WO2016154552A1

WO2016154552A1 - The use of iron oxide nanoparticles in magnetically guided sentinel lymph node biopsy

Info

Publication number: WO2016154552A1
Application number: PCT/US2016/024252
Authority: WO
Inventors: Richard Matthew FERGUSON; Amit Praful KHANDHAR; Scott Jeffrey Kemp; Kannan Manjapra KRISHNAN
Original assignee: Lodespin Labs, Llc
Priority date: 2015-03-25
Filing date: 2016-03-25
Publication date: 2016-09-29

Abstract

Disclosed herein is a method for locating a sentinel lymph node using single-core iron oxide nanoparticles. The method involves injecting iron oxide nanoparticles into tissue and subsequently using a magnetic detection device to locate the lymph node into which the nanoparticle solution drains (the "sentinel node"). The iron oxide nanoparticles described can contain magnetic cores of a preferred size so they generate a strong signal, and be coated with a suitable coating that makes them water soluble. The iron oxide nanoparticles disclosed herein exhibit unexpectedly beneficial magnetic behavior when probed by magnetometers such as those in common use and under development for locating sentinel lymph nodes.

Description

THE USE OF IRON OXIDE NANOPARTICLES IN MAGNETICALLY GUIDED

SENTINEL LYMPH NODE BIOPSY

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Patent Application 62/138,367 filed on March 25th, 2015, the disclosure of which is hereby incorporated by reference in its entirety.

STATEMENT OF GOVERNMENT LICENSE RIGHTS

This invention was made with Government support under NIH No. 2R42EB013520- 02A1, awarded by the National Institutes of Health. The Government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to the use of single core iron oxide nanoparticles used for sentinel lymph node biopsy with a magnetic detection device.

BACKGROUND Sentinel lymph node biopsy (SLNB) is the standard of care for staging disease progression in patients found node-negative by clinical and radiological examinations for breast cancer or melanoma. In clinical practice since the late 1990s, SLNB is a minimally invasive surgery that replaces the more extensive procedure called completion axillary lymph node dissection (ALND), in which all detected axillary nodes are removed. Recommended for most patients, SLNB provides much the same outcome as ALND, but with reduced morbidity. Currently, radioactive tracers and blue dye are used to identify SLNs for biopsy (Figure 1). The so-called combined technique is effective, but suffers from drawbacks related to substantial regulation governing the use of radiotracers and associated costs of handling and disposal, as well as related safety concerns. As a result, SLNB is limited to 66% of patients in the developed world, and used very rarely in low- resource countries. The use of radiation is governed by strict legislation and nuclear medicine personnel are often required to administer injections. Medical personnel and patients are exposed to radiation and the radioactive waste generated during surgery requires temporary storage causing logistical challenges.

In Europe, a first generation magnetic solution for SLNB, the SentiMag^® and tracer Sienna+^®, have been approved for clinical use and were recently determined to be "not- inferior" to the standard method in a multi-center clinical trial. However, despite this achievement, Sienna+^® is a sub-optimal tracer with relatively poor intrinsic signal and uptake into the sentinel lymph nodes. Sienna+^® consists of superparamagnetic iron oxide nanoparticles (SPIONs), which are coated with carboxydextran. Sienna+ is polydisperse, containing a mix of individual and clustered cores. The clusters typically contain about 3 to 7 iron oxide nanoparticles (5-6 nm diameter iron oxide cores) per cluster, which interact to give the net magnetic properties of the cluster. The varying number of particles in each cluster results in a broad range of magnetic responses from the coated clusters. The relatively poor signal from Sienna+^®, observed in magnetic detection devices, is a result of the diverse magnetic properties of the clustered SPION particles in Sienna+^®. Thus, it would be advantageous to have a magnetic tracer with a high intrinsic signal for magnetic sentinel lymph node biopsy. SPIONs composed of magnetite (Fe 0₄), maghemite (Fe₂0 ) or a mixture of magnetite and maghemite, have been used in the clinic to enhance the T2/T2* (negative) MRI contrast [Feridex IV.® and Combidex® - produced by AMAG pharmaceuticals, Resovist®, produced by Bayer Schering Corporation], and more recently for the treatment of iron deficiency anemia in chronic kidney disease (CKD) patients [Feraheme® - produced by AMAG pharmaceuticals]. Experimentally, SPIONs of various compositions have been used for biomedical applications such as cell labeling and separation, drug delivery, magnetic gene transfection (magnetofection), tissue repair and hyperthermia [Gupta et al, Biomaterials 2005;26:3995-4021, Krishnan, IEEE Trans. Mag. 46, 2523-2558 (2010) ].

The unique nonlinear magnetic response of SPIONs can be exploited in alternating magnetic fields to induce a detectable signal that is proportional to the ac-susceptibility (m'(H)). Applications such as magnetic particle imaging (MPI) [Gleich and Weizenecker, Nature 2005;435: 1214-7], magnetic sentinel lymph node biopsy (SLNB) [M. Douek et al, Ann. Surg. Oncol, 21, 1237 (2013)] and magnetic fluid hyperthermia (MFH) [R. K. Gilchrist et al, Ann. Surgery 146, 596 (1957); U. Gneveckow et al, Med. Phys. 31, 1444 (2004)], employ alternating magnetic fields in the radiofrequency range (1-1,000 kHz) applied to SPIONs. In these applications, maximum signal is generated (Figure 2) when SPIONs with core diameters near the superparamagnetic-to-ferrimagnetic transition (i.e. 20-30 nm for Fe 0₄) and uniform size distribution are used.

SUMMARY OF THE INVENTION

This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

The invention relates to the use of single core iron oxide nanoparticles. Methods for magnetically guided lymph-node biopsy using single core iron oxide nanoparticles of the present invention are also provided. The method provides magnetic tracers with a much higher signal in magnet detection devices than magnetic tracers of the prior art.

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

Figure 1. Illustration of sentinel Lymph Node Biopsy of the breast. A radioactive tracer and/or blue dye are injected into the primary tumor and subsequently diffuse into the lymphatic system. A gamma probe and/or visual detection are used to identify the sentinel nodes, which are removed via surgery. Image courtesy of NCI FactSheet on Sentinel Lymph Node Biopsy.

Figure 2. Optimizing SPIO magnetism. Magnetic properties of SPIOs vary with their size. In (a), hysteresis loops measured in VSM at room temperature show increasing susceptibility and nonlinearity with size, (b) shows m'(H), which is proportional to the induced signal voltage in a Differential Magnetometer (DiffMag). (c) Bright field TEM image and HR TEM images of 27 nm particles showing regular particle size and shape, and excellent crystallinity

Figure 3. (a) In vitro results. Dose response curves for LSL tracers and Sienna+ measured with bench top DiffMag (SPAQ) at 5 kHz, showing >3x improvement of LSL tracer over Sienna+^®. (b) Normalized signal intensity measured with AC magnetometer (the SentiMag™) for the same samples at 25 kHz shows up to 4x greater intensity than Resovist

Figure 4. Preliminary efficacy comparison in mini-pigs, (a) Photo of SLNB procedure being performed in a pig; arrow points to a SLN. (b) The LS-6/DiffMag system provides 10-fold improvement in sensitivity over the existing Sienna+^®/SentiMag system. Secondly, Sienna+ provides only 10% increase in sensitivity from SentiMag to DiffMag system; in comparison, LS-6 provides -200% increase.

Figure 5. Sequence of near-infrared images of Mouse 1 taken at t = 15, 18, 23, 28 and 33 minutes after subcutaneous injection of 20 μΐ 9-189 (2 gFe/L) in both rear footpads. Region of interest (ROI) 1 = iliac lymph nodes; ROI 2 = right popliteal node; ROI 3 = left popliteal node

Figure 6. ROI-specific average radiance as a function of time in mouse 1. Nanoparticle uptake in both popliteal nodes reaches maximum in 10-15 minutes after injection. Uptake in the iliac nodes is seen gradually increasing with time.

Figure 7. Near-infrared image sequence of Mouse 2 at t = 10, 12, 17, 22 and 27 minutes after subcutaneous injection of -10 μΐ 9-189 (2 gFe/L) in the rear right footpad. Note injection volume is half of that injected in mouse 1. No injection performed in rear left footpad, which was used as the contralateral control. ROI 1 = right popliteal node; ROI 2 = region around left popliteal node.

Figure 8. ROI-specific average radiance as a function of time in mouse 2. Compared to the control side (left popliteal node), nanoparticle uptake in the contralateral right popliteal node is seen to gradually increase with time.

Figure 9. Vibrating sample magnetometry (VSM) data from lymph nodes excised from mouse 1 (left) and mouse 2 (right).

Figure 10. Vibrating sample magnetometry (VSM) data from lymph nodes excised from mouse 2.

Figure 11. Bright field TEM images of nanoparticles with varying diameter a) 15 nm, b) 20 nm, c) 24 nm, d) 27 nm, e) 30 nm, f) 35 nm. In g), size distributions are represented after fitting histograms to a log-normal distribution function. The legend lists the median diameter, do and the shape parameter, σ.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the use of single core iron oxide nanoparticles for identifying sentinel lymph nodes with a magnetic detection device, for example during the commonly performed procedure known as sentinel lymph-node biopsy. The term "single core iron oxide nanoparticle" refers to an individual iron oxide nanoparticle which is coated with a surfactant or amphiphilic polymer such that it behaves largely independent of the other iron oxide nanoparticles in solution.

In order to be detectable by a magnetic detection device a plurality of nanoparticles must be used in the claimed method.

In one aspect, a plurality of nanoparticles is provided. The nanoparticles are referred to herein as nanoparticles (NP), iron oxide nanoparticles, magnetic nanoparticles (MNP), and superparamagnetic iron oxide nanoparticles (SPIONs).

In one embodiment, the plurality of nanoparticles is 100 or more nanoparticles. In one embodiment, the plurality of nanoparticles is 1,000 or more nanoparticles. In one embodiment, the plurality of nanoparticles is 1,000,000 or more nanoparticles. When considering the number of nanoparticles required for a particular application, the primary factor is the amount of iron oxide, which is defined by the size and number of nanoparticles. As an example, for a mouse study a typical injection is about 0.1 mg of iron oxide, which contains about 3.3 xlO¹² nanoparticles with an iron oxide core diameter of 25 nm.

In one embodiment the single core iron oxide nanoparticles refer to a plurality of iron oxide nanoparticles that are coated with a surfactant or amphiphilic polymer such that they behave largely independently of the other iron oxide nanoparticles in solution. With respect to their magnetic properties, the nanoparticles respond to a magnetic field largely as individual particles, rather than collections of interacting particles. In one embodiment the single core iron oxide nanoparticle refers to an iron oxide nanoparticle which is coated with a surfactant or amphiphilic polymer such that the particles are soluble in an aqueous system.

In one embodiment the single core iron oxide nanoparticle refers to an iron oxide nanoparticle which is coated with a surfactant or amphiphilic polymer such that the particles do not aggregate.

In one embodiment the single core iron oxide nanoparticles are not clusters of two or more particles bound together via a coating such that the cluster acts as an independent group as evident by dynamic light scattering.

The combination of the specific size range of the iron oxide core, coupled with the specific composition of coating, provides the nanoparticles with superior properties. These benefits include, but are not limited to, improving dispersal in water, preventing aggregation, and preserving the nonlinear magnetic response or AC-susceptibility in aqueous media and serum-containing in vivo environments.

Iron oxide core oxidation state

The oxidation state of the iron oxide core refers phase or phases of iron oxide in the cores. The iron oxides contain a mixture of Fe(II) and Fe(III) and the oxidation state can be reported as the percentage of Fe(II) of the total iron (Fe(II) and Fe(III). The synthesis of nanoparticles via the thermolysis of iron oleate as described in the examples below produces particles with a mixture of phases. All the iron oxide cores have a thin shell (~2 nm) of maghemite which forms rapidly in the presence of air. The rest of the core is typically magnetite or magnetite with some wustite. Since magnetite and maghemite contribute significantly to the magnetic moment of the nanoparticles and wustite does not contribute, iron oxide nanoparticle must have a significant amount of magnetite and/or maghemite to give a signal in a magnetic detection device. When iron oxide nanopraticles consist of a magnetitie inner core surround by a maghemite shell the signal in a magnetic detection device increase with diameter up to about 25 nm, at which point the iron oxide nanoparticles begin to display ferromagnetic behavior which has a negative impact on the signal. The presence of wustite in the inner core of the iron oxide nanoparticles can increase the allowed size of the iron oxide nanoparticles up to about 30 to 35 nm before ferromagnetic behavior is observed [PCT/US2015/058425]

In one embodiment the core of the nanoparticles consists of iron oxide; preferably the iron oxide is wustite (FeO), magnetite (Fe₃0₄), maghemite (Y-Fe₂0₃), a mixture of wustite (FeO) and magnetite(Fe₃04), or a mixture of magnetite (Fe₃04) and maghemite (y-Fe₂0₃); more preferably the iron oxide is magnetite (Fe₃0₄), a mixture of wustite (FeO) and magnetite (Fe₃0₄) or a mixture of magnetite (Fe₃0₄) and maghemite (γ- Fe₂0₃); and most preferably the iron oxide is magnetite (Fe₃0₄).

In another embodiment the iron oxide core of the nanoparticle consists of a maghemite shell with a magnetite core.

In another embodiment the iron oxide core of the nanoparticle consists of a maghemite shell with an inner core consisting of mostly magnetite with some wustite.

In another embodiment the Fe(II) percentage of the total iron content in the core of the iron oxide nanoparticle is between 0% and 50%, more preferably between 20% and 40%, and most preferably between 20% and 32%. Determination of Fe(II) by permanganate titration is presented in example 8 below.

Iron oxide core size

The diameter or size of the iron oxide core of the single core iron oxide nanoparticle plays a major role in the resulting signal obtained in a magnetic detection device. As the diameter increases the signal increase until the particles become ferromagnetic at which point the magnetic signal in an AC system decreases. The ideal size is therefore large enough to generate a useful signal but small enough to remain superparamagnetic. Figure 2 illustrates the diameter-dependent variation in SPION magnetic response to a slowly changing field (2a). Size dependence of the SPION response to AC fields is illustrated in figure 2b, where 25 nm SPIONs showed substantially greater response than 14 nm, leading to greater signal intensity. In previous work it has been demonstrated that iron oxide cores of less than 15 nm have significantly lower signal when analyzed by Magnetic Particle Spectrometer (MPS), compared with iron oxide cores larger than 15 nm [US patent 9259492 B2]. In more recent work, the inventors have demonstrated iron oxide cores containing pure (or nearly pure) magnetite (Fe C"4) are suitable in MPS up to about 25 nm diameter and also display superparamagnetic properties when measured with a vibrating sample magnetometer (VSM). Cores containing under-oxidized magnetite (potentially due to a mix of magnetite and some wustite (FeO)) may be suitable up to 33 or 35 nm. [PCT/US2015/058425]

In one embodiment, each nanoparticle includes an iron oxide core comprising iron oxide, wherein the core has a diameter of 15 nm to 35 nm and a coating surrounding the core, the coating comprising a water solubilizing agent including but not limited to a polymer coating.

In one embodiment the mean diameter (as measured by transmission electron microscopy (TEM)) of the iron oxide core of the nanoparticles, is preferably within the following ranges, 15 nm to 35 nm, 18 nm to 30 nm, 20 nm to 30 nm more preferably between 22 nm and 28 nm, and most preferably between 23 and 27 nm.

Polydispersity of iron oxide core diameter

The iron oxide cores of the invention have a measureable distribution. The narrower the size distribution the more similar, the more similar the magnetic response on each nanoparticle in a plurality of nanoparticles. It therefore advantageous to have a narrowly dispersed or monodispersed plurality of iron oxide cores.

Relevant to the monodispersity of the nanoparticles is the large number of nanoparticles that comprise the plurality of nanoparticles. Maintaining monodispersity is important because it provides uniform characteristics that translate to optimized, reproducible and predictable magnetic performance and stability in aqueous, in vitro and in vivo environments. Many physical properties of nanoparticles vary exponentially with particle diameter, with some sizes being well-suited to a particular application and other sizes being ill-suited. Monodisperse samples can be optimized for an application by making all particles very nearly the optimum size. Polydisperse samples cannot be optimized, since they contain both desirable and undesirable sizes. Monodisperse magnetic nanoparticles provide more intense signals, whereas polydisperse magnetic nanoparticles often give broad and lower intensity signal response.

In one embodiment, the cores are monodisperse, as defined by their having a diameter distribution with geometric standard deviation of equal to or less than 1.35 when a log- normal distribution function is used. In another embodiment, the cores are monodisperse, as defined by their having a diameter distribution with geometric standard deviation of equal to or less than 1.22 when a log-normal distribution function is used. In another embodiment, the cores are monodisperse, as defined by their having a diameter distribution with geometric standard deviation of equal to or less than 1.1 when a log- normal distribution function is used. As used herein, the geometric standard deviation of a plurality of nanoparticles is defined as relating to how spread out are the particle diameters in the sample, with 68% of the samples falling between the lower bound set by do/exp (σ) and the upper bound d₀*exp (σ), where d₀ is the median diameter of the distribution and exp (σ) is the geometric standard deviation.

A log-normal distribution may be applied to the data even if the data do not perfectly fit the log-normal distribution. Furthermore, the distribution function may obey other relationships besides a log-normal distribution, including a normal distribution, a bimodal distribution, and any other relationship known to those of skill in the art.

Coating

A coating surrounds the core in order to decrease aggregation between nanoparticles and preserve magnetic characteristics of the core. As used herein, the term "surrounds" includes both complete surface coverage, as well as partial surface coverage. In one embodiment, the coating completely surrounds the core. In another embodiment, the coating partially surrounds the core. In one embodiment, at least a portion of the plurality of nanoparticles comprises a single core surrounded with the coating. The coating provides both physical and magnetic isolation between adjacent nanoparticles. Specifically, the coating minimizes magnetic dipole-dipole interactions between individual nanoparticles, minimizing clustering and aggregation and preserving their nonlinear magnetic response in alternating magnetic fields used in inductive measurement techniques. As a result, the induced signal is quantitative (linear with concentration) and remains unchanged after administration in in vivo environments, thus enabling detection, and quantitation. In one embodiment, the nanoparticle relaxation or magnetic moment reversal of each core is independent of an adjacent nanoparticle.

The migration of the iron oxide nanoparticle through the lymph vessels and into the sentinel lymph node will be affected by the physical characteristics of the coating. In the examples below a coating of PMAO-PEG was utilized for demonstration of the invention. This PMAO-PEG coating has not been fully optimized and it recognized by the inventors that modification to this coating will affect the uptake of the single core iron oxide nanoparticles into the sentinel lymph node. It also is apparent to someone skilled in the art that variations in coating of the iron oxide nanopaticles may increase delivery of iron oxide nanoparticles to the lymph nodes.

In one embodiment, the coating is attached to the core by a mechanism selected from the group consisting of covalent bonding, ionic bonding, van der Waals forces, and hydrophobic/hydrophobic interactions.

In certain embodiments, the core comprises an attachment layer on its surface that provides functionality such that the coating adheres to the core. An exemplary attachment layer is oleic acid, which provides hydrophobic moieties extending from the core surface, which can facilitate hydrophobic-hydrophobic bonding.

The nanoparticles can be formed using any methods known to those of skill in the art. In the exemplary embodiments disclosed herein (in the Examples below), the iron oxide core is synthesized in organic solvents via the thermolysis of iron oleate and then transferred from the organic to aqueous phase using an amphiphilic polymer. Hydrophobic-hydrophilic interactions attach the polymer to the cores in the aqueous phase.

In one embodiment the nanoparticles are coated with an amphiphilic polymer. Those skilled in the art are well able to prepare suitable amphiphilic polymers which are able to solubilize the nanoparticle in an aqueous system. Amphiphilic polymers may include, but are not limited to polymers containing one or more of the following polymers polymaleic anhydride-alt-octadecene, polymaleic anhydride-alt-tetradecene, polymaleic anhydride-alt-isobutylene, polyacrylic acid, polymethacrylic acid, polylactic acid, polyglutamic acid, polyethylene glycol (PEG), methoxy-PEG-OH, methoxyPEG-amine, polypropylene glycol, and polyvinyl alcohol. It is apparent to one skilled in the art that different polymers may be combined to form block polymers. It also is apparent to someone skilled in the art that variations in coating of the iron oxide nanopaticles may increase delivery of iron oxide nanoparticles to the lymph nodes.

In another embodiment the iron oxide nanoparticles can be water solubilized using non- polymeric surfactants which may include, but are not limited to cetyltrimethylammonium bromide (CTAB), polysorbates (Tween™), Sodium dodecyl sulfate (sodium lauryl sulfate), sodium oleate, sodium stearate, lauryl dimethyl amine oxide, polyethoxylated alcohols, poly oxy ethylene sorbitan, octoxynol (Triton XI 00™), N,N- dimethyldodecylamine-N-oxide, hexadecyltrimethylammonium bromide (HTAB), polyoxyl 10 lauryl ether, Brij 721™, bile salts (sodium deoxycholate, sodium cholate), polyoxyl castor oil (Cremophor™), nonylphenol ethoxylate (Tergitol™), cyclodextrins, lecithin, methylbenzethonium chloride (Hyamine™). Additionally, other generic classes of surfactants may be useful, including but not limited to carboxylates, sulphonates, sulfates, alkylbenzenesulphonates, naphthalenesulphonates, alkyl sulphates, pertroleum sulphonates, quarternary ammonium salts, and alkylamines.

In one embodiment the single core iron oxide nanoparticles are a solution or suspension in water. The iron oxide nanoparticles described herein can be formulated in a variety of ways. Those skilled in the art are well able to prepare suitable solutions which control pH, isotonicity and stability. Additives for formulation may include but are not limited to excipients, carriers, buffers, stabilizers, preservatives or anti-oxidants or other materials. It also is apparent to someone skilled in the art that variations in formulation may increase delivery of iron oxide nanoparticles to the lymph nodes. Additionally, it also is apparent to someone skilled in the art that variations in formulation maybe utilized to increase the shelf-life of the nanoparticles.

In one embodiment a solution of the nanoparticles is administered to patients by an injection. The injection of nanoparticle preferably is injected into or near the tumor site.

Magnetic Detection

The following description of magnetic detection is provided to illustrate the invention and does not limit the scope or spirit of the invention to any single detection method or detection device. Magnetic nanoparticles can be detected using a variety of known magnetic devices, which generally follow the principle of applying a magnetic field to magnetize the nanoparticles and subsequently measuring the change in magnetic flux due to the presence of the magnetic nanoparticles. Example magnetic detection devices include, without limitation, vibrating sample magnetometers, AC susceptometers, SQUIDs, the Sentimag^® device, differential magnetometers [S. Waanders, M. Visscher, T. O. B. Oderkerk, H. J. G. Krooshoop, and B. Haken, "Method and apparatus for measuring an amount of superparamagnetic material in an object", Patent application EP20120194029; M. Visscher, S. Waanders, H. J. G. Krooshoop, and B. ten Haken, "Selective detection of magnetic nanoparticles in biomedical applications using differential magnetometry," J Magn Magn Mater ; vol. 365, no. C, pp. 31-39, Sep. 2014.], and magnetic particle spectrometers[S. Biederer, T. Knopp, T. Sattel, Ludtke-Buzug K, B. Gleich, J. Weizenecker, J. Borgert, and T. M. Buzug, "Magnetization response spectroscopy of superparamagnetic nanoparticles for magnetic particle imaging," J Phys D Appl Phys, vol. 42, p. 205007, 2009.], and magnetic particle relaxometers[P. W. Goodwill, A. Tamrazian, L. R. Croft, C. D. Lu, E. M. Johnson, R. Pidaparthi, R. M. Ferguson, A. P. Khandhar, K. M. Krishnan, and S. M. Conolly, "Ferrohydrodynamic relaxometry for magnetic particle imaging," Appl. Phys. Lett., vol. 98, no. 26, pp. 262502-262502, 2011.]. Those of skill in the art will appreciate there are other devices not named herein which may be used, and that innovative magnetometer designs may be developed which fit the description of a magnetic detection device.

In one embodiment the nanoparticles are detected in a patient with a magnetic detection device. In another embodiment the nanoparticles are detected in a patient with a handheld magnetic detection device. In another embodiment the nanoparticles are detected in a patient with magnetic detection device that uses an alternating or AC magnetic field to magnetize the superparamagnetic nanoparticles and measure the resulting change in magnetization (MPS). In another embodiment the nanoparticles are detected in a patient with a magnetic detection device that uses the principle of magnetic susceptometry or AC susceptibility.

In another embodiment the nanoparticles are detected in a patient with a magnetic detection device using the principle of differential magnetometry, wherein a combination of alternating (AC) and static (DC) magnetic fields are applied to magnetize the superparamagnetic nanoparticles. In another embodiment the nanoparticles are detected in a patient using the handheld SentiMag^® device. In another embodiment the nanoparticles are detected in a patient with a handheld device using the differential magnetometry (DiffMag) detection principle.

Dyes

It is common practice to use different dyes during the locating of sentinel lymph nodes. The use of a dye in combination with iron oxide nanoparticles may be useful in the identification of sentinel lymph nodes. In the examples below, the fluorescent dye, Cyanine7.5 was covalently attached to the amphiphilic polymer of the iron oxide nanoparticles. The dye labeled nanoparticles were evaluated in vitro (Table 4) and showed good fluorescence even a low concentration. One of these dye labeled nanoparticle samples (9-189) was evaluated in a mouse model and showed good fluorescence in vivo (Presented in figures 5,6,7 and 8). In one embodiment the solution of nanoparticles may contain a dye. The dye may be detected visually and/or fluorescently and may include, but not limited to the following dyes and their derivatives: isosulphan blue, Patent blue V (PBV), Methylene blue (MB), Indigocarmine (IDC), indocyanine green, IR-820, IR-775, cardiogreen, phthalocyanine, and cyanines. Additionally, one skilled in the art may attach dyes to the nanoparticles via a covalent bond to the amphiphilic polymer, to the iron oxide core via a chelating ligand or utilizing hydrophobic interactions.

In vitro and in vivo evaluation

The present invention demonstrates the potential of superparamagnetic iron oxide nanoparticle (SPION) tracers for SLNB in both in vitro and in vivo studies, the latter using a porcine (mini-pig) model (Figure 4) and a murine model (Figures 5-8)

To demonstrate that single core SPIO tracers are suitable for DiffMag SLNB, a study was performed, in vitro, in which the magnetic signal generated by samples of tracer fluid in the bench-top differential magnetometer was measured. A 3 -fold increase in signal intensity per unit iron compared to Sienna+® was observed. A dilution series was prepared with water and test samples were measured using DiffMag principles, according to a previously published protocol [Visscher M, Waanders S, Krooshoop HJG, Haken ten B. Selective detection of magnetic nanoparticles in biomedical applications using differential magnetometry. J Magn Magn Mater. Elsevier; 2014 Sep 1;365:31—9]. Results of the study are provided in Figure 4a. At all available concentrations, LSL tracers showed 3-4x greater signal than Sienna+^®. These results agree with measurements made using an AC magnetometer (the SentiMag^®), presented in Figure 3, where 4x greater signal intensity per unit iron than Resovist was measured (Resovist^® and Sienna+® are identical and showed the same DiffMag signal in a previous study [Douek M, Klaase J, Monypenny I, Kothari A, Zechmeister K, Brown D, et al. Sentinel Node Biopsy Using a Magnetic Tracer Versus Standard Technique: The SentiMAG Multicentre Trial. Ann Surg Oncol. 2013 Dec 10;21(4): 1237-45]. A preliminary in vivo study in mini-pigs evaluated the suitability of single-core SPIONs for SLNB at the IRCAD Institute, Strasbourg, France. Magnetic tracer (Sienna+^® (Endomagnetics, UK), Resovist^® (Bayer Schering Pharma, DE) or LS-6 (LodeSpin Labs, USA)) was injected subcutaneously into the areola of the left and right 3rd inguinal mammary glands in 13 mini-pigs. The magnetic tracer was injected in 0.5 mL quantities (except for pigs #1, #2, #12 and #13, which were injected with 0.1 mL of tracer material for MRI monitoring of SPIO uptake) with all tracers diluted to an iron concentration of 5 mg/mL. A handheld magnetometer (SentiMag^®, Endomagnetics) was used to perform transcutaneous hotspot measurements prior to surgery. Bilateral sentinel lymph node biopsies were performed 6 hours after injection of the tracer. Using the handheld magnetometer, sentinel lymph nodes containing tracer material were identified and subsequently resected. Signal intensities of these nodes were recorded ex vivo. Resected nodes were fixated in formalin, and the iron content of each sentinel lymph node was quantified according to previously published methods using Vibrating Sample Magnetometry (VSM) and differential magnetometry [Anninga B, et al. Magnetic sentinel lymph node biopsy and localization properties of a magnetic tracer in an in vivo porcine model. Breast Cancer Res Treat. 2013 Aug 17; 141(l):33-42; Douek M, et al. Sentinel Node Biopsy Using a Magnetic Tracer Versus Standard Technique: The SentiMAG Multicentre Trial. Ann Surg Oncol. 2013 Dec 10;21(4): 1237-45; Visscher M, et al. Quantitative Analysis of Superparamagnetic Contrast Agent in Sentinel Lymph Nodes Using Ex Vivo Vibrating Sample Magnetometry. IEEE Trans Biomed Eng. 2013;60(9):2594-602]. Iron amounts were quantified by measuring the magnetic moment of the lymph nodes at saturation (up to 4 Tesla) after subtraction of the linear background. By scaling the recorded saturation magnetization with known reference values, the iron amount present in the lymph node was determined. Signal intensities of the excised nodes were recorded using a standalone Differential Magnetometer (named the superparamagnetic quantifier, or SPAQ), which represents a quantitative measure of the amount of particles present in the lymph node without any necessary background field correction from the (diamagnetic) tissue in which the particles are embedded. In the mini-pig experiments, 6 injections of LodeSpin tracer LS-6 were made and nodes were successfully identified with the SentiMag^® in 5 of the injections. In ex vivo analysis, LS-6 showed lower SentiMag^® signal intensity than controls Sienna+^® and Resovist^® as measured by the SentiMag^® (Figure 5). The LS-6 nodes showed 5-fold higher sensitivity (signal Fe mass) than Sienna+^® with the SentiMag^® device, and 10-fold higher with the DiffMag method. The primary conclusion of this study are the single core SPIO nanoparticle tracers (i.e. LS-6) showed outstanding magnetic behavior for DiffMag detection and SentiMag^® detection, even with lower node uptake.

It is noted that one skilled in the art could modify the coating of the iron oxide nanoparticle to increase lymph node uptake. By modifying the coating, the hydrodynamic size, the surface charge, and hydrophobicity could be varied. These variation would affect the lymph-node uptake.

Iron oxide nanoparticle cores were synthesized by modification of methods according to U.S. Patent Application Publication No. 2013/0149539, the disclosure of which is hereby incorporated by reference in its entirety. The iron oxide nanoparticle cores were coated with amphiphilic polymers using procedures and methods according to U.S. Patent Application Publication No. PCT/US 14/67410 the disclosure of which is hereby incorporated by reference in its entirety.

EXAMPLES

The invention is described in greater detail by the following non-limiting examples.

Materials. 1-Octadecene (tech. 90%), oleic acid (tech. 90%), and iron trichloride hexahydrate (ACS, 97.0-102.0%), triethylamine (99%), and triphenylphosphine (99%) were obtained from Alfa Aesar. Sodium oleate (>97%), benzyltriethylammonium chloride (>98%) and p-toluenesulfonyl chloride (>99%) were obtained from Tokyo Chemical Industry CO, LTD. Poly(maleic anhydride-alt- 1-octadecene) (average Mn 30,000-50,000 Da) was obtained from Sigma-Aldrich. Sodium hydroxide, magnesium sulfate (anhydrous), sodium sulfate (anhydrous) Potassium permanganate (99.2%), and methylene chloride (HPLC grade) were obtained from Fisher Scientific. Hexane (mixture of isomers), chloroform, acetone were HPLC grade and obtained from Sigma- Aldrich. Diethyl ether was obtained from J. T. Baker. Ethanol (200 proof) was obtained from Decon Labs. Phosphoric acid (85%), sulfuric acid were obtained from Macron. Water used in any experiment was purified at 18.2 MOhm-cm. The DigiTrol II was obtained from Sigma-Aldrich. SUBA-SEAL® septum were obtained from Chemglass. Dialysis tubing, Spectra/Por Dialysis Membrane Biotech CE tubing MW Cutoff: 50 kD, flat width = 31 mm, was obtained from Spectrum Laboratories, Inc.

mPEG-NH₂ of various MWs were either purchased from JenKem or produced from the appropriate mPEG-OH by formation of the tosylate, displacement with sodium azide and reduction of the azide to the amine with triphenylphosphine.

Example 1

Synthesis of Iron(III) oleate.

This is a modification of a published procedure ("Ultra-large-scale syntheses of monodisperse nanocrystals" Park, J.; An, K.; Hwang, Y.; Park, J.-G.; Noh, H.-J.; Kim, J - Y.; Park, J.-H; Hwang, N.-M.; Hyeon, T. Nat. Mater. 2004, 3, 891). To a 2-liter three neck round bottom flask equipped with a 1-1/2 x 5/8 inch Teflon coated magnetic stir bar, was added sodium oleate (147.05 g, 483 mmol) and hexanes (500 mL). The flask was equipped with a glass stopper in the left neck, a SUBA-SEAL® septum with a thermocouple in the right neck, and reflux condenser topped with a schlenk line attachment on the center neck. The mixture was stirred to suspend the white powder, then ethanol (300 mL) was added. The slow (30 seconds) addition of water (60 mL) caused nearly all of the solids to dissolve. The reaction vessel was equipped with a heating mantle and heated to 40 °C with stirring, at which point the sodium oleate had completely dissolved. A solution of iron(III) trichloride hexahydrate (43.518 g, 161 mmol) in water (100 mL) was prepared in a 250 mL Erlenmeyer flask with stirring for about 30 minutes, at which time the iron(III) chloride solution had completely dissolved. The iron(III) chloride solution was added to the reaction vessel via a funnel with pre- wetted qualitative filter paper (15 cm) and washed in with water (20 mL). The reaction vessel was purged with argon for 1 minute and heated to a gently reflux (57 °C internal temperature). The reaction was held at reflux and stirring (500 rpm) was maintained for 4 hours. The heating mantle was then removed and the reaction was allowed to cool to 50 °C, then transferred to a 1-liter separately funnel. The bottom layer was drained and the upper red layer was washed with water (3 x 150 mL, 10 second shake period). The organic layer was then transferred to a 1 -liter Erlenmeyer flask containing anhydrous sodium sulfate (50 g). The solution was swirled occasionally for 10 minutes and then filtered through qualitative filter paper into a 2-liter round bottom flask. The solution was concentrated carefully on a rotary evaporator using a water aspirator for vacuum, first at a water bath temperature of 20 °C and then increasing in increments to 30 °C. After solvent removal appeared to have ceased, the vacuum source was switched to high vacuum on the rotary evaporator and concentrating was continued for about 30 minutes at 30 °C bath temperature. After drying on a high vacuum line overnight, the resulting dark red syrup (144.05 g) was deemed to contain 160 mmol of iron(III) oleate and could be divide by mass for use in the nanoparticle synthesis.

Example 2-5 are removed

Example 6

Iron oxide nanoparticle synthesis with high temperature 1% oxygen oxidation and 21.5 hour post-oxidation anneal (9-71).

Oleic acid to Fe ratio: 6.7: 1. To a 1-liter 3-neck heavy walled round bottom flask with 24/40 joints was added the iron(III) oleate (40 mmol, 36.00 g) in 1-octadecene (50 mL), followed by oleic acid (75.70 g, 268 mmol) and 1-octadecene (204 mL). The flask was equipped with a 1-1/2 x 5/8 inch Teflon coated magnetic stir bar, a glass stopper in the center neck, a SUBA-SEAL® septum with a thermocouple in the right neck, and a bump trap topped with an air condenser and schlenk line attachment on the left neck. A DigiTrol II was used to control the heating of the reaction vessel. The glass joints were sealed with a few drops of 1-octadecene. The reaction was heated to 40 °C, held under vacuum and stirred at 450 rpm until bubbling ceased (25 min). The reaction was evacuated and filled with argon five times and then purged with argon for 5 minutes. The upper half of the reaction vessel and the necks were wrapped in foil to reduce water condensation. The set point was changed to 110 °C. After 15 minutes the temperature was 122 °C. The controller was set to ramp at 5 °C/min and the set point was changed to 318 °C. Purging with argon (40 mL/min) was continued via needle through the septum in the right flask neck to aid in the removal of water vapor into the bump trap. When the temperature reached 318 °C the argon purge through the septum was switched to the schlenk line to maintain an atmosphere of argon. The argon purging line and needle were removed from the septum. Approximately 2 to 3 minutes later, the reaction temperature reached 324 °C. Over the next 30 minutes the set point was gradually increased in 2 °C increments to maintain the temperature at 324 °C. After 1 hour 26 minutes since reaching 318 °C, the reaction mixture had darkened and finally turned turbid with the color of milk chocolate indicating particle formation. After an additional 30 minutes the set point was changed to 318 °C. After the reaction had cooled to 318 °C (about 15 minutes), the addition of 1% oxygen in argon was begun at a flow rate of approximately 140 mL/min via a 16 gauge X 6-inch stainless steel needle immersed about ½ inch into the reaction mixture. After 3 hours of 1% oxygen addition the reaction had turned black. The needle was pulled up so the tip of the needle was about 2 inches above the surface of the reaction mixture and the flow rate of 1% oxygen was reduced to about 15 mL/min. The reaction was kept at 318 °C for 26 hours from the time the reaction first reached 318 °C (21.5 hour for the point the 1% oxygen flow was reduced). The heating was turned off and the reaction was allowed to cool to about 300 °C and the heating mantle was then removed to speed cooling. When the reaction mixture was at 50-60 °C the mixture was transfer to a 500 mL bottle with the aid of hexanes (100 mL) which was purge with argon. Analysis of 9-71 : TEM diameter of iron oxide core was 24.3 nm, Fe(II) content was 23.5%. Example 7

Optimized procedure for high quality Magnetite Iron oxide nanoparticle synthesis with high temperature 1% oxygen oxidation and 28.5 hour post-oxidation anneal (9-172). Oleic acid to Fe ratio: 7.3 : 1. To a 1-liter 3-neck heavy walled round bottom flask with 24/40 joints was added the iron(III) oleate (40 mmol, 36.00 g), followed by oleic acid (82.478 g, 304 mmol) and 1-octadecene (200 grams). The flask was equipped with a 1- 1/2 x 5/8 inch Teflon coated magnetic stir bar, a glass stopper in the center neck, a SUBA-SEAL® septum with a thermocouple in the right neck, and a bump trap topped with an air condenser and schlenk line attachment on the left neck. A DigiTrol II was used to control the heating of the reaction vessel. The glass joints were sealed with a few drops of 1-octadecene. The reaction was heated to 50 °C, held under vacuum and stirred at 450 rpm for 18 hours. The reaction was evacuated and filled with argon five times (holding vacuum for 5 minutes each time) and then purged with argon for 5 minutes. The upper half of the reaction vessel and the necks were wrapped in foil to reduce water condensation. The set point was changed to 110 °C. After 15 minutes the internal temperature was 122 °C. The controller was set to ramp at 5 °C/min and the set point was changed to 318 °C. The stir rate was increased to 800 rpm. Purging with argon (40 mL/min) was continued via needle through the septum in the right flask neck to aid in the removal of water vapor into the bump trap. When the temperature reached 318 °C the argon purge through the septum was switched to the schlenk line to maintain an atmosphere of argon. The argon purging line and needle were removed from the septum. Approximately 2 to 3 minutes later, the reaction temperature reached 324 °C. Over the next 30 minutes the set point was gradually increased in 2 °C increments to maintain the temperature at 324 °C. After 1 hour 40 minutes, since reaching 318 °C, the reaction mixture had darkened and finally turned turbid with the color of milk chocolate indicating particle formation. After an additional 30 minutes the set point was changed to 318 °C. After the reaction had cooled to 318 °C (about 15 minutes), the addition of 1% oxygen in argon was begun at a flow rate of approximately 140 mL/min via a 16 gauge X 6-inch stainless steel needle immersed about ½ inch into the reaction mixture. After 3 hours of 1% oxygen in argon addition the reaction had turned black. The needle was pulled up so the tip of the needle was about 2 inches above the surface of the reaction mixture and the flow rate of 1% oxygen was reduced to about 15 mL/min. The stir rate was reduced to about 450 rpm to prevent possible loss of stirring during the night. The reaction was kept at 318 °C for 34 hours from the time the reaction first reached 318 °C (28.5 hours from the point the 1% oxygen flow was reduced). The heating was turned off and the reaction was allowed to cool to about 300 °C and the heating mantle was carefully removed to speed cooling. When the reaction mixture was at 50-60 °C the mixture was transfer to a 500 mL bottle with the aid of hexanes (100 mL) and purged with argon. The above procedure was repeated multiple times (as recorded in Table 1) to provide batches of nanoparticles with a variety of core diameters (Figure 11).

Example 8

Analysis of iron oxide nanoparticle cores

Preparation of samples for TEM analysis and TEM analysis.

A sample for TEM was prepared with 2 mL of reaction mixture added to a 40 mL vial, followed by the addition of hexanes (5 mL) followed by the addition of acetone (10 mL). The vial was placed on the edge of a FeNdB permanent magnet, Grade N51 (3" x 3" x 1") for about 10 minutes. The solution was removed from the resulting black solids and the wash procedure was repeated 2 more times. The black nanoparticles were dissolved in chloroform for preparation for TEM imaging and electron diffraction. TEM size analysis was performed using bright-field imaging, by counting over 1000 particles from several micrographs captured from different regions of the grid. Images were analyzed using the particle size analyzer (PSA rl2) plugin available in the imagej software, to produce histograms of particle diameters, which were fit to a log-normal distribution function. Selected area electron diffraction (SAED) patterns were analyzed using the radial integration tool in imagej .

Titration of iron(II) content in iron oxide nanoparticles.

A washed and dried sample of nanoparticles (40-50 mg) was placed in 40 mL vial that was tightly sealed and purged with argon. To the reaction vessel was added 6 mL of a solution of concentrated sulfuric acid, 85% phosphoric acid and water (1 : 1 : 1). The mixture was sonicated for 5 minutes, then stirred for 30 minutes while resting on a 50 °C hotplate. Hexanes (0.3 mL) was added and the mixture was stirred at 50 °C and periodically sonicated while maintaining positive argon pressure. Once all the nanoparticles had dissolved (2 hours to overnight), additional hexanes (10 mL) was added. The majority of the hexane (upper) layer was carefully removed with a syringe. The remaining aqueous iron solution was titrated with 0.05 N potassium permanganate using a NORM- JET disposable 1 mL syringe. TGA analysis under nitrogen was used to estimate the total iron oxide content of the oleic acid coated nanoparticles, typically about 95%. Note: trace oxygen and undissolved iron oxide nanoparticles both will lower the determined amount of iron (II) in the sample. Nanoparticles titrated: 9-71 : Fe(II) determined to be 23.5% of total Fe

Example 9

Nanoparticle washing procedure in preparation for phase transfer.

Nanoparticles from crude synthesis batch were washed with a mixture of hexanes and ethyl acetate. After separating iron oxide cores with a magnet, supernatant containing excess oleic acid and octadecene was decanted. To iron oxide core pellet, hexane was added and sonicated in water-bath sonicator for 10 minutes. After dispersion in hexanes, acetone and ethyl acetate solvent mixture was added to precipitate nanoparticles and then separated with magnet. Supernatant from the separated nanoparticles was decanted and washing procedure was repeated for an additional 2 times. The final wash was performed using hexanes and acetone. After last wash, iron oxide cores were dried under high vacuum before phase transfer with PMAO-PEG polymer.

Example 10

Synthesis of PMAO (30-50 kDa) loaded 12.5% with mPEG-NFL (20 kDa) (9-04).

To a 25 mL round bottom flask was added PMAO (70 mg), mPEG-NH₂ (MW = 20 kDa, 1.00 g, 0.050 mmol) and dichloromethane (8 mL) followed by triethylamine (0.028 mL). The reaction mixture was stirred for 2 days under argon and then concentrated. The resulting residue was dissolved with water (125 mL) and 30% aqueous sodium hydroxide (0.5 mL) and stirred for 18 hours. Dialysis using 50 kDa MW cut off dialysis tubes was performed against water for 24 hours with 8 water changes. The aqueous polymer solution was lyophilized to give 12.5% (20 kDa) mPEG- H-PMAO (1.143 g).

Example 11

Synthesis of PMAO (30-50 kDa) loaded 25% with mPEG- L (20 kDa) (9-05).

To a 100 mL round bottom flask was added PMAO (70 mg), mPEG-NH₂ (MW = 20 kDa, 2.00 g, 0.100 mmol) and dichloromethane (20 mL) followed by triethylamine (0.056 mL). The reaction mixture became very viscous. The reaction mixture was stirred for 6 days under argon and then concentrated. The resulting residue was dissolved with water (125 mL) and 30% aqueous sodium hydroxide (0.5 mL) and stirred for 18 hours. To get the white residue to completely dissolve an additional portion of water (125 mL) was added and the mixture was stirred for another 2 hours. Dialysis using 50 kDa MW cut off dialysis tubes was performed against water for 24 hours with 8 water changes. The aqueous polymer solution was lyophilized to give 25% (20 kDa) mPEG- H-PMAO (1.895 g).

Example 12

Synthesis of LS-3, 8-107: nanoparticle core 5-87 phase transferred with polymer 9-4. I l l mg of polymer batch 9-04 (PMAO loaded with 12.5% 20 kDa mPEG- H₂; Mn ~ 6.10E5 g/mol) was dissolved in 14.8 mL chloroform (7.5 mg/mL concentration). 10 mg of washed iron oxide nanoparticles (batch 5-87) were dispersed in 1 mL chloroform (10 mg/mL concentration) using a water-bath sonicator. To a 20 mL glass vial containing 6.33 mL of 7.5 mg/mL polymer solution dissolved in chloroform, was added 1 mL of iron oxide nanoparticles dispersed in chloroform. An additional 2.67 mL of chloroform was added to bring the final volume up to 10 mL, which diluted the polymer concentration to 4.75 mg/mL and iron oxide nanoparticle concentration to 1 mg/mL (approximately 234 PMAO-PEG polymer units per iron oxide nanoparticle, assuming 26.3 nm core diameter). The nanoparticle and polymer mixture in chloroform was sonicated for 60 minutes and then stirred using a magnetic stir bar. After 24 hours of stirring, chloroform was evaporated using rotary evaporation, giving a nanoparticle and polymer solid mixture that was dried overnight under high vacuum. To the 20 mL glass vial containing dried nanoparticle and polymer solid mixture, 10 mL of lx TAE buffer was added, and nanoparticle and polymer solid mixture was dispersed by sonication for 60 minutes. After sonication, the solution was checked for any visible aggregates and sonicated for additional time if necessary. The PMAO-PEG coated nanoparticles dispersed in lx TAE buffer were stirred for 24 hours using magnetic stir bar, then filtered with 200 nm nylon syringe filter. To remove excess polymer and salt, filtered PMAO- PEG coated nanoparticles were passed through S-200 sephacryl gel column (GE Healthcare) pre-rinsed with DI water. To remove aggregates, nanoparticles were centrifuged at 5,000 rcf for 15 minutes and supernatant containing aggregate-free nanoparticles was carefully collected. Hydrodynamic diameter (Z-average) = 75 nm; PDI = 0.14

Example 13

Synthesis of LS-4, 8-115: nanoparticle core 9-13 phase transferred with polymer 9-5. 125 mg of polymer batch 9-5 (PMAO loaded with 25% 20 kDa mPEG- H₂; Mn ~ 1.18E6 g/mol) was dissolved in 12.5 mL chloroform (10 mg/mL concentration). 10.0 mg of washed iron oxide nanoparticles (batch 9-13) were dispersed in 1.0 mL chloroform (10 mg/mL concentration) using a water-bath sonicator. To a 40 mL glass vial containing 9.25 mL of 10 mg/mL polymer solution dissolved in chloroform, was added 1.0 mL of iron oxide nanoparticles dispersed in chloroform. The final polymer concentration in the nanoparticle-polymer mixture in chloroform was 9.0 mg/mL and iron oxide nanoparticle concentration was 1.0 mg/mL (approximately 158 PMAO-PEG polymer units per iron oxide nanoparticle, assuming 23.1 nm core diameter). The nanoparticle and polymer mixture in chloroform was sonicated for 60 minutes and then stirred using a magnetic stir bar. After 24 hours of stirring, chloroform was evaporated using rotary evaporation, giving a nanoparticle and polymer solid mixture that was dried overnight under high vacuum. To the 40 mL glass vial containing dried nanoparticle and polymer solid mixture, 10 mL of lx TAE buffer was added, and nanoparticle and polymer solid mixture was dispersed by sonication for 60 minutes. After sonication, the solution was checked for any visible aggregates and, if necessary, sonicated for additional time. The PMAO- PEG coated nanoparticles dispersed in lx TAE buffer were stirred for 24 hours using magnetic stir bar, then filtered with 200 nm nylon syringe filter. To remove excess polymer and salt, filtered PMAO-PEG coated nanoparticles were passed through S-200 sephacryl gel column (GE Healthcare) pre-rinsed with DI water. To remove aggregates, nanoparticles were centrifuged at 5,000 rcf for 15 minutes and supernatant containing aggregate-free nanoparticles was carefully collected. Hydrodynamic diameter (Z- average) = 75 nm; PDI = 0.10

Example 14

Synthesis of LS-5, 8-123 : nanoparticle core 9-32 phase transferred with 9-5.

83.3 mg of polymer batch 9-5 (PMAO loaded with 25% 20 kDa mPEG- H₂; Mn ~ 1.18E6 g/mol) was dissolved in 8.3 mL chloroform (10 mg/mL concentration). 5.0 mg of washed iron oxide nanoparticles (batch 9-32) were dispersed in 0.5 mL chloroform (10 mg/mL concentration) using a water-bath sonicator. To a 40 mL glass vial containing 5.4 mL of 10 mg/mL polymer solution dissolved in chloroform, was added 0.5 mL of iron oxide nanoparticles dispersed in chloroform. The final polymer concentration in the nanoparticle-polymer mixture in chloroform was 9.15 mg/mL and iron oxide nanoparticle concentration was 1.0 mg/mL (approximately 200 PMAO-PEG polymer units per iron oxide nanoparticle, assuming 23.7 nm core diameter). The nanoparticle and polymer mixture in chloroform was sonicated for 60 minutes and then stirred using a magnetic stir bar. After 24 hours of stirring, chloroform was evaporated using rotary evaporation, giving a nanoparticle and polymer solid mixture that was dried overnight under high vacuum. To the 40 mL glass vial containing dried nanoparticle and polymer solid mixture, 5 mL of DI water was added, and nanoparticle and polymer solid mixture was dispersed by sonication for 60 minutes. After sonication, the solution was checked for any visible aggregates and, if necessary, sonicated for additional time. The PMAO-PEG coated nanoparticles dispersed in lx TAE buffer were stirred for 24 hours using magnetic stir bar, then filtered with 200 nm nylon syringe filter. To remove excess polymer, filtered PMAO-PEG coated nanoparticles were passed through S-200 sephacryl gel column (GE Healthcare) pre-rinsed with DI water. To remove aggregates, nanoparticles were centrifuged at 5,000 rcf for 15 minutes and supernatant containing aggregate-free nanoparticles was carefully collected. Hydrodynamic diameter (Z-average) = 74 nm; PDI = 0.18

Example 15

Synthesis of LS-6, 8-144: nanoparticle core 9-71 phase transferred with 9-5.

283 mg of polymer batch 9-5 (PMAO loaded with 25% 20 kDa mPEG- H2; Mn ~ 1.18E6 g/mol) was dissolved in 14.2 mL chloroform (20 mg/mL concentration). 28.3 mg of washed iron oxide nanoparticles (batch 9-71) were dispersed in 2.83 mL chloroform (10 mg/mL concentration) using a water-bath sonicator. To a 40 mL glass vial containing 14.15 mL of 20 mg/mL polymer solution dissolved in chloroform, was added 2.83 mL of iron oxide nanoparticles dispersed in chloroform. An additional 11.3 mL of chloroform was added, which diluted the final polymer concentration in the mixture down to 10 mg/mL and iron oxide nanoparticle concentration to 1.0 mg/mL (approximately 244 PMAO-PEG polymer units per iron oxide nanoparticle, assuming 26 nm core diameter). The nanoparticle and polymer mixture in chloroform was sonicated for 60 minutes and then stirred using a magnetic stir bar. After 24 hours of stirring, chloroform was evaporated using rotary evaporation, giving a nanoparticle and polymer solid mixture that was dried overnight under high vacuum. To the 40 mL glass vial containing dried nanoparticle and polymer solid mixture, 28 mL of lx TAE buffer was added, and nanoparticle and polymer solid mixture was dispersed by sonication for 60 minutes. After sonication, the solution was checked for any visible aggregates and, if necessary, sonicated for additional time. The PMAO-PEG coated nanoparticles dispersed in lx TAE buffer were stirred for 24 hours using magnetic stir bar, then filtered with 200 nm nylon syringe filter. To remove excess polymer and salt, filtered PMAO-PEG coated nanoparticles were passed through S-200 sephacryl gel column (GE Healthcare) pre- rinsed with DI water. To remove aggregates, nanoparticles were centrifuged at 5,000 rcf for 15 minutes and supernatant containing aggregate-free nanoparticles was carefully collected. Hydrodynamic diameter (Z-average) = 100 nm; PDI = 0.14

Example 16

Synthesis of LS-8, 10-6: nanoparticle core 9-71 phase transferred with 9-153.

1.32 g of polymer from batch 9-153 (PMAO loaded with 18.75% 20 kDa mPEG- H2; Mn ~ 895,000 g/mol) was dissolved in 66 mL chloroform (20 mg/mL concentration). 155 mg of washed iron oxide nanoparticles (batch 9-71) were dispersed in 15.5 mL chloroform (10 mg/mL concentration) using a water-bath sonicator. To a single-neck 500 mL round bottom flask containing 66 mL of 20 mg/mL polymer solution dissolved in chloroform, was added 15.5 mL of 10 mg/ml iron oxide nanoparticles dispersed in chloroform. An additional 73.6 mL chloroform was added, which diluted the final polymer concentration in the mixture down to 8.50 mg/mL and iron oxide nanoparticle concentration to 1.0 mg/mL (approximately 207 PMAO-PEG polymer units per iron oxide nanoparticle, assuming 26 nm core diameter). The nanoparticle and polymer mixture in chloroform was sonicated for 120 minutes and then stirred using a mechanical stirrer. After 48 hours of stirring, 10 ml of P+polymer mixture in chloroform was transferred to a single-neck 100 ml pear shaped flask (Chemglass) and the solvent was evaporated using rotary evaporation, giving a nanoparticle and polymer solid mixture that was dried overnight under high vacuum. To the pear flask containing dried nanoparticle and polymer solid mixture, 10 mL of DI water was added, and nanoparticle and polymer solid mixture was dispersed by sonication for 90 minutes at 40-50 °C water bath temperature. After sonication, the solution was checked for any visible aggregates and, if necessary, sonicated for additional time. After completing sonication, PMAO-PEG coated nanoparticles were filtered with 200 nm nylon syringe filter. To remove excess polymer, filtered PMAO-PEG coated nanoparticles were passed through S-200 sephacryl gel column (GE Healthcare) pre-rinsed with DI water. To remove aggregates, nanoparticles were centrifuged at 5,000 rcf for 20 minutes and supernatant containing aggregate-free nanoparticles was carefully collected. Hydrodynamic diameter (Z- average) = 79 nm; PDI = 0.10

Example 17

Cy7.5 fluorescent dye conjugation to sample LS-08 (10-6).

Attachment of the fluorescent dye, cyanine7.5 amine (from lumiprobe), to LS-08. In the following procedures three differently loadings of dye on LS-08 were prepared:

Synthesis of 9-188. Dye to polymer molar ratio (10: 1). To a 1.5 mL vial was added an aqueous solution of LS-008 (480 uL). A solution of cyanine7.5 amine (from lumiprobe) in DMF (75 uL at 2 mg/mL) was added. A solution of EDC in DMF (36 uL at 10 mg/mL) was added. The vial was protected from light with foil and stirred overnight.

Synthesis of 9-189. Dye to polymer molar ratio (20: 1). To a 1.5 mL vial was added an aqueous solution of LS-008 (480 uL). A solution of cyanine7.5 amine (from lumiprobe) in DMF (150 uL at 2 mg/mL) was added. A solution of EDC in DMF (72 uL at 10 mg/mL) was added. The vial was protected from light with foil and stirred overnight.

Synthesis of 9-190. Dye to polymer molar ratio (5: 1). To a 1.5 mL vial was added an aqueous solution of LS-008 (480 uL). A solution of cyanine7.5 amine (from lumiprobe) in DMF (37.5 uL at 2 mg/mL) was added. A solution of EDC in DMF (18 uL at 10 mg/mL) was added. The vial was protected from light with foil and stirred overnight.

Each of the above samples (9-188, 9-189, and 9-190) were purified, to remove any unbound dye, using sephacryl S-200 size exclusion columns and eluted with deionized water. The resulting samples were concentrated by spinning at 5,000 rcf for 30 minutes in 30,000 MWCO centrifuge filtration tubes. After centrifugation, the concentrated nanoparticle supernatant was collected. Serial dilutions of the three samples, including Cy7.5 and sample 10-6 for positive and negative controls, respectively, were analyzed in a phantom (96-well plate; see Table 4) using a Lumina II (Caliper LifeSciences) in vivo imaging system (IVIS) equipped with a near-infrared camera. Sample 9-189 was selected for further evaluation in a murine (mouse) model.

Example 18

Sentinel Lymph Node Biopsy in Porcine model using magnetic tracer, LS-6.

A complete description of the procedure for evaluating test nanoparticles for sentinel lymph node biopsy in mini pigs is described in [Anninga, B., Ahmed, M., Van Hemelrijck, M., Pouw, J., Westbroek, D., Pinder, S., et al. (2013). Magnetic sentinel lymph node biopsy and localization properties of a magnetic tracer in an in vivo porcine model. Breast Cancer Research and Treatment, 141(1), 33-42. doi: 10.1007/sl0549-013- 2657-0] which is hereby incorporated by reference. The following paragraphs summarize the testing procedure.

We performed a preliminary in vivo study in mini-pigs to evaluate the suitability of sample LS-6 for SLNB at the IRCAD Institute, Strasbourg, France. Magnetic tracer (Sienna+^® (Endomagnetics, UK), Resovist^® (Bayer Schering Pharma, Germany) or LS-6 (LodeSpin Labs, USA)) was injected subcutaneously into the areolar of the left and right 3rd inguinal mammary glands in 13 mini -pigs. Bilateral sentinel node biopsies were performed followed by bilateral groin node clearance. We used 3 mini-pigs (6 sentinel node procedures) for each tracer dose evaluated. The iron oxide nanoparticles, LS-6, contained 25nm iron oxide cores and coated with a 20kDa MW PEG-PMAO, with 25% of available carboxylate sites loaded with PEG.

The magnetic tracer was injected in 0.5 mL quantities (except for pigs #1, #2, #12 and #13, which were injected with 0.1 mL of tracer material for MRI monitoring of SPIO uptake), with all tracers diluted to an iron concentration of 5mg/mL (2.5 mgFe injected). A handheld magnetometer (SentiMag^®, Endomagnetics) was used to perform transcutaneous hotspot measurements prior to surgery. Bilateral sentinel lymph node biopsies were performed 6 hours after injection of the tracer. Using the handheld magnetometer, sentinel lymph nodes containing tracer material were identified and subsequently resected. Signal intensities of these nodes were recorded ex vivo. Resected nodes were fixated in formalin and transported to the Universiteit Twente, where the iron content of each sentinel lymph node was quantified using Vibrating Sample Magnetometry (VSM), following the procedure described in [Visscher M, Pouw JJ, van Baarlen J, Klaase JM, Haken ten B. Quantitative Analysis of Superparamagnetic Contrast Agent in Sentinel Lymph Nodes Using Ex Vivo Vibrating Sample Magnetometry. IEEE T Bio-Med Eng; 60(9):2594-602]. Iron amounts were quantified by measuring the magnetic moment of the lymph nodes at saturation (up to 4 Tesla) after subtraction of the linear background. By scaling the recorded saturation magnetization with known reference values, the iron amount present in the lymph node was determined.

Example 19

Evaluation of sample 9-189 in a mouse lymph node model.

We performed a preliminary lymph node uptake study in mice. Sample 9-189 at 2 gFe/L dispersed in IX PBS was injected subcutaneously in the rear footpad of mice. Injection volume per footpad was 10-20 μΐ, delivering approximately 20-40 μgFe.

Experimental procedure: Two female CD-I mice (7 weeks old) were used for this preliminary study. Prior to injection, mice were anesthetized with 3.5% isoflurane and maintained at 2% isoflurane with a nose cone during injection.

Mouse# 1 was subcutaneously injected with 20 μΐ of 9-189 (2 gFe/L) in each rear footpad, delivering 40 μgFe per footpad. The hind legs were gently massaged for approximately 2 minutes to promote lymphatic flow. Mouse 1 was placed in the Lumina II IVIS (Caliper LifeSciences) and imaged at 15, 18, 23, 28 and 33 minutes after the injection time (Figure 5 and Figure 6). Near-infrared imaging was done using a 745 nm excitation wavelength and 810-875 nm emission filter. After the last imaging time-point, mouse was euthanized with C0₂ overdose and cervical dislocation. Popliteal and iliac lymph nodes (both left and right) were collected for magnetometer (Figure 9 and Table 3) and histology analysis. Mouse# 2 was subcutaneously injected with -10 μΐ of 9-189 (2 gFe/L) in the rear right footpad, delivering 20 μgFe in the footpad. The left footpad was not injected and used as a control. The hind legs were gently massaged for approximately 2 minutes to promote lymphatic flow. Mouse 2 was placed in the Lumina II IVIS (Caliper LifeSciences) and imaged at 10, 12, 17, 22 and 27 minutes after the injection time (Figure 7 and Figure 8). A 745 nm excitation wavelength and 810-875 nm emission filter was used for imaging. Popliteal nodes (both left and right) were collected for magnetometer (Figure 10 and Table 3) and histology analysis.

Example 20

Analysis of water soluble, phase transferred iron oxide nanoparticles.

All water soluble phase transferred iron oxide nanoparticles were analyzed using dynamic light scattering (DLS), magnetic particle spectroscopy (MPS) and vibrating sample magnetometry (VMS). Properties of water-dispersible nanoparticles are provided in Table 2. Magnetic diameter of water soluble samples was estimated from magnetic measurements of magnetization vs field, by fitting to a Langevin function and assuming a traditional log-normal distribution function. The method is described in [R. W. Chantrell, J. Popplewell, and S. W. Charles, "Measurements of particle size distribution parameters in ferrofluids," IEEE Transactions on Magnetics, vol. 14, no. 5, pp. 975-977, 1978.]

While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

Table 1. Properties of selected SPION samples

Table 2. Properties of water-dispersible SPIONs with PMAO-PEG coating

Table 3. Vibrating sample magnetometry data for iron quantification in lymph nodes.

Mouse 1 data Mouse 2 data

Table 4. Fluorescent signals (Total flux [p/s]) from 96-well plate imaged in a Lumina II IVIS (Caliper LifeSciences). The plate contained known concentrations of Cy7.5 dye, nanoparticles without dye (sample 10-6), and nanoparticle samples conjugated with various amounts of Cy7.5 dye (9-188, 9-189, and 9-190).

Claims

1. A method for locating a sentinel lymph node, comprising:

a. injection of tissue with an aqueous solution containing a plurality of single core iron oxide nanoparticles, wherein the iron oxide core of the iron oxide nanoparticles has a median diameter between 15 and 35 nm

b. detection of the iron oxide nanoparticles with a magnetic detection device

2. The method of claim 1, wherein the iron oxide nanoparticles median diameter is between 20 and 35 nm

3. The method of claim 1, wherein the iron oxide core of the iron oxide nanoparticles median diameter is between 22 and 32 nm

4. The method of claim 1, wherein the iron oxide core of the iron oxide nanoparticles median diameter is between 23 and 28 nm.

5. The method of claim 1, wherein at least 50% of the mass of the iron oxide core of the nanoparticles is composed of magnetite.

6. The method of claim 1, wherein the iron oxide core of the nanoparticles is composed of between 0 and 50% iron(II) based on total iron.

7. The method of claim 1, wherein the iron oxide core of the nanoparticles is composed of between 20 and 40% iron(II) based on total iron.

8. The method of claim 1, wherein the iron oxide core of the nanoparticles is composed of between 20 and 32% iron(II) based on total iron.

9. The method of claim 1, wherein the single core iron oxide nanoparticles have a hydrodynamic size, of between 30 nm and 150 nm, as measured by dynamic light scattering using the Z average.

10. The method of claim 1, wherein the single core iron oxide nanoparticles has a hydrodynamic size, as measured by dynamic light scattering, of between 40 nm and 110 nm.

11. The method of claim 1, wherein the injection contains a dye.

12. The method of claim 1, wherein the injection contains a dye which is attached to the coating of the iron oxide nanoparticles.

13. The method of claims 11 and 12, wherein the dye is a fluorescent dye.

14. The method of claims 11 and 12, wherein the dye is a fluorescent dye which has an emission wavelength of between 500 nm and 1200 nm.

15. The method of claim 1, wherein the magnetic detection device is a handheld magnetic detection device.

16. The method of claim 1, wherein the magnetic detection device uses an alternating or AC magnetic field to magnetize the superparamagnetic nanoparticles and measure the resulting change in magnetization (MPS).

17. The method of claim 1, wherein the magnetic detection device uses the principle of magnetic susceptometry or AC susceptibility.

18. The method of claim 1, wherein the magnetic detection device uses the principle of differential magnetometry, wherein a combination of alternating (AC) and static (DC) magnetic fields are applied to magnetize the superparamagnetic nanoparticles.

19. The method of claim 1, wherein the magnetic detection device is the handheld SentiMag^® device.

20. The method of claim 1, wherein the magnetic detection device is a handheld device using the differential magnetometry (DiffMag) detection principle.

21. The method of claim 1, wherein the tissue injected is tumor bearing tissue.

22. The method of claim 1, wherein the method is performed on a human subject or patient.

23. The method of claim 1, wherein the method comprises an addition step of surgical biopsy.

24. The method of claim 1, wherein the method comprises an addition step of surgical removal of one or more lymph nodes

25. The method of claim 1, wherein the method comprises an addition step of surgical removal of tumor tissue.