Evaluation Of the Therapeutic Efficacy of Curcumin and Diclofenac in Wound Healing Using Biodegradable and Non-Biodegradable Polymer Films

The process of wound healing constitutes a biological response aimed at the restoration of damaged tissues following an injury and is characterized by a series of interrelated and sequential stages (1). The healing process can be categorized into four principal phases: hemostasis, which is responsible for the regulation of bleeding through the formation of clots; inflammation, a phase in which immune cells are activated to eliminate debris and avert infection; proliferation, during which new tissue and blood vessels are generated to facilitate the closure of the wound; and tissue remodeling, a stage characterized by the strengthening and maturation of the newly formed tissue (2,3). In order to be deemed effective, a wound healing agent must exhibit a combination of properties. Specifically, it should possess antibacterial capabilities to mitigate the risk of infection, demonstrate anti-inflammatory effects to alleviate swelling and redness, and encompass regenerative attributes that promote accelerated cellular growth and tissue repair (4,5). The integration of these characteristics is essential for optimizing the wound healing process.During the wound healing process, a fluid known as exudate is generated and is present throughout nearly all phases of the healing process. This exudate plays a crucial role in maintaining a moist environment, which is essential for optimal healing. However, when produced in excess, it can hinder the healing process(6). Additionally, the presence of pathogenic microorganisms on the wound surface may lead to serious infections, which are a common cause of delayed wound recovery. If these bacteria multiply uncontrollably, it can result in severe conditions such as sepsis, blood poisoning, ordeath(7). Therefore, the development of non-toxic, biocompatible, biodegradable formulations that possess adequate mechanical strength, can absorb excess exudate without causing the wound to dry out, and can prevent or manage infections, is critical for effective wound care and healing(8).An advance class of wound dressing known as bioactive or smart dressings is made to actively interact with the wound environment and deliver therapeutic agents straight to the woundsite(9). Among the many kinds of wound dressing developed with diverse materials and production techniques,polymeric films have gained a lot of interest because of their superior biocompatibility and low immunological response risk. These films are also non-invasive, user-friendly, allow for gas exchange, and can be made transparent to enable wound monitoring(10). Both natural polymers (such as polysaccharides and proteins) and synthetic polymers (polyvinyl alcohol (PVA), polyacrylic acid (PAA), poly- ε-caprolactone (PCL), polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), and polylactic acid (PLA)) are commonly utilized to formulate biodegradable and non-biodegradable wound dressings (11,12).

CURC, a yellow phenolic compound, is the primary active ingredient found in turmeric, the powdered rhizome of Curcuma longa Linn., a plant belonging to the ginger family and commonly grown in South and Southeast Asia(13). The rhizome, often referred to as the root, is widely used in traditional medicine. CURC makes up about 2-5% of turmeric and is responsible for its bright yellow color due to the presence of curcuminoids(14,15). It exhibits various therapeutic effects, including anti-inflammatory, antioxidant, antimicrobial, and chemopreventive properties, and has been studied for its potential in treating diseases like Alzheimer’s and cystic fibrosis (2, 16). Its anti-inflammatory action involves suppressing the production of key cytokines such as TNF-α and IL-1, which are released by immune cells like monocytes and macrophages (17). CURC also affects the NF-κB signaling pathway, a critical regulator of inflammation, by targeting specific kinases and reducing its activation. Since NF-κB can also be influenced by reactive oxygen species (ROS), CURC’s role in modulating both oxidative stress and inflammation makes it particularly useful in promoting wound healing (18,19).DICL is a nonsteroidal anti-inflammatory drug (NSAID) belonging to the phenylacetic acid group. It has anti-inflammatory, pain-relieving (analgesic), and fever-reducing (antipyretic) properties, similar to other drugs in its class (20,21). Although it is available in oral, dermal, and intramuscular forms, oral use is limited because of its low solubility in acidic conditions, which affects its absorption and may cause side effects (22). Therefore, topical application is often preferred. DICL works by inhibiting the enzymes COX-1 and COX-2, which are responsible for producing prostaglandins like PGE2 and thromboxanes that cause inflammation and pain. It is especially effective in reducing PGE2 levels during inflammation. Although DICL does not directly influence the different stages of wound-healing, its anti-inflammatory and antibacterial effects help reduce pain and prevent infection, indirectly promoting the healing process (23,24).In this research work, non-biodegradable (NBFs) (sodium alginate/HPMC/Carbopol) and biodegradable (BDFs) (Pectin/Allantoin) wound healing films of CURC and DICL were optimized. The encapsulation efficiency and release profiles of CURC and DICL, both individually and in combination were evaluated (25). The films loaded with a natural compound CURC and a synthetic drug DICL exhibited enhanced wound healing in experimental rat models.

1. MATERIALS AND METHODS
   1. MATERIALS

Curcumin (purity >95%) was purchased from Saptamveda Pvt. Ltd., Diclofenac, sodium alginate, HPMC E5, starch, and PEG 400 were purchased from S D Fine Chem. Ltd. (Mumbai, India), and carbopol was purchased from Molychem Pvt. Ltd. (Mumbai, India), pectin was purchased from D I Pvt. Ltd. (Mumbai, India), allantoin was purchased from BRM Chem. (New Delhi, India), glutaraaldehyde, gelatin were purchased from NICE CHEMICALS Pvt. Ltd. (Kerala, India).

2. Film Preparation

1. For Biodegradable Film

Polymers and plasticizer-based surgical film were prepared by the solvent casting method, using the procedure described in our research work. 5% w/v polymeric solution of pectin was prepared and physically crosslinked with glutaraldehyde (2% v/v) in aqueous medium. The solution was mixed under constant stirring for 2 hours at 35ºC. Then, the solution was enriched with allantoin to obtain pectin allantoin hydrogel in a 1:1 ratio relative to the pectin used(26). Finally, the solution was plasticized with glycerol with the pectin used, under continuous stirring for 1 h at 35ºC. At the end, the mixture was poured into a mould and dried at ambient temperature to prepare air-dried films.

2. For Non-biodegradable Film

Polymers and plasticizer-based surgical film were prepared by the solvent casting method, using the procedure described in our research work. Appropriate polymer masses (60%-70% w/v) were added to the aqueous solution of PEG 400 (6%-7%). The mixture was stirred on a magnetic stirrer at room temperature for 1 hour, then heated up to 50ºC, the stirring rate was increased to 100 rpm, and the stirring was continued for 30 minutes under the same conditions, with further application of the ultrasonic bath(27). Then, the mixture was poured into a petri dish (d = 8 cm) and dried for 20 h at a temperature of 40ºC. Finally, obtained crosslinked Alg/HPMC/Carbopol films were air-dried. To prepare films containing CURC (Alg/HPMC/Carbopol-CURC), DICL (Alg/HPMC/Carbopol-DICL), and a mixture of CURC and DICL (Alg/HPMC/Carbopol-CURC+DICL), by adding 1.5% w/v CURC (in ethanol); DICL (in water) solutions to the polymer mixture after stirring for 30 min, followed by an additional 30 min of stirring at room temperature.

3. Film Characterization

1. UV- Spectroscopy and Standard Plot

A calibration curve for CURC and DICL was created by measuring their absorbance at 429 nm and 276 nm, respectively, for solutions ranging from 2 µg/ml to 8 µg/ml. The solutions were prepared by diluting a stock solution with a hydroalcoholic mixture and analyzed using UV-Spectroscopy (PERKIN-ELMER LAMBDA 25). The resulting calibration curve was plotted by taking absorbance against concentration, demonstrating the relationship between absorbance and concentration for both compounds.

2. FTIR Studies

The FTIR spectra of pure drugs and physical mixtures were compared to check any interaction between drugs and selected polymers, over a spectral region from 4000 to 4000 cm^-1, using a PERKIN-ELMER FTIR spectrophotometer.

3. Swelling studies

The swelling behavior of the films was assessed using a buffer solution with a pH of 6.8. Each film sample was submerged in 15 mL of the buffer, and at specific time intervals, it was taken out, lightly blotted to remove surface moisture, and then weighed. This process was repeated until no further increase in weight was observed, signifying that the films had reached equilibrium swelling(28). The degree of swelling was computed using the formula:

Swelling Index=W-W0W0  ×100

W is the weight at time t, and W₀ is the initial weight. An increase in weight was observed after 1 min, 10 min, and 20 min.

4. Folding Endurance

Folding endurance was measured by continuously folding the film, and the number of times it could be folded without breaking was recorded as its folding endurance. This method offered a quantitative evaluation of the film’s ability to withstand repeated folding stress. Higher folding endurance values indicate better durability and mechanical strength of the film (29).

5. Moisture Content

To determine the moisture content of a film sample, a specific portion of the film was cut based on its thickness and density. The initial weight of this sample was measured using an analytical balance and recorded as W₁. The sample was then placed in a hot air oven maintained at a consistent temperature, typically between 105ºC and 110ºC, and dried for a set period, usually 24 hours or until a constant weight was achieved, indicating complete moisture removal. After drying, the sample was immediately transferred to a desiccators to cool in a moisture-free environment, preventing it from reabsorbing moisture from the air. Once cooled, the sample was reweighed, and the final weight was recorded as W₂(30,31). The moisture content of the film was then calculated using the formula:

Moisture Content %=W1-W2W1 ×100

W₁: Initial weight of the film, W₂: Final weight of the film.

6. Entrapment Efficiency

The percentage of drug entrapped in the CURC and DICL-loaded wound healing film was determined using UV-visible spectroscopy. The process begins by extracting the unentrapped (free) drug from the film (32).

The procedure was typically carried out by dissolving the film in phosphate buffer of pH 6.8, and the solution was filtered to remove any undissolved particles. The resulting clear supernatant, containing the free drug, was then analyzed using a UV-Vis spectrophotometer. The absorbance of this solution was measured at the drug’s maximum wavelength (λmax), specifically at 425 nm and 272 nm, respectively(33). The concentration of the free drug was calculated using a previously established calibration curve (Absorbance vs. Concentration). Once the concentration was determined, the total amount of free drug was obtained by multiplying it by the volume of the solution. Finally, the entrapment efficiency was calculated using the appropriate formula:

EE%=(Total drug-Free drug)(Total drug) ×100

7. Stability Studies

Stability studies for selected formulations were conducted at ambient temperature and stability chamber for 1 month. The films were evaluated after 5, 10, 15, 20, and 30 days, up to approximately one month. The films were evaluated for the following parameters (34,35).

• Physical appearance

The physical appearance and surface texture of the film were assessed through visual inspection. There was no change in the appearance and texture of the films.

• Uniformity of drug content

This parameter was determined by dissolving a film measuring 1×1 cm in 100 ml of phosphate buffer at pH 6.8 for 30 minutes while continuously shaking. From this, 10 ml was diluted to 50 ml using pH 6.8 buffer solution. The absorbance was then measured and analyzed to determine the drug content.

• Surface pH of films

The surface pH was determined by using a digital pH meter. The film was slightly wet due to the use of water. The pH was measured by bringing the combined glass electrode into contact with the surface of the film.

• Thickness of the film

The thickness of the film was measured using a screw gauge with a least count of 0.01 mm at different spots of the film. The thickness was measured at three different spots of the film, and the average was taken.

• Folding endurance

Folding endurance was determined by repeated folding of the whole film at the sample place till the film breaks. The number of times the film was folded without breaking was computed as the folding endurance value.

• Moisture uptake

The moisture uptake of the films was determined by exposing them to an environment of 40ºC with 75% relative humidity for 1 week. The uptake of moisture by the films was calculated with the percent increase in weight.

• Swelling index

The film was weighed and immersed in 50 ml of pH 6.8 phosphate buffer. At specific time intervals, the film was removed, gently blotted with absorbent tissue to remove excess surface moisture, and reweighed. This process continued until a constant weight was recorded, indicating equilibrium swelling. The degree of swelling was calculated using the formula:

Swelling Index=W-W0W0  ×100

8. In Vitro Release Study Of The Drug-Loaded Films

1. Diffusion Cell Method

Thein vitro release of CURC from the Alg/HPMC/Carbopol-CURC film, as well as DICL from the Alg/HPMC/Carbopol-DICL film, was assessed using a Franz diffusion cell (model 2351- 6C, KSHITIJ INNOVATIONS, HARYANA, INDIA). A cellulose membrane was placed between the donor and receptor compartments to act as a barrier, with the receptor compartment containing phosphate buffer (pH 6.8) to facilitate the analysis of drug permeation(36). Meanwhile, a 2×2 cm film and approximately 5 ml of buffer solution were added to the donor compartment for drug release testing.The amount of drugs released in vitro was estimated using a UV-Vis spectrophotometer at 429 nm and 272 nm for CURC and DICL, respectively.

2. Release kinetic models for the best fit of release

To find out the patterns and best fit for the release of drugs from the films, various mathematical models were applied. These models for data fitting were the zero-order release model, the first-order release model, the Higuchi model, and the Korsmeyer-Peppas model. The best fit was decided based on the highest correlation factor(37).

4. In Vivo Study

The animal research studies were approved by the Institutional Animal Ethics Committee and conducted according to the guidelines of the Committee for Control and Supervision of Experiments on Animals. The use of the prepared films with incorporated CURC and DICL, both individually and in combination, was investigated for in vivo healing of incision-caused wounds (38). For an in vivo study, male Wistar albino rats (6 to 8 weeks old, average body weight 200-250 g) were used. This study involved a total of four groups of animals for NBFs and two groups for BDFs.

For NBFs, the incision was caused for group one (n=6)and was not further treated, the second group (n=6) was treated with the Alg/HPMC/Carbopol+DICL films, the third group (n=6) with Alg/HPMC/Carbopol+CURC films, and the fourth group (n=6) with the Alg/HPMC/Carbopol+DICL+CURC films. For BDFs, the incision was caused in group one (n=6) and was not treated, the second group(n=6) was treated with theAlg/HPMC/Carbopol+CURC+DICL films. The process of causing incisions to rats was performed following the protocol in a previously published study. Before causing incision, the animals were anesthetized in the gluteal region of the right limb with intramuscular (10 mg/kg) or intraperitoneal xylazine (5 mg/kg body weight). Then, the back of the healthy rat was shaved using a razor. On the shaved skin area, the surgical incisions of (2×2 cm) in diameter were made in the dorsal area(39). Wounds caused this way were covered with the prepared NBF and BDF films. The healing process was monitored for two weeks, with the daily replacement of film samples.

1. 3. RESULTS AND DISCUSSION
1. UV-Spectroscopy and Standard Plot

The value of λmax of drugs was determined in phosphate buffer, pH 7.4, and 0.1N HCL by scanning the sample solution in the range 200nm to 800nm at 1cm path length using a UV/ visible spectrophotometer (PERKIN-ELMER LAMBDA 25). CURC and DICL showed maximum absorption at 429nm and 276nm, respectively. The standard plot in phosphate buffer pH 7.4 and ethanol (1:1) of CURC and 0.1N HCL of DICL was prepared.

2. FTIR Studies

FTIR spectroscopy was used to identify functional groups and assess chemical interaction, providing insight into the composition and stability of the films. Spectra of CURC, DICL, and film components were recorded using a PERKIN-ELMER FTIR spectrophotometer over a range of 4000-450 cm^-1 to detect any interaction between drugs and excipients. The FTIR spectra of CURC,DICL,Sodiumalginate,HPMC, Carbopol, Mixture 1(Alg/HPMC/carbopol/CURC+DICL), Allantoin, Pectin, Mixture 2 (pectin/allantoin/CURC+DICL) as shown in Figure 3.

Figure No. 3 FTIR Spectrum of CURC, DICL, Sodium Alginate, HPMC, Carbopol, Mixture 1, Allantoin, Pectin, Mixture 2

The specific peaks in the FTIR spectrum of DICL were observed at: 3322 cm^-1 for N-H stretching of the secondary amine, 1576 cm^-1 for C=O stretching of the carboxyl ion, 1505 cm^-1 for C=C ring stretching, 750 cm^-1 for C-Cl stretching, 1688 cm^-1 for the C=C bond in the benzene ring, 2835.73 cm^-1 for the COOH carboxylic acid peak. Similarly, the characteristic peaks of SA, HPMC, and Carbopol, Pectin, Allantoin, and the FTIR spectra of the mixture of pure drugs and the components of the films, as shown in the figure, exhibited all the characterization  peaks of the individual components. There was no shifting of peaks; hence, no interaction was observed in the spectra.

3. Swelling Studies

The swelling index of the prepared films was evaluated in phosphate buffer (pH 6.8). As shown in Table 1, a slight increase in volume was observed after 1 minute, with a swelling index of 24.24%. However, a consistent and significant increase in swelling was recorded at 10 and 20 minutes, reaching a maximum swelling index of 81.69%.This result was observed for the optimized formulation, indicating a high capacity for fluid uptake. The substantial swelling behavior suggests that the film matrix can effectively facilitate the release of the active ingredients, thereby contributing to an improved therapeutic outcome(28).

Swelling Index=W-W0W0  ×100

W is the weight at time t, and W₀ is the initial weight. An increase in weight was observed after 1 min, 10 min, and 20 min.

Table 1. Swelling data of the film

4. Folding Endurance

The wound healing film was further evaluated by its folding resistance, dry, and wet. When dried, the film was brittle and broke after being folded twice, indicating low flexibility and mechanical strength. However, it was observed that in the wet state, the film withstood more than 50 folds without breaking, demonstrating enhanced flexibility and mechanical strength. This proves that themechanical behavior of the film strongly depends on moisture content. While moisture improves flexibility, the dry state may offer better resistance to bacterial growth, enhancing the film’s microbiological stability.

Enhanced Flexibility: The presence of moisture makes the film much more flexible and durable, allowing it to withstand multiple folds without breaking. This suggests that the material composition benefits from hydration, which likely helps in maintaining its integrity. At the same time, it is indicative of film being resorbable.

Improve Adaptability: It helps ensure that the film stays intact and works properly over time, even when applied to areas of the body that move a lot, like joints. This makes the film more durable and effective during everyday use, providing continuous protection and support for the wound.

5. Moisture Content

The moisture content of the wound healing film was determined to evaluate its ability to retain water, which can influence the film’s flexibility, texture, and overall performance. The optimized formulation showed average moisture content of 14.63%. This level of moisture was considered adequate to maintain the film’s flexibility and structural integrity, which are essential for its effective application in wound healing.

6. Entrapment Efficiency (EE)

The EE of the drug-loaded films was determined to evaluate the percentage of drug successfully incorporated into the polymeric matrix. The results revealed that the optimized formulations exhibited a high EE of 98.91% for CURC and 99.81% for DICL. These high EE values indicate that almost the entire amount of drug added during the formulation process was effectively entrapped within the films. This suggests minimal drug loss during preparation and confirms the efficiency of the film-forming method used. The high EE was also expected to support sustained and localized drug release at the site of application.

7. Stability Studies

Stability studies for selected formulations were conducted at ambient temperature and stability chamber for 1 month. The evaluation of the films was conducted after 5, 10, 15, 20, and 30 days, and continued for approximately one month. The films were evaluated for the following parameters in the given table:

Table 3. In the Stability Chamber

8. In Vitro Release Study of the Drug-Loaded Films

1. Diffusion Cell Method

The in vitro cumulative drug release profile of CURC and DICL was studied over 12 hours to evaluatetheir individual release characteristics and comparativebehavior. After 12 hours, the cumulative percentage release of CURC and DICL from the optimized formulation was found to be 60.14% and 80.11%, respectively.Thedrug release profile indicates that CURC exhibited a gradual and sustained release, starting at approximately 5% within 2 hours and progressively increasing to about 60% by 12 hours. This controlled release is likely due to CURC’s limited solubility and hydrophobic nature. In contrast, DICL exhibited a more rapid and effective release, reaching 15% at 2 hours and attaining 80% by 10 hours, after which the release became stable. The enhanced release of DICL may be attributed to its better solubility and greater compatibility with both the film matrix and the receptor medium.

As observed in the cumulative release profiles (Fig 2), DICL consistently exhibited a faster and higher release than CURC throughout the study duration, with final release values of 80.11% and 60.14% respectively, at 12 hours, indicating that both drugs maintain their distinct release characteristics when incorporated within the same carrier system.

2. Release kinetic models for best fit of release

The results of mathematical models for data fitting of drug release from films were calculated by applying zero-order, first-order, Higuchi, and Korsmeyer-Peppas models. The highest value of the regression coefficient (R²-0.9660) was observed for zero-order, and hence, the best fit for the release profile of CURC-DICL film was explained in terms of the zero-order release models. The highest value was found for CURC and DICL while the zero-order model was applied.

The Higuchi model, which describes a diffusion-controlled release mechanism, showed a moderate correlation for CURC (R² = 0.8840), suggesting that diffusion may play a role in its release. However, DICL exhibited a lower correlation (R² = 0.7638), indicating that diffusion is not the dominant release mechanism for DICL.

The Korsmeyer-Peppas model, often used to identify the involvement of multiple release mechanisms such as diffusion and polymer erosion, showed a better fit for DICL (R² = 0.8686), suggesting that a combination of mechanisms may govern its release. CURC showed a lower correlation (R² = 0.7440), indicating that this model is less applicable to its release profile.Overall, the zero-order model best explains the release behavior of both drugs, especially DICL, indicating a controlled, concentration-independent release pattern(40).

8. In Vivo Study

The effectiveness of the test formulation was assessed using a full-thickness incision wound model in rats.The experimental groups received bioactive drug films (BDFs and NBFs) loaded with CURC (15mg/kg), DICL (15mg/kg), or a combination of CURC+DICL. On the other hand, the control group received no drug treatment. The primary parameters assessed were the rate of wound contraction and the duration required for complete healing. These evaluations helped determine the biological response elicited by each treatment and its potential efficacy as wound-healing agents.

Wound Contraction Rate

Following the injury, wound contraction was observed on days 0, 2, 4, 6, 8, 10, 12, and 14. Compared to the control group, the treatment groups showed a noticeably higher rate of wound contraction. By day 10, the treatment groups achieved approximately 80% wound closure, while the control group reached only 55%. This marked difference indicates that the drug-loaded films substantially accelerated tissue regeneration.

Time to Complete Healing

Complete epithelialization was observed in the treated groups by day 14, indicating full skin regeneration. In contrast, the control group required more than 14 days for complete wound closure. These results suggest that the film formulations significantly reduced the healing duration and enhanced the overall wound repair process. Representative healing images of the wound on different days were given in Tables 5, 6.