WO2020154822A1

WO2020154822A1 - Aerogel based on graphene oxide and chitosan with haemostatic application

Info

Publication number: WO2020154822A1
Application number: PCT/CL2020/050007
Authority: WO
Inventors: Manuel Francisco MELÉNDREZ CASTRO; Claudio AGUAYO TAPIA; Katherina Fabiola FERNÁNDEZ ELGUETA; Toribio Andrés FIGUEROA AGUILAR; Satchary Alexander CARMONA GIACAMAN
Original assignee: Universidad De Concepcion
Priority date: 2019-01-31
Filing date: 2020-01-13
Publication date: 2020-08-06
Also published as: CL2019000254A1

Abstract

The invention relates to an aerogel that allows blood absorption and clot formation for wound healing, which comprises 60-93% w/w graphene oxide noncovalently functionalised with a deacetylated chitin derivative in a proportion of 50-5% w/w and 15-2% w/w proanthocyanidins in the matrix thereof.

Description

AEROGEL BASED ON GRAPHENE OXIDE AND CHITOSAN WITH HEMOSTATIC APPLICATION.

Technical Sector

The technology is aimed at the health area, more particularly, it corresponds to an airgel that allows the absorption of blood and the formation of a clot for wound healing.

Previous Technique

Wounds are common conditions that occur in humans and depending on their magnitude and the place where it occurs -either vein or artery- can lead to profuse bleeding or bleeding, which can lead to the death of a person. As reported by the World Health Organization (WHO), for 2017 the main causes of death were due to cardiovascular conditions and diabetes, both diseases with a direct tendency to the appearance of bleeding.

In general, hemostatic agents (ideal dressings) are used to treat these conditions, which should achieve rapid wound healing at a reasonable cost and with minimal inconvenience to the patient. From the physiological point of view, the dressing used must be compatible with the exposed skin cells, they must also allow good exudation in the wound, correct occlusion of the wound and guarantee the presence of a humid environment for allowing an adequate supply of blood and oxygen to be maintained, an adequate temperature, and thereby avoiding infection (Boateng & Catanzano, 2015).

Depending on the type and magnitude of the wound, level of exudate, presence or absence of infection and stage of healing in which the wound is, several medical products have been used for these purposes. These hemostatic materials can be classified depending on their mode of action in the body into hydrogels, standard hydrocolloids, impregnated compresses, bioactive and carbon dressings, or reabsorbiol materials such as Gelfoam®, Surgicel® and aerogels. An important factor for the use of these materials as hemostatic agents is their surface properties and, in particular, their surface load. Faced with this situation, the need arises to develop formulations with improved properties for the effective and precise delivery of the required therapeutic agents.

In the case of micro- or meso-porous inorganic hemostatic materials, these promote hemostasis based on the rapid absorption of the fluid and the proliferation of blood cells and platelets (Liang, et al., 2018). An example of this is the chitosan bands modified with synthetic polymers (Chan et al., 2016), whose positively charged surface stops wound bleeding through electrostatic interaction with cell membranes. negatively charged with erythrocytes, to cause agglutination and wound sealing through tissue adhesion. Yan et al. (2017) propose a sponge-like material based on collagen reinforced with chitosan and calcium pyrophosphate nanoflowers, which is capable of activating the intrinsic procedure of the coagulation cascade, inducing adhesion of hemocytes and platelets, and promoting the blood coagulation to control bleeding in vitro and in vivo. Similarly, hydrogels have been developed for hemostatic activity (Behrens et al., 2014), from synthetic hydrogel particles with N- (3-aminopropyl) methacrylamide, showing rapid swelling due to the absorption of blood causing local aggregation while delaying coagulation in the fluid, thereby avoiding the risk of distal thrombus formation. This due to the highly positive surface and low crosslink density. These technologies agree on the importance of surface modification for the improvement of its properties and its subsequent platelet aggregation, initiating the coagulation process. However, they do not incorporate active agents that respond to the stimulation of bleeding, promoting the coagulation process.

On the other hand, the progress in nanotechnology has allowed the synthesis of new precursors of nanomaterials and / or intelligent materials in order to be applied in hemostatic processes, considering that these materials have physicochemical and biological properties that support their use in the biomedical area. (Howe & Cherpelis, 2013). Among them, the derivatives of graphene and in particular graphene oxide (GO) stand out, since it is qualified as a potential construction material to produce drug delivery systems due to its large specific surface area, abundant functional groups and its biocompatibility (Mao et al., 2013). The presence of oxygenated functional groups in the GO structure has allowed it to develop various favorable properties, which have been successfully used in the biomedical area (Nezakati et al., 1987). However, in this field of study, this material has presented biocompatibility and toxicity problems at certain concentrations, so it has been linked to polymers, whose functionalization process improves the properties of both materials (Pinto et al., 2013).

On the other hand, composite materials based on GO-functionalized polymers can be transformed into 3D porous structures called aerogels. Aerogels are colloidal materials similar to gels, with three-dimensional structures in which the liquid component is exchanged for gas, and as a result highly porous, low-density structures with a large surface area and good mechanical properties are achieved (Araby et al ., 2016). To obtain it, a sol-gel technique is used, which basically consists of obtaining a highly crosslinked hydrogel, which is subsequently subjected to freeze drying or supercritical drying with CO2 to give rise to obtaining the aerogels (Ma et al., 2015). Many polymers, natural or synthetic, have been used to generate aerogels for these purposes, such as polyethylene glycol (PEG), polyvinyl alcohol (PVA), polylactic acid, chitosan (CS), gelatin, polyacrylamide, among others (Pan et al., 2017; Bai et al., 2010; Bao et al., 201 1; Piao et al., 2015; Scaffaro et al., 2016).

The synthesis process of the aerogels is influenced by variables such as GO oxidation degree and sonication time, reaction temperature of the polymer and GO, pH of the GO / polymer solution, GO / polymer ratio, surface charge, among other. These variables generate significant changes in the structure of the aerogels, hence the importance of considering their study in new formulations.

Additionally, experimental evidence has shown that the inclusion of materials with active properties in the aerogel polymer matrices modify their surface properties, in favor of improving interaction with external media. These materials that are charged to the aerogels can in turn be released into the environment and act as drugs depending on the purpose given to them.

Taking this background into account, a current challenge is the development of intelligent materials whose structural modifications favor the interactions of these biomaterials with external media such as blood; allowing them to act as agents promoting hemostasis, accelerating blood coagulation processes.

References:

• Araby, S., et al., Journal of Materials Science (2016) 51 (20), 9157.

• Aron, P. M., and Kennedy, J. A., Molecular Nutrition & Food Research (2008) 52 (1), 79.

• Bai, H., et al., Chemical Communications (2010) 46 (14), 2376.

• Bao, H., et al., Small (Weinheim an der Bergstrasse, Germany) (201 1) 7 (1 1), 1569.

• Behrens, A. M., et al., Acta biomaterialia (2014) 10 (2), 701.

• Boateng, J., & Catanzano, O., Journal of pharmaceutical Sciences (2015) 104 (1 1), 3653.

• Chan, L. W., et al., Acta biomaterialia (2016) 31, 178.

• Howe, N., and Cherpelis, B., Journal of the American Academy of Dermatology (2013) 69 (5), 659. e1.

• Nezakati, T., et al., Archives of Toxicology (2014) 88 (1 1), 1987.

• Ma, Y., and Chen, Y., National Science Review (2015) 2 (1), 40.

• Li, G. F., et al., Colloids and Surfaces B-Biointerfaces (2018) 161, 27.

• Liang, Y. P., et al., Colloids and Surfaces B-Biointerfaces (2018) 169, 168.

• Pan, C., et al., Macromolecular Materials and Engineering (2017) 302 (10).

• Piao, Y., and Chen, B., Journal of Polymer Science Part B: Polymer Physics (2015) 53 (5), 356.

• Pinto, A. M., et al., Colloids and surfaces. B, Biointerfaces (2013) 1 1 1, 188.

• Scaffaro, R., et al., Composites Science and Technology (2016) 128 (Supplement C), 193. Yan, TS, et al., Carbohydrate Polymers (2017) 170, 271. DOI: 10.1016 / j.carbpol.2017.04.080.

Brief description of the figures

Figure 1: X-ray diffraction for GO and airgel samples GO-MMW-CS at pH 10.

Figure 2: FTIR spectroscopy for GO and GO-MMW-CS at pH 10.

Figure 3: Raman spectrum for GO-MMW-CS sample at pH 10.

Figure 4: Raman spectra for GO-MMW-CS and GO-MMW-CS-Ext samples at pH 10.

Figure 5: Deconvolution of Raman spectra for samples of (A) GO-MMW-CS and (B) GO-MMW-CS-Ext at pH 10.

Figure 6: Elastic module of the synthesized aerogels.

Figure 7: SEM images of the aerogels without load and with load of grape extract (6% and 12%) at pH 4.

Figure 8: SEM images of the aerogels without load and with load of grape extract (6% and 12%) at pH 10.

Figure 9: SEM images and pore and sheet size analysis for GO-MMW-CS at pH 10.

Figure 10: SEM images and pore and sheet size analysis for GO-MMW-CS-Ext at pH 10.

Figure 1 1: Absorption of PBS.

Figure 12: Water absorption.

Figure 13: Percentage of blood absorption in the developed aerogels, gaza and fresh blood were used as controls.

Figure 14: SEM images of aerogels with a load of grape extract of 6% (A) and 12% (B) at pH 10 once the blood was absorbed.

Figure 15. Release of the grape seed extract from the synthesized aerogels.

Disclosure of the Invention

The present technology corresponds to an airgel based on graphene oxide (GO) functionalized with a deacetylated derivative of chitin (CS) not covalently, with the inclusion of proanthocyanidins (PAs) in its matrix, whose physicochemical properties allow blood absorption and clot formation; and in turn, it allows the release of the extracts, which promote the negative charge on the surface of the airgel, promoting the healing of a wound.

This airgel has a high specific surface area (390 - 600 m ² / g), a negative surface charge, which gives it a great capacity to absorb fluids, mainly blood, which makes it suitable as a hemostatic agent. This is due to the fact that the graphene oxide (GO), given its chemical and physical properties, is functionalized with chitosan (CS) allowing the generation of a porous, light and highly versatile structure.

Specifically, the airgel comprises at least the following components: a. graphene oxide (GO) between 60 - 93% w / w;

b. a deacetylated derivative of chitin (CS) between 50-5% w / w; and c. proanthocyanidins (PAs) between 15 - 2% w / w.

Where proanthocyanidins are obtained from grape seed extracts, so that through a sustained release process they are released from the airgel.

This airgel has a pore size between 10.2 - 13.0 pm and manages to absorb between 50 - 70 g / g of water and PBS, respectively, making it a good absorbent.

This technology has the advantage of absorbing profuse bleeding during the injury, and at the same time releasing the bioactive components (natural extract) at the site of the injury, in response to a change in pH that generates the release of blood and exudates from the wound. This compared to common absorbents such as gauze, means a 100% improvement in absorbent performance. This combined effect, on the one hand, of absorbing the blood in case of profuse bleeding, and at the same time releasing an active component from the matrix (grape seed extract), makes it a unique product that is not currently marketed.

Application example

Example 1 . Synthesis of GO-CS aerogels with grape extract.

Firstly, graphene oxide was obtained, then GO-CS aerogels were synthesized, and finally GO-CS aerogels were obtained with grape extract, the details of each are presented below:

eleven . Synthesis of graphene oxide.

GO was synthesized from powdered graphite flakes, for which the Hummers method modified according to the protocol of Marcano et al. (ACS nano (2010) 4 (8), 4806), as it is a recommended method for exfoliation of the superimposed layers of graphene. A solid mixture of KMn04 was prepared (3.6 g) and graphite (0.6 g), then slowly added to a liquid mixture of H2SO4 (72 mL) and H3PO4 (8 mL) with stirring in an ice bath under a hood. After it cooled, it was kept at a temperature of 50 ° C with stirring for 12 hours. The reaction was cooled and quenched with 30% v / v H2O2 and the product was filtered with a large filter. It was washed and centrifuged for 10 minutes at 20,000 rpm, where the supernatant was discarded and the solid was collected. The collected was washed with 30% v / v HCI, filtered and centrifuged at 20,000 rpm for 10 minutes. Then, an ethanol wash was carried out, filtered and centrifuged. These washes and spins were repeated with Mili-Q (MQ) water twice. The resulting material was coagulated with ether and recovering the final solid and then being lyophilized for 3 days.

1 .2.- Synthesis of the aerogels GO-CS.

The GO and CS compounds were lyophilized to prepare the non-covalent functionalization of GO-CS according to the protocol of Yu et al. {Journal of Environmental Chemical Engineering (2013) 1 (4), 1044), modified to assess the influence of pH and molecular weight of chitosan.

A mixture of 0.5 mg / mL GO and 600 mL MQ water was prepared at pH 6 and kept under stirring. A low molecular weight CS solution (LMW-CS) (1.0 mg / mL, pH 2.30 mL) was then prepared, which was added slowly by dripping with a plunger pipette into the GO mixture, observing a brown precipitate. It was maintained by stirring for one hour and then 3 washes with MQ water were carried out at 4500 rpm for 5 minutes, obtaining a bottom product. It was placed in a glass Petri dish sealed with plastic wrap and covered. It was then frozen using CFC FREE equipment and lyophilized for three days.

The same protocol was recreated by adding 1 M NaOFI dropwise to the resulting GO-LMW-CS mixture until reaching pH = 10, observing a darker precipitate. Then the same stirring, spinning and washing steps were followed.

Finally, the same protocol as above was carried out using medium molecular weight CS (MMW-CS). With this, a total of 4 GO-CS was obtained, two using LMW-CS at pH 4 and 10, and two using MMW-CS at pH 4 and 10. Table 1 shows the conditions under which the aerogels were produced. .

Table 1 . Synthesis conditions for aerogels

1 .3.- Synthesis of GO-CS aerogels with grape extract (GO-CS-ext.).

For the loading of the aerogels, the protocol of Yu et al. (Journal of Environmental Chemical Engineering (2013) 1 (4), 1044), with modifications. With the GO-CS mixture being stirred, having already adjusted its pH, 20 or 40 mg of grape seed extract was slowly added. After stirring for one hour, the GO-CS-Ext airgel sample was centrifuged, washed, frozen and lyophilized.

Example 2. Evaluation of the properties of the GO-CS airgel.

Several tests were carried out to demonstrate the properties of the airgel, which are detailed below:

2.1 .- X-Ray Diffraction Spectrum (DRX).

The DRX spectrum was measured using a Bruker AXS model D4 Endeavor X-ray diffractometer with Cu K radiation (l = 1.5541841 A; 2.2kW) as the reference point, a voltage of 40 kV, and a current of 20 mA. The samples were measured from 2 to 50 ° (2Q) with steps of 0.02 ^Q and a measurement time of 141 s per step. The crystallinity of GO and the formed aerogels was analyzed by XRD.

Figure 1 shows the diffractograms for GO (a) and for GO-MMW-CS (b) aerogels at pH 10. For GO, a 10 ° diffraction peak (angle 2Q), typical of this, is observed. type of graphite material. For the airgel sample, a single 10 ° diffraction peak, similar to GO, is shown due to GO sheets stacked on the compound. When incorporating the CS into the GO and forming the airgel, a small decrease in the diffraction peak is observed, added to a decrease in its intensity, since the interlaminar space increases. This effect is attributed to the fact that the CS chains are interspersed between the GO sheets, which is also associated with a correct distribution of the CS chains in the GO sheets.

2.2.- FTIR Spectroscopy.

The samples were evaluated by Fourier transform infrared spectroscopy (FTIR), using a Perkin Elmer brand FTIR spectrometer model Spectrum two with UATR accessory, in a wave number range from 500 to 4000 cnr ¹ .

The presence of functional groups of GO (a) and GO-CS (b) was identified in the corresponding spectra shown in the range between 500 - 4000 cnr ¹ in Figure 2. Eigen traits on the FTIR spectrum of GO ( a) are the absorption bands corresponding to the C-0 bonds in 972 cnr ¹ and C = 0 bonds to 1733 cnr ¹ , which shows the different oxygenated groups present on the GO surface. A broad band with peak 3300 cnr ^{1 is} observed, corresponding to the presence of -OFI bonds, hydroxyl groups, carboxyls and water (corresponding to interlaminar water). In the FTIR spectrum of the GO-CS (b) sample, a large peak is observed at 3550 cnr ¹ , indicating -OH and -NH bonds present in the CS structure. The presence of C = 0 links is demonstrated by a peak at 1628 cnr ¹ .

2.3.- Raman spectroscopy.

The vibrational analysis was carried out by Raman spectroscopy on a Horiba model, Labram HR Evolution model with an excitation line of 633 nm, a power of 13.3 mW and 1.96 eV. The laser location was centered on the sample using an Olympus 100x VIS lens and a NUV camera (B / S UV 50/50 + F125 D25 Lens). The laser intensity was kept constant to minimize any damage to the sample. All samples were measured using an object holder at room temperature and none of them was evaluated in solution.

Figure 3 shows the Raman spectrum of the airgel (GO-MMW-CS) without extract at pH 10, where three main peaks can be seen. The first peak located at 1339 cnr ¹ , called band D, which is associated with the respiration mode of the sp ² carbon rings with free bonds at the plane terminations. The second major peak represents the G-band, located at 1583 cnr ¹ , originates from vibration in the plane of carbon atoms with sp ² hybridization. The third peak, called the 2D band, occurs at 2700 cnrr ¹ . CS is known to have a peak at 2837 cnr ¹ , corresponding to the CH stretching vibration, which is not present in the spectrum for the GO-MMW-CS airgel, indicating that CS was incorporated into the material.

Figure 4 compares the Raman spectra of the airgel samples (GO-CS) without extract (a) and with extract (b), synthesized with CS from MMW and pH 10. When loading the samples with extract (b), observed a slight displacement in bands D and G, positioning them at 1337 and 1574 cnr ¹ , respectively. For the airgel sample without extract (a) a ratio of intensities of ID / IG = 3.57 was calculated, which increased to ID / IG = 4.33 in the airgel loaded with grape seed extract, which shows its presence in the airgel.

Figure 5 presents the deconvolution of the Raman signal for samples of aerogels without (A) and with (B) extract, respectively. In both samples, the D ^* band is observed, which represents the presence of amorphous carbons and their sp ³ hybridization, which did not show variations in their intensity when loading the airgel (<10%). The intensity of the D ^** band, when the airgel was loaded, did not show an appreciable variation (<10%). This result is reasonable, since there was no chemical modification of the GO structure and the vibrations of the C = C, CH and residual graphite bonds are represented there. As the airgel was loaded with grape seed extract, it increased the intensity of the defects in the sample, a phenomenon represented by band D ', which increased by 330%.

2.4.- Surface area and pore distribution.

The surface area of the aerogels GO-CS, GO-CS-extrac (12%) at pH 4 and 10 was estimated using the BET equation with the data obtained from the Micromeritics Gemini Vil team, adsorbing N2 at a temperature of -198.85 ° C from cryogenic bath with manual degassing, obtaining the PPo data up to a saturation pressure of 760 mm Hg.

BET design equation 1 was used, where (v) is the volume of gas adsorbed at pressure (P); (Po) is the saturation pressure; (v _m ) is the volume of the gas absorbed when the adsorbent surface is completely covered by a unimolecular layer; and (c) corresponds to a constant related to the adsorbate-adsorbent system.

The results indicated that the aerogels had a surface area in a range between 390 - 600 m ² / g and an average pore radius of 53 - 40 Á, which generates a large amount of volume to absorb the blood.

2.5.- Elastic Module.

The uniaxial compression tests to obtain the stress versus deformation curves were taken with a universal Instron test machine, model 4468, equipped with Instron Series IX software with a 500 kN cell and a displacement of 1 mm / min.

The elastic modulus of the aerogels was influenced by the pH of the composition synthesis (Figure 6). The compressive strength increased when going from acidic to basic pH, which is related to a greater interaction between the functional groups of CS and GO, when moving away from the respective isoelectric points of the original components (Pl: GO = pH 2 , 0, CS = pH 5.0), they must have their functional groups activated. When adding the extract, the aerogels hardened and the elastic modulus increased even more, this increase being directly proportional to the concentration of the added extract, which would help to maintain the integrity of the composition during its application.

2.6.- Morphology of aerogels.

The morphological and surface properties of the particles were studied using scanning electron microscopy (SEM) (JSM-6380LV, JEOL).

Figures 7 and 8 show the SEM images of the aerogels synthesized without extract (A), and loaded with 6% (B) and 12% (C) of extract at pH 4 and pH 10, respectively. The pH of the synthesis had an effect on the structure of the aerogels, at a basic pH a more intricate and rough structure is observed than at acid pH.

To study the pores and foil of the GO-CS aerogels, the “Digital Gafan Microscopy 3.0” software was used and a magnification of the SEM image, represented in Figure 9, was performed for GO-MMW-CS at pH 10. Then with This image was made a cross-sectional profile to determine how much each sheet and pores measured, determining that the sheets had measurements between 0.38 - 0.62 pm and the pores measured between 10.2 - 13.0 pm.

The loading of the extract on the airgel caused the size of the sheets to increase and the size of the pores to decrease as the load increased. In the case of the GO-CS-Ext airgel, GO sheets were obtained with measurements between 0.9 - 1.4 pm and pores that measured between 2.2 - 7.2 pm (Figure 10). By adding the extract to the airgel, this caused a redistribution in the interaction of CS and GO, which caused a decrease in pore size. This data agrees with what was observed after synthesizing the GO-CS-Ext, which macroscopically presented a denser appearance. Added to this is the greater rigidity observed in the compression analyzes, which is consistent with this analysis.

2.7.- Surface load of the aerogels.

Small pieces of aerogels of about 1 cm ³ were dissolved in 30 ml of water and then sonicated for 30 min. Subsequently, they were shaken vigorously with the help of the vortex, until obtaining a homogeneous solution. Potential Z was measured in the HORIBA SCIENTIFIC equipment (nano parti analyzer), the detail of these results is presented in Table 2.

The results indicate that the synthesized aerogels were negatively charged and when contrasting the samples, it was observed that the addition of the extract caused the aerogels to become even more negative against a simulated blood condition (PBS buffer). The extract concentration and the pH of the synthesis influenced the final surface charge. At higher concentration and at acid synthesis pH, the aerogels were more negatively charged, which would favor the activation of the coagulation cascade in the wound.

Table 2. Z potential of aerogels determining in water.

2.8.- Kinetics of absorption of water and phosphate buffer (PBS).

The absorption kinetics of the LMW-CS and MMW-CS aerogels were evaluated at pH 10, with samples measuring 10mm x 10mm x 7mm, on average, which were immersed in 5 ml of MQ water in a glass plate (100x 15mm) removing the excess of it at different times (5, 10, 15, 30, 60, 300, 900, 2700 seconds). The mass of each sample was measured before (So) and after (Sw) of each dive and the ratio was evaluated according to the following expression:

For the absorption of phosphate buffer (PBS, pH 7.4), the same procedure was performed replacing the MQ water with the phosphate buffer and taking samples after 5, 15, 30, 60, 300, 1200 and 2700 seconds, to LMW-CS at pH 4 and 10, and MMW-CS at pH 4 and 1. The results are presented in Figure 1 1 for the absorption of PBS and in Figure 12 for the absorption of water. One of The main properties of the aerogels is their high absorption capacity, where it was verified that the aerogels reached their maximum absorption capacity independent of the fluid, a few seconds after starting their contact. The aerogels managed to store 70 g / g of PBS and 50 g / g of water, respectively; this is caused by the presence of a more negative charge on the airgel, which allowed for greater attraction of solute molecules. For water, there was no influence on the molecular weight of CS, but there was for the absorption of PBS.

2.10.- Fresh blood adsorption kinetics.

Fresh blood adsorption kinetics of the aerogels GO-CS, GO-CS-extrac (6%) and GO-CS-extrac (12%) were evaluated at pH 4 and 10, in 12-well well plates, at which were added 50 pl of fresh blood leaving it at different times (30, 60, 120, 180 and 240 s); After this period, 3 mL of MQ water were added homogeneously, forming a supernatant.

The supernatant was poured into a glass cell to be analyzed by UV absorption at 540 nm. Where, as in the previous procedure, the absorption capacity was evaluated from a blank corresponding to absorption with gauze and pure blood.

% Absorbance = Average sample * 100% (3)

In Figure 13 it can be seen that the absorption was almost immediate, since in the first 30 seconds of contact between the blood and the airgel, practically 100% of it was incorporated into the matrix. Zooming in the first seconds of the test, it is observed that the aerogels with 12% extract load at pH 4, were the ones that presented a better absorption of blood. These results validate the use of aerogels as hemostatic agents, since their performance is far superior to that of gauze, a dressing traditionally used against bleeding.

Figure 14 shows SEM images of the blood absorption process using aerogels with 6% (A) and 12% (B) of extract. In them it is possible to distinguish the presence of white, red and platelet cells trapped in the pores of the airgel, which accounts for the success of the absorption of blood.

2.1 .1. Extract release kinetics

The aerogels GO-CS, GO-CS-extrac (6%) and GO-CS-extrac (12%) at pH 4 and 10, were subjected to a release in PBS and the concentration of total phenols was determined by the Folin assay Ciocalteau.

A thermostatic bath at 37.5 ° C (Fried Electric TEPS-1 Serial No. 1881) was mounted, and at the working temperature, a piece of airgel of known mass was added in a volume of 250 mL of PBS. The release time began when the piece of airgel of known mass was added to the PBS solution. Aliquots of 1.5 mL were taken at fixed time intervals and the channels were transferred to Eppendorf tubes. Between 0-10 minutes portions were taken at 2.5 min intervals. Then, between 30-60 minutes, extractions were made with intervals of 15 min; Aliquots were also taken at 60 and 120 minutes. After 12 hours, the last aliquot was removed from equilibrium. Obtained the samples, the spectrophotometric analysis was carried out according to the method of determination of total phenols using the Folin-Ciocalteu reagent. Measurements were taken on a Spectroquant Prove 600 spectrophotometer at 765 nm.

The extract release percentage is finally given by the following expression:

Release% = ^Atest x 100 (4)

Acontrol

Where Atest is the absorbance of the sample extracted from the PBS solution and Acontroi is the absorbance of the control sample with% of known extract.

The release data of the aerogels in PBS is shown in Figure 15. In it, a sustained release of the extract is observed during the first minutes, without observing differences between the samples. Upon reaching equilibrium, the release of the extract was directly proportional to its mass, the higher the concentration loaded, the more it was released, releasing about 30% of what was loaded (at 12 h or 720 min). The airgel synthesized at pH 4 with 12% extract, was the one that presented the most release, as a result of which proanthocyanidins are more stable at acidic pH than at basic pH. These results are consistent with that observed in the absorption of blood, where the airgel that absorbed the most was the one with the highest concentration of extract.

Finally, these results allowed to demonstrate the hemostatic capacity of the airgel based on graphene oxide and chitosan, in addition to grape extract.

Claims

1 An airgel that allows blood absorption and clot formation for wound healing CHARACTERIZED because it comprises graphene oxide between 60 - 93% w / w, non-covalently functionalized with a deacetylated derivative of chitin between 50 - 5% w / w and proanthocyanidins between 15 - 2 w / w% in their matrix.

2.- An airgel that allows blood absorption and clot formation according to claim 1, CHARACTERIZED because the proanthocyanidins are obtained from grape seed extracts.

3.- An airgel that allows blood absorption and clot formation according to claim 1, CHARACTERIZED because it has a specific surface area between 390 - 600 m ² / g.

4.- An airgel that allows blood absorption and clot formation according to claim 1, CHARACTERIZED because it has a pore size between 10.2 - 13.0 pm.

5.- An airgel that allows blood absorption and clot formation according to claim 1, CHARACTERIZED because it absorbs between 50 - 70 g / g of water and PBS.

6.- Use of the CHARACTERIZED airgel because it serves as a hemostatic agent.