Technical Field

The present invention relates to a wound dressing that can be applied topically for use in the treatment of skin diseases including skin cancer.

More specifically, the present invention discloses a wound dressing and the production method thereof that is made of a polymer selected from the group of polyvinyl alcohol (PVA), polylactic (PLA) polylactic (PLA), polyglycolic acid (PGA), poly lactic -co-gly colic acid (PLGA), polycaprolactone (PCL), polyurethane (PU), polyhydroxy butyrate (PHB), or materials containing combinations thereof, providing a combined therapy including photodynamic therapy (PDT) and targeted drug systems, that has nanofibers, that comprises a hydrogel containing photodynamic therapy agent and chemotherapeutic drug in order to be used in the treatment of skin cancer, particularly melanoma.

State of the Art

Today, skin cancer is one of the most common types of cancer. Among the types of skin cancer, melanoma is the deadliest one. It is vital for patients to follow a rapid course of treatment for malignant (spreading) melanoma. As a current treatment method, the tumor area is surgically removed and, if necessary, chemotherapy is administered in first-stage melanomas. Methods such as radiotherapy, chemotherapy, immunotherapy, photodynamic therapy, targeted drug systems are used as alternatives for advanced stage melanomas.

However, today, these treatment methods generally cannot individually provide complete recovery. For example, spreading tumor cells to the lower layers of the skin creates a limitation in surgical treatment. Since the light cannot reach the lower layers of the skin in photodynamic therapy, it only provides a superficial solution. In the immunotherapy method, on the other hand, its use is disadvantageous since the treatment response varies according to the person. In chemotherapy, drugs spread throughout the body and damage important organs such as the brain. It has many side effects. Targeted drug systems can also have side effects since they are taken orally in high doses many times.

It is important that the side effects of cancer drugs used during treatment are being at the lowest level and are an easy and economical method to be applied. Treatment methods used today generally cannot individually provide complete recovery. Therefore, patients are exposed to long and difficult treatment processes. In this way, patients are negatively affected by the treatment process psychologically and economically while they are losing time socially. Also, doses of the drugs that are employed chemotherapeutically in order to reduce pain during treatment is one of the factors that negatively affect the patient. High doses of chemotherapeutic drugs both cause side effects and increase the cost of treatment. In addition, due to the method/methods used, the protection of the melanoma area from infection becomes difficult during treatments that do not provide a definite result.

Also, melanoma is the deadliest among skin cancers. Even if a single cancer cell remains in the treated area, tumor growth is observed again.

For the purpose of overcoming the problems and insufficiencies in the above-mentioned state of the art, the inventors have designed a novel wound dressing that allows for providing a combined treatment including photodynamic therapy (PDT) and targeted drug systems to provide a complete cure, namely, to destroy the last remaining tumor cell.

As a result of the search conducted in the state of the art, it is found that the article of “Drug-loaded electrospun materials in wound-dressing applications and in local cancer treatment. Expert Opinion on Drug Delivery, 10(4), 469-483.” by Ignatova, M., Rashkov, I., & Manolova, N., published in 2013. Here, it was stated the antibacterial properties of wound dressings, and that they can be used in cancer treatment. However, a combined cancer treatment was not mentioned. In addition, the invention subject to our patent application comprises photodynamic therapy using GQDs and drug therapy using doxorubicin. Combined therapy is applied by creating an antibacterial environment on the wound dressing. This supports the originality of the present invention.

Unlike the methods used so far, the fact that this new method is a combined method provides a more effective result in the treatment. Targeted drug therapy is helpful in photodynamic therapy to destroy the tumor in the lower layers of the skin where light cannot reach.

Description of the Invention

The object of the present invention is to develop a new treatment method by overcoming the problems in the above-mentioned state of the art. Differently from the state of the art, in the method of the invention, a combined treatment method including photodynamic therapy (PDT) and targeted drug systems was developed by means of using wound dressing. The drugs to be used are administered locally, by means of the wound dressing according to the present invention. In this way, low doses of drugs are sufficient. Therefore, side effects of drugs are reduced. Reducing the side effects with a drug with analgesic/fever-reducing effect and using agents that will increase the effect of photodynamic therapy (GQDs or different photosensitizers) will contribute to the patient's recovery from melanoma with the least side effects.

The wound dressing design of the present invention is a topical treatment formed by adding a gel on a nanofiber wound dressing. Optionally, a nanofiber wound dressing having antibacterial property may be used. Also, the fact that it is a topical treatment is another factor that allows for using low drug doses. In addition, it also acts as a barrier between it and the external environment in order to prevent infection in the area to be treated while creating a suitable environment for a combined treatment.

These methods will be applied simultaneously, and the treatment time of the patient will be shortened by means of the combined treatment. In this way, the treatment methods are applied separately and the loss of time is prevented and it is ensured that it does not negatively affect the patient in a social way. Thus, the patient will be able to spare more time for himself/herself and his/her environment. The proposed design is feasible in terms of applicability due to the biocompatibility of the materials used and the anatomical similarity of the wound dressing with the skin tissue. It provides an advantage over other treatment methods in terms of productability and economy considering the materials used and the production method.

Said method according to the present invention is the first method for cancer treatment combined with a wound dressing. This method can also be modified for different diseases. Its applicability is facilitated by combining different treatment methods on the wound dressing. The method that is very easy to implement will provide convenience for both the patient and the doctor. In addition, a short treatment period will prevent the patient's psychology from being adversely affected.

Another advantage of shortening the treatment period is to provide convenience economically to the patient by preventing the costs incurred by the patient during the treatment. With the decrease in the dose of drugs, which is one of the advantages of combined therapy, the amount of drugs to be used will decrease and this will indirectly provide an economic advantage.

Comparing the present invention with the methods used in the state of the art is given in Table 1 below.

Table 1: Comparison of the state of the art and the properties and effects of the method according to the present invention

Description of the Figures

Figure 1 illustrates the example view of the gel wound dressing on nanofiber material. Figure 2 illustrates the example view of the nanofiber wound dressing with double layer.

Figure 3 illustrates the schematic view of the production method according to the present invention.

Figure 4 illustrates the FTIR Analysis of Augmentin -loaded 35% PVA solution.

Figure 5 illustrates the FTIR Analysis of Drug (ibuprofen, doxorubicin) loaded Collagen Hydrogel.

Figure 6 illustrates the DSC Analysis of Hydrogel.

Figure 7 illustrates the SEM Analysis 5.00k x for 35% w/v PVA Wound Dressing.

Figure 8 illustrates the SEM Analysis 10.00k x for 35% w/v PVA Wound Dressing.

Figure 9 illustrates the DLS Analysis for generated GQDs.

Description of the Reference Numerals

NUMBER Reference Name

1 Antibiotic

2 Polymer solution

3 Electrospinning method

4 Hydrogel

5 Nanofiber

6 Graphene quantum dots (GQDs)

7 Photosensitizer

8 Wound dressing

9 Anti-inflammatory drug

10 Chemotherapeutic drug

Detailed Description of the Invention

The present invention mainly relates to the nanofiber (5) wound dressing (8) made of polymer material and characterized by comprising a hydrogel (4) containing a photodynamic therapy agent and a chemotherapeutic drug (10).

The wound dressing (8) used in the invention can be applied as a gel on the nanofiber (5) material as shown in Figure 1 or as a double-layered nanofiber (5) as shown in Figure 2. The figures represent an exemplary embodiment of the present invention. The nanofiber (5) wound dressing (8) of the present invention is applied topically to be used in the treatment of skin diseases, including skin cancer. The most important feature of the invention is that it is applied topically for use in a combined therapy including photodynamic therapy (PDT) and targeted drug systems.

Here, the polymer material is a polymer selected from the group consisting of polyvinyl alcohol (PVA), polylactic (PLA), polyglycolic acid (PGA), poly lactic -co-gly colic acid (PLGA), polycaprolactone (PCL), polyurethane (PU), polyhydroxy butyrate (PHB), or a combination thereof.

Here, collagen is used as the hydrogel (4); one or more active substances selected from doxorubicin, adriamycin, ibuprofen, vinblastine, dabrafenib are used as the chemotherapeutic drug (10); and as a photodynamic therapy agent, a photosensitizer (7) selected from graphene quantum dots (GQDs) (6), graphene, graphene oxide, and temoporfin is used.

In Figure 3, the configuration of the wound dressing (8) of the present invention and the production method thereof were given schematically.

In stage a), the biomaterial polymer solution (2) that is used for the wound dressing (8) and the antibiotic

(1) for the antibacterial property are mixed (loading the antibiotic (1) into the polymer solution (2)). Antibacterial property is optional. Materials such as antibiotic (1), Zn can be added in order to protect the treated area against the infection risk. The material used for the wound dressing (8) can be changed according to the disease being treated. Amoxicillin (Augmentin) was determined as the polymer solution

(2) and antibiotic (1) for the wound dressing (8) to be used in the experiments.

In stage b), the wound dressing (8) is produced with the specified material (and with materials/drugs to be added) by using an appropriate method (electrospinning method (3) or 3D printer). Electrospinning method (3) was used in the experiments.

In stage c), a hydrogel (4) containing photodynamic therapy agents and a chemotherapeutic drug (10) (drugs according to the purpose such as anti-inflammatory drug (9) can be added for side effects) is prepared to be applied to the cancerous area (or to the disease area to be treated). It can be applied instead of hydrogel (4) as seen in Figure 2 or with different designs. A more effective treatment was aimed by using photodynamic therapy and targeted drug systems together. Different treatment methods can be combined on the wound dressing (8) depending on the drugs to be used.

In stage d), Hydrogel (4) containing drugs is added to the wound dressing (8) (Formation of wound dressing (8) with antibiotic (1) loaded polymer nanofiber (5). For the experiments, collagen as a hydrogel (4), doxorubicin as a chemotherapeutic drug (10), ibuprofen as an anti-inflammatory drug (9), and graphene quantum dots (6) as a photodynamic therapy agent were used. The reason of using graphene quantum dots (GQDs) (6) for photodynamic therapy is that it offers a highly efficient cancer treatment, it increases stability and biocompatibility. Different agents can also be used as photosensitizers (7) in experiments (with or without graphene quantum dots (6)). The reason for the determination of collagen for the hydrogel (4) in the experiments is that it creates a biocompatible environment since it is the main protein of the extracellular matrix (ECM). A natural polymer was selected to mimic the ECM such that healthy cells are not damaged during the treatment. Different materials can be selected and, in these cases, and the fabrication method can be changed accordingly.

In stage e), the developed wound dressing (8) is applied to the cancerous area or the area of the determined disease. It is ensured using different treatments together

The nanofiber (5) wound dressing (8) made of polyvinyl alcohol material of the present invention is basically produced according to the following process steps: a) producing the material of wound dressing (8) with nanofiber (5) by means of electrospinning (3) or 3D printing method by using polymer material, b) Preparing the hydrogel (4) containing the photodynamic therapy agent and the chemotherapeutic drug (10), c) Adding the hydrogel (4) containing drugs to the wound dressing (8).

The production method of the present invention is given in detail below and an exemplary embodiment is made. Proving the effectiveness of the product and characterization studies were carried out by subjecting the produced product to the experimental studies. a) Production of wound dressing (8)

Preparation of polymer solution (2), production of wound dressing (8)

The nanofiber wound dressing (8) was produced by means of using the electro-spinning method (3). With the electrospinning method (3), polymer solutions are obtained as nanofibers (5) in high voltage and electric field. The advantage of this method is that it is easy to apply and suitable for industrial production. The absorption ability of the wound dressing (8) produced by the electrospinning method (3) is higher than the conventional wound dressings. The reason for this is its structure with high pores and large surface area. In addition, porous structure thereof is large enough to allow oxygen exchange, but too small for microorganisms to pass through.

First of all, polymer solutions (2) (preferably PVA solution) at a concentration of 25-35% were prepared. Distilled water is used as the solvent. 30g of PVA per 100g of water is used for solution preparation. The reason for selecting this polymer is that it is easy to print because it is a synthetic polymer. Synthetic polymers have a lower risk of contamination compared to natural polymers. They are less affected by external factors such as temperature, pH, pressure. Nanofiber (5) was produced from PVA (Mw 13.000-23.000, 87-89% hydrolyze) at different rates (35%, 30%, and 25%, respectively) with the electrospinning device. Production parameters were determined as 15kV, 10 cm distance and 1,5 mL/h velocity for 5 hours for all ratios. Augmentin was added to the solution at a rate of 0,1% w/v. After the characterization analysis, a drug-loaded polymer solution (2) at a rate of 35% w/v was found to be ideal.

Augmentin is an antibiotic (1) that is used to treat or prevent infections and approved by the FDA (Food and Drug Administration). It is effective against a wide range of bacteria. For this reason, it was used to prevent the risk of infection. During the treatment, the nanofiber material degrades (dissolves) and protects the treatment area against bacteria. Antibacterial chitosan or AgNP, which are natural polysaccharides, may be used instead of Augmentin. In addition, different antibiotics (1) such as amoxicillin, clavunate, croxilex, alfacillin, bioment, macrocillin, sultamicillin, and duobak can also be used. b) Preparation of the hydrogel (4)

Collagen gel aims to inhibit tumor cells with drugs loaded therein by creating a biocompatible environment such that healthy cells are not damaged. A natural polymer was selected that can mimic the extracellular matrix in order not to damage healthy cells.

As a solvent for the hydrogel (4) 8:2 distilled water: acetic acid, 50% w/w collagen: gelatin to be distilled was used.2 grams of gelatin and 2 grams of collagen were used for 15 mL of solvent. In addition, drugs, 2 mg of ibuprofen and 4 mg of adriamycin (1.5 mg of DOX), 0.2 mL of produced GQDs were used. Since adriamycin contains preservatives as well as chemotherapeutic doxorubicin, the amount used was calculated accordingly. The drugs that are used are in very low doses.

In the gel in the design:

• Graphene quantum dots (GQDs) (6): Photosensitizer (7) substance for Photodynamic Therapy

• Doxorubicin (DOX): Chemotherapeutic drug (10)

• Ibuprofen: Anti-inflammatory (9) and analgesic drugs are loaded.

Synthesis of Graphene Quantum Dots (6)

Graphene quantum dots (GQDs) (6) were used as a photodynamic therapy agent within the scope of the present invention. In photodynamic therapy, the tumor area is sensitized to light by using drugs called photosensitizer (7). When a stimulating light is given to this area, the drug is activated and kills tumor cells locally by generating free radicals and reactive oxygen (ROS). Graphene quantum dots (GQDs)(6) are carbon nanomaterials smaller than lOOnm. In recent years, their use has increased in order to solve the problems of conventional methods for cancer treatment. They are very important in the medical field since they are biologically compatible.

Graphene quantum dots (6) offer a highly efficient cancer treatment. Also, it increases singlet oxygen efficiency (photodynamic therapy efficiency), stability, and biocompatibility. In photodynamic therapy, it reduces toxicity and allows healthy cells to be treated without damage in addition to killing tumor cells.

GQDs synthesis was synthesized from carbon black by means of the hydrothermal method. This technique is the production of crystalline inorganic substances from hot aqueous solution at high vapor pressure. Liquid mixed materials are heated in a sealed, stainless-steel autoclave above the boiling point of water and as a result, the pressure in the reaction autoclave is significantly increased. This synergistic effect of high temperature and pressure provides a one-step process for producing crystalline materials. Hydrothermal strategies are very easy and short-term processes.

First, 0,5 g of waste carbon black and 5% of H2O2 by weight were added to the teflon part of the hydrothermal reactor and heated in an oven at 180°C for 4 hours. After the reaction system was cooled, the solution was transferred to the tubes. It was centrifuged at 10,000 rpm for 2 hours. The supernatant was separated and diluted at a rate of 1/100, and dimensional analyzes were conducted with DLS analysis.

Graphene, graphene oxide, thermoporfin can be used as a photosensitizer (7) agent except for graphene quantum dots (6).

Chemotherapeutic drug (10)

Chemotherapeutic drugs (10) are used in order to cure the disease, to stop or kill uncontrolled growing cells, and to prevent the disease from recurring. They can be taken into the body orally, locally or through the blood. It has an effect on normal cells as well as cancer cells. However, these side effects are not permanent. In the present invention, the chemotherapeutic drug (10), which is one of the drugs that is planned to be released from the hydrogel is adriamycin (Doxorubicin). It is also commonly used in the treatment of stage 2, 3, 4 melanoma cancers.

This drug is delivered through local route. Its side effects are expected to be minimal since it will be applied in very low doses. There is 2mg Adriamycin in the prepared gel. Besides Adriamycin, Vinblastine and Dabrafenib may be used at the same dose as another chemotherapeutic drug (10).

Analgesic and anti-inflammatory drug (9)

In an alternative embodiment of the present invention, analgesic and/or anti-inflammatory drug (9) can be added. Preferably, ibuprofen is added as a further drug. Ibuprofen is generally used as an analgesic. It also helps reduce fever and prevent inflammation. Studies have shown successful effects of ibuprofen on cancer. In some cases, it is among the results obtained in studies that it ceases the growth of cancer cells. 2mg of ibuprofen was used in the prepared gel.

Apart from ibuprofen, all of the drugs containing paracetamol (Aferin, Gripin, Calpol, Minoset, Panadol, Excedrin) may be used with the same doses for the antipyretic and analgesic effects thereof.

Characterization Tests:

In these studies, FT-IR, SEM, DSC, and DLS analysis methods were applied. Degradation testing and cell testing for the gel are not yet complete. The aims of the main methods to be used are described below:

For the SEM process, different fibers produced by the electrospinning method (3) are randomly selected. The diameters of the fibers in the dressings that are produced based on the sem image are found on average.

Whether the drugs are loaded on the dressing material and which functional groups the produced material has can be determined with FTIR. Whether the drugs are loaded properly on the dressing of the present invention will be observed with this process.

DLS will determine the size and size distributions of nanoparticles. The analysis of the products obtained will be performed by means of this method.

DSC will determine the thermal properties of the hydrogel (4).

FT-IR

FTIR analyzes are conducted for the determining chemical structure of drug-loaded polymer solution(2), PVA solution (Figure 4), and the hydrogel (4) (Figure 5) produced. Collagen and gelatin used for the hydrogel (4) are of food grade.

DSC analysis for hydrogel (4)

DSC analysis was performed after the FT-IR analysis. It was aimed to observe thermal properties of the collagen hydrogel (4) such as glass transition temperature and melting point. As seen in Figure 6 in DSC results, it has been obtained that the melting point of the collagen hydrogel (4) is 280°C, and the glass transition temperature is between 130-150°C.

SEM analysis for wound dressing (8)

20%, 25%, 30% and 35% w/v of PVA dressing (8) is produced. Structures produced at 25% and 30% w/v ratios are rigid. 35% w/v ratio was found to be ideal for drug loaded PVA solution.

%35 PVA has headless fibers in SEM images (Figure 7-8). In addition, the fibers have a porous structure, which indicates that the wound dressing (8) allows for gas passage. It was observed that the fiber structure of the solution (2) using 35% PVA is more suitable according to SEM images.

DLS analysis for produced GQDs

Graphene quantum dots (6) as a photosensitizer (7) agent were produced by means of the hydrothermal method. Nanoparticles under 100 nm are counted as QDOT. Dimensional analysis of the GQDs was performed after the experiments. QDOTs under 100 nmhave been successfully produced. The first peak seen in Figure 9 demonstrates that the desired dimensions were obtained.

As seen in part d of the description of the present invention, a more effective treatment method for melanoma skin cancer (materials used for different diseases and design can be modified) is aimed by means of using a combination of photodynamic therapy and targeted drug technology (combined methods may vary depending on the drugs and materials used) and combining it on a wound dressing (8). The design of the wound dressing (8) is easy to produce and apply, and also offers a more economical and effective treatment compared to available treatment methods. The innovative property here is to combine different treatment methods on a wound dressing (8), and to produce a wound dressing (8) that can treat diseases capable of being treated topically (experiments were conducted for melanoma skin cancer).

The present invention aims to treat the cancerous area over the skin locally with drugs by means of creating an antibacterial environment. The drugs in the wound dressing (8) and in the gel are released to the treatment area by the degradation of the materials. (A drug delivery system was not used in the experiments; however, therapy can be extended by binding the drugs to a carrier nanoparticle.)

Drugs are sufficient at low doses since the present invention is a topical treatment. The aforementioned drugs in part c, particularly the drugs with high side effects, such as the chemotherapeutic drug (10), reduce the side effects of the treatment since the doses are low.