WO2021094535A1

WO2021094535A1 - Organometallic salt of nitroprusside acting as an no-releasing agent

Info

Publication number: WO2021094535A1
Application number: PCT/EP2020/082060
Authority: WO
Inventors: Jérôme LONG; Joulia GUARI - LARIONOVA; Yannick Guari; Françoise QUIGNARD LLECHA LLOP; Nathalie TANCHOUX; Saad SENE
Original assignee: Centre National De La Recherche Scientifique; Universite De Montpellier; École Nationale Supérieure de Chimie de Montpellier
Priority date: 2019-11-15
Filing date: 2020-11-13
Publication date: 2021-05-20
Also published as: FR3103104B1; EP4058463A1; FR3103104A1

Abstract

The invention relates to the use of an organometallic salt of nitroprusside as an NO-releasing agent, a composite material comprising such an organometallic salt of nitroprusside, an implant comprising such an organometallic salt of nitroprusside and a method for manufacturing this organometallic salt of nitroprusside. It also relates to a method for releasing NO and a method for decontaminating a substrate which is suspected of being contaminated with bacteria. The organometallic salt of nitroprusside used in the invention has the following formula I: M^II[Fe^II(CN)₅(NO)].xΗ₂Ο formula I, where Mn represents Zn^II or Fe^II, and 2 ≤ x ≤ 18. The invention can be used in the medical field in particular.

Description

ORGAN OMET ALLIC SALT OF NITROPRUSSIDE AS A NO RELEASE AGENT

The invention relates to the use of an organometallic salt of nitroprusside as an agent releasing NO, to a composite material comprising such an organometallic salt of nitroprusside, to an implant comprising such an organometallic salt of nitroprusside and to a process for the manufacture of this organometallic salt of nitroprusside.

It also relates to a method for releasing NO and a method for decontaminating a substrate suspected of being contaminated with bacteria.

Nitric oxide gas, NO gas, has many properties, including antibacterial and healing properties.

In addition, NO gas has been used successfully for the treatment of pulmonary hypertension, where it is delivered directly to the lungs.

However, the delivery of nitrogen monoxide NO in gaseous form is notoriously difficult due to its reactivity with oxygen or nitrogen.

It was therefore proposed to use NO donors, that is to say molecules transporting and releasing NO at the desired site.

These molecules are in solid form and are still widely used today in clinics. These are, in particular, organic nitrates and nitroprusside.

Nitroprusside is generally used in the form of its sodium salt dihydrate of nitroprusside of the formula Na ₂ [Fe (CN) ₅ NO] .2H ₂ O.

However, this nitroprusside sodium salt degrades to give 5 equivalents of cyanide upon release of NO.

Furthermore, a single ferrous nitroprusside of the formula [Fe (CN) ₅ NO] .2H ₂ O is known. This compound has a stable orthorhombic crystal structure. The electrochemical properties of this ferrous nitroprusside salt have been studied by doCarmo et al. in the article entitled "Electrochemical study of Fe [Fe (CN) ₅ NO] in graphite paste electrode, ECLETICA QUIMICA, vol 27, pages 197-210, special issue: SI, 2002".

The invention aims to overcome the problem of the release of five cyanide molecules when the sodium salt of nitroprusside releases NO.

For this purpose, the invention proposes the use of an organometallic salt of nitroprusside of the following formula I:

[Chem.l]

M ^II [Fe ^II (CN) ₅ (NO)]. XH ₂ O

Formula I in which M ^II represents Zn ^II or Fe ^II , and 2 ≤ x≤ 18 as agent releasing nitrogen monoxide NO. The nitroprusside salt is in the form of particles, the size of which (largest dimension of the particle) is between 50 nm and 6 mih.

This size is measured by SEM scanning electron microscopy, up to a dimension of 200 nm and by transmission electron microscopy below 200 nm.

In a preferred embodiment, the organometallic salt of nitroprusside of formula I is coated in a biopolymer such as chitosan, an alginate of the metal M (Zn or Fe), pectin, agar-agar, carrageenan, a copolymer lactic acid and gly colic acid (PLGA), a poly (L-lactide) polymer (PLLA), most preferably chitosan.

In this case, preferably, the organometallic salt of nitroprusside of formula I is coated in the biopolymer to form a film or to form spheres.

When the organometallic salt of nitroprusside of formula I is coated in the biopolymer to form spheres, these spheres preferably have a diameter of between 1.5 mm and 4.5 mm. This diameter is measured using a graduated ruler and by SEM scanning electron microscopy). Still when the organometallic salt of nitroprusside of formula I is coated in the biopolymer to form spheres, the organometallic salt of formula I / biopolymer mass ratio is preferably between 2 and 60%.

When this ratio is greater than 10%, all the particles of organometallic salt of nitroprusside of formula I are not completely coated in the biopolymer: some have a part on the surface, on the outside, of the sphere.

Likewise, when the organometallic nitroprusside salt of formula I is coated in the biopolymer to form a film, this film preferably has a thickness of between 30 mih and 1 mm.

Still when the organometallic salt of nitroprusside of formula I is coated in the biopolymer to form a film, the organometallic salt of formula I / biopolymer mass ratio is preferably between 2 and 60%.

When this ratio is greater than 10%, all the particles of organometallic nitroprusside salt of formula I are not completely coated in the biopolymer: some have a part on the surface, on the outside, of the film.

The invention also provides an organometallic salt of nitroprusside of the following formula I: [Chem.2]

M ^II [Fe ^II (CN) ₅ (NO)]. XH ₂ O

Formula I wherein M ^II is Zn ^II or Fe ^II , and 2 x 18 as an antibacterial agent, or as a wound healing agent, or for use in the treatment of pulmonary hypertension. In this case, preferably, the organometallic salt of nitroprusside of formula I is in the form of particles having a size between 50 nm to 6 μm.

Still in this case and preferably, the organometallic salt of nitroprusside of formula I is coated in a biopolymer, preferably chosen from chitosan, an alginate of the metal M (Zn or Fe), pectin, agar-agar, carrageenan, a copolymer of lactic acid and glycolic acid (PLGA), a poly (L-lactide) polymer (PLLA), most preferably chitosan to form a film or spheres.

The particle size of the organometallic salt of nitroprusside, the diameter of the spheres, the thickness of the film, the biopolymer / organometallic salt of nitroprusside mass ratio are identical to those described above.

The invention also provides a composite material characterized in that it comprises an organometallic salt of nitroprusside of the following formula I:

[Chem.3]

M ^II [Fe ^II (CN) ₅ (NO)]. XH ₂ O Formula I in which M ^II represents Zn ^II or Fe ^II , and 2 ≤ x ≤ 18 coated in a biopolymer.

In a preferred embodiment, the organometallic salt of nitroprusside of formula I is coated in a biopolymer such as chitosan, an alginate of the metal M (Zn or Fe), pectin, agar-agar, carrageenan, a copolymer lactic acid and glycolic acid (PLGA), a poly (L-lactide) polymer (PLLA), most preferably chitosan.

When the organometallic salt of nitroprusside of formula I is coated in the biopolymer to form spheres, these spheres preferably have a diameter of between 1.5 mm and 4.5 mm. This diameter is measured using a graduated ruler and by SEM scanning electron microscopy. Still when the organometallic salt of nitroprusside of formula I is coated in the biopolymer to form spheres, the organometallic salt of formula I / biopolymer mass ratio is preferably between 2 and 60%.

Likewise, when the organometallic nitroprusside salt of formula I is coated in the biopolymer to form a film, this film preferably has a thickness of between 30 μm and 1 mm. Still when the organometallic salt of nitroprusside of formula I is coated in the biopolymer to form a film, the organometallic salt of formula I / biopolymer mass ratio is preferably between 2 and 60%.

The invention also proposes the use of the composite material according to the invention as an agent releasing nitrogen monoxide NO.

Another subject of the invention is the composite material according to the invention for use as an antibacterial agent, or agent for promoting wound healing, or for the treatment of pulmonary hypertension.

Yet another object of the invention is an implant comprising an organometallic salt of nitroprusside of the following formula I:

[Chem.4]

M ^II [Fe ^II (CN) ₅ (NO)]. XH ₂ O

Formula I in which M ^II represents Zn ^II or Fe ^II , and 2 ≤ x ≤ 18

Preferably, in this implant, the organometallic salt of nitroprusside of formula I is in the form of particles having a size (largest dimension) of between 50 nm to 6 μm.

This implant can be made of a composite material according to the invention made to the desired size and shape. Still another object of the invention is a process for releasing gaseous nitrogen monoxide, characterized in that it comprises irradiation, with radiation emitting in the range of visible wavelengths (400 to 800 nm). , and / or the application of an aqueous suspension of at least one amino compound on particles of organometallic salt of nitroprusside of formula I of an organometallic salt of nitroprusside of following formula I:

[Chem.5]

M ^II [Fe ^II (CN) ₅ (NO)]. XH ₂ O Formula I in which M ^II represents Zn ^II or Fe ^II , and 2 ≤ x ≤ 18 optionally coated in a biopolymer.

The aqueous suspension of at least one amino compound is typically a biological fluid, or an aqueous suspension of lysine.

The invention also proposes a method for decontaminating a substrate suspected of being contaminated by bacteria, characterized in that it comprises: a) the application, to the substrate, of particles of organometallic salt of nitroprusside of the following formula I:

[Chem.6]

M ^II [Fe ^II (CN) ₅ (NO)]. XH ₂ O

Formula I in which M ^II represents Zn ^II or Fe ^II , and 2 ≤ x ≤ 18 in the form of particles, optionally coated in a biopolymer, and b) irradiation by radiation emitting in the visible wavelength range and / or the application of an aqueous suspension of an amino compound on the organometallic salt of nitroprusside of formula I, optionally coated in the biopolymer.

In the above processes, the morphological characteristics of the organometallic salt of nitroprusside of formula I and those of this salt when coated in the biopolymer, are as defined above.

Finally, the invention proposes a process for the manufacture of particles of organometallic salt of nitroprusside of the following formula I:

[Chem.7]

Y ^YI [Fe ^II (CN) ₅ (NO)]. XH ₂ O Formula I in which M ^II represents Zn ^II or Fe ^II , and 2 ≤ x ≤ 18 characterized in that it comprises:

- Synthesis of the nitroprusside salt by coprecipitation of sodium nitroprusside Na ₂ [Fe (CN) ₅ NO] .2H ₂ O with a metal salt based on iron or zinc in water. Solutions of nitroprusside and of the metal salt are prepared in water at concentrations varying from 5.10 ^-3 M to 1.10 ^-1 M. These solutions are then added simultaneously at a rate varying from 2 mL / h to 0.8 mL / min in a volume of water varying from 10 to 100 mL. The nitroprusside solution can also be added dropwise to the solution of the metal salt.

- Synthesis of the nitroprusside salt in polymer spheres by successive impregnations of the spheres in solutions of nitroprusside and of a metal salt based on Iron or Zinc in methanol. Solutions of nitroprusside and of the metal salt are prepared in methanol at concentrations varying from 1.10 ^-3 M to 1.10 ^-1 M. The polymer spheres (alcoogels prepared beforehand and suspended in methanol) are impregnated for 24 to 48 hours in a nitroprusside solution. After washing in methanol, these spheres are then impregnated in a solution of the metal salt for 24 to 48 hours. At this point, a cycle has been performed. The number of cycles can thus be increased by carrying out additional impregnations for 24 to 48 hours. - Synthesis of the nitroprusside salt in a polymer film by successive impregnations of the film in solutions of nitroprusside and of a metal salt based on Iron or Zinc in methanol. Solutions of nitroprusside and of the metal salt are prepared in methanol at concentrations varying from 1.10 ^-3 M to 1.10 ^-1 M. The polymer film (film prepared beforehand and suspended in methanol) is impregnated for 24 to 48 hours in a nitroprusside solution. After washing in methanol, the film is then impregnated in a solution of the metal salt for 24 to 48 hours. At this point, a cycle has been performed. The number of cycles can thus be increased by carrying out additional impregnations for 24 to 48 hours.

The invention will be better understood and other characteristics and advantages thereof will emerge more clearly on reading the following explanatory description which is given with reference to the appended figures in which:

FIGURES

[Fig.l] FIG. 1 represents the curve of the release of NO from the organometallic salt of formula I obtained in Example 23, as a function of time, when exposed to light radiation emitting in the wavelength range of visible and suspended in water,

[Fig.2] Figure 2 shows the infrared spectra, before and after irradiation with light radiation emitting in the field of visible wavelengths, of the organometallic salt of formula I obtained in Example 23, in suspension in water,

[Fig.3] Figure 3 shows the NO release curve of the organometallic salt of formula I obtained in Example 3, as a function of time and in suspension in water, when exposed to light radiation emitting in the visible wavelength range,

[Fig.4] Figure 4 shows the infrared spectra, before and after irradiation with light radiation emitting in the field of visible wavelengths, of the organometallic salt of formula I obtained in Example 3,

[Fig.5] FIG. 5 represents the curve of the release of NO from the organometallic salt of formula I coated in chitosan spheres obtained in Example 17, as a function of time and in suspension in water, when exposed to light radiation emitting in the visible wavelength range,

[Fig.6] Figure 6 shows the infrared spectra, before and after irradiation with light radiation emitting in the field of visible wavelengths, of the organometallic salt of formula I obtained in Example 17,

[Fig.7] Figure 7 shows the NO release curve of the dihydrated sodium salt of nitroprusside of the prior art coated in a chitosan film obtained in Comparative Example 1, as a function of time and in suspension in the 'water, when exposed to irradiation (light radiation emitting in the visible wavelength range),

[Fig.8] FIG. 8 represents the curve of release of NO from the organometallic salt of formula I obtained in Example 23, as a function of time, when exposed to light radiation emitting in the visible wavelength domain. This curve is obtained with the film of Example 23 not placed in suspension in water, as is the case for FIG. 1, and

[Fig.9] FIG. 9 shows the infrared spectra, before and after irradiation with light radiation emitting in the field of visible wavelengths, of the organometallic salt of formula I obtained in Example 23. These spectra are obtained with the film of Example 23 not placed in suspension in water, as is the case for Figure 2.

The invention is based on the discovery that organometallic salts of nitroprusside of the following formula I:

[Chem.8] Na ₂ [Fe (CN) ₅ NO]. 2H ₂ O

Formula I in which M ^II represents Zn ^II or Fe ^II , and 2 ≤ x ≤ 18 could release, under various external stimuli, whether in solid phase or in suspension in water, nitrogen monoxide (NO), while releasing cyanide in an amount a thousand times less than that of the dihydrate sodium salt of nitroprusside widely used in the art today.

Without wishing to be bound by this theory, the inventors believe that it is because of the crystallographic structure of the organometallic salt of nitroprusside of formula I that this release of cyanide during the release of NO gas is greatly reduced.

Indeed, during the release of NO, a structural rearrangement occurs, within the crystallographic mesh, in which the cyanides remain strongly bound.

The remaining traces of cyanide that can be released can be captured by vitamin B 12a (hydroxocobalamin) to form vitamin B 12 (cyanocobalamin). Indeed, it has been shown that hydroxocobalamin can be used as a treatment against cyanide poisoning: Petrikovics et al. “Past, present and future of cyanide antagonism research: From the early remedies to the current therapies” World J. Methodol. 2015 June 26; 5 (2): 88-100.

The inventors have also discovered that, surprisingly, when the organometallic salt of nitroprusside of formula I is irradiated at visible wavelengths, that is to say at wavelengths between 400 and 800 nm, NO is released, whether in solid form or in suspension.

Irradiation by a table lamp can therefore be used.

The application of an aqueous solution of an amino compound such as a biological fluid or an aqueous solution of lysine can also be used to release organometallic salt of nitroprusside of formula I used in the invention.

An application of both such irradiation and such an aqueous solution is generally used, in particular during the implementation of the method for decontaminating a substrate according to the invention. Thus, the invention provides the use of an organometallic salt of nitroprusside of formula I as an agent releasing NO.

Therefore, the invention also relates to the organometallic salt of nitroprusside of formula I for use as an antibacterial agent, or as a wound-healing agent, or as an agent for treating pulmonary hypertension.

The organometallic salt of nitroprusside of formula I can also be used in a process for decontaminating a substrate suspected of being contaminated with bacteria, by application of the organometallic salt of nitroprusside of formula I and irradiation at the wavelengths indicated above and / or by applying an aqueous solution of an amino compound.

Advantageously, the organometallic salt of nitroprusside of formula I can also be used in a method of therapeutic treatment for its antibacterial properties, promoting wound healing or pulmonary hypertension. In this method, the organometallic salt of nitroprusside of formula I is brought to the desired site and irradiated or covered with a solution of an amino compound, which in this case is preferably a biological fluid.

The organometallic salt of nitroprusside of formula I is advantageously coated in a biopolymer, preferably chitosan, in order to be able to bring the particles of organometallic salt of nitroprusside of formula I to the particular site to be treated and to keep them in place.

The organometallic salt of nitroprusside of formula I can be coated with chitosan to form a film which will be deposited on the surface or on the site to be treated.

The organometallic salt of nitroprusside of formula I can also be coated in chitosan in the form of spheres.

By “coated in a biopolymer” is meant, in the invention, that the particles of the organometallic salt of formula I are either completely covered with the biopolymer, when the ratio by mass of organometallic salt of nitroprusside of formula I / biopolymer is between 2 and 60%, ie some particles are only partially covered with the biopolymer, when the organometallic salt of nitroprusside of formula I / biopolymer ratio by mass is greater than 10%.

In fact, when the organometallic salt of nitroprusside salt of formula I / biopolymer mass ratio is greater than 10%, the biopolymer is not in sufficient quantity to cover all the particles of the organometallic salt of formula I and those close to the surface, either spheres, or biopolymer film, are not completely covered.

For therapeutic applications in particular, it is advantageous to use particles of the organometallic salt of nitroprusside of formula I having a size of between 50 nm and 6 μm coated in a biopolymer.

Thus, another subject of the invention is a composite material comprising an organometallic salt of nitroprusside of formula I coated in a biopolymer, whether in the form of spheres or in the form of a film. The composite material according to the invention comprising an organometallic salt of nitroprusside of formula I coated in a biopolymer as an agent releasing NO is another subject of the invention. Yet another object of the invention is the composite material according to the invention comprising an organometallic salt of nitroprusside of formula I coated in a biopolymer for use as an antibacterial agent, or as an agent for promoting wound healing, or for use in the treatment of pulmonary hypertension.

The particles of organometallic salt of nitroprusside of formula I, coated or not in a biopolymer can also be placed in or on an implant which will be placed at the desired site.

In a preferred embodiment, the implant is a film formed from particles of organometallic nitroprusside salt of formula I coated in a biopolymer, shaped and sized.

Such an implant can also be a hollow capsule made of a biopolymer in which particles of the organometallic salt of nitroprusside of formula I are placed.

Such an implant comprising the organometallic salt of nitroprusside of formula I is also an object of the invention. The implant can also be a support made of a biocompatible material coated with the organometallic salt of nitroprusside of formula I or containing the organometallic salt of nitroprusside of formula I.

Preferred biopolymers for use in coating the organometallic salt particles of nitroprusside of formula I are chitosan, an alginate of the metal M (Zn or Fe), pectin, agar-agar, carrageenan, a copolymer of lactic acid and gly colic acid (PLGA), a poly (L-lactide) polymer (PLLA), most preferably chitosan.

The size of the particles of organometallic salt of nitroprusside of formula I can be controlled by the operating conditions of manufacture. This size varies according to the concentrations of the nitroprusside solutions and of the metal salt used, or according to the number of impregnation cycles carried out for the spheres and films.

EXAMPLES

In order to better understand the invention, we will now describe, by way of purely illustrative and non-limiting examples, several modes of implementation.

Equipment

All commercial products were used without additional purifications. Chitosan (low molecular weight), sodium nitroprusside Na ₂ [Fe (CN) ₅ NO] .2H ₂ O, 99%), sodium hydroxide (NaOH, 97%), acetic acid (99% ), tetraphenylphosphonium chloride (PPh ₄ Cl, 98%), iron (II) tetrafluoroborate (Fe (BF ₄ ). 6H ₂ O, ascorbic acid (99%), glucose (99%) were purchased from Sigma Aldrich Iron (II) chloride (FeCl ₂ .4H ₂ O, 98%), zinc nitrate (Zn (NO ₃ ) ₂ -6H ₂ O, 98%) were purchased from Alfa Aesar. Absolute ethanol and methanol were obtained from Carlo Erba Distilled water was used during all experiments. Example 1 - Synthesis of Zn [Fe (CN) ₅ NO] .xH ₂ O (2 ≤ x ≤ 18)

This synthesis takes place according to the following reaction:

[Chem.9]

0.594 g (2 mmol) of Zn (NO ₃ ) ₂ .6H ₂ O are dissolved in 20 ml of distilled water.

0.596 g (2 mmol) of Na ₂ [Fe (CN) ₅ NO] .2H ₂ O are dissolved in 20 mL of distilled water.

The Na ₂ [Fe (CN) ₅ NO] .2H ₂ O solution is isolated from light.

Once these precursors are completely dissolved, the colorless Zn (II) solution is added to the orange / red Na ₂ [Fe (CN) ₅ NO] solution which has been isolated from light.

After mixing, a cloudiness begins to appear after a few seconds, and a precipitate after a few minutes. The mixture is left under stirring and isolated from light for two hours. After these two hours, the mixture is centrifuged at 20,000 rμm for 10 minutes. The solution is removed and the resulting solid is washed with three times the volume of water. Another washing is carried out with ethanol in order to facilitate the drying process. Finally, the solid (light pink) is dried under a vacuum of about 10 mbar.

The particles obtained have a size between a few μm and a few tens of mih. Example 2 - Synthesis of Fe [Fe (CN) ₅ NO] .xH ₂ O (2 ≤ x ≤ 18)

This synthesis proceeds according to the following equation:

[Chem.10]

0.33 g (1 mmol) of Fe (BF ₄ ) ₂ -6H ₂ O are dissolved in 20 ml of distilled water.

0.297 g (1 mmol) of Na ₂ [Fe (CN) ₅ NO]. 2H ₂ O are dissolved in 20 ml of distilled water.

After these precursors are completely dissolved, the red / orange solution of Na ₂ [Fe (CN) ₅ NO], which has been isolated from light, is added dropwise to the solution of Fe (BF ₄ ) ₂ ( colorless solution).

Immediately, a brown solid begins to appear. The mixture is stirred and isolated from light for two hours.

After these two hours, the mixture is centrifuged at 20,000 rμm for 10 minutes. The solution is removed and the resulting solid is washed with three times the volume of water. Another washing is carried out with ethanol in order to facilitate the drying process. Finally, the brown solid is dried under a vacuum of about 10 mbar.

The particles obtained have a size (largest dimension) of between a few μm and a few tens of μm.

Example 3 - Synthesis of Fe [Fe (CN) ₅ NO] .xH ₂ O (2 ≤ x ≤ 18) In this example, ascorbic acid is added to prevent oxidation of iron.

This synthesis proceeds according to the following equation:

[Chem.11]

1.49 g (5 mmol) of Na ₂ [Fe (CN) ₅ NO] .2H ₂ O are dissolved in 100 ml of distilled water and isolated from the light. 0.99 g (5 mmol) of FeCl ₂ .4H ₂ O and 0.88 g of ascorbic acid are dissolved in 100 ml of distilled water.

Once these precursors are completely dissolved, the red / orange solution of Na ₂ [Fe (CN) ₅ NO], which has been isolated from light, is added dropwise to the solution of Fe ²⁺ and acid. ascorbic. A brown solid begins to appear gradually during the addition. The mixture is stirred and isolated from light for two hours.

The mixture is then centrifuged at 20,000 rpm for 10 minutes. The supernatant is removed and the resulting solid is washed with 100 mL of distilled water and then centrifuged at 20,000 rμm for 10 minutes. A final wash is carried out under the same conditions and the brown solid is dried under vacuum. The particles obtained have sizes (largest dimension) of between 1, 2 and 5 mih.

Example 4: Synthesis of precursor [PPh ₄ ] ₂ [Fe (CN) ₅ NO] .xH ₂ O (2 ≤ x ≤ 18) [PPh ₄ ] ₂ [Fe (CN) ₅ NO] xH ₂ O (2 ≤ x ≤ 18) is a starting compound used in the synthesis of organometallic salts of formula I.

This synthesis takes place according to the equation:

[Chem.12]

1.50 g (4 mmol) of [PPh ₄ ] Cl are dissolved in 20 ml of ultrapure water (colorless solution).

0.596 g (2 mmol) of Na ₂ [Fe (CN) ₅ NO] are dissolved in 20 ml of water (orange / red solution)

The Na ₂ [Fe (CN) ₅ NO] solution should be isolated from the light with aluminum foil in order to avoid the release of NO.

Once the two solids have dissolved, the Na ₂ [Fe (CN) ₅ NO] solution is added dropwise to the [PPh ₄ ] Cl solution and stirred.

A light brown solid immediately begins to appear. If the mixture becomes too dense and the agitation is not sufficient, it is possible to add more water.

The mixture is left under stirring and isolated from light for two hours.

After these two hours, the mixture is centrifuged at 15,000 or 20,000 rμm for 10 minutes.

The solution is removed and the resulting solid is washed with three times its volume of water.

Finally, the solid is dried under vacuum. Example 5 Synthesis of chitosan spheres lg of chitosan (“chitosan, low molecular weight,” Sigma Aldrich), are dissolved in 40 mL of a 1 vol.% Solution of acetic acid, with magnetic stirring overnight at ambient temperature. The aqueous chitosan solution is then added dropwise using a separating funnel fitted with a 0.8 mm diameter syringe tip, into a crystallizer containing 500 mL of aqueous NaOH solution (2M). with stirring (tip-solution distance of 10 cm). After the addition of 40 ml of chitosan solution, the beads contained in the sodium solution are left under stirring with stirring for 1 night.

The chitosan beads are subsequently washed thoroughly with distilled water (V _H2O = 500 mL) to a pH 6-7. An exchange of solvents is then carried out in increasing solutions of ethanol or methanol so as not to deform the porous structure of the microspheres. The spheres are thus immersed for 15 minutes in Water-Ethanol or Water-Methanol baths of 200 mL of increasing alcohol concentration (10, 30, 50, 70, 90, 100% by volume) in order to exchange the water contained in the spheres by ethanol or methanol.

The beads are kept in this alcoholic state or dried with _{supercritical CO 2} to replace ethanol with liquid _{CO 2.} The CO ₂ is then eliminated under supercritical conditions in an autoclave, above 31 C and 73 bar (typically 39 C and 85 bar), after a controlled expansion (at constant flow, less than 100 L / h) to atmospheric pressure.

The spheres obtained have a size of between 2.6 and 4.7 mm for the alcoogels and between 1.5 and 3.2 mm for the aerogels.

Example 6 - Synthesis of Zn [Fe (CN) ₅ NO] .xH ₂ O (2 ≤ x ≤ 18) in chitosan spheres This synthesis takes place according to the following reaction scheme:

[Chem.13]

In a Schlenk tube, 0.28 g (0.96 mmol) of Zn (NO ₃ ) ₂ are dissolved in 48 ml of deoxygenated methanol (colorless solution).

0.15 g (260 spheres) of chitosan (alcoogels or aerogels) are added to the solution and a flow of argon is passed through the solution for 2 to 10 minutes so as to maintain the solution under an inert atmosphere.

The mixture is stirred mechanically and isolated from the light (the Schlenk tube can be covered with aluminum foil for example) for 48 hours.

After these two days, the methanol solution is removed and new deoxygenated methanol is added (20 ml). The mixture is left under mechanical stirring and isolated from light for at least 15 minutes. After this time, the solvent is removed and 20 ml of deoxygenated methanol are added again. This cycle is repeated three times. Separately, a solution of [PPh ₄ ] ₂ [Fe (CN) ₅ NO] deoxygenated methanol is prepared by dissolving 0.86 g (0.96 mmol) of [PPh ₄ ] ₂ [Fe (CN) ₅ NO] in 48 ml of deoxygenated methanol.

The yellow / brown solution obtained was added to the Zn (NO ₃ ) ₂ -6H ₂ O solution containing the previously prepared chitosan spheres.

After that, a stream of argon is passed through the solution for several seconds through the Schlenk tube containing the mixture so as to keep it under inert conditions.

During this step, it is very important to work with the minimum amount of light, because nitroprusside solutions are sensitive to light: they can start releasing NO just with daylight or a lamp.

After that, the mixture is mechanically stirred and isolated from light for another 48 hours.

After these two days, the solution is removed from the mixture and the chitosan spheres are washed three times with 20 ml of deoxygenated methanol for at least 15 min for each wash.

During the wash cycle, mechanical agitation is used. The spheres are then dried under vacuum or with supercritical _{CO 2.} The chitosan spheres obtained have a size varying from 1.75 to 2.75 mm.

Example 7 - Synthesis of Zn [Fe (CN) ₅ NO] .xH ₂ O (2 ≤ x ≤ 18) in chitosan spheres Example 4 was repeated but using a reaction time of 24 hours for the stirring of the mixture of Zn (NO ₃ ) ₂ -6H ₂ O and chitosan spheres, and also a reaction time of 24 hours for the solution containing chitosan spheres, Zn (NO ₃ ) ₂ -6H ₂ O and [ PPh ₄ ] ₂ [Fe (CN) ₅ NO]. The chitosan spheres obtained have a size varying from 1.75 to 2.75 mm.

Example 8 - Synthesis of Zn [Fe (CN) ₅ NO] .xH ₂ O (2 ≤ x ≤ 18) in chitosan spheres.

In this example, acetonitrile is used as a solvent instead of methanol.

This synthesis takes place according to the following reaction scheme:

[Chem.14]

0.095 g (0.32 mmol) of Zn (NO ₃ ) ₂ .xH ₂ O are dissolved in 16 ml of deoxygenated acetonitrile in a Schlenk tube. A colorless solution is obtained.

0.05 g (100 spheres) of chitosan spheres (alcoogels or aerogels) are added to the solution and an argon flow is increased from 2 to 10 minutes so as to maintain the solution under an inert atmosphere.

The mixture is stirred mechanically and isolated from light (the Schlenk tube has been covered with aluminum foil) for 48 hours.

After these two days, the acetonitrile solution is removed and a new solution of deoxygenated acetonitrile is added (15 ml).

The mixture is stirred mechanically and isolated from light for at least 15 minutes. After this time, the solvent is removed and 20 ml of deoxygenated acetonitrile are added again. This cycle is repeated three times.

Separately, a solution of deoxygenated acetonitrile of [PPh ₄ ] ₂ [Fe (CN) ₅ NO] is prepared by dissolving 0.286 g (0.32 mmol) of [PPh ₄ ] ₂ [Fe (CN) ₅ NO] in 16 ml of deoxygenated acetonitrile.

The resulting yellow / brown solution was added to the solution containing the chitosan spheres.

A flow of argon is passed for several seconds through the Schlenk tube containing the mixture so as to maintain it under inert conditions. It is important here to work with the minimum amount of light.

After that, the mixture was mechanically stirred and isolated from light for another 48 hours.

After these two days, the solution is removed from the mixture and the chitosan spheres are washed three times with deoxygenated acetonitrile for at least 15 min for each wash.

During washing, mechanical agitation is used. The spheres are then dried under vacuum or with supercritical _{CO 2.}

The chitosan spheres obtained have a size varying from 1.75 to 2.75 mm.

Example 9 - Synthesis of Zn [Fe (CN) ₅ NO] .xH ₂ O (2 x 18) in chitosan spheres In this example, water is used as a solvent instead of methanol.

This reaction takes place according to the following reaction scheme:

[Chem.15]

In a Schlenk tube, 0.095 g (0.32 mmol) of Zn (NO ₃ ) ₂ -6H ₂ O are dissolved in 16 ml of deoxygenated water. A colorless solution is obtained.

0.05 g (100 spheres) of chitosan (alcoogels or aerogels) are added to the solution and a flow of argon is passed through the Schlenk tube for 2 to 10 minutes so as to maintain the solution under an inert atmosphere.

The mixture is allowed to stir mechanically and is isolated from light (the Schlenk tube has been covered with aluminum foil) for 48 hours.

After these two days, the aqueous solution is removed and new deoxygenated water is added (15 ml). The mixture is left under mechanical stirring and isolated from light for at least 15 min.

The solvent is then removed and new deoxygenated water (15 mL) is added. The cycle is repeated three times.

Separately, 0.095 g (0.32 mmol) of Na ₂ [Fe (CN) ₅ NO] are dissolved in 16 ml of deoxygenated water. The yellow / brown solution obtained was added to the solution containing the previously prepared chitosan spheres. A flow of argon is then passed through the Schlenk tube for 2 to 10 minutes so as to maintain it under inert conditions. During this step, it is very important to work with the minimum amount of light.

After that, the mixture is left under mechanical stirring and isolated from light for another 48 hours.

After these two days, the solution is removed from the mixture and the chitosan spheres are washed three times with deoxygenated water for at least 15 min for each wash.

During the wash cycle, mechanical agitation is used. The spheres are then dried under vacuum or with supercritical _{CO 2.}

The chitosan spheres obtained have a size varying from 1.75 to 2.75 mm.

Example 10 - Synthesis of Fe [Fe (CN) ₅ NO] .xH ₂ O (2 ≤ x ≤ 18) in chitosan spheres This synthesis takes place according to the following reaction scheme:

[Chem.16]

In a Schlenk tube, 0.4 g (1.2 mmol) of Fe (BF ₄ ) ₂ -6H ₂ O are dissolved in 60 ml of deoxygenated methanol. A colorless solution is obtained.

0.2 g of chitosan spheres (alcoogels or aerogels) are added to the solution and a flow of argon is passed through the Schlenk tube for several seconds so as to maintain the solution under an inert atmosphere. The mixture is left under mechanical stirring and isolated from light (the Schlenk tube has been covered with aluminum foil) for 48 hours.

After these two days, the methanol solution is removed and new deoxygenated methanol is added (25-30 ml). The mixture is left under mechanical stirring and isolated from light for at least 15 min.

After that, the solvent is removed and new deoxygenated methanol is added (25-30 ml). This cycle is repeated three times.

Separately, 1.07 g (1.2 mmol) of [PPh ₄ ] ₂ [Fe (CN) ₅ NO] are dissolved in 60 ml of deoxygenated methanol. The resulting yellow / brown solution was added to the solution containing the chitosan spheres prepared above.

After that, a flow of argon is passed for 2-10 minutes through the Schlenk tube containing the mixture so as to keep it under inert conditions.

During this stage, it is very important to work with the minimum amount of light.

After that, the mixture is placed under mechanical stirring and isolated from light for another 48 hours.

After these two days, the solution is removed from the mixture and the chitosan spheres are washed three times with deoxygenated methanol (25-30 ml), for 15 min for each wash.

During the washings, mechanical agitation is used.

The spheres are then dried under vacuum or with supercritical _{CO 2.} The chitosan spheres obtained have a size varying from 1.25 to 2.25 mm.

Example 11 - Synthesis of Fe [Fe (CN) ₅ NO] xH ₂ O (2 ≤ x ≤ 18) in chitosan spheres In this example, impregnation times of 24 hours are used compared to example 10 (48 hour impregnation time).

This synthesis takes place according to the following reaction scheme:

[Chem.17]

0.2 g of chitosan spheres (alcoogels or aerogels) washed three times with methanol are added to the solution and a flow of argon is passed through the Schlenk tube for 2 to 10 minutes so as to maintain the solution under an inert atmosphere. The mixture is left under mechanical stirring and isolated from light (the Schlenk tube has been covered with aluminum foil) for 24 hours.

After 1 day, the methanol solution is removed and new deoxygenated methanol is added (25-30 ml). The mixture is left under mechanical stirring and isolated from light for at least 15 min. After that, the solvent is removed and new deoxygenated methanol is added (25-30 ml). This cycle is repeated three times.

After that, the mixture is placed under mechanical stirring and isolated from light for another 24 hours.

After these 24 hours, the solution is removed from the mixture and the chitosan spheres are washed three times with deoxygenated methanol (25-30 ml) for 15 min for each wash.

During the washings, mechanical agitation is used. The spheres are then dried under vacuum or with supercritical _{CO 2.} The chitosan spheres obtained have a size varying from 1.25 to 2.25 mm. Example 12 - Synthesis of Fe [Fe (CN) ₅ NO] .xH ₂ O (2 ≤ x ≤ 18) in chitosan spheres In this example, ascorbic acid is used to prevent the oxidation of Fe ²⁺ .

This synthesis takes place according to the following reaction scheme:

[Chem.18]

In a Schlenk tube containing 0.2 g (350 spheres) of chitosan (alcoogels or aerogels), a solution of 0.239 g of Fe (Cl ₂ ) .4H ₂ O and 0.211 g of ascorbic acid dissolved in 60 ml of deoxygenated methanol is added. There is an immediate change in color of the chitosan spheres which turn blue.

A flow of argon is passed for 2 to 10 minutes through the Schlenk tube so as to maintain the solution under an inert atmosphere.

The mixture is allowed to stir mechanically and isolated from light (the Schlenk tube has been covered with aluminum foil) for 48 hours.

After these two days, the deoxygenated methanol solution is removed and a new deoxygenated methanol solution is added (25-30 ml). The mixture is left under mechanical stirring and isolated from light for at least 15 min.

After that, the solvent is removed and new deoxygenated methanol (15-20 mL) is added. This cycle is repeated three times.

Separately, the methanolic solution of [PPh ₄ ] ₂ [Fe (CN) ₅ NO] is prepared by dissolving 1.7 g of [PPh ₄ ] ₂ [Fe (CN) ₅ NO] in 60 ml of deoxygenated methanol. The resulting yellow / brown solution was added to the solution containing the chitosan spheres prepared above.

After that, a flow of argon is passed for several seconds through the Schlenk tube containing the mixture so as to keep it under inert conditions.

During this stage it is very important to work with the minimum amount of light.

After these two days, the solution is removed from the mixture and the chitosan spheres are washed three times with deoxygenated methanol for at least 15 min for each washing. During washing, mechanical agitation is used. The spheres are then dried under vacuum or with supercritical _{CO 2.}

The chitosan spheres obtained have a size varying from 1.25 to 2.25 mm.

Example 13 - Synthesis of Fe [Fe (CN) ₅ NO] xH ₂ O (2 ≤ x ≤ 18) in chitosan spheres This synthesis takes place according to the following reaction scheme:

[Chem.19]

The procedure was as in Example 12 but without adding ascorbic acid and using reaction times of 24 hours instead of 48 hours, in order to avoid the formation of Prussian blue in place of Fe-nitroprusside.

This example shows that the same compound is formed with ascorbic acid or without ascorbic acid: the beads obtained after integration of Fe-nitroprusside have the same dimensions, 1.25 to 2.25 mm. Example 14 - Synthesis of Fe [Fe (CN) ₅ NO] xH ₂ O (2 ≤ x ≤ 18) in chitosan spheres This synthesis takes place according to the following reaction scheme:

[Chem.20]

In this example, the procedure was as in Example 12, that is to say by adding ascorbic acid but using a reaction time of only 24 hours.

This example shows that the same compound is formed using a reaction time of 24 hours or 48 hours: the beads obtained after integration of Fe-nitroprusside have the same dimensions, 1.25 to 2.25 mm

Example 15 - Synthesis of Fe [Fe (CN) ₅ NO] .xH ₂ O (2 ≤ x ≤ 18) in chitosan spheres In this example, glucose is used instead of ascorbic acid to avoid oxidation of Fe ²⁺ . This synthesis takes place according to the following reaction scheme:

[Chem.21]

In a Schlenk tube containing chitosan spheres was added a solution of 0.07 g (0.35 mmol) of Fe (Cl ₂ ) .4H ₂ O and 0.63 g (0.35 mmol) of glucose dissolved in 17 ml of deoxygenated methanol. The amount of chitosan spheres was 0.5 g (100 spheres) (alcoogels or aerogels). A little water should be added because glucose is not completely soluble in deoxygenated methanol. A flow of argon is passed through the Schlenk tube for several seconds so as to maintain the solution under an inert atmosphere. The mixture is allowed to stir mechanically and isolated from light (the Schlenk tube has been covered with aluminum foil) for 48 hours.

After these two days when the chitosan spheres have turned orange, the deoxygenated methanol solution is removed, and new deoxygenated methanol is added (25 to 30 ml). The mixture is left under mechanical stirring and isolated from light for at least 15 min.

After this time, the solvent is removed and new deoxygenated methanol is added. This cycle is repeated three times.

Separately, 0.313 g (0.35 mmol) of [PPh ₄ ] ₂ [Fe (CN) ₅ NO] are dissolved in 17 ml of deoxygenated methanol. The resulting yellow / brown solution was added to the Schlenk tube containing the chitosan spheres. After that, a stream of argon is passed through the Schlenk tube so as to keep the mixture under inert conditions.

After that, the mixture was mechanically stirred and isolated from light for another 48 hours. After these two days, the solution is removed from the mixture and the chitosan spheres are washed three times with deoxygenated methanol for at least 15 min for each washing. During washing, mechanical agitation is used. The spheres are then dried under vacuum or with supercritical _{CO 2.} The chitosan spheres obtained have a size varying from 1.25 to 2.25 mm.

Example 16 - Synthesis of Fe [Fe (CN) ₅ NO] .xH ₂ O (2 ≤ x ≤ 18) in chitosan spheres This synthesis takes place according to the following reaction scheme:

[Chem.22]

The procedure was as in Example 13 but without adding glucose and using a reaction time of 48 hours instead of 24 hours.

Example 17 - Synthesis of Fe [Fe (CN) ₅ NO] .xH ₂ O (2 ≤ x ≤ 18) in chitosan spheres In this example, the sodium nitroprusside precursor is used and impregnation times of 48 hours , then 24 hours are used.

This synthesis takes place according to the following reaction scheme:

[Chem.23]

0.12 g (0.6 mmol) of FeCl ₂ .4H ₂ O and 0.11 g of ascorbic acid are dissolved in 60 ml of distilled water. This solution is added to a pill container containing 0.1 g of chitosan spheres in the form of airgels (blue spheres in contact with the solution), and left under magnetic stirring for 48 hours. The solvent is then removed and the spheres washed in 60 mL of methanol; the operation is repeated three times with removal of the solvent after each washing.

0.18 g (0.6 mmol) of Na ₂ [Fe (CN) ₅ NO] .2H ₂ O are dissolved in 60 ml of distilled water and isolated from light. This solution is then added to the pill container containing the chitosan spheres and left under magnetic stirring for 48 hours. The solvent is then removed and the spheres washed in 60 mL of methanol; the operation is repeated three times with removal of the solvent after each washing. The same cycle (spheres in Fe ²⁺ solutions then in Fe (CN) ₅ NO ^{2- solution} ) is repeated two more times under the same conditions (concentrations, volumes) but for 24 hours:

1) Spheres brought into contact in a solution of FeCl ₂ .4H ₂ O and ascorbic acid for 24 hours and then washed;

2) Beads brought into contact in a solution of Na ₂ [Fe (CN) ₅ NO] .2H ₂ O for 24 hours and then washed;

The spheres are then dried with supercritical _{CO 2.} The chitosan spheres obtained have a size varying from 1.25 to 2.25 mm. Example 18 - Synthesis of Fe [Fe (CN) ₅ NO] .xH ₂ O (2 ≤ x ≤ 18) in chitosan spheres This synthesis takes place according to the following reaction scheme:

[Chem.24]

The procedure was as in Example 15 but using a reaction time of only 24 hours. This example shows that the same compound is formed using a reaction time of 24 h instead of 48 h.

Example 19 - Synthesis of a chitosan film

1.75 g of chitosan and 364 μl of acetic acid are placed in 70 ml of water. The mixture obtained is placed under stirring overnight to dissolve the chitosan.

By the dip-coating method, a glass slide (76 x 26 mm) is introduced into the chitosan solution at the speed which has been chosen (between 2 and 5 mV)

Then, the slide is successively introduced into increasing solutions of ethanol (0, 20, 40, 60, 80, 100%). The film is then air dried.

Example 20 - Synthesis of chitosan films lg of chitosan (chitosan, low molecular weight, Sigma Aldrich), are dissolved in 40 mL of a solution of acetic acid at 1 vol.%, With magnetic stirring overnight at room temperature . A first sheet of filter paper is then impregnated in a 2 M NaOH solution prepared beforehand. The sheet is placed in a 60 mm diameter petri dish.

6.5 mL of the chitosan solution are added to the sheet of filter paper in the petri dish. A second sheet of filter paper is also impregnated in 2M NaOH and placed on top of the chitosan solution in the filter paper. 3 mL of NaOH are also added on top. After 1 hour of gelation, the excess NaOH solution and the filter papers are removed. The chitosan film formed is rinsed in 3 mL of distilled water with orbital stirring for 15 minutes then the solvent removed. The operation is repeated until the washing water reaches a pH 6-7.

The film is then immersed for 15 minutes in water-methanol baths of 3 ml of increasing concentration in methanol (10, 30, 50, 70, 90, 100% by volume) in order to exchange the water contained in the film by the methanol. The film is then stored in methanol.

Example 21: Synthesis of Zn [Fe (CN) ₅ NO] .xH ₂ O (2 ≤ x ≤ 18) in chitosan films This synthesis takes place according to the following reaction scheme:

[Chem.25]

0.28 g (0.96 mmol) of Zn (NO ₃ ) ₂ .6H ₂ O are dissolved in 48 ml of deoxygenated methanol in a beaker.

A colorless solution is obtained and the chitosan film prepared on a glass slide in Example 20 is placed in the solution. The mixture is placed under mechanical stirring and isolated from light (the beaker has been covered with aluminum foil for 24 hours).

After these 24 hours, the methanol solution is removed and new methanol is added (20 ml). The mixture is left under mechanical stirring and isolated from light for at least 15 min. Then the solvent is removed and new methanol is added. This cycle is repeated three times.

Separately, a solution of PPh ₄ [Fe (CN) ₅ (NO)] in methanol is prepared (0.86 g, 48 ml). The yellow / brown solution was added to the beaker containing the film from the previous reaction.

It is very important during this stage to work with the minimum amount of light.

Then the mixture was placed under mechanical stirring and isolated from light for an additional 24 hours.

After these 24 hours, the solution is removed from the mixture and the chitosan film is washed three times with deoxygenated methanol for 15 minutes for each wash. During washing, mechanical agitation is used.

Particles of 1 to 6 mih are obtained on the surface of the chitosan film, the thickness of which is approximately 1 mm.

Example 22 - Synthesis of Fe [Fe (CN) ₅ NO] .xH ₂ O (2 ≤ x ≤ 18) in chitosan films This reaction takes place according to the following reaction scheme:

[Chem.26]

0.338 g (1 mmol) of Fe (BF ₄ ) ₂ -6H ₂ O are dissolved in 50 ml of deoxygenated methanol in a beaker. The colorless solution obtained is added to a Schlenk tube containing a film of chitosan prepared on a glass slide. A flow of argon is passed for several seconds through the Schlenk tube to maintain the solution under an inert atmosphere. The mixture is left under mechanical stirring and isolated from light (the Schlenk tube has been covered with aluminum foil) for 24 hours.

After these 24 hours, the methanol solution is removed and new deoxygenated methanol is added (25-30 ml). The mixture is left under mechanical stirring and isolated from light for at least 15 min.

After that, the solvent is removed and new deoxygenated methanol is added. This cycle is repeated three times.

Separately, 0.895 g (1 mmol) of PPh ₄ [Fe (CN) ₆ (NO)] are dissolved in 50 ml of deoxygenated methanol. The resulting yellow / brown solution was added to the Schlenk tube. After that, a flow of argon is passed for several seconds through the Schlenk tube containing the mixture so as to keep it under inert conditions. During this step, it is very important to work with the minimum amount of light.

After these 24 hours, the solution is removed and the chitosan film is washed three times with deoxygenated methanol for at least 15 min for each wash. During washing, mechanical agitation is used. The film is then dried in air. A film of about 1 mm thick of brown color is obtained.

The particles of the organometallic salt of nitroprusside have a size of 1 to 6 mih at the surface of the film.

Example 23 - Synthesis of Fe [Fe (CN) ₅ NO] .xH ₂ O (2 ≤ x ≤ 18) in chitosan films This reaction takes place according to the following reaction scheme:

[Chem.27]

0.03 g (0.15 mmol) of FeCl ₂ .4H ₂ O and 0.03 g of ascorbic acid are dissolved in 15 ml of methanol. This solution is added to a petri dish containing a chitosan film previously stored in methanol. The mixture is left under orbital stirring for 48 hours. The solvent is then removed and the film washed with 15 mL of methanol; the operation is repeated three times with removal of the solvent after each washing.

0.04 g (0.15 mmol) of Na ₂ [Fe (CN) ₅ NO] .2H ₂ O are dissolved in 15 ml of methanol and isolated from light. This solution is then added to the petri dish containing the chitosan film and left under orbital stirring for 48 hours. The solvent is then removed and the film washed with 15 mL of methanol; the operation is repeated three times with removal of the solvent after each washing. The same cycle (film in Le ²⁺ solutions then in Le (CN) NO ^{2- solution} ) is repeated two more times under the same conditions (concentrations, volumes) but for 24 hours:

1) Lilm brought into contact in a solution of FeCl ₂ .4H ₂ O and ascorbic acid for 24 hours and then washed;

2) Film brought into contact in a solution of Na ₂ [Fe (CN) ₅ NO] .2H ₂ O for 24 hours and then washed; The film is then dried in air. A film of about 1 mm thick of brown color is obtained.

The particles of the organometallic salt of nitroprusside have a size of 1 to 6 mih at the surface of the film. Comparative example 1

A film containing dihydrated sodium salt of nitroprusside was prepared. This reaction proceeds according to the following reaction scheme:

[Chem.28]

0.04 g (0.15 mmol) of Na ₂ [Fe (CN) ₅ NO] .2H ₂ O are dissolved in 15 ml of methanol and isolated from light. This solution is then added to the petri dish containing the chitosan film and left under orbital stirring for 48 hours. The solvent is then removed and the film washed with 15 mL of methanol; the operation is repeated three times with removal of the solvent after each washing. The pink film obtained is dried in air.

Comparative Example 2 - Synthesis of Chitosan Spheres Containing Sodium Salt of Nitroprusside Dihydrate

This reaction takes place according to the following reaction scheme:

[Chem.29]

0.18 g (0.06 mmol) of Na ₂ [Fe (CN) ₅ N0] .2H20 are dissolved in 60 ml of methanol and isolated from light. This solution is then added to a pill container containing 0.1 g chitosan beads (alcoogels) and left under orbital stirring for 48 hours. The solvent is then removed and the films are washed with 60 mL of methanol; the operation is repeated three times with removal of the solvent after each washing. The pink beads obtained are dried with supercritical _{CO 2.}

Example 24 - NO release test

The particles, films and spheres obtained in Examples 3, 17 and 23 as well as in Comparative Example 1 were tested for their property of releasing NO and CN.

The NO release measurement was performed according to either of the following two protocols.

- The measurement of the so-called solid NO release from samples of organometallic salts of nitroprusside coated in a biopolymer (particles, spheres and film obtained respectively in Examples 3, 17 and 23) is the measurement of the release of NO from the samples under irradiation by light radiation emitting in visible wavelengths. This release of NO was measured using an NO analyzer (Sievers NOA280i) after calibrating the device between 0 and 90 pμm of NO in N2. 30 mg of a ground sample are thus placed in a sample holder and subjected to visible irradiation using a desk lamp. The quantity of NO released is thus measured continuously every 5 seconds over 48 hours. The quantities released were subsequently cumulated to plot the NO release kinetics as a function of time. -The measurement of the release of NO, known as liquid, from samples of organometallic salts of nitroprusside is the measurement of the release of NO from samples suspended in water and under irradiation by light radiation emitting in the wavelengths of the visible (compounds of Examples 3, 17, 23). It is carried out as follows.

The formation of NO is measured by the quantification of nitrites (NO ₂ -) which constitute the products of oxidative degradation of NO. This is because NO reacts quickly with molecules such as oxygen or the superoxide anion to give nitrites. These can subsequently be determined by a spectrophotometric method using the Griess reaction: it is a diazotization reaction where the nitrites form a diazonium salt with sulfanilamide which is then coupled with an amine (N-naphthylethylene diamine ) to give a dye absorbent at 540 nm. A calibration line is carried out in water using NaNO ₂ between 0 and 50 μM. Subsequently 10 mg of samples are placed in an eppendorf to which 2 ml of distilled water are added. The eppendorf is subjected to visible irradiation using a desk lamp. At different times, the sample is centrifuged, the supernatant is recovered and 2 ml of distilled water are stirred again, then the mixture returned to irradiation. Each recovered supernatant is then assayed by the Griess reaction using increasing concentrations of Griess reagents until the maximum absorbance value is obtained. The quantities obtained from nitrite concentrations are then cumulated and the change in the quantity of NO released as a function of time is plotted.

The results obtained are shown in the form of curves in Figures 1 to 7.

Figure 1 shows the release curve of NO from the organometallic salt of nitroprusside of formula I coated in a film of chitosan (compound of Example 23) suspended in water as a function of time and Figure 2 shows the infrared spectra before and after irradiation of this compound of Example 23 in suspension in water. The infrared spectrum obtained shows the appearance of the CN vibration band of Prussian blue.

Figure 3 shows the release curve of NO from the organometallic salt of nitroprusside of formula I (compound of Example 3) and Figure 4 shows the infrared spectra before and after irradiation of this compound of Example 3. The infrared spectrum obtained shows the appearance of the CN vibration band of Prussian blue.

Figure 5 shows the release curve of NO from the organometallic salt of nitroprusside of formula I (compound of Example 17) and Figure 6 shows the infrared spectra before and after irradiation of this compound of Example 17. The infrared spectrum obtained shows the appearance of the CN vibration band of Prussian blue.

FIG. 7 shows the NO release curve of the sodium salt dihydrate of nitroprusside of the prior art coated in a chitosan film obtained in Comparative Example 1, as a function of time and in suspension in water, when exposed to irradiation. FIG. 8 shows the curve of the release of NO from the organometallic salt of formula I obtained in Example 23, as a function of time, when exposed to light radiation emitting in the range of visible wavelengths. This curve is obtained with the film of Example 23 not placed in suspension in water, as is the case for FIG. 1.

FIG. 9 shows the infrared spectra, before and after irradiation with light radiation emitting in the field of visible wavelengths, of the organometallic salt of formula I obtained in Example 23. These spectra are obtained with the film of Example 23 not placed in suspension in water, as is the case for Figure 2.

As can be seen, there is a gradual release of NO as a function of time and under irradiation with all the compounds, whether or not they are placed in suspension in water.

However, the quantities of NO released (μmol / mg) under irradiation are greater in the case of organometallic salts alone or coated in the spheres or films of chitosan, compared to nitroprusside coated in chitosan.

Example 25 - CN release test

50 mg of samples are placed in 30 mL of distilled water. The suspension is subjected to irradiation in the visible using a desk lamp. At determined times (1, 2, 4 and 7 days), the suspension is centrifuged, the supernatant is recovered and 30 ml of distilled water are added again, then the suspension is put back under irradiation. The supernatants are then combined (total volume of 120 mL) to quantify the CN- ions.

The quantification of CN- ions is carried out in solution in water by a photometric assay method: the free cyanides are decomposed at pH 3.8. The hydrogen cyanide present at pH 3.8 is separated by gas diffusion at a temperature between 30 ° C and 40 ° C through a hydrophobic membrane. Using the gas diffusion method, hydrogen cyanide is absorbed into sodium hydroxide solution.

[Table 1]

The reference compound of the prior art (sodium salt of nitroprusside di hydrate) alone or coated in chitosan releases little NO (less than 0.03 mmol / mg whatever the conditions) but still releases traces of CN- . The compounds linked to the invention release quantities of NO (between 0.06 and 0.18 depending on the conditions) and release comparable traces of CN. Without irradiation, the reference compound of the prior art (sodium salt of nitroprusside dihydrate) alone or coated in chitosan also releases traces of CN-. The presence of CN in the samples is therefore not linked to the release of NO but intrinsic to the presence of the reference compound of the prior art (sodium salt of nitroprusside dihydrate).

However, these quantities of the order of 100 μg / L are clearly lower than the quantities for which symptoms are observed in humans of 50-100 mg / L, i.e. quantities 500-1000 times lower than the toxic quantities (Meyler's Side Effects of Drugs Sixteenth Edition; The International Encyclopedia of Adverse Drug Reactions and Interactions, Sodium Nitroprusside 2016, Pages 421- 423). Example 26 - Synthesis of Alginate Films

2 g of alginate are dissolved in 100 ml of water with magnetic stirring overnight at room temperature. A first sheet of filter paper is then impregnated in a solution of FeCl ₂ .4H ₂ O / 0.1 M ascorbic acid previously prepared. The sheet is placed in a 60 mm diameter petri dish.

10 mL of the alginate solution is added to the sheet of filter paper in the petri dish. A second sheet of filter paper is also impregnated in a solution of FeCl ₂ .4H ₂ O / 0.1 M ascorbic acid and placed on top of the chitosan solution in the filter paper. 10 mL of FeCl ₂ .4H ₂ O / 0.1 M ascorbic acid are also added on top.

After 1 hour of gelation, the excess solution and the filter papers are removed. The alginate film formed is rinsed in 15 mL of distilled water with orbital stirring for 15 minutes then the solvent removed. The operation is repeated 3 times.

The film is then immersed for 15 minutes in Water-Methanol baths of 15 mL of increasing concentration of methanol (10, 30, 50, 70, 90, 100% by volume) in order to exchange the water contained in the film by methanol. The film is then stored in methanol.

Example 27 - Synthesis of Fe [Fe (CN) ₅ NO] .xH ₂ O (2 ≤ x ≤ 18) in alginate films This reaction takes place according to the following reaction scheme:

[Chem.30]

0.05 g (0.2 mmol) of Na ₂ [Fe (CN) ₅ NO] .2H ₂ O are dissolved in 20 ml of methanol and isolated from light. This solution is then added to the petri dish containing the alginate film and left under orbital stirring for 48 hours. The solvent is then removed and the film washed with 15 mL of methanol; the operation is repeated three times with removal of the solvent after each washing. 0.04 g (0.2 mmol) of FeCl ₂ .4H ₂ O and 0.04 g of ascorbic acid are dissolved in 20 ml of methanol. This solution is added to a petri dish containing an alginate film previously stored in methanol. The mixture is left under orbital stirring for 48 hours. The solvent is then removed and the film washed with 15 mL of methanol; the operation is repeated three times with removal of the solvent after each washing.

The same cycle (film in Fe ²⁺ solutions then in Fe (CN) NO ^{2- solution} ) is repeated two more times under the same conditions (concentrations, volumes) but for 24 hours.

The film obtained is brought into contact in a solution of FeCl ₂ .4H ₂ O and ascorbic acid for 24 hours and then washed; it is then brought into contact in a solution of Na ₂ [Fe (CN) ₅ NO] .2H ₂ O for 24 hours and then washed.

The film is then dried in air. A film of about 1 mm thick of brown color is obtained.

Claims

1. Use of an organometallic salt of nitroprusside of the following formula I: in which: M ^II represents Zn ^II or Fe ^II , and

- 2 ≤ x ≤ 18 as agent releasing nitrogen monoxide NO.

2. Use according to claim 1 characterized in that the organometallic salt of nitroprusside of formula I is coated in a biopolymer, preferably chosen from chitosan, an alginate of the metal M, a copolymer of lactic acid and glycolic acid ( PLGA), a poly (L-lactide) polymer (PLLA), most preferably chitosan.

3. Use according to claim 2 wherein the organometallic salt of nitroprusside of formula I is coated in the biopolymer to form a film.

4. Use of an organometallic salt of nitroprusside of formula I according to claim 2 characterized in that the organometallic salt of nitroprusside of formula I is coated in the biopolymer to form at least one sphere.

5. Use according to any one of the preceding claims, characterized in that the organometallic salt of nitroprusside of formula I is in the form of particles having their largest dimension between 50 nm to 6 μm.

6. Organometallic nitroprusside salt of the following formula I:

M ^II [Fe ^II (CN) ₅ (NO)]. XH ₂ O

Formula I wherein M ^II represents Zn ^II or Fe ^II , and

- 2 ≤ x ≤ 18 as an antibacterial agent.

7. Organometallic nitroprusside salt of the following formula I:

M ^II [Fe ^II (CN) ₅ (NO)]. XH ₂ O

Formula I wherein M ^II represents Zn ^II or Fe ^II , and

- 2 ≤ x ≤ 18 as an agent promoting wound healing.

8. Organometallic nitroprusside salt of the following formula I: M ⁿ [Fe ⁿ (CN) 5 (N0)], xH20 Formula I wherein M ^II represents Zn ^II or Fe ^II , and

- 2 ≤ x ≤ 18 for use in the treatment of pulmonary hypertension.

9. Organometallic nitroprusside salt of the following formula I:

M ^II [Fe ^II (CN) ₅ (NO)]. XH ₂ O

Formula I wherein M ^II represents Zn ^II or Fe ^II , and

- 2 <x <18 for use according to any one of claims 6 to 8 characterized in that it is in the form of particles having a size between 50 nm and 6 μm

10. Organometallic nitroprusside salt of the following formula I:

M ^II [Fe ^II (CN) ₅ (NO)]. XH ₂ O

Formula I wherein M ^II represents Zn ^II or Fe ^II , and

- 2 ≤ x ≤ 18 for use according to any one of claims 6 to 9, characterized in that it is coated in a biopolymer, preferably chosen from chitosan, an alginate of the metal M, a copolymer of lactic acid and glycolic acid (PLGA), a poly (L-lactide) polymer (PLLA), most preferably chitosan.

11. Organometallic nitroprusside salt of the following formula I:

M ^II [Fe ^II (CN) ₅ (NO)]. XH ₂ O Formula I in which M ^II represents Zn ^II or Fe ^II , and

- 2 ≤ x ≤ 18 for use according to claim 10, characterized in that it is in the form of a film.

12. Organometallic nitroprusside salt of the following formula I:

M ^II [Fe ^II (CN) ₅ (NO)]. XH ₂ O

Formula I in which M ^II represents Zn ^II or Fe ^II , and 2 ≤ x ≤ 18 for use according to claim 10 characterized in that it is in the form of at least one sphere.

13. Composite material characterized in that it comprises an organometallic salt of nitroprusside of the following formula I:

M ^II [Fe ^II (CN) ₅ (NO)]. XH ₂ O Formula I in which M ^II represents Zn ^II or Fe ^II , and - 2 ≤ x ≤ 18 coated in a biopolymer, preferably chosen from among chitosan, an alginate of metal M, a copolymer of lactic acid and glycolic acid (PLGA), a poly (L-lactide) (PLLA) polymer, most preferably chitosan.

14. Composite material according to claim 13, characterized in that the organometallic nitroprusside salt of formula I is in the form of particles having a size of between 50 nm to 6 μm.

15. Composite material according to claim 13 or 14, characterized in that the organometallic salt of nitroprusside of formula I is coated in a biopolymer, preferably chosen from chitosan, an alginate of the metal M, a copolymer of lactic acid and of glycolic acid (PLGA), a poly (L-lactide) polymer (PLLA), most preferably chitosan, to form at least one sphere the size of which is between 1.75 and 2.75 mm.

16. Composite material according to claim 13 or 14, characterized in that it is in the form of a film of a biopolymer, preferably chosen from chitosan, an alginate of the metal M, a copolymer of lactic acid and d glycolic acid (PLGA), a poly (L-lactide) (PLLA) polymer, most preferably chitosan, in which the organometallic salt of nitroprusside of formula I is coated.

17. Use of the composite material according to any one of claims 13 to 14 as an agent releasing nitrogen monoxide NO.

18. Composite material according to any one of claims 13 to 16 for use as an antibacterial agent, or agent for promoting wound healing, or for the treatment of pulmonary hypertension.

19. Implant comprising an organometallic salt of nitroprusside of the following formula I:

M ^II [Fe ^II (CN) ₅ (NO)]. XH ₂ 0O Formula I in which M ^II represents Zn ^II or Fe ^II , and 2 ≤ x ≤ 18

20. Implant according to claim 19, characterized in that the organometallic salt of nitroprusside of formula I is in the form of particles having a size of between 50 nm to 6 μm.

21. Implant according to claim 19 or 20, characterized in that the organometallic salt of nitroprusside of formula I is coated in a biopolymer, preferably chosen from chitosan, an alginate of the metal M, a copolymer of lactic acid and of acid. gly colic (PLGA), a poly (L-lactide) polymer (PLLA), most preferably chitosan.

22. Implant according to any one of claims 19 to 21 characterized in that it is covered with particles of organometallic salt of nitroprusside of formula I, optionally coated in a biopolymer, preferably chosen from chitosan, an alginate of the metal M , a copolymer of lactic acid and glycolic acid (PLGA), a poly (L-lactide) polymer (PLLA), most preferably chitosan.

23. Implant according to any one of claims 19 to 21 characterized in that the implant contains the organometallic salt of nitroprusside of formula I, optionally coated in a biopolymer, preferably chosen from chitosan, an alginate of the metal M, a copolymer of lactic acid and glycolic acid (PLGA), a poly (L-lactide) polymer (PLLA), most preferably chitosan.

24. Process for the release of NO, characterized in that it comprises the irradiation of particles of an organometallic salt of nitroprusside of the following formula I:

M ^II [Fe ^II (CN) ₅ (NO)]. XH ₂ O

Formula I wherein M ¹¹ represents Zn ^II or Fe ^II , and

- 2 ≤ x ≤ 18 optionally coated in a biopolymer, preferably chosen from chitosan, an alginate, a copolymer of lactic acid and glycolic acid (PLGA), a poly (L-lactide) (PLLA) polymer, the more preferably chitosan, with radiation emitting in the visible wavelengths.

25. A method of releasing nitrogen monoxide, characterized in that it comprises the application of an aqueous solution of an amino compound on particles of organometallic salt of nitroprusside of the following formula I:

M ^II [Fe ^II (CN) ₅ (NO)]. XH ₂ O

Formula I wherein M ¹¹ represents Zn ^II or Fe ^II , and

- 2 ≤ x ≤ 18 optionally coated in a biopolymer, preferably chosen from chitosan, an alginate of the metal M, a copolymer of lactic acid and glycolic acid (PLGA), a poly (L-lactide) polymer (PLLA) ), most preferably chitosan.

26. Process for decontaminating a substrate suspected of being contaminated by bacteria, characterized in that it comprises: a) the application, on the substrate, of an organometallic salt of nitroprusside of the following formula I:

- 2 ≤ x ≤ 18 in the form of particles, optionally coated in a biopolymer, preferably chosen from chitosan, an alginate of metal M, a copolymer of lactic acid and gly colic acid (PLGA), a poly ( L-lactide) (PLLA), most preferably chitosan, and b) irradiating these particles with radiation emitting in visible wavelengths and / or applying an aqueous solution of a compound amino on the organometallic salt of nitroprusside of formula I.

27. Process for the synthesis of particles of an organometallic salt of nitroprusside of the following formula I:

M ^II [Fe ^II (CN) ₅ (NO)]. XH ₂ O Formate I in which M ^II represents Zn ^II or Fe ^II , and

- 2 ≤ x ≤ 18 characterized in that it comprises the following steps:

-preparation of spheres or polymer film preserved in methanol,

- impregnation of the spheres or film in a nitroprusside solution for 24 to 48 hours followed by washing

-impregnation of the spheres or film in a metal salt solution for 24 to 48 hours followed by washing

- repetition of successive impregnations 1 to 2 times

-drying with _{supercritical CO 2} for the spheres and in air for the films.