Correspondence:
Department of Biology, Science College, King Khalid University, P.O. Box 9004, Abha, Saudi Arabia.
e-mail: [email protected]
P a g e | 5
Contents lists available at Article URL
KKU Journal of Basic and Applied Sciences
Journal homepage: journals.kku.edu.sa/jbas Vol 7, 1 (2021) 5-9
The Ability of Extracellular Vesicles to Select an Active Ingredients from Suaeda Fruticose Plant Extract.
Kamel Ahmad Saleh
1*1.
Department of Biology, Science College, King Khalid University, P.O. Box 9004, Abha, Saudi Arabia
Keywords: Extracellular vesicles, Exosomes, Plant extract, drug delivery
1. INTRODUCTION:
Accumulated knowledge about cancer molecular biology has also facilitated in identifying some biomarkers that are used in cancer screening, diagnosis, and follow-ups [1].
Unfortunately, many cancer screening procedures involve invasive techniques (e.g., endoscopy and catheterization) in order to obtain sample biopsies [2]. Identifying new biomarkers from patients’ liquid biopsies (e.g., Blood, Urine, and saliva) may serve as a non-invasive solution for cancer screening [2].
Extracellular vesicles (EVs) are defined as nano-particles with sizes ranging between 40 and 2000 nm, surrounded by a double phospholipid bilayer membrane [3]. EVs are found in all extracellular fluid including plasma and urine [3]. According to their physical properties, these nano- particles are capable of engulfing and saving their loaded molecular contents (DNA, RNA, and proteins) from micro-environmental effects like the immune system,
plasma pH, and imbalanced ions [3].
A number of attempts have been conducted to utilize EVs as a drug carrier [4-8]. The hydrophobic bilayer phospholipid membrane of the EVs may serve as a physical barrier be tween the loaded drug and water surrounding the EVs. This cellular-like EVs membrane would also facilitate endocytosis by target cells which serving a full discharge of the drug inside the cells [4]. In addition, the natural features of EVs grant it the ability to enter body fluids without induction of immunogenicity reflex [9]. EVs membrane would also protect loaded drugs from degradation in the circulation system and body fluids. Best of all, EVs can overcome natural barriers like the blood-brain barrier, so they possess the ability to de liver anticancer drugs to the brain [10]. On the other hand, clinical trials that used EVs as a drug delivery system, demonstrated the safety of EVs administration [7]. The relatively easy access to EVs by isolating them from different body fluids before they are loaded with a drug and returned to the same patient, gives them an extra advantage [11].
Received: Revised: Accepted: Published:
Abstract: While drug delivery mechanism suffering from various challenges such as (the stability of drug, side effects, immune system induction, more time, and high amount of drug to get the same impact) recent studies showed that normal and cancer cells use safe and more effective special Nano-convoys (50 to 5000nm) called Extracellular Vesicular (EVs) in their communications.
Despite their small size, EVs can contain specific messages "proteins, DNA, or RNA” These messages induce other cells to do a certain function and perhaps several functions depending on the nature of the message they are carrying.
Whereas, EVs surrounded by a bilayer membrane made up of phospholipid revealed the elimination of all disadvantages of traditional drug delivery methods.
This study aimed to confirm the difference of the impact between the extract of Suaeda fruticose plant loaded inside the normal or cancerous EVs, and the impact of the non-loaded (free) extract using colon cancer cell line (HCT116) in vitro by the meaning of MT test.
Results revealed that the impact of loaded EVs presents in reduced the cellular proliferation by the means of MTT 50 times compared to normal extract of the Suaeda fruticose plant, which confirmed that the ability of EVs to be a good and safe delivery system with semi permeable selectivity attribution. In addition, it could be a good sign that the EVs may have the ability to select the active ingredients and their co-factors from the extract.
Further studies are needed to confirm the mechanism of action of how the EVs uptake the active ingredients.
Persistent attempts to obtain pure vesicles from plasma or other body fluids indirectly led to the recognition of surface proteins that distinguish the vesicles from other particles [12]. Later, lead to classifying each type of vesicle accurately which facilitates diagnosis [12]. Where, proteins involved in EVs biogenesis (e.g., Alix, Tsg101 and ESCRT complex) tetraspanins (e.g., CD9, CD37, CD53, CD63, CD81, and CD82), GTPases, annexins ,and flotillin), heat shock proteins (Hsp70, Hsp90) , and epithelial cell adhesion molecules (EpCam) are the most common proteins used in the identification of EVs, later they become the most famous cancer biomarkers as they have found in cancer patient plasma comparing to standard case [13]. On the other hand, generally, the mechanism of action of miRNAs presents the major reason to release them from cancer cells. Whereas, miRNAs are used to downregulate or upregulate specific genes involved in apoptosis, invasion or angiogenesis and even cell proliferation [14].
The ability of vesicles to transfer DNA from cell to cell depends on vesicle size. Therefore, the apoptotic bodies and onco-bodies have the opportunity to enclose portions of DNA up to 250-kilo base pairs (kB) more than exosomes could do which is around 100 base pair [15].
Therefore, it is very logical to predict that large size vesicles were more suspected to transform other cells.
However, recent studies confirmed that the ability of EVs to transform cells is not related to size, but it is related to the nature of their enclosed charge. It seems that what the vesicles carry is a direct reflection of what the cell carries
"negatively or positively." Likewise, using anti-5' methylcytosine antibodies in B16-F10 melanoma cells present a similar signature with their extracellular vesicles signature, suggesting that EVs have comparable DNA status. Regardless of the ability or inability of DNA to transform other cells the pres ence of a sufficient amount of DNA encourages our research to achieve its purpose
"DNA fingerprinting of cancer cells EVs." [15]
There are many types of RNA enclosed by vesicles like (miRNA, tRNA, noncoding RNA). All of them are short not exceed 200 nucleotides. The shortness of RNA presents as a challenge in the process of extraction, as they may decay among other molecules [14]. Despite that, the good news here is that long non-coding RNA, as well as miRNA, are enclosed in large oncosomes, where it is easier to isolate, concentrate and then the extraction will have done.
Moreover, depending on the cell type the amount of RNA enclosed by EVs will vary [14]. Consequently, EVs released from cancer cells are more abundant comparing to normal cells [3]. Indeed, all researchers agree that the vesicles contain different types of RNA, but it is remaining unclear what is the purpose of its existence and what is their function [14].,however, we believe that there is a possibility to classify them among several groups depending on their size.
The presence of protein in the vesicles is often associated with the origin of the endolysoso mal compartment during the biogenesis of the vesicles [16]. In addition, to have a vital role in metabolism pathways, like the endosomal sorting complex required for transport (ESCCRT) is involved in the budding of the internal membrane to form
multivesicular bodies. Thereby, it is not surprising that the inactivation of ESCRT protein is related to some diseases.
Surprisingly, however, the action of this protein needs some auxiliary proteins such as Alix, protein 101 which are present in the exosomes as looks like a "full delivery"
order [16]. Consequently, the cellular transformation done smoothly without arousing the immune system, as the proteins are all enclosed @ in a suitable cell membrane.
There are some differences in content between types of vesicles. Where the MVs contain several modified proteins like glycoprotein or phosphoproteins, which are post-translated proteins, while the exosomes contain only a small amount of them. This may be due to their capacity since the exosomes are much smaller than the MVs [14, 16].
The lipid content in the vesicles was not expected to have a bio-activity. On the contrary, lipids molecules have a vital activity no less than other macromolecules despite them are not involved in biotransformation. For instance, exosomes contain phosphatidylserine in their outer leaflet, which has a role in facilitating the internalization by competent cells [17].
2. MATERIALSANDMETHODS:
Chemical reagent
Hexane, acetic acid, sulphuric acid, chloroform, hydrogen chloride acid, lead acetate, ferric chloride, iodine, potassium iodide, mercuric chloride, sodium hydroxide, PBS, MT stain, DMSO, and doxorubicin purchased from Sigma Chemical Co. (St. Louis, MO, USA). Cell culture media including DMEM, and RPMI, Cell culture supplementary including, FBS, exo-free FBS, penicillin Streptomycin, and trypsin obtained from Gibco/Life Technologies (Carlsbad, CA, USA). Cell culture vessels were obtained from Nunc A/S (Roskilde, Denmark).
Total exosomes isolation reagent form cell culture media were purchased from (Invitrogen, Thermo Fisher Scientific, USA)
Cell line and culture
Common human cancer cell lines HCT 116 representing colon cancer used in this study for the anticancer activity of the different crude fraction, while normal cell line human fibrosarcoma FHC purchased from ATCC (USA) where used in the EVs experiment. Cells routinely maintained in RPMI cell culture media for the cancer cell lines and DMEM for the normal cell line supplemented with 1 mM sodium pyruvate, 2 mM L.glutamine, 100 units/ml penicillin-streptomycin and 10% fetal bovine serum, in case of EVs experiment the exo-free FBS was used and incubated in 5% CO2 humidified incubator at 37
°C (Binder, USA). Each cell line is passaged and subculture weekly after reaching 80% confluence. For sub culturing the cells, first, the confluence of the cells and the absence of contamination is detected by using an inverted microscope (Fisher Scientific, USA). Then, the process of media aspiration is used, and the cells washed with PBS solution. After that, cells are trypsinized with 0.25%
trypsin-EDTA for 2-5 mints and incubated in 5% CO2 humidified incubator at 37 °C. Further, the cells examined again under an inverted microscope to ensure that almost
all cells are separated from the flask surface and appears in a rounded shape. Finally, the cells collected and passed to another cells culture flask and re-suspended with RPMI culture media to inactivate the enzymatic reaction of trypsin. Before starting the passing, all solutions must be pre-heated using a water bath (Fisher Scientific, USA) at 37ºC and all the processes are done under sterilized conditions using biological safety cabinets (Nuaire, USA).
Plant identification, classification, and sample collection
The halophyte plant (Suaeda fruticosa) which was chosen for this experiment were collected from the Red Sea shoreline, Asser region, Southwestern coast of KSA between 17-21 July 2017 during the summer season 36o- 40oC.
Suaeda fruticose
Suaeda fruticose aerial parts were also collected from sebkha at Al Birk region (41°46′38.1"E, 18°33′43.9"N) and put in 2 Kg plastic bags.
Classification of Suaeda fruticose
Kingdom Subkingdom Super division Division Class Subclass Order
Plantae Tracheobionta Spermaophyta Magnoliophyta Magnoliopsida Caryophyllidae Caryophyllales Family: Chenopodiaceae
Genus: Suaeda
Species: forssk (fruticose)
S.fruticosa is salt tolerate perennial shrub that is found in groups with 100-120 cm tall. It has an active roots system sending branching stem from the base. The stem begins green and rough when the leaves are shed turning to thick and brown. The leaf is green succulent 1-2 cm long and linear, terete or some are almost elliptic which grow along the branches in an alternative arrangement [18]. Further, it has inflorescences terminal in simple or branched spikes. The flowers are over three and found in axillary clusters.
Extracellular vesicles Isolation:
The isolation was achieved in the direction of the kit supplier (Giagene- exoEasy Maxi kit) following the steps that started with a collection of cell supernatant by centrifugation (Hermle, Germany), followed by adding r XBP buffer to the sample then the sample was mixed well gently. After that the mixture was kept to warm up until it reached room temperature. The mixture of (Sample/XBP) was added onto the exoEasy spin column then centrifuged at 500 x g for 1 min. 10 ml buffer XWP and Centrifuged at 5000 x g for 5 min to remove the residual buffer from the column. The collected samples Transferred to a fresh collection tube. 400 μl Buffer XE was added to the membrane and incubated for 1 min.
Centrifuge at 500 x g for 5 min to collect the eluate [11].
Extracellular Vesicles loading:
Hexane extract contains hydrophobic ingredients, which means that they have the ability to cross the phospholipid membrane of extracellular vesicles easily [7].
Nevertheless, it has been incubated in different concentrations of plant extract under the same conditions of temperature and pH. Before the loading procedure, the structure and the shape were checked and looked un- affected (Figures 1-3)
Detection of Isolated EVs using scan and transition electron microscope (SEM and TEM)
The degradation and the ability of Extracellular Vesicles to transfer the plant extract verified by photographing the samples using (SEM and TEM) Also, (Figure 1-3).
Apoptotic and Necrotic cells detection
To determine the impact of the EVs (loaded and unloaded) with Suaeda fruticosa extract to induce apoptosis in HCT 116, cancer cell line, untreated and treated HCT116 cells incubated for 24h cells then fixed and stained using dual AO/EtBr fluorescent dye. Which is efficient for monitoring the morphological change of the cell during the apoptosis process including, cell shrinkage, pyknosis, membrane blabbing, and chromatin condensation which is easy to detect under a fluorescent microscope.
MTT assay
Living cells are capable to convert 3-[4,5-dimethylthiazol- 2-yl]-2,5 diphenyl tetrazolium bromide into formazan crystals which are accepted as an indicator for mitochondrial activation. HCT 116 human colon cancer cell lines were evaluated for their sensitivity against Loaded EVs of the normal cell line human fibrosarcoma FHC prepared as described in the material and method by the meaning of MTT assay. Results revealed the low cytotoxicity of EVs, deposit that, the unloaded EVs had very low IC50 compared with the loaded EVs. These results are compatible with the previously done by SulphoRhodamine-B (SRB) assay. [19].
3. RESULTS
The results revealed that there is a clear difference between the impacts of the cells treated with the unloaded EVs compared to the loaded ones. Whereas, the unloaded hexane extract has a weak ability to induce apoptosis, although the HCT 116, cancer cells showed membrane blebs in addition to apoptotic bodies. However, the extract was found not to be necrosis inducer for HCT 116 cells as the counted necrosis cases were not significant.
On the other hand, the EVs isolation methods present sufficient amount of EVs with high accuracy despite their easy modified procedure. While the uploading method clearly affected by pH, where the effect was directly
P a g e | 8 related to the increase in alkalinity, the greater the
alkalinity, the greater the upload thus the effect of loaded vesicles on HCT 116 cells cancer increased also (Figures 1-3).
Figure 1-3: Isolated Exosomes with 57 nm size, scanned using TEM (JEM-F200) showing A) Normal EVs, B) after 12 h incubation with Suaeda fruticosa plant extract C) After 24h Incubation with Suaeda fruticosa plant extract.
4. DISCUSSION:
Harnessing Extracellular vesicles "EVs" in drug delivery presented in many previous studies [5- 8].Whereas, they focused on the ability of EVs to transfer the chosen medicine or drugs to specific organs. These procedures were applied in many diseases and medicine, and showed various impacts depending on the medicine and disease [5-8]. On the other side, different studies attempt to enhance the impact of pure natural molecules like curcumin [20]. Aforementioned studies confirmed that EVs are considered as a reliable Bio-carriers.
Consequently, we tried to go one-step forward to inspect the ability of EVs to select the active ingredients from total extract without going to fractionations.
Current study results revealed that EVs might possesses the ability to select some active secondary metabolites.
However, what might deem as a surprise, the ability of the EVs to allow the activators of the secondary metabolites to cross their membrane and thus enhance their impact, which revealed that the vesicles might have the ability to select the activators of the active secondary metabolites also. This is might be due to their membrane structure, where, the membrane construct of phospholipids.
The results also indicated the close relationship between
the ability of vesicles to extract active substances from plant extracts and the alkaline pH of the medium, as the relationship between them was a direct relationship, the higher the alkalinity, the higher the loading capacity. This is consequent to the chemical-physical nature of the membrane of the vesicles where the rigidity of the membrane increases the alkalinity increase, which is compatible with the previous studies [21, 22]. These information were confirmed in this study, the vesicles were lost their semipermeable specialty where the membrane become more rigid in acid media. This is very compatible with the behavior of the all cancer cells whereas they try to keep their environment in acid value in order to prevent bacterial growth and attacks, in addition to keep their extracellular vesicles membrane more rigid to prevent the lost or gain un-needed molecules.
Using hexane extract allowed the researcher to select the ingredients that have hydrophobic properties and usually these ingredients are more capable to cross EVs membrane due to their membrane structure which looks like cell membrane. The importance of this idea appeared in the results that revealed the entry of a large amount of the extract and faster.
The current study remains in the early stages despite the results are encouraging the researchers to go forward in order to eliminate the many steps needed to isolate active ingredients (secondary metabolites) from extracts.
ACKNOWLEDGMENT
Author would like to thank Ms. Tahani H. A. Al-Bin Hassan and Prof. Dr. Mohammad Ali Alsheri for their valued help in preparing this manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
REFERENCES
[1] Bardella, Chiara, Patrick J. Pollard, and Ian Tomlinson. "SDH mutations in cancer." Biochimica et Biophysica Acta (BBA)- Bioenergetics 1807.11 (2011): 1432-1443.
[2] Henry, N. Lynn, and Daniel F. Hayes. "Cancer biomarkers."
Molecular oncology 6.2 (2012): 140-146.
[3] Borges, Fernanda Teixeira, L. A. Reis, and Nestor Schor.
"Extracellular vesicles: structure, function, and potential clinical uses in renal diseases." Brazilian Journal of Medical and Biological Research 46 (2013): 824-830.
[4] Akers JC, Gonda D, Kim R et al. Biogenesis of extracellular vesicles (EV): exosomes,microvesicles, retrovirus-like vesicles, and apoptotic bodies. J Neurooncol 2013; 113: 1- 11.
[5] Tian Y, Li S, Song J et al. A doxorubicin delivery platform using engineered natural membrane vesicle exosomes for targeted tumor therapy. Biomaterials 2014; 35: 2383-2390.
[6] Jang SC, Kim OY, Yoon CM et al. Bioinspired exosome-mimetic Nano-vesicles for targeted delivery of chemotherapeutics to malignant tumors. ACS Nano 2013; 7: 7698-7710.
[7] Zhuang X, Xiang X, Grizzle W et al. Treatment of brain inflammatory diseases by delivering exosome encapsulated anti- inflammatory drugs from the nasal region to the brain. Mol Ther 2011;
19: 1769-1779.
A
B C
P a g e | 9 [8] Sun D, Zhuang X, Xiang X et al. A novel nanoparticle drug delivery
system: the anti-inflammatory activity of curcumin is enhanced when encapsulated in exosomes. Mol There 2010; 18: 1606-1614.
[9] Dabrowska, Sylwia, et al. "Immunomodulatory and Regenerative Effects of Mesenchymal Stem Cells and Extracellular Vesicles:
Therapeutic Outlook for Inflammatory and Degenerative Diseases."
Frontiers in Immunology 11 (2021): 3809.
[10] Saint-Pol, Julien, et al. "Targeting and crossing the blood-brain barrier with extracellular vesicles." Cells 9.4 (2020): 851.
[11] Zabel, Rachel R., et al. "Enrichment and characterization of extracellular vesicles from ex vivo one‐sided human placenta perfusion."
American Journal of Reproductive Immunology 86.2 (2021): e13377.
[12] Pariset, Eloise, Vincent Agache, and Arnaud Millet. "Extracellular vesicles: isolation methods." Advanced Bio-systems 1.5 (2017): 1700040.
[13] Zaborowski, Mikołaj Piotr, et al. "Methods for systematic identification of membrane proteins for specific capture of cancer-derived extracellular vesicles." Cell reports 27.1 (2019): 255-268.
[14] Shahabipour F, Barati N, Johnston TP et al. Exosomes:
Nanoparticulate tools for RNA interference and drug delivery. J Cell Physiol 2017; 232: 1660-1668.
[15] Elzanowska, Julia, Christine Semira, and Bruno Costa‐Silva. "DNA in extracellular vesicles: Biological and clinical aspects." Molecular Oncology 15.6 (2021): 1701-1714.
[16] Rocha, Sara, et al. "3D cellular architecture affects microrna and protein cargo of extra cellular vesicles." Advanced science 6.4 (2019):
1800948.
[17] Freeman, David W., et al. "Altered extracellular vesicle concentration, cargo, and function in diabetes." Diabetes 67.11 (2018):
2377-2388.
[18] Saleh, Kamel A., et al. "Anticancer property of hexane extract of Suaeda fruticose plant leaves against different cancer cell lines."
Tropical Journal of Pharmaceutical Research 19.1 (2020): 129-136.
[19] Rai, Yogesh, et al. "Mitochondrial biogenesis and metabolic hyper activation limits the application of MTT assay in the estimation of radiation induced growth inhibition." Scientific reports 8.1 (2018): 1-15.
[20] Sun, Dongmei, et al. "A novel nanoparticle drug delivery system: the anti-inflammatory activity of curcumin is enhanced when encapsulated in exosomes." Molecular therapy 18.9 (2010): 1606-1614.
[21] Szigyártó, Imola Cs, et al. "Flow-Alignment of Extracellular Vesicles: Structure and Orientation of Membrane Associated Bio- macromolecules Studied with Polarized Light." Chem Bio Chem 19.6 (2018): 545-551.
[22] Tahani H. Asiri, Anticancer Activities of Some Halophytes Collected from Asser Region in Saudi Arabia, p 70, King Khalid University, 2018.
