Exosome is one of extracellular vesicles secreted by most cells. It has 40-200 nm size and contains lipids, proteins, and RNAs. Exosome can transport its cargo to target cells and is considered to be a key player in intercellular communication. Therefore, potential application of exosome to drug delivery system has attracted considerable interest in recent years. Especially macrophage-derived exosome is known to cross blood-brain barrier and deliver the cargo after intravenous administration. However, it has been challenging to load enough amount of drugs for therapeutic efficacy into exosome without changing original characteristics of exosome, because the drug loading adds physicochemical stress to it.
Purpose of this study is to develop protein-loading method into exosome with high efficiency while keeping innate exosome quality, such as particle-size distribution.
IC-21 (mouse macrophage) cells were cultivated in media containing fetal bovine serum (FBS). FBS-derived exosome had been removed before the cell culture. Exosome was isolated from IC-21 by an ultracentrifuge method. Briefly, cell debris and large vesicles were removed by sequential centrifugation at 1,000 g for 10 min and 10,000 g for 60 min. Supernatant was spun at 120,000 g by ultracentrifuge and pellet was collected. This ultracentrifuge step was repeated twice. The pelleted exosome was resuspended in buffer at specific pH. Particle-size distribution and particle number of the exosome were measured by Nanoparticle Tracking Analysis (NTA). Total protein amount of the exosome was determined using Pierce™ BCA Protein Assay Kit. Expression of exosome-marker protein was confirmed by Simple Western after lysing the exosome and denaturing the proteins under a reducing condition.
Protein drug, leptin, was loaded into exosome by using a probe sonicator, 505 Sonic Dismembrator at 500 W and 2 kHz. The exosome suspension (3-6 x 1011 particle/mL, 250 μL) and leptin solution (0.5-4.0 mg/mL, 125 μL) was mixed and sonicated at 20% amplitude and 6 cycles by 4 s pulse/2 s pause. Then, the mixture was cooled on ice and sonicated in the above-mentioned conditions again. The sample was incubated at 37ºC for 30 min to allow for recovery of the exosomal membrane. Free leptin was removed by gel filtration chromatography. The incubated sample (375 μL) was applied to a column filled with Sepharose® CL-6B and eluted with PBS (600 μL) by gravity flow. Loaded amount of leptin was measured by Enzyme-Linked ImmunoSorbent Assay (ELISA). The exosome suspension was treated with RIPA buffer to lyse the exosomal membrane and collect the leptin before the ELISA. The effect of pH in exosome suspension, cooling conditions during the sonication, and feed leptin concentration on drug-loading efficiency were investigated.
Release profile of leptin from the exosome was evaluated in PBS and mouse serum at 37ºC. Released amount of leptin was determined by ELISA. Particle stability of the exosome stored at 4ºC was also investigated using NTA.
IC-21 derived exosome had 100-110 nm of mean size and 80-90 nm of mode size based on NTA data. It also contained exosomal markers, such as Alix (ESCRT-interacting protein related to formation of multivesicular body) and Rab11A (a protein required to release exosome from multivesicular body).
Leptin-loaded exosome was prepared by the sonication method in various conditions. Drug-loading efficiency of leptin was increased with a decrease of pH from 7.1 to 5.5. The highest efficiency would be due to positive charge of leptin at pH5.5 because isoelectric point of leptin is ~5.8 and exosome has negative charge on its surface. Although many aggregates were observed after the sonication at pH5.5, cooling the sample during the sonication could avoid an increase in its particle size and aggregation of the exosome. The size distribution of the leptin-loaded exosome in this condition was similar to that of bare exosome. Drug-loading efficiency was around 10% at pH5.5 and this value was not significantly changed even if feed leptin concentration was increased to 4 mg/mL. Leptin concentration in exosome suspension reached to ~60 μg/mL in the best condition.
Although ~10% and ~50% of leptin burst from the exosome after dilution with PBS and mouse serum, respectively, the other leptin was considered to be retained in the exosome within 24 hours at 37ºC in the release study. Particle-size distribution and particle number of the leptin-loaded exosome were stable at 4ºC for at least 1 month.
Approximately 10% of leptin was loaded into macrophage-derived exosome without changing its original size by the sonication method under optimized conditions, such as pH, temperature, and drug concentration. This methodology is promising strategy for drug delivery application of exosome to protein drugs.