RU2587902C2 - Detection, measurement and imaging of cells, such as cancer, and other biological substances using targeted nanoparticles and magnetic properties thereof - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely, to the means of detecting and measuring cells or substances via targeted nanoparticles. Apparatus comprises a magnetizing subsystem for magnetization nanoparticles which are associated with one or more specified types of cancer cells, or biological substances in a patient, a sensor subsystem to detect the residual magnetic field in the region of interest of the patient after the unbound nanoparticles back to the random magnetization, and for a time nanoparticles of transition related to the random homogeneous magnetization, and the management and analysis of the residual magnetic field to detect, measure or determine the location of one or more specific types of cancer cells, or biological substances in a patient. Method uses a device and includes placing a plurality of targeted nanoparticles to the patient, wherein each of the plurality of targeted nanoparticles include paramagnetic nanoparticle conjugated to a targeting agent that binds to one or more specified types of cancer cells, or biological substances, the use of the magnetizing subsystem sensor subsystem and control system and analysis.

EFFECT: use of the invention allows early detection of tissue damage.

21 cl, 27 dwg

Description

[0001] FIELD OF THE INVENTION

The present invention relates to the in vivo detection and measurement of cells or substances using targeted nanoparticles and measurements of magnetic relaxation, and is particularly suitable for the detection and measurement of cancer cells in humans.

[0002] BACKGROUND OF THE INVENTION

Early detection of the disease maximizes the likelihood of successful treatment and recovery. Moreover, earlier detection and determination of the localization of the disease allows targeted therapy in relation to the site of the disease, optimizing the effectiveness of the treatment. Using an appropriate detection device, treatment can be monitored, further increasing the effectiveness of the drugs used or other forms of therapy. The ability to target specific diseases can also improve treatment outcomes. Early detection and localization of cancer, the second leading cause of death in the United States, can improve patient outcomes. All the most common methods used for clinical purposes for detecting cancer are non-specific, that is, they cannot distinguish between cancerous or benign tumors, and none of them leads to 100% detection accuracy. All available methods have flaws and weaknesses, leading to high levels of false diagnosis and too low levels of reliable diagnosis, which together leads to increased levels of mortality. The most common clinical methods currently available are: (1) X-ray mammography, (2) magnetic resonance imaging (MRI), and (3) ultrasound scanning with the additional option (4) positron emission tomography (PET), if it is available.

[0003] The X-ray attenuation measurement provides information on the density of the intermediate medium and is the FDA approved and most common device used to detect various forms of the disease and, in particular, cancer. He is also responsible for many false negative and false positive results. Cancers of an early stage can be detected, but without specificity as to whether they are benign or cancerous. Artifacts can be caused by healthy tissue and produce false positive results. Although the dose is low, there is an increasing problem regarding exposure to x-rays and radiation in general.

In general, the number of false positive results in x-ray imaging of cancer remains high, and the x-ray method cannot detect tumors in the early stages.

[0004] Ultrasound is used to provide a second method for imaging tumors. Ultrasound has excellent contrast resolution, but suffers from weakened spatial resolution compared to x-rays and other imaging techniques. Ultrasound is not currently approved by the FDA as the primary method for screening cancer, but is usually used as follow-up medical examination to examine any abnormalities found during standard procedures. This is a method often used to confirm questionable areas in radiographs of breast and ovarian cancer.

[0005] MRI is used for follow-up medical observation on potential problem areas observed during x-ray scans; however, the high cost of MRI scanning often discourages its use. MRI can detect small abnormalities in the tissue, and is also useful in determining whether cancer has metastasized. Dynamic Contrast Enhanced MRI (DCE) potentially distinguishes between benign and cancerous tumors, but yields a series of false positive results. The high cost of MRI limits its use as a screening method. Magnetic nanoparticles are often used as contrast agents in MRI imaging of cancer, and this is a recognized protocol that provides standards for the administration of such nanoparticles. Intravascular contrast agents for MRI at a dose of 2 mg / kg by weight of the nanoparticles were used to detect metastatic lesions.

[0006] Due to the importance of early detection of the disease, there are a variety of other techniques that are currently being studied for imaging. These include scintimammography using PET (positron emission tomography) or SPECT (single-phonon emission computed tomography), impedance tomography and various forms of RF imaging.

[0007] Earlier detection of lesions while they are still localized is key because the cure rate of many forms of cancer detected early is about 100%. Existing visualization methods often do not determine lesions until significant growth has occurred. There is ongoing research into alternative methods, including MRI, PET, ultrasound, scintigraphy, and other methods. Currently, none of these methods has specificity for the type of tumor using differences in tissue properties between cancerous and noncancerous tissue. In particular, a new approach is definitely needed, not based on radiation, or very expensive procedures and offering very early detection of tumors. The present invention provides new opportunities for the detection of cancer in vivo.

[0008] SUMMARY OF THE INVENTION

This application relates to the following applications, each of which is incorporated herein by reference: US 61/259011, filed November 6, 2009; 61/308897, filed February 27, 2010; 61/314392, filed March 16, 2010; 61/331816, filed May 5, 2010; 61/361998, filed July 7, 2010; each of which is incorporated herein by reference.

[0009] The present invention provides devices and methods for detecting cells or substances, such as cancer cells in tissue. The system comprises a magnetic system including a magnetic field generator that acts on a subject tissue by a known magnetic field to magnetize targeted paramagnetic nanoparticles bound to cells or a substance of interest; and including a sensitive magnetic sensor that can detect a residual magnetic field as the magnetization of the nanoparticles decays. An illustrative magnetic system comprises a superconducting quantum interference sensor, comprising a magnetic pulse generator adapted to apply a uniform magnetizing pulsed field to a cancerous tissue of a patient placed on a measurement platform; and a residual magnetic field detector adapted to detect and visualize the residual magnetic field produced by the acting pulsed field. The magnetic pulse generator may include a pair of Helmholtz coils. The residual magnetic field detector may include a set of gradiometers. Another illustrative magnetic system includes an atomic magnetometer and a set of atomic gradiometers — very sensitive magnetic field sensors that can be used to measure very weak magnetic fields based on the Larmor precession of atoms in a magnetic field. In some embodiments, the atomic magnetometer comprises a chipset containing a small cavity containing an atomizer cell. This evaporation cell contains Rb atoms and is optically pumped by a circularly polarized laser beam. Atoms undergo a Larmor precession, and the frequency of this precession causes a change in the refractive index of the vapor in response to the acting magnetic field. The second laser can be used as a measurement field for a given change in refraction, using a set of gratings to measure changes in the interference pattern as the acting magnetic field changes. Evaporation cells can be separate or ordered in a gradiometer configuration to measure field changes as a function of distance.

[0010] The method according to the present invention includes providing a magnetic system; the introduction of a variety of targeted (e.g., labeled with an antibody) paramagnetic nanoparticles in a subject for specific binding to cancer cells or other cells or a substance of interest; exposure to a known (eg, homogeneous) magnetizing pulsed field for magnetizing nanoparticles in the tissue of a subject; and detecting a residual magnetic field of magnetized nanoparticles, thereby providing visualization of the nanoparticles associated with the cancerous tissue of the patient. The targeted paramagnetic nanoparticle may comprise a magnetic core coated with a biocompatible coating to which at least one specific antibody is attached. For example, the magnetic core may contain ferromagnetic material, such as iron oxide. Examples of suitable targeting agents, such as antibodies, are described below.

[0011] A BRIEF DESCRIPTION OF GRAPHIC MATERIALS

The accompanying graphic materials, which are included in and form part of the description, illustrate the present invention and, together with the description, describe the present invention. In graphic materials, the same elements are denoted by the same numbers.

[0012] FIG. 1 is a schematic illustration of an example preparation of tissue of a subject for measurement according to the present invention.

Figure 2 (a, b, c, d) provides a schematic illustration of a measurement example according to the present invention.

Figure 3 is a schematic illustration of measurements from the process described in connection with Figure 2.

Figure 4 is a schematic illustration of a device suitable for use in the present invention.

5 is a schematic illustration of a sample device using magnetic sensors of a superconducting quantum interference sensor (SQUID).

6 is a schematic illustration of a sample SQUID-sensor device that can be used for research on human cancer.

7 is a schematic illustration and photograph of an atomic magnetometer for measuring a weak field.

Fig. 8 is a schematic illustration of a biocompatible coated magnetic nanoparticle and attached antibodies to target specific cells.

Fig. 9 is an image of the number of He2 sites per cell, calculated by comparison with a range of microspheres with known binding abilities. Figure 10 is an illustration of the magnetic moments of two cell lines of breast cancer, MCF7 / HER218 and MDA-MB-231, measured as a function of time after incubation with antibodies to HER2 / neu and nanoparticles.

[0013] FIG. 11 is an illustration of the magnetic moments of cell samples, measured as a function of cell number by double dilution by pipetting. 12 is an illustration of an ampoule phantom with MCF7 cells, left 2E + 06, right = 1E + 06 cells.

Fig.13 is a photograph of a nude mouse under the SQUID system. Fig.14 contains plots of the reliability of the positions obtained from tumors in mice. 15 is an illustration of magnetic contour lines observed at 35 different measurement sites.

Fig.16 is an illustration of the temporal dynamics of measurements for two mice and both tumors of each mouse.

17 is an illustration of the results of these measurements and shows very good agreement with in vivo measurements on a live mouse.

Fig. 18 is an illustration of a 2-dimensional 95% confidence limit for the locations of two tumors superimposed on actual mouse tumors.

Fig. 19 is a photograph of the histology of tumors after isolation.

FIG. 20 is an illustration of ovarian cancer showing tumor growth on the ovary.

[0014] FIG. 21 is a photograph of a full-sized ovarian phantom placed under a SQUID sensor device at a distance that will be typical of a patient subject.

Fig.22 is an illustration of the results of sensitivity studies for live ovarian cells inserted into the phantom shown in Fig.21.

23 is an illustration of confirmation of antibody sites for these cells by flow cytometry.

24 is an illustration of magnetic moments from magnetic nanoparticles (from Ocean Nanotech) attached to human ovarian cancer tumors in a live mouse.

25 is a photograph of a mouse used to confirm that the SQUID-sensor method works in vivo with magnetic contour fields from a mouse. Fig is a graph of measuring the magnetic moment of the SQUID-sensor system as a function of time for incubation of attached magnetic nanoparticles to lymphoma cell lines.

Fig. 27 is an illustration of flow cytometry measurements of RS-cells from the lymphatic system when determining the number of sites available for nanoparticles and detection by SQUID sensors.

Fig. 28 is a histological section from a patient with Hodgkin's disease.

[0015] MODES FOR CARRYING OUT THE INVENTION AND INDUSTRIAL APPLICABILITY

The present invention is described in the context of various illustrative embodiments and applications. In some parts of the description, the expression "detection" is used for brevity; the present invention can provide detection of the presence of cells or substances, measuring the number of cells or quantity of a substance, determining the location of cells or substances, determining the change or rate of change in the previous one, and similar determinations, all of which are included in the expression "detection".

[0016] A simplified example of measuring magnetic relaxation according to the present invention is first described. Figure 1 is a schematic illustration of an example of obtaining tissue of a subject for measurement according to the present invention. The illustrations in the figure are very simplified and are intended only for ease of explanation and are not intended to display the actual shapes, sizes, proportions or complexities of the actual materials involved. A portion of tissue 11, for example, the organ to be examined, or a known or suspected tumor site, contains several cells of type of interest (shown in the figure as circles with “V” shaped perimeter structures) and several cells of other types (shown as ovals in the figure with rectangular perimeter structures). A plurality of magnetic nanoparticles 12 are provided, shown as small circles in the figure. A number of targeting molecules 13 are also provided, shown as small triangles in the figure. Nanoparticles and targeting molecules combine (or conjugate) to form targeted nanoparticles 14.

Targeted nanoparticles can then be introduced into tissue 15. Cells of the type of interest have binding sites or other affinities for the targeting molecule illustrated in the figure by "V" -like structures around the perimeter of such cells.

Targeting molecules attach to cells of interest of the type illustrated in the figure by triangular targeting molecules located within "V'-shaped structures. In general, each cell will have a large number of such binding or affinities. Other types of cells do not have such binding or affinities are illustrated in the figure by ovals without attached targeted nanoparticles. Targeted nanoparticles that do not bind to cells and remain free in the resulting sample are llyustrirovany the figure small circles with attached triangles that are not connected with any particular cell.

[0017] Figure 2 (a, b, c, d) is a schematic illustration of an illustrative measurement according to the present invention. In Fig. 2a, the fabric is as in Fig. 1, with the addition of arrows near each nanoparticle. The arrows represent the magnetization of each nanoparticle, and indicate that the magnetization of the nanoparticles in the tissue is random (in the figure, the arrows are shown in one of four directions just for ease of illustration; in practice, the magnetization can have any direction).

[0018] In FIG. 2b, an external magnetic field is applied (represented by an outlined arrow in the lower right corner of the figure). The magnetization of the nanoparticles in response to the acting magnetic field is now uniform, shown in the figure using all the magnetization arrows pointing in one direction.

[0019] FIG. 2c illustrates tissue shortly after magnetic field is removed. Non-cell nanoparticles are free to move in Brownian motion, and their magnetization quickly returns to arbitrary, shown in the figure by the magnetization arrows of unbound nanoparticles, showing in different directions. The nanoparticles bound to the cells, however, are restrained from such physical movement and, therefore, their magnetization remains essentially the same as in the presence of an acting magnetic field.

[0020] Fig. 2d illustrates the obtained sample over a longer period of time after the exposure to the magnetic field. The magnetization of bound nanoparticles has now also returned to arbitrary.

[0021] FIG. 3 is a schematic illustration of measurements from a process described in connection with FIG. 2. The magnetic field is shown as a function of time in a simplified representation for ease of illustration; in fact, in practice, the units, scales and waveforms can be different and more complex. At the beginning of the process, which corresponds to the state of Fig. 2a, the magnetization of the nanoparticles is arbitrary and is affected by an external magnetic field. From this point in time, the magnetization of the nanoparticles is uniform, which corresponds to the state of Fig.2b. The magnetic field can be ignored for a short period of time until the unbound nanoparticles return to random magnetization, which corresponds to the state of FIG. 2c. Then you can measure the magnetization as the transition of the bound nanoparticles from homogeneous to random magnetization, which corresponds to the state of Fig.2d. The magnetization characteristics when measured from the state of FIG. 2c to the state of FIG. 2d are related to the number of bound nanoparticles in the sample and, therefore, to the number of cells of the type of interest in the sample.

[0022] FIG. 4 is a schematic illustration of a device suitable for use in the present invention. The platform 41 for the subject is configured to place the subject in a coordinated position with respect to the rest of the device. The magnetizing system 42, for example, Helmholtz coils, is mounted relative to the platform for the subject so that the magnetizing system can act on the sample with a magnetic field. The magnetic sensor system 43 is mounted relative to the platform for the subject so that it can detect small magnetic fields associated with magnetized nanoparticles. The system is controlled and data from the sensor is analyzed using a control and analysis system 44; for example, a computer with the appropriate program.

[0023] FIG. 5 is a schematic illustration of a sample device using magnetic sensors of a superconducting quantum interference sensor (SQUID). A Dewar tank 51 for liquid helium at the top of the figure maintains the temperature of the SQUID sensors. 2nd order axial SQUID gradiometers are contained in a white protrusion 52 passing through the support frame 53. There are seven gradiometers contained in this illustrative protrusion; one in the center and 6 in a circle with a radius of 2.15 cm. Each gradiometer is inductively connected to a low-temperature SQUID. Two ring coils 54 form a Helmholtz pair, which can provide a magnetizing pulsed field for nanoparticles. The uniform field produced by these coils can vary, but typically is 40-50 Gauss, and the pulse length is typically 300-800 ms. In this example, the wooden frame supports SQUID and a measurement platform, as well as magnetizing coils. Non-magnetic frame system includes a 3-dimensional platform 55, which can be performed without metal components, for example, plastic. The upper two black handles control x-y movements of the platform in the range of +/- 10 cm, and the lower handle is used to raise and lower the platform for measurement in the range of 20 cm. The sample holder can be placed on the platform, which can contain living subjects, such as mice or other small animals.

[0024] FIG. 6 is a schematic illustration of an illustrative SQUID sensor device that can be used for human cancer research. Wood structure 63 may be similar to the supporting frame shown in FIG. 5. The measurement platform may be replaced with a bed 65 to accommodate a patient. Two large Helmholtz coils 64 include wooden ring shapes above and below the bed. These large coils can be used to create a uniform pulsed field and magnetize magnetic nanoparticles that are administered to the patient. The currents can be modified, for example, increased compared to those used in the device shown in FIG. 5, to again produce fields in the range from 40 to 50 Gauss. Like the device shown in FIG. 5, a Dewar vessel 61 for SQUID with a set of magnetic gradiometers can be used to measure changes in the residual magnetic field produced by magnetized nanoparticles.

[0025] FIG. 7 is a diagram and photograph of an atomic magnetometer for measuring a weak field. This device is miniaturized using microchip manufacturing methods, and multiple units can be placed side by side to form a set of sensors. The magnetometer operates through the use of a laser light beam guided by an optical fiber. This beam pumps the heated Rb gas in the evaporation cell into specific atomic states. The beam is initially elliptically polarized and collimated into the evaporation cell. A mirror reflects this beam back through the cell and lenses into a polarization analyzer. A magnetic field acting perpendicular to the length of the magnetometer measures the refractive index of the gas in the cell, a change in the polarization of the light passing through the cell. A change in polarization results in the magnitude of the acting magnetic field. The pumping laser is fed into multiple fiber optic cables and is thus used for multiple magnetometers. The set of these magnetometers for relaxometric measurements may contain 7 evaporation cells placed with one in the center surrounded by 6 others. The acting field from the magnetizing coils is perpendicular to the arrangement shown in FIG. 5 in order to induce the maximum observed magnetic moments of the nanoparticles. The photograph below FIG. 7 shows an illustrative physical arrangement and size of an atomic magnetometer for use with the present invention. The sensitivity of the device shown is 0.16 ϕT / √Hz compared with the sensitivity of the illustrative SQUID system shown in FIG. 5, 1.0 pT / √Hz (1000 ϕT / √Hz). Atomic magnetometers do not need a cryogenic cooler, which may make them desirable for clinical applications, where such coolers, in particular liquid helium, cannot always be easily obtained.

[0026] Illustrative application for the detection of breast cancer. For breast cancer, mammography is the current preferred method for screening and detection. Despite the fact that mammography has led to a significant improvement in our ability to detect breast cancer at an earlier stage, it still suffers from an inability to distinguish between benign and malignant lesions, difficulties in detecting tumors in dense and scarred breast tissue and cannot detect 10 30% of breast cancers. The use of magnetic nanoparticles conjugated with tumor-specific reagents, combined with the detection of these particles by measuring their relaxing fields, represents a promising new technology that has the potential to improve our ability to detect tumors at an earlier stage. Moreover, the detection of targeted magnetic nanoparticles using weak field sensors is fast and may be more sensitive than MRI detection, since only particles associated with their target cells are detected.

[0027] We have developed conjugated magnetic nanoparticles targeting breast cancer cells that express the HER2 antigen, which is overexpressed in ~ 30% of human breast cancer cases. We characterized the nanoparticles by their magnetic properties and selected those that have the optimal size and magnetic moment per mg of Fe. A number of different cell lines that are specific for HER2 have been studied to determine their site density and sensitivity of the sensory system for detection. A murine SCID model was studied using tumors grown from human cell lines, visualizing the mouse under the influence of the sensory system, followed by confirmation by histological studies. These results show the validity of the magnetic sensory approach for sensitive detection of breast cancer.

[0028] FIG. 8 is a schematic illustration of a biocompatible coated magnetic nanoparticle and attached antibodies to target specific cells. To demonstrate an illustrative embodiment of the present invention, we used antibodies (AB) to HER2, which are specific for 30-40% of breast cancers. Nanoparticles had coatings containing carboxyl groups, and the sulfo-NHS method is used to conjugate nanoparticles with antibodies. Flow cytometry performed for MCF7, MCF7 / Her2-18 breast cancer cell lines (clone MCF7 stably transfected with Her2), BT474 and MDA-MB-231. The number of He2 binding sites was determined by flow cytometry; antibodies to He2 were conjugated to a FITC fluorescence probe. Fig. 9 is an image of the number of He2 sites per cell, calculated by comparison with a range of microspheres with known binding abilities. MCF7 cells designed for overexpression of Ner2-18 have 11 × 106 He2 / cell binding sites, BT-474 have 2.8 × 106, MCF7 0.18 × 106, MDA-MB-231 0.11 × 106. Cell lines that do not belong to the mammary gland have <4000 He2 / cell binding sites.

[0029] FIG. 10 is a graph of magnetic moment measurement in an SQUID sensor system as a function of time for incubating attachable magnetic nanoparticles (from Ocean Nanotech) to breast cancer cell lines. A biocompatible carboxylic coating was applied to the magnetic nanoparticles and then conjugated to an anti-Her2 / neu antibody. This antibody is specific for approximately 30% of human breast cancer cells. Labeled magnetic nanoparticles were injected into ampoules containing live cancer cells, and magnetic moments of the ampoule, measured at various points in time in the range from one minute to 16 minutes. The zero point in time is the magnetic moment of the ampoule with nanoparticles before being added to the cells. The absence of a magnetic moment for unmixed particles is a demonstration that unbound particles do not give a magnetic signal with this SQUID imaging method. When mixed with cells, the magnetic moments increase rapidly and become saturated, indicating that the cells collected on their surfaces the maximum number of possible nanoparticles in one to two minutes. The upper curve is for the breast cancer cell line, MCF7 / Her218, which is known to be very specific for the anti-Her2 / neu antibody, and a large magnetic signal confirms this. The breast cancer cell line, MDA-MB-231, is also Her2 / neu positive, but with much fewer sites for attachment of antibody-targeted nanoparticles to them. Smaller values also indicate this trend. The CHO cell line is non-specific for Her2 / neu and gives significantly lower magnetic moments after incubation. The presence of a magnetic moment indicates some phagocytosis in these cells, in which nanoparticles penetrate into the cells. The curve for the absence of cells is a curve for ampoules containing only nanoparticles, and demonstrates that the particles themselves do not give a signal and, thus, there is no agglomeration with the particles. These results demonstrate the specificity of the antibody for target cancer cells and confirm that only bound particles give magnetic moments. This result is not reliable for other methods, such as MRI, which sees all particles bound or unbound.

[0030] FIG. 11 is an illustration of the magnetic moments of cell samples, measured as a function of the number of cells by diluting twice with pipetting. The demonstrated sensitivity is 100,000 cells for MCF7 cells and Ocean nanoparticles, for cells at a distance of 3.5 cm from the sensor. There are 2.5 × 106 nanoparticles / cell. Linearity demonstrates that the magnetic moment gives the number of cells; MRI contrast is not a linear function of cell number. A typical mammogram requires 10 million cells.

[0031] The mammary gland phantom was constructed using a standard mammogram calibration phantom as a model. The phantom was constructed from clay, a non-metallic material permeable to these fields. Ampoules containing living cells were introduced into the phantom. 12 is an illustration of a phantom with inserted ampoules with MCF7 cells, left 2E + 06, right = 1E + 06 cells. Cells are conjugated to Ocean Nanotech anti-HER2 nanoparticles. Fields were mapped in five 7-channel SQUID positions = 35 sites. 3-D contour maps represent field distributions. The locations and magnitudes of the moment were obtained from the inverse problem. Moments determine the number of cells in the ampoules from the data for the cells shown above.

[0032] A murine model of breast cancer has been developed acceptable for SQUID-sensory measurements. SCID nude mice were used with human breast cancer cell lines. Fig.13 is a photograph of a nude mouse under the SQUID system. To study in vivo processes using the SQUID technique, mice were injected with human MCF7 cells two weeks earlier in two places. These cells then produced human tumors on the lateral surfaces of the mouse body; one such tumor is visible under the right ear of the mouse. Mice injected anesthesia through a tube through its mouth. Labeled magnetic nanoparticles were injected into the mouse on this platform using either tail, interperitoneal, or intratumoral injections. After injection, the mouse was placed under the sensory system, as shown, and a magnetizing impulse was reported, and the obtained magnetic moments of the introduced particles were measured. As in the case of living cancer cells, no moments were observed, unless the particles were attached to the cells within the tumors. In some cases, both tumors were MCF7 cells, and in other cases, two different cell lines were used to develop tumors in mice. The mouse remained on the platform shown in FIG. 5 and could be moved to several positions under the sensor system to obtain more spatial information. Measurements were performed as a function of time to determine how quickly particles were captured from the bloodstream and how quickly phagocytosis occurred with particles entering the liver. The mouse, as a rule, was placed in five positions of the platform under the 7-channel SQUID system to obtain 35 spatial locations. Magnetic fields in all positions were then applied in a special code to solve the inverse electromagnetic problem, using the Levenberg-Markardt theorem to determine the location of all sources of magnetic particles in the mouse. This information was then compared with known mouse geometry from photographs to determine accuracy and sensitivity to locate breast cancers in live animals. Fig.14 contains plots of the reliability of the positions obtained from murine tumors. The left sphere is from the left tumor, which is ~ 2 times larger than the right tumor in the magnetic moment (see below). The positions were calculated using the two-dipole least squares method to highlight magnetic moments and positions. Moments determine the number of labeled cells in tumors.

[0033] The results of the SQUID system for in vivo measurements in live animals are shown in FIG. 16, 17, 18 for animals bearing two different tumors. Each mouse had two tumors, but different cell types. Each of the two tumors absorbed different amounts of nanoparticles. A mouse with MCF7 cells exhibited higher magnetic moments than a mouse with MDA-MB-231 tumors, which is believed to be due to the large number of specific sites for anti-HER2 / neu antibodies in the first one. Fig. 15 is an illustration of magnetic contour lines observed at 35 different measurement locations, as described in Fig. 13. The analysis of these magnetic fields resulted in the spatial positions of the tumors, which were consistent with the measured values of these positions; moreover, SQUID results give higher accuracy than physical measurements of approximately 3 mm. Fig.16 is an illustration of the temporal dynamics of measurements for two mice and both tumors of each mouse. Particle capture occurred rapidly with a signal near the maximum received in the first hour. Nanoparticles remain in the tumors for at least 5 hours for the duration of the experiments. After these measurements, the mice were euthanized and tumors and other organs removed and placed under the sensory system to determine how many introductions of nanoparticles were in the tumors. The graphs in FIG. 17 are illustrations of the results of these measurements and show very good agreement with in vivo measurements on a live mouse. In the lower left figure, a magnetic moment was observed in the liver, indicating that there was some phagocytosis and the particles were delivered to the liver for excretion. Subsequent histology of tumors also showed significant attachment of particles to cells in the tumor by staining with Prussian blue to detect iron in magnetic nanoparticles.

[0034] Confidence areas were calculated to determine the accuracy of the location of tumors for in vivo measurements of mice. Fig. 18 is an illustration of a 2-dimensional 95% confidence limit for the locations of two tumors superimposed on actual mouse tumors. A spatial accuracy of approximately +/- 3 mm is obtained in x and y direction. Fig. 19 is a photograph of the histology of tumors after isolation. Microscopic image of a single section of the tumor MCF-7. Staining with Prussian blue cells reveals iron present in nanoparticles attached to the cells. The arrow indicates a cell coated with nanoparticles.

[0035] A magnetic field sensitive sensor system has been demonstrated for early detection in vivo of breast cancer by detecting magnetic nanoparticles conjugated to antibodies to breast cancer cell lines. Over 1 million nanoparticles attach to each cancer cell. The method is sensitive to <100,000 cells at distances comparable to breast tumors. Standard X-ray mammography typically requires a cell density of ten million cells. The measured moments are linear in relation to the number of cells; that is, measuring the magnetic moment results in the number of cancer cells present. Very high contrast nanoparticles not attached to the cells are not observed. Phantom studies show multiple sources that have an exact location and a specific number of cells per source. A mouse model was developed using multiple tumors of human breast cancer cell lines and in vivo measurements were performed to determine the location and number of cancer cells of these tumors after nanoparticle administration. Solutions to the inverse problem successfully determine the location of tumors and the number of cells. Histology confirms the presence of murine tumors with nanoparticles.

[0036] Illustrative application for the detection of ovarian cancer.

The etiology of ovarian cancer is not fully understood and there is little evidence regarding risk factors that would suggest prophylactic screening. A routine screening test is a pelvic examination to determine if there are any suspicious symptoms, such as an abdominal enlargement, and the results usually show a progressive stage of cancer. Routine screening of women is currently not performed, as there are no reliable screening studies. The great challenge now regarding ovarian cancer is that by the time it is discovered, it has metastasized from the ovary to other organs. For this reason, a hysterectomy is often performed along with removal of the ovary. If the presence of ovarian cancer can be determined at an early stage, and it is contained in the ovary, the five-year survival rate is 95%. However, only 29% are found at this stage. If the disease has spread locally, its survival rate drops to 72%, and if it metastasizes to distant locations, the survival rate is 31%. Thus, the development of early detection methods is an urgent need.

[0037] FIG. 20 is an illustration of ovarian cancer showing tumor growth on the ovary. These tumors are composed of cells with high amounts of receptors for the anti-CA-125 antibody and can be targets for magnetic nanoparticles labeled with this antibody. Fig is a photograph of a full-sized phantom of the ovary, placed under the SQUID-sensor device at a distance that will be typical for the patient subject. The phantom has an ampoule containing live ovarian cancer cells introduced into it. Magnetic nanoparticles labeled with an anti-CA-125 antibody were introduced into this ampoule, and since these antibodies are highly specific to these ovarian cancer cells, they attach to the cell surface in large numbers. These magnetic nanoparticles were then detected using an SQUID sensor device to obtain sensitivity calibrations for in vivo measurements for both animal and human in vivo models.

[0038] The results of sensitivity studies for live ovarian cells inserted into the phantom shown in FIG. 21 are illustrated in FIG. 22 for three different ovarian cancer cell lines; namely tov-112D, Ov-90 and nihovcar-3. The graph shows the minimum number of cells that were detected by this device for three different cell lines as a function of the distance from the sensor to the patient's ovaries. The ov-90 cancer cell line is known to be one of the most aggressive forms of cancer, and these results show that there are many CA-125 receptors on the cell surface. The number of nanoparticles per cell can be estimated from these measurements and corresponds to 20,000 particles per cell for tov-112D, 3400 for ov-90 and 6700 for ovcar-3.

[0039] Fig. 23 is an illustration of confirmation of antibody sites for these cells by flow cytometry. FIG. 23a and 23b show two of the four cell lines analyzed. Only the signal from the cells is shown, and the isotype (using a molecule with non-specific binding, Igg), the anti-Her2 / neu antibody and the anti-CA-125 antibody are shown with an increasing number of sites on the right in these graphs. These figures show that the anti-CA-125 antibody has a large number of sites on these cells, with SK-OV-3 being the largest of the two. The Her2 / pei antibody is also specific for 30% of breast cancer cells.

[0040] Measurements were performed as a function of time to determine how quickly particles were captured from the bloodstream and how quickly phagocytosis of particles entering the liver occurred. The measurement of magnetic moment in a SQUID-sensor device as a function of time for magnetic moments from magnetic nanoparticles (from Ocean Nanotech) attached to human ovarian cancers in a live mouse is shown in FIG. 24. The mouse had two ovarian tumors, one SK-OV-3 and the other NIH-OVCAR3. A magnetic carboxylic biocompatible coating was applied to the magnetic nanoparticles and then conjugated to an anti-CA-125 antibody. This antibody is specific to human ovarian cancer cells. Labeled magnetic nanoparticles were introduced into murine tumors and the magnetic moments of the mouse were measured at various time points ranging from one minute to 300 minutes. Particle capture occurred rapidly, with a signal close to the maximum received in the first hour. Temporal dynamics show that nanoparticles remain in the tumors for several hours. Nanoparticles remained in the tumors for at least 5 hours during the course of the experiments. Each of the two tumors absorbed different amounts of nanoparticles. A murine tumor with SK-OV-3 cells exhibited higher magnetic moments than a mouse with NIH-OVCAR-3 tumors, as expected because of the higher number of specific sites for anti-CA-125 antibodies in the first of the above. Nanoparticles did not give a magnetic moment before administration and give a magnetic signal only when attached to something, for example, tumor cells. Experiments have shown that introductions to sites other than the tumor do not give a signal since the particles do not bind to normal cells. After a period of time, the liver begins to show signs of accumulation of these particles as they phagocytose from the system. After these measurements, the mice were euthanized, tumors and other organs were removed and placed under a sensor device to determine the number of nanoparticle injections into the tumor. These measurements are very well consistent with in vivo measurements on a live mouse. Subsequent histology of the tumors showed significant attachment of particles to the tumor cells by staining with Prussian blue to detect iron in magnetic nanoparticles.

[0041) A photograph of a mouse used to confirm that the SQUID sensor method is applicable in vivo, together with magnetic contour fields from this mouse, is shown in FIG. 25. Human tumors are shown on the lateral surfaces of the mouse; they are tubercles above and on the sides of the tail in FIG. These tumors were obtained by introducing live human ovarian cancer cells into this severely immunocompromised mouse and were allowed to grow for several weeks until a 6-10 mm tumor appeared. Mice were anesthetized with a tube through the mouth during all SQUID-sensory experiments. Labeled magnetic nanoparticles were injected into the mouse on this platform using either tail, interperitoneal, or intratumoral injection. After injection, the mouse was placed under the sensor, as shown in FIG. 1, and a magnetizing pulse was applied, and the obtained magnetic moments of the introduced particles were measured. As in the case of living cancer cells, no moments were observed, unless the particles were attached to the cells within the tumors. In some cases, both tumors were SK-OV-3 type cells, and in other cases, two different cell lines were used to develop tumors in mice.

[0042] A mouse placed on the platform shown in FIG. 5 can be moved to several positions under the sensor to obtain more spatial information. The mouse was typically placed in five positions of the platform under the 7-channel SQUID to obtain 35 spatial locations. Magnetic fields at all positions were then applied in the code to solve the inverse electromagnetic problem using the Levenberg-Markardt theorem to determine the location of all sources of magnetic particles in the mouse. This information was then compared with known mouse geometry from photographs to determine accuracy and sensitivity to locate cancerous tumors in live animals. 25 shows magnetic contour lines observed for 35 different measurements. The analysis of these magnetic fields resulted in the spatial positions of the tumors, which were consistent with the measured values of these positions; moreover, SQUID results give higher accuracy than physical measurements of approximately 3 mm.

[0043] Illustrative application for the detection of Hodgkin lymphoma. Hodgkin lymphoma (HL) accounts for 30% of all lymphomas. HL typically occurs in the lymph nodes, preferably in the cervical regions, and the thymus; but predominantly the disease may involve distant lymph nodes, spleen and bone marrow. The majority of cases occur in young people from 15 to 34, and the second peak of occurrence occurs in people over 55. Currently, a biopsy assessment is needed for diagnosis. A surgical biopsy has complications such as infection and bleeding, and a biopsy evaluation usually takes 3-5 days. Thus, in cases of HL, and of which the tumor mass interferes with the return blood flow in the heart (namely, the superior vena cava syndrome, 10% of cases), significant pain and mortality may occur during this waiting period. Some of the antibodies that target Hodgkin's lymphoma have been identified; namely, CD15, CD30 and CD25. The latter antibody, however, targets many cells and is less specific. Another application where the present invention can have a significant clinical effect is the detection of persistent HL after therapy. If a patient experiencing an exacerbation undergoes high-dose radiation therapy, then there is a good prognosis if the exacerbation is detected at an early stage. Patients with exacerbation will have a prognosis, determined primarily by the duration of the first remission. The persistence of large fibrous nodes, especially in the mediastinum, after therapy leads to confusion in determining whether persistent cancer is present, and surgery for fibrous nodes is fraught with the difficulty of controlling the bleeding problem and pain for the patient.

[0044] The relaxometric method of the present invention can provide a quantitative estimate of the number of lymphoma cells present in organs affected by Hodgkin's disease, such as thymus and spleen. RS cells are giant cells derived from B lymphocytes that contain millions of CD30 and CD15 receptors. Previous results with SQUID sensors targeting T-cell lymphocytes have shown that for smaller cells, approximately one million nanoparticles can be attached to each T-cell. Steric mismatch limits the number of nanoparticles attached to a normal lymphocyte, but much larger RS cells can have 25-50 times more bound nanoparticles. The amount of iron per nanoparticle is 4.4 × 10 ^-6 ng / nanoparticle. Given the large size of the RS cells, there may be several million nanoparticles per cell, so that each cell can have up to 10 ng of iron. One hundred RS cells accumulated in the spleen or thymus may contain micrograms of iron. Less than a microgram is sufficient for SQUID detection, hence the possibility of detecting 100 RS cells is likely. The measured amplitude of the remanent magnetization of antibody-labeled nanoparticles in vivo may provide an important diagnostic tool for lymphoma cancer. The signal strength depends on the density of antigens on the cell surfaces of the tumor, and thus, the strength of the field produced by the nanoparticles is proportional to the numerical density of the antigenic sites on the lymphoma cells. You can determine the number of particles and density in order to obtain the amplitude of the detected magnetic field. This information can be used in planning in vivo detection, and also as an aid in the selection of nanoparticles for use. The SQUID sensor is the optimal sensor system for Hodgkin's disease with high sensitivity to RS cells, and in vivo detection of the disease without biopsy, and the ability to track the treatment of the disease during chemotherapy.

[0045] FIG. 26 is a graph of magnetic moment measurements in an SQUID sensor system as a function of time for incubating attachable magnetic nanoparticles (from Ocean Nanotech) to lymphoma cell lines. A biocompatible carboxylic coating was applied to the magnetic nanoparticles and then conjugated to an anti-CD34 antibody. This antibody is specific for one type of lymphoma cell, namely, acute lymphatic leukemia in humans. Labeled magnetic nanoparticles were introduced into ampoules containing live cancer cells, and the magnetic moments of the ampoule were measured at various points in time ranging from one minute to 16 minutes. The zero point in time is the magnetic moment of the ampoule with nanoparticles before being added to the cells. The absence of a magnetic moment for unmixed particles at time zero indicates that unbound particles do not produce a magnetic signal with this SQUID imaging method. When mixed with cells, the magnetic moments increase rapidly and become saturated, indicating that the cells collected on their surfaces the maximum number of possible nanoparticles in one to two minutes. The upper curve is presented for the U937 lymphoma cancer cell line, which is known to be very specific for the anti-CD34 antibody, and a large magnetic signal confirms this. The lower curve is presented for the same cell line, but a non-specific marker, BSA, and shows significantly lower magnetic moments after incubation. The presence of a magnetic moment for BSA is a sign of some phagocytosis of these cells, in which nanoparticles penetrate into the cells. U937 is a lymphoma of T-lymphocytes, and RS is a lymphoma of B-lymphocytes. Since one of the main goals of lymphocytes is to capture particles that do not belong to the body, this amount of nonspecificity is expected. These results demonstrate the specificity of the antibody for target cancer cells and confirm that only bound particles give magnetic moments. This result is not observed for other methods, such as MRI, which sees all particles bound or unbound.

[0046] Samples of RS cells were obtained from the Tissue Bank facility of the University of New Mexico, a national organization for storing cells and multiple samples. The effectiveness of the SQUID sensor system for detecting RS cells was compared with the number of RS cells in the sample, determined by manual hematocytometric counts. These isolated RS cells were labeled with nanoparticles specifically bound to CD15 and CD30 during the isolation procedure. Sensitivity calibration was performed by serial dilution in the range of 1 in 10 to 1 in 100,000 cells. Density ranges of nanoparticles on malignant cells exceeded 107 nanoparticles / cell. The density of CD15 sites is determined using flow cytometry technique that counts receptors / cell. The number of CD15 and CD30 sites / cell was confirmed using a quantitative immunofluorescence staining technique.

[0047] FIG. 27 is an illustration of measurement results of flow cytometry of RS cells from the lymphatic system when determining the number of sites available for nanoparticles and detection by SQUID sensors. 27A is a photograph showing a morphological view of RS cells isolated from a lymph node sample. 27B shows a flow cytometry analysis of a bone marrow sample, where (B1) is before and (B2) after the enrichment procedure to increase the frequency of RS cells in a flow cytometry sample. Normally, RS cells arise with a frequency of 1 in 104 or 105 normal lymphocytes and should be increased before CD15 and CD30 staining using flow cytometry for detection. The SQUID-sensory system detects all RS cells in vivo and does not need to be sampled; therefore, amplification is not necessary, as is necessary for determination by flow cytometry.

[0048] Lymph nodes are one of the main places where RS cells accumulate, in addition to the thymus. Fig. 28 is a histological section from a patient with Hodgkin's disease. RS cells were stained with immunoperoxidase staining. The CD15 antibody is shown on the right, and the CD30 antibody is shown on the left. The surrounding cells are non-cancerous cells in the lymph node. The SQUID sensor can detect several hundred of these labeled RS cells in the lymph node.

[0049] Illustrative application for the detection of prostate cancer. Prostate cancer has a high mortality rate due to the lack of early detection using standard screening techniques. The number of cases in 2009 in the United States was 192280 with 27360 deaths. Prostate cancer accounts for 9% of male deaths and there is now a 1 in 6 chance of developing prostate cancer over the course of a lifetime. The disease is usually not detected until it causes an enlarged prostate, problems with urination, or spreads to other organs. Asymptomatic detection of the disease is usually carried out using a digital study, the result of an elevated PSA test (prostate specific antigen) or a biopsy. The PSA test is now considered unreliable, leading to many unnecessary biopsies that are associated with a risk of infection. Digital research is also highly subjective. Testing for prostate cancer is very controversial. PSA testing costs more than $ 3 billion in the US alone, and a recent study published in the New England Journal of Medicine found that modern screening methods do not reduce mortality in men over 55. The present invention can detect this cancer before it metastasizes.

[0050] An exemplary method for detecting prostate cancer in tissue includes placing a patient on a platform for measuring a sensor device of a superconducting quantum interference sensor; introducing a plurality of antibody-labeled magnetic nanoparticles into the patient for specific binding to the patient’s tissue; exposure to a uniform magnetizing pulsed field to magnetize nanoparticles introduced into a patient; and detecting a residual magnetic field of magnetized nanoparticles, thereby obtaining an image of the nanoparticles bound to the patient’s tissue. The tissue may include prostate tissue, and antibody-labeled magnetic nanoparticles can specifically bind to antigens of prostate cancer cells. An antibody-labeled magnetic nanoparticle may comprise a magnetic core coated with a biocompatible coating to which specific antibodies are attached. For example, the magnetic core may contain ferromagnetic material, such as iron oxide. For example, the biocompatible coating may contain dextran, carboxyl, or amine. To detect prostate cancer, a specific antibody may be an anti-PSMA antibody.

[0051] Prostate-specific membrane antigen (PSMA) is a transmembrane glycoprotein that is highly expressed by most forms of prostate cancer. It is also called mAb 7E11. It is expressed on the surface of the vascular endothelium of a solid carcinoma tumor, but not on normal prostate cells. The amount of PSMA observed in prostate cancer indicates the severity or extent of the tumor. Flow cytometry showed that there are large numbers of receptor sites for this antibody on some prostate cancer cell lines, including LNCaP and PC-3, while the PSMA-negative cell line DU-145 does not show any expression.

The results of attaching magnetic nanoparticles to these positive cell lines show one million or more nanoparticles per cell. These results are comparable to those for ovarian and breast cancer with respect to nanoparticles per cell and tumor depths in the body, and biomagnetic detection methods using SQUID sensors will have the same sensitivity to prostate cancer as to ovarian cancer (described in one or several of the related applications included in the link above). The results of studies on ovarian cancer can thus be directly applied to detect and locate prostate cancer. Compared to the anti-CA-125 antibody for ovarian cancer, PSMA is even more specific for in vivo strategies for specifically targeting the prostate.

[0052] The SQUID-sensory method can provide a quantitative assessment of the microvascular structure in tumors, leading to a new substitute for vascular formation (angiogenesis) and gradation of a single tumor. It was shown in a study of the microvascular characteristics of a tumor in an experimental model of prostate cancer using nanoparticles that growth and aggressiveness / stage of the tumor are directly related to neovascularization of the tumor. Other studies estimate the concentration of magnetic particles in the tumor as approximately 2.3 mg of nanoparticles per gram of tissue. This concentration is usually achieved in tumors of patients with human liver cancer receiving treatment through intrahepatic arterially administered radioactive microspheres; nanoparticles tend to concentrate in the tumor vessel growth ring. Less than a nanogram is sufficient for SQUID detection. The measured in vivo residual magnetization amplitude of antibody-labeled nanoparticles can provide an important diagnostic tool for prostate cancer. The signal strength depends on the density of antigens on the cell surfaces of the tumor and, thus, the strength of the field produced by the nanoparticles is proportional to the numerical density of antigenic sites on the cells of the prostate tumor. Thus, the number and density of particles provide the amplitude of the detected magnetic field. This information can then be used in in vivo planning, and also as an aid in the selection of nanoparticles for use.

[0053] Illustrative application for the detection of glioblastoma. Brain cancer is particularly deadly and occurs in a number of forms. Cancer involving glial cells is the most common form and also the most aggressive brain tumor in humans. Various glial cells may be involved, causing cancer of the type oligodendrogliomas (involving oligodendrocytes), astrocytomas (involving astrocytes), and glioblastomas. The latter is the most common form of brain cancer. These types of cancer usually lead to death in a very short amount of time. Gliablastoma cells can be targeted using markers, such as antibodies to EGFR, 81C6 and PTN, which can be used to visualize this type of cancer. Mouse models and brain cancer cell lines, such as U-251, are available for testing before human use.

[0054] An important point in targeting brain cancer is the delivery of nanoparticles with attached markers through the blood-brain barrier. This barrier is to some extent open in the vascular system associated with malignant tumors, but still remains an obstacle. The use of nanoparticles coated with lipophilic surfaces and then conjugated to antibodies or peptides increases the ability to cross the barrier. Additionally, the nanoparticles with markers can be encapsulated in a polymer shell with a liposome surface in a micelle, which is a different approach, and releasing conjugated nanoparticles from the polymer after entering the brain using a low-temperature RF or ultrasound pulse.

[0055] An exemplary method for detecting brain cancer includes placing a patient on a platform for measuring a sensor device with a superconducting quantum interference sensor; introducing a plurality of antibody-labeled magnetic nanoparticles into a patient with specific binding to a brain tumor in a patient; exposure to a uniform magnetizing pulsed field with the magnetization of nanoparticles introduced into a patient; and detecting a residual magnetic field of magnetized nanoparticles, thereby providing visualization of the nanoparticles associated with the patient’s tissue. The target is a brain tumor, and antibody-labeled magnetic nanoparticles can specifically bind to brain cancer cell antigens. An antibody-labeled magnetic nanoparticle may comprise a magnetic core coated with a biocompatible coating to which specific antibodies are attached. For example, a magnetic core may include a ferromagnetic material, such as iron oxide. For example, a biocompatible coating may include dextran, carboxyl, or amine. To detect glioblastoma, the specific antibody may be EGFR or a similar antibody.

[0056] EGFR angiogenesis takes several forms and is a variant of the epidermal growth factor receptor (EGFR), which is overexpressed by certain types of cancer cells, including glioblastoma cells, but not normal cells. EGFR is currently undergoing immunotherapeutic clinical trials for patients diagnosed with glioblastoma. It can be conjugated to magnetic nanoparticles suitable for detection using magnetic relaxometry, and introduced into the body. These magnetic nanoparticles may include a coating, such as polyethylene glycol (PEG), which will increase the effectiveness of the targeted nanoparticles to penetrate the blood-brain barrier. In another illustrative embodiment of the present invention, magnetic nanoparticles with attached markers may be contained within polymer coatings that are able to penetrate the blood-brain barrier and then release when a small RF heating pulse is used or ultrasound is used. The results of the attachment of these angiogenesis peptides to magnetic nanoparticles and their attachment to cells are comparable to other results for ovarian and breast cancer antibodies with respect to nanoparticles per cell and the depths of tumors in the body. Biomagnetic detection methods using systems such as SQUID sensors will have the same sensitivity to brain cancer as ovarian cancer (described in one or more of the related applications incorporated by reference above). The results of studies on breast and ovarian cancer can thus be directly applied to the detection and location of brain cancer.

[0057] Illustrative application for the detection of pancreatic cancer. A number of tumor markers are present in pancreatic cancer. CA19-9 is one example of a marker whose level rises with this cancer, but it is not very sensitive (77%) and non-specific (87%). Marker combinations were proposed by the University of Texas Cancer Research Center (M.D. Anderson Cancer Center) and were investigated for screening for pancreatic cancer. These markers are miRNAs and include miR-21, MiR-210, miR-155 and miR-196a. However, this combination also achieves only low sensitivity (64%), but greater specificity (89%) than CA19-9. Additionally, a series of antibodies to specific human pancreatic cancer cell lines has been determined, for example, the FG cell line, and these include S3-15, S3-23, S3-41, S3-60, S3-110 and S3-53. Another marker of determination is the urokinase plasminogen activator receptor (uPAR), which is highly expressed in pancreatic cancer and also in tumor stromal cells. The latter marker was used to deliver magnetic nanoparticles to pancreatic cancer types grown as xenografts in athymic mice. These markers led to MRI detection of tumors in mice when used as labeled contrast agents. The mechanism primarily represents the delivery of nanoparticles to tumor endothelial cells.

[0058] There are no reliable imaging approaches for diagnosing pancreatic cancer. Thus, the development of biomarkers as a targeted visualization tool for MRI or providing a more sensitive magnetic relaxometry technique is a significant advantage. MRI can detect small abnormalities in tumors and is also useful in determining whether cancer has metastasized. Dynamic Contrast MRI (DCE) potentially distinguishes between benign and malignant tumors, but produces a series of false positive results. The high cost of MRI limits its use as a screening tool. MRI imaging of tumors often uses magnetic nanoparticles as contrast agents, as mentioned above, and is an accepted protocol that provides standards for the introduction of such nanoparticles. Intravascular MRI contrast agents at a dose of 2 mg / kg of nanoparticle weight were used to detect metastatic lesions. However, the use of MRI in pancreatic cancer is severely limited.

[0059] The present invention can provide a quantitative assessment of the microvascular structure in tumors, leading to a new substitute for vascular formation (angiogenesis) and gradation of a single tumor. The results of a study of the microvascular characteristics of a tumor in an experimental model of pancreatic cancer using nanoparticles showed that tumor growth and aggressiveness / stage are directly related to tumor neovascularization. Other studies estimate the concentration of magnetic particles in the tumor as equal to about 2.3 mg of nanoparticles per gram of tissue. This concentration is regularly achieved in tumors of patients with human liver cancer receiving treatment through intrahepatic arterially administered radioactive microspheres; nanoparticles tend to concentrate in the tumor vessel growth ring. Nanograms are sufficient for detection using the present invention. The measured amplitude of the remanent magnetization of antibody-labeled nanoparticles in vivo may provide an important diagnostic tool for pancreatic cancer. The signal strength depends on the density of antigens on the surface of the tumor cells and, therefore, the field strength formed by the nanoparticles is proportional to the numerical density of the antigenic sites on the tumor cells of the pancreas. The number of particles and density can be determined by providing the amplitude of the detected magnetic field. This information can be used in planning for in vivo detection, as well as to assist in the selection of nanoparticles for use. Examples of pancreatic cancer cell lines include FG or MIA PaCa-2, which are known to be specific for an anti-uPAR antibody.

[0060] The present invention has been described, as set forth herein, in relation to various illustrative embodiments and design decisions. It will be understood that the foregoing description is illustrative only for applications of the principles of the present invention, the scope of which is defined by the claims considered in light of the description of the invention. Other variations and modifications of the invention will be apparent to those skilled in the art.

Claims (21)

1. Device for detecting, measuring or determining the location of one or more specified types of cancer cells or biological substances in vivo, containing:
a) a magnetizing subsystem configured to magnetize nanoparticles that are associated with one or more specified types of cancer cells or biological substances in a patient; and
b) a sensory subsystem configured to detect a residual magnetic field in a patient region after the unbound nanoparticles return to random magnetization, and during the transition time of the bound nanoparticles from uniform to random magnetization, and
c) a control and analysis system configured to analyze the residual magnetic field to detect, measure or locate one or more specified types of cancer cells or biological substances in a patient. 2. The device according to claim 1, where the magnetizing subsystem contains many Helmholtz coils. 3. The device according to claim 1, where the sensor subsystem contains a set of gradiometers. 4. The device according to claim 1, where the sensor subsystem contains many planar gradiometers. 5. The device according to claim 4, where at least one planar gradiometer has a base of about 2 cm 6. The device according to claim 4, where at least one planar gradiometer is manufactured using photolithography. 7. The device according to claim 4, further comprising a superconducting quantum interference sensor (SQUID) coupled to at least one of a plurality of planar gradiometers, where the SQUID is superconducting at temperatures near the temperature of liquid nitrogen. 8. The device of claim 4, further comprising a plurality of SQUIDs, each associated with one of a plurality of planar gradiometers, where the SQUIDs are superconducting at temperatures near the temperature of liquid nitrogen. 9. The device according to claim 1, where the sensor subsystem contains an atomic magnetometer. 10. The device according to claim 1, where the sensor subsystem is configured to solve an inverse electromagnetic problem. 11. A method for detecting, measuring or locating one or more specified types of cancer cells or biological substances in vivo, including:
a) ensuring the availability of the device according to claim 1;
b) placing a plurality of targeted nanoparticles in a patient, wherein each of the plurality of targeted nanoparticles comprises a paramagnetic nanoparticle conjugated to a targeting agent that preferably binds to one or more predetermined types of cancer cells or biological substances;
c) the use of a magnetizing subsystem to magnetize nanoparticles;
d) the use of a sensor subsystem to detect residual magnetic field in the patient’s area over time after the attenuation of the resulting magnetic moments of nanoparticles not associated with cancer cells or biological substances, and before the magnetic moments of the nanoparticles associated with cancer cells or biological substances decay, and
e) the use of a control and analysis system to analyze the residual magnetic field to detect, measure or locate one or more specified types of cancer cells or biological substances. 12. The method of claim 11, wherein the targeted nanoparticles are labeled with antibodies that specifically bind to a given type of cancer cell. 13. The method of claim 12, wherein the antibody-labeled paramagnetic nanoparticles comprise a magnetic core coated with a biocompatible coating to which at least one specific antibody is attached. 14. The method of claim 13, wherein the magnetic core comprises ferromagnetic material. 15. The method of claim 14, wherein the ferromagnetic material comprises iron oxide. 16. The method of claim 15, wherein the magnetic core is less than 30 nanometers in diameter. 17. The method of claim 13, wherein the biocompatible coating comprises dextran, carboxyl, amine, or a combination thereof. 18. The method of claim 13, wherein the at least one specific antibody comprises one or more of: an antibody specific for prostate cancer, an antibody specific for breast cancer, an antibody specific for ovarian cancer, an antibody specific for Hodgkin’s lymphoma, pancreatic cancer-specific antibodies; antibodies that specifically bind to Reed-Sternberg cells associated with Hodgkin's lymphoma. 19. The method of claim 13, wherein the at least one specific antibody comprises an anti-CD15 or CD30 antibody. 20. The method of claim 13, wherein the at least one specific antibody comprises an anti-CA-125 antibody. 21. The method of claim 13, wherein the at least one specific antibody comprises anti-HER-2 antibodies.

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