Formulating and Characterizing an Exosome-based Dopamine Carrier System

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 exosomes^4,5. Exosomes are also involved in processes such as synaptic plasticity, neuronal stress response, cell-cell communication, and neurogenesis in the brain^6,7. 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 day^11,12,13. It is known that PD is associated with degeneration of dopaminergic neurons in the substantia nigra of the mesencephalon and loss of motor neuron functions^14,15. 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 tremor^12,13. 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 regeneration^21,22,23. In addition, it has been reported that MSC-derived exosome secretomes reduce apoptosis by showing neurotrophic and neuroprotective effects, especially on dopaminergic neurons^24,25. 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 organs^26,27. 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 systems^28,29,30. 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 incubation^30,34. 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.

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: 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 (%) = 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 (%) =  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 nm^42,43.
      NOTE: The MTT viability test is performed in triplicate.

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...

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 fusion^33,44. Compared to other nanocarrier systems, the lipids and cholesterol found on the exosome surface confer the ability to carry both hydrophilic a...

Materials

List of materials used in this article

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