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

Summary

Here, we present a protocol to synthesize the zinc oxide (ZnO) nanoparticles using the polyisoprene-rich aqueous extract obtained from Eucommia ulmoides tree bark. The wound-healing potential exhibited by the synthesized ZnO nanoparticles on human umbilical vein endothelial cells (HUVECs) was evaluated using scratch assay, a simple, cost-effective, and efficient method.

Abstract

The aqueous extract from the bark of Eucommia ulmoides serves as a rich source of bioactive compounds with numerous health benefits. The protocol here aims to explore the preparation of zinc oxide (ZnO) nanoparticles using the Eucommia ulmoides bark-mediated polyisoprene-rich aqueous extract. Meanwhile, the proposed protocol is associated with the preparation of wound healing material by easing the process. In addition, the wound-healing potential of the synthesized nanoparticles (Eu-ZnO-NPs) was evaluated using a simple scratch assay on a human umbilical vein endothelial cell (HUVEC) monolayer. After 24 h of treatment with Eu-ZnO-NPs, the cell proliferation and migration of HUVEC cells were assessed. At the end of the study, cell proliferation and migration were observed in scratched monolayer treated with different concentrations of Eu-ZnO-NPs, whereas poor cell migration and proliferation rates were observed in control cells. Of the chosen concentrations, 20 µg/mL Eu-ZnO nanomaterials showed better cell migration and enhanced wound healing potential.

Introduction

Medicinal plants and plant-derived compounds have been shown to exhibit numerous health benefits¹. The World Health Organization (WHO) reported that 80% of the global population depends upon traditional medicinal plants for primary health care. China is well-recognized and popular for its Traditional Chinese Medicine (TCM) practices. Chinese medicinal herbs have been reported to treat various diseases and utilized for their biological potential. Medicinal plants serve as reservoirs for bioactive compounds and multiple therapeutic roles. Medicinal plants have also been utilized to treat the wounds. There are several types of approaches applied to treat chronic wounds². A recent investigation revealed that medicinal plants were involved in the wound healing process by providing favorable conditions for healing, free from infections, and fastening tissue regeneration³. Meanwhile, the antibacterial and antifungal properties of bioactive compounds present in medicinal plants can help to cure wounds and fasten wound healing efficiency⁴.

Metal-based nanomaterials are gaining attention due to their biocompatible and biodegradable properties. Eucommia ulmoides, commonly called the Chinese rubber tree, is a native species of China. The leaves and bark of the tree are used in medicinal practices. Most importantly, the plant species was cultivated in the central and western provinces of China⁵. Peng et al.⁶reported that leaves, barks, and staminate flowers were edible with therapeutic potential. In addition, E. ulmoides serve as the best source of lignans, phenylpropanoids, iridoids, flavonoids, amino acids, and trace elements. Furthermore, the bark has been utilized for various biomedical applications such as controlling blood pressure, lowering fat, and promoting antiosteroporosis and hypoglycemic activity⁷. Hence, undoubtedly, it has been proven that E. ulmoides bark extract has a long history in traditional Chinese medicine. Earlier reports suggested that natural polymer polyisoprene is rich in the barks of Eucommia ulmoides⁸. Based on the information above, the present work aims to fabricate nanomaterials using Eucommia ulmoides bark extracts. The combination of zinc with bark extract is an attractive choice for preparing nanomaterials. Overall, the ultimate aim of the present investigation was to fabricate a novel hybrid nanomaterial for wound healing applications.

Protocol

NOTE: Before preparing the extract, the obtained bark material was washed twice using deionized water and dried in a shaded place. The shade-dried barks were stored in an airtight container.

1. Preparation of Eucommia ulmoides bark extract

1. Chop the bark collected from the Eucommia ulmoides tree into small pieces using scissors.
2. Wash the chopped bark materials twice with double distilled water.
3. Dry the bark pieces under 37 °C for 24 h at shaded conditions.
   1. Adjust the duration of the drying process based on the quantity of bark utilized for the study. Ensure the barks are completely dried before chopping the barks into small pieces. Avoid direct sunlight.
4. Transfer 20 g of shade-dried barks into a conical flask containing 220 mL of sterile double distilled water and heat at 130 °C for 20 min.
   1. The color of the reaction solution changes to light yellow. The changes in color of the solution occur after 10 min. Allow the solution to heat for another 10 min. Adjust the volume of double distilled water based on the sample quantity.
5. Store the crude extract containing polyisoprene at 4 °C for further use. The formation of a thread-like structure indicates the presence of polyisoprene in the extracts.

2. Biosynthesis of E. ulmoides bark-mediated ZnO nanoparticles

1. Add 1 M Zinc nitrate dihydrate Zn (NO₃)₂ to 50 mL of deionized water in a 500 mL conical flask. Stir continuously using magnetic stirring (60 RPM). The Zn (NO₃)₂ takes 30 min to dissolve completely.
2. Add 15 mL of E. ulmoides bark extract dropwise to 20 mL of 1 M Zinc nitrate dihydrate (Zn (NO₃)₂) solution.
3. Place the covered reaction mixture onto a magnetic stirrer, switch on the stirrer, and spin at (60 RPM) for 3 h.
4. Add 1 N sodium hydroxide NaOH (3 mL) solution dropwise to the reaction mixture to adjust the pH to 9. Add NaOH until the solution mixture turns milky white and the pH is not more than 9. The solution becomes a milky white color when ZnO nanoparticles are formed.
   1. The preparation of 1 M of Zinc nitrate dihydrate Zn (NO₃)₂ solution volume may vary based on the experimental needs. Make sure to use the freshly prepared bark extracts. If the pH exceeds 10, it will result in nanoparticle aggregation.
5. Transfer the synthesized Eu-ZnO-NPs to a 50 mL centrifuge tube and centrifuge it at 100 x g for 5 min at 4 °C.
   NOTE: The impurities can be avoided by immediate washing.
6. Collect the washed Eu-ZnO-NPs in a glass plate and dry them at 45-50 °C for 1 h in a hot air oven.
   NOTE: If the Eu-ZnO-NPs are kept for more than 30 min can affect the physicochemical nature of the nanoparticles

3. Size confirmation using TEM

1. Prepare 1 mg/mL of nanoparticles in DDH₂O and load 5 µL of Eu-ZnO-NP sample in the copper grid and wait until it completely dries. Vortex the sample prior to loading on the copper grid.
2. Load the copper grid containing Eu-ZnO-NPs on the TEM sample holder and acquire images at 50x and 100x magnifications.
   NOTE: Grids must be picked up using tweezers during this step.

4. Cytotoxicity assessment

1. Seed 1 × 10⁴ HUVECs in each well of a 96 well plate and place it in a 5% CO₂ 37 °C incubator.
2. Add 10 µL of various concentrations 0, 10, 20, 30, 40, and 50 µg/mL of Eu-ZnO-NPs into the 90% confluent cells and incubate it for 24 h.
3. After incubation, remove the old medium without disturbing the cells, add 10 µL of CCK-8 solution to each well containing 90 µL of fresh DMEM medium, and incubate in a 5% CO₂, 37 °C incubator.
4. Measure the absorbance of cells treated with CCK8 solution at 450 nm using a spectrophotometer.
   NOTE: The absorbance was measured immediately within 15 min to avoid the changes in absorbance.

5. Preparation of HUVEC cells for scratch assay

1. Seed an appropriate (1 × 10⁵) quantity of HUVECs in 12 well culture plates containing Dulbecco's modified Eagle medium with 10% fetal bovine serum (FBS) and 1% pen-strep and incubate it in a 5% CO₂ 37 °C incubator.
   1. Before performing the scratch assay, check the confluency using the inverted microscope.
      NOTE: For performing the scratch assay 6 well plates or 12 plates will be utilized based on the requirements, and the cell density will vary for different culture plates. The usage of 70%-80% confluent cells is ideal and recommended for scratch assay. The volume of DMEM medium may vary based on the culture plates utilized in the study. For instance, 6 well plates require 1-1.5 mL of culture medium, and 12 well plates require 0.5-1.0 mL of growth medium.
2. Gently make a scratch using a sterile 200 µL pipette tip in the representative wound with a wound width of 200 µm.
3. Ensure that the tip used to make scratches on the cell monolayer makes contact with the surface of the cells.
   NOTE: Every time a scratch is made, use the sterile tip.
4. Remove the complete medium and wash the HUVEC monolayer using 1 mL of 1x PBS to remove the detached cells.
   NOTE: Ensure the detached monolayer cells are completely removed from the respective wells. Confirm that no damage has occurred in the wound-created area.
5. To evaluate the wound healing potential of synthesized Eu-ZnO-NPs, add 0 (Control), 10, and 20 µg/mL concentrations of Eu-ZnO nanoparticles combined with a complete medium with 10% FBS into the wells. Maintain the experimental plates at 37 °C in a 5% CO₂ incubator.
   NOTE: Do not disturb the cell monolayer while adding the fresh, complete medium. Ensure that the monolayer cells remain undisturbed during the incubation.
6. Acquire microphotograph at 0 h and 24 h using an inverted microscope. Use the annotation tool to measure the wound closure in different time intervals.
   1. Calculate the percentage of wound closure using the following formula: Wound closure (%) = [(cell migration (in µm) at 0 h - cell migration (in µm) at 24 h)/ cell migration (in µm) at 0 h] x 100.

Results

The present investigation aims to synthesize nanoparticles using the barks of Eucommia ulmoides tree. The bark material was dried completely in a shaded environment (Figure 1). The bark materials were utilized to prepare the hot water aqueous crude extract by heating the samples at 130 °C for 20 min. A slight alteration in the temperature and duration may disrupt the phytocompounds and make them unsuitable for preparing the aqueous extract. The aqueous crude bark extracts of E. ulmoides were prepared and utilized for the nanoparticle synthesis (Figure 2). The prepared extract can be utilized immediately. The extract stored for a prolonged period may influence the synthesis process. Hence, a freshly prepared extract is recommended for the preparation of nanoparticles.

The successful synthesis of crude bark extract-mediated ZnO NPs was confirmed by the appearance of a milky white color and a change in the pH (Figure 3). The pH is closely linked with the size of the nanoparticles, and a pH exceeding 9 results in agglomeration and uneven size. The structure of synthesized nanomaterial was confirmed using TEM analysis, and the synthesized nanoparticles displayed a particle size of 80-90 nm, as represented in Figure 4. The NaOH should be added dropwise to prevent a sudden change in the reaction mixture. The metal-based nanoformulations are suitable, inexpensive, and least toxic. However, metal-based nanomaterials are well-known for their biological functions.

The cytotoxicity of Eu-ZnO-NPs against HUVECs cells was determined by CCK-8 assay with the exposure of different concentrations of Eu-ZnO-NPs for 24 h. The results indicated that Eu-ZnO-NPs displayed no noticeable changes in cytotoxicity to HUVECs cells at the higher concentration of 50 µg/mL (Figure 5). The appropriate quantity of cells was loaded, and a uniform size of wounds was made using a sterile 200 µL pipette tip. The cell volume exceeding the limit may cause a change in pH and a slower proliferation rate. The migrations of cells and wound closure were observed in the treatment group after 24 h of treatment, whereas no similar changes were observed in the control. The results of the wound healing activity revealed that the synthesized Eu-ZnO-NPs showed wound healing properties on HUVECs cell lines. A concentration of 20 µg/mL of Eu-ZnO nanomaterials promotes faster cell migration and enhanced wound healing properties. Most importantly, the synthesized Eu-ZnO nanoparticles displayed a concentration-dependent mode of action. Furthermore, after 24 h of treatment, cell proliferation and migration were observed in scratched monolayer and, whereas poor cell migration and proliferation rate were observed in control cells (Figure 6). The percentage of wound closure was observed as 16% for control, followed by 51% for 10 µg/mL and 81.5% for 20 µg/mL treated groups after 24 h (Figure 7).

Figure 1: Collection of Eucommia ulmoides bark pieces. Picture of Eucommia ulmoides bark used for preparing the aqueous hot water extract. Before extraction, the barks should be washed thoroughly to remove impurities and completely shade-dried. Please click here to view a larger version of this figure.

Figure 2: Preparation of Eucommia ulmoides bark extract: Picture of Eucommia ulmoides bark extracts utilized for synthesizing the nanoparticles. The Eucommia ulmoides bark extract of 200 mL was prepared using 20 g of bark pieces. Please click here to view a larger version of this figure.

Figure 3: Synthesis of Eucommia ulmoides bark-mediated ZnO nanoparticles. The image shows the formation of Eu-ZnO nanoparticles and the solution with a milky white color represents the formation of Eu-ZnO nanoparticles. Please click here to view a larger version of this figure.

Figure 4: Transmission electron microscopy analysis: The image shows different magnifications of Eucommia ulmoides bark-mediated ZnO nanoparticles (A) 100 nm magnification and (B) 50 nm magnification. Please click here to view a larger version of this figure.

Figure 5: CCK-8 cell viability assay. The bar graphs represent the viability of cells after being treated with different concentrations of 0-50 µg/mL, whereas the cells without treatment serve as the negative control. The data represents mean and standard deviation. Please click here to view a larger version of this figure.

Figure 6: Scratch assay for wound closure assessment. The cells were treated with synthesized EU-ZnO NPs (10 µg/mL and 20 µg/mL) and without EU-ZnO NPs (control group) and incubated for 24 h. Cell proliferation and migration were observed in the cells treated with EU-ZnO NPs. Please click here to view a larger version of this figure.

Figure 7: Wound closure percentage. The bar graphs represent the wound closure percentage on the HUVEC monolayer with and without EU-ZnO NP treatment at 0 h and 24 h. The data represents mean and standard deviation. Please click here to view a larger version of this figure.

Discussion

The E. ulmoides bark, seed, and leaves are believed to exhibit numerous health benefits. Our results have shown that the synthesis of EU-ZnO nanoparticles was achieved using a simple and cost-effective approach. The aqueous extract was utilized to synthesize the nanoparticles. Heating the bark material at high temperatures may cause degradation of some phytoconstituents and lower the extraction efficiency. The phytoconstituents present in the aqueous herbal extract can reduce the metal ions to form nanoparticles. Zinc nitrate dihydrate is utilized for the preparation of ZnO nanoparticles for their biocompatible and highly stable properties⁹. Using minimum to high toxicity causing metal elements for synthesizing or designing the nanomaterials is risky and cannot be used for biomedical claims. This synthesis can be performed within a day (24 h). However, toxic-free metals and biocompatible materials are considered to be safe and utilized to evaluate wound healing efficiency. The main reason behind the agglomeration of synthesized nanoparticles is the phytochemical bonds. The agglomeration of nanoparticles was due to the attractive nature of the Van der Waal interactions and attractive forces between the surface^10,11. The agglomeration of nanoparticles can influence the physical and optical properties. The size and shape of the nanoparticles can be modified using calcination and annealing¹². The resuspension of nanomaterials in deionized water and sonication can help to avoid the agglomeration of nanoparticles. The presence of bioactive compounds or functional groups in the aqueous extract are likely to involve in the reduction process. The nanoparticle yield solely depends upon the volume of the reaction mixture used for the synthesis process. The synthesizing of nanoparticles on a large scale has several limitations, including agglomeration and non-uniform particle size.

Similar to our findings, Aydin Acar et al.¹³ reported that Calendula officinalis flower extract-mediated ZnO nanoparticles exhibited wound healing properties on L929 cells with 69.1% wound closure. Furthermore, Crotalaria verrucosa leaf-mediated ZnO nanoparticles displayed wound healing potential at 5 µg/mL concentration on HeLA cell lines with 40.45% wound closure¹⁴. Zhang et al.¹⁵reported that the ZnO nanoparticles synthesized using Euphorbia fischeriana showed significant wound-healing properties at 10 and 15 µg/mL concentrations. These studies have proven that the synthesized nanoformulation could be used as a wound-healing agent. The research outcomes of these studies showed that the synthesized compounds can repair the wound and help in re-epithelialization in treatment groups.

Hemostasis and cell migration are considered important processes in healing wounds. The scratch created on the monolayer of HUVECs was made using pipette tips to mimic artificial wounds. Further, creating wounds with the same widths is quite tricky and complicated. On the other hand, this is a less expensive method. In this assay, it was observed that the designed nanoparticles showed excellent wound-healing properties on the HUVECs at 10-20 µg/mL concentrations compared to the control. Moreover, the synthesized Eu-ZnO-NPs have the potency to repair the wound created on the cell monolayer without causing any toxic effects. These in vitro experiments could be used to identify the effectiveness of synthesized polymer-based nanomaterials using animal models.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
12 well plate	NEST	703011	Used for cell culture and assay
Centrifuge	SCILOGEX	SC1406	Used to separate the nanoparticles from the colloidal mixture
Centrifuge Tube – 15 mL	BIOFIL	CFT-312150	To centrifuge the synthesized solution
Centrifuge tube – 5 mL	Biosharp	BS-50-CM-S	To store the nanoparticles
CO2 incubator	Thermo scientific	3010	To culture the HUVEC cells
Denoised water	Millipore	Not applicable	For preparation of the extract
DMEM medium	Cytiva	SH30243.01	Used for cell culture work
Funnel	Thermo scientific	42600060	To hold the filter paper during the filtration
Glass beakers	Borosilicate	1102-50	Used to prepare the aqueous extract
Hot air oven	Genetimes	Not Applicable	Used to dry the nanoparticles and collect in the powder form
Magnetic stirrer	KYLIN-BELL	GL-5250-A	Used for nanoparticles synthesis
Microscope	Nikon Eclipse	Ts2	Used to take microphotographs
Petri dish	NEST	753001	Used to collect the nanoparticles
Pipette 1 mL	Lab Science	YEA17AD0055580	To take/add the specific volume of solution/extract
Pipette tips 1 mL	SAINING	3014200-T	To take/add the specific volume of solution/extract
PTFE Magnetic Mixer Stir Bars	LAN RAN	Not applicable	Used for nanomaterial synthesis process
Sodium hydroxide	Sigma Alrich	71690	Used to adjust pH during the synthesis
Stainless Scissor	Deli	6034	Used for chopping the bark materials
T25 tissue culture flask	NEST	707001	Used to maintain the cells
Weighing Balance	Radwag	AS220R2	Used to weigh the chemicals
Whatman filter paper No.1	Newstar	GB/T1914-2017	Used to filter the extract for synthesis
Zinc nitrate	Sigma Alrich	13778-30-8	Used as precursor for the nanoparticle’s synthesis