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

Summary

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.

Abstract

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 Stöber 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.

Introduction

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 bacteria^3,4. 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—the siderophore recognized by Yersinia enterocolitica—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 Stöber 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

Protocol

NOTE: For the reactions performed under inert atmosphere conditions, all the glassware was previously dried in an oven at 65 °C, sealed with a rubber septum and purged with argon three times.

1. Synthesis of magnetic nanoparticles conjugated with feroxamine

1. Synthesis of Fe₃O₄ magnetic nanoparticles (MNPs)
   1. Add 0.5 g of Fe(acac)₃ in a 20 mL glass vial and then mix with 10 mL of benzyl alcohol.
   2. Sonicate this mixture for 2 min, then transfer to a heating block and heat at 180 °C for 72 h.
   3. 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.
   4. Separate the nanoparticles from the supernatant by magnetic attraction using a neodymium (NdFeB) magnet and discard the residual solvent.
   5. 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.
2. Magnetic nanoparticles SiO₂ coating (MNP@SiO₂)
   1. 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.
   2. Heat the mixture at 40 °C for 2 h with continuous stirring and then sonicate for 1 h.
   3. Separate the MNP with a magnet, discard the supernatant, and disperse it in 30 mL of isopropanol.
   4. Repeat steps 1.2.1. and 1.2.2.
   5. Remove and wash the magnetic stir bar with 96% ethanol to recover all the material.
   6. Separate the nanoparticles from the supernatant by magnetic attraction using a magnet.
   7. Discard the supernatant and rinse the nanoparticles with 96% ethanol three times alternating with sonication.
   8. Dry the nanoparticles under vacuum at room temperature for 12 h.
3. Functionalization of MNP@SiO₂ with (3-aminopropyl)triethoxysilane (APTES)
   1. 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.
   2. Re-suspend the particles in a round bottom flask, under stirring with a magnetic stir bar and add 9 mL of APTES.
   3. Stir the mixture at 60 °C for 12 h.
   4. Discard the supernatant and rinse the nanoparticles with 96% ethanol three times alternating with sonication.
4. Synthesis of feroxamine
   1. 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.
   2. 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.
   3. Freeze-dry the aqueous phase to afford feroxamine as a red solid.
5. Synthesis of N-succinylferoxamine
   1. 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.
   2. 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.
   3. Dissolve the reaction crude in 3 mL of methanol.
   4. 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.
   5. Collect the red fraction and remove the methanol under vacuum using a rotatory evaporator.
6. Synthesis of the conjugate MNP@SiO₂@NH@Fa
   1. 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.
   2. 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.
   3. 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).
   4. Add mix A to mix B dropwise.
   5. Shake the final mixture using an orbital shaker at room temperature overnight.
   6. Separate the resulting conjugate (MNP@SiO₂@NH@Fa) from the suspension using a magnet.
   7. Rinse the resulting solid and then, sonicate it five times with 10 mL of ethanol.
   8. Dry the solid under vacuum for 24 h.

2. Bacterial assay with Y. enterocolitica strains to quantify the capture of pathogenic bacteria with nanoparticles

1. Prepare a suspension of all the intermediate nanoparticles and the final conjugate in PBS at 1 mg/mL in sterile 2 mL tubes.
2. Prepare a culture of Y. enterocolitica in 5 mL of Luria Bertani (LB) broth overnight incubating at 37 °C.
3. Prepare a 5 mL of iron deficient tryptic soy broth (TSB) by adding 50 µL of 10 mM 2,2′-bipyridyl.
4. Inoculate the 5 mL of iron deficient TSB with 50 µL of the overnight culture of Y. enterocolitica and then, incubate at 37 °C with agitation until an OD₆₀₀ = 0.5‒0.8 is reached.
5. Take 100 µL of the culture obtained in the step 2.4 and dilute in a 2.0 mL tube containing 900 µL 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.
6. Add 100 µL 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.
7. Incubate the culture at 20 °C for 1 h.
8. Separate the MNP/bacteria aggregates by using a magnet and carefully discard the supernatant.
9. Rinse the separated nanoparticles twice with 1 mL PBS using a vortex.
10. Suspend the nanoparticles in 1 mL of PBS to count the amount of bacterial capture in CFU/mL.
11. Prepare four successive 1/10 dilutions from the former suspension until a 1 x 10^-4 dilution is reached.
12. Plate 10 µL of each dilution onto TS agar plates and incubate them at 37 °C overnight.
13. 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.

Results

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...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1-Hydroxybenzotriazole hydrate HOBT	Acros	300561000
2,2′-Bipyridyl	Sigma Aldrich	D216305
3-Aminopropyltriethoxysilane 99%	Acros	151081000
Ammonium hydroxide solution 28% NH3	Sigma Aldrich	338818
Benzotriazol-1-yloxytris(dimethylamino)-phosphonium hexafluorophosphate BOP Reagent	Acros	209800050
Benzyl alcohol	Sigma Aldrich	822259
Deferoxamine mesylate salt >92,5% (TLC)	Sigma Aldrich	D9533
Ethanol, anhydrous, 96%	Panreac	131085
Ethyl Acetate, Extra Pure, SLR, Fisher Chemical
Iron(III) acetylacetonate 97%	Sigma Aldrich	F300
LB Broth (Lennox)	Sigma Aldrich	L3022
N,N-Diisopropylethylamine, 99.5+%, AcroSeal	Acros	459591000
N,N-Dimethylformamide, 99.8%, Extra Dry, AcroSeal	Acros	326871000
Pyridine, 99.5%, Extra Dry, AcroSeal	Acros	339421000
Sephadex LH-20	Sigma Aldrich	LH20100
Succinic anhydride >99%	Sigma Aldrich	239690
Tetraethyl orthosolicate >99,0%	Sigma Aldrich	86578