Molecular and Cellular Biochemistry

https://doi.org/10.1007/s11010-024-05191-x

Exosome isolation based on polyethylene glycol (PEG): a review

Qionglian Huang1 · Jue Wang1 · Hanjuan Ning1 · Weiwei Liu1 · Xianghui Han1

Received: 6 September 2024 / Accepted: 7 December 2024

© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2024

Abstract
Exosome acts as an outstanding biomarker for ongoing studies, diagnosis and prognosis of multiples diseases. Therefore,
the call for economically and efficiently isolating a large number of exosomes is an active area of investigation. However, to
date, the challenges including complex isolated procedure, uneconomical equipment, low protein content and distinct loss
in the particle number of exosomes etc. still encounter in exosome isolation. Polyethylene glycol (PEG)-induced exosome
isolation increasingly attracts wide attention of scientists. PEG precipitation reveals higher performance in the yield of
exosomes among multiple common isolation techniques. PEG-based precipitation is a temporarily low-purity, but inexpen-
sive, time-save, labor-less, convenient and high-yield technique to gain exosomes with high biological activities. Hence, the
PEG-based exosome isolation approach wins the endorsement of experimental workers. Herein, we summary the existing
knowledge on procedures of PEG-based exosomes separation from different biospecimens, the binding process of PEG to
exosomes, some notices, demerits, merits of PEG-based exosome isolation, and at last the advantages by combining PEG-
precipitation to other techniques for exosome isolation, with a view to eliciting profound insights for investigators who recruit
PEG for exosome separation, and advancing references for the standardization of PEG-based exosome isolation in future.

Keywords Exosome isolation · PEG precipitation · Advantages · Disadvantages · High-yield · Inexpensive

Abbreviations UF Ultrafiltration
CUC​ Cushioned-UC WB Western blot
DC Differential centrifugation
DG Density gradient centrifugation
EXM ExoMir™ Introduction
EXQ Exo-spin™ exosome precipitation
FBS Fetal bovine serum Exosome, with bilayer membrane, is a type of unique bio-
LPS Lipopolysaccharide logical container that carries nucleic acids, all kinds of pro-
NaCl Sodium chloride teins, lipids, sugar chains, phospholipids and transcription
NTA Nanoparticle tracking analysis factors etc. [1]. Exosomes present in various body fluids, i.e.,
PC Protamine chloride blood, sweat, tears, urine, swab, joint fluid, breast milk, and
PEG Polyethylene glycol saliva [2]. Secreted by living cells, exosomes play as crucial
PROSPR Protein organic solvent precipitation roles in intercellular communication to endow recipient cells
SC Sucrose cushion with exogenous properties in various biological processes.
SEC Size-exclusion chromatography What’s more, exosomes are dramatically essential players in
TEI Total exosome isolation complicating the development of various diseases, including
TEM Transmission electron microscopy the diagnosis and treatment of diseases on account of the
UC Ultracentrifugation abundant bio-cargoes that they carry [3, 4]. For example,
most researchers found that exosomes contributed to regu-
lating tissue healing, suppressing immune system as well
* Xianghui Han as tolerating treatment, and they performed other pivotal
[email protected] biological processes including cellular communication,
1
Institute of Chinese Traditional Surgery, Longhua Hospital,
growth, and differentiation [5]. Otherwise, exosomes have
Shanghai University of Traditional Chinese Medicine, been extensively subjected as drug carriers to treat cancer,
Shanghai, China

Vol.:(0123456789)

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 Molecular and Cellular Biochemistry

neurodegenerative diseases, and hemiplegia [6–8]. Accord- for 5–10 min to deplete dead cells and cellular debris. Next,
ingly, on account of the extensively assessed bio-function the acquired supernatant is centrifugated at 2000–6800×g
of exosomes, economically and efficiently harvesting a for 10–45 min to deplete larger vesicles and apoptotic vesi-
large quantity of high-quality exosomes warrants thorough cles. Then, the supernatant is collected and passed through a
investigation. 0.22 μm porous filter to further deplete non-exosome mate-
Polyethylene glycol (PEG), formed by ethylene oxide and rials. And then, the resulted solution is mixed with aqueous
ethylene glycol or hydrate, shares advantageous properties of PEG solution. Given a coincubation of exosomes and PEG for
being non-toxic, non-irritating and high scalability. Accord- more than 12 h (To create a setting for the thorough binding
ingly, it is used for early virus enrichment and commonly of PEG and exosomes or the self-assembly of exosomes to
recruited as therapeutic tools [7, 9, 10]. Indicatively, it is enhance the weight of conglomerates, thus harvesting a con-
developed for exosome isolation through dehydrogenation siderable number of exosomes at a relatively low centrifuga-
or dehydration [11, 12]. The combination of PEG and dif- tion speed.), a centrifugation at 1500–16,000×g for 10–60 min
ferential ultracentrifugation (UC) only needs smaller volume to the mixed solution is carried out. Usually, a centrifugation
of starting samples and less preparation time before the UC at 1,00,000–1,30,000×g for 30–130 min or again is needed
step, and captures a larger population of exosomes compared to purify the exosomes. All performances are based on 4 °C.
to differential UC alone. This also reduces the potential dam- Figure 1 shows the schematic representation of PEG-based
age to instruments. Based upon of these, PEG was widely exosome isolation procedure. Obviously, the protocol of PEG
employed to isolate exosomes from various bio-samples capturing exosomes can be easily realized in common labo-
[13, 14]. However, there are still absent criteria on exosome ratories due to its alleviation of resource constraints. Of note,
extraction in terms of PEG strategies. Thus, this paper con- 1 M sodium chloride (NaCl) should be added when dissolving
cludes with a summary of exosome isolation instructions PEG due to several fascinating reasons. Firstly, NaCl affects
involved in PEG, aiming to illuminate the application value the stability of exosomes, beneficial in optimizing the sepa-
of PEG-participation exosome separation and lay down the ration of exosomes from other small molecules or undesired
foundation of optimizing exosome isolation. proteins during coincubation. Secondly, it adjusts the ionic
strength of solution, aiding in concentrating and purifying
exosomes. Thirdly, it regulates the solubility and interactions
PEG‑based exosome isolation of proteins and other molecules, driving the separation of
exosomes from non-exosomal components. Specifically, a cer-
PEG, a highly transformable polymer, has good biocompat- tain concentration of NaCl can reduce the electrostatic interac-
ibility and biodegradability [15]. Consequently, PEG and its tions among proteins [39, 40], conferring the dissociation of
derivatives are generally utilized in pharmacokinetic process impurities from exosome surface, which thereby increases the
or drug delivery, which has been approved by FDA of USA purity of exosomes.
[15]. PEG aids in maintaining or even enhancing therapy At the same time, dynamic light scattering, tunable resis-
efficacy via improving pharmacokinetic parameters, or the tive pulse sensing and nanoparticle tracking analysis (NTA)
formulation of long circulating nanoparticle drugs [15]. are employed to detect the particle size distribution and con-
All these approve the non-toxic and bio-safe properties of centration identification of the achieved exosomes. Trans-
PEG. In addition, PEG has been well developed to enrich mission electron microscopy (TEM) and scanning electron
virus. Hence, a growing body of institutes exploit PEG for microscopy are adoptable for morphological characteristics
exosome isolation from all kinds of biospecimens includ- identification. And western blot (WB) or nanoscale liquid
ing animals and plants (cellular cultured supernatant [7, 10, chromatography coupled to tandem mass spectrometry is
16–24], urine [13, 14, 25], plasma [2, 13, 23, 25, 26], serum commonly employed to confirm the expression level of exo-
[24, 27–30], blood [31], cerebral spinal fluid [13], saliva some markers [5]. Additionally, green fluorescent protein
[13, 24], pleural effusions [32], gouty arthritis synovial fluid has been developed to determine whether exosomes gained
[33], synovial tissue [34], Leishmania spp. [35], and aloe by PEG can be well taken up by the recipient cells [13, 17,
saponaria [36], fetal bovine serum (FBS) [37, 38]). 41, 42].

The procedure of PEG‑precipitation exosome The formation of PEG‑exosome

isolation aggregations

Table 1 summaries the major protocols of PEG-based exosome Obtained by water and any diols, or polymerizable epox-
isolation in terms of different biofluids or biospecimens. In yethane, PEG exhibits a wide range of colloidal behaviors
summary, the bio-samples are centrifugated at 300–1000×g based upon different conditions, primarily related to its

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 Table 1  Representative literatures employed PEG for exosome isolation from different bio-samples
Exosome source Main isolation procedure Advantages Disadvantages
1. Deplete cellular debris and 2. Co-incubation 3. Get primary precipitation
larger vesicles and wash

HeLa cells [7] 0.22 μm 10% PEG6000; co-incubated 3000×g (10 min); 1,20,000 g High yield; convenient and NR
for 30 min (120 min) efficient; high purity; inex-
pensive
Prostate cancer cell lines [16] 500×g; 2000×g 8% PEG; co-incubated for 16,000×g (60 min) Its purity is similarly equal to NR
overnight that of commercial kits
Molecular and Cellular Biochemistry

4T1; MCF-7 cells [41] 300×g (10 min); 2000×g V1:1; 8% PEG 10,000×g (60 min); 1,20,000×g High yield; biological function NR
(20 min); 10,000×g (30 min); (120 min) is guaranteed
0.22 μm
BMSCs [17] 500×g (5 min); 2000×g 8%, 16% PEG; ­V1:1; co-incu- 10,000×g (60 min); drain High yield; high purity; bio- NR
(10 min); 10,000 g (30 min) bated for overnight for 5 min; vortex for 5 s; logical function is guaran-
1,00,000×g (120 min) teed; high specificity
MSCs [18] 10,000×g (45 min) PEG20000; co-incubated for 10,000×g (20 min) High yield NR
12 h
MSCs [19] 6800×g (45 min); 0.22 μm 12.5% PEG6000; co-incubated 1500 g (30 min); washed by High yield; convenient and effi- NR
for 8–16 h 0.9% NaCl; 1,00,000×g cient; inexpensive; scalable
(130 min)
RPE; NPCE cell [20] 1500×g (15 min); 0.22 μm 8% PEG8000; ­V1:5 (PEG: sample) 1500×g (30 min); 1,00,000×g Successfully applied for down- NR
(70 min) stream analysis
Murine macrophage cell [21] 400×g (10 min); 2000×g 8% PEG6000; ­V1:5 (PEG: sample); 1500×g (30 min); + iodixanol High yield NR
(20 min); 0.22 μm co-incubated for 18 h
HEK293T cell [10] 10,000×g 6%, 8%, 10%, 12%, 15% PEG; 1500×g (30 min); 1,10,000×g High yield; high purity; high NR
co-incubated for overnight (120 min) reproducibility; biological
function is guaranteed
HEK293; SH-SY5Y cell [22] NR 10% PEG8000; ­V1:1 16,000×g (60 min); again High yield; inexpensive Low purity and quality
HEK293 cell; plasma; cerebral 500×g (5 min); 2000×g 5–15% PEG; ­V1:1, co-incubated 3214×g (60 min); drain for Convenient and efficient; inex- NR
spinal fluid; urine; saliva [13] (30 min) for overnight 5 min; 1,00,000×g (70 min); pensive; high reproducibility
shaking up to 30 min
HGSCC; plasma [23] 500/850 g (30 min); 2000×g 8% PEG; ­V1:1; co-incubated for 1000×g (30 min); 1000×g High yield NR
(10 min); 1000×g (30 min) overnight (10 min); 1,10,000×g
(70 min); 1,10,000×g
(70 min)
Plasma [2] 1500×g (15 min) 5%, 10%, 15%, 20% PEG8000; 1500×g (30 min) High yield Low purity

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­V1:1
Plasma [26] NR Co-incubated on ice for 30 min 1500×g (30 min) High yield NR
Plasm; urine [25] 800×g (20 min at 24 °C ); 1.5% PEG2000 + dextran blue; 17,000×g (20 min at 24 °C ) Convenient and efficient; inex- NR
17,000×g (20 min at 24 °C ) co-incubated for 30 min pensive; high reproducibility
Urine [14] 3000×g (10 min) 8% PEG8000; co-incubated for 4000×g (60 min) Biological function is guar-
overnight anteed
 Table 1  (continued)
Exosome source Main isolation procedure Advantages Disadvantages
1. Deplete cellular debris and 2. Co-incubation 3. Get primary precipitation
larger vesicles and wash

Blood [31] 3000×g (15 min) 10% PEG8000; co-incubated 16,100×g (60 min) High yield; inexpensive; less Low purity
for overnight time–cost
Serum; saliva; liver stem cells 3000×g (25 min); again; PEG35,000 + protamine chlo- 15,000×g (30 min at 22 °C); Convenient and efficient; NR
[24] 0.22 μm ride; ­V1:1; co-incubated for 1000×g (1 min) biological function is guaran-
overnight teed; inexpensive
Serum [27] NR V1:5 (PEG: sample); co-incubated 7000 g NR NR
for 60 min
Serums [28] 3000×g (15 min) 10% PEG6000; co-incubated 1500×g (30 min) Inexpensive NR
for 30 min on ice
Serum [29] 12,000×g (30 min) 12% PEG20000; co-incubated 12,000×g (10 min); + magnetic Suitable for the noninvasive NR
for 60 min on ice beads diagnosis of lung cancer
Serum from chickens [30] 500×g (5 min); 2000×g 8% PEG6000; ­V1:1 15,000 rpm (60 min) High yield; convenient and NR
(30 min); 12,000×g (45 min) efficient; inexpensive; high
reproducibility
Pleural effusions [32] + proteinase K; 16,000×g 12% PEG8000; a tube shaker 400 rpm (60 min); 23,000 g Reliable and sensible NR
(30 min) overnight (60 min)
Gouty arthritis synovial fluid 3000×g (20 min); 0.45 μm; 8% PEG6000 12,000×g (45 min) High yield Low purity; loss of IL-1β
[33] 0.22 μm
Synovial tissue [34] 0.22 μm 8% PEG; V­ 1:1; co-incubated for 12,000×g (30 min); again NR Low purity
overnight
Leishmania spp. [35] 1000×g (5 min at RT); 1000×g 8% PEG; co-incubated for 3000×g (60 min); 100,000 g NR NR
(5 min at RT); 0.45 μm; overnight (70 min)
0.22 μm
Aloe saponaria [36] 1000×g (10 min); 3000 g PEG6000; co-incubated for 1500×g (30 min) High yield; less time–cost; Low purity
(30 min); 10,000×g (60 min); 16 h inexpensive
0.45 μm
Depletion of exosome in FBS NR co-incubated for overnight 1500×g (30 min) Friendly to guarantee the bio- NR
[37] peculiarity of FBS
Depletion of exosome in FBS PEG-coated ­Fe3O4 MNPs 15%, 30% PEG4000; 3000 rpm (30 min) Deplete exosome in FBS NR
[38] PEG6000; PEG8000

Unless specially illustrated, all operations are performed at 4 °C. 0.22 μm: means that samples need to be passed through 0.22 μm filter
BMSCs bone marrow mesenchymal stem cell, FBS fetal bovine serum; HEK293 human embryonic kidney cells, HGSCC high-grade serous carcinoma cell, MSCs mesenchymal stem/stromal

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cells, NPCEC non-pigmented ciliary epithelium cell, NR none report, PCC pancreatic cancer cell, PEG-coated Fe3O4 MNPs polyethylene glycol-coated ­Fe3O4 magnetic nanoparticles, RPEC
retinal pigment epithelium cell, SH-SY5Y human neuroblastoma cells
Molecular and Cellular Biochemistry
Molecular and Cellular Biochemistry

Fig. 1  The schematic diagram of isolating exosomes from cell culture obtain PEG-exosomes aggregations. D A centrifugation at 1,00,000–
supernatant based on PEG. A A centrifugation at 300–1000×g for 1,30,000×g for 30–130 min to purify exosomes. All protocols are
5–10 min to deplete dead cells and cellular debris. B A centrifugation carried out at 4 °C. Different bio-samples, matching to different
at 2000–6800×g for 10–45 min to deplete larger vesicles and apop- extracted parameters, are listed in Table 1
totic vesicles. C A centrifugation at 1500–16,000×g for 10–60 min to

molecular weight, environmental conditions, and interac- exosomes in aqueous DMEM [45]. This process helps to
tions with other substances [15]. It increases the dielectric form PEG-exosomes micelles or microcapsules by self-
constant of water by binding to biomolecules, and thus assembly (seen in right of Fig. 2), indicating PEG as a
facilitates the innate interaction forces [43]. Therefore, it is fascinating carrier to assemble a considerable number of
an optimal selection to capture and pull exosomes. Mean- exosomes [46]. In addition, the hydroxy (–OH), exoge-
while, high molecular weight PEGs (PEG4000-20000) are nously added carboxyl (–COOH), possible thiol (–SH) or
often used as stabilizers and dispersants for nanoparticles amine (–NH2) et al. in PEG chains support the opportunity
due to their brilliant biocompatibility and aqueous char- of PEG linking to the collagen matrix or other biomol-
acteristic [15]. It acts as a dispersant to stabilize exosomes ecules on exosome surface by favorable reducibility, oxi-
and a solubilizing agent to help other substances insolu- dation dehydrogenation or dehydration [7, 11, 12, 27, 47].
ble or slightly soluble in water, thus forming a stable col- Consequently, PEG chains recruit exosomes and physi-
loidal dispersion system [44]. In consequence, PEG can cally wrap abundant free exosomes (shown in Fig. 2). This
create a hydrophobic microenvironment that accelerates process confers the formation of aggregations containing
the agglutination of exosomes. This greatly highlights adequate exosomes, which in turn augments the weight
the probability that PEG, dissolved in water, reduces of exosomes. Predictably, PEG-based isolation is strongly
the solubility of exosomes and binds to the hydrophobic achievable even at a low centrifugation speed. Overall,

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 Molecular and Cellular Biochemistry

Fig. 2  The schematic process of forming micelle or microcapsule aggregations containing PEG and exosomes during coincubation. PEG chains
provide a hydrophobic microenvironment beneficial in aggregating exosomes

the various properties of PEG herald the scheme of PEG- results among various concentrations of PEG [13, 40]. How-
based exosome isolation. ever, recent data showed that the exosomes derived from
various body fluids or cell culture medium require a specific
average molecular weight and concentration range of PEG. It
Some notices for PEG‑precipitation exosome is a prompted need of investigation that higher concentration
isolation of exosomes is closely related with the specific concentra-
tion of PEG. (3) The molecular weight of PEG should be
The colloidal behaviors of PEG yet rely on several fac- suitably selected. PEGs with different molecular weights
tors, which should be brought to the attention of scientific are matched with different physical and chemical proper-
workers. (1) Reactant ratio determines the equilibrium state ties. And a reaction system given lower molecular weight
of the reaction as well as the distribution of reactants and of PEG offers preferentially rapid cross-linking reactions.
products [48]. Therefore, exploring appropriate reactant Nevertheless, it is inappropriate to opt PEG with a too small
ratio for PEG-based exosome isolation can ameliorate the molecular weight for exosome isolation. This is because the
quality of exosomes. It seems that, as for exosome isolation, principle of PEG-based exosome isolation method depends
­V1:1 (PEG: sample) is a better choice for separating exosomes on that macromolecular PEG aggregates certain number
from larger-volume specimen (i.e. cell supernatants) [22, of exosomes to increase weight, only in which allows the
41], while ­V1:5 (PEG: sample) is recommendatory for the smaller- separation even if applying a low-speed centrifugation. PEG
volumes (i.e. serum) [27]. (2) Appropriate concentration of with molecular weight less than 4000 is thereby not appli-
PEG should be employed. Compelling evidence shows that cable for exosome isolation. However, a higher molecular
8% co-incubated concentration of PEG is an ideal choice. weight of PEG is also not recommended. This is because
For example, the authors mixed different-concentration PEG the higher molecular weight of PEG causes the formation
solutions with specific body fluids or cell supernatants in of PEG-autologous aggregations, which in turn inhibits the
1:1 volume to extract exosomes, and found that scheme of autologous agglutination of exosomes around PEG. What’s
8% PEG solution to incubate exosomes displayed optimum more, higher molecular weight of PEG will enhance the

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Molecular and Cellular Biochemistry

proportion of PEG in final product and lower the exoso- protein signatures may be altered by PEG [26]. But how does
mal purity, adverse to exosome isolation. Accordingly, the PEG affect the structure and expression of related proteins
molecular weight of PEG ranges from 6000 to 20,000 is in exosomes needs further evidences. Actually, WB or TEM
more considerable. Particularly, in terms of the principle of result implicates that the wrapping of PEG also can avoid the
lower viscosity and cost, PEG6000 is the most widely used interference of serum or cellular proteins to exosomes. Oth-
[15, 40, 49]. (4) Appropriate incubated time and temperature erwise, though PEG is possibly co-isolated along with final
should be established. Actually, incubating exosomes at 4 °C product, some technological approaches have been devel-
for more than 12 h is needed (identified by the evidences oped to avoid PEG. For instance, barium iodide staining
from NTA and WB), and increasing the basic area of sample technique validated that the residual PEG could be efficiently
as soon as possible is also indispensable [10, 40]. (5) The depleted after the last step [10].
suitable pH stabilizing PEG ranges from 4 to 9 [50]. Nota- Using PEG, less volume of bio-specimen is prepared for
bly, the pH values ranging 7.2–7.4 ensures the integrity of the UC step, giving the remarkable convenience and high
exosome, included in 4–9. (6) In the step of depleting cell throughput compared to differential UC alone. Besides,
debris and larger cellular vesicles, different biological sam- more than 5–14 times total protein and more exosome char-
ples should be applied with different centrifugation param- acteristics were seen in NTA, WB and TEM technology plat-
eters or co-precipitating agents. For instance, unlike most forms by this methodology [18, 19, 26, 37]. Intriguingly,
samples, the sample of aloe saponaria is centrifugated for 5%–20% concentration of PEG is prepared coupled with
three times at a progressively rising centrifugation speed commercial kits, and PEG with higher molecular weight of
and passed through a 0.45-μm strainer rather than the com- is prone to obtain smaller exosomes [2].
monly used 0.22-μms before co-incubated [36]. (7) In case Except for a larger number of exosomes acquired by PEG
of damaging the completed package that contains exosomes precipitation, the foundational bio-function of exosomes also
and PEG, rotation or agitation is forbidden during coincuba- could be ensured, possibly due to the protective wrapping of
tion [35]. However, gently agitating and thorough mixing PEG. In a recent study, fluorescence microscopy and flow
before coincubation is essential. (8) The volume of PBS to cytometry were carried out to detect the integrality and
resuspend precipitation should be tightly controlled to main- biology properties of exosomes harvested by UC, density
tain exosome concentration. (9) Blow the precipitation at a gradient centrifugation (DG) and PEG6000 or PEG8000 pre-
fixed site as much as possible after UC. (10) Although NaCl cipitation. In result, better incorporation efficiencies were
assists in purifying exosomes, excessive NaCl concentration observed in terms of large yield of eGFP-labelled exosomes
or improper equilibrium solubility may adversely affect the obtained by PEG6000 or PEG8000 precipitation, implicat-
structure or activity of exosomes [40]. Therefore, precise ing favorably defended biology properties of exosomes [10].
weight of NaCl is required in practice. Based upon these, Simultaneously, confocal microscopy analysis uncovered
scientists explored an economical, time-saving, convenient that more ­CD63+ exosomes twinkling with green fluores-
and clinically friendly exosome isolation avenue based on cence within recipient cells were observed with PEG pre-
PEG. cipitation than that with sucrose cushion (SC) or differen-
tial centrifugation (DC) [13]. The fact that no interference
occurs in exosomal biological properties using PEG-based
Disadvantages and advantages exosome isolation were elucidated in aspects of not only
of PEG‑precipitation exosome isolation phenotypes but also molecular levels such as protein, miR-
NAs, DNA, and related therapeutic effects [10, 28, 51, 52].
The sizes of exosomes obtained by PEG range in similarity It seems that PEG-precipitation exosomes are more
with that obtained by other methods such as differential UC suitable for downstream RNA analysis [25, 28, 53]. For
(30–200 nm) [24]. Nonetheless, some cell fragments, large- example, the quantification of exosomal DNA obtained by
size extracellular vesicles, non-exosomal proteins, immu- Exoquick®, PureExo®, UC and PEG have been performed.
noglobulins, particles of virus and other contaminants are Surprisingly, PEG-precipitation exosomes emerged highest
also observed at the final particles precipitated by PEG [26]. total DNA recovery, indicating the value of PEG-precipi-
Actually, PEG is a non-ionic polymer that easily alters the tation exosomes for DNA downstream analysis relevant in
dielectric constant of water, thus causing the non-specific malignant solid neoplasms [31]. In addition, the bio-func-
aggregation and precipitation of pellets. Fortunately, this tional properties of exosomes are strengthened by PEG. For
issue may be solvable by combining with UC. For exam- instance, Warren et.al illustrated that PEG coating remark-
ple, Ludwig et.al documented that the final particles from ably improved the mucin permeability and particle stability
PEG precipitation plus UC performed purer than that from of exosomes derived from milk even in a setting of strong
UC alone, which was validated by the calculated ratio of stomach acid [42]. Table 2 summarizes the quantitative com-
particles per mg protein [10]. What’s more, some exosome parison of exosome characteristics using PEG precipitation

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 Molecular and Cellular Biochemistry

and other methods in terms of recovery rates, protein con- purpose. For example, the adding of a dendrimer to PEG
centration, functional activity, which relatively supports the and aEpCAM, a full antibody, effectively fueled the bind-
superiority of PEG precipitation. ing tropism of aEpCAM to exosomes secreted by MCF-7
cells, thus minimizing the non-specific binding, and rais-
ing the number of aEpCAM-contained exosome [57]. Since
The combination of PEG‑precipitation citrate-capped gold nanoparticles unstably coexist in buffer
exosome isolation and other techniques solutions, Pammi Guru et al. [29] introduced citrate-capped
gold nanoparticles into PEG to form PEG-gold nanoparti-
Remedy the defects of other exosome isolation cles, and then incubated them with serum for exosomes iso-
techniques lation. Interestingly, with centrifugating the solution within
2 h and then applying the achieved exosomes for dynamic
Even though the investigators do not succumb to the light scattering, NTA, TEM and WB characterization tech-
extensive challenges of exosome isolation caused by the niques, the authors have confirmed excessively intensive sta-
physicochemical and biochemical properties of exosomes bility of citrate-capped gold nanoparticle in buffer solution
(i.e., being ultra-small size, lower-density, and exposed to conferred by PEG. Therefore, a conclusion is underlined that
complex biological environment) by developing multiple the steric repulsion of PEG chain functionalizes the citrate-
technologies for exosome separation (differential UC, size- capped gold nanoparticles. Otherwise, exosomes, attained
exclusion chromatography (SEC), immunoaffinity capture, by combining protamine chloride (PC) to PEG, have higher
microfluidics and PEG-based precipitation et al.) to optimize biological function integrality. And more labelled exosomes
the efficacy of exosome preparation, there are still deficien- within the recipient cells by this method compared to that by
cies to be figured out. Since other traditional techniques are without PC have been proven. Moreover, through comparing
low-yield, expensive, labor-intensive, complexity-procedure to the results from DC, SC and PEG6000 techniques, Rider
and time–cost (Table 3 lists more details), the introduction of et al. [13] demonstrated higher-quality RNA in PEG-precip-
PEG largely remedies the drawbacks of other exosome isola- itation group. More surprisingly, to deplete exosomes from
tion techniques primarily owing to the advantage of higher FBS, PEG precipitation is a winner choice among isolation
yield. (e.g., elevates exosome yield, provides assistance in technologies of UC, ultrafiltration (UF) and PEG precipita-
strengthening the affinity of antibodies to exosomes or pro- tion [37].
tects exosome bio-function).
For instance, the authors compared the extraction effi- Remedy the defects of PEG‑precipitation exosome
ciency and exosomes purity of DC and PEG-precipitation isolation
method. In consequence, even though the purer and higher
biological-characteristics exosomes were proven using DC The shortcomings of PEG-based exosome precipitation
method, massive membrane fragments were also observed can be eradicated by combining with other techniques. On
under electron microscopy. And damaged bonds that bind one hand, it seems that the PEG-precipitation exosomes are
exosome and glycoproteins (i.e., Wnt3a) were demonstrated impure, but purer by combining to other techniques. For
by NTA and WB, suggesting the losing guarantee to the example, the combined utility of PEG precipitation and UC
integrality of exosomes due to the directly physical expo- becomes a preferential scheme, which apparently circum-
sure to mechanical force or higher viscosity [24, 54–56]. vents the appearance of some contaminant proteins like
Interestingly, no similar results were found in PEG-based BSA in final pellets. This is due to the fact that though UC
separating strategy due to the protective wrapping of PEG. is effective in separating exosomes, it ubiquitously leads to
Furthermore, PEG was able to enhance the stability of contaminants. While the combination of UC and PEG-based
Wnt3a-containing exosome membrane, or augment the num- precipitation makes exosomes become more stable aggrega-
ber of Wnt3a-containing exosome [17]. And PEG plus UC tions. This minimizes the background impurities of achieved
presumably avoids the demand of preparing excessive start- exosomes, which is beneficial in functional analysis, prot-
ing volume of sample [54–56]. Deregibus et al. [26] verified eomic analysis and molecular downstream analysis [7, 10].
that protamine-combined PEG precipitation produced higher This process has less impacts on the biological activity of
recovery rate, more regular particle size morphology and exosomes. Dextran, a high molecular weight polysaccha-
more uniform distribution of exosomes than UC. Apparently, ride [58], also has been added for exosome isolation based
introducing PEG into exosome isolation makes up for the on PEG. The participation of dextran in exosome isolation
defects of poorly low-yield. increases the viscosity and molecular crowding. Firstly, this
What’s more, PEG is an attractive vehicle to be remolded efficacy contributes to separating exosomes from other small
owing to the amazing scalability of PEG surface configura- molecules or hetero-proteins. The presence of dextran cre-
tions, thus easily making it match the exosomes analysis ates a crowded microenvironment, conducive to the binding

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 Table 2  Quantitative comparison of exosome characteristics isolated by PEG participation and other strategies
References Exosomal particle amount Purity Obtained exosomal proteins Quantity of being taken up

2015 [60] PEG: 68.3; ExoQuick: 38.8; UC: .15.2 NR PEG: 22.5; ExoQuick: 4.7 UC: 0.5; (pro- NR
(recovery efficiency, %) tein index, %)
2016 [13] DC: 0.2–0.3 × ­109; 8% PEG: 15–23 × ­109; No difference in purity: 8% PEG vs. UC 95% (492 of 517), (Mass spectrometry)# PEG > DC; (MFI of CD63-GFP in HEK293
ExoQuick: 15–20 × ­109; (NTA)* (p = 0.344); PEG vs. SC (p = 0.169) cells, Confocal microscopy)*
2016 [7] With exosomal size range: 50–150 nm, NR 97% (6299 of 5120), (Mass spectrometry)# PEG-participation exosomes were success-
(NTA) fully applied for downstream proteomic
analysis
Molecular and Cellular Biochemistry

2016 [26] SEC: 1.9 × ­1012; PEG: 1.0 × ­1012; NR PEG: 21.1 ± 10; PROSPR: 3.9 ± 1.1; SEC: NR
PROSPR: 3.3 × ­1011; (NTA) 0.3 ± 0.3; (mg, BCA kit)
2016 [28] NR NR PEG: (33) > EXS (27) > EXM (26) > EXQ PEG-participation exosomes from serum of
(20) > TEI (19) > UC (18); (MFI of 10 human were successfully applied for
CD63-GFP in HEK293 cells, Flow the assessment of miRNAs level
cytometry)*
2018 [41] IS-NPs: 8.8 ± 1.3 × ­109; PEG: NR Kit: 35; PEG: 33; IS-NPs: 17; UC: 10; (μg/ NR
3.7 ± 0.8 × ­109; Kit: 3.2 ± 0.8 × ­109; UC: mL)
1.1 ± 0.4 × ­109; (Particles/μg protein,
NTA)
2018 [14] With exosomal size range less than NR NR PEG-participation exosomes were suc-
200 nm, (NTA) cessfully applied for assessment of renal
fibrosis diagnostics based on the level of
miR-29c or miR-21 in urinary exosomes
for patients
2018 [10] PEG: 1 × ­109; UC: 0.90 × ­109; DG: NR NR NR
0.25 × ­109; DC: 0.60 × ­109; SEC:
0.20 × ­109; (NTA)
2019 [21] PEG≈ CUC > UC; (NTA)* Described in literature: PEG≈ CUC≈ UC PEG≈ CUC > UC; (Quantified by Qubit) * NR
2019 [31] PEG: 3 × ­109; Exoquick: 2 × ­109; UC: UC = PureExo > Exoquick≈ PEG, (Purity Exoquick (3.8) > PEG (3.5) > UC PEG (0.65) > Exoquick (0.5) > UC
0.23 × ­109; PureExo: 0.2 × ­109; (/mL, index, p < 0.01)* (2) > PureExo (0.3), (ExoELISA CD9 (0.35) > PureExo (0), (DNA copies score,
NTA)* kit, × ­108/mL) *; Exoquick (30)≈ ng/μL, p < 0.01, Genomic DNA quantifica-
PEG > UC (20)≈ PureExo (0.3) (μg/μL, tion)*
p < 0.01)
2019 [75] PEG: 5.43 ± 1.12 × ­1010; PEG-UC: NR NR NR
1.63 ± 0.34 × ­1010; UC: 4.17 ± 1.12 × ­108;
(particles/mL, NTA); PEG: 65.8 ± 3%;
PEG-UC: 86.0 ± 2.3%; UC: 89.3 ± 3.6%;
(p < 0.05, proportion of exosomes less

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than 150 nm)
2020 [34] NR NR PEG > UC (protein of synovial tissue- NR
derived exosomes, p > 0.05)
 Table 2  (continued)
References Exosomal particle amount Purity Obtained exosomal proteins Quantity of being taken up

2021 [82] NR NR NR Higher miR-19b and miR-425 Cp values in

exosomes using PEG than UC *
PEG-participation exosomes were applied
for the downstream miRNAs detection of
prostate cancer or heathy patients
2022 [18] PEG: 4 × ­109; UC: 0.5 × ­109; (p = 0.023, NR PEG (450) > UC (80); (μg/mL, p = 0.0013) Higher enhanced ability of exosome to
NTA) inhibit LPS-induced inflammatory-asso-
ciated cytokine increases using PEG than
using UC (e.g. TNF-α)
2022 [74] SEC: 96.03 ± 4.46%; PEG: 91.03 ± 9.07%; NR PEG: 24.14 ± 3.90; SEC: 1.14 ± 0.17; UC: Visually more PKH67 within cells in PEG
UC: 45.08 ± 10.22%; (Proportion of 0.11 ± 0.04; (p < 0.0001, g/L, BCA kit) group than UC group, (p < 0.05, confocal
exosomes ranging 30–150 nm) microscope)
2023 [37] UC: 1,16 × ­1010; Commercial: 1.11 × ­1010; NR Commercial: 38,140 ± 580; PEG: Commercial: 1694 ± 165; PEG: 1629 ± 83;
PEG: 2.99 × ­109; UF: 2.43 × ­109; (/mL, 30,472 ± 2548; UC: 27,792 ± 182; UF: UC: 1478 ± 36; UF: 934 ± 72; (pg/mL,
NTA) 7435 ± 389, (μg/mL, BCA kit) TGFβ activity)
2024 [40] NR NR NR No significant difference of exosomes
by UC, PEG and ExoQuick in promoting
cell proliferation and migration
2024 [22] Smaller particles from SH-SY5Y NR NR NR
cells: PEG (27,666 ± 2186) > UC
(26,133 ± 633) > UF (25,766 ± 966);
Larger particles from SH-SY5Y cells
(per mL): PEG (5566 ± 1934) > UC
(3933 ± 317) > UF (3400 ± 458); (/mL,
Flow cytometry)
2024 [83] NR NR NR UC: 33, 1.8; PEG: 33, 1.9; SEC: 8.65, 1.7;
(ng/μL, absorbance ratio 260/280)

*The illustrated numbers were approximate numbers visualized from bar graphs reported in corresponding literature. #The proportion of PEG-precipitation proteins that were identified as
exosome proteins, officially recorded in the ExoCarta and Vesiclepedia databases. Except for special illuminated, the sizes of harvested exosomes were less than 200 nm. Several literatures
described that different exosomal RNAs by different isolated methods exhibited diverse levels
CUC​ cushioned-UC, DC differential centrifugation, DG density gradient centrifugation, EXM ExoMirTM, EXQ Exo-spinTM exosome precipitation, LPS lipopolysaccharide, NR none report,
PROSPR protein organic solvent precipitation, SC sucrose cushion, SEC size-exclusion chromatography, UC ultracentrifugation, UF ultrafiltration, TEI total exosome isolation

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Molecular and Cellular Biochemistry
Molecular and Cellular Biochemistry

Table 3  Disadvantages of current methodologies for exosome isolation

Methodologies Disadvantages

PEG-based precipitation Low purity (coprecipitation of protein and PEG), no specificity (undetermined)
Differential ultracentrifugation Low purity (coprecipitation of protein), no specificity, low yield*, large starting sample volume*, specialized
equipment requirement, high time cost (pre-preparation)*, complexity procedure, labor-consuming*, poten-
tial of damage and deformation to exosome*
Size-exclusion chromatography No specificity, low yield*, high economy cost (device)*, high time cost (running)*, semi-tortuous procedure
Ultrafiltration Low purity (non-exosome peptides), no specificity, low yield*, large starting sample volume*, damage and
deformation to exosome*, highly frequent filter clogging and membrane trapping, high economy cost (filter
membrane), low potential for downstream analysis*
Immunoaffinity capture Low yield*, high starting sample volume*, specialized equipment requirement, high time cost (running),
potential of damage to exosomal bioaction*, limited application due to the limited-expression antigen in exo-
some, dramatically high economy cost (antibodies)*
Other methodologies Specialized equipment requirement, complexity procedure, dramatical economy cost*, low sample capacity

*Means high potential settlement of issues by combining to PEG-based precipitation due to its merit of high yield

of PEG to exosomes through non-specific interactions. Sec- utility of PEG precipitation and immunoprecipitation can
ondly, it optimizes the conditions for separation by reducing acquire more immunolabel-based but also purer exosomal
uninteresting clumping, and this improves the accuracy of particles [65]. Moreover, the combination of PEG and other
separation process. Therefore, the ligation of PEG to aque- techniques prospectively gains exosomes within a desired
ous dextran is an ideal scheme to remove the contaminants in range of diameter. Several literatures outlined that the com-
final particles. Surprisingly, it is proven that dextran is prone bined adoption of PEG precipitation, UC and 30% SC or
to pull non-exosomal proteins, while PEG has higher affinity PC not only excessively purified the enriched exosomes but
with exosomes in the next step, thus obviously decreasing also concentrated the exosomes with size < 100 nm [17, 24].
the contaminants [59–61]. Furthermore, PC is a cationic What’s more, a study regarding exosome separation achieved
protein rich in arginine [62]. It hugs interesting properties, specific exosomes with size less than 50 nm by conjugating
and thus being added into PEG-based exosome isolation. citrate-capped gold nanoparticles and anti-CD6 [27]. Over-
Firstly, it charges neutralization. It is well known that PC is a all, on the basis of experimental purposes, the researchers
positively charged polymer [63], prone to bind to negatively could design combined schemes for PEG precipitation so as
charged proteins, including some plasma proteins. This to harvest specialized exosomes and simultaneously avoid
reduces the competitive binding of non-specific proteins the disadvantages of PEG precipitation.
to exosomes, which in turn purifies exosomes. Secondly,
it improves the selectivity of precipitation. The addition of Other reagents for exosome precipitation
PC modifies the balance of particle interactions in solution,
leading to easier recognition and separation of exosomes In order to skip sophisticated instruments and tedious opera-
during PEG-induced precipitation. Additionally, PC reduces tions, there are also some other methods that sperate exo-
non-specific bindings on exosome surface, lessening the co- some from bio-samples via precipitation. Notably, these
precipitation of uninteresting materials. In response to this, strategies present a common disadvantage of higher cost
lipoprotein ApoB100 and ApoA1 were almost absent in ulti- compared to PEG-based exosome isolation. And this deci-
mate exosomes that were pulled down by PC and PEG, but phers the phenomenon that PEG is the most commonly used
presented in final pellets that were gained by UC technique, for exosome isolation among precipitation methods.
indicating the guarantee of both pure and functionally active
exosomes by PEG plus PC [24]. At last, a tangential-flow
filtration (TFF) was also recommended for purifying PEG- Lectin affinity
elicited exosomes [64].
On the other hand, the jointly exploiting of PEG precipi- Lectin, a kind of protein widely presents in animals or
tation and other techniques is viable to harvest a certain type plants, is prone to specifically bind to diverse sugar moie-
of exosome in abundance (e.g. only containing a specific ties [66]. Thus, similar to antibody-capture methods, lectin-
antibody or with a certain range of size). Exosomes with affinity exosome precipitation depends on the high affinity of
different diameters seem to express different proteins, while lectin to saccharide residues situating on exosome surface.
PEG is amenable to bind with specific antibodies to pull Usually, with two steps of centrifugation (2000×g, at 4 °C
down more desired exosomes. For example, the simultaneous for 20 min, then 15,000×g, at 4 °C for 30 min) to deplete

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 Molecular and Cellular Biochemistry

cellular debris and large membrane vesicles, concanavalin A match different experimental occasions, which needs diverse
is used to co-culture with the resulted supernatant for over- procedures. Different commercial kits are beneficial in exo-
night at 4 °C. And then a relatively high speed of centrifu- some isolation from small-volumes (about 0.5–1 mL) of bio-
gation (20,000×g, at 4 °C for 60 min) is carried out [66]. samples including serum, plasma and CSF et al. [71, 72].
The exosomes are obtained in the precipitation. Notably, This is conducive to high integrity of particle morphology,
the exosomes from different bio-samples matched differ- excellent exosome quality and efficient exosome isolation
ent procedures. For example, to obtain exosomes from cell [71, 72]. Nevertheless, less flexibility remains in experimen-
cultured medium, 1 mg/mL concanavalin A and a centrifu- tal design using kits, and the unavoidably differences among
gated speed of 20,000 g for 60 min should be applied. While batches, brands and models cause the heterogeneous of
from urine, 2 mg/mL concanavalin A and a centrifugated exosomes, impacting the consistency of experiment and the
speed of 20,000×g for 90 min should be employed [66]. reliability of results. In spite of the great progress made by
This method hugs merits of technical simplicity, handling bio-companies and the availability of exosome commercial
large initial volumes of biological fluid, but demerits of low kits on the market, these kits remain excessive cost, which
purity, uncontrolled sizes of particles (gain conglomerate thus causes the limited applications (seen corresponding cost
with sizes of 600 nm) [66], difficult to be standardized and in various market platforms).
apparently higher cost relative to PEG-based method (seen
corresponding cost in official Sigma-Aldrich). It was unable
to hold task of exosome isolation alone so some scholars Conclusion and perspectives
combined lectin-affinity and other methods such as PEG pre-
cipitation or SEC to attain exosomes [67]. Intriguingly, the Due to the ability of keeping molecular markers from the
carbohydrates adhering to exosome surface imply the types released cells, transforming into a perspective diagnostic
of the released cells [66, 67]. tool, and acting as a hitherto optimum selection of “natural
delivery systems”, exosomes have been generally applied as
attractive instruments for understanding on diseases devel-
Protein organic solvent precipitation opment and treatment. Thus, harvesting exosomes in a high-
(PROSPR) yield way is one of directions that scientists pursue in recent.
What’s more, although the exosome-acquired strategies have
This method for exosome isolation depends on the been extensively studied, the excavation of optimized iso-
enhanced ion-pairing effects of presented salts (elicited lation methodology and making it standardization are still
by acetone) and proteins (residing in exosomes). Usually, in progress. On the one hand, the scientists are troubled
the samples and chilled acetone are thoroughly mixed as by labor-intensive, low exosome production, high price of
­V1:4 (samples: acetone). Next, the supernatant is centrifugated at equipment, large sample volume demand and duration of the
5000×g less than 1 min for depleting hydrophilic species procedures using current exosome isolated techniques. On
or other contaminants. And then, the collected superna- the other hand, due to diverse complex parameters including
tant is dehydrated using a vacuum concentrator. The dehy- centrifugal radius, centrifugal speed, rotor type, solutions’
drated exosomes are then applied for subsequent analyses. viscosity and unchangeable shape of pores, UC, SEC and
This method is more suitable for exosome isolation from UF hold inferiorities in exosome separation. It seems that
small-volumes (0.5–1 mL) bio-samples including plasma the demand of morphological characteristics, size distribu-
and human brain tissue [68–70]. This method encompasses tion, marker protein expression and downstream-analysis
merits of large yield, stable exosome, less time and labor, but purposes fate the option to techniques for exosome isolation.
demerits of low purity, resolution difficulty, higher cost rela- And the exosomes by different extraction techniques match
tive to PEG-based method (also seen corresponding cost in different peculiarities.
Sigma-Aldrich). Otherwise, acetone may affect the biologi- PEG, confirmed as biosafety by FDA [73], is an out-
cal activity of exosomes, especially with potential damage to standing precipitation agent for exosome preparation due to
the structure and function of biomolecules within exosomes, its multiple colloidal behaviors. It is highlighted that PEG
limiting the downstream analyses [68–70]. precipitation is comparable to the standard or commer-
cial method. Furthermore, this methodology outperforms
other methods in terms of exosomes throughput. Exosomes
Commercial kits extracted from PEG-based precipitation were mainly 5–14
times more than that extracted by UC [19, 74, 75]. Indica-
To date, the biotech-companies have developed a wide range tively, the large-scale output of exosomes by this technique
of kits (ExoPrep, miRCURY Exosome Isolation Kit, TEI and can provide a great convenience for exosome-based clinical
ExoQuick [31, 71, 72]) based on precipitating exosomes to study and animal models. Furthermore, the exosomes coated

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Molecular and Cellular Biochemistry

with PEG are favorably protected from force destruction, Wang, Hanjuan Ning, Weiwei Liu, Xianghui Han: contributed to the
and have more compatibility for subsequent analysis regard- critical revision of the manuscript for important intellectual content.
Xianghui Han: edited, supervision. All authors read and approved the
ing to DNA, RNA and protein. Therefore, applying PEG final manuscript.
for exosome preparation ranks the top of the isolated lists.
However, this summary paper captures that most scholars Funding This work was funded by Xianghui Han (Grant No.
are plagued with remaining PEG or contaminant proteins in 82174016).
final particles. Employing multiple wash steps after precipi- Data availability No datasets were generated or analysed during the
tation is a commonly accepted scheme. Moreover, to deplete current study.
the undesired components, more other techniques have been
introduced into PEG-based exosome precipitation. Notably, Declarations
PEG precipitation is more suitable for research with a large
Conflicts of interest The authors declare no competing interests.
demand for exosomes, but not the purpose of specific exo-
some analysis. Even though, it is yet achievable by remod-
eling the biologically tunable PEG in future. Totally, the
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