Selective reduction of the interaction of magnetic nanoparticles with leukocytes and tumor cells by human plasma

Carboxymethyl-dextran coated magnetic nanoparticles can interact with viable human cells. The interaction of the nanoparticles is cell-type specific. The addition of human plasma led to a dramatic reduction of magnetically separable leukocytes in comparison to tumor cells. We conclude that low plasma concentrations might support an efficient enrichment of circulating epithelial cells from the peripheral blood of tumor patients.     

2-NBDG Fluorescence Imaging of Hypermetabolic Circulating Tumor Cells in Mouse Xenograft model of Breast Cancer

Objectives

To determine use of 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG) as a tracer for detection of hypermetabolic circulating tumor cells (CTC) by fluorescence imaging.

Procedures

Human breast cancer cells were implanted in the mammary gland fat pad of athymic mice to establish orthotopic human breast cancer xenografts as a mouse model of circulating breast cancer cells. Near-infrared fluorescence imaging of the tumor-bearing mice injected with 2-DeoxyGlucosone 750 (2-DG 750) was conducted to assess glucose metabolism of xenograft tumors. Following incubation with fluorescent 2-NBDG, circulating breast cancer cells in the blood samples collected from the tumor-bearing mice were collected by magnetic separation, followed by fluorescence imaging for 2-NBDG uptake by circulating breast cancer cells, and correlation of the number of hypermetabolic circulating breast cancer cells with tumor size at the time when the blood samples were collected.

Results

Human breast cancer xenograft tumors derived from MDA-MB-231, BT474, or SKBR-3 cells were visualized on near-infrared fluorescence imaging of the tumor-bearing mice injected with 2-DG 750. Hypermetabolic circulating breast cancer cells with increased uptake of fluorescent 2-NBDG were detected in the blood samples from tumor-bearing mice and visualized by fluorescence imaging, but not in the blood samples from normal control mice. The number of hypermetabolic circulating breast cancer cells increased along with growth of xenograft tumors, with the number of hypermetabolic circulating breast cancer cells detected in the mice bearing MDA-MB231 xenografts larger than those in the mice bearing BT474 or SKBR-3 xenograft tumors.

Conclusions

Circulating breast cancer cells with increased uptake of fluorescent 2-NBDG were detected in mice bearing human breast cancer xenograft tumors by fluorescence imaging, suggesting clinical use of 2-NBDG as a tracer for fluorescence imaging of hypermetabolic circulating breast cancer cells.     

Theoretical analysis of ferromagnetic microparticles in streaming liquid under the influence of external magnetic forces

The microsphere based detoxification system (MDS) is designed for high specific toxin removal in extracorporeal blood purification using functionalized microparticles. A thin wall hollow fiber membrane filter separates the microparticle-plasma suspension from the bloodstream. For patient safety, it is necessary to have a safety system to detect membrane ruptures that could lead to the release of microparticles into the bloodstream. A non-invasive optical detection system including a magnetic trap is developed to monitor the extracorporeal venous bloodstream for the presence of released microparticles. For detection, fluorescence-labeled ferromagnetic beads are suspended together with adsorbent particles in the MDS circuit. In case of a membrane rupture, the labeled particles would be released into the venous bloodstream and partly captured by the magnetic trap of the detector.A physical model based on fluidic, gravitational and magnetic forces was developed to simulate the motion and sedimentation of ferromagnetic particles in a magnetic trap. In detailed simulation runs, the concentrations of accumulated particles under different applied magnetic fields within the magnetic trap are shown. The simulation results are qualitatively compared with laboratory experiments and show excellent accordance. Additionally, the sensitivity of the particle detection system is proofed in a MDS laboratory experiment by simulation of a membrane rupture.     

纳米磁性药物靶向动力学模拟

基于Navier-Stokes方程与Maxwell-Ampere理论建立了肿瘤细动脉中血液的非定常脉动模型,在磁场和流场耦合状态下研究引入纳米磁性药物后磁场对血液流场的影响.把离散模型和单相模型耦合,用有限元方法对纳米磁性药物在细动脉中的传输进行动力学模拟.模拟结果表明,磁场对纳米尺度磁性粒子的俘获形式明显不同于对微米尺度粒子,纳米磁性粒子运动轨迹与血液流线基本重合.由于磁性纳米粒子的存在,磁场影响了血液的流场分布,在磁体附近形成涡流区并长期滞留,以此方式实现对磁性药物的俘获.     

In vivo monitoring of multiple circulating cell populations using two-photon flow cytometry

To detect and quantify multiple distinct populations of cells circulating simultaneously in the blood of living animals, we developed a novel optical system for two-channel, two-photon flow cytometry in vivo. We used this system to investigate the circulation dynamics in live animals of breast cancer cells with low (MCF-7) and high (MDA-MB-435) metastatic potential, showing for the first time that two different populations of circulating cells can be quantified simultaneously in the vasculature of a single live mouse. We also non-invasively monitored a population of labeled, circulating red blood cells for more than two weeks, demonstrating that this technique can also quantify the dynamics of abundant cells in the vascular system for prolonged periods of time. These data are the first in vivo application of multichannel flow cytometry utilizing two-photon excitation, which will greatly enhance our capability to study circulating cells in cancer and other disease processes.     

1H NMR Detection of superparamagnetic nanoparticles at 1T using a microcoil and novel tuning circuit

Magnetic beads containing superparamagnetic iron oxide nanoparticles (SPIONs) have been shown to measurably change the nuclear magnetic resonance (NMR) relaxation properties of nearby protons in aqueous solution at distances up to approximately 50 microm. Therefore, the NMR sensitivity for the in vitro detection of single cells or biomolecules labeled with magnetic beads will be maximized with microcoils of this dimension. We have constructed a prototype 550 microm diameter solenoidal microcoil using focused gallium ion milling of a gold/chromium layer. The NMR coil was brought to resonance by means of a novel auxiliary tuning circuit, and used to detect water with a spectral resolution of 2.5 Hz in a 1.04 T (44.2MHz) permanent magnet. The single-scan SNR for water was 137, for a 200 micros pi/2 pulse produced with an RF power of 0.25 mW. The nutation performance of the microcoil was sufficiently good so that the effects of magnetic beads on the relaxation characteristics of the surrounding water could be accurately measured. A solution of magnetic beads (Dynabeads MyOne Streptavidin) in deionized water at a concentration of 1000 beads per nL lowered the T(1) from 1.0 to 0.64 s and the T2 * from 110 to 0.91 ms. Lower concentrations (100 and 10 beads/nL) also resulted in measurable reductions in T2 *, suggesting that low-field, microcoil NMR detection using permanent magnets can serve as a high-sensitivity, miniaturizable detection mechanism for very low concentrations of magnetic beads in biological fluids.     

微流体芯片中的癌症生物物理研究

文章从生物物理的新角度出发,介绍了如何利用微流体技术研究癌症的一系列重大问题,其中包括：构建三维微型结构体,用于在体外模拟和研究肿瘤细胞侵袭组织的细胞生物行为;开发新型微流体芯片,以检测血液中循环肿瘤细胞,并分析将其应用于临床中的可能性。文章还展望了飞秒激光三维直写技术构建肿瘤细胞转移模型的应用前景。     

A sandwiched flexible polymer mold for control of particle-induced defects in nanoimprint lithography

Particle related defects are one of the key concerns for nanoimprint lithography, since the particle can amplify the defect to become much larger than the particle itself. We developed a flexible tri-layer mold for control of particle-induced defects. The mold was composed of a PDMS cushion layer sandwiched between a rigid imprint pattern layer and a plastic polyethylene terephthalate (PET) backplane. The PET foil was used as the backplane of the mold to protect the sticky PDMS surface. The PDMS as a cushion layer could locally deform to conform the shape of substrate due to its high elasticity. The multifunctional epoxysiloxane was used for the formation of an imprint layer because of its insensitivity toward oxygen during curing, high transparency, excellent mechanical strength and high resistance to oxygen plasma after cross-linking. Nanostructures with different geometries and sizes were faithfully duplicated by this mold through a UV-curing imprint process. The particle-induced defectivity was dramatically improved by the deformation of the PDMS cushion layer with a slight external pressure. 500 nm pitch grating structures were successfully imprinted on a microposts array surface, both the top and the intervening bottom portions between the microposts.     

磁免疫和荧光免疫传感器的构建及其在检测甲胎蛋白中的应用

甲胎蛋白(AFP)是常见的肝癌肿瘤标志物,在早期诊断方面起到重要作用。分别设计构建了磁免疫和荧光免疫传感器并将其应用于AFP的定量检测。在磁免疫传感器中,采用免疫磁珠代替传统固相载体,实现了目标物的快速分离;利用标记抗体上的辣根过氧化物酶(HRP)催化底物显色,根据底物吸光度值的大小进行定量检测。构建的AFP检测方法的检出限为3.6 ng·mL-1,线性范围为10~80 ng·mL-1。在荧光免疫传感器中,将碲化镉量子点(CdTe QDs)的荧光作为信号输出,并通过同时将多个CdTe QDs连接在纳米硅球表面实现信号放大,通过测量量子点荧光强度实现定量检测。该方法AFP检出限为4.2 ng·mL-1,线性范围为5~150 ng·mL-1。所设计的两种传感器均具有特异性强、灵敏度高的特点,为AFP的检测提供了新的思路和方法。     

Quantification of blood flow velocity in stenosed arteries by the use of finite elements: an observer-independent noninvasive method

Interventions for peripheral arterial disease should be designed to treat a physiological rather than an anatomic defect. Thus, for vascular surgeons, functional information about stenoses is as important as the anatomic one. In case of finding a stenosis by the use of magnetic resonance angiography, it would be a matter of particular interest to derive automatically and directly objective information about the hemodynamic influence on blood flow, caused by patient-specific stenoses. We developed a methodology to noninvasively perform numerical simulations of a patient's hemodynamic state on the basis of magnetic resonance images and by the means of the finite element method. We performed patient-specific three-dimensional simulation studies of the increase in systolic blood flow velocity due to stenoses using the commercial computational fluid dynamic software package FIDAP 8.52. The generation of a mesh defining the flow domain with a stenosis and some simulation results are shown.     

Detoxification of blood using injectable magnetic nanospheres: A conceptual technology description

We describe injectable magnetic nanospheres as a vehicle for selective detoxification of blood borne toxins. Surface receptors on the freely circulating nanospheres bind to toxins. A hand-held extracorporeal magnetic filter separates the toxin-loaded nanospheres from the clean blood, which is returned to the patient. Details of the technology concept are given and include a state-of-knowledge and research needs.     

Detection of circulating tumor cells via an X‐ray imaging technique

Detailed information on the location and the size of tumor cells circulating through lymphatic and blood vessels is useful to cancer diagnosis. Metastasis of cancers to other non‐adjacent organs is reported to cause 90% of deaths not from the primary tumors. Therefore, effective detection of circulating tumors cells (CTCs) related to metastasis is emphasized in cancer treatments. With the use of synchrotron X‐ray micro‐imaging techniques, high‐resolution images of individual flowing tumor cells were obtained. Positively charged gold nanoparticles (AuNPs) which were inappropriate for incorporation into human red blood cells were selectively incorporated into tumor cells to enhance the image contrast. This approach enables images of individual cancer cells and temporal movements of CTCs to be captured by the high X‐ray absorption efficiency of selectively incorporated AuNPs. This new technology for in vivo imaging of CTCs would contribute to improve cancer diagnosis and cancer therapy prognosis.     

Rapid extraction of genomic DNA from saliva for HLA typing on microarray based on magnetic nanobeads

A series of simplified protocols are developed for extracting genomic DNA from saliva by using the magnetic nanobeads as absorbents. In these protocols, both the enrichment of the target cells and the adsorption of DNA can be achieved simultaneously by our functionally modified magnetic beads in one step, and the DNA–nanobeads complex can be used as PCR templates. HLA typing based on an oligonucleotide array was conducted by hybridization with the PCR products. The result shows that the protocols are robust and sensitive.     

In vivo photoacoustic time-of-flight velocity measurement of single cells and nanoparticles

Optical techniques for in vivo measurement of blood flow velocity are not quite applicable for determination of velocity of individual cells or nanoparticles. Here, we describe a photoacoustic time-of-flight method to measure the velocity of individual absorbing objects by using single and multiple laser beams. Its capability was demonstrated in vitro on blood vessel phantom and in vivo on an animal (mouse) model for estimating velocity of gold nanorods, melanin nanoparticles, erythrocytes, leukocytes, and circulating tumor cells in the broad range of flow velocity from 0.1?mm/s to 14?cm/s. Object velocity can be used to identify single cells circulating at different velocities or cell aggregates and to determine a cell's location in a vessel cross-section.     

Platelet aggregation in the formation of tumor metastasis

Metastasis is the major cause of death from cancer, yet the optimal strategy against it remains uncertain. The pathogenesis of hematogenous metastasis is dynamic and consists of the following steps: 1) detachment of tumor cells from the primary site, 2) invasion into the host's blood vessels, 3) migration in the host's blood stream, 4) transport along the circulation, 5) arrest in or adhesion to the capillary in a distant organ, 6) extravasation, and 7) proliferation within the foreign tissues. A key to successful hematogenous metastasis is tumor survival in the bloodstream because most circulating tumor cells are rapidly destroyed by the shear forces or are attacked by the immune system. Less than 0.01% of these cells result in metastasis. Tumor cell-induced platelet aggregation has been reported to facilitate hematogenous metastasis by increasing the arrest of tumor cell emboli in the microcirculation. Platelet aggregation is also believed to protect tumor cells from immunological assault in the circulation. We have identified Aggrus as a platelet-aggregating factor expressed on a number of human cancers. Because hematogenous metastasis is reduced when neutralizing antibodies or eliminating carbohydrates attenuates Aggrus function, Aggrus's main contribution to hematogenous metastasis of Aggrus-expressing cells, then, is by promoting platelet aggregation. Aggrus could serve as an ideal target for drug development to block metastasis.     

Photoacoustic detection of metastatic melanoma cells in the human circulatory system

Detection of disseminating tumor cells among patients suffering from various types and stages of cancer can function as an early warning system, alerting the physician of the metastatic spread or recurrence of the disease. Early detection of such cells can result in preventative treatment of the disease, while late stage detection can serve as an indicator of the effectiveness of chemotherapeutics. The prognostic value of exposing disseminating tumor cells poses an urgent need for an efficient, accurate screening method for metastatic cells. We propose a system for the detection of metastatic circulating tumor cells based on the thermoelastic properties of melanoma. The method employs photoacoustic excitation coupled with a detection system capable of determining the presence of disseminating cells within the circulatory system in vitro. Detection trials consisting of tissue phantoms and a human melanoma cell line resulted in a detection threshold of the order of ten individual cells, thus validating the effectiveness of the proposed mechanism. Results imply the potential to assay simple blood draws, from healthy and metastatic patients, for the presence of cancerous melanoma providing an unprecedented method for routine cancer screening.     

非线性传输线产生射频脉冲原理研究

介绍了非线性传输线的工作原理和色散特性,给出了用于模拟交叉耦合磁饱和非线性传输线的计算方法。算法基于传输线各节点的时域差分方程组进行步进迭代,其中利用J-A模型描述传输线中NiZn铁氧体磁芯的非线性磁化行为,并模拟了非线性传输线的工作方式。实验获得了中心频率165 MHz的宽带脉冲输出,初步验证了用于产生宽带电磁脉冲的非线性传输线关键技术。     

非线性传输线产生射频脉冲原理研究

介绍了非线性传输线的工作原理和色散特性,给出了用于模拟交叉耦合磁饱和非线性传输线的计算方法。算法基于传输线各节点的时域差分方程组进行步进迭代,其中利用J-A模型描述传输线中NiZn铁氧体磁芯的非线性磁化行为,并模拟了非线性传输线的工作方式。实验获得了中心频率165MHz的宽带脉冲输出,初步验证了用于产生宽带电磁脉冲的非线性传输线关键技术。     

Dynamical properties of magnetized two-dimensional one-component plasma

Molecular dynamics simulation are used to examine the effect of a uniform perpendicular magnetic field on a two-dimensional interacting electron system. In this simulation we include the effect of the magnetic field classically through the Lorentz force. Both the Coulomb and the magnetic forces are included directly in the electron dynamics to study their combined effect on the dynamical properties of the 2D system. Results are presented for the velocity autocorrelation function and the diffusion constants in the presence and absence of an external magnetic field. Our simulation results clearly show that the external magnetic field has an effect on the dynamical properties of the system.     

Site-directed research of magnetic nanoparticles in magnetic drug targeting

The targeting of ferrofluids composed of 20 nm magnetic particles was studied through simulation and animal experiment. The results showed that some magnetic particles were concentrated in the target area depending on the applied magnetic field. Through theoretical analysis, the retention of the magnetic nanoparticles in a target area is due to large magnetic liquid beads formed by the magnetic field.