Formulation Of Plant Extract Loaded Electrospun Bandages for Wound Healing Applications

Biodegradable electrospun bandages are emerging as a revolutionary approach to wound healing, offering an advanced alternative to traditional wound dressings. These bandages are fabricated using electrospinning, a technique that produces nano- and microfibrous scaffolds, mimicking the extracellular matrix (ECM) of natural skin. The high surface-area-to-volume ratio of electrospun fibers enhances cell adhesion, proliferation, and migration, accelerating tissue regeneration. A key advantage of biodegradable electrospun bandages is their ability to degrade naturally, eliminating the need for frequent dressing changes and minimizing patient discomfort. Common biodegradable polymers used include polycaprolactone (PCL), polylactic acid (PLA), gelatin, chitosan, and sodium alginate, which offer biocompatibility, antibacterial properties, and moisture retention—essential for an optimal wound-healing environment. Some formulations also incorporate growth factors, antimicrobial agents, or nanoparticles to further promote healing and prevent infections. These bandages are particularly beneficial for chronic wounds, diabetic ulcers, and burn injuries, where prolonged healing is a concern. Electrospun fibers loaded with antibacterial agents (e.g., silver nanoparticles) or bioactive molecules enhance wound repair by reducing infection risks. Additionally, drug-loaded electrospun fibers can provide controlled drug release, ensuring sustained therapeutic effects at the wound site. The future of biodegradable electrospun bandages lies in smart wound dressings, incorporating stimuli-responsive materials that change properties based on environmental factors like pH, temperature, or bacterial presence. With continued advancements, electrospun biodegradable bandages hold significant potential to transform wound management, reduce healing time, and improve patient outcomes in clinical settings.

MATERIALS AND METHODOLOGY:

Millipore water was collected from Ramnarain Ruia College, Sodium alginate was purchased from HIMedia Laboratories, Chitosan was purchased from HIMedia laboratories, Egg was purchased from local market and the albumin was separated out from it. Gelatin was procured from HIMedia laboratories. Centella Asiatica were purchased from plant nursery. The 4% w/v acetic acid was added in millipore water and then chitosan was added for dissolution of chitosan flakes by application of mild heating. After the chitosan gets mixed completely, the Ph was adjusted to 7.5 and 2% w/v egg albumin was added into the chitosan and mixed well, then 3%w/v sodium alginate was added in it and kept it for stirring overnight. After the mixture formulation got homogenised, addition of gelatin was done for clarification process. Once the formulation was prepared freeze-thaw cycles were given for further polymerization process and after that the gel was taken out and kept for electrospinning and the bandage was formed which was used for further characterization.

Electrospinning:

Centella asiatica were collected from local shop. The leaves were dried in aspetic condition and the moisture was allowed to get evaporated. The remained dried leaves were put it into Isopropyl alcohol (absolute) and it was kept in shaker at a speed of 40rpm for 14 h. After that the extract was filtered out with a cheesecloth and kept it in a glass container.

Preparation of plant loaded electrospun fibers:

Electrospinning is a straightforward yet highly effective method for fabricating ultrafine fibers. The preparation process involves several critical steps:

 Polymer Solution Preparation

Selection of biodegradable polymers like Chitosan, egg albumin, sodium alginate and gelatin. And addition of Centella asiatica plant extract with a concentration of 10mg/ml. Dissolve the polymer in an appropriate solvent (e.g., millipore water) to create a homogenous solution with an optimized viscosity and conductivity. Stir the solution at a suitable temperature to ensure uniform polymer dispersion.

 Electrospinning Setup

Load the polymer solution into a syringe fitted with a blunt needle or metallic spinneret. Connect the syringe to a syringe pump, which regulates the flow rate of the solution. Position a high-voltage power supply, typically in the range of 5–30 kV, to create an electric field. Set up a grounded collector (flat plate or rotating drum) to collect the electrospun fibers(3).

Electrospinning Process

Apply a high voltage to induce electrostatic repulsion within the polymer droplet at the needle tip. The polymer solution forms a Taylor cone and ejects a charged jet when the electrostatic forces overcome surface tension. As the polymer jet travels toward the collector, rapid solvent evaporation leads to solid fiber formation. The fiber deposition pattern depends on parameters like voltage, flow rate, humidity, and collector type(4).

Characterizations

Scanning electron microscopy:

Scanning Electron Microscopy (SEM) is a potent imaging method for examining the composition and morphology of surfaces at the nanoscale. In order to produce detailed images with high resolution and depth of field, a concentrated beam of high-energy electrons is scanned over a material, producing secondary and backscattered electrons. SEM is frequently used in biology, material science, and nanotechnology to examine topographical and structural characteristics and it is mainly used to assess the electrospun materials . To improve imaging quality, the sample is prepared by coating it with a conductive substance, such as carbon or gold. Energy-dispersive X-ray spectroscopy (EDS) and other advanced SEM techniques enable elemental composition measurement, which makes SEM indispensable for both industrial and research applications(5).

Spreadability test:

Spreadability refers to how effortlessly a cream or gel distributes when applied to the skin or wound surface. It is determined by measuring the time required for a gel to spread without resistance between two glass slides under a specified force. In the Spreadability test, a 2 cm radius is marked on the bottom of a petri plate, and 200 ?L of gel is placed at the center. Another petri plate is positioned on top in an inverted manner. A predetermined weight is applied for 5 minutes, allowing the gel to disperse. After removing the weight, the spread diameter is measured to evaluate spreadability(6).

Hemolysis test:

Hemolysis refers to the breakdown of red blood cells (RBCs). To assess hemolysis, the samples were rinsed with 0.9% saline solution before being immersed in 10 mL of saline inside a centrifuge tube and incubated at 37°C for 30 minutes. The samples were then introduced into the tube and further incubated at 37°C for one hour. After incubation, centrifugation was performed to separate the components. The supernatant was carefully collected, and its absorbance was measured at 541 nm using UV-visible spectroscopy to quantify hemoglobin release(7).

The Hemolysis rate was calculated by the given formula,

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FT-IR:

Fourier Transform Infrared (FTIR) spectroscopy was utilized to analyze the physical and chemical composition of the formulated gel. This technique employs infrared spectroscopy and is advantageous as it requires minimal sample preparation for both liquid and solid states. The collected raw data is processed mathematically to generate a spectral output, which facilitates the identification of molecular bonds and chemical components present in the sample. The infrared absorption frequency spans from 600 to 4000 cm??1;, allowing for the detailed examination of functional groups. The spectral data is then analyzed to characterize the molecular interactions within the gel formulation(8).

Tensile stress:

A tensile stress tester is a mechanical device used to measure the tensile strength, elasticity, and deformation of materials under applied force. It operates by applying controlled tension to a sample until it breaks, recording key parameters such as ultimate tensile strength (UTS), elongation, and Young’s modulus. Commonly used in material science, biomedical research, and engineering, it evaluates the mechanical properties of polymers, metals, textiles, and biomaterials like wound dressings and scaffolds. Advanced models incorporate digital load cells and real-time data analysis for precise measurement, ensuring quality control and product reliability in research and industrial applications(9).

CAM assay:

We bought fertilized eggs from the Hatchery. The eggs were carefully delivered, cleaned, and incubated for three days at 37°C in an egg incubator. The assay was run on day four. To prevent contamination, the egg shell was cleaned with alcohol, and a syringe was used to remove any extra albumen. The CAM area window was left open for operation, and the upper section of the shell was fractured. The window was sealed using adhesive tape once the work was completed. Every two days, the infected eggs were monitored to assess the toxicity on the embryonic survival rate(10).

Histological studies:

Histological analysis is conducted to examine the microstructure of cells and tissues, aiding in the identification of anatomical details, functional properties, and pathological alterations. It plays a crucial role in disease diagnosis and medical research by providing insights into cellular changes. The hematoxylin and eosin (H&E) staining technique is widely used to highlight cellular components—nuclei appear blue, while the cytoplasm is stained pink. This contrast enhances the visualization of tissue abnormalities, aids in evaluating disease progression, and provides a deeper understanding of tissue organization in both healthy and diseased conditions(11).

In-vitro studies:

The MTT assay is a colorimetric method used to assess cell viability and proliferation during wound healing. It measures metabolic activity, where viable cells reduce MTT (yellow) to formazan (purple). This assay helps evaluate cell growth, toxicity, and the effectiveness of treatments promoting wound repair by quantifying cellular regeneration over time(12).

RESULTS:

Scanning electron microscopy:

       
           
       
Fig. 5

By performing scanning electron microscopy we can clearly state that there is a mesh structure formed in the electrospun fiber which helps in cell proliferation and also can help to absorb and hold the moisture which will eventually help it for wound health and faster recovery.

Spreadability Test:

Hemolysis Test:

Tensile tester (Load displacement):

       
           
       
Fig.8

CAM assay:

Histological studies:

Histological studies of CAM area was done. Hematoxylin and eosin staining was carried out to examine the nucleus availability and cell structure. In comparison to topical cream sample, the control sample has less amount of nucleus present which was confirmed by hematoxylin staining and the there was also less staining of eosin seen as compared to topical cream sample. It concludes that the prepared topical cream is biocompatible and supports angiogenesis

FT-IR analysis:

MTT assay: