Name: formulating and characterizing an exosome-based dopamine carrier system
Uploaded: 2022-04-04T14:00:00
Description: Watch this Scientific Journal Video about Formulating and Characterizing an Exosome-based Dopamine Carrier System at JoVE.com

The work was primarily supported by research funding provided by Y&#305;ld&#305;z Technical University Scientific Research Projects (TSA-2021-4713).

NOTE: See the Table of Materials for details related to all materials and equipment used in this protocol.
1. Culturing and cryopreservation of Wharton&#39;s jelly mesenchymal stem cells
<ol>
	<li>Remove the WJ-MSCs (from ATCC) from the -80 &#176;C freezer. Seed the cells into a flask containing DMEM-F12 medium supplemented with 10% fetal bovine serum (FBS). Incubate them at 37 &#176;C in an incubator containing 5% CO2.</li>
	<li>Change the cultur...

Exosomes are small membrane vesicles with dimensions of 40-200 nm secreted by most cell types, e.g., MSCs1. Capable of enabling communication between cells, exosomes can enter cells in different ways such as endocytosis, phagocytosis, micropinocytosis, lipid-mediated internalization, and fusion33,44. Compared to other nanocarrier systems, the lipids and cholesterol found on the exosome surface confer the ability to carry both hydrophilic a...

Exosomes, bioactive vesicles with significant features, range in size from 40 nm to 200 nm. Exosomes originate from the cell membrane and are formed because of the release of the endosomes1. These structures serve as cell-to-cell communicators and interact with neighboring cells to facilitate the transfer of active molecules. Exosomes can be isolated from many different sources. These include body fluids such as plasma, urine, cerebrospinal fluid, saliva, as well as cell lines cultured under in vitro conditions. Exosomes have an important role in the elimination of nerve damage, thanks to the biomacromolecules they contain, such as lipids, proteins, and nucleic acids2. Glia, which are the supporting cells of the nervous system3, transfer proteins and micro RNAs to the axons of neurons via exosomes4.
Lipids forming the myelin sheath, which are a characteristic feature in nerve conduction, are also released from oligodendrocytes via exosomes4,5. Exosomes are also involved in processes such as synaptic plasticity, neuronal stress response, cell-cell communication, and neurogenesis in the brain6,7. The fact that exosomes possess nano-dimensions enables them to pass through the BBB. There is a special transition route from the interstitial fluid to the cerebrospinal fluid after penetrating this membrane8. Thanks to their surface properties, exosomes can interact efficiently with target cells as a drug delivery system and actively deliver the loaded drugs.
Due to the expression of various adhesive proteins (tetraspanins and integrins) on the surface of exosomes, these extracellular vesicles can easily interact and fuse with host cell membranes9. It is thought that exosomes can be used as a drug delivery system, especially in the treatment of central nervous system diseases due to their ability to penetrate the BBB and their surface properties. Mesenchymal stem cell (MSC)-derived exosomes have a lower risk of immune rejection compared to allogeneic cellular therapies, and in this respect, they can be an important component of cell-free treatment applications10.
Dopamine is a molecule whose deficiency in the brain is the characteristic feature of Parkinson&#39;s disease (PD), worsening day by day11,12,13. It is known that PD is associated with degeneration of dopaminergic neurons in the substantia nigra of the mesencephalon and loss of motor neuron functions14,15. The death of dopaminergic neurons prevents the supply of the neurotransmitter dopamine to the brain striatum. This, in turn, results in the emergence of PD-specific symptoms16. These symptoms of PD are bradykinesia, postural instability, rigidity, and especially resting tremor12,13. Although PD was first described more than two centuries ago, studies to understand the pathology and etiology of the disease are still ongoing and it is currently accepted that PD is a complex systemic disease17. It is predicted that dopamine deficiency occurs, and clinical PD symptoms are observed when more than 80% of neurons degenerate18. In the treatment of the disease, incomplete dopamine supplementation is preferred to reduce motor symptoms. In vivo studies have shown that direct infusion of dopamine into the brain significantly reduces symptoms in animals19. Dopamine precursors such as L-DOPA (L-3,4-dihydroxyphenylalanine) and dopamine receptor drugs are used in the clinic because the direct infusion of dopamine into the brain is not possible in humans and dopamine entering the system cannot cross the BBB20. These types of drugs lose their effectiveness over time. However, there is still no curative treatment approach for PD. Hence, there is a huge necessity to develop new therapeutic strategies and treatment modalities to reveal the pathophysiology of the disease and reduce the impact of PD on patients.
Exosome-based studies have recently attracted attention for gathering information about both therapeutic approaches and pathologies of nervous system diseases. MSC-derived exosomes have been shown to reduce inflammation in nerve damage and contribute to neuronal regeneration21,22,23. In addition, it has been reported that MSC-derived exosome secretomes reduce apoptosis by showing neurotrophic and neuroprotective effects, especially on dopaminergic neurons24,25. Research on platforms in which exosomes are used as therapeutic drug delivery systems have intensively accelerated in recent years. In numerous studies, it has been observed that relevant drugs can be easily encapsulated into exosomes and delivered safely into target cells, tissues, and organs26,27. Different methods such as incubation, freeze/thaw cycles, sonication, and extrusion could be used for drug loading into exosomes28.
Coincubation with exosomes or exosome-like vesicles allows lipophilic small molecules to be passively encapsulated into these delivery systems28,29,30. In particular, various molecules such as curcumin31, catalase30, doxorubicin32, and paclitaxel33&#160;were effectively loaded into exosomes. It has been observed that catalase-containing exosomes, which have antioxidant activity, efficiently accumulated in the neurons and microglial cells in the brain and exhibited strong neuroprotective activities30. In the same study, saponin, added into the complex to increase the loading efficiency, was found to increase the drug loading percentage during incubation30,34. However, further studies are needed to establish the standards for drug loading into exosomes.
This paper describes the development of a nanocarrier system by encapsulation of dopamine into exosomes that were isolated from WJ-MSCs. All steps, including the cultivation of WJ-MSCs,&#160;isolation, and characterization of exosomes, drug loading experiments, characterization of dopamine-loaded exosomes with various techniques, and in vitro cytotoxicity analysis are explained in detail.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#181;m membrane filter</td>
			<td>Aisimo</td>
			<td></td>
			<td>Used for the sterilization process</td>
		</tr>
		<tr>
			<td>0.45 &#181;m syringe filter</td>
			<td>Aisimo</td>
			<td></td>
			<td>Used for the sterilization process</td>
		</tr>
		<tr>
			<td>15 mL Falcon tube</td>
			<td>Nest</td>
			<td></td>
			<td>Used in cell culture step</td>
		</tr>
		<tr>
			<td>25 cm2 cell culture flasks (Falcon, TPP tissue culture flasks</td>
			<td>Nest</td>
			<td></td>
			<td>Used in cell culture step</td>
		</tr>
		<tr>
			<td>50 mL Falcon tube</td>
			<td>Nest</td>
			<td></td>
			<td>Used in cell culture step</td>
		</tr>
		<tr>
			<td>75 cm2 cell culture flasks (Falcon, TPP tissue culture flasks</td>
			<td>Nest</td>
			<td></td>
			<td>Used in cell culture step</td>
		</tr>
		<tr>
			<td>96 well plates (Falcon, TPP microplates)</td>
			<td>Merck Millipore</td>
			<td></td>
			<td>Used in cell culture step</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma</td>
			<td>271004-1L</td>
			<td>Used for HPLC analysis</td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td>NUVE-OT 90L</td>
			<td></td>
			<td>Used for the sterilization process</td>
		</tr>
		<tr>
			<td>Cell Culture Cabin</td>
			<td>Hera Safe KS</td>
			<td></td>
			<td>Used for the cell culture process</td>
		</tr>
		<tr>
			<td>Centrifugal</td>
			<td>Hitachi</td>
			<td>CF16RN</td>
			<td>Used in the exosome isolation step</td>
		</tr>
		<tr>
			<td>CO2 incubator with Safe Cell UV</td>
			<td>Panasonic</td>
			<td></td>
			<td>Used for the cell culture process</td>
		</tr>
		<tr>
			<td>Dopamine hydrochloride H8502-10G</td>
			<td>Sigma</td>
			<td>H8502-10G</td>
			<td>Used in exosome dopamine loading</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium/Nutrient Mixture-F12</td>
			<td>Sigma</td>
			<td>RNBJ7249</td>
			<td>Used as cell culture medium</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum-FBS</td>
			<td>Capricorn</td>
			<td>FBS-16A</td>
			<td>It was used by adding to the cell culture medium.</td>
		</tr>
		<tr>
			<td>Freezer -80 &#176;C</td>
			<td>Panasonic</td>
			<td>MDF-U5386S-PE</td>
			<td>To store cells and the resulting exosomes</td>
		</tr>
		<tr>
			<td>Fridge</td>
			<td>Panasonic</td>
			<td>MPR-215-PE</td>
			<td>Used to store cell culture and other materials</td>
		</tr>
		<tr>
			<td>High performance liquid chromatography-HPLC</td>
			<td>Agilent Technologies</td>
			<td></td>
			<td>The presence of dopamine from the content of the obtained formulation was investigated.</td>
		</tr>
		<tr>
			<td>Microscope- Primovert</td>
			<td>Zeiss</td>
			<td></td>
			<td>Used to observe cells in cell culture.</td>
		</tr>
		<tr>
			<td>MTT Assay</td>
			<td>Biomatik</td>
			<td></td>
			<td>Used to measure cell viability</td>
		</tr>
		<tr>
			<td>NanoSight NS300</td>
			<td>Malvern panalytical</td>
			<td>Malvern panalytical</td>
			<td>Used for exosome characterization</td>
		</tr>
		<tr>
			<td>Optima XPN-100 Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>Used in the exosome isolation step</td>
		</tr>
		<tr>
			<td>PBS tablet</td>
			<td>Biomatik</td>
			<td>43602</td>
			<td>In the preparation of the PBS solution</td>
		</tr>
		<tr>
			<td>Penicilin/Streptomycin Solution</td>
			<td>Capricorn</td>
			<td>PB-S</td>
			<td>It was added to the medium to prevent contamination in cell culture.</td>
		</tr>
		<tr>
			<td>Pipette Aid</td>
			<td>Isolab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Precision balance-Kern</td>
			<td>Kern-ABJ220-4NM</td>
			<td></td>
			<td>Used in the preparation of solutions</td>
		</tr>
		<tr>
			<td>Q500 Sonicator</td>
			<td>Qsonica, LLC</td>
			<td></td>
			<td>Used to digest exosomes in HPLC analysis</td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma</td>
			<td>47036-50G-F</td>
			<td>It was used by adding it to the total solution in the exosome dopamine loading process.</td>
		</tr>
		<tr>
			<td>Spectrostar-Nano-Spectrophotometry</td>
			<td>BMG LABTECH</td>
			<td></td>
			<td>Used for MTT and drug release analyzes</td>
		</tr>
		<tr>
			<td>SPSS 22</td>
			<td></td>
			<td></td>
			<td>statistical package program</td>
		</tr>
		<tr>
			<td>Vorteks-FinePCR</td>
			<td>FinePCR-FineVortex</td>
			<td></td>
			<td>Used to mix solutions homogeneously</td>
		</tr>
		<tr>
			<td>Water Bath 37 &#176;C-Senova</td>
			<td>Senova</td>
			<td></td>
			<td>Used in cell culture step</td>
		</tr>
		<tr>
			<td>Wharton&#8217;s jelly mesenchymal stem cells</td>
			<td>ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ZetaSizer</td>
			<td>Malvern Nano ZS</td>
			<td>Malvern Nano ZS</td>
			<td>Used for exosome characterization</td>
		</tr>
	</tbody>
</table>

formulating and characterizing an exosome-based dopamine carrier system

Exosomes between 40 and 200 nm in size constitute the smallest subgroup of extracellular vesicles. These bioactive vesicles secreted by cells play an active role in intercellular cargo and communication. Exosomes are mostly found in body fluids such as plasma, cerebrospinal fluid, urine, saliva, amniotic fluid, colostrum, breast milk, joint fluid, semen, and pleural acid. Considering the size of exosomes, it is thought that they may play an important role in central nervous system diseases because they can pass through the blood-brain barrier (BBB). Hence, this study aimed to develop an exosome-based nanocarrier system by encapsulating dopamine into exosomes isolated from Wharton's jelly mesenchymal stem cells (WJ-MSCs). Exosomes that passed the characterization process were incubated with dopamine. The dopamine-loaded exosomes were recharacterized at the end of incubation. Dopamine-loaded exosomes were investigated in drug release and cytotoxicity assays. The results showed that dopamine could be successfully encapsulated within the exosomes and that the dopamine-loaded exosomes did not affect fibroblast viability.

Exosome isolation and characterization 
Wharton jelly stem cells are cultured and incubated in a serum-free medium for 48 h when the culture reaches sufficient density. After the end of the incubation, the supernatant is stored at -20 &#176;C. The collected supernatants are diluted with PBS and subjected to ultracentrifugation (Figure 1). The obtained solution is analyzed by NTA and DLS analyses. The exosomes are sterilized by passing through a 0.22 &#181;m filter. The ...

Watch this Scientific Journal Video about Formulating and Characterizing an Exosome-based Dopamine Carrier System at JoVE.com

Formulating and Characterizing an Exosome-based Dopamine Carrier System

Here, we aimed to obtain a formulation by dopamine loading of the isolated exosomes of stem cells from Wharton's jelly mesenchymal stem cells. Exosome isolation and characterization, drug loading into the resulting exosomes, and the cytotoxic activity of the developed formulation are described in this protocol.

Exosomes between 40 and 200 nm in size constitute the smallest subgroup of extracellular vesicles. These bioactive vesicles secreted by cells play an ...

Exosomes might react to vesicles secreted by a cell between 14 and 200 nanometer in size, and split from in the intracellular cargo and communication, which comes to the smallest subgroup of extracellular vesicles. Exosomes which are formed as a result of the differentiation of the endosome, originate from the cell membrane. The stem cells can be isolated from different sources such as bone marrow or adipose tissues.but adult tissue dried stem cells have higher immunogenic potential Cons springing the size of exosomes, it's told that it's made an important role in central nervous system disease because it can pass through to the blood brain barrier. To begin, wash the seeded Wharton's jelly mesenchymal stem cells with five milliliters of PBS. Then add five milliliters of trypsin EDTA solution before placing the flask in an incubator.After incubation, collect the cells and centrifuge at 1, 500 G for five minutes. Then remove the supernatant and add one milliliter of DMEM-F12 containing 10%FBS to the tube. Wash cultured cells with PBS.After washing, add serum free DMEM-F12 medium to the cells and place it in the incubator. To isolate the exosomes, collect the serum free media and centrifuge at 300 G at four degrees Celsius for 10 minutes. Transfer the supernatant to another tube and process it by serial centrifugation at higher speeds.After the final centrifugation, slowly remove the supernatant and dissolve the pellet in one milliliter PBS. Mix by vortexing and proceed with ultracentrifugation at 110, 000 G for 70 minutes at four degrees Celsius. Add three milliliters of PBS to the exosome suspension collected earlier by ultracentrifugation and sterilize the diluted suspension by filtration using a 0.22 micron filter.Then transfer 500 microliters of the sterilized exosome suspension to another tube. Next sequentially, add 500 microliters of 0.5 milligrams per milliliter dopamine solution and saponin into the sterilized suspension. After incubation ultracentrifuge the suspension.The sample can either be used for NTA and DLS analysis to characterize dopamine loaded exosomes or stored at minus 20 degrees Celsius until further use. In the NTA analysis, the average size of Wharton jelly derived exosomes was determined to be 98 nanometers and dopamine loaded exosomes to be 110 nanometers. The numbers of nanoparticles revealed by NTA and DLS analyses were consistent with each other.The zeta potential of Wharton jelly derived exosomes was measured as minus 15.7 and dopamine loaded exosomes as minus 17.6 millivolts. The HPLC analysis of the dopamine loaded exosomes detected the peak for dopamine at 6.45 minutes. The cumulative drug release profile revealed that 74.8%of encapsulated dopamine was released from the exosomes within the first eight hours.The cytotoxicity of the dopamine loaded and free exosomes on fibroblasts was investigated. Although the dopamine loaded exosomes did not demonstrate any cytotoxic effects, saponin decreased the fibroblast viability. The MTT assay showed increased cell viability when the fibroblasts were treated with the dopamine loaded exosomes.The statistical significance of the results was evaluated by performing one-way anova. Additionally, the cytotoxic effects of different concentrations of free exosomes were investigated on the fibroblasts. The fibroblast viability was higher with free exosomes at the concentration of 25 microliters per milliliter.It's really important for the central nervous system is the patient sizes, the play role in the processes such as plasticity, neuro response, side to side communication, and neurogenesis in the brain. In addition, thanks to surface of exosomes, it says, has been observed that they can in interact with with target cell drug and activity delivered during the drug Is a completion. For the first time in the study, we developed a new drug formulation by encapsulating dopamine into stem cell derived exosomes.He proposed that this formulation will be so promising for treatment of Parkinson's disease.

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