Tango of dual nanoparticles: Interplays between exosomes and nanomedicine

Abstract Exosomes are lipid bilayer vesicles released from cells as a mechanism of intracellular communication. Containing information molecules of their parental cells and inclining to fuse with targeted cells, exosomes are valuable in disease diagnosis and drug delivery. The realization of their clinic applications still faces difficulties, such as lacking technologies for fast purification and functional reading. The advancement of nanotechnology in recent decades makes it promising to overcome these difficulties. In this article, we summarized recent progress in utilizing the physiochemical properties of nanoparticles (NPs) to enhance exosome purification and detection sensitivity or to derive novel technologies. We also discussed the valuable applications of exosomes in NPs‐based drug delivery. Till now most studies in these fields are still at the laboratory research stage. Translation of these bench works into clinic applications still has a long way to go.

biopsy biomarkers that are easily accessible for early disease diagnosis and prognosis. 3,4 Because of these reasons, exosomes are extensively studied in recent years for potential clinical applications.
As an important mechanism of intercellular communication, exosomes mediate material exchange among cells by fusing with recipient cells and transmit their cargos into them, a process leading to disease advancing or restraining. 5,6 Because of the homology, exosomes fuse with target cells in a high efficiency without causing undesirable side effects. Moreover, since they are highly biocompatible, there is little concern about the consequence of their ADME (absorption, distribution, metabolism, and excretion), 7 which is usually an important safety issue when artificial nanoparticles (NPs) like polymers are used for the same aim. 8 Therefore, exosomes are also regarded as intriguing natural drug delivery carriers. [9][10][11][12] The studies of exosomes confront technique difficulties. Analysis techniques with high sensitivity and specificity are currently lacking, thus in order to achieve quantitative analysis for clinic applications, a large pool of purified exosomes is usually required. However, the exosome purification technologies available now are far from satisfactory. Some studies showed that the methods used for exosome purification affect their heterogeneity, raising the concern about the fidelity of the purified exosomes to reflect the in vivo natural status. [13][14][15] Moreover, some widely used methodologies based on the recognition of surface markers are recently found to as well enrich other multiple vesicle bodies like ectosomes. 16 Therefore, more efficient and exosome-specific purification and highly sensitive detection methods are eagerly required. An intriguing clinical application of exosomes is for drug delivery. 17 For this aim, adequate exosomes must be produced from source cells and appropriate surface modifications are usually desired. 18 Till today, approaches used for realizing these aims are unmature and have much room for improvement.
Nanotechnology has been widely explored in different fields of biomedicine in recent decades. 19,20 Properties of varieties of NPs, like high specific surface area, 21 unique optical effects, 22 and superparamagnetism, 23 make them highly promising to revolutionize exosome studies. On one hand, nanotechnology provides novel strategies for exosome biogenesis regulation, purification, and analysis, making more sensitive and accurate functional readings possible. 24 On the other hand, it helps to broaden the scope of exosome applications in biomedicine, which is impractical by traditional technologies. 25,26 Some significant progresses have been made in these fields in recent years. In this article, we will summarize the recent progress in NPs' applications for exosome biogenesis control, purification, quantitative detection, and manipulation for medical aims.

| NPS FOR EXOSOME BIOGENESIS REGULATION
Recent studies indicated that exosomes play critical roles in regulation of normal physiological processes like pregnancy, 27,28 development and immune response, 29 as well as in pathogenesis 30 and disease progression. 31,32 Exosomes are also found to be associated with diseases like neurodegenerative and cardiovascular diseases and tumors. 5 Because of this, modification of exosome biogenesis appears as a way of maintaining healthy physiology and preventing disease progression. The accumulated knowledge about the exosome biogenesis opens novel ways to manipulate major steps and factors in this process. 1,[33][34][35] NPs have been reported to modulate exosome attributes. In one study, gold NPs (AuNPs) of 5 nm stimulated mouse embryonic stem cells to secrete exosomes which are distinct from the natural ones since they have higher rigidity and different protein expression profile. 36 Moreover, unlike those produced in nontreated cells, these exosomes did not promote tumor cell migration in breast tumor cell model 4 T1, probably because they did not stimulate the expression or phosphorylation of metastasis-promoting proteins like cofilin and extracellular regulated protein kinases (ERKs). 36 This study suggests the possibility of employing NPs to host cells to produce safer exosomes as drug delivery carriers.
One widely used approach for exosome biogenesis regulation is to manipulate protein expression of important regulator genes involved in the process of exosome generation and conveyance. 37 To do this, silencing molecules like siRNAs or antisense oligonucleotides or exogenous DNAs are usually applied. 38 However, delivery of these molecules confronted low efficiency due to the lack of appropriate carriers.
NPs have a high drug loading capability and are promising to break this limit. GTPase family proteins play important roles in multiple steps of vesicle trafficking including budding, docking, and fusion, thus are important regulators of exosome secretion. 39 By carrying antisense oligonucleotides targeting a GTPase family member RAB27A, AuNPs significantly lessened exosome release from MCF-7 and MDA-MB-453 cells. 40 And the functionalized AuNPs can be carried over to the recipient cells by the secreted exosomes and continue to execute gene silencing. These pilot studies highlight the possibility of using NPs to regulate exosome biogenesis aiming to limit tumor metastasis.
Naturally, cells produce a small number of exosomes, which forms the major limitation of their therapeutic applications. In order to enhance the production, some mechanical methods, like iterative physical extrusion or freeze/thaw cycle have been reported. 41,42 A disadvantage of these methods is that the integrity of exosome membrane may be damaged. NPs have been enforced to stimulate higher exosome yield in cells. In one study, platinum NPs treatment induced six folds higher exosome production in cancer cells via an oxidative stress-mediated mechanism. 43 In another study, porous silicon NPs promoted autophagosome formation in human hepatocarcinoma cell line when used as a drug delivery carrier and stimulated 34 times more exosomes release. 44 Positively charged iron oxide NPs entered mesenchymal stem cells via a dynamin-dependent endocytosis mechanism, and in cells, they enhanced exosome secretion by stimulating the expression of factors involved in autophagosome and autolysosome formation including Beclin-1 and Rab7. 45 These studies showed that application of NPs is an easy and highly efficient way to improve exosome production.
Although the above success, when NPs were applied to cells, the increased exosome yield is often accompanied by cellular toxicity mediated by induction of oxidative stress and autophagy. 43,44 This cellular toxicity consequently decreased source cell viability and the alteration of exosome attributes, as well as leave stress markers in exosome cargos which may affect recipient cell physiology when exosomes are used as theragnostic delivery carriers. 45 So in future, one of the study aims is to develop efficient NPs to stimulate exosome release but with minimal side effects to exosome-derived cells. Second, varieties of NPs with different functions can be combined to form a multiply functionalized nano-platform for realization of simultaneous isolation and quantification. Because of these advantages, exosomes can be purified under a mild condition in a more efficient manner. For example, by applying magnetic NPs, folate functionalized exosomes can be isolated with a high specificity in less than 2 h, compared to at least 4 h by applying ultracentrifugation. 52 Another example is that Fe 3 O 4 @TiO 2 NPs were used for exosome purification taking advantage of the binding between phospholipids of exosomes and TiO 2 shell. By applying an exogenous magnetic field, 96.5% of exosomes can be purified within 5 min, an efficiency higher than any reported traditional approaches. And after simple wash step, the high enriched exosomes can be used for the following-up quantification step. 53 By using antibody-conjugated magnetic nanowires, exosomes were isolated in less than 1 h and the purification yield reached more than 10 times than that obtained by ultracentrifugation ($150 Â 10 8 vs. $10 Â 10 8 exosomes/ml). 54 The most widely used property of NPs is their superparamagnetism produced by magnetic NPs (Figure 1a). 52 The targeting moieties can be any molecules natural to cells, like transferrin receptors from blood cells, 55 or those designed to be carried by exosomes by a donor cellassisted membrane modification strategy, 52  Difficultly in handling large sample NA [51] also developed. In this composite, gold shell surface was conjugated with aptamers to specifically recognize exosome surface markers, and also serves as a SERS test tag. 56 Moreover, detection tags, like

| NPS FOR EXOSOME PURIFICATION
AuNPs for surface-enhanced Raman scattering (SERS) immunoassay, can be added to NPs surface for in situ quantification without the requirement of elution, a critical step to lose the isolated samples.
Magnetic nanowires doped inside with magnetic nanospheres were used to efficiently catch circulating exosomes (Figure 1c). 54 The thermal responsibility of magnetic NPs was also used for exosome purification. 57 NPs may also aid exosome purification by other mechanisms.
Recent studies find that nanometer scale topography can be used for tumor cells or cellÀ/sub-cell size organisms (like bacteria) capture based on cell-nanostructure interaction. 58 This emerges as an important mechanism underlying microfluidic chip technology. 59-61 By using manufacturable silicon processes, an array with uniform nanoscale gap sizes can be produced, which has been reported to efficiently separate exosomes. 62 NPs like graphene oxide are good candidates to be used as coating materials to make nano-interfaces with enhanced capture area while suppressing nonspecific adsorption. 63 applications, like lower cost and ease of modifications, are attracting more interests in recent years. 91 To quantitatively monitor in vivo distribution, exosomes can also be labeled by internalizing contrast agents. An example is that AuNPs were in vitro encapsulated inside exosomes. After administration into animals, both distribution and accumulation quantity in tissues can be monitored by in vivo CT imaging. 92 Some other information molecules like microRNAs that reside in exosome compartment are also reported as target for quantitative detection aim. 24

| Fluorescence-based detection
Because of their excellent properties like size-regulable emission spectra and stronger photoluminescence performance compared to con-   Some NPs are also used in a similar technology named luminescence resonance energy transfer (LRET) for exosome quantification. 74 In this strategy, upconversion and AuNPs are used and a sensitivity of 1000 particles/μl was reached by using CD63 as a test aptamer. Besides, DNA nanotetrahedron-modified electrode employed aptamer to capture more exosomes and, therefore, enhanced the detection sensitivity. 103 81,108,109 According to these studies, a limit of detection as low as 500 exosomes/μl sample can be achieved, which is much lower compared to that obtained by other protocols like colorimetry-and fluorescence-based ones. This mechanism was also applied for in vivo exosome imaging. 92 The higher sensitivity of AuNPs-based plasmon biosensor attributes to the fact that a high density of local AuNPs on exosomes can be achieved by different methods. First, AuNPs conjugated with different antibodies/aptamers can be used to recognize different surface ligands simultaneously.

| EXOSOMES FOR NPS-BASED DISEASE THERAPY
The requirements for an ideal chemotherapy strategy, such as Exosomes as natural drug delivery carriers have limitations like low payload capability, which is often blamed to be the reason of low F I G U R E 3 A surface plasmon resonance (SPR) strategy for exosome detection based on a three-step process. In step 1, aptamer 1 is linked to a DNA tetrahedron probes (DTPs), which are supported on an Au film to prevent gold deposition on the surface. In step 2, exosomes are captured by the aptamer 1 which is complementary to DTPs, and aptamer 2 (here it is CD63)-linked Au@PDA NPs recognize and bind exosomes. In step 3, HAuCl 4 is introduced and in situ reduced to AuNPs by polydopamine coated on the AuNP surface. The reduction results in a further enhanced SPR signal (image reprinted with permission from Reference [110]) therapeutic efficiency after in vivo administration. 111 This limitation can be overcome by NPs, which can load significant amount of drugs, protect them from degradation, and realize controllable drug release by proper chemical modifications. 112 Although the targeting capability of NPs can be entitled by conjugation of targeting ligands on NP surface, it is not uncommon to find that the targeting ligands deteriorate the stability and change in vivo distribution of NPs after administration by a mechanism involving corona formation on a NP surface. 112 The use of exosomes as targeting moieties will help avoid such problems. The NPs-exosome hybrids showed considerably increased therapeutic efficiency in different biomedical applications (Table 3).
Two different approaches have been reported to prepare NPsexosome hybrids. In the first approach, NPs are added to the cell culture to stimulate cellular endocytosis, by which NPs are loaded inside exosomes. In the second approach, NPs are encapsulated into the iso- were also reported in an exosome-mediated tumor photothermal therapy. In this study, after intravenous injection into mice, BP carrying exosomes were accumulated in tumors because of their homing selection, 122 and application of near-infrared laser irritation efficiently produced local heat and ablated tumors ( Figure 4). 114 Second, NPs work as a drug deliverer. In one study, Dox/siNPsencapsulated exosomes were successfully produced by a pre-loading approach, in which doxorubicin loading porous silicon NPs were exposed to cultured hepatocarcinoma cells and they were released by cells in the form of Dox/siNP-loaded exosomes. When administrated by intravenous injection to mice bearing hepatocarcinoma tumors, the exosomes accumulated in a high efficiency in tumors and doxorubicin were released from NPs killing both bulk tumor cells and cancer stem cells. 44 The targeting capability of exosomes can be endowed by genetically engineering of parental cells. For example, by fusing neuronspecific peptide gene to Lamp2b (a ubiquitous exosome membrane protein) in dendritic cells, a neuron-targeting exosome pool was achieved. 123 In another study, an Arg-Gly-Asp (RGD) peptide, which specifically recognizes the integrin α v β 3 of tumor cells and vasculatures, was incubated with cancer cell line to generate RGD expressing exosomes (Exo-RGD). QDs coated with TAT peptide (for nucleus targeting) were encapsulated into Exo-RGD by electroporation. When applied to animal models, this theragnostic platform showed good biocompatibility and long circulation time, and targeted cancer cells with a high transfection efficacy. 124 In BALB/c mice, the exposure to magnetic NPs generated exosomes in the alveolar region, which consequently stimulated T-cell activation. 125 This provides a mechanistic explanation for toxicity caused by NPs administration, but meanwhile, it also implicated the possibility of using this strategy for tumor nano-vaccine development. 114

| In therapy for other diseases
The combination of NPs and exosomes was also exploited for treatment of other diseases. In a notable study, core-shell magnetic NPs were conjugated with dual antibodies, with one (CD63) against antigens on exosome surface and the other (myosin-light-chain, MLC) targeting recipient cells (the injured cardiomyocytes). These dual functionalized NPs were used as vesicle shuttle of capturing and delivering endogenous exosomes to the targeted area, where exosomes were selectively released from NPs to generate the therapeutic effect for myocardial infarction, including promoted angiogenesis, reduced F I G U R E 5 Treatment of infarcted heart tissue via the capture and local delivery of circulating exosomes through antibody-conjugated magnetic nanoparticles (NPs). The magnetic NPs, designated as GMNP EC , consist of a Fe 3 O 4 core and a silica shell that is decorated with poly (ethylene glycol) conjugated to two antibodies respectively recognizing CD63 and myosin-light-chain (MLC) via hydrazone bonds. While anti-CD63 antibodies capture and attach to endogenous circulating exosomes, anti-MLC antibodies lead exosomes to target MLC on the damaged cardiomyocytes when GMNP EC are enriched in the infarct area by the application of a local magnetic field. In the infarct area, exosomes are released from GMNP EC due to the acidosis-induced cleavage of hydrazone bonds. In animal models of myocardial infarction, the accumulation of CD63-expressing exosomes in infarcted tissue leads to reductions in infarct size as well as improves left-ventricle ejection fraction and angiogenesis (Image reprinted with permission from Reference [126]) infarct size, and improved left-ventricle ejection fraction in animal model. 126 Because the selection of targeted antigens on exosome surface can be further optimized, the specificity and efficiency to deliver exosomes still has room of improvement. Thus, this approach has great promises of clinic applications for myocardial infarction treatment. Studies have shown that exosomes containing pro-coagulation and pro-inflammatory factors contribute to thrombosis in myocardial infarction. 127 This finding justifies a strategy for infarction treatment, in which exosomes containing these factors can be recognized and tethered by NPs with conjugation of appropriate antibodies, thus reducing their accumulation in the infarct site and ameliorating infarct size. The same strategy was also used for cerebral disease treatment 92,128 and monitoring and improving fetal health. 129 In both applications, the capability of exosomes to breakdown physiological barriers (blood-brain barrier [BBB] 129 and placenta barrier 130 ) was taken use of. Drugs carried by this system can be more efficiently delivered to targeted sites to realize the maximal therapy efficacy. [131][132][133] Moreover, therapeutic properties of NPs, like the photothermal effect, capability to activate immunity of targeted cells, and long retention time in tumors, can synergize drug therapy efficiency

CONFLICT OF INTEREST
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
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