Formulation of Zinc-Based Nanomaterials using the Eucommia ulmoides Bark Extract and their Wound Healing Potential

Our research focuses on uncovering the wound healing potential of bioengineered metal nanoparticles. We aim to understand how these nanoparticle influence the healing process and their mechanism of action to address the key questions about their therapeutic application in tissue repair. We have successfully developed various nano-based wound dressings using natural polymers, demonstrating their potential for diverse wound healing application, and advancing innovative approaches in tissue repair.

This research will facilitate the identification of potential mechanisms in in vivo models and contribute to developing wound healing materials that possess biocompatible properties, enhancing their effectiveness and safety for use in various medical applications. To begin, chop the bark collected from the Eucommia ulmoides tree into small pieces using scissors. Wash the chopped bark materials twice with double-distilled water.

Then, dry the bark pieces at 37 degrees Celsius for 24 hours in shaded conditions. Transfer 20 grams of shade-dried bark into a conical flask containing 220 milliliters of sterile, double-distilled water. Then, heat the flask at 130 degrees Celsius for 20 minutes.

Note that the solution's color changes to light yellow after 10 minutes. Afterward, store the crude extract containing polyisoprene at four degrees Celsius for further use. The formation of a thread-like structure indicates the presence of polyisoprene in the extracts.

Add one molar zinc nitrate dihydrate to 50 milliliters of deionized water in a 500 milliliter conical flask. Stir the solution continuously at 60 RPM using a magnetic stirrer until the zinc nitrate dihydrate dissolves completely. Then, add 15 milliliters of Eucommia ulmoides bark extract dropwise to 20 milliliters of one molar zinc nitrate dihydrate solution.

Place the covered reaction mixture onto a magnetic stirrer. Switch it on and set it to spin for three hours. Next, add one normal sodium hydroxide solution dropwise to the reaction mixture to adjust the pH to nine.

Add sodium hydroxide until the solution turns milky white, keeping the pH below nine, indicating the formation of zinc oxide nanoparticles. Then, transfer the synthesized Eucommia ulmoides zinc oxide nanoparticles to a 50 milliliter centrifuge tube and centrifuge at 100 G for five minutes at four degrees Celsius. Collect the washed Eucommia ulmoides zinc oxide nanoparticles on a glass plate and dry them at 45 to 50 degrees Celsius for one hour in a hot air oven.

To begin, seed 1 times 10 to the power of 4 human umbilical vein endothelial cells in each well of a 96-well plate. Place the plate in an incubator set to 5%carbon dioxide and 37 degrees Celsius. For cytotoxicity assessment, add 10 microliters of various concentrations of Eucommia ulmoides zinc oxide nanoparticles to the 90%confluent cells.

Incubate the plate for 24 hours at 37 degrees Celsius in a 5%carbon dioxide incubator. After incubation, carefully remove the old medium without disturbing the cells. Add 10 microliters of CCK-8 solution to each well containing 90 microliters of fresh DMEM medium and incubate the plate as shown earlier.

Then, measure the absorbance of the cells treated with CCK-8 solution at 450 nanometers using a spectrophotometer. Seed 1 times 10 to the power of 5 human umbilical vein endothelial cells in 12-well culture plates containing DMEM, supplemented with 10%FBS and 1%penicillin streptomycin. Place the plates in an incubator set at 5%carbon dioxide and 37 degrees Celsius.

Gently make a scratch across the cell monolayer using a sterile 200 microliter pipette tip to create a representative wound with a width of 200 micrometers. After removing the complete medium from the wells, wash the monolayer gently using one milliliter of PBS to remove detached cells. Now, add Eucommia ulmoides zinc oxide nanoparticle solutions of 0, 10, and 20 micrograms per milliliter, combined with complete medium containing 10%FBS, to the wells.

Incubate the plates at 37 degrees Celsius in a 5%carbon dioxide incubator. Acquire micro-photographs of the scratch wound at zero hours and 24 hours using an inverted microscope. Calculate the percentage of the wound closure using the formula shown here.

Eucommia ulmoides zinc oxide nanoparticles showed no cytotoxicity on human umbilical vein endothelial cells at concentrations up to 50 micrograms per milliliter after 24 hours. Wound closure was significantly enhanced in cells treated with 20 micrograms per milliliter Eucommia ulmoides zinc oxide nanoparticles, showing 81.5%closure after 24 hours compared to 16%in the control group. Microscopic images showed reduced wound gap widths in the 20 micrograms per milliliter treatment group compared to control after 24 hours.