Ximei Qian

Targeted magnetic iron oxide nanoparticles for tumor imaging and therapy

Magnetic iron oxide (IO) nanoparticles with a long blood retention time, biodegradability and low toxicity have emerged as one of the primary nanomaterials for biomedical applications in vitro and in vivo. IO nanoparticles have a large surface area and can be engineered to provide a large number of functional groups for cross-linking to tumor-targeting ligands such as monoclonal antibodies, peptides, or small molecules for diagnostic imaging or delivery of therapeutic agents. IO nanoparticles possess unique paramagnetic properties, which and generate significant susceptibility effects resulting in strong T2 and T2* contrast, as well as T1 effects at very low concentrations for magnetic resonance imaging (MRI), which is widely used for clinical oncology imaging. We review recent advances in the development of targeted IO nanoparticles for tumor imaging and therapy.

SERS Nanoparticles in Medicine: From Label-Free Detection to Spectroscopic Tagging

Spectroscopic Tagging Lucas A. Lane,† Ximei Qian,† and Shuming Nie*,†,‡ †Departments of Biomedical Engineering and Chemistry, Emory University and Georgia Institute of Technology, Health Sciences Research Building, Room E116, 1760 Haygood Drive, Atlanta, Georgia 30322, United States ‡College of Engineering and Applied Sciences, Nanjing University, 22 Hankou Road, Nanjing, Jiangsu Province 210093, China

Detection of Circulating Tumor Cells in Human Peripheral Blood using Surface-Enhanced Raman Scattering Nanoparticles

The detection and characterization of circulating tumor cells (CTC) holds great promise for personalizing medicine and optimizing systemic therapy. However, low specificity, low sensitivity, and the time consuming nature of current approaches have impeded clinical adoption. Here we report a new method using surface-enhanced Raman spectroscopy (SERS) to directly measure targeted CTCs in the presence of white blood cells. SERS nanoparticles with epidermal growth factor peptide as a targeting ligand have successfully identified CTCs in the peripheral blood of 19 patients with squamous cell carcinoma of the head and neck (SCCHN), with a range of 1 to 720 CTCs per milliliter of whole blood. Our technique may provide an important new clinical tool for management of patients with SCCHN and other cancers.

Surface-enhanced Raman nanoparticle beacons based on bioconjugated gold nanocrystals and long range plasmonic coupling.

We have developed a new class of surface-enhanced Raman scattering beacons (SERS beacons) that can be turned on and off by long-range plasmonic coupling, induced by biomolecular recognition and binding events. The beacons are based on colloidal gold nanocrystals in two sizes (40 and 60 nm) and are prepared by spectral encoding with a Raman reporter molecule, functionalized with thiolated DNA probes, and stabilized and protected by low molecular weight poly(ethylene glycol)s (PEGs). The results show the SERS signal intensities increase by 40-200-fold when the nanoparticle beacons are activated by plasmonic coupling, much higher than the bright-to-dark intensity ratios reported for traditional molecular beacons. Multivalent gold nanoparticles also have exquisite specificity and are able to recognize single-base mismatches or mutations. This class of SERS nanoparticle beacons has novel mechanisms for molecular detection and signal amplification, and its long-range coupling nature raises new opportunities in developing plasmonic probes to detect proteins, cells, and intact viruses.

Physical Chemistry of Nanomedicine: Understanding the Complex Behaviors of Nanoparticles in Vivo

Nanomedicine is an interdisciplinary field of research at the interface of science, engineering, and medicine, with broad clinical applications ranging from molecular imaging to medical diagnostics, targeted therapy, and image-guided surgery. Despite major advances during the past 20 years, there are still major fundamental and technical barriers that need to be understood and overcome. In particular, the complex behaviors of nanoparticles under physiological conditions are poorly understood, and detailed kinetic and thermodynamic principles are still not available to guide the rational design and development of nanoparticle agents. Here we discuss the interactions of nanoparticles with proteins, cells, tissues, and organs from a quantitative physical chemistry point of view. We also discuss insights and strategies on how to minimize nonspecific protein binding, how to design multistage and activatable nanostructures for improved drug delivery, and how to use the enhanced permeability and retention effect to deliver imaging agents for image-guided cancer surgery.

Abstract 1460: Quantification of circulating tumor cells using multiplexed Surface-Enhanced Raman Scattering Nanoparticles.

Background: Detection, quantification, and molecular characterization of CTCs may aid physicians in treatment decision making and optimizing. However, the major challenge in the detection and characterization of CTCs is the lack of a highly specific and sensitive assay to isolate and identify the rare population of CTCs in complex blood samples. Moreover, current approaches for CTC detection including CellSearch (FDA approved CTC detection method) which primarily relays on single biomarker are suboptimal because of tumor heterogeneity. Here we develop a multiplexed single-tube assay for directly detection of CTCs in blood sample using two targeted Surface-Enhanced Raman scattering (SERS) nanoparticles. Methods: SERS nanoparticles (60 nm) were prepared by spectral encoding with a Raman reporter molecule (QSY or BHQ) and then stabilized and protected by low molecular weight polyethylene glycols (PEGs). A targeting ligand, epidermal growth factor (EGF) peptide or epithelial cell adhesion molecule (EpCAM) antibody, was covalently conjugated to PEG layer of SERS nanoparticle. The functionalized EGF- or EpCAM- SERS nanoparticle was able to identify CTCs in the peripheral blood with high specificity and affinity. Results: We collected and examined fresh blood samples from 20 SCCHN patients with various histologies and successfully detected positive SERS signals that were greater than background in 9 of 12 (75%) of patients. This technique was demonstrated to be capable of identifying rare CTCs by two biomarkers simultaneously, with a range of 5-720 cells per ml of whole blood. Blood samples from 3 normal controls did not show any CTCs. In one patient who had 0 count of CTC detected by EGFR-targeting SERS tag showed 170 CTCs/ml of blood detected by EpCAM-targeting SERS tags. Conclusion: Special design of the two SERS nanoparticles, distinguished with unique spectral signatures and bio-conjugation of EGF or EpCAM antibody as targeting molecule, allowed simultaneous detection of epidermal growth factor receptor (EGFR) or EpCAM on CTCs in one blood sample. Our technique based on two biomarkers rather than those relayed on single biomarker may improve CTC assessment in clinical settings. (This research was supported by the National Cancer Institute award 5 P50 CA128613 and U54CA119338-04). Citation Format: Xu Wang, Ximei Qian, Jonathan J. Beitler, Zhuo (Georgia) Chen, Fadlo R. Khuri, Melinda M. Lewis, Hyung J. Shin, Shuming Nie, Dong M. Shin. Quantification of circulating tumor cells using multiplexed Surface-Enhanced Raman Scattering Nanoparticles. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1460. doi:10.1158/1538-7445.AM2013-1460

S028) Measurement of Circulating Tumors Cells in Squamous Cell Carcinoma of the Head and Neck and Patient Outcomes

radiation treatment schedule remains unknown. The National Comprehensive Cancer Network (NCCN) guidelines recommend both hypofractionated radiotherapy (HFX) or CFX. We compared overall survival (OS) and treatment patterns among patients treated with HFX versus CFX for ESGC using a large national database. Materials/Methods: We identified patients diagnosed with stage I-II (cT12N0M0) glottic cancer from 2004-2013 in the National Cancer Data Base. Patients were treated with either HFX (2.25Gy/fraction to 63-65.25Gy) or CFX (2.0Gy/fraction to 66-70Gy). Multivariable logistic regression was used to determine factors associated with the receipt of HFX versus CFX. OS of patients receiving HFX versus CFX was compared using the logrank test, multivariable Cox proportional-hazards regression, and propensity-score matching. Results: 4,030 patients (39.5%) received HFX and 6,182 patients (60.5%) received CFX. Predictors for receipt of HFX included cT1 disease, recent year of diagnosis, and treatment at academic and highervolume centers (all P<0.001). Patients treated with HFX increased from 22.1% in 2004 to 58.0% in 2013. HFX was associated with improved OS compared with CFX on univariable (5-year OS, 77.0% vs 73.5%; logrank P<.001) and multivariable analysis (HR 0.89, PZ.01), a finding confirmed on propensity-score matching. On subset analyses, an improvement in OS was borderline significant for patients with cT1 disease (5-year OS, 78.2% vs 76.0%; log-rank PZ.053) and statistically significant for patients with cT2 disease (5-year OS, 70.8% vs 64.5%; log-rank PZ.02). Conclusions: HFX is associated with improved survival compared to CFX among patients treated with definitive radiotherapy for ESGC, particularly among patients with cT2 disease. HFX utilization increased over the study period; however, 40% of patients in our cohort did not receive HFX in the most recent year of our analysis.

Abstract 4829: Evaluation prognostic significance of circulating tumor cells (CTCs) using multiplexed gold nanoparticles in patients with head and neck cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Detection, quantification, and molecular characterization of CTCs may aid physicians in optimizing treatment. However, current approaches for CTC detection, including CellSearch (FDA approved CTC detection method) rely on a single biomarker, whic may not be appropriate for heterogeneous tumors. Methods: We developeda multiplexed single-tube assay using two targeting Surface-Enhanced Raman scattering (SERS) nanoparticles for directdetection of CTCs in blood sample. The two SERS nanoparticles were distinguished with unique spectral signatures were targeted for simultaneous detection of the biomarkers epidermal growth factor receptor (EGFR) and epithelial cell adhesion molecule (EpCAM). The correlation of CTC level detected by two biomarkers with clinical characteristics was evaluated and compared. Results: CTCs were present in 60.9% of patients (39 of 64 patients) with a range of 5-1751 cells per ml of whole blood. CTC levels detected by EGFR-tag (CTC-EGFR) and EpCAM-tag (CTC-EpCAM) were strongly cross-correlated (P<0.001). CTC levels detected by both biomarkers correlated significantly with the presence of disease (P=0.005 and P<0.0001 for CTC-EGFR and CTC-EpCAM, respectively). In univariate analysis, CTC level detected by both biomarkers associated significantly with disease free survival (hazard ratio: 2.135; 95%CI: 1.028 to 4.435; P=0.042 for CTC-EGFR, and hazard ratio: 6.028; 95%CI: 2.294 to 12.84; P=0.0003 for CTC-EpCAM). In multivariate analysis, CTC level detected by EpCAM-tag but not by EGFR-tag was significant prognostic factor for disease free survival (hazard ratio: 6.771; 95%CI: 2.069 to 22.151; P=0.0016). Thus, the CTC level detected by EpCAM-tag has stronger association with disease free survival than that by EGFR-tag. Conclusions: We have developed two SERS nanoparticles, allowed the simultaneous detection of EGFR or EpCAM on CTCs from one blood sample. CTC levels detected by both biomarkers associated significantly with disease statue and disease free survival. CTC level detected by EpCAM-tag but not by EGFR-tag was significant prognostic factor for disease free survival. Our technique based on two biomarkers, compared with those relying on a single biomarker, may improve CTC assessment in clinical settings. (This research was supported by the National Cancer Institute awards 5 P50 CA128613 and U54CA119338-04). Citation Format: Xu Wang, Ximei Qian, Jonathan J. Beitler, Jun Zhang, Hyunseok Kang, Zhuo (Georgia) Chen, Kelly R. Magliocca, Xiaojing Wang, Zhengjia Chen, Shuming Nie, Dong M. Shin. Evaluation prognostic significance of circulating tumor cells (CTCs) using multiplexed gold nanoparticles in patients with head and neck cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4829. doi:10.1158/1538-7445.AM2014-4829

Abstract 1155: Quantification of circulating tumor cells in peripheral blood using EGFR-targeting gold nanoparticles

Background: The prognostic significance of circulating tumor cells (CTCs) in patients with squamous cell carcinoma of the head and neck (HNSCC) is of great clinical interest. HNSCCs are overwhelmingly EGFR positive. Objective: Our goal was to pilot nanotechnology to identify CTCs in human xenograft mouse model as a prelude to clinical use. Methods: Surface-enhanced Raman Scattering (SERS)-gold nanocrystals (60 nm) were prepared by spectral encoding with a Raman reporter molecule and then stabilized and protected by low molecular weight polyethylene glycols (PEGs). Heterofunctional PEG was covalently conjugated to epidermal growth factor (EGF) peptide, a ligand that binds to EGFR with high specificity and affinity. To identify CTC in normal mouse blood, varying quantities of Tu212 cells were added into mice blood samples, then our nanoparticle was added to the sample and the presence of Tu212 cells were detected in vitro using laser excitation of the Raman reporters. Different types of cancer cells with variable EGFR expression were also examined in blood to confirm the specificity of the EGF-targeted SERS nanoparticles. Normal mouse blood served as the experimental control to account for non-specific binding. Results: The cellular uptake of the EGFR-targeting nanoparticle in high EGFR expression Tu212 cells was about 10-fold higher than that in low EGFR expression H460 cells in vitro, suggesting a specific EGFR targeting of the gold nanoparticle. Tu212 cells could be selectively labeled and quantified (5.6 × 10 4 nanoparticles/cell) at a concentration of approximately 10 cells per ml of normal mouse blood. Comparing the labeling of Tu212 cells by our nanoparticles with H460 cells in the mouse blood samples showed that the signal intensity in Tu212 cells was about 9-fold higher than that in H460 cells ((3750 ± 230 vs. 350 ± 120, respectively, p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1155.