WO2023170558A1

WO2023170558A1 - Method for preparing a system for controlled release of antibiotics and bioactive factors, formed by microspheres in a hydrogel

Info

Publication number: WO2023170558A1
Application number: PCT/IB2023/052113
Authority: WO
Inventors: Cristhian Leonardo Pinto Mora; Ingrid Zulay SILVA COTE
Original assignee: Instituto Distrital De Ciencia Biotecnologia E Innovación En Salud - Idcbis
Priority date: 2022-03-10
Filing date: 2023-03-07
Publication date: 2023-09-14
Also published as: CO2022002809A1

Abstract

The present invention relates to a method for preparing a system for controlled release of antibiotics and bioactive factors, which is formed by microspheres in a hydrogel, wherein the method comprises synthesising W1/O/W2 microspheres that contain a bioactive factor, which are included in a hydrogel in a proportion selected from 2:1:1, 1:1:1, 1:2:1 or 1:1:2 (PVA:Cs:ALG) containing an antibiotic, thus obtaining a controlled release system with potential for healing chronic wounds.

Description

METHOD FOR PREPARATION OF A CONTROLLED RELEASE SYSTEM OF ANTIBIOTICS AND BIOACTIVE FACTORS CONSISTING OF MICROSPHERES INCLUDED IN A HYDROGEL

FIELD OF INVENTION

The present invention refers to a method of preparing a controlled release system for antibiotics and bioactive factors that is based on the combination of two types of scaffolds: microspheres included in a hydrogel, with potential for wound healing.

The invention has application in the pharmaceutical field, specifically in the technical field of methods for preparing wound dressings.

STATE OF THE TECHNIQUE

In many cases, the acceptable time for wound healing is usually affected by various factors, among them is the generation of infections by multiple pathogens and also underlying diseases such as diabetes, which affects the production of certain bioactive factors essential for the healing processes.

There are traditional treatments that are applied to combat infections and subsequently close the wound, but the associated problem is the application of drugs and bioactive factors at very high concentrations, which can be harmful to patients. Furthermore, these treatments require frequent application and their effectiveness depends largely on the discipline that the patient has after starting the treatment.

An alternative option to traditional treatments are controlled release systems of various bioactive molecules. This release can be achieved by incorporating or encapsulating the bioactive molecules with a polymeric matrix or scaffolds using various techniques, such as microspheres and hydrogels. GD Mogosanu and AM Grumezescu in "Natural and synthetic polymers for wounds and burns dressing" says that depending on the application of these controlled release systems, various biomaterials can be used, whether synthetic or natural, thus we can find PLA (polylactic acid), PLGA (polylactic acid poly(lactic-co-glycolic)), PCL (polycaprolactone) and PVA (polyvinyl alcohol), while in the second group are collagen, fibroin, alginate, chitosan and hyaluronic acid.

Likewise, the release of bioactive molecules is governed by phenomena such as diffusion and degradation of the polymer matrix or scaffolds that contain them. From this, systems can be generated that allow predicting the release rate over a period. of a certain time, and at the same time ensure the delivery of the biomolecules at concentrations that ensure desired therapeutic effects.

There are several techniques used to manufacture scaffolds, including methods to produce microspheres and hydrogels. AA Chaudhari et al in “Future prospects for scaffolding methods and biomaterials in skin tissue engineering; A review'' indicates that microspheres are easy to manufacture, with controlled physical characteristics and are suitable for encapsulating almost any type of molecules, although one of the problems associated with this type of technology is explosive release and its limited range of applicability. when they are not applied with a matrix that supports them. For their part, hydrogels are 3D networks that are made up of hydrophilic biomaterials, whose main characteristics are high water retention, efficient mass transfer, similarity to natural tissues, soft nature that minimizes the friction of surrounding tissues and sensitivity to physiological changes; These properties make them attractive to be used as supports in release systems. Furthermore, the combination of scaffolds is currently of great interest since it allows the generation of dual systems, which allow the release of more than one bioactive molecule at the same time. In this way, the present invention was born in response to the need to find a method of preparing a controlled release system that consists of two types of combined scaffolds: microspheres included in a hydrogel, which guarantees optimal encapsulation of the bioactive factor in microspheres and produce a hydrogel with excellent release capacity of bioactive molecules and antibiotics that prevents an explosive release of the bioactive factor, as well as optimal degradation values and mechanical properties for use as a controlled release dressing in chronic skin wounds.

In search of a technical response to this need, different related patent documents were consulted, such as document CN107496976A that describes a method to prepare a chitosan hydrogel dressing with antibacterial and healing action, where chitosan microspheres are prepared that are then They are uniformly dispersed in an aqueous solution with a mass-volume ratio of 1 to 4% (w/v), and cross-linked with glutaraldehyde at a volume ratio of 1% (v/v). After 3 hours, it was soaked in deionized water, the excess cross-linking agent was removed, and a chitosan hydrogel dressing was obtained, which was stored under sterile conditions at 4 °C.

Another technical solution considered very close to the identified need is found in document CN1 13244444A, which refers to a wound dressing based on microspheres composed of hydrogel. The wound dressing is prepared from composite microspheres and smart response type hydrogel, and the mass ratio of the composite microspheres to smart response type hydrogel is (1:100)-(1:200); The prepared hydrogel is a composite aqueous solution containing pluronic F68 and pluronic F127; and the composite microsphere comprises a nanoscale sodium alginate single-layer microsphere loaded with a vascular endothelial growth factor (VEGF) and a nanoscale sodium alginate single-layer microsphere loaded with leptin (LP). Hydrogel Composite Microspheres Based Wound Dressing Can Be Applied to Wound Treatment combined, and two drugs with different effects are used, so that the prepared dressing has the dual effects of regulating and controlling the immunoreaction process and promoting angiogenesis.

Another related document is CN1 11228565A, which refers to a hydrogel composed of hyaluronic acid and gelatin loaded with PLGA microspheres and Gentamicin sulfate. Furthermore, this document describes the method of preparation of both the microspheres and the hydrogel. The preparation method of the microspheres consists of: dissolving the polylactic acid-glycolic acid copolymer in dichloromethane to prepare an oil phase, and the Gentamicin sulfate is dissolved in deionized water to prepare an aqueous phase. The oil phase is mixed with the water phase, stirred and sonicated to obtain a W ₁ /O solution; the W ₁ /O solution is added to the polyvinyl alcohol solution, stirred, mixed and sonicated, forming a double emulsion; The double emulsion is added to the polyvinyl alcohol solution to form a W ₁ /O/W ₂ solution. The method to prepare the hydrogel consists of: combining aminoethyl methacrylate hyaluronic acid, methacrylated gelatin and microspheres composed of PLGA and Gentamicin sulfate. The photoinitiator is added to deionized water and irradiated with ultraviolet light to obtain a hyaluronic acid-gelatin composite hydrogel containing PLGA microspheres with Gentamicin sulfate.

Furthermore, in the study carried out by C. Xiao et al in “Synthesis and properties of degradable poly (vinyl alcohol) hydrogel”, a new degradable hydrogel has been prepared by condensation of polyvinyl alcohol (PVA) with ELDA, a di-acid. derived from lactic acid.

Likewise, in the study carried out by S. Lin et al in “Evaluation of PVA/dextran/chitosan hydrogel for wound dressing”, poly(vinyl alcohol) (PVA), dextran and chitosan are integrated to produce an ideal dressing for wounds in which glutaraldehyde (GA) is used as a cross-linking agent. The result showed that the 6% PVA hydrogel with 0.25% chitosan provides antimicrobial capacity. The PVA/chitosan hydrogel combined with 4% dextran using GA cross-linking also exhibits high cell proliferation capacity, suggesting that the hydrogel has potential as a wound dressing.

On the other hand, in the study carried out by Shamloo et al in “Fabrication and evaluation of Chitosan/Gelatin/PVA hydrogel incorporating Honey for wound healing applications: An In Vitro, In Vivo Study” physically cross-linked hydrogels were developed using the freezing method and thawing, while different concentrations of honey were included in the hydrogels to accelerate wound healing. The hydrogel was composed of chitosan, polyvinyl alcohol (PVA), and gelatin in a ratio of 2:1:1 (v/v), respectively. In addition, the effect of honey concentrations on antibacterial properties and cellular behavior was investigated.

Additionally, in the study carried out by A. Luciani et al in “PCL microspheres based functional scaffolds by bottom-up approach with predefined microstructural properties and release profiles” a procedure was described to create scaffolds that are obtained through the thermal assembly of microspheres of Protein-activated poly(3-caprolactone) (PCL) prepared with double emulsion and larger protein-free PCL microspheres obtained with single emulsion. In this study it is shown that the pore dimension, interconnectivity and mechanical properties in compression of the scaffold could be predefined by an appropriate choice of the size of the protein-free microparticles and the process conditions. The protein-loaded microparticles were successfully embedded within the scaffold and provided sustained delivery of a model protein (BSA).

However, none of the previous inventions manages to completely solve the problem posed, this is because, although they manage to develop methods for preparing microsphere dressings included in a hydrogel, none of the methods describe the addition of microspheres that guarantee optimal encapsulation of a bioactive factor within a hydrogel, in which an antibiotic is already incorporated, this ensures an improvement in the release capacity of both the bioactive molecules and the antibiotic, preventing an explosive release of the bioactive factor. Finally, thanks to this innovative preparation method, a dual-release dressing can be obtained that releases both bioactive factors of interest and antibiotics, allowing the fight against infections and in turn promoting the healing processes of chronic wounds.

DETAILED DESCRIPTION OF THE FIGURES

Figure 1 shows a Scanning Electron Microscopy (SEM) micrograph of the microspheres, where it can be seen that these microspheres have micrometer-sized spherical structures, with a smooth surface without defects.

Figure 2 shows a graph of the size distribution for Polycaprolactone (PCL) Microspheres.

Figure 3 shows an SEM micrograph for a hydrogel containing a 2:1:1 ratio (PVA:Chitosan:Alginate), where it can be seen that said hydrogel has high porosity and diverse pore sizes.

Figure 4 shows a graph of the cumulative release of hepatocyte growth factor (HGF), with Mes (green) being only microspheres containing said HGF and HgA-Mcs (light blue) being the release system composed of microspheres included in a hydrogel.

Figure 5 shows a graph of the cumulative release for three different concentrations of the antibiotic Meropenem, where as the concentration of the antibiotic increases, the release time of therapeutic concentrations also increases. Figure 6 shows the inhibition zones by the agar diffusion method to verify the effect of the biomatehales used in the prepared hydrogel and the concentration of Meropenem (10X and 100X) on the inhibition of the bacteria S. aureus, E. coli , K. pneumoniae P. aeruginosa. The results of the hydrogel without antibiotic (left halo) and the hydrogel with 10X Meropenem (right halo) are shown at the top. Likewise, the results of the hydrogel with Meropenem 100X are shown at the bottom.

Figure 7 shows a graph that indicates the percentage of cell proliferation of the controlled release system of antibiotics and bioactive factors that is based on two types of combined scaffolds: microspheres included in a hydrogel (green bars), prepared by the novel method described. in this technical document. As a control (brown bars), only a hydrogel with a 2:1:1 ratio (PVA:Chitosan:Alginate) was used.

Figure 8 shows a photo of the controlled release system for antibiotics and bioactive factors that is based on two types of combined scaffolds: microspheres included in a hydrogel, prepared by the novel method described in this technical document.

DETAILED DESCRIPTION OF THE INVENTION

The present invention refers to a method of preparing a controlled release system that is based on two types of combined scaffolds, which are microspheres and hydrogel. The microspheres are responsible for encapsulating the bioactive factors and are subsequently incorporated into the hydrogel, in which the antibiotic will also be incorporated.

The preparation method of said controlled release system consists of two phases, the first is related to the synthesis of microspheres containing bioactive factors, using the W ₁ /O/W ₂ emulsion technique. that is, a water-organic phase-water method. In this methodology, adjustments were made to some parameters, in order to guarantee greater encapsulation of the bioactive factors of interest. For the Wi phase, a percentage of polyvinyl alcohol (PVA) in solution at 1% and 3% w/v was used. The same occurred in the organic phase (O), which is a solution of polycaprolactone (PCL) in chloroform at 3% and 5% w/v. It should be noted that the higher values for phase W ₁ and O do not appear indicated in other state-of-the-art methodologies.

The preparation method of the microspheres is described below:

Preparation of microspheres containing bioactive factors

For the synthesis of microspheres, a W1/O/W2 method is used, where the W ₁ (aqueous) phase is composed of PVA at 3% w/v, bovine serum albumin (BSA) and the bioactive factor of interest, The O (organic) phase is composed of PCL in solution with 5% w/v chloroform and the W ₂ phase is composed of a surfactant, in this case it is 5% w/v PVA.

Preparation of initial solutions:

Preparation of the 3% and 5% w/v PVA solution: for both cases, the PVA is dissolved in phosphate buffer solution (PBS) at pH 7.4, these are left sealed in a container stirring at 700 rpm. for 24 hours at 135°C.

Preparation of 5% w/v PCL solution: PCL is dissolved in chloroform and left stirring at 700 rpm for 30 minutes at room temperature, completely sealed. The container containing the solution should be amber in color since chloroform is a photosensitive solvent.

Synthesis process:

Preparation of the bioactive factor solution: a 10pg/mL solution of the bioactive factor of interest is prepared (for example, a cationic charge bioactive factor, selected from: HGF, TGF-β1, TGF-β2, TGF-β3, TGF -β4, VEGF, IGF, NGF and BPM), where the solvent is 1X PBS supplementing with 1% w/v bovine serum albumin (BSA). Throughout the process, the temperature of the solution at 4°C, this in order to preserve the bioactive factor used.

Preparation of the Wi phase: Initially, 0.1% w/v BSA and the bioactive factor solution are added to the PVA solution (3% w/v) until a final factor concentration of 1 pg is obtained for each mL of W ₁ . Subsequently, the solution is homogenized for 1 minute at 2000rpm. The solutions must be at 4°C.

Preparation of the W ₁ /O phase: to obtain the emulsion, for every 10mL of PCL solution in chloroform, 1 mL of the Wi phase is added, then the mixture is homogenized for 1 minute at 2000 rpm using a homogenizer. It must be guaranteed that the solutions are at 4°C.

Synthesis of W ₁ /O/W ₂ microspheres: The Wi/O solution is added to the W2 phase (5% w/v PVA) by continuous dripping at a flow rate of 1 mL per minute with an injector. The mixture is homogenized at 2000rpm for 1 min and then magnetically stirred for 12h at 700rpm, to ensure that the chloroform present in the organic phase (O) is completely evaporated.

Collection, lyophilization and storage: The microspheres are collected by centrifugation at 2500 gravities for 5 min, the supernatant is discarded, and the precipitate is washed with deionized water, the process is repeated 3 times. Finally, a selection of microspheres is made by size; for this, microsieves with a pore size of 40 pm are used, this ensures the elimination of unwanted residues. The product was lyophilized at -47°C and 0.0125 mBar for 24 hours. The lyophilized product obtained was stored at -20°C.

As we can see, regarding the critical parameters that have a high influence on the encapsulation process, a number of revolutions for homogenization of 2000 rpm for 1 minute was used. The temperature of the solutions to be used in each stage of the production process was also taken into account. synthesis, which is not contemplated in the state of the art. This factor is very important since it allows us to guarantee that the bioactive factors will not suffer inactivation processes due to sudden changes in temperature. On the other hand, a stirring time of 12 hours was used in order to ensure total evaporation of the solvent used in the organic solution (chloroform).

Regarding the methodology related to the preparation of the hydrogel, the combination of PVA (synthetic polymer), chitosan (Cs) and sodium alginate (ALG) (natural polymers) stands out. The combination of these materials had not previously been recorded in the state of the art.

The hydrogel preparation method is described below:

Preparation of hydrogel incorporating microspheres and antibiotics

Preparation of initial solutions:

5% w/v PVA solution: PVA is dissolved in phosphate buffer solution (PBS) at pH 7.4, this is left sealed in a container stirring at 700 rpm for 24 hours at 135°C.

2% w/v chitosan solution: chitosan is dissolved in acetic acid solution (0.1 M), the mixture is left sealed in a container under stirring at 700 rpm for at least 2 hours at room temperature.

2% w/v alginate solution: the alginate is dissolved in phosphate buffer solution (PBS) at pH 7.4, leaving the container sealed and stirring at 700 rpm for at least 2 hours at room temperature

Obtaining the hydrogel

The biomaterials PVA, chitosan and alginate are mixed in proportion: 50% PVA, 25% chitosan and 25% alginate, an initial stirring is carried out at 700 rpm at room temperature for 1 hour. Then an adjustment of the pH of the solution between 7.2 and 7.4 using sodium hydroxide (NaOH) at 1 M concentration, pH adjustment should be done using a pH meter. The solution continues to stir for 2 more hours before adding the previously synthesized PCL-HGF microspheres (100 mg of microspheres per mL of hydrogel), in turn adding the antibiotic of choice at a concentration of 1 mg per mL of hydrogel, and left stirring constantly for 1 hour or more, then the mixture is poured into molds. The hydrogel obtained with the microspheres and the antibiotic is subjected to 3 cycles of freezing for 20 hours at -30°C and thawing for 4 hours at room temperature. After completing the 3 cycles, the hydrogels are stored frozen between -20°C and -30°C.

It can be seen that the joint incorporation of microspheres and antibiotics in the same hydrogel is registered as a novelty of the method described. Likewise, we see that a hydrogel based on PVA/chitosan/alginate (PVA/Cs/ALG) was prepared to take advantage of the antimicrobial and biocompatible effect of chitosan and alginate, and to complement them with the good mechanical properties of PVA. Furthermore, in previous trials different proportions of PVA/Cs/ALG (1/1/1, 2/1/1, 1/1/2 and 1/2/1) were used to evaluate the synergistic effect of their combination, finding that the most suitable candidate to be used in the release system was the hydrogel with a 2/1/1 ratio. The expected result of the study was the improvement of the properties of the hybrid in terms of its mechanical, chemical, microstructural and biological properties.

The tests shown below seek to demonstrate that the choice of biomaterials for the manufacture of the hydrogel and the variations in the parameters in the preparation method of the microspheres that lead to the formation of a release system with optimal characteristics to combat infections and in turn promoting the healing processes of chronic wounds. Encapsulation efficiency and microsphere morphology

As for the microspheres, an encapsulation percentage test was carried out, using Bovine Serum Albumin (BSA) as a model molecule, which is also used in this experiment as a stabilizing agent for bioactive factors. An approximate encapsulation efficiency of 70% was obtained for PCL microspheres whose formulation was 3% PVA (W1), 5% PCL (O) and 5% PVA (W2), which meant an increase in encapsulation compared to various works with PCL microspheres whose synthesis principle is based on a W1/0/W2 method.

In the SEM micrograph of Figure 1, it is observed that the microspheres have micrometer-sized spherical structures, with a smooth surface without defects. On the other hand, in Figure 2 we can see that the average diameter of the measured microspheres was 23.75 ± 4.73 μm.

These results, when contrasted with what is disclosed in the state of the art, for example, the test described by A. Luciani et al, show that the variations made in the described method do not represent considerable changes in the size or morphology of the microspheres. but it does represent a substantial increase in encapsulation, which results in better use of bioactive molecules (for example, HGF).

Hydrogel morphology

Another key component in the preparation method of the controlled release system described is the method of preparation of the hydrogel that contains the PCL microspheres with the bioactive factor and the antibiotic of interest, in addition to being the support that will be in direct contact with the area. of the wound. Said hydrogel will be responsible for releasing the molecules to the environment where they are required and that is why it is important to develop a method that allows optimal morphological characterization. The hydrogel obtained by means of the described method is the one that preferably contains a 2:1:1 ratio (PVA:Cs:ALG). Figure 3 shows the SEM obtained for said scaffold.

From the SEM micrographs of the hydrogel, its pore size was found to be 15.05 ± 12.16 μm. Furthermore, its relative porosity is 73.57%; which makes it a hydrogel with high porosity and diverse pore sizes, making it a scaffold with excellent capacity for releasing bioactive molecules and antibiotics.

Degradation and mechanical properties of the controlled release system obtained by the described method

Other properties to take into account in the manufacture of controlled release systems that include the use of hydrogels are degradation and mechanical properties. Regarding the degradation of the developed system, values of 63.73 ± 1.96% were obtained after two months of study. The use of natural biomaterials such as chitosan and alginate, which do not cross-link in the hydrogel, and the incorporation of microspheres results in the release system degrading faster compared to hydrogels containing only PVA. Studies such as that of C. Xiao, et al., show that hydrogels composed only of PVA show low degradation after 25 days of testing (12.5%).

Regarding the mechanical properties, a Young's modulus value of 28.14 ± 4.40 KPa was obtained for the controlled release system obtained by the described method. This value turned out to be higher compared to research such as that of S. Lin et al., 2019; and Shamloo et al., 2021 ; where they obtained modules of 2.5 Pa and 6.8 ± 0.82 KPa, respectively; for hydrogels whose composition included PVA. In the patents found in the state of the art, there are no relevant results that allow a comparison to be made with respect to the results obtained for the controlled release system prepared by the method described in the degradation and mechanical properties items.

Release of heoatocyte growth factor (HGF):

One of the most common problems when using microspheres is their explosive release, this prevents optimal use of the molecule with therapeutic action during the first hours of release. To solve this problem, it has been decided to incorporate the microspheres in a PVA/Chitosan/Alginate (HgA) hydrogel. In Figure 4, the effect of incorporation of the microspheres into the hydrogel is observed; Mes being only microspheres and HgA-Mcs the release system composed of the microspheres included in the hydrogel.

After 15 days, the average percentage released of HGF in the medium was 74.88 ± 3.86% for the microspheres (Mes), while for the hydrogel-microspheres system (HgA-Mcs) a average value of 56.28 ± 0.56%.

The HgA-Mcs release system after the first 24 hours releases HGF at an approximate rate of 3 to 4% per day. This indicates that for every 100 mg of microspheres (approximately 700 ng encapsulated HGF) incorporated into 1 mL of hydrogel, between 21 to 28 ng of HGF is released daily, which is an optimal amount to stimulate cell proliferation and migration in vitro. .

There is no report in the literature on the release profiles of HGF which is encapsulated in microspheres and embedded in hydrogels, nor is there a release profile that involves said factor in the patent review. The patent CN1 13244444A, indicated in the state of the art, refers to the encapsulation of VEGF (Vascular Endothelial Growth Factor), using other biomaterials (Alginate for microspheres and Pluronic F127 + F68 for hydrogels) for its encapsulation, due to the difference in the nature of the biomaterials, factors and techniques used for the generation of the dressing, cannot be considered valid to make a comparison with what is proposed in this document.

Antibiotic release

To the controlled release system that comprises microspheres of bioactive factors included in a hydrogel, antibiotics are incorporated (for example, a Carbapenem selected from: Meropenem, Doripenem, Ertapenem, Imipenem, among others), in order to create a synergistic effect. time to treat chronic skin wounds. Thus, the method described creates a dual release system with the potential to combat infections by various pathogens and at the same time promote cell proliferation through the action of bioactive factors.

For this specific trial it was decided to use Meropenem, which is an antibiotic that belongs to the Carbapenem family. Before carrying out the release profile for Meropenem, a Minimum Inhibitory Concentration (MIC) test was carried out for four bacterial strains: S. aureus, E. coli, K. pneumoniae and P. aeruginosa. After the test, it was determined that, to guarantee inhibition of the 4 strains, for at least one day, the minimum inhibitory concentration (MIC) incorporated into the hydrogels should be 10 μg/mL.

The concentration and type of antibiotic that is incorporated into the controlled release system prepared by the described method can be adjusted according to the type of bacteria and stipulated treatment time. In Figure 5, for example, the cumulative release is observed for three different concentrations of the antibiotic Meropenem (X=10μg/mL). As the concentration of the antibiotic increases, the time at which therapeutic concentrations are released over a longer period of time also increases. In the case of the 1X hydrogel, it released all of its contents after 24 hours. In the case of the hydrogels with antibiotic at 10X and 100X, after 24 hours, the release that was recorded was 55.20 ± 2.43 and 276.491 ± 26.82 μg/mL, respectively. After the first day and until the seventh day, the hydrogels with 10X and 100X antibiotic recorded a sustained release of 4.66 and 55.93 pg/mL, on average per day, these concentrations are within the concentration range considered susceptible to bacterial inhibition, according to the Clinical and Laboratory Standards Institute (CLSI) (Manual M-100, table 5a).

Antibiochemistry:

One of the advantages of the biomaterials used for the preparation of hydrogels (chitosan and alginate) is that they have an inherent antibacterial character; this, combined with some antibiotic, generates a synergistic effect that enhances the inhibition of pathogenic agents.

To verify the effect of the biomaterials used and the concentration of Meropenem (10X and 100X) on the inhibition of bacteria: S. aureus, E. coii, K. pneumoniae and P. aeruginosa, the inhibition zone was measured by the method diffusion in agar (Figure 6). It was found that the hydrogel (PVA/Cs/ALG) without antibiotic presented an inhibition zone for the strains of S. aureus and E. Coii, which can be attributed to a bacteriostatic effect due to the presence of chitosan and alginate in the hydrogel structure. The S. aureus strain was the one with the highest inhibition (16.0 mm), while for E. coli it was 15.3 mm, in the case of K. pneumoniae and P. aeruginosa no inhibitory halo was recorded. A comparison was made between the halos obtained in our PVA/Cs/ALG hydrogel and the chitosan hydrogel manufactured in patent CN107496976A, and it was found that our hydrogel without any extra component has a higher inhibition capacity than the hydrogel manufactured in said patent. On the other hand, when incorporating antibiotics into the hydrogel, for example: Meropenem, an increase in the inhibition zone can be seen as the concentration of the antibiotic increases. As seen in the following table 1, for antibiotic treatments (II and III), in all cases, the bacteria are susceptible to inhibition, as contemplated by the Clinical and Laboratory Standards Institute (CLSI) ( Manual M-100, table 4A-I), although it should be noted that in the case of E. Coli and S. aureus the zone of inhibition is greater compared to the other two strains.

Table 1 shows the halos obtained for treatments I (only hydrogel), treatment II (hydrogel-Meropenem 10X) and treatment III (hydrogel-Meropenem 100X).

Table 1. Inhibition zones obtained for 4 bacterial strains (mm)

Cell proliferation assay

Cell proliferation was evaluated using the Resazurin quantification method, in order to observe the effect of the controlled release system prepared by the described method (with 100X concentration of Meropenem) on Wharton's gelatin mesenchymal stromal cells. As a control, only a hydrogel with a 2:1:1 ratio (PVA:Cs:ALG) was used. It was observed that cell proliferation in the hydrogel was greater than 70%, which makes it suitable for its application, since it meets the minimum cytotoxicity requirements. Likewise, it is evident that the encapsulation and release of bioactive factors (for example, HGF) increases cell proliferation, achieving approximate maximum values of 125% after 48 hours, this in turn proves that the addition of antibiotics has no adverse effect. about proliferation. These cell proliferation results are normalized to a standard, which is plate cell culture.

As we can see, the present invention describes a novel method to develop a dual release system that releases so many bioactive factors of interest and antibiotics, allowing to fight infections and in turn promote the healing processes of chronic wounds. The in vitro experiments carried out demonstrated that the release system promotes cell proliferation, thanks to the addition of bioactive factors (for example, HGF). The hydrogel itself was also shown to have inherent antimicrobial properties, allowing the effect that antibiotics can have in treating infections to be enhanced. The addition of PCL microspheres with a bioactive factor in the hydrogel allowed obtaining a release profile without a significant explosive effect. Degradation values and acceptable mechanical properties were also obtained to be used as a release dressing in chronic skin wounds.

Claims

1. Method for preparing a controlled release system for antibiotics and bioactive factors made up of microspheres included in a hydrogel, where the method includes the following steps: a) prepare a solution of 10pg/mL of a bioactive factor of interest, in where the solvent used is a 1X phosphate buffer solution (PBS) supplemented with 1% w/v bovine serum albumin (BSA), at a preparation temperature of 4°C; b) add 0.1% w/w BSA to a 3% w/v polyvinyl alcohol (PVA) solution and then add the bioactive factor solution prepared in step a) until obtaining a solution Wi with a final concentration of the bioactive factor 1 μg for each mL of the solution obtained, homogenizing said solution for 1 minute at 2000 rpm at 4°C; c) add 1 mL of the W ₁ solution obtained in step b) for every 10 mL of a 5% solution of polycaprolactone (PCL) in chloroform, homogenizing the mixture for 1 minute at 2000 rpm at 4°C until obtaining a emulsion W- ₁ /O; d) add the Wi/O emulsion obtained in step c) to a 5% w/v PVA solution (W ₂ phase) by continuous dripping at a flow of 1 mL per minute with an injector, where the mixture is homogenize at 2000 rpm for 1 minute and then stir magnetically for 12 hours at 700 rpm to ensure that the chloroform present is completely evaporated and the microspheres are formed; e) collect the microspheres by centrifugation at 2500 gravities for 5 minutes, discarding the supernatant and washing the precipitate with deionized water, repeating said process 3 times; f) select the microspheres by size using microsieves with a pore size of 40 pm; g) freeze-dry the microspheres between -45°C and -55°C, and at 0.0125 mBar for 24 hours, and store them between -20°C and -30°C; h) to obtain the hydrogel, mix a 5% w/v PVA solution, a 2% w/v chitosan solution and a 2% w/v alginate solution in a selected ratio of 2:1:1, 1:1:1, 1:2:1 or 1:1:2, with initial stirring at 700 rpm at room temperature for 1 hour; i) adjust the pH of the solution obtained in step h) using sodium hydroxide (NaOH) at a 1 M concentration until reaching a pH between 7.2 and 7.4; j) shake the hydrogel obtained in step i) for 2 more hours and then add the microspheres previously synthesized and stored as described in step g), where 100 mg of microspheres are added for each mL of hydrogel obtained; k) add the antibiotic of choice at a concentration of 1 mg for each mL of hydrogel and leave it under constant stirring for 1 hour; and l) pour the mixture into molds and subject the hydrogel obtained with the microspheres and the antibiotic to 3 cycles of freezing for 20 hours at -30°C and thawing for 4 hours at room temperature.

2. The method of claim 1, wherein the 3% and 5% w/v PVA solutions are prepared by dissolving PVA in a phosphate buffer solution (PBS) at pH 7.4, wherein the solutions obtained are left sealed in a container stirring at 700 rpm for 24 hours at a temperature of 135°C.

3. The method of claim 1 or 2, wherein the 5% polycaprolactone (PCL) solution is prepared by dissolving PCL in chloroform with stirring at 700 rpm for 30 minutes at room temperature in a completely sealed amber container.

4. The method of any of claims 1 to 3, wherein the 2% w/v chitosan solution is prepared by dissolving the chitosan in a solution of acetic acid (0.1 M) leaving the mixture sealed in a container in stirring at 700 rpm for at least 2 hours at room temperature.

5. The method of any of the preceding claims, wherein the 2% w/v alginate solution is prepared by dissolving the alginate in a phosphate buffer solution (PBS) at pH 7.4, wherein the solution obtained is left sealed. in a container stirring at 700 rpm for at least 2 hours at room temperature.

6. The method of any of the preceding claims, wherein the proportion of PVA:chitosan:ALG is 2:1:1.

7. The method of any of the preceding claims, wherein the bioactive factor of interest has a cationic charge.

8. The method of claim 7, wherein the bioactive factor of interest is hepatocyte growth factor (HGF).

9. The method of any of the preceding claims, wherein the antibiotic of choice is a Carbapenem.

10. The method of claim 9, wherein the antibiotic of choice is Meropenem.

11. The method of any of the preceding claims, wherein the hydrogel obtained that includes the microspheres and the antibiotic is preserved between -20°C and -30°C.