A novel aptamer-based histochemistry assay for specific diagnosis of clinical breast cancer tissues

Pathological detection using immunohistochemistry (IHC) has become an indispensable process in the diagnosis confirmation of various cancers. However, the production of monoclonal antibodies is always very complex, expensive and time-consuming, and the batch differences are significant due to the corporeity and health statuses of animals may be different. In this work, an aptamer-based histochemistry (aptahistochemistry) assay was developed using a DNA aptamer for specific diagnosis of clinical breast cancer tissue sections. This aptahistochemistry assay can specifically distinguish Luminal A breast cancer molecular subtype from Luminal B (HER2+), HER2-enriched, and triple-negative breast cancer molecular subtypes, as well as para-carcinoma tissue, mastitis tissue and normal breast tissue. The accuracy of this aptahistochemistry assay for the diagnosis of Luminal A breast cancer was as high as 80%, which showed a great potential for clinical pathological diagnosis applications.     

Site-specific conjugation of ScFvs antibodies to nanoparticles by bioorthogonal strain-promoted alkyne-nitrone cycloaddition

Particularly suitable: An N-terminal serine mutant of anti-HER2 scFv antibody was conjugated to polymer-coated magnetofluorescent nanoparticles by strain-promoted alkyne-nitrone cycloaddition. The resulting nanoparticles (see scheme) proved effective in targeting and labeling HER2-positive breast cancer cells.     

Imaging of epidermal growth factor receptor on single breast cancer cells using surface-enhanced Raman spectroscopy

Epidermal growth factor receptor (EGFR) is widely used as a biomarker for pathological grading and therapeutic targeting of human cancers. This study investigates expression, spatial distribution as well as the endocytosis of EGFR in single breast cancer cells using surface-enhanced Raman spectroscopy (SERS). By incubating anti-EGFR antibody conjugated SERS nanoprobes with an EGFR-over-expressing cancer cell line, A431, EGFR localization was measured over time and found to be located primarily at the cell surface. To further validate the constructed SERS probes, we applied this SERS probes to detect the EGFR expression on breast cancer cells (MDA-MB-435, MDA-MB-231) and their counterpart cell lines in which EGFR expression was down-regulated by breast cancer metastasis suppressor 1 (BRMS1). The results showed that SERS method not only confirms immunoblot data measuring EGFR levels, but also adds new insights regarding EGFR localization and internalization in living cells which is impossible in immunoblot method. Thus, SERS provides a powerful new tool to measure biomarkers in living cancer cells.     

Aptamer based fluorescent probe for serum HER2-ECD detection: The clinical utility in breast cancer

HB5 aptamer-based probe has been developed for serum HER2-ECD test in auxiliary clinical diagnosis and treatment for HER2-positive breast cancer patients.     

A functional comparison between the HER2(high)/HER3 and the HER2(low)/HER3 dimers on heregulin-β1-induced MMP-1 and MMP-9 expression in breast cancer cells

Overexpression of HER2 correlates with more aggressive tumors and increased resistance to cancer chemotherapy. However, a functional comparison between the HER2(high)/HER3 and the HER2(low)/HER3 dimers on tumor metastasis has not been conducted. Herein we examined the regulation mechanism of heregulin- β1 (HRG)-induced MMP-1 and -9 expression in breast cancer cell lines. Our results showed that the basal levels of MMP-1 and -9 mRNA and protein expression were increased by HRG treatment. In addition, HRG-induced MMP-1 and -9 expression was significantly decreased by MEK1/2 inhibitor, U0126 but not by phosphatidylinositol 3-kinase (PI-3K) inhibitor, LY294002. To confirm the role of MEK/ERK pathway on HRG-induced MMP-1 and -9 expression, MCF7 cells were transfected with constitutively active adenoviral- MEK (CA-MEK). The level of MMP-1 and -9 expressions was increased by CA-MEK. MMP-1 and -9 mRNA and protein expressions in response to HRG were higher in HER2 overexpressed cells than in vector alone. The phosphorylation of HER2, HER3, ERK, Akt, and JNK were also significantly increased in HER2 overexpressed MCF7 cells compared with vector alone. HRG-induced MMP-1 and -9 expressions were significantly decreased by lapatinib, which inhibits HER1 and HER2 activity, in both vector alone and HER2 overexpressed MCF7 cells. Finally, HRG-induced MMP-1 and MMP-9 expression was decreased by HER3 siRNA overexpression. Taken together, we suggested that HRG-induced MMP-1 and MMP-9 expression is mediated through HER3 dependent pathway and highly expressed HER2 may be associated with more aggressive metastasis than the low expressed HER2 in breast cancer cells.     

Design of Silica@Au Hybrid Nanostars for Enhanced SERS and Photothermal Effect

Core-shell nanostructures of silicon oxide@noble metal have drawn a lot of interest due to their distinctive characteristics and minimal toxicity with remarkable biocompatibility. Due to the unique property of localized surface plasmon resonance (LSPR), plasmonic nanoparticles are being used as surface-enhanced Raman scattering (SERS) based detection of pollutants and photothermal (PT) agents in cancer therapy. Herein, we demonstrate the synthesis of multifunctional silica core – Au nanostars shell (SiO₂@Au NSs) nanostructures using surfactant free aqueous phase method. The SERS performance of the as-synthesized anisotropic core-shell NSs was examined using Rhodamine B (RhB) dye as a Raman probe and resulted in strong enhancement factor of 1.37×10⁶. Furthermore, SiO₂@Au NSs were also employed for PT killing of breast cancer cells and they exhibited a concentration-dependent increase in the photothermal effect. The SiO₂@Au NSs show remarkable photothermal conversion efficiency of up to 72 % which is unprecedented. As an outcome, our synthesized NIR active SiO₂@Au NSs are of pivotal importance to have their dual applications in SERS enhancement and PT effect.     

Photocatalytically Powered Matchlike Nanomotor for Light‐Guided Active SERS Sensing

Surface enhanced Raman spectroscopy (SERS) is a powerful optical sensing technique that can detect analytes of extremely low concentrations. However, the presence of enough SERS probes in the detection area and a close contact between analytes and SERS probes are critical for efficient acquisition of a SERS signal. Presented here is a light‐powered micro/nanomotor (MNM) that can serve as an active SERS probe. The matchlike AgNW@SiO₂ core–shell structure of the nanomotors work as SERS probes based on the shell‐isolated enhanced Raman mechanism. The AgCl tail serves as photocatalytic nanoengine, providing a self‐propulsion force by light‐induced self‐diffusiophoresis. The phototactic behavior was utilized to achieve enrichment of the nanomotor‐based SERS probes for on‐demand biochemical sensing. The results demonstrate the possibility of using photocatalytic nanomotors as active SERS probes for remote, light‐controlled, and smart biochemical sensing on the micro/nanoscale.     

Synthesis and Feasibility Evaluation of a new Trastuzumab Conjugate Integrated with Paclitaxel and 89Zr for Theranostic Application Against HER2-Expressing Breast Cancers

The preparation and in vitro evaluation of a theranostic conjugate composed of trastuzumab, paclitaxel (PTX), and deferoxamine (DFO)-chelated ⁸⁹Zr have been reported. These comounds have potential applications against HER2 receptor positive breast cancers. We conjugated DFO and PTX to trastuzumab by exploiting simple conjugation chemistry. The conjugate (DFO-trastuzumab-PTX) showed excellent radiolabeling efficiency with ⁸⁹Zr and the labeled conjugate had high in vitro stability in human serum. Furthermore, DFO-trastuzumab-PTX displayed comparable cytotoxicity with PTX and ⁸⁹Zr-DFO-trastuzumab-PTX exhibited HER2 receptor-mediated binding on HER2-positive MDA-MB-231 breast cancer cells. The results of our in vitro study indicate high potential of ⁸⁹Zr-DFO-trastuzumab-PTX to be utilized in the theranostic application against HER2-postive breast cancers.     

Review: Radionuclide Molecular Imaging Targeting HER2 in Breast Cancer with a Focus on Molecular Probes into Clinical Trials and Small Peptides

As the most frequently occurring cancer worldwide, breast cancer (BC) is the leading cause of cancer-related death in women. The overexpression of HER2 (human epidermal growth factor receptor 2) is found in about 15% of BC patients, and it is often associated with a poor prognosis due to the effect on cell proliferation, migration, invasion, and survival. As a result of the heterogeneity of BC, molecular imaging with HER2 probes can non-invasively, in real time, and quantitatively reflect the expression status of HER2 in tumors. This will provide a new approach for patients to choose treatment options and monitor treatment response. Furthermore, radionuclide molecular imaging has the potential of repetitive measurements, and it can help solve the problem of heterogeneous expression and conversion of HER2 status during disease progression or treatment. Different imaging probes of targeting proteins, such as monoclonal antibodies, antibody fragments, nanobodies, and affibodies, are currently in preclinical and clinical development. Moreover, in recent years, HER2-specific peptides have been widely developed for molecular imaging techniques for HER2-positive cancers. This article summarized different types of molecular probes targeting HER2 used in current clinical applications and the developmental trend of some HER2-specific peptides.     

Silica-coated gold nanostars for combined surface-enhanced Raman scattering (SERS) detection and singlet-oxygen generation: a potential nanoplatform for theranostics

This paper reports the synthesis and characterization of surface-enhanced Raman scattering (SERS) label-tagged gold nanostars, coated with a silica shell containing methylene blue photosensitizing drug for singlet-oxygen generation. To our knowledge, this is the first report of nanocomposites possessing a combined capability for SERS detection and singlet-oxygen generation for photodynamic therapy. The gold nanostars were tuned for maximal absorption in the near-infrared (NIR) spectral region and tagged with a NIR dye for surface-enhanced resonance Raman scattering (SERRS). Silica coating was used to encapsulate the photosensitizer methylene blue in a shell around the nanoparticles. Upon 785 nm excitation, SERS from the Raman dye is observed, while excitation at 633 nm shows fluorescence from methylene blue. Methylene-blue-encapsulated nanoparticles show a significant increase in singlet-oxygen generation as compared to nanoparticles synthesized without methylene blue. This increased singlet-oxygen generation shows a cytotoxic effect on BT549 breast cancer cells upon laser irradiation. The combination of SERS detection (diagnostic) and singlet-oxygen generation (therapeutic) into a single platform provides a potential theranostic agent.     

Multiplex cancer cell detection by SERS nanotags with cyanine and triphenylmethine Raman reporters

SERS nanotags have been prepared to accomplish the multiplex detection of cancer cells. Herein we evaluated the adequacy of lipoic acid-containing cyanine derivatives (Cy3LA and Cy5LA) to function as multiplex partners with a triphenylmethine Raman reporter (B2LA) under a single excitation wavelength. SERS experiments enabled the multiplex recognition of two different cancer cells with antibody-conjugated nanotags that were derivatized with optimized cyanine and triphenylmethine reporters.     

Adenovirus adenine nucleotide translocator-2 shRNA effectively induces apoptosis and enhances chemosensitivity by the down-regulation of ABCG2 in breast cancer stem-like cells

Cancer stem cells (CSCs) are resistant to chemo- and radio-therapy, and can survive to regenerate new tumors. This is an important reason why various anti- cancer therapies often fail to completely control tumors, although they kill and eliminate the bulk of cancer cells. In this study, we determined whether or not adenine nucleotide translocator-2 (ANT2) suppression could also be effective in inducing cell death of breast cancer stem-like cells. A sub-population (SP; CD44+/ CD24-) of breast cancer cells has been reported to have stem/progenitor cell properties. We utilized the adeno- ANT2 shRNA virus to inhibit ANT2 expression and then observed the treatment effect in a SP of breast cancer cell line. In this study, MCF7, MDA-MB-231 cells, and breast epithelial cells (MCF10A) mesenchymally-transdifferentiated through E-cadherin knockdown were used. ANT2 expression was high in both stem-like cells and non-stem-like cells of MCF7 and MDA-MB-231 cells, and was induced and up-regulated by mesenchymal transdifferentiation in MCF10A cells (MCF10A(EMT)). Knockdown of ANT2 by adeno-shRNA virus efficiently induced apoptotic cell death in the stem-like cells of MCF7 and MDA-MB-231 cells, and MCF10A(EMT). Stem-like cells of MCF7 and MDA-MB-231, and MCF10A(EMT) cells exhibited increased drug (doxorubicin) resistance, and expressed a multi-drug resistant related molecule, ABCG2, at a high level. Adeno-ANT2 shRNA virus markedly sensitized the stem-like cells of MCF7 and MDA-MB-231, and the MCF10A(EMT) cells to doxorubicin, which was accompanied by down-regulation of ABCG2. Our results suggest that ANT2 suppression by adeno-shRNA virus is an effective strategy to induce cell death and increase the chemosensitivity of stem-like cells in breast cancer.     

A microfluidic surface-enhanced Raman scattering (SERS) sensor for microRNA in extracellular vesicles with nucleic acid-tyramine cascade amplification

Exosomal microRNA (miRNA) is an ideal candidate of noninvasive biomarker for the early diagnosis of cancer. Sensitive and accurate sensing of abnormal exosomal miRNA plays essential role for clinical promotion due to its close correlation with tumor proliferation and progression. Herein, a microfluidic surface-enhanced Raman scattering (SERS) sensor was proposed for an on-line detection of exosomal miRNA based on rolling circle amplification (RCA) and tyramine signal amplification (TSA) strategy. The microfluidic chip consists of a magnetic enrichment chamber, a serpentine fluidic mixer and a plasmonic SERS substrate functionalized with capture probes. The released miRNA activates the capture probe, triggers RCA reaction, and generates a large number of single-stranded DNA products to drive the catalysis of nanotags deposition via TSA, producing numerous “hot spots” to enhance the SERS signals. In merit of the microfluidics chip and nucleic acid-tyramine cascade amplification, the developed SERS sensor significantly improves the sensitivity for the exosomal miRNA assay, resulting in a limit of detection (LOD) as low as 1 pmol/L and can be successfully applied in the analysis of exosomes secreted from breast tumor cells, which demonstrates the potential utility in practical applications.     

A Multi-action PtIV Conjugate with Oleate and Cinnamate Ligands Targets Human Epithelial Growth Factor Receptor HER2 in Aggressive Breast Cancer Cells

HER2-positive breast cancer is an aggressive subtype that typically responds poorly to standard chemotherapy. To design an anticancer drug selective for HER2-expressing breast cancer, a Pt^IV prodrug with axial oleate and cinnamate ligands was synthesized. We demonstrate its superior antiproliferative activity in monolayer and 3D spheroid models; the antiproliferative efficiency increases gradually with increasing expression of HER2. The results also suggest that the released Pt^II compound inhibits the proliferation of cancer cells by a DNA-damage-mediated mechanism. Simultaneously, the released oleic and cinnamic acid can effectively inhibit HER2 expression. To our knowledge, this is the first platinum-based complex inhibiting HER2 expression that does not contain protein or peptide. Moreover, this Pt^IV prodrug is capable of overcoming the resistance of cancer stem cells (CSCs), inducing death in both CSCs and differentiated cancer cells. Thus, the results substantiate our design strategy and demonstrate the potential of this approach for the development of new, therapeutically relevant compounds.     

SERS纳米探针对电刺激过程中细胞内产生活性氧的检测

电刺激是用于细胞内紊乱电活动引起疾病的一类重要治疗方式. 在电刺激过程中是否会诱导细胞内活性氧(ROS)水平的改变, 以及常规抗氧化抑制药物与电刺激治疗同时运用带来的影响, 目前尚未有相关研究. 本文设计了一种具有较好生物相容性的金/银核壳纳米棒表面增强拉曼(SERS)活性探针, 用于电刺激过程中细胞内产生ROS的检测. 将该探针与细胞共孵育, 使其内化入细胞, 对细胞进行不同时间的电刺激, 利用拉曼光谱对SERS探针的信号进行检测. 实验结果表明, 随着电刺激时间的延长, SERS信号减弱, 说明细胞内产生ROS的量明显增加. 该传感机制是利用ROS能刻蚀金/银核壳纳米棒的银壳, 从而使其变薄引起SERS信号减弱. 抗坏血酸(AA)和谷胱甘肽(GSH)两种抗氧化抑制剂类药物与电刺激同时运用时, 可观察到它们会对电刺激过程产生的ROS有清除作用. 该研究发展了一类用于细胞内ROS检测的光谱方法, 也为异常的氧化应激和肿瘤治疗过程中的组合用药提供了建议.     

Radio-immunoconjugated,Dox-loaded,surface-modified superparamagnetic iron oxide nanoparticles (SPIONs) as a bioprobe for breast cancer tumor theranostics

In this research, we develop dual modality molecular imaging and also radio-immunotherapy (RIT) bioprobes, in the form of modified superparamagnetic iron oxide nanoparticles (SPIONs) conjugated to radiolabeled antibodies, for PET and MRI of HER2 expressing cancers as well as a PH sensitive drug carrier by embedded an anticancer agent for cancer therapeutic applications. The bioprobes were developed by conjugating ⁶⁴Cu labeled trastuzumab (herceptin) and rituximab (Anti CD-20) antibodies to modified SPIONs. The SPIONs were modified with carboxymethyl chitosan and functionalized with acrylic acid (AA). Also, with the purpose of identifying more effective bifunctional chelator (BFC), we compared the properties of novel BFC, p-NO₂-Bn-PCTA with the commonly used DOTA-NHS for radio-immunoconjugate preparations. Moreover, a chemotherapy drug, doxorubicin, was then loaded onto engineered nanoparticles for targeted intracellular drug delivery and selective cancer cell killing. Resulting radio-immunoconjugated-SPIONs were evaluated for molecular imaging and effective targeting of the HER2+ receptors in SKBR3 cell lines and breast tumor bearing Balb/C mice. Therefore, our biocompatible SPIONs could serve as a promising multifunctional theranostics nanoplatform in dual modality imaging guided RIT of HER2 overexpressing cancer applicable to drug delivery and controlled drug release for trigger both intrinsic and extrinsic pathways of apoptosis.     

Development of a ZHER3‐Affibody‐Targeted Nano‐Vector for Gene Delivery to HER3‐Overexpressed Breast Cancer Cells

Despite the initial successes of gene delivery applications, they faced on several intrinsic drawbacks including toxicity and immunogenicity. Therefore, alternative gene‐delivery systems derived from recombinant peptides have emerged and is rapidly developing. Human epidermal growth factor receptor‐3 (HER3) shows high activity in tumor resistance to anti‐human epidermal growth factor receptor 2 (HER2) therapies. In this study, an affibody molecule against HER3 is conjugated to a biomimetic peptide RALA (an amphipathic and cationic peptide enriched with arginine) and the ability of the fusion vector for targeting HER3 and afterward delivering specific genes in breast cancer cells is evaluated. The results demonstrate that the biopolymeric platform, which contains an affibody‐conjugated RALA peptide, can effectively condense DNA into nanoparticles and target the overexpressed HER3 receptors in breast cancer cells and transfer specific genes. The use of such a recombinant biopolymer may pave the way for the development of sensitive and effective diagnostic and treatment tool for breast cancer.     

水溶液中碳纳米管的表面增强拉曼光谱研究

合成了碳纳米管和金纳米颗粒的复合物, 测量了水溶液相中复合物的表面增强拉曼光谱, 结果表明, 碳纳米管的巯基化修饰可以提高碳纳米管与金纳米颗粒复合的效率, 随着金纳米颗粒负载量的增加, 碳纳米管的拉曼信号逐渐增强. 加入己二胺分子可以减小金纳米颗粒之间的距离使表面增强效应更显著, 碳纳米管的拉曼光谱得到进一步的增强. 还可进一步在复合体系中加入对巯基苯胺和罗丹明B等小分子拉曼探针, 利用金纳米颗粒的表面增强效应, 这种多元复合体系有望作为多通道拉曼成像探针材料.     

Droplet formation via flow-through microdevices in Raman and surface enhanced Raman spectroscopy--concepts and applications

This review outlines concepts and applications of droplet formation via flow-through microdevices in Raman and surface enhanced Raman spectroscopy (SERS) as well as the advantages of the approach. Even though the droplet-based flow-through technique is utilized in various fields, the review focuses on implementing droplet-based fluidic systems in Raman and SERS as these highly specific detection methods are of major interest in the field of analytics. With the combination of Raman or SERS with droplet-based fluidics, it is expected to achieve novel opportunities for analytics. Besides the approach of using droplet-based microfluidic devices as a detection platform, the unique properties of flow-through systems for the formation of droplets are capitalized to produce SERS active substrates and to accomplish uniform sample preparation. Within this contribution, previous reported applications on droplet-based flow-through Raman and SERS approaches and the additional benefit with regard to the importance in the field of analytics are considered.     

Development of bioorthogonal SERS imaging probe in biological and biomedical applications

Living-cell imaging demands high specificity,sensitivity,and minimal background interference to the targets of interest.However,developing a desirable imaging probe that can possess all the above features is still challenging.The bioorthogonal surface-enhanced Raman scattering(SERS) imaging has been recently emerged through utilizing Raman reporters with characteristic peaks in Raman-silent region of cells(1800-2800 cm~(-1)),which opens a revolutionary avenue for living-cell imaging with multiplexing capability.In this review,we focus on the recent advances in the technology development and the biological and biomedical applications of the living-cell bioorthogonal SERS imaging technique.After introduction of fundamental principles for bioorthogonal tag or label,we present applications for visualization of various intracellular components and environment including proteins,nucleic acids,lipids,pH and hypoxia,even for cancer diagnosis in tissue samples.Then,various bioorthogonal SERS imaging-guided thera py strategies have been discussed such as photothera py and surge ry.In conclusion,this strategy has great potential to be a flexible and robust tool for visualization detection and diseases diagnosis.