2.2.1. Culturing of UCT-MSCs using LHPL as a culture medium

The study started with the collection of human umbilical cord tissue (hUCT) and culturing the MSCs by an explant method. The study protocol was approved by the Institutional Ethics Committee (Pre-Clinical Studies), Department of Stem Cell Biology, Acadicell Innovations International Pvt. Ltd, Tamil Nadu, India with approval number IEC/ C-P/18/2021, umbilical cord samples were collected from SLIMS, Pondicherry, the cord tissue was cut into smaller segments longitudinally, and the blood vessels were removed. The platelet unit collected by apheresis was subjected to subsequent freeze and thaw cycles at −80°C, to induce platelet lysis for the release of growth factors, and the preparation of LHPL was done. These segments were cultured using basal media supplemented with LHPL as culture medium. The explant was incubated in 5% CO2, at 37°C. Media change was done once every 3 days until the emergent of the MSC from the tissue. The MSCs were passaged after removing the segmented cord tissue.

2.2.2. Priming of Eugenol with EVs and collection of EVs

The cultured cells were passaged and subcultured in a T-75 a cell culture flask at cell seeding density of 4000 cells/cm². Meanwhile, Eugenol was dissolved using an organic solvent. Cells were rinsed with PBS when the confluency reached 70%-80% and preconditioned with Eugenol for 48 h. After preconditioning for 48 h, to obtain EVs, the conditioning medium was collected. The conditioned media was subjected to centrifugation at 300x g for 10 mins and 2000x g for 10 mins for the removal of the cellular debris. Later, the supernatant was centrifuged 10,000x g for 30 mins and the cell-free supernatants were ultracentrifuged at 100,000x g for 70 mins, twice – for collection of cell free supernatant and further washing to proceed with the collection of EVs. As a final step, PBS was used to suspend the EVs, and the collected EVs were further used for analysis.

2.2.3. Characterization of EVs

a) Transmission electron microscopy (TEM) analysis

TEM analysis for Eugenol primed EVs and MSC-EVs was performed using High Resolution TEM (HR-TEM), JEOL Japan. Prior to imaging EVs, the samples were fixed with paraformaldehyde and loaded into a carbon-coated copper grid – 200 mesh. The excess EVs sample was blotted using tissue paper and allowed to dry completely. The grid was placed in the vacuum chamber for image acquisition and evaluation.

b) Nano sight tracking analysis (NTA)

To measure the particle size and concentration, NTA was performed. Eugenol primed EVs and MSC -EVs were diluted in 1 mL PBS and loaded into the sample receiver at the appropriate temperature. Samples were measured at a size distribution of 2 cycles in 11 positions, with a laser wavelength of 520 nm. ZetaView (Version 8.05.16.SP3) was used to analyze the measurements.

c) FTIR spectroscopy

The EVs sample—Eugenol treated and non-treated, were dissolved in a PBS solution for FTIR analysis. The FTIR spectra of these EVs samples were recorded in the wavenumber between 500-4000 cm⁻¹ with a spectral resolution of 4 cm⁻¹.

d) Dynamic light scattering (DLS) analysis

The size distribution of EVs was measured by DLS at the 633 nm wavelength of a solid state He-Ne laser. The intensity of the scattered light was measured at 173°. The measurements were taken at 25°C. For the data processing and analysis, Zetasizer software version 7.13 was used.

e) Western blotting

The marker expression was studied using Zelle Biotech’s Western Blotting Kit. The membrane is incubated with the diluted primary antibodies – Anti-rabbit IgG, HRP-linked antibody, and anti-biotin to detect the biotinylated protein markers in 5% w/v Bovine Serum Albumin (BSA), Tris Buffered Saline (TBS) with Tween at 4°C with gentle shaking overnight. 20 μL of collected and purified exosome samples were loaded onto SDS-page and then electrotransfered to a nitrocellulose membrane. Post transfer, the membrane is washed with TBS and incubated in blocking buffer at room temperature for 1 h. For the detection of proteins, the membrane bound HRP is washed, and the substrate is incubated with the membrane and exposed to X-ray film.

f) Protein estimation – microplate BCA method

Eugenol EVs and MSC-EVs samples were incubated with RIPA buffer overnight to isolate the proteins from EVS. A microplate BCA protein kit was used to estimate the concentration of proteins. The manufacturer protocol was followed for assaying the sample. The samples were suspended in double distilled water prior to measurement. The readings were taken using a microplate reader, and the protein estimation graph was plotted.

2.2.4. Invitro analysis using the colon cancer cell line – HCT 116

a) Cytotoxicity assay

The MTT assay was done to analyze the effectiveness of EVs on HCT 116 cells. In a 96 well plate, each well had a seeding density of 5000 cells. After 24 h cells were treated with varying concentrations of Eugenol primed EVs: 1, 5, and 10 μL and incubated for 48 h. After the incubation period, MTT reagent was added and kept in the incubator for 4 h. Finally, DMSO was added and incubated in the dark for 30 mins. Then the reading was measured at 570 nm.

b) Scratch assay

The scratch assay was done to measure the migratory properties of the cells. i.e., the rate of cell proliferation when a scratch (wound) is created by using a pipette tip. This test was performed to test and assess cell proliferation and migration after treating the cells in different cases. The HCT 116 cells were grown in a 12-well culture plate. Observations of the plate were taken at the 0^th and 24^th hour and the distance of the scratch was analyzed using ImageJ software.

c) Colony formation assay

The assay was performed with HCT 116 cell line. About 500 cells/well were seeded in six well plates and grown overnight. 24 h later, the plates were treated in different cases. After 48 h of incubation, the medium was removed and allowed to incubate for 10 days. After which, the colonies were fixed and stained with formalin and 1% crystal violet, respectively. The number of colonies was estimated, against which the graphs were plotted.

d) AO EtBr staining

The procedure used acridine orange/ethidium bromide (AO/EB) staining on HCT 116 cell lines to identify cancer cell apoptosis. Cells were added to a 96-well plate to a final concentration, and the wells were divided for different cases and cultured until the cells reached confluency of 60 to 70%. A 150 μL dual fluorescent staining solution was pipetted into each well and kept at ambient temperature for 10 mins. After the incubation period, the cells were visualized under an inverted fluorescent phase contrast microscope.

3.7.1. Cytotoxicity assay

MTT was done to identify the inhibitory effects of the drug - Eugenol and EVs in HCT 116 and HEK293 cells. The cells were exposed to varying concentrations of MSC-EVs (A1-A3) in comparison to drug- B1-B3 (Eugenol primed MSC derived EVs). When compared to drugs, the proliferative index of EVs treated cells was identified to be lower in a dose-dependent [Figure 7]. Statistical analysis was performed to confirm the role of EVs which influence the cell viability rate in HCT116 and HEK293. The IC50 value of EVs was calculated and represented in a graph. In our study, we found that the proliferation of HCT116 cells was drastically reduced by EVs.

Figure 7: Cytotoxicity assay of HCT 116 cell line with drugs and EVs. [Click here to view]

3.7.2. Scratch assay

The concentration of MSC-EVs and Eugenol primed MSC - EVs was 5 μL based on the proliferative effect, and the same concentration was used in the scratch assay. The different cases and samples used in the assay, along with the proliferation rate (%), are given in the table below. The rate of proliferation in terms of scratch length was observed at the 0^th and 24^th h.The proliferation rate (%) is calculated using:

The results as depicted in Table 1 suggest a positive effect of decreased development of colon cancer cells in case the of Eugenol primed MSC derived EVs with the least proliferation rate. This rate is lower than that of the MSC derived EVs, which shows the effectiveness of the drug priming with EVs.

Table 1: Results of the scratch assay – rate of proliferation (%).

3.7.3. Colony formation assay

The long-term effects of EVs were noted in HCT116 cells. The cells were treated with drugs and EVs and incubated for 10 days. The determined drug dosage was given, and a lesser number of colonies were identified in EVs treated compared to the drug. Both drug-treated and EVs treated was compared to untreated HCT116 cells. A marked reduction in the number of colonies in treated samples was noted [Figure 8]. This finding clearly shows that EVs have a greater effect on HCT116cells. Therefore, EVs are potently inhibiting the cell proliferation and colony formation in colon cancer cells.

Figure 8: Colony formation analysis between drug treated colonies and EVs treated colonies. [Click here to view]

3.7.4. AO EtBr staining

After the application of AO EtBr stain to MSC - EVs and Eugenol primed MSC -EVs, the apoptotic cells were stained in a yellow – orangish red color. The resultant images are depicted above in Figure 9, and we observed the following: In the control sample, we observed live colon cancer cells. In the MSC-EVs treated group of cells, a mixed population of late apoptotic and necrotic stages of cells was found. Whereas in the Eugenol MSC-EVs treated cells, most of the cell population was in the necrotic stage.

Figure 9: AO EtBr staining of HCT 116 cell line; (A) – Control, (B)- Cells with MSC -EVs, (C) – Cells with Eugenol primed MSC - EVs. [Click here to view]

Understanding the role of EVs is a must in the current healthcare scenario, and it has a major impact on our understanding of intracellular and cell-cell communication, tumor and stem cell biology, and other fields [18]. To date, there are many studies and experiments being performed to support the role of EVs in every aspect of cancer formation and prognosis. On the other hand, anti-tumor activity of EVs is also observed for breast cancer, gastric cancer, liver, and lung cancer [19]. Almost every cell releases EVs, and these are in general classified as nanoparticles. These extracellular vesicles (EVs) do participate in both proximal as well as distal cell-cell communications [18]. MSCs have an increasing ability to treat various diseases due to their paracrine effects that are mediated by extracellular vesicles (EVs). Bioactive substances like lipids, proteins, DNA, RNA, and other metabolites that plays a vital role in cellular communication are found in EVs [20] MSC-EVs help inhibit the development of the tumor in a paracrine manner. This is closely associated with the tumor – related miRNAs that are present in the MSC derived EVs [21]. Also, proteins and long non-coding RNA (lnc RNA) in the EVs are attributed to suppressing the proliferation of the cancer cell and thus initiating the apoptosis of the cancer cell [22, 23]. Angiogenesis is the vital factor for tumor establishment, which ensures an adequate supply of oxygen and nutrients to the growing cancer cells [24]. Studies have reported that MSC derived EVs reduce the rate of angiogenesis by downregulating the expression of VEGF and CD31 [25]. On the other hand, EVs have the ability to act as antigen presenters, stimulating the adaptive and innate responses of the immune system [26]. MSCs have different triggering actions in the signaling pathways in terms of blocking and activating. Studies reported that MSCs block the Wnt signaling pathway by modulating the Dickkopf-related protein 1(DKK1) that is released by the malignant cells, downregulating c-Myc and Cyclin D2, and upregulating P21CIP1 and P27KIP1, thus resulting in the suppression of cancer cells [27]. MSC-EVs are also beneficial due to their properties of immunosuppression and immune evasion in the tumor microenvironment (TME) [28]. In accordance with the therapeutic benefits of EVs, we tend to isolate hUCT-MSC -EVs for treating colorectal cancer due to the tumor homing and immunomodulatory properties they exhibit, thus providing new modalities for cancer treatment [29].

We characterized EVs based on ISEV and the results we encountered in NT analysis, wherein the particle size was < 200 nm in accordance with the classification given by Jeppesen and team [30]. The morphology of the EVs was also confirmed by TEM, and the heterogenous population of EVs was confirmed by the varying size distribution in DLS, in accordance with the EVs characterization [31]. The FT-IR spectra result of our study, which ranged between 400-1608 cm^-1 is in par with the results obtained from the study of osteosarcoma cell derived EVs and other suspended cells [32]. The Western blotting results of our study showed positive markers for CD9, ALIX, and flotillin which are on par with other studies [33]. Furthermore, the BCA protein assay shows the same protein levels for both Eugenol primed EVs vs. non-primed EVs, indicating no change or alteration in the proteins. This finding also matches the lack of significant results in the case of other engineered EVs [34].

The anti-cancer effects of the EVs and Eugenol primed EVs were found using various ^{in vitro} studies. Although the cancer cell proliferation of EVs has been studied, the contradictory nature of the same, i.e., the use of EVs in cancer therapy, must be studied, and we found that the final concentration of 5 μL of MSC derived EVs is sufficient to kill the cells, which was replicated as a percentage of proliferation rate, cytotoxicity assay, and AO/EtBr staining. This also proved the non-promoting effect of EVs on cancer cell lines [35].

With respect to Eugenol, the addition of this phytochemical to the HCT-116 cell line has been studied, and it was found that the cells showed signs of autophagy and apoptosis, thus inhibiting proliferation [36]. As per the earlier studies, it is seen that the antioxidant effect of stem cells can be promoted by preconditioning the EVs with Eugenol [36]. Our study combined the anti-cancer effects of both MSC derived Eugenol primed EVs and found positive results in the ^{in vitro} analysis.