RO138006A2 - Polymeric support - medicament/essential oil for dressings to be used in treatment of bedsore wounds - Google Patents

Abstract

The invention relates to a polymeric support - medicament / essential oil to be used in dressings for treatment of bedsore wounds and to a process for preparing the same. According to the invention, the polymeric support comprises the following components, expressed as weight percentage: 40...80% poloxamer F127 or 20...75% polyvinyl alcohol (PVA) or 35...65% polyvinyl pyrrolidone or 30...70% alginate or 28...49% chitosan, in admixture with 2...10% caboxymethyl cellulose (CMC), chitin, hyaluronic acid, starch, gelatin, polylactic acid, polycaprolactone, plasticizer as glycerol or polyethylene glycol or citrate, vitamin A and E, stabilizers, such as zinc stearate, calcium stearate, emulsifiers, such as Tween 80, acacia gum, cross-linking agent, such as glutaraldehyde, Ag nanoparticles as active substances, antibiotic (tetracycline, gentamicin) and essential oils. The preparation process, as claimed by the invention, consists in preparing an antimicrobial polymeric formulation by introducing the essential oil or the tetracycline as the active pharmaceutical substance, either by physical mixing in the polymeric support, or by encapsulating the essential oil in micro capsules using the emulsion method and charging the micro capsules in the polymeric support.

Description

POLYMERIC SUPPORT-MEDICINE/ESSENTIAL OIL FOR DRESSINGS USED IN THE TREATMENT OF SCARA-TYPE WOUNDS

DESCRIPTION

The present invention refers to the components of the polymeric support that can be loaded with drugs or essential oils in order to create a dressing-type medical device with properties to inhibit or reduce the development of bacterial cultures, during use in contact with areas of the human body affected by open injuries such as bedsores.

Currently, due to stress and inadequate nutrition, the health of a considerable number of people is seriously affected by cardiovascular diseases and diabetes, which in advanced stages can lead to uncontrollable complications. The appearance of bedsores in the case of stroke or pressure ulcers due to diabetes is the third most expensive disease worldwide. Patients face reduced physical activity, reduced ability to feed, urinary and fecal incontinence, as well as decreased consciousness. Last but not least, bedsores and pressure ulcers lead to almost unbearable pain, especially in immobilized patients. Pressure ulcers or bedsores are skin injuries that can be classified into types 1, 2, 3, 4, depending on the depth of the tissue injury. Type 1 represents the least severe stage, and type 4 represents complete tissue destruction. It is estimated that annually the mortality due to the complications of these diseases increased 2-6 times more than due to other diseases. For these reasons it is important to manage bedsores and pressure ulcers in order to estimate the duration of wound closure, as many wounds do not close during hospitalization. Therefore, by controlling the affected area of the skin, objective and reproducible information can be obtained, which can provide the prognosis of healing.

Pressure ulcers are an internationally recognized patient safety problem, estimated to affect 2.5 million people annually.

The development of pressure ulcers in any patient is a serious complication that results in pain, decreased quality of life, and significant costs of both time and money to the healthcare industry. Also known as a pressure ulcer, pressure ulcers or bedsores are a localized lesion of the skin, underlying tissue, or both, usually occurring over a bony prominence as a result of pressure or pressure in combination with shear stress. Intrinsic risk factors for the development of pressure ulcers include aging, poor nutrition, poor perfusion and oxygenation, while extrinsic risk factors include increased humidity, shear and friction. Irreversible tissue damage can occur in a vulnerable patient with continuous pressure for up to 30 minutes. In addition, excessive contact of the skin with fluids affects its barrier function, causes maceration and an increased risk of pressure ulcers.

The overall prevalence rate of pressure ulcers varies from 8% to 30%, depending on patient factors and treatment setting. There are a number of systems for describing the amount of tissue damage, but pressure ulcers are generally classified as 1, 2, 3 and 4, depending on the depth of the tissue damage, with category/stage 1 being the least severe and category/stage 4 indicating complete tissue destruction, as illustrated in Table 1. Most pressure ulcers occur on the sacrum or heel, but they also frequently occur over the elbow, hip, ischium, shoulder, spinous process, ankle, toe, head or face.

Table 1. Classification system: National Pressure Ulcer Advisory Panel (NPUAP)ZEuropean Pressure Ulcer Advisory Panel (EPUAP) (2009)

Stage of eschar Definition Stage 1 At this stage, the skin is intact, but red in color, a color that does not fade when the pressure is removed. The affected area may be painful and may show changes in hardness (firmer or softer) or temperature (warmer or colder) compared to the surrounding area. Stage 2 At this stage, the surface layer of the skin is damaged, forming a superficial wound. These bedsores are often red or pink. At this stage, the eschar can also appear in the form of a blister, intact or broken, with or without secretions, without dead tissue. Stage 3 At this stage, the wound is deeper, it affects the dermis (the second layer of the skin) reaching the subcutaneous adipose tissue. In this stage, necrosis (black dead tissue or in the form of a crust) can appear and deep subcutaneous extensions of the wound (also called pockets) can form. Stage 4 In stage IV of the eschar, the wound extends to the level of the muscles or even to the bone, which is visible or palpable directly. As a rule, the wound has a discharge, is infected and has an abundance of necrotic tissue (dead tissue, under the appearance of a black crust). Internal pockets of the wound are frequently present at this stage and there is a high possibility of infection with risk of general transmission (sepsis).

Currently, there are numerous examples of dressings used to treat skin lesions, which come into contact with the human body for a short period of time.

Numerous and various dressings for the treatment of pressure ulcers are promoted and sold worldwide. Among these, we describe below the important types of dressings and their properties.

Absorbent dressings are applied directly to the wound and can be used as secondary absorbent layers in the management of heavily exuding wounds. Examples include Primapore (Smith & Nephew), Mepore (Molnlycke) and absorbent cotton gauze (BP 1988).

Alginate dressings are highly absorbent fabrics/threads that are presented in the form of calcium alginate or calcium and sodium alginate and can be combined with collagen. Alginate forms a gel upon contact with the wound surface; it can be removed with the dressing or rinsed with sterile saline. Alginate deposition on a secondary viscose pad increases absorption. Examples include: Curasorb (Covidien), SeaSorb (Coloplast) and Sorbsan (Unomedical).

Capillary action dressings consist of an absorbent core of hydrophilic fibers held between two contact layers with low adhesion. Examples include: Advadraw (Advancis) and Vacutex (Protex).

Films, for example permeable film and membrane dressings, are permeable to water vapor and oxygen, but not to water or microorganisms. Examples include Tegaderm (3M) and OpSite (Smith & Nephew).

Foam dressings contain hydrophilic polyurethane foam and are designed to absorb wound exudate and maintain a moist wound surface. There are a variety of versions and some include additional absorbent materials, such as viscose and acrylate fibers, or superabsorbent polyacrylate particles, which are coated with silicone for non-traumatic removal. Examples include: Allevyn (Smith & Nephew), Biatain (Coloplast), Tegaderm (3M), Askina Dressil Border (Braun).

Honey-impregnated dressings contain medicinal honey that has antimicrobial and anti-inflammatory properties and can be used for acute or chronic wounds. Examples include: Medihoney (Medihoney) and Activon Tulle (Advancis).

Hydrocolloidal dressings are usually composed of an absorbent, hydrocolloidal matrix, on a vapor permeable film or foam support. Examples include: Granuflex (ConvaTec) and NU DERM (Systagenix). Fibrous alternatives that resemble alginates and are non-occlusive have also been developed: Aquacel (ConvaTec).

Iodine-impregnated dressings act as a wound antiseptic when exposed to wound exudates. Examples include lodoflex (Smith & Nephew) and lodozyme (Insens).

Dressings with low adhesion consist of cotton pads that are placed directly in contact with the wounds. These may be non-medicated (eg, paraffin gauze dressing, saline gauze dressing) or medicated (eg, containing povidone-iodine or chlorhexidine). Examples include paraffin gauze dressing, BP 1993 dressing, and Xeroform (Covidien)—a mixture of nonadherent petroleum jelly with 3% bismuth tribromophenate on fine mesh gauze.

Odor-absorbing dressings contain charcoal and absorb the smell of wounds. Often this type of dressing is used together with a secondary dressing to improve absorption. An example is CarboFLEX (ConvaTec), Askina Carbosorb.

Other antimicrobial dressings consist of a gauze or loosely adherent dressing impregnated with an ointment loaded with antimicrobial actives (antibiotics, etc.). Examples include: chlorhexidine gauze dressing (Smith & Nephew) and Cutimed Sorbact (BSN Medical).

Protease-modulating matrix dressings modify the activity of proteolytic enzymes in chronic wounds. Examples include: Promogran (Systagenix).

Dressings impregnated with silver ions are used to treat infected wounds, as silver ions are known to have antimicrobial properties. Many versions of silver dressing types are marketed (eg, hydrocolloidal silver, etc.). Examples include: Acticoat (Smith & Nephew), Urgosorb Silver (Urgo), Askina Calgitrol (Braun).

Soft polymer dressings are composed of a soft silicone polymer deposited on a non-adherent layer; they are moderately absorbent. Examples include: Mepitel (Molnlycke) and Urgotul (Urgo); Mepilex Border Sacrum is a soft silicone dressing, created by Safetac® technology, consisting of five layers, intended for the prevention and management of wounds in the sacral area; HARTMANN RespoSorb silicone dressing 8x8cm.

The use of biopolymers to make antimicrobial dressings and the progress made in the biomedical industry market have led to new challenges regarding the continuous improvement of their biocompatibility and biofunctionality. The dressings must not cause discomfort to the patient through changes in the tissue with which they come into contact, such as thrombogenic, allergic and toxic reactions. Over time, numerous researches have been carried out regarding the minimization of these unwanted effects.

The treatment of infected wounds is a major concern in medical care, as these types of conditions create pain and suffering for the patients involved. The resulting complications can be very expensive due to the fact that the patients' stay in the hospital is greatly extended. And yet, the treatment of open wounds is limited by the growing number of antibiotic-resistant strains of bacteria. As such, the prescription of antibiotics is reduced, but this type of treatment is not completely abandoned. Therefore, an alternative treatment is adopted by using dressings loaded with active principles with the role of tissue growth stimulation and antimicrobial agents such as: essential oils or silver nanoparticles.

Thus, the patent reports on attempts to combat infections in the area of pressure sores, by using essential oils or medicinal active substances.

The objective of the invention is the realization of the polymeric support for incorporating the active substance of the medicinal type or essential oil, as a component part in the dressings for the treatment of bedsore type wounds.

The most prevalent compounds when referring to functional wound dressings are antimicrobial agents. These agents give antimicrobial properties to dressings and are divided into three groups: antibiotics (for example: tetracycline, gentamicin), natural biological materials (for example: essential oils, honey) and nanoparticles (for example: silver, gold). Natural and synthetic biomaterials are two main categories of biomaterials used for wound dressings. The most common natural biomaterials used for dressings are collagen, hyaluronic acid, chitin, chitosan, starch, gelatin and alginate. These types of dressings are better in terms of biocompatibility, antibacterial activity, antioxidation, hemostasis and promotion of healing. Caridade et al. have developed thick self-standing membranes made of multilayer films of alginate and chitosan. They concluded that these membranes are biocompatible and very stable in a physiological buffer, offering new perspectives for wound healing and tissue engineering applications. However, dressings based on synthetic polymers can offer a wider spectrum of mechanical properties compared to natural wound dressings. Polylactic acid (PLA), polycaprolactone (PCL) and polyethylene glycol (PEG) are examples of synthetic polymers that have been extensively studied for wound dressing applications. In a study developed by Bardania et al., the new strategy of using synthesized silver nanoparticles (AgNP) incorporated in PLA/PEG nanofilm showed promising results. Biocompatible silver nanoparticles were synthesized using Teucrium polium extract as a reducing agent, an approach that proved to be efficient and cost-effective. The nanofilm displayed promising antimicrobial and antioxidant properties, having strong potential as a wound dressing. Both natural and synthetic biomaterials have advantages and disadvantages and therefore research studies are now focused on combining different types of polymers to improve wound healing properties, control biodegradation and drug release. Amalraj et al. developed biocomposite films by incorporating black pepper essential oil and ginger essential oil into polyvinyl alcohol (PVA), gum arabic (GA) and chitosan (CS). Obtained by the solvent casting method, the biocomposite films showed improved mechanical properties with improved heat stability, as well as antibacterial activity against gram-positive and gram-negative bacteria.

Biomaterials for wound healing applications can be enriched with various bioactive compounds, essential oils, which can accelerate the regeneration process. Bioactive dressings have the ability to release active substances (antibiotics, peptides, drugs, vitamins, /3 growth factors, etc.) into the wound environment to improve the healing process. The dressings interact directly with the wound area, promoting the regeneration process. These interactions include removing excessive exudate, providing a moist wound environment and preventing infection. Importantly, interactive dressings are favorable for the re-epithelialization process due to better oxygen concentration and pH control. All the mentioned characteristics of the dressings optimize the skin regeneration process.

Medical devices are known in the form of cross-linkable, water-swellable microgel particles, consisting of proteins and protein-based biopolymers, which are pseudoplastics, flow in an aqueous environment under shear forces and cover the empty spaces of tissues, organs and wounds . Microgel particles can be injected, sprayed, coated or implanted and can also surround a substitute tissue, and microgel particles refer to microgel particles that aggregate as a single microgel group when shear forces are removed . Microgel particles function as a viscoelastic matrix that supports cell growth, viability and proliferation.

The use of essential oils and medicinal active substances is known for the prevention and/or treatment of 5. epidermitis infections, especially from invasive medical procedures, for example the insertion of catheters. Also in the respective patent it is shown that the use of chlorhexidine in combination with eucalyptus essential oil shows a surprisingly good antimicrobial activity against S. epidermidis bacteria and S. Epidermidis biofilms. It is considered that this combination is useful for preventing and/or treating infections caused by S. Epidermidis bacteria, especially in invasive medical procedures, e.g. insertion of catheters.

The use of essential oils in different compositions for treating wounds is known. Thus, patent CN 109745175 presents a method of preparing the dressing with nanofibres. The method includes the steps of mixing and dissolving gelatin, an acetic acid solution, peppermint essential oil and chamomile essential oil to obtain a spinning solution. Patent CN 104784743 presents a preparation method for dressing based on aromatic and bacteriostatic chitosan. The preparation method includes the following steps: mixing tea tree essential oil, menthol essential oil and folium artemisiae argiyi according to a certain proportion to obtain a naturally aromatic bacteriostatic agent. US patent 2011104243 presents a composition of the consistency of a paste made from natural substances, for healing cuts, bruises, wounds and the like on the skin. The components are a very fine powder of curcuma longa (turmeric), lavender essential oil and glycerol. US Patent 2015030708 discloses a composition for treating wounds, sores, burns and other traumatized dermal tissue and skin lesions comprising Boswellia gum, gel, resin or extract, tea tree oil (Melaleuca oil), an aloe gel, resin, latex or extract and lavender oil. The composition can be incorporated into a medical device such as a wound dressing or bandage, or formulated into a topical preparation such as an ointment, lotion or cream.

It is known to use polyethylene glycol, glycerol, citrate plasticizers (e.g. poly (polyethylene glycol citrate - co - N - isopropylacrylamide)) in polymer compositions for applications in dressings for the treatment of various open wounds. Patent DE102004047115, (Method for producing a wound dressing) refers to methods for producing a dressing, with the help of which the permeability of the dressing can be adjusted. With this method, dressings can be produced for a wide range of therapeutic applications.

There are known uses of essential oils as antimicrobial agents for use in tissue engineering and wound healing (table 2).

Table 2. Various applications of essential oils

Essential oils Polymer matrices applications Lavender Polyurethane Bone tissue engineering Lavender Π Polycaprolactone / Polyethylene glycol Treatment of skin lesions due to its antimicrobial properties, against Staphylococcus aureus and Escherichia coli Thyme Silk fibroin / Gelatin Treatment of skin lesions due to the antimicrobial properties, against Staphylococcus aureus and Klebsiella pneumoniae. Thyme Polycaprolactone / Polyvinyl alcohol Treatment of wounds and surfaces containing pathogenic microorganisms Thyme Potato starch The antioxidant properties of the mentioned oil are due to the significant content of phenolic compounds Cinnamon Polyurethane Antibacterial fibers, facial masks and air purifiers Cinnamon Polyvinyl alcohol Biodegradable matrices/cinnamon essential oil encapsulated in cyclodextrin show excellent antimicrobial properties against Escherichia coli and Staphylococcus aureus. Cinnamon Polyurethane Treatment of skin lesions due to the antimicrobial properties, against Staphylococcus aureus and Klebsiella pneumoniae. Cinnamaldehyde Chitosan / polyethylene oxide Eliminates Pseudomonas infections. Cinnamon Polyvinyl pyrrolidone Good antibacterial owners against Staphylococcus aureus, Escherichia coli and Candida albicans Tea tree Chitosan Membranes with continuous liposomes and encapsulated essential oil show the maximum inhibition zone against Salmonella enteritidis and Salmonella typhimurium.

The present invention offers solutions for obtaining polymeric support type devices for incorporating active substances such as medicine or essential oil as a component part for dressings used in the treatment of bedsore type wounds, with antimicrobial and biocompatibility properties. These characteristics are intended to be preserved over the long-term storage of the devices. Improved recipes are presented by introducing components for medical use such as: biocompatible plasticizers, vitamins and antimicrobial active substances such as antibiotics and essential oils, other additives. The dressings made under the patent can be used for the treatment of open skin lesions such as eschar, pressure ulcers, incisions, etc.

The process of making the polymer support consists of mixing the components in solution and offers the advantage of using multiple materials to generate superior physico-chemical and biological properties necessary for proper use. Also, the mixing of the components takes place effectively until the complete homogenization of the newly obtained material.

According to the invention, the polymeric support for encapsulating the active substances can be composed of a water-soluble polymer type: poloxamer FI27 which can be used in a proportion of 40...80% gr, or polyvinyl alcohol (PVA) which can be used in a proportion of 20. ..75% gr, or polyvinyl pyrrolidone (PVP) which can be used in a proportion of 35...65% gr, or alginate which can be used in a proportion of 30...70% gr, or chitosan which can be used in a proportion of 28...49% gr, carboxymethyl cellulose (CMC) which can be used in a proportion of 2...10% gr, chitin, hyaluronic acid, starch, gelatin, polylactic acid, polycaprolactone, glycerol-type plasticizer, or polyethylene glycol, or citrate, vitamins: vitamin A, vitamin E, stabilizers such as zinc stearate, calcium stearate, emulsifier type Tween 80, gum arabic, crosslinking agent such as glutaraldehyde, active substances such as silver nanoparticles, antibiotic (tetracycline, gentamicin ) and essential oils.

The purpose of the present invention is to create a polymeric support for encapsulating medicinal active substances and essential oils, with improved properties of biocompatibility, antibacterial activity, antioxidation, hemostasis and initiation of healing, as an integral part of dressings for the treatment of open wounds such as eschar and pressure ulcers .

The technical problem that the invention solves refers to obtaining the polymer support made up of some recipes based on polyvinyl alcohol, polyvinyl pyrrolidone, carboxymethyl cellulose with improved biocompatibility due to the use of plasticizers such as glycerol and polyethylene glycol, which shows reduced diffusion through the mass of the polymer, the use of vitamins and the use of medicinal active substances and essential oils as antimicrobial agents to prevent the adherence and formation of colonies of microorganisms from the biological environment with which they come into contact. In the present invention, the essential oils were introduced into the polymeric support by physical mixing with the material of the composition or were encapsulated in sodium alginate and then were introduced into the polymeric mass by physical mixing. the encapsulation of essential oils aimed at their controlled release into the environment they come into contact with.

The degree of novelty is the creation of two types of polymeric support:

- polymeric support in which the active substances: tetracycline, essential oils were added to the mass of the material by physical mixing

- polymeric support in which microcapsules with essential oil were added and gently mixed with the mass of the material.

The recipes made and comparatively studied were coded El - E4, and their compositions are presented in table 3:

Table 3. Experimental compositions E1-E4

Recipe code/ Component He [%1 E2 [%1 E3 [%] E4 [%] PVA 25 2. 3 2. 3 2. 3 PvP 15 10 16 10 Tween 80s + CaCh - Cross-linking agent + cc 2 2 2 2 Pine essential oil 12 12 Tetracycline 7 Vitamin A 3 3 3 3 Vitamin E 2 2 2 2

Deionized water 50 50 38.5 44.5 Glycerol 2.5 2.5 3 3 Glutaraldehyde 0.5 0.5 0.5 0.5

+ represents continuous materials that are not included in the composition of polymer matrices or capsules loaded with essential oils

The polymeric support obtained on the basis of biopolymers, plasticizers, additives and antimicrobial active substances is intended for the manufacture of dressings for the treatment of eschar-type skin lesions.

PROCEDURE

The creation of the polymer support requires the following steps:

- weighing raw materials on the analytical balance;

- solubilization of PVA / PVP (40:60 % gr ...60/40 % gr) and CMC polymers in deionized water by mixing with a magnetic stirrer, at a temperature of 90 °C, speed 500 rpm;

- after complete dissolution, cool the mixture to 40 °C and add glycerol, PEG, vitamin A, vitamin E in turn and mix physically until complete homogenization at room temperature;

- after deaeration, pour the mixture into a Petri dish and dry it in an oven with air circulation at 50 °C for 8 hours; the obtained film constitutes the witness evidence;

- after deaeration, the antimicrobial agent is introduced (e.g. tetracycline dissolved in deionized water or essential oil, or capsules with essential oil)

The obtained recipes were placed in a Petri dish and left to dry by evaporating the water in an oven with air circulation, at a temperature of 50 °C, for 8 hours. After drying, the formed films are detached and samples are taken for analysis.

Realizing the encapsulation of essential oils by the emulsion method involves going through the following stages:

- making the emulsion based on essential oil and ethyl alcohol in a ratio of 50:50, to which Tween 80 surfactant additive is added to stabilize the emulsion, in a Berzelius glass by mixing with a TURAX mixer at 23000 rpm for half an hour at room's temperature,

- making a mixture called MIX 1 by dissolving 1 g of sodium alginate polymer in 100 ml of distilled water in a Berzelius glass on a magnetic stirring plate, at a temperature of 60 °C, 700 rpm until the process is complete,

- making a mixture called MIX 2 by mixing MIX 1 with the emulsion obtained of the essential oil, on the magnetic stirring plate, at room temperature, 700 rpm, for half an hour,

- preparation of the cross-linking agent by dissolving 30 g of calcium chloride in 100 ml of distilled water, in a 200 ml capacity Berzelius glass, on an ice bath, mixing at 200 rpm, until complete dissolution,

- the syringe is filled with the MIX 2 mixture and dripped into the Berzelius glass with calcium chloride solution, while mixing with a magnetic stirrer at 200 rpm,

- microcapsules filled with essential oil are formed, the obtained microcapsules are added to the polymer support material by gentle mixing,

- the obtained composition is poured into a Petri dish to obtain a layer with a uniform thickness,

- let it dry by evaporating the water in an oven with air circulation, at a temperature of 50 °C, for 8 hours,

- after drying, peel off the film and take samples for analysis.

The samples are analyzed from the point of view of the degree of dissolution, transmission properties and biological properties. Biocompatibility properties are demonstrated by cytotoxicity tests. Antimicrobial tests are performed according to EN ISO 22196, using as test microorganisms Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853.

The in vitro evaluation of the cytotoxicity of the initial plasticized polymer recipes and those with an antimicrobial additive is performed on cell cultures, using the technique of cultivating cells in suspension, with DME medium (Dulbecco's Medium Essential), containing 10% fetal bovine serum. The viability of the cells is evaluated by performing the MTT test and observing the morphological aspect of the cells.

EXAMPLE 1

The control sample called El is made, according to the working method presented above and the content presented in table 3. It is placed in a Petri dish and dried in the oven with air circulation at 50 °C for 8 hours. A polymeric composition E2 is made by introducing one gram of tetracycline dissolved in deionized water, in a composition identical to El and mixing until homogenized. Place in a Petri dish and dry in an oven with air circulation at 50 °C for 8 hours. The El and E2 films obtained are analyzed physico-chemically and biologically.

EXAMPLE 2

It is made as in example 1 a control sample EL A polymer composition E3 is made (according to table 3), in which the essential pine oil is introduced drop by drop and mixed until homogenized. Place in a Petri dish and dry in an oven with air circulation at 50 °C for 8 hours. The El and E3 films obtained are analyzed physico-chemically and biologically.

EXAMPLE 3

As in example 1, a control sample EL is made. As in example 2, a sample called E4 is made, in which the essential pine oil is introduced encapsulated. According to the work method described above, microcapsules are made with pine oil. The microcapsules with essential pine oil are inserted into the E4 composition and mixed gently until homogenized. Place in a Petri dish and dry in an oven with air circulation at 50 °C for 8 hours. The El and E4 films obtained are analyzed physico-chemically and biologically.

The testing of films taken from samples El, E2, E3, E4 includes:

Inflating capacity

To calculate the degree of swelling of the films, samples were cut from each recipe in the form of discs with a diameter of 3 cm and were brought to constant weight by drying. The dry samples were further immersed in deionized water at room temperature (20 °C). The experiments were performed in triplicate, and the measurements were performed until the constant mass of the inflated samples (2 to 5 hours). Since the time required to reach equilibrium was very short, the mass of dissolved polymer was considered negligible.

Also, to keep the calculation simple, the amount of active principle contained in the samples and released into the experimental aqueous environment was considered to be insignificant compared to the total amount of water that was absorbed. Swelling degree (SD) was determined with

equation (1), where mi (g) is the initial weight of the sample and m _s (g) represents the weight of the inflated sample.

5Ό = _{x 100} ^mi (1)

Solubility in water

Water solubility measurements were performed in triplicate, according to the method of Shen et al., with some modifications, as will be detailed. The film samples with dimensions (2x2 cm), previously dried to constant weight, were introduced into conical flasks that contained the same measured amount of deionized water (100 ml). The flasks were kept at room temperature and stirred continuously for 24 hours. The remaining undissolved pieces of the films were removed from the water and then dried again to constant weight.

The water solubility (WS) of the composite materials was calculated with the weight of the initial dry sample, mi (g) and the weight of the undissolved dry sample, mf (g), using equation (2):

m.: -Wlf

WS = — ^! ---- x 100 m _f (2)

Water vapor permeability

For each film sample, the water vapor permeability (WVP) is calculated using equation (3), following the method described above. The films are cut in the form of rounds with a diameter of 3 cm and sealed on polypropylene cups containing silica gel (0% RH). The glasses are then placed in a desiccator containing distilled water, at room temperature (20 °C) and 95% relative humidity, for six days. The desiccator chamber is equipped with temperature and relative humidity sensors. Periodically, the absorbed moisture is determined gravimetrically. The test was performed in triplicate:

WVP=WVTR x δ/Δρ (3)

Where:

- WVTR is water vapor permeability (g m' ² day ¹ ),

- δ is the film thickness (m),

- Δρ represents the partial pressure difference of water vapor on the two sides of the coatings (Pa).

The results obtained for the four samples are presented in table 4.

Solubility in water is characteristic of biodegradable films and offers potential benefits. Ideally, the speed of wound healing should be equal to that of degradation of the polymer matrix. All the films kept their original shape during the water solubility experiments. A good dressing should allow the evaporation of exudates to facilitate the healing process and at the same time should prevent dehydration of the wound. A high WVTR will induce dehydration and adhesion of the dressing to the wound surface, while a WVTR that is too low will cause the accumulation of exudates, allowing microbial growth and wound infection. Considering the generally accepted WVTR values in the range of 76-9360 g m' ² day' ¹ depending on the condition of the skin and the type of wound and a specific range of 904-1447 g m' ² day' ¹ , which characterizes wounds with moderate exudates, we can concluded that our films could be applied to wounds with small to moderate exudates. Other researchers have determined the WVTR for PVA in different compositions together with chitosan or collagen, and the values obtained were higher than our compositions. Low WVP values are known to provide a good healing environment by maintaining optimal moisture content of dressing materials. Our obtained values for WVP are lower than those reported for carrageenan-based functional hydrogel films for example.

Table 4. Film thickness (δ), degree of swelling (SD), solubility in water (WS), water vapor transmission rate (WVTR), water vapor permeability of polymer recipe films (WVP)____________________

Sample δχ 10 ³ , (m) SD WS, (%) WVTR, (gm^zi ¹ ) WVP x IO ¹⁰ , (g m' ¹ s' ¹ Pa' ¹ ) He O.238±O.OO3 4.847±0.518 59.27±0.68 578.5±10.4 6.816±0.123 E2 0.307±0.030 6.472±0.325 59.34±0.61 493.3±7.4 7.496±0.112 E3 0.247±0.003 5.775±0.624 59.69±0.97 575.9±14.4 7.041 ±0.176 E4 0.237±0.003 6.048±0.831 66.02±1.05 521.2±5.1 6.115±0.596

Antimicrobial efficiency tests

Antimicrobial tests were performed using standard strains of Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, in accordance with "SREN 14885 - Chemical disinfectants and antiseptics - Application of European standards for chemical disinfectants and antiseptics" . The qualitative screening was performed using an adapted spot diffusion method. Microbial suspensions, with a standard density of 0.5 McFarland (corresponding to 1.5x10 ⁸ CFU/mL), were obtained from fresh bacterial cultures for 24-48 hours. The microbial inoculums were seeded on Petri dishes containing Muller Hinton agar (for bacterial strains) or Sabouraud agar, according to the CLSI diffusion method (Clinical Laboratory Standard Institute, 2022), and the samples of sterile material, with an area of 1 cm ² , were placed on the surface of the inoculated medium and incubated for 20 hours at 37 °C. To evaluate the antimicrobial activity using the Viable Cell Count (VCC) method, the materials were immersed in 1 ml of appropriate broth medium, which was later inoculated with microbial suspension at a final density of 1.5x10 ⁵ CFU/ml. After 20 hours of incubation at 37 °C, the activity of the microbicide was evaluated by the ten-fold serial microdilution technique. Serial dilutions and plate counts were performed in duplicate after inoculation and incubation at 37 °C for 24 hours, microbial colonies were counted and converted to colony forming units per milliliter (CFU/mL). Each test was performed in triplicate. The result is presented in figures 1 - 4. Comparing with the growth control sample (microbial strain culture in normal condition) and the control sample (microbial strain culture in the presence of the control sample El), all the tested materials showed an inhibitory effect, with the drastic attenuation of logarithmic values corresponding to CFU/mL.

The exception was observed for the strain of Pseudomonas aeruginosa ATCC 27853 Gram negative, confirming the qualitative results regarding the high resistance of this strain to the inhibitory activity of essential oils.

Analysis of biocompatibility properties by performing the MTT test

L929 fibroblasts (5x10 ⁵ cells/well) were cultured in DMEM medium (Dulbecco's Modified Eagle Medium, Sigma-Aldrich) supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 1% Pen/Strep (penicillin/streptomycin solution, Sigma Aldrich) for 24 h at 37 °C, 95% humidity with 5% CO2. Samples were co-cultured with fibroblasts for 24 hours (37 °C, 95% humidity, 5% CO2). The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was used to evaluate cell viability and proliferation in the presence of the materials. Cells were incubated for 4 hours with MTT reagent (Vybrant® MTT Cell Proliferation Assay Kit, V13154) at 37 °C, 95% humidity with 5% CO2. After incubation, the formazan crystals were solubilized with DMSO for 10 min at room temperature. The absorbance was measured at λ= 540 nm using the Mulsiskan FC device (Thermo Scientific). Cell morphology was assessed using an Olympus 1X73 inverted microscope after 24 hours of incubation with the biomaterials. According to Figure 5, samples E1-E4 were not toxic and did not lead to cell death, as shown by phase contrast microscope analysis.

Very importantly, none of the evaluated samples induced significant changes in cell morphology.

BIBLIOGRAPHY

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Claims (4)

1. Biocompatible polymer recipes, based on PVA / PVP, CMC characterized by the fact that it results from mixing the components in an aqueous solution according to table 3 and the work method presented, to obtain the control sample. 2. Antimicrobial, biocompatible polymeric recipes, based on PVA / PVP, CMC, as in claim 1, for use as a polymeric support in the production of dressings for the treatment of bedsores, characterized by the fact that they have antimicrobial properties, due to the use of the active medicinal substance tetracycline. 3. Process for obtaining the antimicrobial and biocompatible polymeric recipe for use as a polymeric support in the production of eschar treatment dressings, defined in example 2, characterized in that the essential oil is introduced by physical mixing in a polymeric matrix according to claim 1. 4. Process for obtaining an antimicrobial and biocompatible polymeric recipe for eschar treatment dressings, characterized in that the essential oil is encapsulated in microcapsules by the emulsion method, and the microcapsules are loaded into a polymeric matrix according to claim 1, for use as a polymeric support in the production of dressings for the treatment of bedsores.