Hong Yan Liu

Estrogen inhibition of EAE involves effects on dendritic cell function

Estrogen has been found to have suppressive effects on the induction of experimental autoimmune encephalomyelitis (EAE), an animal model for the human disease multiple sclerosis. We have investigated the effects of 17β‐estradiol (E2) treatment on dendritic cells (DCs) in two different mouse models of EAE. The frequency of CD11b+/CD11c+ DCs was significantly decreased in the brain of mice protected from EAE induction by E2 treatment. In addition, the frequency of CD11c+/CD8α+ DCs producing tumor necrosis factor (TNF)α and interferon (IFN)γ in the spleen of E2‐treated mice was dramatically decreased compared to that in control mice with EAE, demonstrating an effect of E2 on DC function. In order to examine E2 effects on DCs in more detail, splenic DCs were cultured in the presence of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and interleukin (IL)‐4to promote maturation. E2 pretreatment was found to suppress the ability of cultured DCs bearing a mature phenotype to present Ag to myelin basic protein (MBP)‐specific T cells. Analysis of cytokine production demonstrated that E2 decreased TNFα, IFNγ and IL‐12 production in mature DCs. In addition, MBP‐specific T cells cocultured with E2‐pretreated mature DCs in the presence of antigen demonstrated a shift towards production of Th2 cytokines IL‐4 and IL‐10 and a concomitant decrease in the production of Th1 cytokines TNFα and IFNγ. Thus, E2 treatment appears to have multiple effects on the DC population, which may contribute to a down‐regulation or block in the activation of Th1 cells involved in the induction of EAE.

Ligand-bound quantum dot probes for studying the molecular scale dynamics of receptor endocytic trafficking in live cells.

Endocytic receptor trafficking is a complex, dynamic process underlying fundamental cell function. An integrated understanding of endocytosis at the level of single or small numbers of ligand bound-receptor complexes inside live cells is currently hampered by technical limitations. Here, we develop and test ligand nerve growth factor-bound quantum dot (NGF-QD) bioconjugates for imaging discrete receptor endocytic events inside live NGF-responsive PC12 cells. Using single particle tracking, QD hybrid gel coimmunoprecipitation, and immuno-colocalization, we illustrate and validate the use of QD-receptor complexes for imaging receptor trafficking at synchronized time points after QD-ligand-receptor binding and internalization (t = 15-150 min). The unique value of these probes is illustrated by new dynamic observations: (1) that endocytosis proceeds at strikingly regulated fashion, and (2) that diffusive and active forms of transport inside cells are rapid and efficient. QDs are powerful intracellular probes that can provide biologists with new capabilities and fresh insight for studying endocytic receptor signaling events, in real time, and at the resolution of single or small numbers of receptors in live cells.

Engineering Monovalent Quantum Dot−Antibody Bioconjugates with a Hybrid Gel System

Monovalent nanoparticles are of strong current interest in biological imaging and detection due to their potential for stoichiometric binding with target molecules. We report the preparation of monovalent quantum dot-antibody bioconjugates using a high-resolution hybrid gel system specially designed for fractionation of nanoparticle bioconjugates. A key feature of this technology is that it is broadly applicable to many types of nanoparticle-antibody complexes without the need of genetically engineered proteins. This is particularly important because antibodies are still the dominant molecular targeting probes, despite new discoveries made with other targeting probes such as aptamers and peptides. Furthermore, we show experimental evidence of improved quantification capability using the monovalent probes, whose advantages over their multivalent counterparts had largely been a theoretic prediction previously. This new class of nanoprobe should find broad application in quantitative biological detection and imaging in vitro and in vivo.

Single particle quantum dot imaging achieves ultrasensitive detection capabilities for Western immunoblot analysis

Substantially improved detection methods are needed to detect fractionated protein samples present at trace concentrations in complex, heterogeneous tissue and biofluid samples. Here we describe a modification of traditional Western immunoblotting using a technique to count quantum-dot-tagged proteins on optically transparent PVDF membranes. Counts of quantum-dot-tagged proteins on immunoblots achieved optimal detection sensitivity of 0.2 pg and a sample size of 100 cells. This translates to a 10(3)-fold improvement in detection sensitivity and a 10(2)-fold reduction in required cell sample, compared to traditional Westerns processed using the same membrane immunoblots. Quantum dot fluorescent blinking analysis showed that detection of single QD-tagged proteins is possible and that detected points of fluorescence consist of one or a few (<9) QDs. The application of single nanoparticle detection capabilities to Western blotting technologies may provide a new solution to a broad range of applications currently limited by insufficient detection sensitivity and/or sample availability.

Co-targeting EGFR and survivin with a bivalent aptamer-dual siRNA chimera effectively suppresses prostate cancer

Current targeted therapies using small kinase inhibitors and antibodies have limited efficacy in treating prostate cancer (PCa), a leading cause of cancer death in American men. We have developed a novel strategy by engineering an RNA-based aptamer-siRNA chimera, in which a bivalent aptamer specifically binds prostate-specific membrane antigen (PSMA) via an antibody-like structure to promote siRNA internalization in PCa cells, and two siRNAs specific to EGFR and survivin are fused between two aptamers. The chimera is able to inhibit EGFR and survivin simultaneously and induce apoptosis effectively in vitro and in vivo. In the C4-2 PCa xenograft model, the treatment with the chimera significantly suppresses tumor growth and angiogenesis. The inhibition of angiogenesis is mediated by an EGFR-HIF1α-VEGF-dependent mechanism. Our results support that the bivalent aptamer-driven delivery of two siRNAs could be a new combination therapeutic strategy to effectively inhibit multiple and conventionally “undruggable” targets.

Simultaneous targeting of CD44 and EpCAM with a bispecific aptamer effectively inhibits intraperitoneal ovarian cancer growth

CD44 and EpCAM play crucial roles in intraperitoneal ovarian cancer development. In this study, we developed an RNA-based bispecific CD44-EpCAM aptamer that is capable of blocking CD44 and EpCAM simultaneously by fusing single CD44 and EpCAM aptamers with a double stranded RNA adaptor. With the aid of a panel of ovarian cancer cell lines, we found that bispecific CD44-EpCAM aptamer was much more effective than either single CD44 or EpCAM aptamer in the ability to inhibit cell growth and to induce apoptosis. When these aptamers were tested in intraperitoneal ovarian cancer xenograft model, bispecific CD44-EpCAM aptamer suppressed intraperitoneal tumor outgrowth much more significantly than single CD44 and EpCAM aptamer either alone or in combination. The enhanced efficacy of bispecific CD44-EpCAM aptamer is most likely to be attributed to its increased circulation time over the single aptamers. Moreover, we showed that bispecific CD44-EpCAM aptamer exhibited no toxicity to the host and was unable to trigger innate immunogenicity. Our study suggests that bispecific CD44-EpCAM aptamer may represent a promising therapeutic agent against advanced ovarian cancer.

Solid-Phase Bioconjugation of Heterobifunctional Adaptors for Versatile Assembly of Bispecific Targeting Ligands

High-throughput generation of bispecific molecules promises to expedite the discovery of new molecular therapeutics and guide engineering of novel multifunctional constructs. However, high synthesis complexity and cost have hampered the discovery of bispecific molecules in drug development and biomedical research. Herein we describe a simple solid-phase bioconjugation procedure for preparation of Protein A(G,L)-PEG-Streptavidin heterobifunctional adaptors (with 1:1:1 stoichiometry), which enable self-assembly of unmodified antibodies and biotinylated molecules into bispecific targeting ligands in a versatile mix-and-use manner. Utility of such adaptors is demonstrated by assembly of anti-CD3 and anti-Her2 antibodies into bispecific CD3xHer2 targeting ligands, which efficiently drive T-cell-mediated lysis of Her2-positive cancer cells. In comparison to bioconjugation in solution, the solid-phase procedure described here offers precise stoichiometry control, ease of purification, and high yield of functional conjugates. Simplicity and versatility should prove this methodology instrumental for preparation of bispecific ligands, as well as for high-throughput screening of bispecific combinations, before proceeding to synthesis of lead candidates via recombinant engineering or chemical cross-linking.

Quantum dot hybrid gel blotting: a technique for identifying quantum dot-protein/protein-protein interactions.

We describe an alternative to the molecular biology technique of polyacrylamide gel electrophoresis-based Western blotting and immunoprecipitation, which is an extensively used method for separating target proteins from complex cellular mixtures and for identification of protein expression and protein-protein interactions. This novel method, called quantum dot (QD) hybrid gel blotting, allows the purification and analysis of the action of QD bioconjugate-protein complexes in live cells. Moreover, these identified interactions can be correlated with spatial location in cells. QD hybrid gel blotting will be useful in the growing fields of molecular biology/proteomics and nanobiotechnology development in several respects: (1) as a method for identifying specific QD-protein interactions in cells, (2) as a method for correlating QD-protein interactions with their spatial location in live cells, (3) as a means to study the size and composition of QD bioconjugate probes/complexes; and, finally, (4) as an improvement over traditional bead-based immunoprecipitation methods for directly isolating and visualizing proteins from complex mixtures.

Targeting EGFR/HER2/HER3 with a Three-in-One Aptamer-siRNA Chimera Confers Superior Activity against HER2+ Breast Cancer

HER family members are interdependent and functionally compensatory. Simultaneously targeting EGFR/HER2/HER3 by antibody combinations has demonstrated superior treatment efficacy over targeting one HER receptor. However, antibody combinations have their limitations, with high immunogenicity and high cost. In this study, we have developed a three-in-one nucleic acid aptamer-small interfering RNA (siRNA) chimera, which targets EGFR/HER2/HER3 in one molecule. This inhibitory molecule was constructed such that a single EGFR siRNA is positioned between the HER2 and HER3 aptamers to create a HER2 aptamer-EGFR siRNA-HER3 aptamer chimera (H2EH3). EGFR siRNA was delivered into HER2-expressing cells by HER2/HER3 aptamer-induced internalization. HER2/HER3 aptamers act as antagonist molecules for blocking HER2 and HER3 signaling pathways and also as tumor-targeting agents for siRNA delivery. H2EH3 enables down-modulation of the expression of all three receptors, thereby triggering cell apoptosis. In breast cancer xenograft models, H2EH3 is able to bind to breast tumors with high specificity and significantly inhibits tumor growth via either systemic or intratumoral administration. Owing to low immunogenicity, ease of production, and high thermostability, H2EH3 is a promising therapeutic to supplement current single HER inhibitors and may act as a treatment for HER2+ breast cancer with intrinsic or acquired resistance to current drugs.

Ligand-bound quantum dots for intracellular imaging of neural receptors

Quantum dots (QDs) may serve as improved platforms for the complex modulation and ultra-sensitive imaging of molecular signaling in cells. The time course and spatial localization of activated ligand-receptor complexes and their trafficking within cells is becoming increasingly understood as vital for propagating cell signals. However, the movement and fate of ligand-receptor pairs inside cells is difficult to define with current technologies. We have studied the intracellular trafficking of TrkA receptors using QDs conjugated with nerve growth factor, a neuropeptide ligand critical for nervous system development and regulation. We find that NGF-QDs bind and activate TrkA surface receptors in PC12 neurons. Spatiotemporal maps of TrkA-NGF-QD endocytosis and translocation can be directly visualized with single QD resolution. Moreover, single molecule tracking experiments indicates that QDs complexes are actively shuttled over long distances within newly-sprouted neuronal processes. These results indicate that QDs can serve as effective high-resolution probe to track ligand-receptor function in the interior of cells.