Zhaogang Yang

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.

Targeted Delivery of Tumor Suppressor microRNA-1 by Transferrin- Conjugated Lipopolyplex Nanoparticles to Patient-Derived Glioblastoma Stem Cells

OBJECTIVE Among heterogeneous glioblastoma multiforme (GBM) cells, glioblastoma stem cells (GSCs) is a subpopulation having a critical role in tumor initiation and therapy resistance. Thus targeting GSCs would be an essential step to completely eradicate this lethal disease. MicroRNA-1 (miR-1) expression is deregulated in GBM patients and restoration of miR-1 by viral-vector in GBM cells has been demonstrated to inhibit tumor initiation and attenuate cell migration. Here, we show that a transferrin-targeting non-invasive nanoparticle delivery system (Tf-NP) can efficiently deliver miR-1 to GBM patient-derived GSC-enriched sphere cultures (GBM spheres). METHODS Delivery efficiency of the transferrin- targeting non-invasive nanoparticle was investigated by flow cytometry and further confirmed by confocal microscopy. The levels of miR-1 and its target molecules in GBM spheres were measured by qRT-PCR and immunoblotting. Migration capacity of Tf-NP-miR-1 treated GBM spheres were evaluated by transwell migration assay. RESULTS Tf-NPmiR- 1 treatment resulted in an over 200-fold increase of mature miR-1 compared to free miR-1 and Tf-NP-miR negative control (Tf-NP-miR-NC). Transferrin-mediated NP delivery resulted in a 3-fold higher delivery efficiency compared to NP without transferrin modification. Tf-NP-miR-1 treatment on GBM spheres significantly inhibited migration of GBM spheres by 30-50% with associated decline of MET and EGFR expression. Our data supported that Tf-NP could be used as an efficient and effective delivery system which has high potential to benefit the development of miR-based therapeutics for GBM treatment.

A Polyethylenimine-Linoleic Acid Conjugate for Antisense Oligonucleotide Delivery

A novel antisense oligonucleotide (ASO) carrier, polyethylenimine conjugated to linoleic acid (PEI-LA), was synthesized and evaluated for delivery of LOR-2501 to tumor cells. LOR-2501 is an ASO targeting ribonucleotide reductase R1 subunit (RRM1). In this study, PEI-LA was synthesized by reacting PEI (Mw ~ 800) with linoleoyl chloride. Gel retardation assay showed complete complexation between PEI-LA and LOR-2501 at N/P ratio above 8. No significant cytotoxicity was observed with these complexes at the tested dosage levels. Interestingly, at N/P ratio of >6, levels of cellular uptake of PEI-LA/LOR-2501 were double that of PEI/LOR-2501 complexes of the same N/P ratio. PEI-LA/LOR-2501 induced downregulation of 64% and 70% of RRM1 at mRNA and protein levels, respectively. The highest transfection activity was shown by PEI-LA/LOR-2501 complexes at N/P ratio of 10. Finally, using pathway specific inhibitors, clathrin-mediated endocytosis was shown to be the principle mechanism of cellular internalization of these complexes. In conclusion, PEI-LA is a promising agent for the delivery of ASOs and warrants further investigation.

Adenosine A1 receptors selectively target protein kinase C isoforms to the caveolin-rich plasma membrane in cardiac myocytes

Adenosine is a naturally occurring nucleoside that has been shown to regulate a variety of functions in the cardiovascular system. However, the mechanisms in adenosine receptor signaling are not completely understood. Given that adenosine receptors have been linked to protein kinase C (PKC) in cardioprotection and caveolae is critical for receptor signaling, we sought to determine whether activation of adenosine A1 receptors induces selective translocation of PKC isoforms to the membrane from the cytosol and whether activated PKC is targeted to the caveolin-rich plasma membrane microdomains. The freshly isolated adult rat cardiac myocytes were used to examine PKC isoforms including PKCalpha, PKCbeta, PKCvarepsilon, PKCdelta and PKCzeta. Immunoblot analysis revealed that the immunoreactivity for PKCvarepsilon or PKCdelta but not for PKCalpha, PKCbeta or PKCzeta increased significantly in the membrane fractions from cells pretreated with the selective adenosine A1 receptor agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA, 100 nM) when compared with non-stimulated cells. The effect of CCPA on PKCvarepsilon or PKCdelta translocation was blocked by adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 100 nM). When Western blot was performed from the caveolin-enriched plasma membrane fractions, the immunoreactivity for PKCvarepsilon or PKCdelta but not PKCalpha, PKCbeta or PKCzeta was enhanced significantly by CCPA. Furthermore, PKCvarepsilon and PKCdelta were detected in the anti-caveolin-3 immunoprecipitates but not in the samples without primary antibody. Immunofluorescence staining further indicates increased colocalization of PKCvarepsilon or PKCdelta with caveolin-3 at cell peripheral region and T-tubular-like structures in response to adenosine A1 receptor activation. In conclusion, we demonstrate that activation of adenosine A1 receptors promotes the selective translocation of PKCvarepsilon and PKCdelta to the caveolin-enriched plasma membrane microdomains in cardiac myocytes.

Nanomedicine based on Nucleic Acids: Pharmacokinetic and Pharmacodynamic Perspectives

Nanoparticles have shown great promise for improving the efficacy of nucleic acid drugs that lack permeability and bioavailability needed for reaching their intracellular site of action. The pharmacokinetics of the nucleic acid nanomedicines contributes greatly to their pharmacodynamic and toxicity characteristics. The pharmacokinetics of nanomedicines is determined by their physiochemical properties, such as particle size, shape, surface charge, and surface modifications. This review presented the pharmacokinetic and pharmacodynamic perspectives of nucleic acid nanomedicines. The cellular trafficking of the nucleic acid nanomedicines after cellular uptake is also discussed. In addition, the review puts forward potential strategies for circumventing biological barriers to delivering and releasing nucleic acid drugs into their intracellular site of action, and perspectives in this rapidly evolving field.

Insight into Mechanisms of Cellular Uptake of Lipid Nanoparticles and Intracellular Release of Small RNAs

PurposeUnderstanding mechanisms of cellular uptake and intracellular release would enable better design of nanocarriers for delivery of nucleic acids such as siRNA and microRNA (miRNA).MethodIn this study, we investigated cellular pharmacokinetics of siRNA by co-encapsulating fluorescently labeled siRNA and molecular beacon (MB) in four different formulations of cationic lipid nanoparticles (LNPs). A miRNA mimic was also used as a probe for investigating cellular pharmacokinetics, which correlated well with RNAi activities.ResultsWe tried to find the best LNP formulation based on the combination of DOTMA and DODMA. When the DOTMA/DODMA ratio was at 5/40, the LNP containing a luciferase siRNA produced the highest gene silencing activity. The superior potency of DOTMA/DODMA could be attributed to higher uptake and improved ability to facilitate siRNA release from endosomes subsequent to uptake.ConclusionsOur findings may provide new insights into RNAi transfection pathways and have implications on cationic LNP design.

Molecular mechanism underlying adenosine receptor-mediated mitochondrial targeting of protein kinase C.

Activation of protein kinase C (PKC) via adenosine receptors is known to be involved in the cardioprotection of ischemic preconditioning (IPC). Specifically, activation of PKCε is critical for cardioprotection. There is ample evidence that PKCε resides in cardiac mitochondria. However, the signals that promote translocation of PKCε are largely unknown. The present study was designed to determine whether and how adenosine receptor activation induces translocation of PKCε to mitochondria. Freshly isolated adult rat cardiac myocytes and rat heart-derived H9c2 were used in the study. Immunofluorescence imaging of isolated mitochondria showed that PKCε but not PKCδ was localized in mitochondria and this mitochondrial localization of PKCε was significantly increased by adenosine treatment. The adenosine-induced increase in PKCε-positive mitochondria was largely prevented not only by PKC inhibitor chelerythrine, but also by the HSP90 inhibitor geldanamycin and by siRNA targeting HSP90. Immunoblot analysis from percoll-purified mitochondria further demonstrated that adenosine mediated a significant increase in mitochondrial PKCε but not PKCδ. This effect was blocked by inhibiting PKC activity with chelerythrine and bisindolylmaleimide. Furthermore, co-immunoprecipitation data showed that PKCε but not PKCδ was associated with TOM70 and HSP90, and this association was enhanced by adenosine treatment. Moreover, adenosine-induced association of PKCε with TOM70 was reduced by suppressing HSP90 expression with siRNA. In conclusion, we demonstrate that adenosine induces HSP90-dependent translocation of PKCε to mitochondria, possibly through mitochondrial import machinery TOM70. These results point out a novel mechanism in regulating PKC in mitochondria and suggest an important implication in ischemic preconditioning or postconditioning.

Near infrared spectroscopic (NIRS) analysis of drug-loading rate and particle size of risperidone microspheres by improved chemometric model

Microspheres have been developed as drug carriers in controlled drug delivery systems for years. In our present study, near infrared spectroscopy (NIRS) is applied to analyze the particle size and drug loading rate in risperidone poly(d,l-lactide-co-glycolide) (PLGA) microspheres. Various batches of risperidone PLGA microspheres were designed and prepared successfully. The particle size and drug-loading rate of all the samples were determined by a laser diffraction particle size analyzer and high performance liquid chromatography (HPLC) system. Monte Carlo algorithm combined with partial least squares (MCPLS) method was applied to identify the outliers and choose the numbers of calibration set. Furthermore, a series of preprocessing methods were performed to remove signal noise in NIR spectra. Moving window PLS and radical basis function neural network (RBFNN) methods were employed to establish calibration model. Our data demonstrated that PLS-developed model was only suitable for drug loading analysis in risperidone PLGA microspheres. Comparatively, RBFNN-based predictive models possess better fitting quality, predictive effect, and stability for both drug loading rate and particle size analysis. The correlation coefficients of calibration set (Rc(2)) were 0.935 and 0.880, respectively. The performance of optimum RBFNN models was confirmed by independent verification test with 15 samples. Collectively, our method is successfully performed to monitor drug-loading rate and particle size during risperidone PLGA microspheres preparation.

Indole-3-carbinol inhibits tumorigenicity of hepatocellular carcinoma cells via suppression of microRNA-21 and upregulation of phosphatase and tensin homolog

A major obstacle to successful treatment of hepatocellular carcinoma (HCC) is its high resistance to cytotoxic chemotherapy due to overexpression of multidrug resistance genes. Activation of the AKT pathway is known to be involved in chemoresistance in HCC; however, the underlying mechanisms modulating the AKT pathway by chemopreventive agents remain unclear. In the present study, we found that indole-3-carbinol (I3C) treatment for tumor cells repressed the AKT pathway by increasing the expression of phosphatase and tensin homolog (PTEN) in HCC xenograft tumor and HCC cell lines. qRT-PCR data showed that the expression of miR-21 and miR-221&222 was significantly reduced by I3C in HCC cells in vitro and in vivo. Reactivation of the AKT pathway via restoration of miR-21 was reversed by I3C. Ectopic expression of miR-21 mediated-accelerated wound healing was abrogated by I3C. Moreover, reducing the expression of miR-21 by anti-miR decreased the resistance of HCC cells to I3C. These results provide experimental evidences that I3C could function as a miR-21 regulator, leading to repression of the PTEN/AKT pathway and opening a new avenue for eradication of drug-resistant cells, thus potentially helping to improve the therapeutic outcome in patients diagnosed with HCC.

Functional exosome-mimic for delivery of siRNA to cancer: in vitro and in vivo evaluation.

Exosomes, the smallest subgroup of extracellular vesicles, have been recognized as extracellular organelles that contain genetic and proteomic information for long distance intercellular communication. Exosome-based drug delivery is currently a subject of intensive research. Here, we report a novel strategy to produce nanoscale exosome-mimics (EMs) in sufficient quantity for gene delivery in cancer both in vitro and in vivo. Size-controllable EMs were generated at a high yield by serial extrusion of non-tumorigenic epithelial MCF-10A cells through filters with different pore sizes. siRNA was then encapsulated into the EMs by electroporation. Biosafety and uptake efficiency of the EMs were evaluated both in vitro and in vivo. The mechanism underlying their cellular endocytosis was also studied.