ region of the antisense strand, and that such activity was largely abolished by mutations in this region of the saRNA. We found markedly reduced RNAa effects when we created mutations in the genomic target site of saRNA PR-1611, thus providing evidence that RNAa depends on the integrity of the DNA target. We further demonstrated that this saRNA bound the target site on promoter DNA. These results demonstrated that saRNAs work via an on-site mechanism by binding to target genomic DNA in a seed-region-dependent manner, reminiscent of miRNA-like target recognition.

The drug development of siRNA has been seriously hindered by the lack of an effective, safe and clinically applicable delivery system. The cyclic NGR motif and its isomerization product isoDGR recruit CD13 and integrin as their specific receptors, both of which are overexpressed by tumor and neovascular cells. In this study, a bi-functional peptide, named NGR-10R, was designed and tested for siRNA delivery in vitro and in vivo. Through the formation of peptide/siRNA nanoparticles, RNase resistance was greatly enhanced for the siRNAs. Both FACS and confocal assays revealed that the peptide/siRNA complexes were effectively internalized by MDA-MB-231 cells. Gene silencing assays indicated that anti-Lamin A/C siRNA delivered by NGR-10R robustly repressed gene expression in MDA-MB-231 and HUVEC (a CD13⁺/α_vβ₃ ⁺ cell). Importantly, the siRNAs were efficiently delivered into tumor tissues and localized around the nuclei, as revealed by in vivo imaging and cryosection examination. In summary, NGR-10R not only efficiently delivered siRNAs into MDA-MB-231 cells in vitro but also delivered siRNAs into tumor cells in vivo, taking advantage of its specific binding to CD13 (neovascular) or α_vβ₃ (MDA-MB-231). Therefore, the NGR-10R peptide provides a promising siRNA delivery reagent that could be used for drug development, particularly for anti-tumor therapeutics.

Continuous cell electroporation is an appealing non-viral approach for genetically transfecting a large number of cells. Yet the traditional macro-scale devices suffer from the unsatisfactory transfection efficiency and/or cell viability due to their high voltage, while the emerging microfluidic electroporation devices is still limited by their low cell processing speed. Here we present a flow-through cell electroporation device integrating large-sized flow tube and small-spaced distributed needle electrode array. Relatively large flow tube enables high flow rate, simple flow characterization and low shear force, while well-organized needle array electrodes produce an even-distributed electric field with low voltage. Thus the difficulties for seeking the fine balance between high flow rate and low electroporation voltage were steered clear. Efficient in vitro electrotransfection of plasmid DNA was demonstrated in several hard-to-transfect cell lines. Furthermore, we also explored ex vivo electroporated mouse erythrocyte as the carrier of RNA. The strong ability of RNA loading and short exposure time of freshly isolated cells jointly ensured a high yield of valid carrier erythrocytes, which further successfully delivered RNA into targeted tissue. Both in vitro and ex vivo electrotransfection could be accomplished at high cell processing speed (20 million cells per minute) which remarkably outperforms previous devices.