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In Vivo imaging characterization and anticancer efficacy of a novel HER2 Affibody and Pemetrexed conjugate in lung Cancer model
摘要： Introduction: In this study, a new agent consisting of HER2-specific affibody ZHER2:V2 and chemotherapy drug pemetrexed was synthesized to develop a new targeted drug. Its biological characteristics and anticancer efficacy were assessed in cells level and xenografts models by radiolabeling with technetium-99m. Methods: After the ZHER2:V2-pemetrexed conjugate was synthesized, radiolabeling of the conjugate was performed using its C-terminal 4 amino acids (Gly-Gly-Gly-Cys) as the chelating moiety. The radiochemical yield of the [99mTc]Tc-ZHER2:V2-pemetrexed was identified by instant thin-layer chromatography (ITLC). Stability of the radiolabeled conjugate was investigated both in vitro and in vivo. In vitro binding affinity and cell internalization study of the probe were performed in A549 cells (HER2-positive). Tumor uptake was evaluated by in vitro uptake assay in A549 cells and H23 cells (HER2-negative), and by in vivo biodistribution and SPECT imaging in A549 and H23 tumor-bearing mice. The antitumor efficacy of the ZHER2:V2-pemetrexed conjugate was evaluated in cells and xenograft models. Results: The ZHER2:V2-pemetrexed was successfully synthesized and conjugated with technetium-99m, and acquired the radiochemical yield of 97.0 ± 0.3%. The stability of [99mTc]Tc-ZHER2:V2-pemetrexed was good in both physiological saline and human serum. The radiolabeled agent displayed excellent HER2-binding specificity and affinity in vitro ， and was gradually internalized into the cells. Biodistribution study revealed obvious tumor uptake in A549 xenografts (percentage injected dose per gram, 2.6 ± 1.0 %ID/g at 4 h postinjection), while the uptake in HER2-negative H23 tumors was much lower (0.2 ± 0.1 %ID/g at 4 h postinjection, P < 0.01). SPECT imaging exhibited an intensity in the A549 xenograft which could be blocked by excess ZHER2:V2-pemetrexed. Treatment with ZHER2:V2-pemetrexed significantly impaired the tumor growth (P < 0.05), with less weight loss than pemetrexed. Conclusion: [99mTc]Tc-ZHER2:V2-pemetrexed showed desirable property and HER2-specificity. The ZHER2:V2-pemetrexed conjugate could inhibit tumor growth of HER2-positive lung adenocarcinoma and may have the potential to become a targeted drug for lung cancer. Advances in knowledge and implications for patient care: The compound described herein performs HER2-targeting with favorable anticancer efficacy and offers the potential of novel targeting strategies for further tumor therapy.
关键词： pemetrexed,lung cancer,radionuclide imaging,HER2
更新于2025-09-23 15:21:21
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European Microscopy Congress 2016: Proceedings || Studying membrane proteins in intact cells using nanoparticle labels and liquid-phase electron microscopy
摘要： Cells have receptor proteins in their plasma membranes ‘listening’ to chemical signals from the outside world. These signals consist of ligands, small molecules that bind specifically to a receptor. But how those signals are interpreted and lead to decisions is incompletely understood mainly on account of limitations of present analytical methods. It is typically extremely difficult to directly see how endogenously expressed individual proteins respond to ligand binding in the intact cell, which can lead, for example, to the formation of protein complexes triggering signaling processes. Much knowledge about cellular function has been obtained via biochemical methods but these analyze pooled material from many thousands of cells and the knowledge is thus based on population averages. But we need to look at the individual cell in order to understand the fundamentals of how a cell interprets a signal. Studying membrane proteins at the nanoscale in intact eukaryotic cells is now possible using liquid-phase scanning transmission electron microscopy (STEM) [1, 2]. The key step is to specifically label the proteins of interest in a one-to-one ratio with small probes combined with nanoparticles, for example, gold nanoparticles or quantum dots. Cells in liquid are then placed in a microfluidic chamber enclosing the sample in the vacuum of the electron microscope, and are imaged with STEM. It is not always necessary to enclose the cells in the microfluidic chamber. For some studies, it is sufficient to obtain information from the thin outer regions of the cells, and those can be imaged with high resolution using environmental scanning electron microscopy (ESEM) with STEM detector [3]. Liquid-phase STEM was used to explore the formation of the epidermal growth factor HER2 at the single-molecule level in intact SKBR3 breast cancer cells in liquid state [4]. HER2 is a membrane protein and plays an important role in breast cancer aggressiveness and progression. Data analysis based on calculating the pair correlation function from individual HER2 positions revealed remarkable differences in functionality between different cellular regions, and between cells with possible relevance for studying cancer metastasis and drug response.
关键词： quantum dots,STEM,ESEM,whole cells,liquid-phase,EGFR,HER2
更新于2025-09-23 15:21:21
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Immunomagnetic bead-based bioassay for the voltammetric analysis of the breast cancer biomarker HER2-ECD and tumour cells using quantum dots as detection labels
摘要： An electrochemical magnetic immunosensing strategy was developed for the determination of HER2-ECD, a breast cancer biomarker, and breast cancer cells in human serum. A sandwich assay was performed on carboxylic acid-functionalized magnetic beads (MBs) using a screen-printed carbon electrode (SPCE) as transducer surface. The affinity process was detected using electroactive labels; core/shell streptavidin-modified CdSe@ZnS Quantum Dots (QDs). Cd2+ ions, released from the QDs, were determined by differential pulse anodic stripping voltammetry (DPASV). An assay time of 90 min, with an actual hands-on time of about 20 min, a linear range between 0.50–50 ng·mL?1 of HER2-ECD and a limit of detection of 0.29 ng·mL?1 were achieved. Analysis of live breast cancer cells was also performed using the optimized assay. Breast cancer cell lines SK-BR-3 (a HER2-positive cell line), MDA-MB-231 (a HER2-negative cell line) and MCF-7 (a cell line with low HER2 expression) were tested. The selectivity of the assay towards SK-BR-3 cells was confirmed. A concentration-dependent signal that was 12.5× higher than the signal obtained for the HER2-negative cells (MDA-MB-231) and a limit of detection of 2 cells·mL?1 was obtained.
关键词： Breast cancer,Electrochemical immunoassay,Quantum dots,Cancer cells,Magnetic beads,HER2-ECD
更新于2025-09-23 15:19:57
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Quantitative analyses of amount and localization of radiosensitizer gold nanoparticles interacting with cancer cells to optimize radiation therapy
摘要： Previous studies showed that gold nanoparticles (AuNPs) are useful radiosensitizers which optimize radiation therapy under low-dose radiation. However, the mechanisms of AuNP radiosensitization, including the amount and localization of the AuNPs interacting with cancer cells, has not yet been quantified. To answer these questions, we prepared AuNPs conjugated with anti-human epidermal growth factor receptor type 2 (HER2) antibody via polyethylene glycol (PEG) chains (AuNP-PEG-HER2ab). AuNP-PEG-HER2ab specifically bound to the HER2-expressing cancer cells and entered the cells via endocytosis. Whether endocytosis of AuNP-PEG-HER2ab occurred had no effect on radiosensitization efficacy by AuNP-PEG-HER2ab in vitro. The radiosensitization efficacy in vitro depended on dose of AuNP-PEG-HER2ab or dose of X-ray. Moreover, AuNP-PEG-HER2ab administrated into tumor-bearing mice was localized to both the periphery of the tumor tissue and near the nuclei in cancer cells in tumor deep tissue. The localization of AuNP-PEG-HER2ab in tumor tissues was important factors for in vivo powerful radiosensitization efficacy.
关键词： Radiation therapy,Radiosensitizer,Low-dose radiation,Gold nanoparticle,HER2
更新于2025-09-23 15:19:57
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Her2-Functionalized Gold-Nanoshelled Magnetic Hybrid Nanoparticles: a Theranostic Agent for Dual-Modal Imaging and Photothermal Therapy of Breast Cancer
摘要： Targeted theranostic platform that integrates multi-modal imaging and therapeutic function is emerging as a promising strategy for earlier detection and precise treatment of cancer. Herein, we designed targeted gold-nanoshelled poly (lactic-co-glycolic acid) (PLGA) magnetic hybrid nanoparticles carrying anti-human epidermal growth factor receptor 2 (Her2) antibodies (Her2-GPH NPs) for dual-modal ultrasound (US)/magnetic resonance (MR) imaging and photothermal therapy of breast cancer. The agent was fabricated by coating gold nanoshell around PLGA nanoparticles co-loaded with perfluorooctyl bromide (PFOB) and superparamagnetic iron oxide nanoparticles (SPIOs), followed by conjugating with anti-Her2 antibodies. Cell-targeting studies demonstrated receptor-mediated specific binding of the agent to Her2-positive human breast cancer SKBR3 cells, and its binding rate was significantly higher than that of Her2-negative cells (P < 0.001). In vitro, the agent had capabilities for contrast-enhanced US imaging as well as T2-weighted MR imaging with a relatively high relaxivity (r2 = 441.47 mM?1 s?1). Furthermore, the Her2 functionalization of the agent prominently enhanced the US/MR molecular imaging effect of targeted cells by cell-specific binding. Live/dead cell assay and targeted photothermal cytotoxicity experiments confirmed that Her2-GPH NPs could serve as effective photoabsorbers to specifically induce SKBR3 cell death upon near-infrared laser irradiation. In summary, Her2-GPH NPs were demonstrated to be novel targeted theranostic agents with great potential to facilitate early non-invasive diagnosis and adjuvant therapy of breast cancer.
关键词： Anti-Her2 antibody,Photothermal therapy,Ultrasound imaging,Magnetic resonance imaging,Theranostic agent,Breast cancer
更新于2025-09-16 10:30:52
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Activated Plasmonic Nanoaggregates for Dark-Field in Situ Imaging for HER2 Protein Imaging on Cell Surfaces
摘要： Dark-?eld microscopy (DFM) based on localized surface plasmon resonance (LSPR) was used for observation of experimental phenomena, which is a hopeful nondamaging and non-photobleaching biological imaging technique. In this strategy, plasma nanoaggregates with stronger scattering e?ciency were formed in the presence of the target, causing a “turn-on” phenomenon, when asymmetry modi?ed AuNPs were introduced as probes with zero LSPR background. First, ?CC probe were designed for the cycloaddition between azide and alkyne Au1 to form AuNP dimers under catalytic action by Cu+, which was obtained from the reduction of Cu2+ by sodium ascorbate. The two kinds of probes were successfully used for the detection of Cu2+ in rat serum. Then, to apply this concept to protein on cells, DNA and antibody were modi?ed on the ?CC probe were proposed for HER2 protein DFM on cells. By designing an aptamer sequence in primer, the rolling circle ampli?cation (RCA) was introduced in HER2 DFM on cells, and the image signal was much brighter than that from no-RCA. The unique design made it easier to discriminate the target signal from background noise in cell DFM. This method might be used in the ?elds of molecular diagnostics and cell imaging.
关键词： click chemistry,rolling circle ampli?cation,localized surface plasmon resonance,AuNPs,HER2 protein,Dark-?eld microscopy
更新于2025-09-16 10:30:52
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Labeling Single Domain Antibody Fragments with Fluorine-18 Using 2,3,5,6-Tetrafluorophenyl 6-[ <sup>18</sup> F]Fluoronicotinate Resulting in High Tumor to Kidney Ratios
摘要： ImmunoPET agents are being investigated to assess the status of epidermal growth factor receptor 2 (HER2) in breast cancer patients with the goal of selecting those likely to benefit from HER2-targeted therapies and monitoring their progress after these treatments. We have been exploring the use of single domain antibody fragments (sdAbs) labeled with 18F using residualizing prosthetic agents for this purpose. In this study, we have labeled two sdAbs that bind to different domains on the HER2 receptor — 2Rs15d and 5F7 — using 2,3,5,6-tetrafluorophenyl 6-[18F]fluoronicotinate ([18F]TFPFN) and evaluated their HER2 targeting properties in vitro and in vivo. The overall decay-corrected radiochemical yield for the synthesis of [18F]TFPFN-2Rs15d and [18F]TFPFN-5F7 was 5.7 ± 3.6% and 4.0 ± 2.0%, respectively. Radiochemical purity of labeled sdAbs was >95%; immunoreactive fractions were about 60% and affinity was in the low nanomolar range. Intracellularly trapped activity from [18F]TFPFN-2Rs15d and [18F]TFPFN-5F7 in HER2-expressing SKOV-3 ovarian and BT474M1 breast carcinoma cells were similar to the sdAbs labeled using the previously validated radioiodination residualizing prosthetic agents N-succinimidyl 4-guanidinomethyl-3-[125I]iodobenzoate ([125I]SGMIB) and N-succinimidyl 3-guanidinomethyl-5-[125I]iodobenzoate (iso-[125I]SGMIB). Intracellular activity was about 2-fold higher for radiolabeled 5F7 compared with 2Rs15d for both 18F and 125I. While tumor uptake of both [18F]TFPFN-2Rs15d and [18F]TFPFN-5F7 was comparable to those for the co-administered 125I-labeled sdAb, renal uptake of the 18F-labeled sdAbs was substantially lower. In microPET images, tumor was clearly delineated in SKOV-3 and BT474 xenograft-bearing athymic mice with low levels of background activity in normal tissues except bladder. These results indicate that the [18F]TFPFN prosthetic group could be a valuable reagent for developing sdAb-based immunoPET imaging agents.
关键词： N-Succinimidyl 4-fluorobenzoate (SFB),2,3,5,6-etrafluorophenyl 6-fluoronicotinate (TFPFN),single domain antibody fragment,immunoPET,fluorine-18,HER2
更新于2025-09-11 14:15:04
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The challenges and opportunities in functional imaging HER2 positive breast cancers
摘要： Breast cancer is the most common malignancy in women and 20‐30% of these cancers overexpress the HER2 protein making them candidates for HER2‐directed therapies. Trastuzumab (Herceptin?), the first HER2‐directed therapy, is a humanized monoclonal antibody that binds to the extracellular domain of HER2 preventing downstream signaling and cell proliferation. Trastuzumab is also an immunologic agent stimulating antibody‐dependent cytotoxicity. Investigators have developed radiolabeled trastuzumab as a PET imaging agent for use in HER2‐positive breast cancer patients. However, its clinical role has yet to be established. In the Journal, Dr. Woo and his colleagues report the use of NOTA as a chelator for 64Cu labeling of trastuzumab and claim more favorable pharmacokinetics than 64Cu DOTA trastuzumab [1]. But is NOTA a better chelator for 64Cu than DOTA? They base their comparison on a report by Paudyal et al. (Cancer Sci 2010; 101: 1045‐1050) in which the uptake of 64Cu‐DOTA‐trastuzumab in the liver was 26.9 ±7.4% ID/g at 24 hours[12], while in contrast, the uptake of 64Cu‐NOTA‐trastuzumab was 5.44 ±1.84%D/g in the liver at 24 hours (Figure 4)[1]. They conclude the difference in liver uptake between the two studies was due to release of 64Cu from the DOTA but not from the NOTA chelate. However, the two studies cannot be compared since they were not performed using the same tumor models. Paudyal et al. (Cancer Sci 2010; 101: 1045‐1050) performed PET imaging on Her2+ non‐small cell xenografts, while Woo et al. performed PET imaging on Her2+ breast cancer xenografts. The difference in liver uptake may simply reflect differential shedding of Her2 antigen to the liver between the two types of tumors. To make their point, Woo et al. should have compared DOTA vs NOTA conjugated trastuzumab in the same tumor model. This is a clear example of confusing the chemical stability of metal chelates with their metabolic clearance in different tumor models.
关键词： PET imaging,breast cancer,HER2,DOTA,trastuzumab,NOTA
更新于2025-09-04 15:30:14