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 lip....

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: Wash....

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