The authors gratefully acknowledge Professor Klaus Hantke (University of T&#252;bingen, Germany) for kindly supply the Yersinia enterocolitica strains used in this work. This work was supported by grants AGL2015-63740-C2-1/2-R and RTI2018-093634-B-C21/C22 (AEI/FEDER, EU) from the State Agency for Research (AEI) of Spain, co-funded by the FEDER Programme from the European Union. Work in University of Santiago de Compostela and University of A Coru&#241;a was also supported by grants GRC2018/018, GRC2018/039, and ED431E 2018/03 (CICA-INIBIC strategic group) from Xunta de Galicia. Finally, we want to thank to Nuria Calvo for her great collaboration doing the voice-off this video protocol.

NOTE: For the reactions performed under inert atmosphere conditions, all the glassware was previously dried in an oven at 65 &#176;C, sealed with a rubber septum and purged with argon three times.
1. Synthesis of magnetic nanoparticles conjugated with feroxamine
<ol>
	<li>Synthesis of Fe3O4 magnetic nanoparticles (MNPs)
	<ol>
		<li>Add 0.5 g of Fe(acac)3 in a 20 mL glass vial and then mix with 10 mL of benzyl alcohol.</li>
		<li>Sonicate this mixture for 2 min, then transfer to a heating block and heat at 180 &#176;C for 72 h.</li>
		<li>After the reaction is completed, allow the vials to cool down, rinse the nanoparticles with 96% ethanol and centrifuge at 4000 x g for 30 min. Repeat the centrifugation at least twice.</li>
		<li>Separate the nanoparticles from the supernatant by magnetic attraction using a neodymium (NdFeB) magnet and discard the residual solvent.</li>
		<li>Rinse with 96% ethanol repeating step 1.1.4. and discard the supernatant alternating with sonication in a bath for 1 min at 40 kHz until the solvent looks clear.</li>
	</ol>
	</li>
	<li>Magnetic nanoparticles SiO2 coating (MNP@SiO2)
	<ol>
		<li>Prepare a suspension of 2 g of MNP in 80 mL of isopropanol and then add 4 mL of 21% ammonia, 7.5 mL of distilled water and 0.56 mL of tetraethyl orthosilicate (TEOS) (in this order) in a round bottom flask with a magnetic stir bar.</li>
		<li>Heat the mixture at 40 &#176;C for 2 h with continuous stirring and then sonicate for 1 h.</li>
		<li>Separate the MNP with a magnet, discard the supernatant, and disperse it in 30 mL of isopropanol.</li>
		<li>Repeat steps 1.2.1. and 1.2.2.</li>
		<li>Remove and wash the magnetic stir bar with 96% ethanol to recover all the material.</li>
		<li>Separate the nanoparticles from the supernatant by magnetic attraction using a magnet.</li>
		<li>Discard the supernatant and rinse the nanoparticles with 96% ethanol three times alternating with sonication.</li>
		<li>Dry the nanoparticles under vacuum at room temperature for 12 h.</li>
	</ol>
	</li>
	<li>Functionalization of MNP@SiO2 with (3-aminopropyl)triethoxysilane (APTES)
	<ol>
		<li>Rinse 500 mg of the MNP@SiO2 obtained from the previous step with N,N-dimethylformamide (DMF) under inert atmosphere and then sonicate for 1 min at 40 kHz. Then, discard the supernatant and repeat this process three times.</li>
		<li>Re-suspend the particles in a round bottom flask, under stirring with a magnetic stir bar and add 9 mL of APTES.</li>
		<li>Stir the mixture at 60 &#176;C for 12 h.</li>
		<li>Discard the supernatant and rinse the nanoparticles with 96% ethanol three times alternating with sonication.</li>
	</ol>
	</li>
	<li>Synthesis of feroxamine
	<ol>
		<li>Dissolve 100 mg (0.15 mmol) of deferoxamine mesylate salt and 53.0 mg (0.15 mmol) of Fe(acac)3 in 5 mL of distilled water and stir the mixture overnight at room temperature.</li>
		<li>Wash the resulting product three times with 20 mL of EtOAc in a separation funnel and then, remove the organic solvent under vacuum using a rotatory evaporator.</li>
		<li>Freeze-dry the aqueous phase to afford feroxamine as a red solid.</li>
	</ol>
	</li>
	<li>Synthesis of N-succinylferoxamine
	<ol>
		<li>Add 350 mg (3.50 mmol) of succinic anhydride to a solution of 100 mg (0.17 mmol) of feroxamine in 5 mL of pyridine in a 50 mL round bottomed flask under inert atmosphere.</li>
		<li>Stir the resulting mixture at room temperature for 16 h. After that time, remove the excess of pyridine under reduced pressure in a rotatory evaporator to give a dark red solid.</li>
		<li>Dissolve the reaction crude in 3 mL of methanol.</li>
		<li>Transfer the methanolic solution into a Sephadex column (20 cm of Sephadex in a 20 mm diameter column) and elute at 0.5 mL/min.</li>
		<li>Collect the red fraction and remove the methanol under vacuum using a rotatory evaporator.</li>
	</ol>
	</li>
	<li>Synthesis of the conjugate MNP@SiO2@NH@Fa
	<ol>
		<li>Rinse 30 mg of dry MNP@SiO2@NH2 twice with DMF and sonicate the nanoparticles in a 100 mL Erlenmeyer flask for 30 min under inert atmosphere.</li>
		<li>Prepare a solution of N-succinylferoxamine (200 mg, 0.30 mmol), benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP, 173 mg, 0.45 mmol), 1-hydroxybenzotriazole (HOBt, 46 mg, 0.39 mmol) and N,N-diisopropylethylamine (DIPEA, 128.8 mg, 1.21 mmol) in 10 mL of DMF (Mix A) in a 50 mL round-bottomed flask under inert atmosphere.</li>
		<li>Suspend the previously rinsed MNP@SiO2@NH2 in 3 mL of DMF under sonication in dry under oxygen free conditions using an argon gas atmosphere (Mix B).</li>
		<li>Add mix A to mix B dropwise.</li>
		<li>Shake the final mixture using an orbital shaker at room temperature overnight.</li>
		<li>Separate the resulting conjugate (MNP@SiO2@NH@Fa) from the suspension using a magnet.</li>
		<li>Rinse the resulting solid and then, sonicate it five times with 10 mL of ethanol.</li>
		<li>Dry the solid under vacuum for 24 h.</li>
	</ol>
	</li>
</ol>
2. Bacterial assay with Y. enterocolitica strains to quantify the capture of pathogenic bacteria with nanoparticles
<ol>
	<li>Prepare a suspension of all the intermediate nanoparticles and the final conjugate in PBS at 1 mg/mL in sterile 2 mL tubes.</li>
	<li>Prepare a culture of Y. enterocolitica in 5 mL of Luria Bertani (LB) broth overnight incubating at 37 &#176;C.</li>
	<li>Prepare a 5 mL of iron deficient tryptic soy broth (TSB) by adding 50 &#181;L of 10 mM 2,2&#8242;-bipyridyl.</li>
	<li>Inoculate the 5 mL of iron deficient TSB with 50 &#181;L of the overnight culture of Y. enterocolitica and then, incubate at 37 &#176;C with agitation until an OD600 = 0.5&#8210;0.8 is reached.</li>
	<li>Take 100 &#181;L of the culture obtained in the step 2.4 and dilute in a 2.0 mL tube containing 900 &#181;L of PBS to obtain a first 1/10 dilution. Then, prepare a 1/100 dilution from the first dilution using the same procedure to get a concentration of bacterial cells at 1 x 106 Colony Forming Units (CFU)/mL approximately.</li>
	<li>Add 100 &#181;L of nanoparticles suspension at 1 mg/mL to 1 mL of the 1/100 dilution of bacterial suspension in a 2.0 mL tube, and homogenize with vortex.</li>
	<li>Incubate the culture at 20 &#176;C for 1 h.</li>
	<li>Separate the MNP/bacteria aggregates by using a magnet and carefully discard the supernatant.</li>
	<li>Rinse the separated nanoparticles twice with 1 mL PBS using a vortex.</li>
	<li>Suspend the nanoparticles in 1 mL of PBS to count the amount of bacterial capture in CFU/mL.</li>
	<li>Prepare four successive 1/10 dilutions from the former suspension until a 1 x 10-4 dilution is reached.</li>
	<li>Plate 10 &#181;L of each dilution onto TS agar plates and incubate them at 37 &#176;C overnight.</li>
	<li>Photograph the plate with a gel digitalizer in epi white mode. Process the image with an appropriate software to amplify a spot to count the number of individual colonies. 
	NOTE: Each MNP intermediate was characterized to follow up the progress of the synthesis. Firstly, bare MNPs were studied by XRD to check the crystalline structure. Then, the FT-IR spectrum of each intermediate was run to check the changes that occurred in the corresponding reaction. Raman spectroscopy analysis of each intermediate was also carried out in order to confirm the conclusions deduced from FT-IR spectra. TGA analysis allowed us to estimate the loss weight of the intermediates bearing organic material in its structure. The morphology and size of each intermediate was studied by TEM. Finally, XPS analysis was critical to determine the atom oxidation states at each MNP intermediate surface and to confirm the covalent bond formation in the conjugate MNP@SiO2@NH@Fa.</li>
</ol>

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We have nothing to disclose.

This protocol describes the synthesis of a conjugate between magnetic nanoparticles and the siderophore feroxamine by covalent bonding. The synthesis of magnetite was carried out using the protocol reported by Pinna et al.5 followed by silica coating to protect the magnetic core of corrosion in aqueous systems, to minimize the aggregation and to provide a suitable surface for functionalization6. The silica coating process was modified. Instead of carrying out three coatings...

The bacteria detection methods using MNP are based on the molecular recognition of antibodies, aptamers, bioprotein, carbohydrates conjugated to MNP by the pathogenic bacteria1. Taking into account that siderophores are recognized by specific receptors on the outer membrane of bacteria, they could also linked to MNP to increase their specificity2. Siderophores are small organic molecules involved in the Fe3+ uptake by bacteria3,4. The preparation of conjugates between siderophores and MNP along with their evaluation for the capture and isolation of bacteria has not yet been reported.
One of the crucial steps in the synthesis of conjugates of magnetic nanoparticles with small molecules is the selection of the type of bond or interaction between them to ensure that the small molecule is attached to the surface of the MNP. For this reason, the procedure to prepare the conjugate between magnetic nanoparticles and feroxamine&#8212;the siderophore recognized by Yersinia enterocolitica&#8212;was focused at the generation of a modifiable surface of the MNP to allow linking it covalently to the siderophore by carbodiimide chemistry. In order to get an uniform magnetite nanoparticles (MNP) and to improve nucleation and size control, a solvolysis reaction with benzyl alcohol was carried in a thermal block without shaking5. Then, a silica coating was generated by St&#246;ber method to confer protection and improve the stability of the nanoparticles suspension in aqueous media6. Taking into account the structure of the feroxamine, the introduction of amine groups is necessary to produce suitable nanoparticles (MNP@SiO2@NH2) to be conjugated with the siderophore. This was achieved by condensation of (3-aminopropyl)triethoxysilane (APTES) with the alcohol groups present on the surface of the silica modified nanoparticles (MNP@SiO2) using a sol-gel method7.
In parallel, the feroxamine iron(III) complex was prepared by complexation of the commercially available deferoxamine with iron acetyl acetonate in aqueous solution. N-succinylferoxamine, bearing succinyl groups that will act as linkers, was obtained by the reaction of feroxamine with succinic anhydride.
The conjugation between MNP@SiO2@NH2 and N-succinylferoxamine to give MNP@SiO2@NH@Fa was carried out through carbodiimide chemistry using as coupling reagents benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP) and 1-hydroxybenzotriazole (HOBt) in a soft basic media to activate the terminal acid group in N-succinylferoxamine8.
Once the MNPs were characterized, we evaluated the capabilities of bare and functionalized magnetic nanoparticles to capture wild type (WC-A) and a mutant of Y. enterocolitica lacking feroxamine receptor FoxA (FoxA WC-A 12-8). Plain MNPs, functionalized MNPs and the conjugate MNP@SiO2@NH@Fa were allowed to interact with each Y. enterocolitica strain. The bacteria-conjugate aggregates were separated from the bacteria suspension by the application of a magnetic field. The separated aggregates were rinsed twice with phosphate buffered saline (PBS), re-suspended in PBS to prepare serial dilutions and then, they were plated for colony counting. This protocol demonstrates each step of the synthesis of MNP@SiO2@NH@Fa, the structural characterization of all the intermediates and the conjugate, and a bacterium capture assay as an easy way to evaluate the specificity of the conjugate in relation to the intermediates.9

<table><tbody><tr><td>1-Hydroxybenzotriazole hydrate&lt;br/&gt; HOBT</td><td>Acros</td><td>300561000</td><td></td></tr><tr><td>2,2&amp;prime;-Bipyridyl</td><td>Sigma Aldrich</td><td>D216305</td><td></td></tr><tr><td>3-Aminopropyltriethoxysilane 99%</td><td>Acros</td><td>151081000</td><td></td></tr><tr><td>Ammonium hydroxide solution 28% NH&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>338818</td><td></td></tr><tr><td>Benzotriazol-1-yloxytris(dimethylamino)-phosphonium hexafluorophosphate BOP Reagent</td><td>Acros</td><td>209800050</td><td></td></tr><tr><td>Benzyl alcohol</td><td>Sigma Aldrich</td><td>822259</td><td></td></tr><tr><td>Deferoxamine mesylate salt &gt;92,5% (TLC)</td><td>Sigma Aldrich</td><td>D9533</td><td></td></tr><tr><td>Ethanol, anhydrous, 96%</td><td>Panreac</td><td>131085</td><td></td></tr><tr><td>Ethyl Acetate, Extra Pure, SLR, Fisher Chemical</td><td></td><td></td><td></td></tr><tr><td>Iron(III) acetylacetonate 97%</td><td>Sigma Aldrich</td><td>F300</td><td></td></tr><tr><td>LB Broth (Lennox)</td><td>Sigma Aldrich</td><td>L3022</td><td></td></tr><tr><td>N,N-Diisopropylethylamine, 99.5+%, AcroSeal</td><td>Acros</td><td>459591000</td><td></td></tr><tr><td>N,N-Dimethylformamide, 99.8%, Extra Dry, AcroSeal</td><td>Acros</td><td>326871000</td><td></td></tr><tr><td>Pyridine, 99.5%, Extra Dry, AcroSeal</td><td>Acros</td><td>339421000</td><td></td></tr><tr><td>Sephadex LH-20</td><td>Sigma Aldrich</td><td>LH20100</td><td></td></tr><tr><td>Succinic anhydride &gt;99%</td><td>Sigma Aldrich</td><td>239690</td><td></td></tr><tr><td>Tetraethyl orthosolicate &gt;99,0%</td><td>Sigma Aldrich</td><td>86578</td><td></td></tr></tbody></table>

synthesis of functionalized magnetic nanoparticles, their conjugation with the siderophore feroxamine and its evaluation for bacteria detection

In the present work, the synthesis of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with (3-aminopropyl)triethoxysilane (APTES) and its conjugation with deferoxamine, a siderophore recognized by Yersinia enterocolitica, using a succinyl moiety as a linker are described.
Magnetic nanoparticles (MNP) of magnetite (Fe3O4) were prepared by solvothermal method and coated with SiO2 (MNP@SiO2) using the St&#246;ber process followed by functionalization with APTES (MNP@SiO2@NH2). Then, feroxamine was conjugated with the MNP@SiO2@NH2 by carbodiimide coupling to give MNP@SiO2@NH2@Fa. The morphology and properties of the conjugate and intermediates were examined by eight different methods including powder X-Ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM) and energy dispersive X-Ray (EDX) mapping. This exhaustive characterization confirmed the formation of the conjugate. Finally, in order to evaluate the capacity and specificity of the nanoparticles, they were tested in a capture bacteria assay using Yersinia enterocolitica.

An exhaustive structural characterization is carried out in order to determine the morphology and the properties of each intermediate and the final conjugate. For this purpose, the techniques XRD, FT-IR, Raman spectroscopy, TGA, TEM, EDX mapping and XPS are used in order to demonstrate the formation of the conjugate. The oxidation states of the atoms at the surface of the nanoparticles acquired by X-ray photoelectron spectroscopy (XPS) are the most relevant data to confirm the formation of covalent bonds between the nano...

Watch this Scientific Journal Video about Synthesis of Functionalized Magnetic Nanoparticles, Their Conjugation with the Siderophore Feroxamine and its Evaluation for Bacteria Detection at JoVE.com

Synthesis of Functionalized Magnetic Nanoparticles, Their Conjugation with the Siderophore Feroxamine and its Evaluation for Bacteria Detection

This work describes protocols for the preparation of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with (3-aminopropyl)triethoxysilane (APTES) and its conjugation with deferoxamine using a succinyl moiety as a linker. A deep structural characterization description and a capture bacteria assay using Y. enterocolitica for all the intermediate nanoparticles and the final conjugate are also described in detail.

In the present work, the synthesis of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with ...

synthesis-functionalized-magnetic-nanoparticles-their-conjugation

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9.2K Views.  Universidade da Coruña. Magnetische nanodeeltjes zijn ideale instrumenten voor analytische wetenschap en nanogeneeskunde vanwege hun sterke magnetische eigenschappen en de eenvoudige functionalisering van hun oppervlak. We rapporteren hier de eerste instantie van een conjugaat tussen aminofunctionaliseerde silica magnetische deeltjes en de siderophore feroxamine samen met evaluatie voor het vastleggen van fotogeniek materiaal. Siderophores zijn kleine, organische moleculen die een belangrijke rol spelen in het ijzer...

Video: Synthesis of Functionalized Magnetic Nanoparticles, Their Conjugation with the Siderophore Feroxamine and its Evaluation for Bacteria Detection

Synthesis of Immunotargeted Magneto-plasmonic Nanoclusters

Magnetic and plasmonic properties combined in a single nanoparticle provide a synergy that is advantageous in a number of biomedical applications including contrast enhancement in novel magnetomotive imaging modalities, simultaneous capture&#160;and detection of circulating tumor cells (CTCs),&#160;and multimodal molecular imaging combined with photothermal therapy of cancer cells. These applications have stimulated significant interest in development of protocols for synthesis of magneto-plasmonic nanoparticles with optical absorbance in the near-infrared (NIR) region and a strong magnetic moment. Here, we present a novel protocol for synthesis of such hybrid nanoparticles that is based on an oil-in-water microemulsion method. The unique feature of the protocol described herein is synthesis of magneto-plasmonic nanoparticles of various sizes from primary blocks which also have magneto-plasmonic characteristics. This approach yields nanoparticles with a high density of magnetic and plasmonic functionalities which are uniformly distributed throughout the nanoparticle volume. The hybrid nanoparticles can be easily functionalized by attaching antibodies through the Fc moiety leaving the Fab portion that is responsible for antigen binding available for targeting.

Here, we describe a protocol for synthesis of magneto-plasmonic nanoparticles with a strong magnetic moment and a strong near-infrared (NIR) absorbance. The protocol also includes antibody conjugation to the nanoparticles through the Fc moiety for various biomedical applications which require molecular specific targeting.

Magnetic and plasmonic properties combined in a single nanoparticle provide a synergy that is advantageous in a number of biomedical applications ...

Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. The most common techniques utilized are imaging modalities such as Magnetic Resonance Imaging (MRI), Positron Emission Topography (PET), and Computed Topography (CT) and are optimal for understanding the physical structure of the disease but can only be performed once every four to six weeks due to the use of imaging agents and overall cost. With this in mind, the development of &#8220;point of care&#8221; techniques, such as biosensors, which evaluate the stage of disease and/or efficacy of treatment in the clinician&#8217;s office and do so in a timely manner, would revolutionize treatment protocols.1 As a means to exploring ferrocene based biosensors for the detection of biologically relevant molecules2, methods were developed to produce ferrocene-biotin bio-conjugates described herein. This report will focus on a biotin-ferrocene-cysteine system that can be immobilized on a gold surface.

The synthesis of asymmetric species of ferrocene is challenging using solution techniques. This report focuses on the methods carried out to produce a ferrocene-biotin bioconjugate using facile and clean reactions accomplished via solid-phase synthesis. Incorporation of a thiolate moiety is shown to impart the ability for immobilization on gold surfaces.

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. ...

Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo kinetics of transplanted cells. A non-invasive method to repetitively monitor transplanted stem cells in vivo would allow investigators to directly monitor stem cell transplants and identify successful or unsuccessful engraftment outcomes.
A wide range of stem cells continues to be investigated for countless applications. This protocol focuses on 3 different stem cell populations: human embryonic kidney 293 (HEK293) cells, human mesenchymal stem cells (hMSC) and induced pluripotent stem (iPS) cells. HEK 293 cells are derived from human embryonic kidney cells grown in culture with sheared adenovirus 5 DNA. These cells are widely used in research because they are easily cultured, grow quickly and are easily transfected. hMSCs are found in adult marrow. These cells can be replicated as undifferentiated cells while maintaining multipotency or the potential to differentiate into a limited number of cell fates. hMSCs can differentiate to lineages of mesenchymal tissues, including osteoblasts, adipocytes, chondrocytes, tendon, muscle, and marrow stroma. iPS cells are genetically reprogrammed adult cells that have been modified to express genes and factors similar to defining properties of embryonic stem cells. These cells are pluripotent meaning they have the capacity to differentiate into all cell lineages 1. Both hMSCs and iPS cells have demonstrated tissue regenerative capacity in-vivo.
Magnetic resonance (MR) imaging together with the use of superparamagnetic iron oxide (SPIO) nanoparticle cell labels have proven effective for in vivo tracking of stem cells due to the near microscopic anatomical resolution, a longer blood half-life that permits longitudinal imaging and the high sensitivity for cell detection provided by MR imaging of SPIO nanoparticles 2-4. In addition, MR imaging with the use of SPIOs is clinically translatable. SPIOs are composed of an iron oxide core with a dextran, carboxydextran or starch surface coat that serves to contain the bioreactive iron core from plasma components. These agents create local magnetic field inhomogeneities that lead to a decreased signal on T2-weighted MR images 5. Unfortunately, SPIOs are no longer being manufactured. Second generation, ultrasmall SPIOs (USPIO), however, offer a viable alternative. Ferumoxytol (FerahemeTM) is one USPIO composed of a non-stoichiometric magnetite core surrounded by a polyglucose sorbitol carboxymethylether coat. The colloidal, particle size of ferumoxytol is 17-30 nm as determined by light scattering. The molecular weight is 750 kDa, and the relaxivity constant at 2T MRI field is 58.609 mM-1 sec-1 strength4. Ferumoxytol was recently FDA-approved as an iron supplement for treatment of iron deficiency in patients with renal failure 6. Our group has applied this agent in an &ldquo;off label&rdquo; use for cell labeling applications. Our technique demonstrates efficient labeling of stem cells with ferumoxytol that leads to significant MR signal effects of labeled cells on MR images. This technique may be applied for non-invasive monitoring of stem cell therapies in pre-clinical and clinical settings.

We describe a technique for labeling and tracking stem cells with FDA-approved, superparamagnetic iron oxide (SPIO), ferumoxytol (Feraheme). This cellular imaging technique that utilizes magnetic resonance (MR) imaging for visualization, is readily accessible for long-term monitoring and diagnosis of successful or unsuccessful stem cell engraftments in patients.

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo ...

Medicine

Surface-enhanced Resonance Raman Scattering Nanoprobe Ratiometry for Detecting Microscopic Ovarian Cancer via Folate Receptor Targeting

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic spread has already occurred. However, ovarian cancer has a unique pattern of metastatic spread, in that tumor implants are initially contained within the peritoneal cavity. This feature could enable, in principle, the complete resection of tumor implants with curative intent. Many of these metastatic lesions are microscopic, making them hard to identify and treat. Neutralizing such micrometastases is believed to be a major goal towards eliminating tumor recurrence and achieving long-term survival. Raman imaging with surface enhanced resonance Raman scattering nanoprobes can be used to delineate microscopic tumors with high sensitivity, due to their bright and bioorthogonal spectral signatures. Here, we describe the synthesis of two &#39;flavors&#39; of such nanoprobes: an antibody-functionalized one that targets the folate receptor &#8212; overexpressed in many ovarian cancers &#8212; and a non-targeted control nanoprobe, with&#160;distinct spectra. The nanoprobes are co-administered intraperitoneally to mouse models of metastatic human ovarian adenocarcinoma. All animal studies were approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center. The peritoneal cavity of the animals is surgically exposed, washed, and scanned with a Raman microphotospectrometer. Subsequently, the Raman signatures of the two nanoprobes are decoupled using a Classical Least Squares fitting algorithm, and their respective scores divided to provide a ratiometric signal of folate-targeted over untargeted probes. In this way, microscopic metastases are visualized with high specificity. The main benefit of this approach is that the local application into the peritoneal cavity &#8212; which can be done conveniently during the surgical procedure &#8212; can tag tumors without subjecting the patient to systemic nanoparticle exposure. False positive signals stemming from non-specific binding of the nanoprobes onto visceral surfaces can be eliminated by following a ratiometric approach where targeted and non-targeted nanoprobes with distinct Raman signatures are applied as a mixture. The procedure is currently still limited by the lack of a commercial wide-field Raman imaging camera system, which once available will allow for the application of this technique in the operating theater.

Ovarian cancer forms metastases throughout the peritoneal cavity. Here, we present a protocol to make and use folate-receptor targeted surface-enhanced resonance Raman scattering nanoprobes that reveal these lesions with high specificity via ratiometric imaging. The nanoprobes are administered intraperitoneally to living mice, and the derived images correlate well with histology.

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic ...

Cancer Research

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.

A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin.

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide ...

Bioengineering

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and water-screening methods used currently. While conventional bacterial detection methods, such as polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays (ELISA) can specifically detect pathogenic contaminants, they require extensive sample preparation and lengthy waiting periods. In addition, these practices demand sophisticated laboratory instruments and settings, and must be executed by trained professionals. Herein, a protocol is proposed for a simpler diagnostic technique that features the unique combination of magnetic and fluorescent parameters in a nanoparticle-based platform. The proposed multiparametric magneto-fluorescent nanosensors (MFnS) can detect E. coli O157:H7 contamination with as little as 1 colony-forming unit present in solution within less than 1 h. Furthermore, the ability of MFnS to remain highly functional in complex media such as milk and lake water has been verified. Additional specificity assays were also used to demonstrate the ability of MFnS to only detect the specific target bacteria, even in the presence of similar bacterial species. The pairing of magnetic and fluorescent modalities allows for the detection and quantification of pathogen contamination in a wide range of concentrations, exhibiting its high performance in both early- and late-stage contamination detection. The effectiveness, affordability, and portability of the MFnS make them an ideal candidate for point-of-care screening for bacterial contaminants in a wide range of settings, from aquatic reservoirs to commercially packaged foods.

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

The overall goal of this protocol is to synthesize functional nanosensors for the portable, cost-effective, and rapid detection of specifically targeted pathogenic bacteria through a combination of magnetic relaxation and fluorescence emission modalities.

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and ...

Immunology and Infection

Using Magnetometry to Monitor Cellular Incorporation and Subsequent Biodegradation of Chemically Synthetized Iron Oxide Nanoparticles

Magnetic nanoparticles, made of iron oxide, present a peculiar interest for a wide range of biomedical applications for which they are often internalized in cells and then left within. One challenge is to assess their fate in the intracellular environment with reliable and precise methodologies. Herein, we introduce the use of the vibrating sample magnetometer (VSM) to precisely quantify the integrity of magnetic nanoparticles within cells by measuring their magnetic moment. Stem cells are first labeled with two types of magnetic nanoparticles; the nanoparticles have the same core produced via a fast and efficient microwave-based nonaqueous sol gel synthesis and differ in their coating: the commonly used citric acid molecule is compared to polyacrylic acid. The formation of 3D cell-spheroids is then achieved via centrifugation and the magnetic moment of these spheroids is measured at different times with the VSM. The obtained moment is a direct fingerprint of the nanoparticles&#8217; integrity, with decreasing values indicative of a nanoparticle degradation. For both nanoparticles, the magnetic moment decreases over culture time revealing their biodegradation. A protective effect of the polyacrylic acid coating is also shown, when compared to citric acid.

Iron oxide nanoparticles are synthesized via a nonaqueous sol gel procedure and coated with anionic short molecules or polymer. The use of magnetometry for monitoring the incorporation and biotransformations of magnetic nanoparticles inside human stem cells is demonstrated using a vibrating sample magnetometer (VSM).

Magnetic nanoparticles, made of iron oxide, present a peculiar interest for a wide range of biomedical applications for which they are often internalized ...

Biofunctionalization of Magnetic Nanomaterials

Magnetic nanomaterials have received great attention in different biomedical applications. Biofunctionalizing these nanomaterials with specific targeting agents is a crucial aspect to enhance their efficacy in diagnostics and treatments while minimizing the side effects. The benefit of magnetic nanomaterials compared to non-magnetic ones is their ability to respond to magnetic fields in a contact-free manner and over large distances. This allows to guide or accumulate them, while they can also be monitored. Recently, magnetic nanowires (NWs) with unique features were developed for biomedical applications. The large magnetic moment of these NWs enables a more efficient remote control of their movement by a magnetic field. This has been utilized with great success in cancer treatment, drug delivery, cell tracing, stem cell differentiation or magnetic resonance imaging. In addition, the NW fabrication by template-assisted electrochemical deposition provides a versatile method with tight control over the NW properties. Especially iron NWs and iron-iron oxide (core-shell) NWs are suitable for biomedical applications, due to their high magnetization and low toxicity.
In this work, we provide a method to biofunctionalize iron/iron oxide NWs with specific antibodies directed against a specific cell surface marker that is overexpressed in a large number of cancer cells. Since the method utilizes the properties of the iron oxide surface, it is also applicable to superparamagnetic iron oxide nanoparticles. The NWs are first coated with 3-aminopropyl-tri-ethoxy-silane (APTES) acting as a linker, which the antibodies are covalently attached to. The APTES coating and the antibody biofunctionalization are proven by electron energy loss spectroscopy (EELS) and zeta potential measurements. In addition, the antigenicity of the antibodies on the NWs is tested by using immunoprecipitation and western blot. The specific targeting of the biofunctionalized NWs and their biocompatibility are studied by confocal microscopy and a cell viability assay.

In this work, we provide a protocol to biofunctionalize magnetic nanomaterials with antibodies for specific cell targeting. As examples, we utilize iron nanowires to target cancer cells.

Magnetic nanomaterials have received great attention in different biomedical applications. Biofunctionalizing these nanomaterials with specific targeting ...

Synthesis, Characterization, and Application of Superparamagnetic Iron Oxide Nanoprobes for Extrapulmonary Tuberculosis Detection

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was synthesized to enhance imaging sensitivity for extrapulmonary tuberculosis (ETB). An SPIO nanoprobe was synthesized and conjugated with MtbsAb. The purified SPIO-MtbsAb nanoprobe was characterized using TEM and NMR. To determine the targeting ability of the probe, SPIO-MtbsAb nanoprobes were incubated with Mtb for in vitro imaging assays and injected into Mtb-inoculated mice for in vivo investigation with magnetic resonance (MR). The contrast enhancement reduction on magnetic resonance imaging (MRI) of Mtb and THP1 cells showed proportional to the SPIO-MtbsAb nanoprobe concentration. After 30 min of intravenous SPIO-MtbsAb nanoprobe injection into Mtb-infected mice, the signal intensity of the granulomatous site was enhanced by 14-fold in the T2-weighted MR images compared with that in mice receiving PBS injection. The MtbsAb nanoprobes can be used as a novel modality for ETB detection.

To improve serological diagnostic tests for Mycobacterium tuberculosis antigens, we developed superparamagnetic iron oxide nanoprobes to detect extrapulmonary tuberculosis.

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was ...

Antibody-Functionalized SERRS Nanoprobe Based Imaging: A Highly Sensitive Raman-imaging Technique to Detect Metastatic Ovarian Cancer In Vivo

Source: Andreou, C. et al. <a href="https://www.jove.com/t/58389">Surface-enhanced Resonance Raman Scattering Nanoprobe Ratiometry for Detecting Microscopic Ovarian Cancer via Folate Receptor Targeting.</a> J. Vis. Exp. (2019)
This video describes the surface-enhanced resonance Raman scattering technique to detect metastatic ovarian cancer using targeted nanoprobes specific anti-receptor antibodies and non-targeted control nanoprobes. This method helps to determine the relative abundance of the two nanoprobes and differentiate the cancer cells from the visceral background.

Source: Andreou, C. et al. Surface-enhanced Resonance Raman Scattering Nanoprobe Ratiometry for Detecting Microscopic Ovarian Cancer via Folate Receptor ...

Synthesis of Functionalized Magnetic Nanoparticles, Their Conjugation with the Siderophore Feroxamine and its Evaluation for Bacteria Detection

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