RGD/FA双靶纳米金棒的制备及其在肿瘤细胞协同靶向成像与光热治疗中的应用

目的:通过制备RGD/FA双靶纳米金考察其与高表达整合素与叶酸受体B16细胞的协同靶向成像与热疗作用;方法:采用功能化PEG分子将靶向小分子RGD与叶酸通过强健Au-S键连接至纳米金棒表面,利用激光共聚焦与808 nm近红外激光器评价修饰纳米金的协同靶向作用;结果:RGD与叶酸分子被成功连接于纳米金表面,且双靶纳米金对小鼠黑色素瘤细胞具有较好的协同靶向作用;结论:同时靶向同一肿瘤细胞的不同表位,可克服单一靶向功能化纳米粒子难以在肿瘤位点有效积累的问题,本研究为多功能纳米金棒在临床肿瘤早期诊断与光热治疗中的应用提供研究基础。     

Targeting of ovarian cancer cell through functionalized gold nanoparticles by novel glypican-3- binding peptide as a ultrasound contrast agents

Gold nanoparticles (AuNPs) are widely studied nanomaterials for their potential employment in advanced biomedical applications, such as selective molecular imaging and targeted drug delivery. AuNPs are generally low cost and highly biocompatible, can be easily functionalized with a wide variety of functional ligands, and have been demonstrated to be effective in enhancing ultrasound contrast at clinical diagnostic frequencies. Therefore, AuNPs might be used as contrast agents in echographic imaging. In this work, we have developed a AuNPs -based system for the in vitro molecular imaging of ovarian carcinoma cells that express high levels of glypican-3 protein (GPC-3) on their surface. In this regard, a novel GPC-3 targeting peptide was designed and conjugated to fluorescent AuNPs nanoparticles. The physicochemical properties, acoustic behavior, and biocompatibility profile of the functionalized AuNPs were characterized. Then, the binding and uptake of both naked and functionalized AuNPs were analyzed by laser scanning confocal microscopy in human HeLa cells (ovarian carcinoma) cell line. The results obtained showed that GPC-3-functionalized fluorescent AuNPs significantly enhanced the ultrasound contrast and were effectively bound and taken up by HeLa cells without affecting their viability.     

Controlled release of PEG chain from gold nanorods: Targeted delivery to tumor

Gold nanorods exhibit strong absorbance of light in the near infrared region, which penetrates deeply into tissues. Since the absorbed light energy is converted into heat, gold nanorods are expected to act as a contrast agent for in vivo bioimaging and as a thermal converter for photothermal therapy. To construct a gold nanorod targeted delivery system for tumor a peptide substrate for urokinase-type plasminogen activator (uPA), expressed specifically on malignant tumors, was inserted between the PEG chain and the surface of the gold nanorods. In other words, we constructed PEG–peptide-modified gold nanorods. After mixing the gold nanorods with uPA, the PEG chain was released from the surface of the gold and subsequently nanorod aggregation took place. The formation of the aggregation was monitored as a decrease in light absorption at 900 nm. Tumor homogenate induced a significant decrease in this absorption. Larger amount of the PEG–peptide-modified gold nanorods bound to cells expressing uPA in vitro compared with control gold nanorods, which had scrambled sequence of the peptide. The PEG–peptide-modified gold nanorods showed higher accumulation in tumor than the control after they were injected intravenously into tumor-bearing mice, however, the density of the peptide on the surface of the gold nanorods was a key factor of their biodistributions. This targeted delivery system, which responds to uPA activity, is expected to be a powerful tool for tumor bioimaging and photothermal tumor therapy.     

Laser-induced gene expression in specific cells of transgenic zebrafish

Over the past few years, a number of studies have described the generation of transgenic lines of zebrafish in which expression of reporters was driven by a variety of promoters. These lines opened up the real possibility that transgenics could be used to complement the genetic analysis of zebrafish development. Transgenic lines in which the expression of genes can be regulated both in space and time would be especially useful. Therefore, we have cloned the zebrafish promoter for the inducible hsp70 gene and made stable transgenic lines of zebrafish that express the reporter green fluorescent protein gene under the control of a hsp70 promoter. At normal temperatures, green fluorescent protein is not detectable in transgenic embryos with the exception of the lens, but is robustly expressed throughout the embryo following an increase in ambient temperature. Furthermore, we have taken advantage of the accessibility and optical clarity of the embryos to express green fluorescent protein in individual cells by focussing a sublethal laser microbeam onto them. The targeted cells appear to develop normally: cells migrate normally, neurons project axons that follow normal pathways, and progenitor cells divide and give rise to normal progeny cells. By generating other transgenic lines in which the hsp70 promoter regulates genes of interest, it should be possible to examine the in vivo activity of the gene products by laser-inducing specific cells to express them in zebrafish embryos. As a first test, we laser-induced single muscle cells to make zebrafish Sema3A1, a semaphorin that is repulsive for specific growth cones, in a hsp70-sema3A1 transgenic line of zebrafish and found that extension by the motor axons was retarded by the induced muscle.     

Near-infrared fluorescence-labeled anti-PD-L1-mAb for tumor imaging in human colorectal cancer xenografted mice

The expression of programmed death ligand-1 (PD-L1) in tumor has been used as a biomarker to predict the anti-PD-L1 immunotherapy response. To develop a noninvasive imaging technique to monitor the dynamic changes in PD-L1 expression in colorectal cancer (CRC), we labeled an anti-PD-L1 monoclonal antibody with near-infrared (NIR) dye and tested the ability of the NIR-PD-L1-mAb probe to monitor the PD-L1 expression in CRC-xenografted mice by performing optical imaging. Consistent with the expression levels of PD-L1 protein in three CRC cell lines in vitro by flow cytometry and Western blot analyses, our in vivo imaging showed the highest fluorescence signal of the xenografted tumors in mice bearing SW620 CRC cells, followed by tumors derived from SW480 and HCT8 cell lines. We detected the highest fluorescent intensity of the tumor at 120 hours after injection of NIR-PD-L1-mAb. The highest fluorescence intensity was seen in the tumor, followed by the spleen and the liver in SW620 xenografted mice. In SW480 and HCT8 xenografted mice, however, the highest fluorescent signals were detected in the spleen, followed by the liver and the tumor. Our findings indicate that SW620 cells express a higher level of PD-L1, and the NIR-PD-L1-mAb binding to PD-L1 on the surface of CRC cells was specific. The technique was safe and could provide valuable information on PD-L1 expression of the tumor for development of a therapeutic strategy of personized targeted immunotherapies as well as treatment response of patients with CRC.     

1064‐nm‐resonant gold nanorods for photoacoustic theranostics within permissible exposure limits

Therapeutic and diagnostic methods based on photomechanical effects are attracting much current attention in contexts as oncology, cardiology and vascular surgery, for such applications as photoacoustic imaging or microsurgery. Their underlying mechanism is the generation of ultrasound or cavitation from the interaction of short optical pulses with endogenous dyes or targeted contrast agents. Among the latter, gold nanorods are outstanding candidates, but their use has mainly been reported for photoacoustic imaging and photothermal treatments. Conversely, much less is still known about their value as a precision tool for photomechanical manipulations, such as to impart local damage with high spatial resolution through the expansion and collapse of microbubbles. Here, we address the feasibility of gold nanorods exhibiting a distribution of surface plasmon resonances between about 900 to above 1100 nm as a contrast agent for photoacoustic theranostics. After testing their cytotoxicity and cellular uptake, we discuss their photostability and use to mediate cavitation and the photomechanical destruction of targeted cells. We find that the choice of a plasmonic band peaking around 1064 nm is key to enhance the translational potential of this approach. With respect to the standard alternative of 800 nm, at 1064 nm, relevant regulations on optical exposure are less restrictive and the photonic technology is more mature.      

Tumor angiogenic endothelial cell targeting by a novel integrin-targeted nanoparticle

Angiogenesis is an important process in cancer growth and metastasis. During the tumor angiogenic process, endothelial cells express various cell surface receptors which can be utilized for molecular imaging and targeted drug delivery. One such protein receptor of interest is the integrin alphav beta3. Our group is involved in the development of molecular imaging probes and drug delivery systems targeting alphav beta3. Based on extensive lead optimization study with the integrin antagonist compounds, we have developed a new generation of integrin alphav beta3 compound (IA) which has superior binding affinity to alphav beta3. Utilizing this IA as a targeting agent, we have developed a novel integrin-targeted nanoparticle (ITNP) system for targ alphav beta3 was observed. These ITNPs also were rapidly taken up by cells that express alphav beta3. The ITNPs accumulated in the angiogenic vessels, after systemic administration in a murine squamous cell carcinoma model. This novel intergrin targeted ITNP platform will likely have an application in targeted delivery of drugs and genes in vivo and can also be used for molecular imaging.     

Optimization of protein production in mammalian cells with a coexpressed fluorescent marker

The expression of mammalian proteins in sufficient abundance and quality for structural studies often presents formidable challenges. Many express poorly in bacterial systems, whereas it can be time consuming and expensive to produce them from cells of higher organisms. Here we describe a procedure for the direct selection of stable mammalian cell lines that express proteins of interest in high yield. Coexpression of a marker protein, such as green fluorescent protein, is linked to that of the desired protein through an internal ribosome entry site in the vector that is transfected into cells in culture. The coexpressed marker is used to select for highly expressing clonal cell lines. Applications are described to a membrane protein, the 5HT2c serotonin receptor, and to a secreted cysteine-rich protein, resistin. Besides providing an expeditious means for producing mammalian proteins for structural work, the resulting cell lines also readily support tests of functional properties and structure-inspired hypotheses.     

Transient expression in Nicotiana benthamiana fluorescent marker lines provides enhanced definition of protein localization,movement and interactions in planta

Here, we report on the construction of a novel series of Gateway‐compatible plant transformation vectors containing genes encoding autofluorescent proteins, including Cerulean, Dendra2, DRONPA, TagRFP and Venus, for the expression of protein fusions in plant cells. To assist users in the selection of vectors, we have determined the relative in planta photostability and brightness of nine autofluorescent proteins (AFPs), and have compared the use of DRONPA and Dendra2 in photoactivation and photoconversion experiments. Additionally, we have generated transgenic Nicotiana benthamiana lines that express fluorescent protein markers targeted to nuclei, endoplasmic reticulum or actin filaments. We show that conducting bimolecular fluorescence complementation assays in plants that constitutively express cyan fluorescent protein fused to histone 2B provides enhanced data quality and content over assays conducted without the benefit of a subcellular marker. In addition to testing protein interactions, we demonstrate that our transgenic lines that express red fluorescent protein markers offer exceptional support in experiments aimed at defining nuclear or endomembrane localization. Taken together, the new combination of pSITE‐BiFC and pSITEII vectors for studying intracellular protein interaction, localization and movement, in conjunction with our transgenic marker lines, constitute powerful tools for the plant biology community.     

Specific capture of mammalian cells by cell surface receptor binding to ligand immobilized on gold thin films

Aldehyde-terminated self-assembled monolayers (SAMs) on gold surfaces were modified with proteins and employed to capture intact living cells through specific ligand-cell surface receptor interactions. In our model system, the basic fibroblast growth factor (bFGF) binding receptor was targeted on baby hamster kidney (BHK-21) cells. Negative control and target proteins were immobilized on a gold surface by coupling protein primary amines to surface aldehyde groups. Cell-binding was monitored by phase contrast microscopy or surface plasmon resonance (SPR) imaging. The specificity of the receptor-ligand interaction was confirmed by the lack of cell binding to the negative control proteins, cytochrome c and insulin, and by the disruption of cell binding by treatment with heparitinase to destroy heparan sulfate which plays an essential role in the binding of bFGF to FGF receptors. This approach can simultaneously probe a large number of receptor-ligand interactions in cell populations and has potential for targeting and isolating cells from mixtures according to the receptors expressed on their surface.     

Peptide-conjugated gold nanorods for nuclear targeting

Resonant electron oscillations on the surface of noble metal nanoparticles (Au, Ag, Cu) create the surface plasmon resonance (SPR) that greatly enhances the absorption and Rayleigh (Mie) scattering of light by these particles. By adjusting the size and shape of the particles from spheres to rods, the SPR absorption and scattering can be tuned from the visible to the near-infrared region (NIR) where biologic tissues are relatively transparent. Further, gold nanorods greatly enhance surface Raman scattering of adsorbed molecules. These unique properties make gold nanorods especially attractive as optical sensors for biological and medical applications. In the present work, gold nanorods are covalently conjugated with a nuclear localization signal peptide through a thioalkyl-triazole linker and incubated with an immortalized benign epithelial cell line and an oral cancer cell line. Dark field light SPR scattering images demonstrate that nanorods are located in both the cytoplasm and nucleus of both cell lines. Single cell micro-Raman spectra reveal enhanced Raman bands of the peptide as well as molecules in the cytoplasm and the nucleus. Further, the Raman spectra reveal a difference between benign and cancer cell lines. This work represents an important step toward both imaging and Raman-based intracellular biosensing with covalently linked ligand-nanorod probes.     

Development and validation of algorithms for measuring G-protein coupled receptor activation in cells using the LSC-based imaging cytometer platform.

BACKGROUND: A cell-based assay system (Transfluor) has been developed for measurement of G-protein coupled receptor (GPCR) activity by using cells transfected to express a fusion protein of arrestin plus green fluorescent protein (GFP) and the target GPCR. Upon agonist stimulation, the arrestin-GFP translocates to and binds the activated GPCR at the plasma membrane. The receptor/arrestin-GFP complexes then localize in clathrin-coated pits and/or intracellular vesicles. This redistribution of arrestin-GFP into condensed fluorescent spots is useful for visually monitoring the active status of GPCRs and its quantitation is possible with certain types of digital image analysis systems. METHODS: We designed two lines of image processing algorithms to carry out quantitative measurement of the arrestin-GFP movement on an inverted version of laser scanning cytometry (iCyte) as an imaging platform. We used a cell line expressing arrestin-GFP and the wild-type beta2-adrenergic receptor or a modified version of this receptor with enhanced affinity for arrestin. Each cell line was challenged with various concentrations of agonist. RESULTS: A dose-dependent signal was measured and half-maximal effective concentration values were obtained that agreed well with results determined by other methods previously reported. CONCLUSIONS: The results indicate that the combination of Transfluor, iCyte, and our algorithms is suitable for robust and pharmacologically relevant GPCR ligand exploration.     

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see ¹). Compound transgenic fish which express different fluorescent reporters in neighboring cell types provide a means of following cellular interactions ² and/or tissue-level responses to experimental manipulations over time. In this video, we demonstrate methods that can be used for imaging multiple transgenically labeled cell types serially in individual fish over time courses that can span from minutes to several days. The techniques described are applicable to any study seeking to correlate the "behavior" of neighboring cells types over time, including: 1) serial ''catch and release'' methods for imaging a large number of fish over successive days, 2) simplified approaches for separating fluorophores with overlapping excitation/emission profiles (e.g., GFP and YFP), 3) use of hypopigmented mutant lines to extend the time window available for high-resolution imaging into late larval stages of development, 4) use of membrane targeted fluorescent reporters to reveal fine morphological detail of individual cells as well as cellular details in larger populations of cells, and 5) a previously described method for chemically-induced ablation of transgenically targeted cell types; i.e., nitroreductase (NTR) mediated conversion of prodrug substrates, such as metronidazole (MTZ), to cytotoxic derivatives ^3,5.As an example of these approaches, we will visualize the ablation and regeneration of a subtype of retinal bipolar neuron within individual fish over several days. Simultaneously we will monitor several other retinal cell types, including neighboring non-targeted bipolar cells and potential degeneration-stimulated retinal stem cells (i.e., Mϋller glia). This strategy is being applied in our lab to characterize cell- and tissue-level (e.g., stem cell niche) responses to the selective loss and regeneration of targeted neuronal cell types.     

Production of cell lines secreting TAT fusion proteins.

Transduction of proteins and other macromolecules constitutes a potent technology to analyze cell functions and to achieve therapeutic interventions. In general, fusion proteins with protein transduction domains, such as TAT, are produced in a bacterial expression system. Here we describe the generation of a mammalian expression vector coding for TAT-EGFP fusion protein. Transfection of CHO-K1 cells by this vector and subsequent selection by Zeocin resulted in cell lines that express and secrete EGFP, a variant of the green fluorescent protein GFP. The ultimate cell line was produced by first cloning the stable integrants and subsequent selection of EGFP-expressing cells by flow cytometric sorting. In the resulting cell line approximately 98% of cells express EGFP. Using the same methodology, we generated cell lines that express DsRed fluorescent protein. The advantages of using such a mammalian expression system include the ease of generating TAT fusion proteins and the potential for sustained production of such proteins in vitro and, potentially, in vivo.     

A new Trichoplusia ni cell line for membrane protein expression using a baculovirus expression vector system

A new cell line, MSU-TnT4 (TnT4), was established from Trichoplusia ni embryos for use with baculovirus expression vectors and evaluated for its potential for membrane protein production. To evaluate membrane protein synthesis, recombinant baculoviruses were constructed to express the human neurotensin receptor 1 as an enhanced green fluorescent protein (GFP) fusion. TnT4 cells had a doubling time of 21 h and expressed the membrane-GFP fusion protein at approximately twice the level as Sf21 cells from the p10 promoter, as evaluated by GFP intensity. Expression of secreted alkaline phosphatase (SEAP) was similar to that of Sf21 cells. Expression of membrane-GFP fusion proteins in recombinant baculoviruses provides a rapid method for evaluating the potential of new cell lines for the production of membrane proteins using a baculovirus expression vector system (BEVS).     

Targeted photodynamic therapy agent with a built-in apoptosis sensor for in vivo near-infrared imaging of tumor apoptosis triggered by its photosensitization in situ

Imaging apoptotic cells or tissues after cancer therapy in situ would be a very useful tool for assessing proper treatment conditions and therapeutic outcome. By combining therapeutic and imaging functions, we have designed a multifunctional, membrane-permeable, and cancer-specific agent that triggers and images apoptosis in targeted cells. We chose photodynamic therapy (PDT) as an appropriate cancer treatment modality and caspase 3 as an apoptosis-specific imaging target. This targeted photodynamic therapy agent with a built-in apoptosis sensor (TaBIAS) induces photodamage only to target cells and simultaneously identifies those that are apoptotic by its near-infrared fluorescence. It contains a fluorescent photosensitizer used as an anticancer drug and a cancer-associated folate receptor homing molecule connected to a caspase 3 cleavable peptide linker that has a fluorescence quencher on the opposing site. We demonstrated that PDT-triggered cleavage of the peptide linker by caspase 3, one of the key executioner caspases, results in a detectable increase in fluorescence in folate receptor-overexpressing cancer cells and tumors. The presence of apoptosis was confirmed in vitro by flow cytometry and ex vivo by Apoptag assay, supporting the ability of TaBIAS to specifically induce and image apoptosis in situ.     

Functionalized Plasmonic Anisotropic Nanocrystals for Multimodal Imaging of Cancer Cells

Nanoparticles internalized by cells are valuable probes for bioimaging. In particular, nanoparticles can be detected in “biological transmission window,” i.e., near infrared region. Here, we report a preparation of biotargeting diethylthiatricarbocyanine iodide (DTTC)-functionalized gold nanorods, utilized for detection of malignant cells. These biotargeting DTTC-functionalized gold nanorods are efficiently internalized into cultured cells and can serve as probes for surface-enhanced Raman scattering (SERS) and dark-field imaging. The robust SERS signal from malignant cells has clearly demonstrated a signature peak of DTTC in the presence of our formulation. A short acquisition time, we used in this experiment, is able to exclude bulk of Raman signal from natural cellular constituents. This signature peak will be a key of identifying cancer due to cancer-specific property of biotargeted molecule. The results are leading to promising real-time cancer detection. In addition, these multimodal probes demonstrated low toxicity in cell viability studies which enables a broad range of multiplex imaging applications.     

Color-coded imaging of splenocyte-pancreatic cancer cell interactions in the tumor microenvironment

In spite of advances in surgical and medical care pancreatic cancer remains a leading cause of cancer-related death in the United States. An understanding of cancer cell interactions with host cells is critical to our ability to develop effective antitumor therapeutics for pancreatic cancer. We report here a color-coded model system for imaging cancer cell interactions with host immune cells within the native pancreas. A human pancreatic cancer cell line engineered to express green fluorescent protein (GFP) in the nucleus and red fluorescent protein (DsRed2) in the cytoplasm was orthotopically implanted into the pancreas of a nude mouse. After 10-14 days red or green fluorescent splenocytes from immune-competent donors were delivered systemically to the pancreatic cancer-bearing nude mice. Animals were imaged after splenocyte delivery using high-resolution intravital imaging systems. At 1 day after iv injection red or green fluorescent spleen cells were found distributed in lung, liver, spleen and pancreas. By 4 days after cell delivery, however, the immune cells could be clearly imaged surrounding the tumor cells within the pancreas as well as collecting within lymphatic tissues such as lymph nodes and spleen. With the high-resolution intravital imaging afforded by the Olympus IV100 and OV100 systems the interactions of the dual-colored cancer cells and the red fluorescent spleen cells could be clearly imaged in this orthotopic pancreatic cancer model. This color-coded in vivo imaging technology offers a novel approach to imaging the interactions of cancer and immune cells in the tumor microenvironment (TME).     

金纳米棒的表面修饰及其应用于肿瘤诊疗的研究进展

金纳米棒具有独特的光学性质、表面易修饰性、较低的生物毒性和良好的生物相容性,因而在成像、光热治疗和药物载带等方面具有极高的潜在应用价值.本文综述了典型的金纳米棒表面修饰方法及其在生物成像、光热治疗和药物治疗中的应用,重点阐述了通过金纳米棒同时实现肿瘤诊断和治疗相结合的研究进展.     

Matrix metalloproteinase sensitive gold nanorod for simultaneous bioimaging and photothermal therapy of cancer

Herein, we developed matrix metalloprotease (MMP) sensitive gold nanorods (MMP-AuNR) for cancer imaging and therapy. It was feasible to absorb NIR laser and convert into heat as well as visualize MMP activity. We showed the possibility of gold nanorods as a hyperthermal therapeutic agent and MMP sensitive imaging agent both in vitro and in vivo condition. The results suggested potential application of MMP-AuNR for simultaneous cancer diagnosis and therapy.