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JoVE Journal Bioengineering

Bioengineering

Formulating and Characterizing an Exosome-based Dopamine Carrier System

Published: April 4, 2022 doi: 10.3791/63624
Automatic Translation
1,2, 3,4,5,6, 2,7, 2, 2, 8, 8, 3,4,5,6,9
1Vocational School of Health Services/İstanbul, Altinbas University, 2Chemical Metallurgy Faculty, Department of Bioengineering, Yildiz Technical University, 3Faculty of Medicine, Department of Histology & Embryology, Istinye University, 43D Bioprinting Design & Prototyping R&D Center, Istinye University, 5Stem Cell and Tissue Engineering R&D Center, Istinye University, 6Medicine Faculty, Istinye University, 7Vocational School of Health Services, Istinye University, 8The V. Akhundov National Scientific Research Medical Prophylactic Institute, 9Center for Stem Cells and Regenerative Medicine (LivMedCell), Liv Hospital

Summary

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.

Abstract

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.

Introduction

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'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 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, 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.

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Protocol

NOTE: See the Table of Materials for details related to all materials and equipment used in this protocol.

1. Culturing and cryopreservation of Wharton's jelly mesenchymal stem cells

  1. Remove the WJ-MSCs (from ATCC) from the -80 °C freezer. Seed the cells into a flask containing DMEM-F12 medium supplemented with 10% fetal bovine serum (FBS). Incubate them at 37 °C in an incubator containing 5% CO2.
  2. 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.
  3. Remove the PBS and add 5 mL of trypsin-EDTA solution to the flask. Incubate the flask for 5 min at 37 °C in an incubator containing 5% CO2.
  4. 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.
  5. 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 °C.

2. Production of exosomes from Wharton'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.

  1. 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 °C in a 5% CO2 incubator. Following incubation, collect the serum-free medium in the tube and store at -20 °C.
    NOTE: In step 2.1, 180 mL of serum-free medium is collected and stored at -20 °C. After thawing, a batch centrifugation process is started, as described below.
  2. Isolation of exosomes
    1. Centrifuge the collected serum-free medium at 300 x g for 10 min at +4 °C. Carefully withdraw the supernatant and transfer it to another tube.
    2. Centrifuge the collected supernatant at 2,000 × g for 10 min at +4 °C. Carefully withdraw the supernatant and transfer it to another tube.
    3. Centrifuge the collected supernatant at 10,000 × g for 30 min at +4 °C. Carefully withdraw the supernatant and transfer it to another tube.
    4. Transfer the collected supernatant to ultracentrifuge tubes and ultracentrifuge at 110,000 × g for 130 min. Slowly remove the supernatant and add 1 mL of PBS to the pellet.
    5. Vortex the pellet and ultracentrifuge at 110,000 × g for 70 min at +4 °C36​ (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 °C. The obtained pellet is now ready for characterization.

Figure 1
Figure 1: Exosome isolation. Please click here to view a larger version of this figure.

3. Characterization of exosomes

  1. 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.
    1. Dilute the exosomes 100-fold by adding 990 µL of PBS to 10 µL of exosomes. Take the diluted suspension into a disposable syringe.
    2. Turn on the NTA device and connect the computer. Open the software.
    3. Inject the sample in the syringe into the tubing in the cassette section of the device. Close the cassette cover of the device.
    4. In the software that opens, click on the Start Analysis button. Save the results obtained by pressing the Record button.
  2. Dynamic light scattering analysis
    NOTE: Exosomes isolated for zeta potential and size measurement are also diluted in the same manner as for NTA analysis.
    1. Add 980 µL of PBS to 20 µL of the exosomes.
    2. Open the zeta sizing device and the connected computer. Open the software.
    3. 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.
    4. Select the cuvette type in the device'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.

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.

  1. Add 3 mL of PBS to the exosome suspension from step 2.2.5. Sterilize the diluted suspension by filtration using 0.22 µm filters. Transfer 500 µL of the sterilized exosome suspension to another tube.
  2. Prepare dopamine solution (0.5 mg/mL) with distilled water. Add 500 µL of dopamine solution into the tubes containing the exosomes.
  3. Add saponin to the suspension in step 4.2. Incubate the prepared exosome-dopamine suspension for 24 h at 37 °C.
    NOTE: Saponin concentration should not exceed 0.002% of the total solution.
  4. Ultracentrifuge the suspension at 90,000 × g for 70 min to remove free dopamine and saponin from the medium. Store the isolated exosomes at -20 °C until use for further analysis37.
    NOTE: Add 0.02% ascorbic acid for the stability of the prepared dopamine solution.

5. Characterization of dopamine loaded exosomes

  1. Nanoparticle tracking analysis (NTA)
    NOTE: Nanoparticle tracking analysis is performed to determine the size and concentration of isolated exosomes.
    1. Follow steps 3.1.1-3.1.4 to dilute the exosomes and perform NTA.
  2. Dynamic light scattering analysis
    NOTE: Exosomes isolated for zeta potential and size measurement are also diluted similarly to NTA analysis.
    1. 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.

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.

  1. Put the formulation in a 75 °C heater to evaporate. Add acetonitrile (50:50 ratio) to the suspension and vortex. Sonicate the solution for 10 min.
  2. Centrifuge the solution for 10 min at 10,000 × g. Filter the supernatant through a 0.22 µm filter.
  3. Analyze all analytes using a C18 column at 30 °C at a flow rate of 1 mL/min (the mobile phase H2O/acetonitrile). Measure the absorbance at 230 nm38.

7. Drug loading capacity (DL) measurement and in vitro drug release kinetics

  1. 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.
    1. 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.
    2. Generate a standard calibration curve by measuring the absorbance of each dilution of dopamine at 280 nm in a UV-Spectrophotometer.
    3. Measure the absorbance of the supernatant at 280 nm.
    4. Calculate the drug loading capacity for dopamine using Eq (1)39.
      DL (%) = Equation 1 100 (1)
      NOTE: The analysis is performed in triplicate.
  2. 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.
    1. 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.
    2. 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.
    3. Calculate the results using Eq (2)40.
      Release (%) = Equation 2  100  (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.

8. In vitro cytotoxicity test

  1. Revitalization and culture of fibroblasts
    1. Remove the fibroblasts from the -80 °C freezer. Seed the cells into a flask containing DMEM-F12 + 10% FBS.
    2. Incubate the cells at 37 °C in an incubator containing 5% CO2. Change the culture medium every 2 days.
    3. Passage the cells until they reach 80% confluence. Follow steps 1.3-1.5. After centrifugation at 1,500 × 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.
      1. Add DMEM-F12 medium + 10% FBS and incubate the cells at 37 °C for 18 h in an incubator containing 5% CO2. Change the medium of the wells at the end of the incubation.
    4. Prepare the stock of dopamine-loaded exosome suspension. Dilute the dopamine-loaded exosome formulation with medium to obtain concentrations of 100 µL/mL, 250 µL/mL, 500 µL/mL, and 1,000 µL/mL.
    5. Add the prepared concentrations onto the cells within each well of the 96-well plate. Incubate the plates at 37 °C for 18 h in an incubator containing 5% CO241.
  2. MTT cell viability assay
    NOTE: At the end of the incubation, the viability ratios of cells are determined by the MTT test.
    1. Prepare the MTT solution (5 mg/mL) with PBS. Add 10 µL of MTT solution to each well. Incubate for 4 h at 37 °C.
    2. At the end of the incubation, add 100 µL 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.

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Representative Results

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 °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 µm filter. The average size of the obtained exosomes was determined to be 98 nm (Video 1). Approximately 10 billion exosome particles were obtained at the end of centrifugation. The numbers of nanoparticles revealed by NTA and DLS analyses are consistent with each other. In addition, the zeta potential of exosomes originating from WJ-MSCs was measured as -15.7 mV (Figure 2 and Figure 3).

Video 1: Nanoparticle tracking analysis of Wharton Jelly-derived exosomes. Please click here to download this Video.

Loading of dopamine into exosomes
As calculated in NTA analysis, there were approximately 1.5 billion nanoparticles in 500 µL of the isolated samples from Wharton's jelly. Dopamine solution (5 mg/mL) was mixed with 500 µL of exosomes, and the suspension was incubated for 24 h at 37 °C. Following incubation, the obtained solution was characterized by NTA and DLS analyses. According to the NTA results, the average particle size of the solution was found to be 110 nm. A comparison of the particle sizes of exosomes and dopamine-loaded exosomes reveals that there was a significant increase in the dimensions of the dopamine-loaded exosomes. The numbers of nanoparticles revealed by NTA and DLS analyses were consistent with each other. In addition, the zeta potential of dopamine-loaded exosomes was measured as -17.6 Mv (Figure 2 and Figure 3).

Video 2: Nanoparticle tracking analysis of dopamine-loaded exosomes. Please click here to download this Video.

Sample Zeta potential
Exosome -15.7 mV ± 1.1
Saponin -12 mV ± 0.7
Dopamine -14.4 mV ± 1.3
Dopamine- loaded exosome -17.6 mV ± 0.8

Table 1: Zeta potential of all solutions.

DSL analyzes, the Zeta sizer, and Zeta potential table of the dopamine, saponin, and dopamine-loaded exosome solution are shown in Table 1.

Figure 2
Figure 2: Nanoparticle tracking analysis of exosomes. (A) Exosomes; (B) dopamine-loaded exosomes. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Zetasizer analysis. (A) Exosomes, (B) dopamine-loaded exosomes, (C) Saponin, (D) Dopamine. Abbreviation: d = size. Please click here to view a larger version of this figure.

The presence of dopamine in the solution was determined by HPLC analysis. Dopamine-loaded exosomes were ultracentrifuged and filtered to remove free dopamine. If dopamine loading into exosomes is successful at the end of incubation, it can be detected by direct measurement of dopamine following the rupture of the exosomes. For this purpose, the dopamine-loaded exosome suspension was heated with steam, acetonitrile added, and the suspension was sonicated. Absorbance was measured at 230 nm to assess the amounts of dopamine released from the disrupted exosomes.

Figure 4
Figure 4: Presence of dopamine in the formulation determined by HPLC analysis. All analyses were performed using a C-18 column with a mobile phase of H2O/Acetonitrile at a flow rate of 1 mL/min at 30 °C. Absorbance is measured at 230 nm to monitor the elution of dopamine. (A) Dopamine, (B) Saponin, (C) Dopamine-loaded exosomes. Abbreviation: HPLC = high-performance liquid chromatography. Please click here to view a larger version of this figure.

As a result of HPLC analysis, the resulting peak for dopamine was observed at 6.45 min. The presence of saponin was also detected after exosome fragmentation (Figure 4).

Drug loading capacity and in vitro drug release kinetics
Dopamine loading capacity was calculated using UV spectrophotometry measurements and Eq (1) and Eq (2). Loading capacity percentage was found to be 10.89 ± 0.33. The cumulative drug release profile is shown in Figure 5. Drug release kinetics monitored for 8 h revealed that 74.8% of encapsulated dopamine was released from exosomes within the first 8 h (Figure 5).

Figure 5
Figure 5: Cumulative drug release profile. Please click here to view a larger version of this figure.

In vitro cytotoxicity test
The cytotoxicity of dopamine-loaded and free exosomes were investigated on fibroblasts. Fibroblasts were exposed to different concentrations of formulations (100 µL/mL, 250 µL/mL, 500 µL/mL, and 1,000 µL/mL) and incubated for 18 h. Although dopamine-loaded exosomes did not demonstrate any remarkable cytotoxic effects, saponin added to the formulation to increase the encapsulation of dopamine decreased fibroblast viability (p < 0.05, Figure 6). There was an increase in cell viability when the fibroblasts were treated with dopamine-loaded exosomes up to 500 µL/mL concentration. At the end of the 18 h incubation, cell viability was determined by the MTT test (Figure 7). The statistical significance of the results was evaluated by performing one-way ANOVA.

Figure 6
Figure 6: Microscope images (10x) after incubation of the formulation with the cells. (A) Dopamine, (B) Saponin, (C) Dopamine-loaded exosomes 100 µL/mL, (D) Dopamine-loaded exosomes 250 µL/mL, (E) Dopamine-loaded exosomes 500 µL/mL, (F) Dopamine-loaded exosomes 1,000 µL/mL. Scale bars = 100 µm. Please click here to view a larger version of this figure.

Figure 7
Figure 7: Statistical analysis of MTT viability test results in cells incubated for 18 h with different concentrations of dopamine, saponin, and dopamine-loaded exosomes. (A) Cytotoxicity results, (B,C) Statistical analyses. Abbreviations: D = Dopamine; S = Saponin; Exo-DA1 = Dopamine-loaded exosomes 100 µL/mL; Exo-DA2 = Dopamine-loaded exosomes 250 µL/mL; Exo-DA 3 Dopamine-loaded exosomes 500 µL/mL; Exo-DA 4 = Dopamine-loaded exosomes 1,000 µL/mL; MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Please click here to view a larger version of this figure.

Additionally, the cytotoxic effects of free exosomes were also investigated on fibroblasts at similar concentrations (Figure 8). Before the experiment, exosomes were diluted with PBS to 60 million particles per mL. Then, 10 µL, 25 µL, 50 µL, and 100 µL of the exosome suspension was added to each well of a 96-well plate.

Figure 8
Figure 8: Statistical analysis of MTT viability test results in cells incubated for 18 h with different concentrations of exosomes. (A) Cytotoxicity results, (B,C) statistical analyses. Abbreviations: EXO = exosomes; MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Please click here to view a larger version of this figure.

Fibroblast viability was found to be higher with 500 µL/mL dopamine-loaded exosomes and 25 µL/mL free exosomes compared to other concentrations. However, no significant cytotoxicity was observed at any concentration.

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Discussion

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 and hydrophobic molecules, with the hydrogen bonding of the cholesterol ends allowing tight packaging45.

The isolation of MSC-derived exosomes, drug loading experiments, and cytotoxicity assays were performed in the present study. It is known that exosomes have an important role in neurogenesis, particularly in the brain, in addition to their significant functions such as cargo and communication between cells6,7. Furthermore, MSC-derived exosomes are relatively well tolerated, have inherent therapeutic properties, and show high stability and homing ability46,47. In recent years, research on nanoscale treatment methods such as exosome applications have attracted extensive attention as alternative treatments against neurodegenerative diseases since there is no cure for these disorders caused by neuronal damage. In addition, the ability of exosomes to pass through the BBB makes them excellent platforms as a drug carrier system.

Although there are several methods for exosome isolation, herein, we used the ultracentrifuge isolation method, which is accepted as one of the most effective methods. In the current study, WJ-MSCs, selected as donor cells, are known for their low immunogenic potential47 and are used as a source of exosomes in clinical trials48. The obtained exosomes were then incubated with dopamine solution during which saponin is added to the solution to increase the success of drug loading. At the end of the incubation, free dopamine and saponin were removed from the medium. Saponin is expected to selectively remove the membrane-bound cholesterol of exosomes and create pores in the exosomal lipid bilayers, promoting the entry of dopamine molecules. As a result, it is observed that the dimensions of the exosomes increased slightly, probably because of dopamine loading.

The zeta potential shows greater electrostatic repulsion between particles and their tendency to aggregate. The zeta potential of the obtained formulation was compatible with the results of previous studies49. In the HPLC analysis performed at the end of the incubation period, the presence of dopamine was detected after fragmentation of the exosomes. As mentioned above, saponin promotes encapsulation of dopamine. The drug loading capacity was measured by UV-vis spectrophotometry based on absorbance values39. Although the incubation method for drug loading is simple and less expensive than other methods, the loading efficiency is low in this method26. In this study, the loading capacity was approximately 11%. Saponin is an effective permeabilizing agent for the cytoplasmic membrane50 and appears to increase the loading capacity of exosomes with dopamine.

In future studies, it may be more effective to prefer the sonication method to enhance exosomal drug loading efficiency38. The cumulative drug release profile revealed that large amounts of dopamine were released at the end of 8 h of incubation. An explosive release was observed in the first 5 h of the in vitro release study. Since the release profile of exosome formulations depends on many parameters such as drug properties, bilayer fluidity, and molecular weight, differences can be observed in the amounts of released drugs per hour in many studies51. In addition, no cytotoxic effects of the formulations were observed on fibroblasts in this study. However, the toxic effect of saponin added to increase dopamine loading into exosomes was detected on fibroblast cells. According to these data, the endogeneity of exosomes is a natural and unique advantage when compared to liposomes, microspheres, microemulsions, and other synthetic drug delivery systems52. To reduce the disadvantages of the current method, different transfection reagents, electroporation techniques, or endogenous dopamine-producing cells, preferably genetically modified MSCs, can be used as the source of exosomes.

Exosome isolation was conducted with this protocol. However, the isolation step is a very long and difficult process. Care should be taken to ensure that the supernatant collected from cell culture is in the serum-free medium. In addition, to date, the amounts of exosomes have been associated with protein concentration. However, we propose that calculating and applying the number of exosome particles can help increase accuracy in clinical studies. The incubation method used for drug loading of isolated exosomes was found to be low in terms of drug loading capacity. The sonication method, which has a high drug loading capacity, may help obtain more effective results.

These findings showed that WJ-MSC-derived exosomes are powerful carriers for dopamine delivery. Along with their ability to pass the BBB and with their immunomodulatory properties, targeted therapies for Parkinson's or any other CNS diseases can be developed with drug-carrying exosomes. However, studies should be carried out in vivo and supported with clinical trials.

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Disclosures

The authors declare that they have no competing financial interests.

Acknowledgments

The work was primarily supported by research funding provided by Yıldız Technical University Scientific Research Projects (TSA-2021-4713).

Materials

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

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References

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Exosome-based Dopamine Carrier System Extracellular Vesicles Intercellular Cargo Communication Body Fluids Blood-brain Barrier Nanocarrier System Encapsulating Dopamine Wharton's Jelly Mesenchymal Stem Cells Drug Release Cytotoxicity Assays Fibroblast Viability
Formulating and Characterizing an Exosome-based Dopamine Carrier System
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Yavuz, B., Darici, H., Zorba Yildiz, … More

Yavuz, B., Darici, H., Zorba Yildiz, A. P., Abamor, E. Ş., Topuzoğullari, M., Bağirova, M., Allahverdiyev, A., Karaoz, E. Formulating and Characterizing an Exosome-based Dopamine Carrier System. J. Vis. Exp. (182), e63624, doi:10.3791/63624 (2022).

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