Design of a water-soluble chitosan-based polymer with antioxidant and chelating properties for labile iron extraction

Synthesis of Chito@DOTAGA@DFO

Chemical substances: Freeze-dried Chito@DOTAGA was supplied by MexBrain (Villeurbanne, France). It has been synthetized as described in Natuzzi et al.²³. Briefly, the beginning materials is a medical grade chitosan with a low diploma of acetylation (4.5% ± 0.5%, decided by ¹H NMR spectroscopy by the Hirai methodology)²⁴ and with Mw = 2.583 10⁵ g.mol⁻¹ (decided by dimension exclusion chromatography coupled with refractive index and multi-angle laser gentle scattering measurements)⁴⁷. Acetylation of this preliminary chitosan was carried out by addition of pure acetic anhydride in a water/1,2-propanediol combination chitosan resolution. After this primary step, re-acetylated chitosan (Reac-Chito) was obtained and a pattern of the response combination was purified by tangential filtration and freeze-dried for evaluation of acetylation diploma by ¹H NMR spectroscopy. Functionalization with DOTAGA was obtained by direct addition of DOTAGA anhydride (1,4,7,10-tetra-azacyclododecane-1-glutaric anhydride-4,7,10-triacetic acid) to the answer of Reac-Chito in water/1,2-propanediol. p-NCS-Bz-DFO (N1-hydroxy-N1-(5-(4-(hydroxy(5-(3-(4-isothiocyanatophenyl)thioureido)pentyl)amino)-4-oxobutanamido)pentyl)-N4-(5-(N-hydroxyacetamido)pentyl)succinamide) was furnished by Chematech (Dijon, France). 1,2-propanediol, DMSO and acetic anhydride had been bought from Sigma-Aldrich (Saint-Quentin-Fallavier, France). Glacial acetic acid (AnalR NormaPur) grade was furnished by VWR (Rosny-sous-Bois, France).

Grafting of DFO

20 g of freeze-dried Chito@DOTAGA had been dissolved in 2 L of ultra-pure water. This resolution was stirred at room temperature till full dissolution of the polymer. The pH was measured to be 6.3. A second resolution containing p-NCS-Bz-DFO was then ready by dissolving 3.8 g of p-NCS-Bz-DFO in 380 mL of DMSO. The answer was stirred at room temperature for at the least 30 min. Then the answer of Chito@DOTAGA in water was poured in a ten L reactor and 80 mL of ultrapure water had been added. Then, 1.2 L of 1,2-propanediol was added underneath stirring and at managed temperature (30 °C). 380 mL of DMSO had been then added slowly into the reactor. The combination was stirred at 30 °C for 1 h, earlier than gradual addition of the answer of p-NCS-Bz-DFO within the reactor utilizing a peristaltic pump at a movement charge of 400 µL min⁻¹. The reactor was maintained at 30 °C throughout the response and stirred for 16.5 h. After this, the synthetized product was purified by tangential filtration utilizing a 100 kDa cut-off membrane (Sartocon Slice 200, PES membrane) with a Sartoflow Superior equipment. Briefly, the combination was diluted by 5 in acetic acid (0.1 M) then concentrated to achieve a dilution issue of two in comparison with the preliminary quantity. Then, the product was purified utilizing acetic acid 0.1 M then ultra-pure water and re-concentrated to the preliminary quantity.

Characterization of DFO grafting by UV–Vis titration

The quantity of DFO grafted was decided by titration with Fe(III) by measuring the absorption at 429 nm (most wavelength of DFO-Fe(III) advanced)²⁷. Polymer is first dissolved in water at 5 g L⁻¹. Then numerous options of 0.1 g L⁻¹ polymer in ammonium acetate (0.1 M)/acetic acid (0.1 M) buffer had been ready with completely different concentrations of FeCl₃, starting from 0 to 80 µM. The absorption at 429 nm was then plotted versus the iron content material in resolution, calculated in mmol per gram of polymer. After linear regression, the iron chelation capability by DFO is decided because the focus on the noticed change of slope.

Characterization of DFO grafting by ¹H NMR

¹H NMR spectra had been carried out on the Polymer NMR Platform of Institut de Chimie de Lyon (Axel’One Campus-Lyon) on a Bruker Avance III spectrometer working at 400.1 MHz for ¹H statement and geared up with a 5 mm BBFO + probe. Samples had been dissolved in D₂O at 5 g.L⁻¹. ¹H spectra had been recorded with a 30° flip angle, 6.0 s recycle delay and 512 scans at 343 Okay. TopSpin software program was used for processing. Baseline correction, part adjustment, and integration had been processed manually.

Evaluation of antioxidant energy

Chemical substances: 2,2-diphenyl 1-picrylhydrazyle (DPPH, CAS 1898-66-4), L-ascorbic acid (99%, CAS 50-81-7), glacial acetic acid (99.5%, CAS 64-19-7), methanol (98.5%, CAS 67-56-1), EDTA calcium disodium salt (≥ 97%, CAS 62-33-9), iron sulfate heptahydrate (≥ 99.5%, CAS 7782-63-0), (±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox, 97%, CAS 53188-07-1), HEPES (≥ 99.5%, CAS 7365–4569), H₂O₂ 30% (v/v) (Reagent grade, CAS 7722-84-1), sodium benzoate (≥ 99.5%, CAS 532-32-1), sodium phosphate dibasic (≥ 99.0%, CAS 7558-79-4) and potassium phosphate monobasic (≥ 99.0%, CAS 7778-77-0) had been bought from Sigma-Aldrich (Saint-Quentin-Fallavier, France).

Preparation of antioxidant options

Chito@DFO (re-acetylated chitosan solely grafted with DFO, x = 0.41 and z = 0.027) was synthetized as described in Supporting Information. Briefly, acetylation of chitosan was carried out by addition of pure acetic anhydride in a water/1,2-propanediol combination. Functionalization with DFO was obtained by gradual addition of an answer of p-NCS-Bz-DFO in DMSO to the earlier resolution in a water/1,2-propanediol/DMSO combination. Re-acetylated chitosan (Reac-Chito, x = 0.29) was purified following the re-acetylation step of Chito@DOTAGA synthesis as beforehand described. For DPPH assay, a polymer inventory resolution was ready at 20 g L⁻¹ then this resolution was diluted at numerous concentrations and the pH was adjusted to 4 with acetic acid. Ascorbic acid resolution was ready equally.

DPPH scavenging capability

The willpower of two,2-diphenyl 1-picrylhydrazyle (DPPH) scavenging by chitosan-based polymers was tailored from a technique beforehand described by Avelelas et al.³¹. Samples had been ready by mixing 1 mL of DPPH resolution (0.2 mM in methanol) and 1 mL of antioxidant resolution beforehand ready. All samples had been ready in triplicates, protected against gentle and stirred. Absorbance at 517 nm is measured after 1 h and 19 h with a Cary 50 Scan UV–seen spectrophotometer utilizing plastic cuvettes with optical path size of 10 mm. Scavenging capability (SA) was calculated as observe:

the place Abs(pattern) is the absorbance measured for samples containing antioxidants and Abs(management) the absorbance of management pattern with solely DPPH within the combination methanol/water at pH 4 after pH adjustment with acetic acid.

Fenton-generated hydroxyl radical scavenging

Hydroxyl radical scavenging was decided with a technique impressed by Feng et al.⁴⁰. An answer of FeSO₄ at 5 mM was ready in 10 mM EDTA resolution (pH adjusted to three). 0.6 mL of this resolution was added dropwise to 4.2 mL of antioxidant resolution (chitosan-based polymers or Trolox as constructive management) beforehand solubilized in 0.1 M HEPES buffer (pH 7.4). Then 0.6 mL of 10 mM sodium benzoate (NaBz) and 0.6 mL of 10 mM H₂O₂ had been added and the pattern was stirred at 37 °C for two h. After centrifugation (4000 rpm, 15 min), fluorescence from the supernatant was analyzed with a Cary Eclipse Fluorescence Spectrophotometer (excitation at 310 nm, emission at 407 nm) utilizing plastic cuvettes with optical path size of 10 mm. Fluorescence depth was additionally measured for a clean pattern containing only one mM NaBz in 0.1 M HEPES. Scavenging capability (SA) was calculated as observe:

the place ({I}_{F}left(sampleright)) is fluorescence depth (310/407 nm) of samples containing antioxidants and ({I}_{F}(management)) is fluorescence depth of unfavorable management (with out antioxidants).

TiO₂/UV-generated hydroxyl radical scavenging: The assay was based mostly on hydroxyl radical measuring methodology developed by Xiang et al.⁴⁸. 5 mg of TiO₂ (85% anatase, 15% rutile) had been launched in vials and 4.8 mL of antioxidant resolution (chitosan-based polymers or Trolox) in 0.1 M phosphate buffer (pH 7.4) had been added. Below stirring, 0.6 mL of 10 mM sodium benzoate (NaBz) and 0.6 mL of 30% w/w H₂O₂ had been added to the pattern. Samples are positioned underneath UV-radiation at 375 nm and stirred for two h. The sunshine supply (ThorLabs M375L3) was positioned 10 cm above pattern floor. After centrifugation (4000 rpm, 10 min), the fluorescence depth from supernatant was analyzed with a Cary Eclipse Fluorescence Spectrophotometer (excitation at 310 nm, emission at 407 nm) utilizing plastic cuvettes with optical path size of 10 mm. Fluorescence depth was additionally measured for a clean pattern containing only one mM NaBz in 0.1 M phosphate buffer. Scavenging capability (SA) was calculated equally to Fenton-generated hydroxyl radical scavenging.

Analysis of iron extraction in competitors with Deferiprone

Chemical substances

Normal iron options for ICP-MS had been supplied by SCP-Science (ICP Normal 50,000 µg mL⁻¹, 140-041-265 and ICP Normal 1000 µg mL⁻¹, 140-051-260). Deferiprone (purity 98%, CAS 30652-11-0), sodium phosphate dibasic (ACS reagent ≥ 99.0%, CAS 7558-79-4) and potassium phosphate monobasic (Reagent Plus ≥ 99.0%, CAS 7778-77-0) had been bought from Sigma-Aldrich (Saint-Quentin-Fallavier, France).

Preparation of inventory options

Freeze-dried Chito@DOTAGA and Chito@DOTAGA@DFO had been each dissolved in ultra-pure water at a focus of 1 g L⁻¹. An answer of Fe(III) at 1 mg.L⁻¹ in 10^–2 M HCl was ready by dilution of the usual iron resolution at 50,000 mg.L⁻¹. Deferiprone was first solubilized at 10 g.L⁻¹ in ultra-pure water then diluted to achieve a focus of 10 mg L⁻¹. Phosphate buffer (10 mM, pH 7.7) was ready from sodium phosphate dibasic and potassium phosphate monobasic dissolved in ultra-pure water.

Experimental protocol

From all beforehand described inventory options, samples (20 mL) had been ready in 10 mM phosphate buffer with the next concentrations: 0.45 µM of iron (III), 1.8 µM or 14.4 µM of deferiprone, 10 mg.L⁻¹ of polymer (Desk 2). Reference samples had been additionally ready with solely polymer in phosphate buffer or with solely iron(III) and deferiprone however with out polymer. Samples had been stirred at room temperature for one hour, then centrifuged utilizing 20 mL Vivaspin with a 100 kDa PES cut-off membrane. The precept of the experiment is described in Fig. 5A. Briefly, because of the bigger dimension of the polymer in comparison with membrane cut-off, the polymer can’t undergo the membrane whereas small molecules, equivalent to deferiprone, will cross the membrane. Centrifugation was carried out at 2000 rpm till round 2 mL of supernatant was recovered.

Iron quantification by ICP-MS

The ⁵⁶Fe content material within the preliminary resolution (earlier than polymer addition), within the undernatant and within the supernatant after centrifugation had been measured by ICP-MS. ICP-MS evaluation was carried out utilizing a Perkin Elmer NexION2000 geared up with Syngistix software program (Licensed software program by Perkin Elmer. Model: 2.3 (Construct 2.3.7916.0)) and a ESI SC-FAST Pattern Introduction in Kinetic Power Discrimination (KED) mode. The samples had been ready by dilution in an aqueous resolution of 1% (v/v) HNO₃.

Ex vivo efficacy of iron extraction by hemodialysis

Chemical substances

Hemosol B0 dialysate resolution was produced by Baxter. The usual Cu and Fe options for the ICP-MS are supplied by SCP-Science: ICP Normal Cu 1000 µg.mL⁻¹ (SCP Science, 14-051-290) and ICP Normal Fe 1000 µg.mL⁻¹ (SCP Science, 14-051-260).

Preparation of Chito@DOTAGA@DFO and Hemosol B0 dialysate

2.2 L or 1.8 L of reconstituted Hemosol B0 had been manually withdrawn from a 5 L Hemosol B0 bag and launched right into a clear dialysis bag utilizing a peristaltic pump. 2.2 L of Hemosol B0 had been used to conduct the experiment with out polymer and 1800 mL of Hemosol B0 had been accomplished with 400 mL of sterile liquid formulation of Chito@DOTAGA@DFO (5 g.L⁻¹ in 0.7% NaCl resolution) to acquire a remaining focus of 0.9 g.L⁻¹ of Chito@DOTAGA@DFO.

Preparation of bovine plasma bag

4 500-mL bottles of frozen bovine plasma with sodium citrate (EUROBIO SCIENTIFIC, reference S0260-500, hematocrit 18.72 mg/100 mL) had been positioned in a single day at 4 °C to thaw. Within the morning, the 500-mL bottles of bovine plasma had been positioned in a dry oven at 37 °C till temperature stabilization. The bovine plasma was then filtered on a large mesh (1.5 mm) to take away protein clumps and transferred in a 5 L effluent bag utilizing a peristaltic pump. 100 µL of heparin sodium (Heparin Choay®, 5000 U.I./mL, Sanofi) was added to the plasma bag to forestall coagulation.

Experimental protocol

Dialysis protocol is illustrated in Fig. 6A utilizing a Prismaflex monitor and two forms of dialyzers: HF1400 set (Excessive-flux membrane, 15–20 kDa, 1.4 m²) or SepteX set (Excessive cut-off, < 45 kDa, 1.1 m²). The dialysate movement charge was fastened at 500 mL.h⁻¹ and the blood movement charge was maintained at 100 mL.min⁻¹. For every dialyzer sorts, two dialysis had been carried out: reference experiment with Hemosol B0 alone or experiment with Hemosol B0 and Chito@DOTAGA@DFO. The period of all dialysis experiments was 4 h. Sampling for iron quantification was carried out at time zero from the dialysate and each hour from the effluent bag. All samples had been obtained in duplicates.

Metallic quantification by ICP-MS

ICP-MS evaluation was carried out utilizing a Perkin Elmer NexION2000 geared up with Syngistix software program (Licensed software program by Perkin Elmer. Model: 2.3 (Construct 2.3.7916.0)) and an ESI SC-FAST Pattern Introduction in Kinetic Power Discrimination (KED) mode. The samples had been ready by dilution in an aqueous resolution of 1% (v/v) HNO₃. 250 µL of dialysate samples had been diluted in 4.7 mL of 1% (v/v) HNO₃ resolution and 50 µL of 200 µg.L⁻¹ indium resolution was added as inside customary.

Information evaluation

The metallic extraction from the effluent bag at every time level was corrected with the preliminary metallic content material of Chito@DOTAGA@DFO or Hemosol B0 within the dialysate bag at time zero (t0) as observe: effluent pattern (ppb, Tx)−dialysate pattern (ppb, t0) = effluent pattern corrected (ppb, Tx). The metallic content material (µg) was obtained by multiplying the effluent pattern focus (corrected, ppb) by the actual quantity of dialysate (L) for the time focal point (T1 hour: 0.5 L, T2 hours: 1 L, T3 hours: 1.5 L, T4 hours: 2L). Normal deviations of uncooked information had been calculated from the 4 replicates (two isotopes (⁶³Cu and ⁶⁵Cu for copper, ⁵⁶Fe and ⁵⁴Fe for iron) sampled in duplicate). Normal deviations for corrected values had been calculated as observe: ({RSD}_{Corrected values}=sqrt{frac{RSD1}{N1}+frac{RSD2}{N2}}) the place RSD1 and RSD2 are the usual deviations of the 2 values which might be subtracted and N1 and N2 are the variety of replicates of the 2 values which might be subtracted. RSD for metallic content material (µg) had been obtained by multiplying the pattern focus RSD (corrected, ppb) by the precise quantity of dialysate (L) on the involved time level. All calculations and information are detailed in Supporting Information.