Chongyang Liang

In situ surface-enhanced Raman scattering spectroscopy exploring molecular changes of drug-treated cancer cell nucleus.

Investigating the molecular changes of cancer cell nucleus with drugs treatment is crucial for the design of new anticancer drugs, the development of novel diagnostic strategies, and the advancement of cancer therapy efficiency. In order to better understand the action effects of drugs, accurate location and in situ acquisition of the molecular information of the cell nuclei are necessary. In this work, we report a microspectroscopic technique called dark-field and fluorescence coimaging assisted surface-enhanced Raman scattering (SERS) spectroscopy, combined with nuclear targeting nanoprobes, to in situ study Soma Gastric Cancer (SGC-7901) cell nuclei treated with two model drugs, e.g., DNA binder (Hoechst33342) and anticancer drug (doxorubicin, Dox) via spectral analysis at the molecular level. Nuclear targeting nanoprobes with an assembly structure of thiol-modified polyethylene glycol polymers (PEG) and nuclear localizing signal peptides (NLS) around gold nanorods (AuNRs) were prepared to achieve the amplified SERS signals of biomolecules in the cell nuclei. With the assistance of dark field/fluorescence imaging with simultaneous location, in situ SERS spectra in one cell nucleus were measured and analyzed to disclose the effects of Hoechst33342 and Dox on main biomolecules in the cell nuclei. The experimental results show that this method possesses great potential to investigate the targets of new anticancer drugs and the real-time monitoring of the dynamic changes of cells caused by exogenous molecules.

Recombinant Lz-8 from Ganoderma lucidum induces endoplasmic reticulum stress-mediated autophagic cell death in SGC-7901 human gastric cancer cells

In Asia, the mushroom of the fungus Ganoderma lucidum has been widely used as a traditional medicine for the past two millennia. The aim of this study was to investigate the anticancer activity of recombinant Lz-8 (rLz-8), a protein belonging to a family of fungal immunomodulatory proteins. We report that rLz-8 induces endoplasmic reticulum (ER) stress-mediated autophagic cell death in the human gastric cancer cell line SGC-7901. Our results show that rLz-8 induces autophagic cell death by aggregating in the ER, triggering ER stress and the ATF4-CHOP pathway. A foreign protein, in the ER rLz-8 causes the activation of the ubiquitine/proteasome ER-associated degradation (ERAD) system. The autophagic arm of this system is then overstimulated by an excessive abundance of rLz-8 and causes the cell’s death through an over-autophagic response. We also found that caspase inhibitors do not prevent rLz-8-induced cell death, and therefore the autophagic response induced by rLz-8 is independent of caspase activation.

Tracing the Therapeutic Process of Targeted Aptamer/Drug Conjugate on Cancer Cells by Surface-Enhanced Raman Scattering Spectroscopy

Targeted delivery of chemotherapeutic agents to pathology areas can improve drug efficiency and reduce serious side effects on normal regions. However, their treatment mechanism on cells or cell nuclei is still mysterious due to the lack of in situ characterization methods. In this paper, the specific diagnosis and treatment processes of a targeted antitumor agent (doxorubicin, Dox) functionalized aptamer complex (TLS11a-GC-Dox) toward HepG2 cells, a human hepatocellular carcinoma cell line, were tracked in real time by the surface-enhanced Raman scattering (SERS) spectroscopic technique and dark-field imaging with the assistance of gold nanorod-based nuclear targeted probes, which possess remarkable SERS enhancement ability, specific targeting, and excellent biological compatibility. This is the first time to explore the acting mechanism of an aptamer-based targeted drug on cell nucleus based on the spectral information on components inside the cell nucleus. The results demonstrate that this aptamer/drug conjugate has targeting and sustained-release actions and its therapeutic effect is achieved by the gradual damage of relevant proteins and DNA in nuclei. Better understanding of the mechanism of aptamer-drug conjugates acting on cancer cells is conductive to increasing cancer therapy efficiency and is also helpful for the design of highly effective drug delivery methods.

Note: Raman microspectroscopy integrated with fluorescence and dark field imaging

A Raman detection platform integrated with both fluorescence and dark field microscopes was built for in situ Raman detection with the assistance of fluorescence and dark field imaging to locate the target micro regions. Cells and organelles can be easily found via fluorescence imaging with labeling techniques. Besides, nano-sized particles could be observed and located by dark field microscopes. Therefore, comparing with the commercial Raman spectrometers, much more researches based on Raman spectroscopy could be carried out on this integrated Raman platform, especially in the fields of analyzing biological tissues and subwavelength samples.

Effect of recombinant Ganoderma lucidum immunoregulatory protein on cyclophosphamide-induced leukopenia in mice

Abstract The aim of the study is to investigate the effect of recombinant Ganoderma lucidum immunoregulatory protein (rLZ-8) on mouse models of cyclophosphamide-induced leukopenia, which we have established with both single-phase and multi-phase administration methods. Treatment with rLZ-8 had a strong effect on both models of cyclophosphamide-induced leukopenia. In particular, it increased the number of neutrophils, lymphocytes and monocytes. rLZ-8 treatment also increased the percentage of CD4+ T cells and the levels of secreted IL-3 and IL-4, which contributed to the cyclophosphamide-induced immune dysfunction and immune system imbalance. In conclusion, rLZ-8 treatment benefitted mice with cyclophosphamide-induced leukopenia by improving overall immune function and by specifically increasing the number of white blood cells.

Enriching PMMA nanospheres with adjustable charges as novel templates for multicolored dye@PMMA nanocomposites

Multicolored fluorescent dye loaded PMMA nanospheres were synthesized by the electrostatic adsorption of dye molecules on the charged PMMA nanospheres, whose charges were adjusted by choosing different initiators. The charged PMMA nanospheres have a wider capacity and advantage for combining the charged dyes. The fluorescent dye@PMMA composite nanospheres possess the advantages of higher brightness, longer lifetime and stronger resistance to photobleaching relative to dye molecules. Dye leakage remained lower than 5% over one week. These fluorescent nanospheres have been used in biological labels in cell imaging. They can easily stain blood cancer cells without further surface modification.

Tracing sialoglycans on cell membrane via surface-enhanced Raman scattering spectroscopy with a phenylboronic acid-based nanosensor in molecular recognition

Sialoglycan expression is critical for assessing various diseases progression. Especially, its abnormal levels are commonly believed to be associated with tumor and metastatic cancer types. While, complicated structures, multiple types and dynamic distributions make it challenging for in situ investigating sialoglycans at the physiological status. Herein, we developed a 4-mercaptophenylboronic acid (MPBA)-based surface-enhanced Raman scattering (SERS) nanosensor to in situ study sialoglycan levels and dynamic expression processes of different cell types based on molecular recognition between phenylboronic acid and sialoglycans at physiological condition. This nanosensor is designed by the MPBA decorated silver nanoparticle (AgNP), which is unique and multifunctional because of its three-in-one role involving the Raman signal enhancer (AgNP), the sensing reporter of MPBA and the target receptor based on the recognition of phenylboronic acid and sialoglycans. When this nanosensor binds to sialoglycans, the molecular vibrational modes of MPBA will change, which can be traced by ultrasensitive SERS technique. The superiority of this study is that we built the relation between the spectral changes of MPBA (relative intensities) in molecular recognition with the sialoglycan dynamic expression of cells. We believe that our SERS strategy could be further extended to explore crucial physiological processes and significant biological system that glycans are involved in.

Recombinant Ganoderma lucidum immunomodulatory protein modified with polyethylene glycol

Recombinant Ganoderma lucidum immunomodu-latory protein (rLZ-8) expressed using the Pichia yeast eukaryotic expression system is a potential new drug for cancer therapy; however, it has a short half-life in the body. In order to optimize the potency and stability of rLZ-8, we modified the recombinant protein chemically using methoxy-PEG-succinimidyl propionate (mPEG-SPA). The results indicated that several parameters, including pH, the molar ratio of rLZ-8 to mPEG-SPA and time, played crucial roles in the modification process. In particular, when the molar ratio of rLZ-8 to mPEG-SPA was 1:1, rLZ-8 was modified by a single mPEG-SPA moiety. In addition, MALDI-TOF/TOF and ESI Q-Trap results revealed that the difference in molecular weight (MW) between the peptide-linked mPEG-SPA and the mPEG-SPA closely matched the MW of a methionine amino acid. Taken together, these data suggest that modification of mPEG-SPA occurred on the N-terminal helix of rLZ-8. This modification method has laid a foundation for the development of long-acting formulations of rLZ-8.

A novel protein expression system-PichiaPink™- and a protocol for fast and efficient recombinant protein expression

Pichia pastoris is a eukaryote and has many of the advantages of higher eukaryotic expression systems, such as protein processing, protein folding, and the availability of posttranslational modifications. It is as easy to manipulate as Escherichia coli or Saccharomyces cerevisiae . However, some serious and unavoidable problems occur with the Pichia pastoris system that is difficult to overcome, such as low transforming efficiency and degradation of protein. Recently, a new protein expression system named PichiaPink™ was introduced. We have used the pichia-based system to express some proteins successfully, such as human serum albumin, Ganoderma lucidum immunoregulatory protein (Lz-8), and found that the system can improve the transformation of the Pichia stain, the low yields and the degradation of the protein of interest. In the example of human serum albumin (HSA), the results indicated two facts: (1) the protease knockout PichiaPink™ strains are quite helpful in decreasing the degradation of HSA without affecting the yield of HSA or the strain growth, and the high-copy expression vector worked better than the low-copy expression vector in terms of the HSA yield. The example of rLz-8 showed that compared with the traditional Pichia strain Gs115, the new system provided an easier selection method for screening correct and higher level of expression transformants.

A strategy of designing the ligand of antibody affinity chromatography based on molecular dynamics simulation.

Designing affinity ligands has always been the development focus of affinity chromatography. Previous antibody affinity ligand designs were mostly based on the crystal structure of protein A (UniProt code number: P38507), and the antibody-binding domains were modified according to the properties of amino acid residues. Currently, more effective bioinformatic prediction and experimental validation has been used to improve the design of antibody affinity ligands. In the present study, the complex crystal structure (the domain D of protein A and the Fab segment of IgM, PDB code: 1DEE) was used as the model. The vital site that inhibits the binding between domain D and IgM was estimated by means of molecular dynamics (MD) simulation, then MM-GBSA calculations were used to design a mutant of domain D (K46E) for improving affinity on the above vital site. The binding analysis using Biacore showed the association and dissociation parameters of K46E mutant that were optimized with IgM. The affinity increase of K46E mutant preferred for IgM, the affinity order is K46E tetramer (KD=6.02×10(-9)M)>K46E mutant (KD=6.66×10(-8)M)>domain D (KD=2.17×10(-7)M). Similar results were obtained when the optimized ligands were immobilized to the chromatography medium. A complete designing strategy was validated in this study, which will provide a novel insight into designing new ligands of antibody affinity chromatography media.