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 culture medium every 2 days. Passage the cells when they reach 80% confluence. Wash the cells to be passaged with 5 mL of phosphate-buffered saline (PBS). 
	NOTE: Washing with PBS before adding trypsin ensures easy separation of cells from the flask surface.</li>
	<li>Remove the PBS and add 5 mL of trypsin-EDTA solution to the flask. Incubate the flask for 5 min at 37 &#176;C in an incubator containing 5% CO2.</li>
	<li>Collect the supernatant into the tube and centrifuge at 1,500 x g for 5 min. After centrifugation, remove the supernatant and add 1 mL of DMEM-F12 + 10% FBS to the tube by pipetting.</li>
	<li>Transfer the cells to a larger flask and continue the culture by adding DMEM-F12 + 10% FBS until sufficient exosomes are obtained35. 
	NOTE: Cultured cells are frozen at a density of 1 million/mL with 10% dimethyl sulfoxide (DMSO) and stored at -80 &#176;C.</li>
</ol>
2. Production of exosomes from Wharton&#39;s Jelly mesenchymal stem cells
NOTE: Exosomes are isolated from cultured WJ-MSCs. Exosome isolation is performed when cells cover the flask surface and reach approximately 80% density.
<ol>
	<li>Remove the supernatant medium containing 10% FBS from the flask and wash the cells with 5 mL of PBS. Add serum-free DMEM-F12 medium to the cells after rinsing with PBS. Incubate flasks for 48 h at 37 &#176;C in a 5% CO2&#160;incubator. Following incubation, collect the serum-free medium in the tube and store at -20 &#176;C. 
	NOTE: In step 2.1, 180 mL of serum-free medium is collected and stored at -20 &#176;C. After thawing, a batch centrifugation process is started, as described below.</li>
	<li>Isolation of exosomes
	<ol>
		<li>Centrifuge the collected serum-free medium at 300 x g for 10 min at +4 &#176;C. Carefully withdraw the supernatant and transfer it to another tube.</li>
		<li>Centrifuge the collected supernatant at 2,000 &#215; g for 10 min at +4 &#176;C. Carefully withdraw the supernatant and transfer it to another tube.</li>
		<li>Centrifuge the collected supernatant at 10,000 &#215; g for 30 min at +4 &#176;C. Carefully withdraw the supernatant and transfer it to another tube.</li>
		<li>Transfer the collected supernatant to ultracentrifuge tubes and ultracentrifuge at 110,000 &#215; g for 130 min. Slowly remove the supernatant and add 1 mL of PBS to the pellet.</li>
		<li>Vortex the pellet and ultracentrifuge at 110,000 &#215; g for 70 min at +4 &#176;C36&#8203; (Figure 1). Slowly remove the supernatant and add 1 mL of PBS to the pellet. 
		NOTE: After the ultracentrifugation steps are completed, the exosomes obtained can be stored at -80 &#176;C. The obtained pellet is now ready for characterization.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63624/63624fig01.jpg" /> 
Figure 1: Exosome isolation. <a href="https://www.jove.com/files/ftp_upload/63624/63624fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Characterization of exosomes
<ol>
	<li>Nanoparticle Tracking Analysis (NTA) 
	NOTE: Nanoparticle tracking analysis is performed to determine the size and concentration of the isolated exosomes. Tablets used for 1x PBS solution must be in the range of pH 7.3-7.5. 1x PBS Solution contains 10 mM phosphate buffer, 137 mM sodium chloride, and 2.7 mM potassium chloride. The solution is prepared by adding 1 PBS tablet in 100 mL of distilled water. This prepared solution is sterilized by autoclaving.
	<ol>
		<li>Dilute the exosomes 100-fold by adding 990 &#181;L of PBS to 10 &#181;L of exosomes. Take the diluted suspension into a disposable syringe.</li>
		<li>Turn on the NTA device and connect the computer. Open the software.</li>
		<li>Inject the sample in the syringe into the tubing in the cassette section of the device. Close the cassette cover of the device.</li>
		<li>In the software that opens, click on the Start Analysis button. Save the results obtained by pressing the Record button.</li>
	</ol>
	</li>
	<li>Dynamic light scattering analysis 
	NOTE: Exosomes isolated for zeta potential and size measurement are also diluted in the same manner as for NTA analysis.
	<ol>
		<li>Add 980 &#181;L of PBS to 20 &#181;L of the exosomes.</li>
		<li>Open the zeta sizing device and the connected computer. Open the software.</li>
		<li>Add the suspension from step 3.2.1 to the disposable cuvette. Open the lid of the device, place the cuvette in the cassette, and close the lid.</li>
		<li>Select the cuvette type in the device&#39;s software. Click on the Start Analysis button for zeta potential and dimensional analysis. 
		NOTE: Analyses are performed separately for dimensional analysis and zeta potential.</li>
	</ol>
	</li>
</ol>
4. Dopamine loading into exosomes
NOTE: After the characterization of WJ-MSCs exosomes is completed, dopamine-loaded exosomes were obtained as a drug delivery system. Drug loading into exosomes is performed using the incubation method.
<ol>
	<li>Add 3 mL of PBS to the exosome suspension from step 2.2.5. Sterilize the diluted suspension by filtration using 0.22 &#181;m filters. Transfer 500 &#181;L of the sterilized exosome suspension to another tube.</li>
	<li>Prepare dopamine solution (0.5 mg/mL) with distilled water. Add 500 &#181;L of dopamine solution into the tubes containing the exosomes.</li>
	<li>Add saponin to the suspension in step 4.2. Incubate the prepared exosome-dopamine suspension for 24 h at 37 &#176;C. 
	NOTE: Saponin concentration should not exceed 0.002% of the total solution.</li>
	<li>Ultracentrifuge the suspension at 90,000 &#215; g for 70 min to remove free dopamine and saponin from the medium. Store the isolated exosomes at -20 &#176;C until use for further analysis37. 
	NOTE: Add 0.02% ascorbic acid for the stability of the prepared dopamine solution.</li>
</ol>
5. Characterization of dopamine loaded exosomes
<ol>
	<li>Nanoparticle tracking analysis (NTA) 
	NOTE: Nanoparticle tracking analysis is performed to determine the size and concentration of isolated exosomes.
	<ol>
		<li>Follow steps 3.1.1-3.1.4 to dilute the exosomes and perform NTA.</li>
	</ol>
	</li>
	<li>Dynamic light scattering analysis 
	NOTE: Exosomes isolated for zeta potential and size measurement are also diluted similarly to NTA analysis.
	<ol>
		<li>Follow steps 3.2.1-3.2.4 to dilute the exosomes and perform dynamic light scattering analysis. 
		NOTE: Size and zeta potentials for each solution (saponin, dopamine, exosome-dopamine) are analyzed separately.</li>
	</ol>
	</li>
</ol>
6. High-Performance Liquid Chromatography (HPLC)
NOTE: The amounts of dopamine loaded into exosomes are measured by the high-performance liquid chromatography (HPLC) method. To detect the presence of dopamine within the obtained formulation, exosomes are detonated by a special process.
<ol>
	<li>Put the formulation in a 75 &#176;C heater to evaporate. Add acetonitrile (50:50 ratio) to the suspension and vortex. Sonicate the solution for 10 min.</li>
	<li>Centrifuge the solution for 10 min at 10,000 &#215; g. Filter the supernatant through a 0.22 &#181;m filter.</li>
	<li>Analyze all analytes using a C18 column at 30 &#176;C at a flow rate of 1 mL/min (the mobile phase H2O/acetonitrile). Measure the absorbance at 230 nm38.</li>
</ol>
7. Drug loading capacity (DL) measurement and in vitro drug release kinetics
<ol>
	<li>Drug loading capacity (DL) 
	NOTE: The amount of dopamine loaded into exosomes is quantified using UV-Vis spectroscopy. The absorbance is read at 280 nm. The collected supernatants during synthesis are used to measure the amount of unloaded drug. The dopamine concentration in the supernatant is determined via a standard calibration curve for dopamine.
	<ol>
		<li>Prepare a stock solution of 1 mg/mL of dopamine. Prepare concentrations of 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg/mL from the stock solution using distilled water. 
		NOTE: Add 0.02% ascorbic acid for the stability of the prepared dopamine solution.</li>
		<li>Generate a standard calibration curve by measuring the absorbance of each dilution of dopamine at 280 nm in a UV-Spectrophotometer.</li>
		<li>Measure the absorbance of the supernatant at 280 nm.</li>
		<li>Calculate the drug loading capacity for dopamine using Eq (1)39. 
		DL (%) = <img alt="Equation 1" src="/files/ftp_upload/63624/63624eq01.jpg" style="margin: auto;" /> 100 (1) 
		NOTE: The analysis is performed in triplicate.</li>
	</ol>
	</li>
	<li>In vitro drug release kinetics 
	NOTE: Drug release kinetics of dopamine-loaded exosomes are performed using a dialysis membrane. PBS, pH 7.4, is used as the release medium to simulate a physiological microenvironment.
	<ol>
		<li>Add 1 mL of dopamine-loaded exosomes to the dialysis membrane. Place the membrane in a beaker. Add 15 mL of PBS to the beaker.</li>
		<li>Sample 1 mL of release medium in a beaker at 0.5, 1, 2, 4, 6, and 8 h. At each time point, replace the volume of the sample with fresh PBS. Analyze the samples taken at specified intervals at 280 nm using a UV-Vis spectrometer.</li>
		<li>Calculate the results using Eq (2)40. 
		Release (%) = <img alt="Equation 2" src="/files/ftp_upload/63624/63624eq02.jpg" style="margin: auto;" /> &#160;100&#160; (2) 
		NOTE: A calibration curve is prepared for the determination of in vitro drug release kinetics. Dopamine solution is prepared at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, and 5.0 mg/mL concentrations. The UV absorbance value of each concentration is determined at 280 nm with a UV-Vis spectrophotometer.</li>
	</ol>
	</li>
</ol>
8. In vitro cytotoxicity test
<ol>
	<li>Revitalization and culture of fibroblasts
	<ol>
		<li>Remove the fibroblasts from the -80 &#176;C freezer. Seed the cells into a flask containing DMEM-F12 + 10% FBS.</li>
		<li>Incubate the cells at 37 &#176;C in an incubator containing 5% CO2. Change the culture medium every 2 days.</li>
		<li>Passage the cells until they reach 80% confluence. Follow steps 1.3-1.5. After centrifugation at 1,500 &#215; g for 5 min, remove the supernatant, add 1 mL of DMEM-F12 + 10% FBS, and seed 10,000 cells per well in a 96 well plate.
		<ol>
			<li>Add DMEM-F12 medium + 10% FBS and incubate the cells at 37 &#176;C for 18 h in an incubator containing 5% CO2. Change the medium of the wells at the end of the incubation.</li>
		</ol>
		</li>
		<li>Prepare the stock of dopamine-loaded exosome suspension. Dilute the dopamine-loaded exosome formulation with medium to obtain concentrations of 100 &#181;L/mL, 250 &#181;L/mL, 500 &#181;L/mL, and 1,000 &#181;L/mL.</li>
		<li>Add the prepared concentrations onto the cells within each well of the 96-well plate. Incubate the plates at 37 &#176;C for 18 h in an incubator containing 5% CO241.</li>
	</ol>
	</li>
	<li>MTT cell viability assay 
	NOTE: At the end of the incubation, the viability ratios of cells are determined by the MTT test.
	<ol>
		<li>Prepare the MTT solution (5 mg/mL) with PBS. Add 10 &#181;L of MTT solution to each well. Incubate for 4 h at 37 &#176;C.</li>
		<li>At the end of the incubation, add 100 &#181;L of DMSO to each well. Incubate the plates for 30 min at room temperature in the dark. Measure the absorbance values in ELISA reader at 570 nm42,43. 
		NOTE: The MTT viability test is performed in triplicate.</li>
	</ol>
	</li>
</ol>

The authors declare that they have no competing financial interests.

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><tbody><tr><td>0.22 &amp;micro;m membrane filter</td><td>Aisimo</td><td></td><td>Used for the sterilization process</td></tr><tr><td>0.45 &amp;micro;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 cm&lt;sup&gt;2&lt;/sup&gt; 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 cm&lt;sup&gt;2&lt;/sup&gt; 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>CO&lt;sub&gt;2&lt;/sub&gt; 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&#039;s Modified Eagle&#039;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 &amp;deg;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 &amp;deg;C-Senova</td><td>Senova</td><td></td><td>Used in cell culture step</td></tr><tr><td>Wharton&amp;rsquo;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 ...

formulating-characterizing-an-exosome-based-dopamine-carrier

Research

JoVE Journal

Bioengineering

3.0K Views.  Altinbas University. Exosomen kunnen reageren op blaasjes die worden uitgescheiden door een cel tussen 14 en 200 nanometer groot, en splitsen zich af in de intracellulaire lading en communicatie, die naar de kleinste subgroep van extracellulaire blaasjes gaat. Exosomen die ontstaan als gevolg van de differentiatie van het endosoom, zijn afkomstig uit het celmembraan. De stamcellen kunnen worden geïsoleerd uit verschillende bronnen, zoals beenmerg of vetweefsel.maar gedroogde stamcellen van volwassen weefs...

Video: Formulating and Characterizing an Exosome-based Dopamine Carrier System

Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is terminated by diffusion2-3, extracellular enzymes4, and membrane transporters5. The dopamine transporter, located in the peri-synaptic cleft of dopamine neurons clears the released amines through an inward dopamine flux (uptake). The dopamine transporter can also work in reverse direction to release amines from inside to outside in a process called outward transport or efflux of dopamine5. More than 20 years ago Sulzer et al. reported the dopamine transporter can operate in two modes of activity: forward (uptake) and reverse (efflux)5. The neurotransmitter released via efflux through the transporter can move a large amount of dopamine to the extracellular space, and has been shown to play a major regulatory role in extracellular dopamine homeostasis6. Here we describe how simultaneous patch clamp and amperometry recording can be used to measure released dopamine via the efflux mechanism with millisecond time resolution when the membrane potential is controlled. For this, whole-cell current and oxidative (amperometric) signals are measured simultaneously using an Axopatch 200B amplifier (Molecular Devices, with a low-pass Bessel filter set at 1,000 Hz for whole-cell current recording). For amperometry recording a carbon fiber electrode is connected to a second amplifier (Axopatch 200B) and is placed adjacent to the plasma membrane and held at +700 mV. The whole-cell and oxidative (amperometric) currents can be recorded and the current-voltage relationship can be generated using a voltage step protocol. Unlike the usual amperometric calibration, which requires conversion to concentration, the current is reported directly without considering the effective volume7. Thus, the resulting data represent a lower limit to dopamine efflux because some transmitter is lost to the bulk solution.

The amperometric technique measures dopamine release from a single cell by detecting the oxidative current produced by spontaneous dopamine oxidization. Simultaneous voltage clamp and amperometry methodology reveal the mechanistic relationship between the overall "activity" of dopamine transporter and the regulatory role of this activity on the reverse transport of dopamine.

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is ...

Neuroscience

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

Preparation and Characterization of Lipophilic Doxorubicin Pro-drug Micelles

Micelles have been successfully used for the delivery of anticancer drugs. Amphiphilic polymers form core-shell structured micelles in an aqueous environment through self-assembly. The hydrophobic core of micelles functions as a drug reservoir and encapsulates hydrophobic drugs. The hydrophilic shell prevents the aggregation of micelles and also prolongs their systemic circulation in vivo. In this protocol, we describe a method to synthesize a doxorubicin lipophilic pro-drug, doxorubicin-palmitic acid (DOX-PA), which will enhance drug loading into micelles. A pH-sensitive hydrazone linker was used to conjugate doxorubicin with the lipid, which facilitates the release of free doxorubicin inside cancer cells. Synthesized DOX-PA was purified with a silica gel column using dichloromethane/methanol as the eluent. Purified DOX-PA was analyzed with thin layer chromatography (TLC) and 1H-Nuclear Magnetic Resonance Spectroscopy (1H-NMR). A film dispersion method was used to prepare DOX-PA loaded DSPE-PEG micelles. In addition, several methods for characterizing micelle formulations are described, including determination of DOX-PA concentration and encapsulation efficiency, measurement of particle size and distribution, and assessment of in vitro anticancer activities. This protocol provides useful information regarding the preparation and characterization of drug-loaded micelles and thus will facilitate the research and development of novel micelle-based cancer nanomedicines.

A protocol for the preparation and characterization of lipophilic doxorubicin pro-drug loaded 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG) micelles is described.

Micelles have been successfully used for the delivery of anticancer drugs. Amphiphilic polymers form core-shell structured micelles in an aqueous ...

Phase Change Dimethyldioctadecylammonium-Shelled Microdroplets as a Promising Drug Delivery System: Results on 3D Spheroids of Mammalian Tumor Cells

Significant improvement of phase-change perfluorocarbon microdroplets (MDs) in the vast theranostic scenario passes through the optimization of the MDs composition with respect to synthesis efficiency, stability, and drug delivery capability. To this aim, decafluoropentane (DFP) MDs stabilized by a shell of dimethyldioctadecylammonium bromide (DDAB) cationic surfactant were designed. A high concentration of DDAB-MDs was readily obtained within a few seconds by pulsed high-power insonation, resulting in low polydisperse 1 &#181;m size droplets. Highly positive &#950;-potential, together with a long, saturated hydrocarbon chains of the DDAB shell, are key factors to stabilize the droplet and the drug cargo therein. The high affinity of the DDAB shell with cell plasma membrane allows for localized chemotherapeutics delivery by increasing the drug concentration at the tumor cell interface and boosting the uptake. This would turn DDAB-MDs into a relevant drug delivery tool exhibiting high antitumor activity at very low drug doses.
In this work, the efficacy of such an approach is shown to dramatically improve the effect of doxorubicin against 3D spheroids of mammalian tumor cells, MDA-MB-231. The use of&#160;three-dimensional (3D) cell cultures developed in the form of multicellular tumor spheroids (i.e., densely packed cells in a spherical shape) has numerous advantages compared to 2D cell cultures: in addition to have the potential to bridge the gap between conventional in vitro studies and animal testing, it will improve the ability to perform more predictive in vitro screening assays for preclinical drug development or evaluate the potential of off-label drugs and new co-targeting strategies.

Decafluoropentane microdroplets developed with a shell of dimethyldioctadecylammonium bromide exhibited an exceptional colloidal stability and an actractive biointerface. DDAB-MDs proved to be efficient drug reservoirs characterized by a high affinity to plasma membranes together with enhanced uptake and antitumor activity of Doxorubicin against human triple-negative breast cancer (MDA-MB-231) 3D model.

Significant improvement of phase-change perfluorocarbon microdroplets (MDs) in the vast theranostic scenario passes through the optimization of the MDs ...

Chemistry

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Strategies for Expressing and Purifying Solute Carrier Transporters

High-Throughput Expression and Purification of Human Solute Carriers for Structural and Biochemical Studies

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, metabolites, neurotransmitters, and pharmaceuticals. Despite having emerged as attractive therapeutic targets and markers of disease, this group of proteins is still relatively underdrugged by current pharmaceuticals. Drug discovery projects for these transporters are impeded by limited structural, functional, and physiological knowledge, ultimately due to the difficulties in the expression and purification of this class of membrane-embedded proteins. Here, we demonstrate methods to obtain high-purity, milligram quantities of human SLC transporter proteins using&#160;codon-optimized gene sequences. In conjunction with a systematic exploration of construct design and high-throughput expression, these protocols ensure the preservation of the structural integrity and biochemical activity of the target proteins. We also highlight critical steps in the eukaryotic cell expression, affinity purification, and size-exclusion chromatography of these proteins. Ultimately, this workflow yields pure, functionally active, and stable protein preparations suitable for high-resolution structure determination, transport studies, small-molecule engagement assays, and high-throughput in vitro screening.

Author Spotlight: Expression and Purification of Human Solute Carrier Transporters Using Codon-Optimized Genes 

Structural and biochemical studies of human membrane transporters require milligram quantities of stable, intact, and homogeneous protein. Here we describe scalable methods to screen, express, and purify human solute carrier transporters using codon-optimized genes.

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, ...

Biochemistry

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...

Assessment of Dopaminergic Homeostasis in Mice by Use of High-performance Liquid Chromatography Analysis and Synaptosomal Dopamine Uptake

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) neurotransmission is strongly associated with several central nervous system-associated diseases such as Parkinson&#39;s disease, attention-deficit-hyperactivity disorder and drug addiction1,2,3,4. Delineating disease mechanisms involving DA imbalance is critically dependent on animal models to mimic aspects of the diseases, and thus protocols that assess specific parts of the DA homeostasis are important to provide novel insights and possible therapeutic targets for these diseases.
Here, we present two useful experimental protocols that when combined provide a functional read-out of the DAergic system in mice. Biochemical and functional parameters on DA homeostasis are obtained through assessment of DA levels and dopamine transporter (DAT) functionality5. When investigating the DA system, the ability to reliably measure endogenous levels of DA from adult brain is essential. Therefore, we present how to perform high-performance liquid chromatography (HPLC) on brain tissue from mice to determine levels of DA. We perform the experiment on tissue from dorsal striatum (dStr) and nucleus accumbens (NAc), but the method is also suitable for other DA-innervated brain areas.
DAT is essential for reuptake of DA into the presynaptic terminal, thereby controlling the temporal and spatial activity of released DA. Knowing the levels and functionality of DAT in the striatum is of major importance when assessing DA homeostasis. Here, we provide a protocol that allows to simultaneously deduce information on surface levels and function using a synaptosomal6 DA uptake assay.
Current methods combined with standard immunoblotting protocols provide the researcher with relevant tools to characterize the DAergic system.

Synaptosomal dopamine uptake and high-performance liquid chromatography analysis represent experimental tools to investigate dopamine homeostasis in mice by assessing the function of the dopamine transporter and levels of dopamine in striatal tissue, respectively. Here we present protocols to measure dopamine tissue content and assess the functionality of the dopamine transporter.

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) ...

Experimental Quantification of Interactions Between Drug Delivery Systems and Cells In Vitro: A Guide for Preclinical Nanomedicine Evaluation

A major component of designing drug delivery systems concerns how to amplify or attenuate interactions with specific cell types. For instance, a chemotherapeutic might be functionalized with an antibody to enhance binding to cancer cells (&#34;targeting&#34;) or functionalized with polyethylene glycol to help evade immune cell recognition (&#34;stealth&#34;). Even at a cellular level, optimizing the binding and uptake of a drug carrier is a complex biological design problem. Thus, it is valuable to separate how strongly a new carrier interacts with a cell from the functional efficacy of a carrier&#39;s cargo once delivered to that cell.
To continue the chemotherapeutic example, &#34;how well it binds to a cancer cell&#34; is a separate problem from &#34;how well it kills a cancer cell&#34;. Quantitative in vitro assays for the latter are well established and usually rely on measuring viability. However, most published research on cell-carrier interactions is qualitative or semiquantitative. Generally, these measurements rely on fluorescent labeling of the carrier and, consequently, report interactions with cells in relative or arbitrary units. However, this work can be standardized and be made absolutely quantitative with a small number of characterization experiments. Such absolute quantification is valuable, as it facilitates rational, inter- and intra-class comparisons of various drug delivery systems-nanoparticles, microparticles, viruses, antibody-drug conjugates, engineered therapeutic cells, or extracellular vesicles.
Furthermore, quantification is a prerequisite for subsequent meta-analyses or in silico modeling approaches. In this article, video guides, as well as a decision tree for how to achieve in vitro quantification for carrier drug delivery systems, are presented, which take into account differences in carrier size and labeling modality. Additionally, further considerations for the quantitative assessment of advanced drug delivery systems are discussed. This is intended to serve as a valuable resource to improve rational evaluation and design for the next generation of medicine.

Experimental Quantification of Interactions Between Drug Delivery Systems and Cells In Vitro: A Guide for Preclinical Nanomedicine Evaluation

A workflow is demonstrated for the absolute quantification of drug carrier-cell interactions using flow cytometry to allow better rational evaluation of novel drug delivery systems. This workflow is applicable to drug carriers of any type.

A major component of designing drug delivery systems concerns how to amplify or attenuate interactions with specific cell types. For instance, a ...

Microneedle Patches for Sustained Delivery of Exosomes in Biomedical Applications

Fabrication and Characterization of Microneedle Patches for Loading and Delivery of Exosomes

Exosomes, as emerging &#34;next-generation&#34; biotherapeutics and drug delivery vectors, hold immense potential in diverse biomedical fields, ranging from drug delivery and regenerative medicine to disease diagnosis and tumor immunotherapy. However, the rapid clearance by traditional bolus injection and poor stability of exosomes restrict their clinical application. Microneedles serve as a solution that prolongs the residence time of exosomes at the administration site, thereby maintaining the drug concentration and facilitating sustained therapeutic effects. In addition, microneedles also possess the ability to maintain the stability of bioactive substances. Therefore, we introduce a microneedle patch for loading and delivering exosomes and share the methods, including isolation of exosomes, fabrication, and characterization of exosome-loaded microneedle patches. The microneedle patches were fabricated using trehalose and hyaluronic acid as the tip materials and polyvinylpyrrolidone as the backing material through a two-step casting method. The microneedles demonstrated robust mechanical strength, with tips able to withstand 2 N. Pig skin was used to simulate human skin, and the tips of microneedles completely melted within 60 s after skin puncture. The exosomes released from the microneedles exhibited morphology, particle size, marker proteins, and biological functions comparable to those of fresh exosomes, enabling dendritic cells uptake and promoting their maturation.

Author Spotlight: Innovative Microneedle-Based Strategies for Enhanced Exosome Delivery and Stability

Exosomes possess significant clinical potential, but their practical application is limited due to easy in vivo clearance and poor stability. Microneedles present a solution by enabling localized delivery by puncturing physiological barriers and dry-state preservation, thereby addressing the limitations of exosome administration and expanding their clinical utility.

Exosomes, as emerging &#34;next-generation&#34; biotherapeutics and drug delivery vectors, hold immense potential in diverse biomedical fields, ranging ...

Formulating and Characterizing an Exosome-based Dopamine Carrier System

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