WO2023129070A2

WO2023129070A2 - Biocompatible and biodegradable hydrogels suitable for use in wound and burn treatment and showing drug delivery system properties

Info

Publication number: WO2023129070A2
Application number: PCT/TR2022/051642
Authority: WO
Inventors: Amitav SANYAL; Rana SANYAL; Ismail ALTINBASAK
Original assignee: Bogazici Universitesi
Priority date: 2021-12-30
Filing date: 2022-12-28
Publication date: 2023-07-06
Also published as: WO2023129070A3

Abstract

The present invention relates to hydrogels for the treatment of wounds and burns, especially wounds and burns with irregular surfaces. These hydrogels are biocompatible and biodegradable and can also work as a drug delivery system and provide controlled drug release. By means of the hydrogels, which can be used as both a wound-burn dressing and a drug delivery system, wound-burn healing is accelerated, and an effective treatment is provided.

Description

BIOCOMPATIBLE AND BIODEGRADABLE HYDROGELS SUITABLE FOR USE IN WOUND AND BURN TREATMENT AND SHOWING DRUG DELIVERY SYSTEM PROPERTIES

Technical Field of the Invention

The present invention relates to hydrogels for the treatment of wounds and bums, especially wounds and burns with irregular surfaces. These hydrogels are biocompatible and biodegradable and can also work as a drug delivery system and provide controlled drug release.

State of the Art

The skin is the largest organ of the human body, which is responsible for the protection, temperature regulation and sensitivity of the organism, and consists of three layers: epidermis, dermis, and hypodermis. The epidermis is the outermost layer of the skin and is mostly consists of cells we call keratinocytes. The dermis is the second layer of the skin, below the epidermal layer. The main cells in the dermis are fibroblasts and make the extracellular supporting matrix tissue of the dermis. The hypodermis is the bottom layer of the skin and consists of cells we call lipocyte.

Wound is defined as the temporary or complete loss of the existing physiological properties of the tissues that forms the skin or mucous membrane due to the deterioration or loss of their integrity due to different reasons such as trauma, surgical intervention, or diseases [1 ], and wounds are divided into acute or chronic according to their etiology. Acute wounds are wounds caused by a temporary agent and heal in an acceptable time. There are few factors that prevent the healing of these wounds. Chronic wounds, on the other hand, are non-healing wounds that are caused by a continuous factor or that heal very slowly and often recur. There are many systemic and local factors that prevent healing [2],

A burn is a type of injury that results in the deterioration of skin integrity due to high heat, extreme cold, electricity, friction, radiation, or chemical substances. Bums may occur due to many environmental factors. It can be caused by too much exposure to the sun, electric shocks, boiling water, fire, or many other external factors. Most bums today are caused by home accidents or the sun. Bums can occur in various types such as thermal bums (flame, steam bums), chemical bums (with the contact of acid or base products), radiation bums (sun rays, solarium, etc.), and electrical bums. Bum symptoms vary according to the seventy of the bum. Peeling, skin discoloration, and pain may occur. The most common complication in bums is infection. Electrical bums cause compartment syndrome (edema in the area where the muscles are gathered) and rhabdomyolysis (sudden and rapid damage to the muscle tissues in the body for any reason) due to damage to the muscles. Depending on the degree of bum, it may cause a decrease in bone density and loss of muscle mass. In large bums, the patient may be affected psychologically and experience post-traumatic stress disorder. Depending on the degree and size of the bum, disturbing scars may remain. Sunburns may cause skin cancer. Bums are observed in different intensities as first, second-, and third-degree bums. Treatment is determined by the degree of the bum. First and second-degree bums heal within 2-3 weeks without leaving a trace with drug treatment. If more than 2/3 of the dermis is affected, scars and loss of function are observed. Therefore, it may require surgical intervention. In third-degree bums, since the dermis and epidermis are completely burned, scars and loss of function occur, and surgical intervention is required.

In case any wound or bum, the healing of the skin is a systemic process involving the stages of, hemostasis (coagulation), inflammation (mononuclear cell infiltration), proliferation (epithelialization, fibroplasia, angiogenesis, and formation of granulation tissue), and maturation (collagen deposition or tissue formation) respectively, at the wound region. Wound dressings have an important place in research and applications made for wound treatment. Wound care products contribute to the healing process by covering the wound area, protecting the damaged tissue from external effects, and activating cell production if its structure is appropriate [3], In the past, conventional wound dressings such as natural and synthetic bandages, hydrophilic cotton, bandages, and gauze with varying absorption capacities were used for wound care. The anticipated primary function of these dressings is to prevent bacteria from multiplying in the wound environment by drying the wound in a way that allows the wound exudate to evaporate [4], Today, it has been observed that a warm and moist environment created around the wound provides faster and more successful wound healing. This understanding is based on the creation of ideal environmental conditions that will allow the movement of epithelial cells during the wound healing process. The ideal conditions for wound treatment are summarized as an environment that is moist enough to allow the regeneration of cells and tissues around the wound, effective oxygen circulation, and low bacterial contamination. In order to achieve these ideal conditions, modem wound dressings are being developed.

There are many local and systemic factors that affect wound and bum healing. Local factors can be listed as infection in the wound area, insufficient blood circulation, hypoxia, tissue necrosis, presence of foreign particles, repetitive traumas, and mobility of the wound area. While many wounds heal without problems, in most of the chronic wounds, with the prolongation of the repair time, problems in healing are observed and these appear as open wounds.

Hydrogels are cross-linked polymer structures that can swell by absorbing a high percentage of water, and these systems release molecules trapped in the network structure into the solution environment in a controlled (slow) manner. Hydrogels are three-dimensional structures and are formed by holding hydrophilic polymer chains together with cross-links, and these hydrogels are used as three-dimensional scaffolds in tissue culture studies. Three-dimensional scaffolds provide structural support to cells, enabling cells to attach and proliferate. These dressings have a high absorption capacity and do not adhere to the wound surface. In addition, they have pain-relieving properties and, by means of moist structure thereof, they create a cooling effect by adjusting the temperature of the wound.

The patent application numbered US2016101176A1 in the state of the art discloses hydrogels obtained with monomers of polyethylene imine and 1 -vinyl imidazole and optionally polyacrylic acid and/or polyvinyl alcohol and N,N-methylene bisacrylamide as crosslinking agent. These hydrogels are used for the delivery of therapeutic agents. When desired, drug release is provided from the hydrogels described here, by electrical stimulation. However, although these hydrogels have drug delivery and controlled release properties, they do not appear to be biodegradable and not biocompatible. The use of such systems, which do not have biodegradability, for wound and bum treatment or drug delivery is very limited.

In another study in the prior art, hydrogels with antibacterial properties, which are formed by combining N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride (HTCC), an antibacterial polymer, and polydextran aldehyde, which has bioadhesive properties, in the wound site are described [5], It is observed that the system described in this study by Hoque et al. does not have the property of biodegradability. This situation limits the application area of said gel.

In another study by Altmba§ak et al., redox sensitive hydrogels are described [6], The hydrogels described in this publication are obtained by reacting a methacrylate backbone polymer with furan molecule and PEG chains in its side branches, and a crosslinking agent containing a maleimide group at both ends and a disulfide group in its structure. Hydrogels prepared in this way gain the property of decomposition in the physiological environment by means of the disulfide group in their structure. It is observed that the hydrogel included here has many desirable properties such as biocompatibility, biodegradability, and drug delivery properties required for drug delivery and use in wound treatment. However, the formation of the hydrogel described here takes a certain amount of time after the components that form the gel have been combined. In other words, the hydrogels formed in this manner are not injectable. Also, the crosslinking agent used here is insoluble in water, therefore, using these crosslinking agents requires the use of toxic organic solvents. Since these toxic solvents will harm the human body, they prevent the use of these hydrogels as injectables. Therefore, it is not possible to use these crosslinking agents for the fabrication of many gels.

It is necessitated to develop a biocompatible and biodegradable wound-burn dressing to be applied in wound and burn treatments, especially in wound and bum treatments with irregular surfaces due to the reasons such as the inadequacy of the wound dressings applied in wound and bum treatments, the inability of the available woundburn dressings to provide an effective healing, not being biocompatible and biodegradable, not eliminating the contact of wound-burn with the external environment sufficiently in the existing wound-burn dressings, and that additional complications and infections may occur, in the state of the art. Brief Description and Objects of the Invention

The present invention discloses biocompatible and biodegradable hydrogels for use in wound and bum treatments, particularly wound and bum treatments with irregular surface. Said hydrogels cover wounds and bums with irregular surfaces and remove their contact with the external environment, or by injecting these hydrogels to a point in the body, instant gelation is provided in the wound area or target area.

An object of the present invention is to provide a biocompatible and biodegradable wound and bum dressing that provides effective healing for use in wound and bum treatments. The hydrogels of the present invention are obtained by combining polymers that can react quickly and gel. In this way, both rapidly forming hydrogels are obtained and the biocompatibility of said hydrogels is ensured by means of the selected maleimide and thiol-containing polymers. Polyethylene glycol homopolymers containing maleimide and thiol ensure both rapid gelation and biocompatibility of the system. Also, these polymers (i.e. polymers containing both maleimide and thiol) are sensitive to both pH and redox, and deteriorates faster than normal healthy tissues, especially in tissues such as tumor tissue. In addition, polymers with different branch number and polymer molecular weight can be used in the present invention, and thus the mechanical properties (hardness, softness, flexibility) of hydrogels can be changed. Thus, hydrogels can withstand the effects of mechanical stress on the applied surfaces without degradation. In addition, as mentioned above, different drug release profiles (rapid drug release, controlled drug release, long-term drug release) can be obtained by changing the properties of the polymer. In addition thereto, said hydrogels also show biodegradable properties since they are degradable in the physiological environment in the body, by means the redox sensitivity provided by the disulfide bonds in the structure of the polymer forming the gel.

Another object of the present invention is to provide a wound and burn dressing that can also be used as a drug delivery system. The hydrogels obtained in the present invention not only remove the contact of the wound with the external environment as a wound dressing, but also provide the delivery of drugs or any other therapeutic agent loaded therein and a controlled release of drugs in the area where it is located. In the present invention, drugs are mixed with polymer solutions prior to gelation. Later, when the polymers are combined with each other and hydrogels are formed, the drugs are physically trapped inside these hydrogels.

In cases where the aforementioned hydrogels are preferred to be used as a drug delivery system, hydrogels are loaded with various therapeutic agents and the hydrogel is placed in the body without the need for invasive methods.

Another object of the present invention is to provide a wound and bum dressing to be applied in wound and burn treatments with irregular surfaces. The hydrogels of the present invention ensure that the wounds with irregular surfaces are covered and their contact with the external environment is removed. Although the hydrogels of the present invention are liquid as of the moment they are injected into the wound, they take the form of a gel very soon after contact with the wound, and this ensures that these gels cover a wound with an irregular surface exactly according to its shape and remove its contact with the external environment.

Another object of the present invention is to obtain hydrogels with adjustable water swelling capacity, flexibility and protein release profile, decomposition rate to be applied in wound and bum treatments. The hydrogels of the present invention can absorb water up to 20 times their own weight and swell at 35-37 °C between pH 5 and pH 9. The swelling capacity, flexibility, degradation rate, and protein release profile of hydrogels depend entirely on the properties of the polymers to be used initially (Polymers with branch numbers between 3 and 64, polymer molecular weight between 1 kDa and 100 kDa, and having a thiol (-SH) group at each end) Hydrogels obtained with polymers with different arm numbers or different lengths will also have different mechanical properties, such as different flexibility, softness, hardness, drug loading capacity (0-20 mg protein/100 mg hydrogel), allowing this hydrogel platform to be modified for different purposes or use in different parts of the body.

The importance of this for controlled drug release is that the drug molecules that are loaded into the hydrogels move from the inside to the outside only by means of the liquid medium in this hydrogel.

Another object of the present invention is to provide hydrogels that can instantly gel in the wound area or target area in applications such as placing the hydrogel at any point in the body. The hydrogels obtained in the present invention are injectable, and after injection, they form a gel in the target area and shows a high level of compatibility with the surface of the target area. Said hydrogels can be injected into a targeted point in the body by means of this property and can instantly gel in the wound area or in the target area. The functional groups, thiols and maleimides on the polymers react very quickly with each other and transform the polymer solutions into hydrogels. Since the initially injected polymer solutions take the form of the wound surface where they are injected, this form is preserved even after gelation occurs.

Description of the Figures

Figure 1 : Graph of release of albumin protein from hydrogels.

Figure 2: The method for preparing the hydrogels of the present invention.

Detailed Description of the Invention

The present invention relates to biocompatible and biodegradable hydrogels for use in wound and burn treatments, especially wound and bum treatments with irregular surface. These hydrogels can be used as wound and bum dressings or as a drug delivery system by loading any therapeutic agent and provide controlled release.

The hydrogels of the present invention are cross-linked polymers formed by covalent bonding of the 3-64-armed (Formula II) or 3-64-branched (Formula III) polymer with - SH group at the end of each arm or branch, and the crosslinking agent that has the chemical structure shown in formula I and that has a maleimide group at both ends.

The hydrogels of the present invention are obtained as a result of the reaction of the 3-64-armed polymer with -SH group at the end of each arm or branch with the crosslinking agent, which has the chemical structure shown in Formula 1 and has a maleimide group at its both ends.

Formula I In formula 1 ; it can be x=0-20, y=1 -20 and n=0-2270. In the preferred embodiment of the present invention; the value of x specified in formula 1 is between 1 and 18, preferably between 2 and 16. The value of x can be, for example, a value between 3 and 15, or a value between 4-13, or 0, or 1 , or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11 , or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20. Examining the structure of formula I, this x value is defined only for the carbon in the middle of a 3- carbon chain. In this case, if the value of x is 0, this chain is 2 carbons, not 3 carbons. In other words, when x is 0, there is a -CH2-CH2 structure between COO- and maleimide. In the preferred embodiment of the present invention; the value of y specified in formula 1 is between 1 and 18, preferably between 2 and 16. The value of y can be, for example, a value between 3 and 15, or a value between 4-13, or 1 , or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11 , or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20 In a preferred embodiment of the present invention, the value of n is between 0-460, preferably between 1-455. In the preferred embodiment of the present invention, the x and y values specified in Formula 1 are 1 and the n value is 45. In the preferred embodiment of the present invention, said polymer is polyethylene glycol bisulfide bismaleimide.

The most important property of the crosslinking agent of the present invention is that it comprises both bisulfite and maleimide groups and is water-soluble. By means of the above-mentioned property, the crosslinking agent of the present invention can both perform the maleimide-thiol reaction, which provides injectability, and a controlled degradation of the hydrogels obtained with said crosslinking agent is provided since it contains bisulfite groups thereon.

3-64-armed polymers that have -SH group at the end of each arm or branch, and that are used to obtain the hydrogel of the present invention are indicated by Formula II, and the 3-64 branched polymers are indicated by Formula III.

Formula III

In Formula II, it can be Z=1 -62, and the polymer used in the thiol molecule can be biocompatible polymers with thiol groups attached to their ends. In a preferred embodiment of the present invention, z=1 -16, preferably 1-10, in particular, preferably 1 -5. Group A in Formula II and Formula III refers to biocompatible polymers. Said biocompatible polymer may be selected from a group consisting of polyethylene glycol, polylactic acid, polyglycolic acid, polycaprolactone, poly(lactic-co-glycolic) acid, poly(N-isopropyl acrylamide), and poly((DL-lactic acid-co-glycolic acid)-g-ethylene glycol. In a preferred embodiment of the present invention, polyethylene glycol is used as group A biocompatible polymer.

The group A biocompatible polymer may be selected from a group consisting of polyethylene glycol, polylactic acid, polyglycolic acid, and polycaprolactone. In a particularly preferred embodiment of the present invention, polyethylene group A with a z-value of 2 in Formula II or III is selected as polyethylene glycol.

In the present invention, biocompatible polymers that have a number of arms or branches between 3-64, preferably between 3-18, preferably 3-12, in particular, preferably 3-7, that have a polymer molecular weight between 1 kDa and 100 kDa, preferably between 2 kDa and 50 kDa, in particular, preferably between 5 kDa and 20 kDa, and that have a thiol (- SH) group at each end thereof, are used as a biocompatible polymer for obtaining hydrogels. In an embodiment of the present invention, Biocompatible polymers with molecular weights of 5 kDa and 10 kDa and 20 kDa can be used as polymers.

In a preferred embodiment of the present invention, a 4-armed polyethylene glycol having a polymer molecular weight of 10 kDa and a thiol (-SH) group at each end thereof is used. The hydrogels, which are the subject of the invention, are degradable in the physiological environment of the body and thereby gaining biodegradable properties by means of the redox sensitivity provided by the disulfide bonds in the structure of the polymers forming the gel.

It can be observed that SH groups can react with both maleimide groups and disulfide groups (-S-S-), while -SH groups found on the polymers used in the present invention in the analyzes carried out in the present invention surprisingly react selectively only with maleimide groups during gelling, and the (-S-S-) groups did not degrade.

The hydrogel obtained in the present invention was loaded with albumin labeled with fluorescein dye, and fluorescein bound serum albumin-bearing hydrogel (F-BSA hydrogel) was kept in buffer solution between pH 6-8 and in 1 .4-dith ioerythritol (DTT) solution separately for 10 minutes. There is no degradation in the hydrogel kept in the buffer solution, which does not contain any thiol (-SH) group. On the other hand, DTT is a molecule containing free thiol (-SH) groups, and When the hydrogel containing fluorescein bound albumin, which is the subject of the present invention, comes into contact with solutions containing DTT, the disulfide structures (-S-S-) present in the gel entered into a redox reaction in the presence of the thiol group (-SH), causing the gel structure to completely degrade in a very short time such as 10 minutes. This degradation is evident from the dispersion of the dye in the solution. As a result of the analysis, It has been proven that fluorescein-labeled albumin is released when the albumin-loaded hydrogel labeled with fluorescein dye is degraded in the presence of - SH molecules, which are also present in the physiological environment of the body, and it has been shown that the hydrogels of the present invention are suitable for use in delivering drugs or any other therapeutic agent (protein, growth factor, etc.).

The hydrogels obtained in the present invention not only remove the contact of the wound with the external environment as a wound dressing, but also provide the delivery of drugs or any other therapeutic agent loaded therein and a controlled release of drugs in the area where it is located. In the present invention, drugs are mixed with polymer solutions prior to gelation. Later, when the polymers are combined with each other and hydrogels are formed, the drugs are physically trapped inside these hydrogels. Figure 1 illustrates the release of albumin protein through hydrogels. Proteins mixed with polymer solutions before gelling are loaded into hydrogels after gelling. Subsequently, these hydrogels are immersed in phosphate-buffered saline (PBS, phosphate buffered saline solution) with or without dithiothreitol (DTT) molecule (a molecule that causes gels to break down) and the release of proteins is observed. In the first twenty hours, while the proteins come out quickly from the hydrogels in the solution containing the DTT molecule (black circle), the protein release from the hydrogels in the solution without DTT molecule (black square) remains at 15%. After 20 hours, a high concentration of DTT molecule was added to the solution that did not contain DTT molecule in order to control protein release, and as can be seen in the figure, a rapid protein release was obtained.

Method for preparing hydrogels to be applied in wound and burn treatments, particularly wound and bum treatments with irregular surfaces, which is described in the present invention comprises the process steps of; i. Dissolving the crosslinking agent indicated by formula I in a buffer solution at pH 6-8, ii. Dissolving the polymer that has 3 to 64 arms, that has a polymer molecular weight between 1 kDa and 100 kDa, and that has thiol (-SH) group at each end thereof in the same volume of buffer solution as used in the process step (i), with the same number of -SH groups as the maleimide groups present in the crosslinking agent, iii. Obtaining the hydrogel by mixing the solution comprising the crosslinking agent and the solution comprising the polymer.

In an embodiment of the present invention, method for preparing hydrogels to be applied in wound and burn treatments, particularly wound and burn treatments with irregular surfaces comprises the process steps of; i. Dissolving the crosslinking agent indicated by formula I in a buffer solution at pH 6-8, ii. Dissolving the polymer that has 4 to 16 arms, that has a polymer molecular weight of 5 kDa, 10 kDa, or 20 kDa, that has thiol (-SH) group at each end thereof in the same volume of buffer solution as used in the process step (i), with the same number of -SH groups as the maleimide groups present in the crosslinking agent, iii. Obtaining the hydrogel by mixing the solution comprising the crosslinking agent and the solution comprising the polymer. In the preferred embodiment of the present invention, method for preparing hydrogel to be applied in wound and bum treatments comprises the process steps of; i. Dissolving 10 mg of crosslinking agent indicated by formula I in 50 mcl of buffer solution, ii. Dissolving 10 mg polyethylene glycol that has a molecular weight of 10 kDa and that has 4 arms with a thiol (-SH) group at each end thereof in 50 mcl of buffer solution, iii. Obtaining the hydrogel by mixing the solution comprising the crosslinking agent and the solution comprising the polymer

In another aspect, the present invention relates to hydrogels according to the present invention for use as wound and burn dressings in wound and burn treatments.

For use as a drug delivery system in wound and burn treatments, antibiotics and therapeutic proteins, anti-inflammatory agents, analgesic agents, therapeutic proteins, and/or antibacterial agents can be loaded into the hydrogels of the present invention, and these drugs are released in a controlled manner where they are delivered.

In the present invention, any buffer solution with a pH value in the range of 6-8, preferably a phosphate buffered saline solution (PBS) with a pH value in the range of 6-8 is used as a buffer solution to dissolve the crosslinking agent or polymer.

A polymeric composition to obtain a hydrogel suitable for use as both a dressing and a drug delivery system in wound and bum applications comprises;

- A crosslinking agent indicated by Formula I according to any one of claims 1 to 8, and optionally Composition 1 containing buffer solution

Formula I - 3-64-armed biocompatible polymer (A) with at least three -SH groups, indicated by Formula II, and/or 3-64 branched biocompatible polymer (A) indicated by formula III, and optionally a composition containing buffer solution

Formula II Formula III wherein z=1-62. In an embodiment of the present invention, z=1-16, preferably 1-10. In the preferred embodiment of the present invention, in polymeric composition, 4- armed polyethylene glycol that has a molecular weight of 10 kDa, and that has a thiol (-SH) group at each end thereof is used.

The present invention also relates to a kit comprising the hydrogel and crosslinking agent of the invention. A kit for use in wound and burn applications comprises;

A first composition comprising a crosslinking agent indicated by formula I

Formula I

- 3-64-armed biocompatible polymer (A) with at least three -SH groups, indicated by Formula II, and/or a second composition comprising 3-64 branched biocompatible polymer (A) indicated by Formula III

Formula III at least one injector.

Here, in Formula II or Formula III, z = 1 to 62.

In the kit according to the present invention, buffer solution may also be present in the first composition and the second composition. That is, said kit may comprise two different powder compositions and injectors, or it may be a kit comprising two different solutions and injectors. The second composition comprising the polymer indicated by formula II or III may be available as a powder suitable for solution by dissolving with a solution, or may be a solution with an optional buffer solution having a pH value of 6- 8. The kit contains one or more injectors that will allow these two compounds to be mixed and applied to the surface. Injectors are separate from each other and can be connected to each other with an extra apparatus, or it can be a double chamber injector. Any injector ensuring that both polymer solutions can be added to its chamber without mixing with each other and that these solutions are mixed with each other at the time of exit from the injector can be included in the kit. However, preferably, an injector with double chamber is used. In case of using powder rather than solution in the kit, a buffer solution with a pH between 6 and 8 may also be included in the kit as a part of the kit.

In the preferred embodiment of the present invention, the kit comprises two vials and an injector comprising 50mg crosslinking agent and 50 mg PEG thiol polymer with 4 arms and molecular weight of 10 kDa. The polymers in the vials are individually dissolved in 250 pl of water. Then, the polymer solutions formed are taken into separate injectors and applied to the desired area with the help of an injector. Before leaving the injector, the solutions are mixed in the injector and injected in this manner.

REFERENCES

1. Degim, Z. “Use of microparticulate systems to accelerate skin wound healing” J. Drug Target. 16(6), 437-448 (2008).

2. Percival, J.N. “Classification of wounds and their management” Surgery, 20, 114-117 (2002).

3. Valenta, C., Auner, B.G. “The use of polymers for dermal and transdermal delivery” Eur. J. Pharm. Biopharm., 58, 279-289 (2004).

4. Zahedi, P., Rezaeian, I., Ranaei-Siadat, S.O., Jafari, S.H., Supaphol, P. “A review on wound dressings with an emphasis on electrospun nanofibrous polymeric bandages” Polym. Adv. Technol., 21 , 77-95 (2010)

5. Hoque, J.; Prakash, R. G.; Paramanandham, K.; Shome, B. R.; Haidar, J. Biocompatible Injectable Hydrogel with Potent Wound Healing and Antibacterial Properties. Mol. Pharmaceutics 2017, 14, 1218.

6. Altinbasak, I.; Sanyal, R.; Sanyal, A. Best of Both Worlds: Diels-Alder Chemistry towards Fabrication of Redox-Responsive Degradable Hydrogels for Protein

Release. RSC Adv. 2016, 6, 74757.

Claims

CLAIMS A water-soluble crosslinking agent for use in the preparation method of a hydrogel for use as both a wound dressing and a drug delivery system in wound and bum applications;

Formula I wherein x=0-20, y=1 -20, and n=0-2270. A crosslinking agent according to Claim 1 , wherein, n=0-460, or n=1 -455. A crosslinking agent according to Claim 2, wherein, x=1 -18, or x=2-16, or x=3- 15, or x=4-13. A crosslinking agent according to Claim 3, wherein, x value is 0, or 1 , or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11 , or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20. A crosslinking agent according to Claim 2, wherein y=1 -18, or y=2-16, or y=3- 15, or y=4-13. A crosslinking agent according to Claim 5, wherein y value is 0, or 1 , or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11 , or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20. A crosslinking agent according to Claim 1 , wherein, x = 1 -16, y = 1-18, and n = 1 -455.

. A crosslinking agent according to Claim 1 , wherein x and y values are 1 , and n is 45. . A polymeric composition to obtain a hydrogel suitable for use as both a dressing and a drug delivery system in wound and bum applications, characterized by comprising;

- A crosslinking agent indicated by Formula I according to any one of claims 1 to 8, and optionally a first composition containing buffer solution

Formula I

- 3-64-armed biocompatible polymer (A) with at least three -SH groups, indicated by Formula II, and/or 3-64 branched biocompatible polymer (A) indicated by formula III, and optionally a second composition containing buffer solution

wherein z=1 -62. 0.A polymeric composition according to Claim 9, wherein z=1 -16.

11. A polymeric composition according to Claim 10, wherein z=1-10.

12. A polymeric composition according to any one of claims 9-11 , characterized in that, said biocompatible polymer is selected from the group consisting of polyethylene glycol, polylactic acid, polyglycolic acid, polycaprolactone, poly(lactic-co-glycolic) acid, poly(N-isopropyl acrylamide), and poly((DL-lactic acid-co-glycolic acid)-g-ethylene glycol.

13. A polymeric composition according to any one of claims 9-12, characterized in that, the buffer solution has a pH value in the range of 6-8.

14. A polymeric composition according to Claim 13, characterized in that, said buffer solution is a phosphate buffered saline solution (PBS) with a pH value in the range of pH 6-8.

15. A hydrogel for use as both a dressing and a drug delivery system in wound and bum applications, characterized by comprising a polymeric composition according to any one of claims 9-14.

16. A kit for use in wound and burn applications, characterized by comprising;

- A first composition comprising a crosslinking agent indicated by Formula I according to any one of claims 1 to 8

Formula I

18

- at least one injector, and wherein in Formula II or Formula III, z = 1-64

17. A kit according to Claim 16, characterized in that, the first composition and the second composition further comprises a buffer solution having a pH value in the range of 6-8.

18. A kit according to Claim 17, characterized in that, said buffer solution is a phosphate buffered saline solution (PBS) with a pH value in the range of pH 6- 8.

19.A polymeric composition according to Claim 17, wherein z=1 -16.

20. A polymeric composition according to Claim 17, wherein z=1 -10.

21. A kit according to Claim 17, characterized in that, said biocompatible polymer is selected from the group consisting of polyethylene glycol, polylactic acid, polyglycolic acid, polycaprolactone, poly(lactic-co-glycolic) acid, poly(N- isopropyl acrylamide), and poly((DL-lactic acid-co-glycolic acid)-g-ethylene glycol.

22. A kit according to Claim 17, characterized by comprising an apparatus for interconnecting separate injectors in case having more than one injector.

23. A kit according to Claim 17, characterized in that, said injector is an injector with double chamber.

19

24. A method for preparing hydrogels for use as both a dressing and a drug delivery system in wound and bum applications, characterized by comprising the process steps of; i. Dissolving the crosslinking agent indicated by formula I in a solvent with a pH value of 6-8;

Formula I wherein x=0 - 20, y=1 -20, and n=0 - 2270, ii. dissolving the polymer that has 2 to 64 arms, that has a polymer molecular weight between 1 kDa and 100 kDa, and that has thiol (- SH) group at each end thereof in the same volume of buffer solution as used in the process step (i), with the same amount as the crosslinking agent, iii. Obtaining the hydrogel by mixing the solution comprising the crosslinking agent and the solution comprising the polymer

25. A method according to Claim 24, characterized by comprising the process steps of; i. dissolving the crosslinking agent indicated by formula I in a buffer solution at pH 6-8, ii. dissolving the polymer that has 4 to 16 arms, that has a polymer molecular weight of 5 kDa, 10 kDa, or 20 kDa, that has thiol (-SH) group at each end thereof in the same volume of buffer solution as used in the process step (i), with the same number of -SH groups as the maleimide groups present in the crosslinking agent,

20 iii. Obtaining the hydrogel by mixing the solution comprising the crosslinking agent and the solution comprising the polymer.

26. A method according to Claim 24, characterized by comprising the process steps of; i. Dissolving 10 mg of crosslinking agent indicated by formula I in 50 mcl of buffer solution,

Formula I wherein x=0 - 20, y=1 -20, and n=0 - 2270, ii. dissolving 10 mg polyethylene glycol that has a molecular weight of 10 kDa and that has 4 arms with a thiol (-SH) group at each end thereof in 50 mcl of buffer solution, iii. Obtaining the hydrogel by mixing the solution comprising the crosslinking agent and the solution comprising the polymer.

27. Hydrogel prepared by a method according to any one of claims 24-26, having functions of both wound and bum dressing, drug delivery system, and controlled drug release system.

28. A hydrogel according to claim 27 for use as a wound and burn dressing in wound and bum treatments.

29. Use of a hydrogel according to claim 27 as a drug delivery system in wound and bum treatments.

21