Burns are injuries of our skin caused by heat, chemicals, electricity or radiation. They present significant dangers and complications, ranging from immediate physical damage to long-term health issues.

Standard treatments are quite expensive and include wound cleaning, debridement to remove dead tissue, and the application of topical antibiotics and dressings. The risk of infection remains high, necessitating frequent monitoring and dressing changes, which can be painful and distressing for the patient. The healing process can be slow, and there is a significant risk of scarring, which may require further surgical intervention. Some patients may even experience allergic reactions or sensitivities to certain topical agents or dressings, complicating treatment further. Emerging treatments, such as skin substitutes and growth factor therapies, show promise, but are often expensive and not widely accessible [7].
The depth and percentage of burned area of the body is the guide for the treatment plan. There are very different approaches to treat a burn, so each therapy plan has to be personalized for the patients specific injuries.

Resilin, a natural protein with outstanding mechanical properties found for example in dragonflies, is the foundation of our reSkin hydrogel. We want to crosslink our resilin with hyaluronic acid, which is an important component for skin hydration and has anti-inflammatory properties [9]. This combination forms a unique dressing that has extraordinary mechanical properties withstanding physical stress. It is the perfect basis for a new wound dressing, as it is a customizable drug-delivery system. The hydrogel will protect the burned skin, keep it moist and absorb wound fluids which furthermore prevents an infection.
The hydrogel can be loaded with active ingredients. reSkin contains three of them: the pigment astaxanthin, vitamin E and the enzyme bromelain.

The integration of active ingredients provides additional support through enzymatic and autolytic debridement, thus increasing wound healing. The drugs diffuse from the gel into the wound and take their effects, which can aid in the overall recovery and prevention of complications.

Compound	Description	Effect on burn wound healing
Astaxanthin	A carotenoid pigment with potent antioxidant properties, found in algae and seafood.	- Reduces oxidative stress and inflammation, helps to prevent wound deepening - Promotes tissue repair by supporting fibroblast activity and collagen production
Vitamin E	A strong antioxidant commonly in foods like nuts and seeds - often used in skincare products.	- Reduces scar formation by promoting collagen synthesis - Neutralizes free radicals to protect cells in the wound bed - Moisturizes skin and improves its elasticity
Bromelain	A proteolytic enzyme derived from pineapple stems.	- Removes necrotic tissue selectively through enzymatic debridement, allowing healthy tissue to regenerate - Decreases inflammation and swelling - Reduces scarring, because the eschar is broken down - Stimulates blood circulation by breaking down fibrin, which helps to differentiate between healthy and dead tissue and is therefore a tool to assess the burn degree

Our reSkin hydrogel combines different treatment methods as well as the depth assessment in one step.

1. First 48 hours: During this time, the burn wound can deepen due to excess thermal energy caused by the burn. reSkin keeps the wound moist and cool, and the incorporated astaxanthin helps to contain the wound from deepening. It limits the need for surgery and therefore also the risk of complication [2].
2. Depth Assessment: Stimulating blood circulation in the burned skin with bromelain helps to assess the burn depth and therefore the diagnosis of burn degree. While 2a burns, which do not need surgery, will turn a rosy colour when treated with reSkin, the skin cells of 2b burns will not change colour as they are already dead. The colour change of the healthy skin is also promoted by astaxanthin and can therefore be used on all skin tones and types. Using reSkin, all 2a and 2b burns can be more easily differentiated in the whole wound area, and further treatment can be planned quicker und more precisely [5][7].
3. Debridement: reSkin combines enzymatic and autolytic debridement and therefore reduces the need for surgical interventions. Bromelain cleanses the wound by enzymatic degradation of necrotic tissue. The hydrogel promotes autolytic debridement by being moisture-retentive. Our hydrogel base with hyaluronic acid can absorb large amounts of water, keeping the wound moist so the body’s natural enzymes can help with the wound cleansing. It is important to keep as much healthy tissue as possible, so the skin can heal faster on its own. Surgical or mechanical debridements often injure healthy tissue, which leads to more stress in the wound healing process. If cut too deep, the skin may require an initially unnecessary graft, raising the risks for complications [9].
4. Dressing: reSkin is put directly on the wound and serves as a dressing. It keeps the burn cool and moist while absorbing wound excudates. In combination with our active ingredients, reSkin helps to prevent infections of the wound and promotes wound healing [6].

In comparison to existing treatments, reSkin hydrogel represents a superior option for burn patients. Its advantages can be enumerated as follows:

Pros	Explanation
Saving Lives	By preventing infections through wound containment and reducing the need for surgery, reSkin can significantly lower complications and risks associated with burn wounds. Because it is cheaper than other treatments, can be used for all skin tones, and is easily applied and removed, reSkin is usable in every clinic, including low-income countries, helping where it is most needed.
Depth Assessment	reSkin helps to differentiate between 2a and 2b burns across all skin types, using astaxanthin for better visual assessment and bromelain to evaluate tissue viability. The whole wound area is inspected and is not limited by depth.
Surgery prevention	By combining enzymatic and autolytic debridement, the need for surgical intervention is reduced, protecting healthy tissue and limiting complications from grafting. The removal of eschar reduces scarring and therefore also prevents surgeries later on.
Pain reduction	Minimized amounts of dressing changes reduce patient discomfort and stress by combining multiple treatment methods (wound containment, depth assessment, debridement, regeneration, wound-fluid absorption, infection prevention) in one step. The non-invasive enzymatic debridement with a lower bromelain concentration leads to a less painful wound cleaning.
Enhanced healing	Promotes faster wound healing by supporting collagen synthesis, reducing oxidative damage, and preserving healthy tissue, leading to reduced scarring and less complications.
Flexibility & Strength	The resilin-based hydrogel offers superior mechanical properties, making it suitable for various body areas, including those requiring flexibility and durability like the muscoskeletal system.
Customizable	reSkin can be customized to include additional drugs tailored to individual patient needs, expanding its versatility for different burn types and conditions.
Accessibility	Our hydrogel is cost-effective, easy to apply without a specially trained nurse, and suitable for all skin types, making it widely accessible for clinics worldwide not contigent on social or economic status.
Environmentally friendly	It is sustainable and biodegradable, reduces waste by combining treatment steps into one, reducing the environmental impact.

Our reSkin hydrogels have the potential to transform the treatment and assessment of burn wounds worldwide, offering a simpler and more effective solution.

Our aim was to create super-long resilin filaments of 32 to 64 repeats to enable crosslinking. Since repetitive sequences of this extend cannot be synthesized, we had to develop a new method - Repeatigo. This enables us to generate repeats of the same gene sequence.

We used Repeatigo to clone 32 repeats of resilin into the pET28c(+) vector. Six oligos were designed and ordered:

Complementary oligos are annealed separately and then ligated. To prevent false binding, the annealed start and middle oligos are first ligated, and then the annealed end oligos are added. The ligase can chain them together properly because of their 5'-phoshorylation. To get the desired fragment length of 1.2 to 3 kilobases, it is necessary to run an agarose gel and cut and clean up the specific product sizes.
A polymerase-chain-reaction amplifies the cleaned ligation, which needs to be again checked using an agarose gel. After a double digest of the resilin repeat ligation product and our pET28c(+) vector with NdeI and SacI, the restricted products can be ligated and transformed.

Our pET28c(+)_Resilin_Re32 needs to be treated with care. Most E. coli strains have naturally occurring recombinases like recA, which tend to delete repeating sequences. We decided to use the E. coli strain JM109, which is recA-deficient and, therefore, less likely to destroy our plasmid. pET28c(+) has a lac operon, so resilin production can be easily started by adding IPTG [19].

Since our resilin is expressed with a C-terminal His-tag comprising six histidines, we purify it by affinity chromatography using a magnetic beads kit with nickel ions bound to nitrilotriacetic acid [20].
When adequately purified and detected via SDS-PAGE, the resilin polymer can be used for crosslinking with hyaluronic acid. The lysate is then added to an equilibrated magnetic beads solution, incubated, and washed according to the kit protocol. The final elution holds the purified resilin, which can be detected by running an SDS-PAGE. Depending on the number of repeats, the molecular weight will vary.

Some specific strains of E. coli are predestined to use for the synthesis of hyaluronic acid, as they already have an incomplete pathway for the synthesis. By using metabolically engineered E. coli, we can synthesize hyaluronic acid naturally without depending on chemical synthesis or the extraction from animals. The E. coli strain Top10 already owns most of the genes for this synthesis. By transforming the HasA gene, which codes for hyaluronic acid synthetase, we can complete the pathway and synthesize hyaluronic acid in the bacteria [25].

To determine how well the HasA expression in our E. coli is working, we added the sequence for enhanced green fluorescent protein (eGFP): If the expression is successful, UV light will induce fluorescence [26].
To do this, we first insert the sequence for eGFP and a linker behind the HasA gene in our pUC_HasA plasmid. For this, both vector and eGFP have to be linearized by conducting a PCR. Afterwards, they can be merged by a SLiCE homologous recombination [27]. Our plasmid is now ready to express the synthetase marked by eGFP and is transformed in the competent Top10 E. coli. The cells can be multiplied by incubation (overnight culture) at 37 degrees Celsius, the optimum temperature for our bacteria [28].

The expression of hyaluronic acid synthetase can then be induced by adding arabinose, as our vector has an arabinose promotor.
All the steps have to be continuously checked by running gel electrophoreses. For a successful insertion of eGFP into the pUC_HasA plasmid, we expect to see bands at 5982 base pairs. The expression of the synthetase can be shown by testing fluorescence, but also by running an SDS-PAGE.

To extract hyaluronic acid (HA), the sample must be prepared in advance. For this, a fermentation broth sample that contains HA-producing cells is diluted with an equal volume of 0.1% w/v SDS and incubated for 10 minutes at room temperature. The SDS helps to dissolve the cell membrane and releases the HA. To precipitate the HA and to purify it, 1.5 to 2 volumes of ethanol are added to the sample and incubated at 4 degrees Celsius for 1 hour. After the incubation, the sample is centrifuged at 3000 rpm for 20 minutes at room temperature. HA will form a pellet at the bottom of the tube, and the supernatant will be carefully discarded. The HA pellet must be resuspended with the same volume of 0.1 M NaCl used for the sample. The pellet is incubated in NaCl for 10 minutes to fully dissolve the HA [25].

A modified carbazole assay is used to measure hyaluronic acid: In this assay, one of the critical components, glucuronic acid, is measured to quantify HA. For this, 0.25 ml of our resuspended sample are mixed with 1.5 ml chilled sulphuric acid reagent containing sodium borate decahydrate. This will hydrolyze the HA into glucuronic acid and N-acetylglucosamine. The mixture is placed in a boiling water bath for 20 minutes to break down the HA into measurable components. After 20 minutes the sample has to be cooled immedeatly, as this prevents further reactions and stabilizes the glucoronic acid.
Next, 50 µl of carbazole reagent are added to the cooled sample. Carbazole reacts with glucuronic acid to form a purple-colored complex. Placed back in boiling water for 15 minutes, the sample will develop the purple complex with an enhanced color. Afterwards, it needs cooling again to stop the reaction. Finally, the absorbance can be measured using a spectrophotometer at the appropriate wavelength (530 nm). The HA titer can be calculated as 2.05 times the glucuronic acid concentration. A standard curve of known glucuronic acid concentration can be used to calculate the amount of glucuronic acid in the sample [25].
Since our hyaluronic acid needs to be modified to form crosslinks, we have to add a functional group. We chose methacrylic anhydride (MA) for that purpose. Our HA is incubated for 24 hours at 5 degrees Celsius in a pH 8 solution with an excess of MA to produce MA-HA [29].

Gif 7: Crosslinking of resilin polymer and hyaluronic acid via crosslinking.

We chose riboflavin phosphate (RFP) as our photo-initiator, an electron-donor that produces reactive oxygen species (ROS) and thus starts the crosslinking reaction [31]. According to recent studies, riboflavin as photo-initiator is not cytotoxic, which makes it ideal for our hydrogel [32]. RFP has similar properties, but it is better soluble in water and has been approved by the US-American Food and Drug Administration (FDA) for crosslinking of several substances.
RFP has its absorption maxima at 265 nm (UV light) and 445 nm (blue light) and is photochemically active in the pH-range of 4 to 10 [33]. The degree of crosslinking depends on the amount of photo-initiator as well as on the amount of functional groups in our two components that can form the according bonds [32]. Also, it is important to have oxic conditions for the formation of dityrosine bonds in our hydrogel (photo-crosslinking reaction type II) [33].

We chose phosphate-buffered saline (PBS) as a buffer, because, like RFP, it produces no cytotoxicity and is already a proven component in crosslinking reactions [33]. PBS matches the osmolarity and ion concentrations of the human body and keeps the pH level at 7.4.

For the irradiation, we chose two lamps with wavelengths of 450 nm and 365 nm. Since the reaction is started by blue light in the visible range as well as UV light, we wanted to observe the effect of both to determine which one works best for our purposes. The distance between the light source and the crosslinking tray should not be too long, as differing amounts of photo-initiator and components influence the light penetration of our solution and, therefore, the result [34].

We dissolve MA-HA (2.5% w/v) and RE (20 % w/v) in PBS and mix them together in differing ratios. Then, we dissolve RFP in PBS as well and add it to our solutions according to the RFP concentration to be tested.
Mixing ratios for MA-HA/RE assays:

1. 1 : 1
2. 1 : 1.3
3. 1 : 3.2

Concentration for RFP:
0.01 % (w/v) (if it fails 0.005 % (w/v) or 0.1 % (w/v))
After mixing the components, they have to be used immediately to guarantee maximum reactivity. Since visible light will start the reaction, exposure to light and oxygen can otherwise negatively affect the experiment. Depending on the results, concentrations and ratios need to be adjusted.

1. 1 minute (minimum)
2. 15 minutes
3. 30 minutes (maximum)

The irradiation times should be adapted according to the results. The degree of gelation can be tested simply by turning the tray a little and checking whether liquid accumulates on one end [34].

Fig. 6: reSkin components astaxanthin, vitamin E and bromelain. [12]

One of the components of our hydrogel is astaxanthin, a powerful antioxidant with properties that promote wound healing. It can reduce inflammation and promote the regeneration of skin cells. Furthermore, it supports the repair of damaged tissue and helps to improve skin elasticity. It can also minimize the deepening and spread of the burn in the first 48 hours [35].
Astaxanthin is a pigment belonging to the xanthophyll group. It is a carotenoid, reddish in color, giving our hydrogel a light pink shade. As an antioxidant, it reduces oxidative stress on the wound [36].
Since astaxanthin is a lipophilic substance, we aim to enhance solubility by utilizing a polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer. This is a polymeric solubilizer with an amphiphilic chemical structure specifically developed for solid solutions of active pharmaceutical ingredients. Due to its bifunctional character, it can serve as a matrix polymer for solid solutions and dissolve poorly soluble drugs in aqueous media [37].

The second component we chose is vitamin E. Vitamin E is another powerful antioxidant that protects the skin from harmful free radicals. Free radicals are present in higher concentrations in burn wounds and increase the risk of infection, so neutralizing them is an essential aspect of an efficient treatment. Vitamin E also stabilizes astaxanthin and can promote collagen formation, which further supports the skin structure and accelerates the healing of wounds [38]. Since vitamin E is also a lipophilic substance, we want to use a polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer.

Bromelain is the third pharmaceutical agent to be incorporated into the reSkin hydrogel. It is a cysteine protease derived from pineapple (Ananas comosus). The proteolytic activity of this substance contributes to the regulation of cell proliferation, the growth of healthy cells, and the degradation of debrided wounds [39]. This process can facilitate the gentle cleansing of the wound by removing dead skin cells, thereby reducing the risk of inflammation, particularly in cases of grade 2 to 3 burns [40],[41]. Additionally, bromelain functions as an antioxidant capable of scavenging free radicals such as ROS. These are produced by oxidative stress on cells and are increased in burn wounds. By neutralizing these radicals, bromelain protects the wound from further tissue damage caused by oxidative damage [42].
Furthermore, bromelain acts as an immunosuppressor, inhibiting the nuclear factor-kappa B pathway, for example. This pathway is a key regulator of the inflammatory response. Therefore, bromelain has the potential to attenuate the subsequent inflammatory cascade [43]. Bromelain has applications beyond its use as a therapeutic agent, as it can also be employed in diagnostic procedures. Given these characteristics, bromelain is utilized to ascertain the necessity of a skin transplant. Four hours after application, the necessity for surgery will become apparent, given that it promotes blood circulation. If the skin assumes a rosy color, it can be concluded that no surgical intervention is required since the tissue in question should recover. However, if the skin remains white, the patient requires surgical intervention and a transplant [44]. The implementation of this test allows for the avoidance of unnecessary or belated surgical procedures, thereby ensuring the optimal treatment of burn wound patients. It is recommended that the application period for open burn wounds be limited to four hours, with a daily dose of up to a maximum of 12 mg. However, using too-high concentrations of bromelain can be counterproductive [45]. This underscores the importance of ensuring that the concentration range used for wound application is appropriate.
Bromelain can be isolated from pineapples. The parts of the iGEM team Tongji China from 2022 with the project Bplant can be utilized for the synthetic expression of bromelain. Bromelain must then be expressed in E. coli, whereby purification using an His-tag via a Ni-column is possible.
In order to prevent the degradation of the primary component of our hydrogel, resilin, it is necessary to inactivate the protease prior to applying the hydrogel to the wound. Three different methods may be employed to achieve the inactivation of bromelain. Firstly, bromelain can be inhibited by a peptide inhibitor, bromein, which is also derived from pineapples [46]. Moreover, the enzymatic activity of bromelain can be diminished by reducing the temperature. Bromelain is a thermostable enzyme and exhibits the highest efficacy at 50 degrees Celsius. A temperature of 25 degrees Celsius has been demonstrated to reduce its efficiency by 40 percent. It can be posited that storing the hydrogel prior to its application to burn wounds would prevent the degradation of resilin [47].
An alternative approach would be to reduce the effectiveness by utilizing EDTA, a chelating agent. EDTA can be linked to an amino acid of resilin using click chemistry. EDTA can then be used to remove metal ions that are essential for the catalytic function of bromelain, thereby reducing its activity by 88.5 percent [48]. Following the application of the hydrogel to the wound, bromelain diffuses into the wound, resulting in a reduction of the concentration and overall effectiveness of bromelain within the hydrogel. Bromelain has been approved by the US Food and Drug Administration and is recognized as a safe and effective therapeutic agent that is already used in medical applications worldwide [49].

In order to determine which concentrations and irradiation times work best to form our hydrogel, it is necessary to conduct a number of tests to evaluate the properties of the crosslinking products. Each test helps to optimize our hydrogel and ensure it is safe for use by examining both physical and biological properties.

Gif. 8: Application and effects of reSkin component.