Advanced Micro :nanotechnologies For Exosome Encapsulation and Targeting in Regenerative Medicine

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Clinical and Experimental Medicine

https://doi.org/10.1007/s10238-023-00993-7
REVIEW
Advanced micro‑/nanotechnologies for exosome encapsulation

and targeting in regenerative medicine
Hasti Tashak Golroudbari1 · Seyedeh Parnian Banikarimi1,2 · Aryan Ayati1 · Alireza Hadizadeh1 ·
Khorasani Zavareh Zahra 1 · Kiana Hajikhani1 · Asieh Heirani‑Tabasi1 · Ahmadi Tafti Mohsen 3 · Saeed Davoodi1 ·
Hossein Ahmadi Tafti1
Received: 26 November 2022 / Accepted: 5 January 2023

© The Author(s), under exclusive licence to Springer Nature Switzerland AG 2023
Abstract
Exosomes, a subset of vesicles generated from cell membranes, are crucial for cellular communication. Exosomes' innate
qualities have been used in recent studies to create nanocarriers for various purposes, including medication delivery and
immunotherapy. As a result, a wide range of approaches has been designed to utilize their non-immunogenic nature, drug-
loading capacity, or targeting ability. In this study, we aimed to review the novel methods and approaches in exosome engi-
neering for encapsulation and targeting in regenerative medicine. We have assessed and evaluated each method's efficacy,
advantages, and disadvantages and discussed the results of related studies. Even though the therapeutic role of non-allogenic
exosomes has been demonstrated in several studies, their application has certain limitations as these particles are neither fully
specific to target tissue nor tissue retainable. Hence, there is a strong demand for developing more efficient encapsulation
methods along with more accurate and precise targeting methods, such as 3D printing and magnetic nanoparticle loading
in exosomes, respectively.
Hasti Tashak Golroudbari and Seyedeh Parnian Banikarimi have

contributed equally to this manuscript.
* Hossein Ahmadi Tafti

hosseinahmaditafti@yahoo.com
1
Research Center for Advanced Technologies
in Cardiovascular Medicine, Tehran Heart Center,
Cardiovascular Diseases Research Institute, Tehran
University of Medical Sciences, Tehran, Iran
2
Department of Tissue Engineering and Regenerative
Medicine, School of Advanced Technologies in Medicine,
Mazandaran University of Medical Sciences, Sari, Iran
3
Colorectal Surgery Research Center, Imam Hospital
Complex, Tehran University of Medical Sciences, Tehran,
Iran
13
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Graphical abstract
Keywords Exosome · Encapsulation · Targeting · Biomaterials · Biologic scaffolds
Introduction lipids [4, 5]. Moreover, three studies have reported specific
functions for exosomes, including immunoregulation, repair,
Regenerative medicine is a novel approach for repairing and and regeneration [6–8]. Also, the application of these par-
regenerating injured cells, tissues, and organs using biomate- ticles as a cell-free therapeutic option in regenerative medi-
rials and various cell sources, mainly stem cells. The appli- cine has been investigated by previous studies, reporting
cation of stem cells for tissue regeneration is associated with their remarkable therapeutic advantages [8, 9].
immune rejection, which is a considerable challenge [1]. Given their liposome-like structure and targeting abilities,
However, cell-free tissue engineering eliminates the need the use of exosomes for several specific drug delivery sys-
for externally seeded stem cells and is performed using scaf- tems has been extensively investigated [10, 11]. The intrinsic
folds containing certain particles as the main requirement for ability of these particles to cross various biologic barriers
tissue regeneration. is helpful in reaching the target cells [12]. However, several
Exosomes are a type of extracellular vesicle (EV) pro- steps are needed to establish an efficient exosome-based
duced by various cells, including neoplastic, immune, or therapeutic system. One of the primary steps is loading the
stem cells. These particles have been identified in multi- desired bioactive molecules into the exosome-forming cells
ple biologic fluids and cell culture media and rarely cause or purified exosomes. Moreover, exosome engineering to
immune rejection in the recipients’ bodies [2, 3]. Studies increase their affinity to the desired tissue is another issue.
have shown that exosomes are involved in several paracrine One of the main challenges in the therapeutic applica-
and intracellular signaling pathways, including transcription, tion of exosomes is their low retention in the injured area.
proliferation, and differentiation. Their contents can be dif- Therefore, increasing their half-life, retention, and stabil-
ferent based on their cellular origin. However, they generally ity usually necessitates encapsulation methods [13]. These
contain proteins, nucleic acids (especially miRNAs), and techniques vary from conventional methods, such as loading
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the exosomes in hydrogels, to more advanced and novel their structures, these materials can absorb and retain large
methods. amounts of water [21]. It is believed that hydrogels are a
The present paper has made a comprehensive review promising vehicle for exosome delivery, considering their
of the current techniques used for exosome encapsulation, porosity and swelling degradation. They can reserve several
their underlying mechanisms, related pros and cons, and exosomes in their networks and release them in the injury
examples of the techniques used for treating various dis- site based on a sustained release profile [16, 22]. Moreover,
eases and related outcomes. In addition to the encapsulation these hydrogels have antibacterial and self-healing proper-
methods, targeting methods are of utmost importance in the ties, as well as high biocompatibility [23–25].
use of exosomes as a therapeutic agent or carrier with a Recent studies have investigated the use of exosome-laden
high affinity to a specific tissue. These procedures include hydrogels in various disorders, including wound healing and
several delivery methods for introducing exosomes to the bone, cartilage, and cardiovascular regenerations [26–32].
injured area, such as the surface modification or loading of A summary of these investigations is presented as follows:
the exosomes with targetable particles. The present review
will discuss the benefits and drawbacks of these methods.
Wound healing
Exosome encapsulation technologies Wound healing, especially in diabetes, is a tremendous

challenge for the affected patients and healthcare providers.
In the past decades, several research teams have tried to Interestingly, two separate studies have shown the benefits of
implement exosomes in scaffolds for tissue engineering topical exosome-laden hydrogels in improving wound condi-
purposes [14]. Exosome-laden scaffolds are a new genera- tions in diabetic patients.
tion of cell-free scaffolds with several benefits. For example, Yong et al. investigated the in vitro and in vivo effects
they support the regeneration of the injured tissue/organ by of exosomes derived from the human Umbilical Cord Mes-
releasing biological cues, such as growth factors, cytokines, enchymal Stem Cells (hUC-MSCs). These exosomes were
and interleukins [15], and can decrease the risks of cell encapsulated into the Pluronic F-127 (PF-127), a triblock
implementation for regenerative purposes, such as cancer co-polymer hydrogel made of polypropylene glycol (PPG)
or other disorders [14]. Moreover, these scaffolds prolong and polyethylene glycol (PEG) with several unique proper-
the life, retention duration, and regenerative properties of ties, such as biocompatibility and thermosensitive behavior,
exosomes by encapsulation, causing their slow release to making it a promising hydrogel for wound healing. Given
the injury site [16]. Scaffolds can be used with both cells its suitable pore size, this hydrogel could effectively absorb
and exosomes. They can be used for exosome encapsulation the wound exudates while maintaining sufficient moisture
or protection against the dynamic circulation of tissue fluids in the wound bed. Moreover, its topical application could
and other environmental factors. There are several scaffold enhance angiogenesis and improve the collagen formation
fabrication technologies used for exosome encapsulation, and orientation of collagen fibers in rat models with strep-
from bulk to micro-/nanofabrication methods, such as 3D tozotocin-induced diabetic wounds [33]. Also, Wang et al.
printers [17]. The following sections will make a compre- made an ultraviolet (UV)-protective hydrogel of PF-127,
hensive review of the mentioned methods and their applica- polyethylene imine (PEI), and aldehyde pullulan to deliver
tion in similar research works. the exosomes derived from the adipose stem cells (ASCs) to
the wound site, reporting its exceptional tissue adhesion and
self-healing properties caused by the Schiff-base bonds in its
Bulk methods (hydrogels) structure. In addition, the mentioned hydrogel had consider-
able bactericidal and hemostatic properties, which made it a
Following systemic injection, exosomes are estimated to favorable exosome vehicle [24].
have a half-life of 2–4 min in the plasma [18]. After two Zhao et al. investigated the effects of a gelatin methacry-
hours, most of their content is accumulated in the phago- loyl (GelMA) hydrogel for delivering the exosomes derived
cytes of the spleen and liver [19, 20]. Therefore, it is essen- from the human umbilical vein endothelial cells (HUVECs)
tial to choose a suitable biomaterial to deliver the exosomes to cutaneous wounds, revealing prolonged exosome release,
to the defective sites. As a type of exosome encapsulation improved angiogenesis and decreased granulation tissue for-
method, bulk methods are used for selecting these suitable mation in rat models [26]. Moreover, Li et al. investigated
biomaterials and combining them with the exosomes based the effect of a chitosan-based hydrogel on wound healing in
on their characteristics. rat models with full-thickness cutaneous wounds. Chitosan
Hydrogels are water-swollen, gel-like materials that can is a linear semi-crystalline polysaccharide produced by the
be natural or synthetic. Using several cross-linking bonds in partial deacetylation of chitin with characteristic peaks at
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20 °C, which is highly biocompatible and antibacterial, and groups. The researchers embedded the exosomes derived from
shows a sustained release profile. The encapsulated synovial human placenta mesenchymal stem cells (HP-MSCs) into the
mesenchymal stem cell (SMSC)-derived exosomes loaded mentioned MC-Ch hydrogel, which showed enhanced angio-
with MiR-126 improved the angiogenesis and re-epithelial- genesis and neo-epithelialization in diabetic wounds of mouse
ization of the wound [34–36]. models [42].
Chitosan can also be combined with other biomaterials
using various methods, such as simple blending or nanocom- Periodontitis
posite formation. Shi et al. embedded the exosomes derived
from the gingival mesenchymal stem cells (GMSCs) into Rather than wound healing, chitosan-based hydrogels can be
a silk-chitosan hydrogel and applied it to streptozotocin- used for exosome delivery in other pathological conditions,
induced diabetic cutaneous wounds. The silk-chitosan including periodontitis. This disorder and subsequent alveolar
hydrogel has a sponge-like porosity with pore diameters loss are mainly caused by the excessive inflammatory response
of 50–150 μm, which absorbs and retains a large amount of the immune system, which is usually caused by bacterial
of water, improving exudate-absorbing properties, epithe- infections and mediated by macrophages [43, 44]. Shen et al.
lialization, angiogenesis, and neural development in the prepared an injectable chitosan-based hydrogel by mixing a
injured area [37]. Also, Nooshabadi et al. incorporated the solution containing 2% chitosan and another containing 50%
exosomes derived from the human endometrial stem cells β-glycerophosphate. Then, the exosomes derived from the den-
(HEnSC) into a chitosan-based hydrogel containing chitosan tal pulp stem cells (DPSCs) were added. The resultant mixture
(1% in acetic acid) and glycerol (60% in water), which also was liquid at 4 °C and gradually turned into a gel by increasing
improved the healing process by increasing the fibroblast temperature [45]. Therefore, it completely filled the defected
migration [38]. areas, delivering the DPSC-derived exosomes to the injured
Li et al. introduced another modified chitosan hydrogel to tissues. These exosomes could ameliorate the periodontitis by
deliver the hUC-MSC-derived exosomes to the injured tissues. making the macrophages shift from the pro- to anti-inflamma-
The hydrogel was made by adding genipin (a cross-linker) tory phase, leading to inflammation alleviation [46].
and poloxamer 407 (a thermo- and pH-sensitive agent) to a
carboxymethyl chitosan solution, leading to the formation of
a mesh-structured exosome-loaded hydrogel, the resultant Neurological disorders
hydrogel promoted the healing process by releasing exosomes
within 3 days [39]. Moreover, another study introduced a chi- Exosome therapy is a promising technique for treating neu-
tosan-based hydrogel by adding Ca (NO)2 and N a2HPO4 to rological disorders [47]. As a decisive pathological condi-
chitosan. Also, the exosomes were formed by preparing miR- tion, spinal cord injury (SCI) can result from various factors,
126-3p-overexpressing SMSCs through transfection with len- such as trauma. This disorder is associated with several disa-
tiviruses. The exosome-laden hydrogel enhanced angiogenesis bilities, such as motor and urinary dysfunctions, and severely
and wound healing in diabetic cutaneous wounds of rat models affects the patient's quality of life [48]. According to studies,
[34]. exosomes derived from different stem cell sources, such as
As another natural hydrogel, alginate is derived from human placenta amniotic membrane MSCs, can promote
brown algae and is commonly used in biomedical sciences. tissue regeneration in various SCI models. Li et al. modified
Considering its biocompatibility, relative cost-effectiveness, a hyaluronic acid hydrogel with laminin-derived peptides
low toxicity, and structural resemblance to the extracellular to enhance its affinity for the MSC-derived exosomes. The
matrix (ECM), the material is an excellent platform for exo- interactions between the modified hydrogel and exosomes
some encapsulation and release [40]. Shafei et al. developed led to the considerable accumulation and controlled release
an alginate-based hydrogel containing 3% (w/v) alginate and of the exosomes in the injury site, which improved the
1% (w/v) C aCl2 solution as a cross-linker for alginate conver- microenvironment needed for regeneration, facilitated motor
sion to the hydrogel. Afterward, the hydrogel was added to and sensory recovery, and enhanced the urinary system func-
the ASCD-derived exosomes (100 µg/ml), and the resultant tion in the animal models [49].
exosome-laden hydrogel was applied to the cutaneous wounds Moreover, it has been shown that hydrogels with electri-
of rat models. The hydrogel showed a stable exosome release cal conductive characteristics made from synthetic or natural
during 7 days, promoting tissue regeneration [41]. In another biomaterials can be promising scaffolds in spinal cord regen-
study, Wang et al. used the methylcellulose-chitosan (MC- eration [50, 51]. For example, Fan et al. loaded BM-MSC-
Ch) hydrogel as an exosome carrier for wound healing. The derived exosomes into a conductive hydrogel consisting of
hydrogel exerted shear-thinning and self-healing properties a gelatin-methyl acrylic anhydride, tannic acid, and methyl
because of its structural Schiff-base bonds. Also, it had proper acrylic anhydride-pyrrole hydrogel controlled the rate of
adherence to the wound site due to the aldehyde functional
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exosome release, enhancing axonal outgrowth and reducing Zhang et al. encapsulated the HP-MSC-derived exosomes
inflammatory responses [52]. into a chitosan-based hydrogel containing chitosan solution,
acetic acid, and β-glycerophosphate. The resultant hydrogel
was applied in mouse models with hind limb ischemia and
slowly released the exosomes at a speed of 0.25 µg in an
Cardiovascular disorders hour. The models showed improved angiogenesis and mus-
cle formation [58].
According to the literature, myocardial injury caused by car-
diovascular diseases is almost never fully repaired. Thus,
the use of regenerative medicine may be effective in repair- Skeletal defects
ing the lost myocardial tissue [7, 30, 53–55]. In a study by
Chen et al., the endothelial progenitor cell (EPC)-derived Repairing the defected areas of large bones is a consider-
exosomes were loaded into an injectable shear-thinning gel able challenge in the field of orthopedic surgery [59]. Such
(STG) to prolong the exosome accumulation duration in the problems can be addressed using bone tissue engineering, a
ischemic zones of rat models with myocardial infarction, valuable method for bone repair using scaffolds made of nat-
reporting improved ventricular compliance and hemody- ural or synthetic substances. However, this method is asso-
namic condition in the treated animals [30]. In another study ciated with low regenerative effectiveness due to the lack
by Derkus et al., the human MSCs were treated with cardi- of angiogenesis within the scaffolds [60]. Exosome therapy
omyocyte-derived exosomes to induce the cardiomyocyte has been a favorable treatment for skeletal defects [61–63].
differentiation of MSCs. Exosomes were encapsulated in a Fan et al. encapsulated the exosomes derived from human
triamine-functionalized hyaluronic acid hydrogel with high MSCs with noggin knockdown (noggin is a bone formation
biocompatibility and controllable porous structure, which antagonist) into a hydrogel made from glycidyl methacrylate
led to improve cardiomyocyte regeneration [28]. Moreover, and glycol chitosan (GCS). The mentioned hydrogel showed
Han et al. introduced a synthetic hydrogel to deliver hUC- a sustained exosome release and could induce the expres-
MSC-derived exosomes to the ischemic areas in myocardial sion of osteogenic genes (e.g., Runx-2, Osterix, OCN) and
infarction. The base of the gel was made by blending two bridge the bone defect within 8 weeks [27]. Considering the
main synthetic peptides of GHRPS (His-D-2-methyl-Trp- potential mechanical insults to hydrogels, this material has
Ala-Trp-D-Phe-Lys) and PA (C16-GTAGLIGQ). Afterward, the prominent advantage of self-healing. Wang et al. made
another synthetic peptide, NapFF, was added to improve the a hydrogel from coralline hydroxyapatite (CHA), SF, GCS,
gelation of the PGN hydrogel, a synthetic amphiphilic pep- and de-functionalized polyethylene glycol (DF-PEG) with
tide hydrogel. The researchers reported the sustained release excellent self-healing characteristics. Also, the parts of this
profile of exosomes, improved angiogenesis, increased left hydrogel could fuse without leaving any incision mark after
ventricular ejection fraction, and predominantly alleviated being cutoff. The hydrogel was then loaded with hUC-MSC-
myocardial function [29]. derived exosomes and applied to an animal model of femoral
The application of alginate-based hydrogels for exosome condyle defect, leading to relative healing within 3 months
delivery in cardiovascular diseases has been investigated. In [64].
a recent study, researchers stirred a 2% alginate solution with Given the poor vascularization of the cartilage and the
the exosomes derived from the bone marrow MSCs, fol- low number of chondrocytes in this tissue, treating the car-
lowed by adding 1% calcium solution as a cross-linker. The tilage defects is a clinical challenge [65]. Liu et al. encap-
resultant hydrogel increased the exosome which enhanced sulated the exosomes derived from the human-induced
angiogenesis, and controlled inflammation and apoptosis in pluripotent stem cells (hiPSCs) into a hydrogel glue with
the infarcted area [56]. photo-triggered imine cross-links. Moreover, this hydro-
Age-related vascular dysfunction is associated with gel contained hyaluronic acid modified with o-nitrobenzyl
miR-675 expression, leading to the attenuation of the tissue alcohol, which generated aldehyde groups solidifying under
growth factor β1 (TGF-β1) senescence pathway. Han et al. light emission. These aldehyde groups could react with the
derived exosomes from the miR-675-overexpressing hUC- amino group of various biomaterials, including chitosan,
MSC using the lentivirus plasmid transfection technique. gelatin, and chondroid tissue, forming a seamless and inte-
Afterward, they encapsulated the resultant exosomes in a grated patch in the defected site. The researchers injected
4% SF injectable hydrogel to slow the rapid washout of the the hydrogel directly into the defected cartilage of rabbit
exosomes and used the mixture in aged mouse models with models with hind limbs to turn into its gel phase in place.
ischemia induced by femoral ligation. The improved perfu- The gel was integrated into the defected tissue, which was
sion could increase the levels of miR675, leading to attenu- surrounded by mature intact tissue, resulting in facilitated
ated levels of TGF-β expression following 28 days [57]. cell migration and proliferation, increased deposition of
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Table 1 Different exosome–hydrogel combinations and their applications
Polymers Exosome cell source Application Exosome release Advancements References
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Alginate Adipose-derived stem cells Wound-healing 50% of exosome capacity in 72 h High biocompatibility [41]
and almost all of the exosome Low toxicity
component in 172 h Low price
ECM resemblance
Alginate Bone marrow mesenchymal stem Myocardial Infarction Cumulative release in about Biocompatible [56]
cell 10 days with a burst release in Non-immunogenic
the first days
Chitosan MiR-126-3p over-expressing syn- Wound-healing 80% in 6 days Antibacterial [23]

ovium mesenchymal stem cells Anti-fungal
Hemostatic characteristics
Chitosan Dental pulp stem cell Periodontitis Released most of the loaded Porous gel structure at 37 °C with [46]
exosomes within a week an average pore size of 80 µm
Hydrogel properly fills the
defected area and provides DPSC
exosomes to the injured tissue
Chitosan Human placental mesenchymal Hind limb ischemia 0.25 µg/h release rate for the first Thermosensitive [58]
stem cells 12 h
Chitosan-silk Gingival mesenchymal stem cell Wound-healing _ High capacity for water retention [37]
Induces cell proliferation and ECM
formation
Chitosan-glycerol Human endometrial stem cell Wound-healing _ Synergic application of HEnSC- [38]
Exo increases cell growth and
proliferation
Injectable hydrogel that properly
fills the defect
GP-chitosan Human umbilical cord mesenchy- Wound-healing Released near 85% in 72 h Thermo- and pH-sensitive [39]
mal stem cell High biocompatibility and proper
biodegradability (slowly degrade
in 240 h)
Hydroxyapatite/chitosan miR-126-3p-overexpressed syn- Wound-healing 6 days Non-toxic [34]
ovium MSC Hemostatic
Methylcellulose/Chitosan Human placenta mesenchymal Wound-healing _ Self-healing [42]
stem cell Shear-thinning
Pluronic F127 hydrogel Umbilical cord mesenchymal stem Wound-healing _ Thermosensitive [33]
cell Congeals gradually and fills the
wound defect properly
Pluronic F127 hydrogel Adipose-derived stromal cells Wound-healing 80–90% cumulative release in Self-healing [24]
https://www.tarjomano.com
21 days pH-dependent exosome release

UV-protective
Anti-bacterial
Gelatin-methacrylate Human umbilical vein endothelial Wound-healing Cumulative release in 7 days Biocompatible [26]
cell Skin repair induction
Table 1 (continued)
Polymers Exosome cell source Application Exosome release Advancements References
Gelatin-methyl acrylic anhydride- Bone marrow mesenchymal stem Spinal cord injury 90% in 14 days Electro-conductive [52]
pyrrole cell Adequate tissue adhesion
Biocompatible
Biodegradable
Hyaluronic acid hydrogel with Human placenta amniotic mem- Spinal cord injury 90% in 11 days High degree of accumulation, [49]
laminin-derived peptides brane mesenchymal stem cell Controlled release of exosomes in
the injured site
Improved the microenvironment

needed for regeneration and cell

growth
Tyramine-functionalized hyalu- Primary human cardiomyocyte* Induction of MSC differentia- 50% in 2 days and cumulative Adequate biocompatibility [28]
ronic acid tion into cardiac cells** release in 5 days Sustained release of exosome from
the 3D structure
Hyaluronic acid and gelatin Human-induced pluripotent stem Cartilage defect Preserves more than 90% of its Biocompatible [31]
cells exosome capacity for 14 days Injectable glue that fills the defect
of joint cartilage
High tissue adhesion
Ad-HA and CD-HA Endothelial progenitor cell Myocardial infarction Cumulative release in 21 days Shear-thinning [30]
Self-healing
Synthetic peptide hydrogel Human umbilical cord mesenchy- Myocardial infarction Near 80% in 22 days Biocompatible [29]
mal stem cell Cell proliferation induction
Silk fibroin Human umbilical cord mesenchy- Senescent vascular dysfunction 70% cumulative release in 36 days Biocompatible [57]
mal stem cell Biodegradable
Sustained release rate
CHA/SF/GCS/DF-PEG Human umbilical cord mesenchy- Bone (femur condyle defect) 87% in 30 days Self-healing quality [68]
mal stem cell Improved osteogenic effects
MeGC Genetically modified human mes- Bone (Calvarial) defect _ Cell-viable hydrogel [27, 69]
enchymal stem cells Biodegradable
ECM Extracellular matrix; hEnSC Human endometrial stem cell; MSC Mesenchymal stem cell; GP-chitosan Genipin and poloxamer 407 added to carboxymethyl chitosan; FEP Pluronic F127,
Polyethylenimine (PEI), and Aldehyde Pullulan (APu); MeGC Glycidyl methacrylate blended with Glycol Chitosan (GC); CHA/SF/GCS/DF-PEG Coralline Hydroxyapatite, Silk Fibroin, Gly-
col Chitosan, De-Functionalized Polyethylene Glycol; hUC-MSC Human umbilical cord mesenchymal stem cell; FHE Pluronic F127, oxidized hyaluronic acid, and Poly-ε-L-Lysine; Ad-HA
Adamantine-modified Hyaluronic acid; CD-HA β-Cyclodextrin-modified hyaluronic acid
*In this study, the cardiomyocyte-derived exosomes and human adipose MSCs were encapsulated into a HA/Tyr hydrogel
**This study only included in vitro investigations and did not investigate the in vivo effects
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collagen I and II, and formation of new hyaline cartilage in 3D Bioprinting

the injury site [31].
In general, hydrogels are excellent exosome carriers 3D bioprinting is a modern technology with extensive
owing to their biocompatibility and sustained-releasing applications in tissue engineering. This method can be used
effects. However, the clinical application of these hydrogels for encapsulating various stem cells, drugs, nanoparticles,
can be accompanied by several challenges. For example, and biologic agents, such as exosomes [69]. Moreover, it
there is a chance of toxicity induced by unreacted cross-link- is a promising and revolutionary technique for generating
ers, especially in injectable hydrogels. Moreover, the physi- tissue-like structures for repairing different tissues, such as
cal aspects of different hydrogels vary in various pH and bone, cutaneous, and cardiac tissues [70]. It is performed
temperature conditions. Also, there is a chance of premature by depositing a bioink on a surface to form a 3D, heteroge-
gelation during administration. Finally, injectable hydrogels neous, functional structure. This so-called bioink is a sus-
should be sterilized, typically using gamma irradiation or pension of living cells, biomolecules, growth factors, and
alcohol treatment. However, possible alterations in the phys- biological agents inside a biomaterial platform that can be
ical features of hydrogels or unwanted chemical reactions used by 3D printers to form spatial patterns [70, 71]. 3D bio-
may impede the function of these biomaterials [66]. printing methods can be classified into four main domains:
In conclusion, despite the above challenges, the use of extrusion bioprinting, inkjet-based bioprinting, laser-assisted
a combination of different hydrogels and exosomes from bioprinting, and stereolithography [72].
distinct cell sources has shown promising results in treating Given its availability, simple use, and cost-effectiveness,
several diseases (see Table 1). However, there is a need for extrusion-based bioprinting is a popular technique. These
further investigations to improve the desired characteristics bioprinters can use an extensive range of bioinks with
of these hydrogels [67]. A summary of the mentioned stud- various viscosities to fabricate well-organized construc-
ies on this topic is presented in Table 1. tions with clinically appropriate sizes in a reasonable time.
However, the fast speed of these devices usually decreases
their resolution. On the other hand, inkjet-based bioprint-
Advanced techniques for exosome ers have a higher cell density capacity compared to other
encapsulation methods, while they only print bioinks with low viscosities
and specific electromagnetic properties. Another method,
Considering the challenges in exosome encapsulation using laser-assisted bioprinting, is an expensive option but is
bulk methods such as probable toxicity, undesirable physical associated with the highest cell viability. The last class, ste-
changes, and chemical reactions, it is necessary to design reolithography, has recently acquired popularity because of
an applicable microfabrication system with a controllable its ability in omnidirectional printing and creating support-
and designable releasing profile as an advanced platform free complicated structures. However, this method is often
for delivering exosomes. Encapsulating bioactive molecules, limited to the same vat of photo-curable resins, leading to
such as exosomes, inside 3D scaffolds made using micro- or single-material printing [70, 71]. The characteristics of the
nanofabrication techniques has several advantages. It can mentioned methods are presented in Table 2 in detail.
help in preserving the structure, stability, and function of
these molecules for extended periods. Advanced techniques
like 3D bioprinting can be a great help for tissue engineers Extrusion‑based bioprinting
in encapsulating exosomes or their cell sources into scaffolds
with microscale intricacy. These methods can be useful for Extrusion-based bioprinting is a technique including layer-
fabricating various cell-laden or exosome-laden scaffolds by-layer extrusion or dispensing of continuous strands or
with different polymer sources, shapes, and architectures biomaterial fibers to create 3D scaffold structures. The force
[68]. The following section discusses the 3D bioprinting required for bioink ejection can be provided by air pres-
methods and their pros and cons in detail. sure (pneumatic devices), piston, or screw. In this method,
Table 2 Characteristics of Bioprinting methods Viscosity (mPa.s) Cell density Cell viability (%)
different bioprinting methods
Extrusion 30 − 6 × 107 High 80–90
Inkjet-based < 10 < 16 × 106 cells/mL 90
Laser-assisted 1–300 108 cells/mL > 95
Stereolithography 250 − 104 NA NA
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a continuous flow of bioink is deposited on the substrate. for bone tissue engineering. Also, the combination of
Moreover, the bioink's rheological properties, extruding exosomes derived from umbilical MSCs and a 3D-printed
temperature, and applied pressure can affect the biological scaffold could improve the angiogenesis and osteogenesis in
and physical properties of the printed structure [72]. Another the defected models [77].
technique in this category is cryogenic extrusion bioprint-
ing. In this method, the water freezes in the extrusion slurry,
leading to phase separation and rapid 3D forming. Following Inkjet‑based bioprinting
freeze-drying, the frozen water is removed from the fila-
ments, leaving micropores in the structure. This can increase Inkjet-based bioprinting is a non-contact method in which
the scaffold's surface roughness and porosity, augmenting the bioink is deposited onto the substrate under the control
cellular adhesion, growth, and proliferation. of computer software. This method can be performed using
Hu et al. worked on a strategy for facilitating the heal- the continuous or drop-on-demand modes. In the continuous
ing process of diabetic wounds using a 3D printed scaffold. mode, a coalescent flow of bioink is deposited from a nuzzle
The researchers decellularized the submucosa of the small and reaches a substrate with an electrostatic field. However,
intestine, which was then added with mesoporous bioac- the drop-on-demand mode is based on thermal or piezoelec-
tive glasses to form a sustained-releasing carrier for bio- tric technology. In this method, pressure is applied to the ink
active exosomes. Afterward, a 3D scaffold was fabricated container to deposit the bioink if needed [78].
using cryogenic extrusion bioprinting, and the exosomes Exosomes exist in two states: a “liquid phase”, dispers-
derived from bone marrow stem cells with a mean diameter ing in body fluids, and a “solid phase”, immobilized to the
of 75 nm were added. The resultant scaffold had a proper extracellular matrix [79]. Localized exosome-based micro-
3D structure, high biocompatibility, appropriate porosity, environments are formed by solid-phase exosomes [80]. A
and homeostasis-maintaining effects and was used to cover study by Saigopalakrishna et al. investigated the interac-
the dorsal cutaneous wounds with full thickness in diabetic tion between the ECM components and exosomes during
rats. Finally, the researchers reported an increased migra- intercellular communication. The researchers fabricated a
tion, proliferation, and angiogenesis of the HUVECs. Also, specific pattern of solid-phase exosome microenvironments
the scaffolds could increase wound perfusion, decrease its using an inkjet-based bioprinter. Given the role of exosomes
length, and increase the deposition of collagen fibers [73]. in cell reprogramming, they derived exosomes from differ-
Given the lack of appropriate biomaterials, regenerating ent macrophage subsets. The bioink formulation was esti-
the long-segment trachea has remained a complex medical mated to contain 10 µg/mL of exosomes, in a PBS buffer
issue with no satisfactory answers. Therefore, it is suggested with 10% glycerol, which was added to prevent exosome
to use 3D-printed scaffolds to maintain enough mechani- agglomeration as a humectant. The bioprinter had a speed
cal support to avoid tracheal collapse. Zhang et al. used the of 1.8 ± 0.52 m/s, thus generating approximately 70 ± 4.87
exosomes derived from mouse embryonic fibroblast cells pL droplets and forming a deposited splat with a diameter
(3T3-J2 cells) and the tracheal basal cells (TBCs) isolated of 75 μm. The effect of physical forces during inkjet-based
from the autologous tracheal mucosae of rabbits to build bioprinting on exosome membrane integrity was negligi-
scaffolds using a 3D multi-axis printing system. A double- ble. Thus, no difference was observed between the native
layer method was used to form these scaffolds in order to and bioprinted exosomes. According to the results of this
seed two types of cells simultaneously, with the inner layer in vivo study, inflammatory M1 macrophages induced the
containing TBCs and the outer layer containing autologous angiogenesis and proliferation of myogenic precursor cells
chondrocytes. Afterward, the scaffolds were used to restore in the early phase of muscle regeneration. Moreover, the M2
extended segmental defects of the trachea in New Zealand macrophages promoted the differentiation of mesangiogenic
white rabbits. Following two weeks, the lumen surface of progenitor cells in the last stage of regeneration [79].
the tissue-engineered trachea was entirely covered with cili-
ated epithelium, improving the prognosis of the recipient
animals [76]. Stereolithography
Moreover, the 3D bioplotting method has been used to
fabricate nanohydroxyapatite and PCL scaffolds. In this In this method, a photo-curable liquid or polymer composite
method, nanohydroxyapatite and PCL were mixed with a material is exposed to light to form cross-links. When a sin-
ratio of 3:7, and the resultant paste was put into a polyethyl- gle layer is formed by light exposure, the sample is translated
ene injection cartridge and underwent layer-by-layer extru- to form the next layer [81]. Moreover, the bioprinter contains
sion to fabricate a scaffold used for repairing cranial defects a digital light processing chip, XYZ stages, and a visible
in rat models. This carrier showed a sustained exosome light source with a wavelength of 405 nm.
release for an extended period, achieving the requirements
13
Several studies have used stereolithography 3D bioprint- Moreover, CP05, a highly polar polycationic peptide, was
ing to fabricate scaffolds for exosome encapsulation and used to modify the PCL scaffold substrate to increase its
release. Chen et al. fabricated a 3D scaffold with radially exosome-attaching properties [79]. Afterward, exosomes
oriented channels composed of cartilage ECM, GelMa, and were encapsulated into the 3D scaffold. 3D-printed porous
exosomes derived from MSCs with a diameter of 40–140 nm scaffolds provided a 3D environment for vascular remod-
and an initial concentration of 200 μg/mL in the hydrogel. eling [80]. Also, exosomes acted as an osteogenic matrix and
The researchers used stereolithography for scaffold printing a carrier for the genes promoting vascular osteogenesis in
due to the high-resolution and photo-curable nature of the bone defects. This scaffold can be used as a cell-free tissue
used biomaterial. The generated exosome-laden scaffolds, engineering for vascularized bone remodeling [79].
which had a dimension of 4 × 4 mm, were transplanted into Exosomes derived from MSCs play a crucial role in the
the damaged knee cartilage of rabbit models with osteo- formation, extension, and differentiation of blood vessels in the
arthritis. According to their findings, the scaffolds were regeneration of diverse tissues, including cartilage, skin, and
degraded in less than three weeks after transplantation and tendon [81, 82]. In diseases like myocardial ischemia–reper-
could effectively retain the exosomes for at least seven days. fusion, osteoporosis, and hematological malignancies, which
Moreover, they enhanced the chondrocyte migration, M2 are systemic diseases, exosomes are usually directed into the
macrophage polarization, and regeneration of the cartilage injured area [53, 83, 84]. However, repairing the damaged tis-
and subchondral bone in animal models [54]. sue, especially bone tissue, is a long-term process with differ-
ent phases. Due to the fast loss of exosomes in the body fluids,
unloaded exosomes do not have a continuous effect, which
Laser‑assisted bioprinting restricts their effects. As a result, choosing a suitable carrier
for the sustained release of exosomes at the injured site while
In this method, a laser beam with a high-power density is maximizing their retention and stability can be a promising
devised to melt and fuse a metallic powder. In each layer, technique for repairing bone tissue [85]. A recent study by
the cross-links are created by selectively melting and re- Zhang et al. encapsulated the exosomes derived from umbilical
solidifying the metallic powders. When a layer is completed, MSCs (uMSCs) with a mean diameter of 60–120 nm into the
the building platform moves downward and levels a new hyaluronic acid gel and injected the resultant mixture into the
layer of powders [74]. pores of a bioprinted scaffold. Given their suitable mechani-
Zhai et al. investigated the application of these bioprinters cal properties, biocompatibility, and corrosion resistance,
in cell-free bone regeneration. The researchers pre-differ- titanium alloys are widely used in orthopedic implants [86,
entiated the MSC-derived exosomes in an osteogenic dif- 87]. 3D-printed titanium implants contain porous structures
ferentiation medium for 0, 4, 10, 15, and 20 days. Moreover, and can improve osteoinduction and osteointegration [88].
they fabricated a titanium scaffold with a 3 mm diameter The titanium scaffolds can be fabricated using electron beam
and 8 mm length from T i6Al4V powder as a carrier for melting, another method used for 3D printing. In this method,
exosomes using the laser melting 3D printing method. Then, the scaffolds are designed using a 3D CAD program. After-
they seeded the scaffold with exosomes and used them in rat ward, they are fabricated and sliced into multiple thin layers.
models with a defect in the radial bone. According to their An electron beam selectively melts the desired material, such
findings, the exosome-laden scaffolds pre-differentiated for as metal, alloy, polymer, or ceramic powders, to generate a
10 and 15 days showed osteogenic differentiation, and cell- compact layer with a general thickness of several powder par-
free bone regeneration was achieved within 12 weeks [75]. ticles. Finally, the melted particles solidify quickly in an inert
According to experience, treating large sectional bone environment, such as pure argon, forming a 3D scaffold [89].
defects is a great challenge because the bones newly gener- A recent study by Wu et al. studied the exosomes derived from
ated using tissue engineering have no vascularization [76]. the Schwann cells (SCs) for their in vitro ability to promote
A recent study by Zha et al. used fused deposition modeling proliferation, migration, and differentiation of BMSCs. The
(FDM) to fabricate a 3D-printed exosome-laden polycapro- researchers loaded exosomes into Matrigel and injected them
lactone (PCL) scaffold. In this method, thermoplastic fila- into a porous Ti6Al4V scaffold fabricated using the EBM 3D
ments are melted in a heated nozzle and extruded to con- printing technique. The scaffolds had a diameter of 5 mm and a
struct a layer-by-layer structure [77]. The newly deposited height of 10 mm and were implanted in 36 rabbit models with
material joins the previous layers. Afterward, the head moves bone defects. According to their results, this composite implant
in the X–Y plane, depositing material in the shape of each showed a solid biological activity and provided simultaneous
layer [78]. The researchers used the exosomes derived from mechanical support and open pores, making it a reliable strat-
mouse chondrogenic progenitor cells (ATDC5) containing egy for treating bone defects [90].
the gene of vascular endothelial growth factor (VEGF).
13
Exosome‑modifying methods for targeting plasmid. Then, the CTP was fused to LAMP2b. Moreo-
purposes ver, a glycosylation sequence was attached to the CTP-
LAMP2b to stabilize the resultant peptide on the surface
Exosomes have demonstrated innate targetability which is of exosomes. According to their results, the number of
made possible by their surface antigens that are presented on CTP-expressing exosomes delivered to the cardiac tis-
the surface of these vesicles; however, as these antigens are sue was 15% higher in the intervention group compared
specific to the cells that express the appropriate ligands, other to the control group [97]. Also, Wang et al. used a new
techniques are required for more accurate targeting. Moreover, peptide sequence, CSTSMLKAC (IMTP), for targeting
the short half-life of exosomes minimizes the efficacy of sys- the ischemic heart tissue. The researchers synthesized
temic administration [19, 91]. Although local administration the LAMP2b and IMTP gene sequences and transfected
of exosomes aims to overcome these limitations, studies have the MSCs with them, leading to the fusion of IMTP and
demonstrated the majority of the exosomes are washed off LAMP2b on the surface of exosomes. According to their
before enacting to their full potential. On the other hand, local findings, the number of engineered exosomes targeted to
administration requires invasive and direct approach to target the ischemic tissue increased [98].
tissue. [31, 92]. A study by Bellavia et al. tried to target the cancer-
In recent years, studies have investigated the targeted deliv- ous cells in chronic myelogenous leukemia (CML) with
ery of exosomes to the desired area using a systemic infusion. exosomes containing imatinib or siRNA. Since the inter-
There are three approaches used in the targeted delivery of leukin 3 (IL3) is overexpressed in CML, the researchers
exosomes, including genetic engineering, chemical surface used this protein as an ideal receptor for targeting vehicles
modification, and exosome loading with specific types of parti- and engineered the HEK293T cells to produce exosomes
cles showing targeting properties. Engineering the donor cells expressing IL3-LAMP2b by fusing the human IL3 fragment
by loading ligand-producing genes on the surface of extracted to human LAMP2b. According to their results, reformed
exosomes is an indirect method for increasing exosome tar- exosomes could successfully target cancerous cells [99].
getability. Moreover, it is possible to directly add functional Moreover, Limoni et al. used exosomes for target therapy of
groups to the exosome surface via chemical reactions. Finally, HER2-positive breast cancer. The researchers modified the
the last technique is to load particles, such as magnetic nano- HEK293T cells to produce exosomes with LAMP-DARPin
particles, into the exosomes using physical, chemical, and bio- ligand loaded with siRNA, reporting the successful delivery
logical approaches [93–95]. of therapeutic agents by exosomes to the tumor cells [100].
Cell engineering for making surface-modified exosomes
is a promising method to increase the exosome targeting
Exosome targeting ability. However, this method is time-consuming, and the
cell sources used for exosome extraction should be acces-
Genetic engineering sible. On the other hand, these limitations can be over-
come using chemical surface modification methods.
In the genetic engineering of exosomes, the genes express-
ing the goal ligand are present on the exosome surface.
The cells that are secreted exosomes have to be transfected Chemical surface modification
by ligand expressing gene. Given the expression of the
desired gene in these cells, the extracted exosomes will The surface of exosomes can be directly engineered using
have ligands required for targeting specific cells on their various chemical modifications to increase their targetability
surfaces. The most common protein ligands on exosome [101, 102]. In chemical modification methods, dissimilar
surfaces are adhesion molecules, such as the tetraspanin ligands from synthetic or natural sources can be applied on
and integrin families [96]. exosome surfaces via covalent or non-covalent chemical
Lysosome-associated membrane protein 2b (LAMP2b) reactions. Here we point out various methods for chemi-
is the most abundant protein applied on the exosome cally modifying exosome surfaces.
surface for targeting purposes. Using this protein, the
ligand–receptor interaction on the exosome surface is
mediated via the N-terminus of LAMP2b [93]. Kim et al. Covalent chemical surface modification
produced cardiac-targeted exosomes to deliver therapeutic
agents to the heart tissue. A combination of cardiac-target- Surface modification via covalent interactions can be per-
ing peptide (CTP) and LAMP2b was made by transfecting formed using two main approaches: click chemistry and
the HEK 293 cells using the GNSTM-FLAG-LAMP2b-HA polyethylene glycol modifications.
13
Click chemistry is commonly used to prevent phagocytosis and extend the

circulation half-life of liposomes and synthetic nanoparti-
Click Chemistry, also known as the azide-alkyne cycloaddi- cles [115]. Myung Soo et al. developed a targeted exosome-
tion, is a method for covalent chemical modification. In this based formulation with systemic delivery by integrating the
method, an alkyne group is added to the amine group on the exosomes loaded with paclitaxel, a potent anticancer agent,
exosome surface. Then, it covalently conjugates to the azide and an aminoethyl anisamide-polyethylene glycol (AA-
group of the targeting moiety in the presence of copper [103, PEG) vector moiety. This combination was used to target the
104]. Moreover, the conjugation process does not affect the sigma receptor, which is overexpressed by the lung cancer
size of exosomes or their absorption into the target cells, cells. According to their results, these exosomes showed a
proving that the reactions are acceptable for exosome surface higher absorption capacity, aggregation in malignant cells
modification [111]. after systemic administration, and better therapeutic results.
Thang et al. used click chemistry to chemically modify Exosome-based medication formulations combine targeting
exosomes in order to target neuropilin-1 (RGERPPR, RGE). capabilities with biocompatibility to provide a robust and
Afterward, the exosomes were loaded with superparamag- innovative delivery platform for anticancer treatments. Thus,
netic iron oxide nanoparticles (SPIONs) and curcumin (Cur) these systems might be the next generation of drug delivery
using electroporation and were used for imaging and treating carriers, showing high targetability and a low immunogenic
glioma cells and orthotopic xenografts. According to their profile [116].
results, these exosomes demonstrated good stability, bio-
compatibility, and acceptable glioma-targeting ability. They Non‑covalent chemical surface modification
could pass the blood–brain barrier easily, which facilitated
their glioma-detecting ability and improved their therapeutic These methods include electrostatic, receptor-ligand, and
impact. Moreover, SPION-mediated magnetic flow hyper- hydrophobic interactions.
thermia (MFH) and Cur-mediated treatment had a significant
synergistic anticancer effect. As a result, the diagnostic value
of these methods for glioma detection and their therapeutic Electrostatic interactions
effects were improved considerably, while side effects were
significantly reduced. These modified exosomes might be The use of electrostatic interactions is a non-covalent chemi-
beneficial in early glioma detection and analyzing the treat- cal method for weak attachment of targeted moieties to exo-
ment effectiveness [105]. some surfaces. It is performed by adding cationic species on
Tian et al. applied copper-free click chemistry to conju- the surfaces of exosomes to increase their interaction with
gate a cyclopeptide (cyclo-Arg-Gly-Asp-D-Tyr-Lys) with a negatively charged biological membranes [109]. Nakase
high affinity for integrin v3 onto the exosome surfaces. The and Futaki used this approach to link cationic lipids and a
integrin v3 is found on the reactive vascular endothelial cells pH-sensitive fusogenic peptide (GALA) to the negatively
following cerebral ischemia. Afterward, curcumin was loaded charged membranes of HeLa-derived exosomes. The cati-
onto the surface-modified exosomes. Finally, exosomes were onic lipid lipofectamine (LTX) was attached to the exo-
administered intravenously to target the cerebral ischemic some surface, forming exosomes with positively charged
lesion, resulting in a considerable decrease in the lesion size, surfaces and improved binding and absorption into recipi-
inflammatory responses, and cellular death compared to only ent cells [117]. However, some cationic nanomaterials can
curcumin or curcumin-free exosomes. These findings suggest cause cytotoxicity because of their membrane-weakening
that the exosomes modified with the mentioned cyclopeptide properties. Cationic nanoparticles often enter the cells via
are a potent drug carrier for ischemic brain disorders [106]. endocytosis, which may lead to their lysosomal breakdown.
Tabata et al. suggested that cationic pullulan, which can
Polyethylene glycol modification bind to the asialoglycoprotein receptors of hepatocytes,
might target the damaged liver and increase the therapeutic
Polyethylene glycol modification (PEGylation) is another efficacy of exosome-based treatments. According to their
method for chemical surface modification that adds covalent hypothesis, exosomes modified with cationic pullulan had
links of PEG to the surface of exosomes. PEGylation of a a better internalization into human hepatoma (HepG2) cells
particle reduces the chance of identification by the mononu- because they could internalize using the asialoglycoprotein
clear phagocyte cells. Exosomes are commonly PEGylated receptor, which was exclusively expressed on HepG2 cells
to improve the in vivo stability, circulation duration, and tar- and hepatocytes. In conclusion, the researchers reported
getability to specific tissues and cells [114]. This technique that surface modification with cationic pullulan resulted in
has been successfully applied to extracellular vesicles and a higher therapeutic impact by promoting exosome forma-
tion and subsequent hepatic activity [118].
13
Receptor–ligand interaction ability without compromising the biological activities of

exosome membrane proteins [104].
Receptor–ligand interaction is another method for non-
covalent chemical modification that uses natural recep-
tors on the surface of exosomes for binding to the targeted Tannic acid surface modification
ligands [104]. Using this method, Qi et al. used transferrin
to conjugate SPIONs to the surface of reticulocyte-derived As mentioned in the previous parts, surface engineering of
exosomes. Transferrin receptors were expressed on exosome exosomes is performed using two main methods: genetic
surfaces [107]. Thus, multiple SPIONs were anchored onto manipulation and chemical addition of functional groups on
each reticulocyte-derived exosome via interaction with the exosome membranes. However, the application of these meth-
transferrin receptor, leading to the formation of vehicles ods in biological systems has been associated with certain limi-
with superparamagnetic behavior. According to findings, tations, including the unavoidable use of harsh, complex, and
the response of SPION-containing exosomes to an exter- time-consuming manipulation procedures that may damage the
nal magnetic field was significantly enhanced compared to exosomes. On the other hand, using phenol-containing agents,
individual SPIONs. such as tannic acid (TA), and Fe3+ to form coordination com-
Methods using receptor–ligand interaction can separate plexes on the surface of exosomes is a mild and biocompatible
and purify the circulating exosomes, which can be valuable method for exosome coating. These complexes include an array
for detecting diseases like malignancies. These magnet- of molecules or ions that bind a central metallic atom through
containing exosomes may have an increased range of bio- coordinate bonds [121].
logical applications. Wang et al. introduced a 3-dimensional TA is a polyphenol found in different plant, fruit, and veg-
nanostructured microfluidic device that combined CD63, etable sources. Given its high affinity to proline-rich proteins
as a recognition molecule, and the distinctive topography in ECM, such as elastin and collagen, it can be an effec-
of the nanomaterials to produce exosomes with excellent tive agent for targeting cardiac tissue. TA contains moieties
efficiency. When exosomes, or vesicles, were exposed to rich in phenolic hydroxyl and five gallol groups, interacting
recipient cells, their nanostructure enabled effective drug with the ECM proteins through hydrophobic interactions and
administration and receptor-mediated cellular absorption. hydrogen bonds [122].
Chemical detection of the donor cells was performed Recently, TA has been used as a multifunctional coating
using the biotin and avidin, as dual ligands, in the phos- molecule for various substrates and shapes, including films and
pholipid membrane and encapsulated medicines in the cyto- particles. However, only limited studies have investigated the
plasm. The modified donor cells produced exosomes, and surface modification of exosomes with TA. Kumar et al. devel-
the exosomes inheriting the dual ligand and medicines were oped a droplet-based microfluidic system for coating exosomes
separated using a microfluidic chip. Given the dual ligand with a thin layer of TA with a thickness of 10 nm. This system
targeting and receptor-mediated endocytosis, this exosome was based on linking gold nanoparticles to exosomes via esteri-
delivery method showed increased anticancer effects and tar- fication. Moreover, it did not include nanoparticle aggregation
geting capabilities. It was suggested that this approach might and gold staining. The researchers loaded the coated exosomes
be beneficial for the efficient extraction of intact exosomes with doxorubicin, a chemotherapeutic agent, to target the cancer
and using their natural carrier role to deliver chemothera- cells. Using this simple, quick, and substrate-independent coat-
peutics to tumor cells with enhanced efficacy and targeting ing approach, they produced a consistent and powerful protec-
capabilities [108]. tive layer [TA-Fe3+] on the exosome surfaces. Moreover, the
coated exosomes could be targeted to the diseased area and
benefit treatment goals [109].
Hydrophobic interaction Shin et al. applied TA for modifying proteins in order to
increase their targetability to the cardiac tissue. This process is
Another innovative technique of chemical modification is called TANNylation and is relatively similar to the PEGylation
the application of hydrophobic interactions or membrane approach in the stability manner. However, its principles are
engineering. In this method, the liposomes with membranes different at the molecular level due to the affinity of TA to the
already functionalized with a peptide, antibody, or PEG ECM proteins of cardiac tissue. The researchers reported that
are fused with exosomes using a freeze–thaw process. The TANNylated proteins were accumulated in the heart. There-
resultant exosome–liposome hybrid has a practical targeting fore, this approach can be applied to target different therapeutic
agents in the heart tissue [110].
13
Exosome delivery methods based was applied under the injury site for 30 min to guide the
on magnetic nanoparticles exosomes toward the desired area, leading to the increased
accumulation of magnetic exosomes in the targeted area.
The use of magnetic power is a novel delivery method for Moreover, by applying HUVECs, improved proliferation,
targeting the drug to the desired area. This method is based migration, and angiogenesis were reported in the in vitro
on using magnetic nanoparticles (MNPs) and an external studies. Also, in vivo studies demonstrated an increased
magnetic field. The therapeutic agent is paired with MNPs wound healing rate in the treated group [116].
and guided to the targeted area via a powerful external mag-
net [111].
SPIONs have been widely studied as contrast agents Magnetic nanoparticle incorporation
for various imaging methods, including CT scan or MRI. into purified exosomes
However, given their magnetic features, these nanoparticles
can be used for other purposes, including cancer treatment, In this method, MNPs are added to the already formed and
disease detection techniques, and targeted delivery of drugs purified exosomes. Several methods can be used for incor-
[112]. Moreover, MNPs have been used in different drug porating the MNPs into purified exosomes.
delivery systems at the microscopic and macroscopic levels
and can be merged with the drugs directly or embedded in
a carrier [113]. Also, a novel advancement in SPION-based Surface attachment
delivery systems is using exosomes as vehicles in combina-
tion with SPIONs as targeted delivery agents. MNPs can attach directly to the external membrane of
Different methods have been developed to merge MNPs exosomes. However, this attachment needs different modi-
to exosomes, ranging from indirect incubation of MNPs with fications, including adding covalent binding molecules,
exosome-producing cells to direct attachment of these parti- such as transferrin, or hydrophobic tails to the exosome
cles to the exosome surface or loading them into exosomes. lipid membrane [117]. The final product of this procedure
The following sections provide details of the mentioned is usually called MNP- or SPOIN-decorated exosomes
methods [114]. [117, 118]. Zhuang et al. used SPION-coated exosomes
as a carrier to deliver TNF-α to melanoma cells. Exosomes
were achieved from MSCs containing a TNF-α-producing
Magnetic nanoparticle incubation plasmid. Afterward, the purified exosomes were deco-
with exosome‑producing cells rated with SPIONs using the interaction of transferrin
and its receptor (Tf-TfR). Moreover, the researchers used
In this method, MNPs, such as SPIONs, are incubated with external magnets (1 Tesla) and a Steri-Strip tape to guide
exosome-producing cells during exosome formation, lead- the exosomes to the targeted area for 24 h. According to
ing to the production of SPOIN-containing exosomes by the in vitro results, TNF-SPION-exosomes could inhibit can-
mentioned cells. Lee et al. incubated iron oxide nanoparti- cerous cells. Also, in vivo tests revealed substantial attenu-
cles (IONPs) with bone marrow MSCs at a concentration of ation of TNF-SPION-decorated exosomes in the tumor site
40 µg/mL for 24 h to generate IONP-containing exosomes, and malignant cell attenuations. Decreased toxicity was
which were subsequently used for reducing ischemic heart achieved due to the active targeting of exosomes to the
damage. Moreover, cellular internalization of exosomes tumor site [119].
was confirmed using fluorescent and TEM microscopes. Another study by Zhuang et al. used the exosomes
According to their results, external magnetic guidance decorated with SPION-TF-TFR to deliver a therapeutic
could increase the retention of IONP-containing exosomes. peptide to pancreas islets. Exosomes extracted from the
Also, in vitro studies showed local inflammation allevia- serum were loaded with BAY-9837, a potential treatment
tion, increased viability, and attenuated apoptosis of these agent for type 2 diabetes. Afterward, transferrin-modified
exosomes under hypoxic conditions, while in vivo investiga- SPIONs were obtained by adding carboxylated chitosan to
tions showed improved vascularization and cardiac function SPIONs. These SPIONs were subsequently coated on the
in the treated group [115]. membranous TF receptors of the blood-derived exosomes
In another study in 2020, Li et al. incubated the IONPs at using TF-TFR connections. Moreover, acquired exosomes
a concentration of 50 µg/ml with umbilical cord MSCs for were administered intravenously to rat models with type
16 h to produce exosomes targeting the injured spot. After- 2 diabetes and guided toward the pancreas with the help
ward, the exosomes were administered intravenously into of an external magnet (1 Tesla) on the surface of the pan-
the rat models with cutaneous wounds. A 1.2-Tesla magnet creas for 1 h. The results showed a higher concentration
of exosomes in the targeted area and increased insulin
13
secretion due to the enhanced delivery of BAY-9837 with distortion [129]. The following method is electroporation,
targeted exosomes [120]. which has been reported to be the most efficient method
Wang et al. used a novel targeting method for doxoru- in this category [125]. This method can partially disrupt
bicin delivery. First, the drug was loaded into the engineered the exosome membrane by introducing electrical pulses,
exosomes using electroporation. Using surface modification creating a decent opportunity to load the desired particles
methods, the surface of DOX-loaded and biotin-labeled into the exosomes [130].
exosomes was coated with SPIONs and avidin molecules The thermal shock technique includes repeated cycles
paired with specific molecular beacons. These molecular of freezing and thawing to introduce cargos into the exo-
beacons could pair with the miR-21 of the malignant cells. some. However, these thermal cycles may lead to slight
Afterward, external magnetic guidance was used to gather membrane disturbances [11]. Moreover, this method has a
the engineered exosomes toward a flank tumor in mouse moderate yield in cargo loading through the phospholipid
models inoculated by HeLa (MCF-7) malignant cells. The bilayer of the exosome [129]. Another method of parti-
loaded drug and molecular beacon were released from the cle internalization, the extrusion method, is performed
exosomes into the target tumor using infrared-induced by inducing the rapid movement of exosomes and their
hyperthermia, leading to the considerable size reduction of desired cargos through filters with specific pore dimen-
the tumor [121]. sions, which leads to membrane breaking and reassem-
Liu et al. introduced a novel double targeting mecha- bling of exosomes after passing through these filters [131].
nism for rabbit models with myocardial infarction. They This method introduces the cargos into the newly formed
synthesized a vesicle shuttle consisting of Fe3O4 nanopar- exosomes with relatively high efficiency [126]. However,
ticles covered by a silica shell and decorated with ethylene the breakage and reformation of exosomes can alter their
glycol. Afterward, the anti-CD63 and anti-MLC antibodies signaling and targeting capabilities by chaining their pro-
were attached to the shuttle. The anti-CD63 antibody was tein content [132].
used to attach the shuttle vesicle to circulating endogenous To the best of our knowledge, particle internalization
exosomes, while the anti-MLC antibodies acted as the first into exosomes has not yet been used in exosome targeting
targeting mechanism and attracted the shuttle vesicle and by MNPs. However, considering other similar work, we
connected exosomes toward the myocardial cells. Then, the suggest that the incorporation of MNPs with exosomes
synthesized shuttle was injected into the animal models with using these methods can be a promising technique for tar-
myocardial infarction. Following the injection, the second geting studies.
targeting mechanism was achieved by placing a 1.3-Tesla
magnet on the hearts of the models for 10 min. Reduced pH
in the infarct area triggered the local release of connected
exosomes. Afterward, the accumulation of exosomes in the Discussion and future trends
infarcted area reduced the infarct size, improved the left ven-
tricular ejection fraction, and increased the angiogenesis in Considering the safety and efficacy of exosomes reported
the animal models [122]. by the preclinical studies, there are a growing number of
recent clinical trials investigating the benefits of exosomes
as a therapeutic agent for various conditions. In May
Particle internalization into exosomes 2022, fifty-three interventional clinical trials investigating
exosomes are registered in the NIH clinical trials (www.
The second method in this category is loading the mag- clinicaltrials.gov). However, several limitations still exist
netic nanoparticles into the exosomes rather than their sur- for using exosomes in a potential large-scale clinical trial.
faces. These methods include incubation [123], extrusion Therefore, despite their promising effects in recent stud-
[124], sonication [125], thermal shock, and electroporation ies, exosome treatments have not yet been approved by
[11]. Co-incubation is the simplest method for incorporat- the FDA.
ing desired materials into purified exosomes. However, The development of an applied and clinical exosome-
the integration rate of this method is lower compared to based treatment necessitates large-scale production and
other methods. Moreover, molecular size, hydrophobic- suitable storage conditions. Given the low yield of exosome
ity, and particle charge are the main factors affecting the production and high therapeutic doses, there is a need for
loading speed [126, 127]. Another method, sonication, effective large-scale production [133]. A storage temperature
can increase the rate of cargo insertion compared to sim- of − 80 °C has been recommended for isolated exosomes
ple incubation, which is achieved by creating temporary because higher temperatures can gradually damage the
openings in the exosome membrane [128]. However, this exosomes [134]. And this storage condition requires special
method is associated with the possibility of exosome equipment. Thus, achieving more feasible storage conditions
13
Fig. 1 Different hydrogels including alginate, chitosan and some other biomaterials are applied with the combination of exosomes (from dissimi-
lar cell sources) with the proposal tissue regeneration in various injuries
is critical for large-scale clinical studies. Therefore, it has achieve an effective treatment system. Moreover, the type of
been shown that lyophilized exosomes have demonstrated the original cell can directly affect the exosome interactions
higher stability and efficacy in room temperature. This with the targeted tissue. Therefore, selecting an appropriate
method can extensively expand the clinical application of cell line for exosome extraction is essential to the outcomes
exosomes [135]. [140–142].
Another concern in exosome production is heterogene- The nucleic acid contents of exosomes, especially the
ity [136]. It is necessary to reach a reproducible protocol miRNAs, play a fundamental role in the signaling pathways
for exosome production, isolation, and storage to produce a [143]. Therefore, several studies have tried to produce modi-
consistent product from different cell types [137]. Moreover, fied exosomes with specific miRNAs to increase their thera-
there is a need for developing accurate product evaluation peutic efficacy [142, 144].
methods after exosome production to achieve reliable effi- Different modification methods have proven helpful in
cacy [137, 138]. Also, developing guidelines on the dosage increasing the effectiveness of drug delivery and treatment.
of the treatment regime is another crucial factor [139]. Among these methods, bulk encapsulation, surface modi-
On the other hand, further extended studies are needed fication, targeting systems, 3D-printing techniques, and
for an adequate understanding of the possible reactions or microfluidic procedures were discussed in this review. These
consequences of long-term exosome administrations. Sev- methods can help resolve the mentioned limitations in devel-
eral characteristics of exosomes need to be addressed to oping exosomes as a legitimate clinical treatment option.
13
Fig. 2 Exosome engineering techniques aiming to increase its targetability
The final characteristic of any exosome treatment system is Conclusion

the delivery method. Systemic delivery using IV injections is
the main delivery method in clinical studies. However, disper- Exosomes are a group of natural nanovesicles with pivotal
sion of the injected exosomes and their hepatic and pulmonary roles in intracellular communication. Given their content
can limit the therapeutic effects of systemic delivery [145]. of proteins, nucleic acids, and metabolites, they can be
Therefore, several other roots of administration have been stud- used as a treatment agent for various diseases. However,
ied as well, including oral [146], intraperitoneal [147], intramu- it is worth mentioning that the cell source is essential for
ral [148], intradermal [149], and inhalation [150] administra- exosome extraction in the presence of different agents
tions. As discussed, targeting methods can also help direct the determining the effects that the exosomes present. Given
injected exosomes toward the desired area despite a systemic the similarity between the exosome surface membrane and
injection (Figs. 1 and 2). cell membrane, exosomes can serve as a natural vehicle for
13
drug delivery since they do not elicit any immunological down-regulation of endothelial RAB11FIP2 expression. Car-
responses as the synthetic substitutes do. In this study, we diovasc Res. 2017;113(5):440–52.
8. Jing H, He X, Zheng J. Exosomes and regenerative medicine:
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exosome retention and targetability, hence improving the 9. Moghadasi S, Elveny M, Rahman HS, Suksatan W, Jalil AT,
exosome therapeutic capacity. Abdelbasset WK, Yumashev AV, Shariatzadeh S, Motavalli R,
Behzad F. A paradigm shift in cell-free approach: the emerg-
Acknowledgements We would like to acknowledge all the contribu- ing role of MSCs-derived exosomes in regenerative medicine. J
tions from the Research Center for Advanced Technologies in Cardio- Transl Med. 2021;19(1):1–21.
vascular Medicine, Cardiovascular Diseases Research Institute, Tehran 10. Sun D, Zhuang X, Xiang X, Liu Y, Zhang S, Liu C, Barnes
University of Medical Sciences. S, Grizzle W, Miller D, Zhang H-G. A novel nanoparticle
drug delivery system: the anti-inflammatory activity of cur-
Author contributions HTG did manuscript composure, conceptualiza- cumin is enhanced when encapsulated in exosomes. Mol Ther.
tion, and visualization. SPB done manuscript composure and visualiza- 2010;18(9):1606–14.
tion. AA provided manuscript composure, revision and editing. ZKZ, 11. Wang J, Chen D, Ho EA. Challenges in the development and
AH-T, and KH were involved in manuscript composure. AH revised establishment of exosome-based drug delivery systems. J Control
and edited the manuscript. HAT contributed to editing, revision, sci- Release. 2021;329:894–906.
entific supervision, and conceptualization. SD and MAT edited and 12. Elliott RO, He M. Unlocking the power of exosomes for
revised the article. crossing biological barriers in drug delivery. Pharmaceutics.
2021;13(1):122.
Funding This study was carried out in authors own capacity, and this 13. Khayambashi P, Iyer J, Pillai S, Upadhyay A, Zhang Y, Tran
study was not funded by any governmental organization and the authors SD. Hydrogel encapsulation of mesenchymal stem cells and
themselves financed this study. their derived exosomes for tissue engineering. Int J Mol Sci.
2021;22(2):684.
Data availability Data available on request due to privacy/ethical 14. Shafiei M, Ansari MNM, Razak SIA, Khan MUA. A compre-
restrictions. hensive review on the applications of exosomes and liposomes
in regenerative medicine and tissue engineering. Polymers.
Declarations 2021;13(15):2529.
15. Bai Q, Han K, Dong K, Zheng C, Zhang Y, Long Q, Lu T. Poten-
Conflict of interest The authors declare that they have no conflict of tial applications of nanomaterials and technology for diabetic
interest. wound healing. Int J Nanomed. 2020;15:9717.
16. Huang J, Xiong J, Yang L, Zhang J, Sun S, Liang Y. Cell-
Consent for publication All authors have consent for publication of free exosome-laden scaffolds for tissue repair. Nanoscale.
the article. 2021;13(19):8740–50.
17. Foyt DA, Norman MD, Yu TT, Gentleman E. Exploit-
Ethical approval Not applicable. ing advanced hydrogel technologies to address key chal-
lenges in regenerative medicine. Adv Healthcare Mater.
2018;7(8):1700939.
18. Saunderson SC, Dunn AC, Crocker PR, McLellan AD. CD169
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