Exosomes for Drug Delivery: History
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Particular interest among the scientific community is focused on exploring the use of exosomes for several pharmaceutical and biomedical applications. This is due to the identification of the role of exosomes as an excellent intercellular communicator by delivering the requisite cargo comprising of functional proteins, metabolites and nucleic acids. Exosomes are the smallest extracellular vesicles (EV) with sizes ranging from 30–100 nm and are derived from endosomes. Exosomes have similar surface morphology to cells and act as a signal transduction channel between cells. They encompass different biomolecules, such as proteins, nucleic acids and lipids, thus rendering them naturally as an attractive drug delivery vehicle. Like the other advanced drug delivery systems, such as polymeric nanoparticles and liposomes to encapsulate drug substances, exosomes also gained much attention in enhancing therapeutic activity. Exosomes present many advantages, such as compatibility with living tissues, low toxicity, extended blood circulation, capability to pass contents from one cell to another, non-immunogenic and special targeting of various cells, making them an excellent therapeutic carrier. Exosome-based molecules for drug delivery are still in the early stages of research and clinical trials. The problems and clinical transition issues related to exosome-based drugs need to be overcome using advanced tools for better understanding and systemic evaluation of exosomes.

  • exosomes
  • biogenesis
  • isolation
  • drug delivery

1. Isolation and Separation Techniques for Exosomes

Exosomes have been separated from various fluids (biological) like cerebrospinal fluid (CSF), saliva, urine, amniotic fluid, semen, breast milk, tears, etc. [1][2]. It can also be separated from conditioned mediums like tissues, cultured organs, and cell lines [3]. In most cases, exosomes are obtained from conditioned medium and biological fluids by suitable laboratory techniques such as ultrafiltration, immunological separation, ultracentrifugation, size exclusion chromatography (SEC), precipitation based on polymer, magnetic separation, acoustic fluidic separation, dielectrophoretic separation, deterministic Lateral Displacement (DLD) separation and currently microfluidic devices [4][5]. The methods used to isolate and separate the exosomes are discussed below.

1.1. Ultrafiltration

The separation of exosomes is a simple, size-dependent, and convenient technique [6]. Ultracentrifugation is a slower, more complex, and less productive process. The advantage of this process is that it only takes a small sample to get the required production.
Cheruvanky et al. showed that exosomes could be separated successfully using 0.5 mL of the urine sample by ultrafiltration, indicating its high efficiency [7]. Direct flow filtration and tangential flow filtration techniques were used to perform ultrafiltration. Direct flow filtration is the commonly used technique for small-volume sample filtration (up to 30 mL). But this filtration method suffers from membrane fouling problems and poor particle separation. For large-scale exosome extraction, tangential flow filtration is much more effective and practical in preventing clogging and cake formation; the sample stream runs over the ultrafilter membrane in TFF [8].

1.2. Immunological Separation

Immunoaffinity is another approach for exosome purification. Affinity purification employing antibodies to CD63, CD81, CD82, CD9, EpCAM, and Rab5 is better for specific exosome isolation. These could be used separately or in combination. Magnetic beads, chromatography matrices, plates, and microfluidic devices could all be used to immobilize antibodies for this application [9][10]. Although this approach is scalable, most extracellular exosome-associated antigens are not specific to exosomes. Protein complexes and other particulates, including antigen-carrying non-exosome vesicles like cell debris, could be purified using immunoaffinity-based isolation techniques. However, this procedure may isolate exosomes exclusive to a particular biomarker [11][12].

1.3. Ultracentrifugation

The traditional and gold standard method for isolating exosomes is ultracentrifugation. Based on density, size, and form, this technique uses centrifugal force to remove cells, and the significant cell remains out of biological fluids [13] héry et al. published the following experimental methodology to extract and separate exosomes via ultracentrifugation [10].
Exosome isolation should be improved by ultracentrifugation with density gradient separation as centrifugation technology improves [14]. Although ultracentrifugation is a well-established method for isolating exosomes, prolonged usage can cause the exosome membrane to rupture due to centrifugal force’s effects on the exosome [15].

1.4. Size Exclusion Chromatography

The chromatographic technique known as size exclusion chromatography (SEC) traps particles smaller than the pore in a column with porous beads. Particles that are larger than the pore are quickly eluted in contrast. Exosomes have a diameter of 40–100 nm, although most proteins have a diameter of less than 10 nm. Using centrifugation, exosomes can be separated from proteins to remove more significant cell remains, accompanied by beads of suitable size. Exosomes have been extracted from protein using Sepharose CL-4B, Sephacryl S-400, and Sepharose CL-2B beads [16][17], having an apparent pore size distribution of 42, 31, and 75 nm, respectively.
SEC has several advantages. The vesicular structure of exosomes is highly intact since they are not exposed to any extreme circumstances, such as centrifugal forces [18]. Because of its relatively inexpensive and quick separation, SEC is considered a feasible choice for bulk-scale exosome production.

1.5. Polymer-Based Precipitation Separation

This approach includes charge-based exosome precipitation using hydrophilic polymers like polyethylene glycol (PEG). PEG reduces exosome solubility by capturing water molecules and forcing exosomes to sink at a relatively low centrifugal force. Exosomes are precipitated when exosome-containing samples co-incubate with PEG solution (MW: 8000 Da). After incubating at 4 °C overnight, the deposited exosome can also be recovered or isolated by filtering or centrifugation. The following approach does not need advanced equipment and is reasonably simple to use with minimal downtime [19][20].

1.6. Magnetic Separation

Clayton et al. proposed a simple and quick approach for routine exosome separation and analysis based on immuno-magnetic isolation of exosomes containing human primary histocompatibility complex class II moieties [12]. This approach employs magnetic beads bounded with antibodies directed against proteins expressed on the exosomal surface. This method offers several benefits. Flow cytometry with fluorophore-conjugated antibodies can be utilized to investigate the bead-exosome complexes, allowing for fast quantitative analysis of the exosome surface make-up. On the other hand, these complex systems can be immunoblotted, and their composition is determined by treating them with an SDS sample buffer [10].

1.7. Acoustic Fluidic Separation

Acoustic fluidic separation is highly scalable, allowing for the manipulation of bioparticles ranging from nanoscale to microscale. Transducers built of piezoelectric materials are a practical approach to creating acoustic waves. When mechanical tension is applied, piezoelectric materials can generate electrical polarization, or mechanical deformation can be caused by electrical polarization [21]. Lee et al. used a standing surface acoustic wave separation instrument operating at 38.6 MHz to separate extracellular vesicles [22]. Exosomes were isolated from other extracellular vesicles using a cut-off size of 300 nm. Because of its biological acceptance also contactless separation advantages, the acoustic-fluidic separation technique has tremendous potential. The process output is limited to 0.40–0.43 L/min [22].

1.8. Dielectrophoretic Separation

Dielectrophoretic separation depends on the dielectric force acting on polarized particles in a non-uniform electric field. Nanoscale particles are attracted to the dielectrophoretic (DEP) high-field zones surrounding the circular microelectrode edge. The DEP low field zones amongst the electrodes attract cells and more prominent entities. Finally, the DEP field has little effect on tiny biomolecules, cations, and anions. A difference in dielectric properties between nanoparticles and the surrounding fluid (blood, plasma, serum, etc.) causes the DEP force [23]. The dielectric constant governs how rapidly charges travel via a medium when the external electric field changes. Transient dipoles form across the nanoparticles due to charges in the fluid medium and nanoparticles realigning themselves at various speeds in response to the shifting external electric field. These dipoles produce the forces that drag nanoparticles into the DEP high-field areas in a nonuniform electric field [24]. Although the requirement for electrothermal heating constraints this technique’s application, its advantages in speed and output might be considered.

1.9. Deterministic Lateral Displacement (DLD) Separation

Huang et al. pioneered DLD, a robust passive microfluidic particle separation approach that uses pillar arrays to separate particles depending on the dimensions [25]. DLD offers promise because of its low cost, robustness, and ability to precisely manipulate particles and efficiently separate them. DLD works at a low Reynolds number compared to other isolation methods and allows high dynamic size isolation, from millimeters to micro and nano sizes. It was recently shown that DLD could identify molecules according to morphologies, compressibility, and electrical characteristics. Because of separation’s excellent responsiveness, with a resolution limit of 20 nm. In microfluidic DLD, tilted pillar arrays are employed to create a fluid bifurcation and different streamlines between the gaps. The total fluid flux across each gap can be grouped by the periodicity (N), resulting in the conclusion that the DLD array’s periodicity equals the number of streamlines between each pillar. Fluidic forces and pillar barriers impact the particle fluxes in the DLD array. After a molecule enters the gap, any molecule with a small radius, when compared to the first streamline width, will follow the first streamline and travel in a zigzag pattern.
On the other hand, the molecule of size more significant than the 1st streamline moves straight and is forced against a pillar and displaced to the following streamline laterally [25]. Researchers encounter separation and clogging issues despite its ease of use and lack labels [26].

1.10. Microfluidic Devices

Using small samples and microfluidic devices, it is possible to automate the exosome separation and purification process [27]. These devices possess the unique feature of purifying EVs and their classes faster with high precision. Depending on their size, charge, surface properties, and interactions, these devices may combine sieving barriers, mixing chambers, and antibody-coated and functionalized layers to enable the separation of EV subclasses. These approaches are affinity-based [28][29]. Microfluidic devices are mainly used as diagnostic instruments because they specialize in small volumes (L).
Example: It finds cell-specific EV enrichments associated with particular disease states.
Though it has excellent output in diagnostics, this method is suffering from significant limitations of non-scalability on large-scale diagnostics due to the absence of standardization of components and benchmarking [30].

2. Characterization of Exosomes

Exosomes are characterized based on their physical, chemical, functional, structural and biological [31]. Exosomes may need more characterization techniques for specific uses, for example, in enzyme replacement therapy (ERT), where they play the role of the vehicle to transport drug substances or biological compounds to the site of action. Exosomes must be created using precise procedures that consistently produce exosomes with composition, structural, and functional properties that fall within a specific range of acceptable ranges. The techniques used for the characterization of exosomes are described below.

2.1. Nanoparticle Tracking Analysis (NTA)

The nanoparticle tracking analysis (NTA) approach uses the particle light scattering and Brownian motion to quantify particle diameter. The hydrodynamic diameters are determined by following particles’ simultaneous, individual Brownian motion. Because each particle is photographed in distinct areas, it may be possible to discriminate between different sample sizes. NTA can also use fluorescent labeling to measure fluorescence and estimate the presence of antigens on exosomes [32][33].

2.2. Dynamic Light Scattering (DLS)

The physical technique of Dynamic Light Scattering (DLS), also known as “Photon Coherence Spectroscopy”, is used to determine particle size and dispersion. It works based on detecting particle light scattering and analyzing an optical signal to detect particles. This approach is suitable for predicting exosome size but can’t get source data. Moreover, the major shortcoming of this technique is for formulation containing particles of various sizes, significantly larger size particles that interfere with the detection of smaller particles. Hence, this technique’s obtaining size distribution is not perfect [34][35].

2.3. Atomic Force Microscopy (AFM)

Determining vesicle diameters using cryogenic transmission electron microscopy (cryo-TEM) is considered a gold standard technique, but problems are associated with it. The instrument’s cost, the knowledge necessary for sample preparation, imaging, and data interpretation, and a tiny number of particles consistently visible in images are all obstacles. Hence, the available and accessible substitute is atomic force microscopy (AFM) to overcome these challenges. AFM can provide valuable data on extracellular vesicles’ three-dimensional geometry, size, and other biophysical characteristics [36]. It also maps exosome mechanical properties and estimates their relative size distribution. It achieves sub-nanometer resolution imaging by scanning the surface with the tip of the cantilever beam. Structure, biomechanics, and abundance may be quantified and detected using AFM [37].

2.4. Microscopy Study

2.4.1. Transmission Electron MICROSCOPY (TEM)

Transmission electron microscopy (TEM) examines the form and structure of particles using an accelerated electron beam. The images of exosomes obtained by TEM can estimate their size. Moreover, the morphology of exosomes can be impacted while preparing of TEM sample. To overcome this technique’s limitation, the advanced process “Cryo-TEM” was invented by scientists. Cryo-TEM is an advanced version that avoids sample preparation effects [31][38].
Spherical exomes and exosomes are different (heterogeneous) in shape, according to Colombo et al. [39]. However, the electron beam may be responsible for destroying biological samples in a few circumstances. The cup-shaped structure of isolated exosomes analyzed by TEM is the most prominent feature, and frozen exosomes are examined using cryo-TEM techniques to reveal round forms [40].

2.4.2. Scanning Electron Microscopy (SEM)

Scanning Electron Microscopy (SEM) detects low-energy electrons ejected from only form proximity to the sample surface. It can give valuable sample data, including morphology, composition, surface texture, and roughness [41]. Thus, SEM provides information regarding exosome surface characteristics comprising size, shape and morphology. In a few cases, backscattered electrons (BSE) detector could also be helpful, especially for exosomes containing surfaces labeled with heavy metal [42]. It gathers electrons from more depths below the sample’s surface than scattered electrons and gives information about the composition and topography [43].

2.5. Enzyme-Linked Immunosorbent Assay (ELISA)

A plate-based enzyme-linked immunosorbent test identifies and measures proteins, peptides, hormones, and antibodies. It has been used to detect exosomes but needs samples in larger quantities and suffers from less responsiveness. With ELISA’s help, this assay is designed to accurately provide cancer exosome numbers [44]. It also determines exosomes from plasma, serum, and urine using different antibodies [45].

2.6. Fluorescence Correlation Microscopy (FCM)

Exosomes are captured using a particular antibody, tagged with a fluorescent dye and measured by a plate reader in microfluidic-dependent Fluorescence Correlation Microscopy (FCM). A microfluidic chip used for immunocapture and quantitative exosome analysis can be developed using FCM [46].

2.7. Colorimetric Detection

A chromogenic substance’s color intensity determines the particles in calorimetric detection. Exosomes were captured using microfluidic technologies, and exomes were detected and quantified using ELISA. Scientists and researchers have successfully utilized this technology to detect exosomes from cancer cells [47][48][49].

2.8. Surface Plasmon Resonance (SPR) Detection

Scientists and researchers have successfully utilized this technology to detect exosomes from cancer cells. Exosomes are studied using a nano-plasmonic exosome (nPLEX) created by modifying a nano substrate to improve detection performance.

2.9. Nuclear Magnetic Resonance (NMR) Detection

Nuclear Magnetic Resonance (NMR) is mainly utilized to analyze chemicals. To evaluate the quantity and presence of proteins in exosomes, micro-NMR techniques have also been developed. Systems can detect exosomes after concentrating microvesicles containing immunogenic nanoparticles via filtering. The number of exosomes is quantified using a signal-to-noise ratio [50][51].

3. Exosomes as Drug Delivery Vehicle

Many advanced drug delivery systems like polymeric nanoparticles and liposomes encapsulate drug substances. They are most frequently used to deliver anticancer, antiviral, and antifungal drugs. Still, the significant concerns are their compatibility with living tissue, extended stability, and the potential to attack the host’s immune system for extended periods [52]. A recent advancement was made in the drug delivery science is exosomes; it is a novel nanoscale delivery system with many advantages, such as biocompatibility, biodegradability, less toxic, specificity to the target cells, small size, promotes plasma membrane fusion, among different cells, longer half-life, low-uptake machinery, capability to pass contents from one cell to another cell, and do not produce an immune reaction and the unique feature that they have more tendency to accumulate in the cancerous cell than normal cells make it a good choice.

3.1. Small Molecules

Exosomes are also attractive small-molecule drug delivery carriers because of their small size, lower toxicity, and biocompatibility than other nanocarriers wiz, liposomes, niosomes, etc. Exosomes also act as nanoparticles that can carry their cargo to receptor cells and also helps in cellular exchange that can be used as a carrier for drug delivery. Exosome-encapsulated APIs show superior PKPD features and in vivo anti-neoplastic activity compared to free drugs. Exosome-loaded small compounds have been reported to offer superior therapeutic advantages in a few investigations; for example, exosomes containing doxorubicin were developed and tested for distribution along with fast cellular absorption to the intracellular fluid [53]. Compared to free doxorubicin and its liposomal forms, exosomes of the doxorubicin demonstrated better in vitro potency in various cell types and increased cell absorption and biodistribution. Several tests were then conducted to demonstrate that curcumin incorporation within the exosome enhances solubility, stability, and bioavailability [54].
Exosomal curcumin’s anti-inflammatory efficacy was also tested in vitro and in vivo. In vitro, exosome containing curcumin inhibited the release of inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor (TNF-), implying that exosome containing curcumin has anti-inflammatory characteristics. Animal model of lipopolysaccharides the septic shock created in vivo, and mice treated with exosomal curcumin outlived those treated with just curcumin. Finally, exosomal curcumin was found to lower the number of CD11bGr-1 cells in the lungs, which improves the response to lipopolysaccharide-induced septic shock and causes acute lung inflammation. The following work demonstrated that exosomes might transport extremely hydrophobic medicines like curcumin, boosting their anti-inflammatory properties [55]. Exosomes transport small molecular medications transverse to the blood-brain barrier (BBB) and increase therapeutic characteristics. Indeed, 98 percent of powerful centrally-acting drugs cannot pass through BBB, and their in-vitro efficacy has not been replicated in clinical trials. Most of the nano-formulations have been used to tackle difficulties related to medication permeability across the BBB. Nanotoxicity and fast drug elimination by the mononuclear phagocyte system (MPS) were additional problems. Polyethylene glycol (PEG) reduces MPS medication absorption to compensate for these issues. This, however, resulted in less contact between target cells, resulting in decreased drug distribution throughout the brain. In this scenario, exosomes, naturally produced by the body’s cells substance, may be made to pass the BBB, increasing drug delivery to the brain while lowering MPS drug clearance [56].

3.2. Large Molecule (Protein and Peptide Delivery)

Large molecules, such as proteins and peptides, are delivered using exosomal drug delivery systems. Exosomes were researched to transmit biological materials for diverse tasks such as therapeutic and diagnostic reasons, as they were found to remove the protein, lipids, and nucleic acid unwanted for cells [57]. Since research has shown that exosomes separated from most cells are intrinsic carriers for endogenous protein molecules, exosomes are currently portrayed as ideal carriers for protein and peptide molecules [55]. Recent research, for example, in diseases like Parkinson’s, have shown that exosomes containing the antioxidant protein catalase were effectively carried across the BBB, resulting in a better disease state [52].

3.3. Nucleic Acids

Exosomes have also been shown to transport nucleic acids like DNA, RNA, mRNA, miRNA etc., to specific cells in the body, producing genetic alterations in both normal and pathological processes. They target cytoplasm and alter cell function that can be used for the treatment of various diseases. Exosomes piqued researchers’ interest in gene therapy treatment alternatives because of their natural carrying capacity of genetic material. Exosomes have been used in experiments to administer therapeutic genetic substances that influence gene expression in specific illnesses and promote genetic treatment [57]. It also can carry different biological components such as lipids, proteins and nucleic acids. It contributes to the intercellular communication process [58][59][60][61][62].

3.4. Small Interfering RNAs (siRNAs)

SiRNA is a kind of RNA that disrupts genes of interest in genetic treatments. However, it has limited stability and degrades systemic circulation. On the other hand, storing and giving siRNA to specific cells can act as a therapeutic carrier and helps to overcome this barrier. Most research has been done to evaluate effective siRNA carriers to target cells [63]. Exosomes were employed to distribute siRNA because they show no immune response and naturally transport RNA from cell to cell. Researchers discovered that endothelial exosomes could deliver siRNA to endothelial cells. Some experiments used exosomes from dendritic cells to electroporate loaded exogenous siRNA to achieve tissue-specific targeting.
In contrast, dendritic cells were modified to produce exosomal surface component Lamp2b. Several research in recent years has revealed the effective transport of siRNA to target cells. It has also been discovered that the loaded siRNA is delivered to cancer cells through exosomes. Exosomes obtained by centrifugation by HEK-293 cells were tagged with fluorescent dye before being electroporated into exosomes. After loading it into the exosomes, gel filtering was used to remove the excess siRNA. The findings showed that siRNA encapsulation was adequate, with a high exosome production and successful transport into cancer cells [50].

3.5. MicroRNA (miRNA)

miRNA is a short non-coding RNA in eukaryotic cells and contains non-proteinaceous nitrogenous bases. miRNAs regulate post-translational gene expression by binding to complementary sites on targeted mRNA. Because exosomes naturally transport miRNA, they can be used to therapeutically transfer miRNA to selected cells [64]. The research used exosomes to deliver miRNA to breast cancer cells targeting the epidermal growth factor receptor (EGFR). EGFR expression was high in various human cancers derived from epithelial cells, indicating that EGFR ligands might be used as cancer treatment targets. Exosomes were created to deliver 5-fluorouracil (5-FU) with microRNA-21 inhibitor oligonucleotide (miR-21i) to HER2, expressing cancerous cells resulting in cell cycle arrest and decreased tumor growth and increased apoptosis. They restored PTEN and hMSH2 levels, which are miR-21 regulatory targets. In 5-FU tolerant colon cancerous cells, combining miR-21i with 5-FU delivery in exosomes successfully overcame drug resistance and efficiently boosted cytotoxic activity [65]. Exosomes generated from MDA-MB-231 cells were employed in another investigation to treat NSCLC (Non-Small-Cell Lung Cancer). Because of a link between the cancer cell surface protein C (SPC) and the overexpressed integrin 4 (found on exosomes), NSCLC absorbed exosomes (231-Exo) preferentially. The antisense oligonucleotide of miRNA-155 was incorporated into exosomes by RBCs with the help of an electroporator, which resulted in enhanced therapeutic activity on lung diseases. Exosomes (miR-126:231-Exo) were loaded with miRNA-126, which decreased cell proliferation and migration by interfering with the PTEN/PI3K/AKT signaling pathway. An in vivo investigation revealed that it inhibited lung cancer metastasis in mice [63].

3.6. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR Associated Protein 9 (Cas9) System

Scientists recently employed CRISPR/Cas9 systems to cure numerous hereditary disorders like cancer by repairing, eliminating, or suppressing disease-related genomic abnormalities. It is a two-part system for the deletion, insertion and modification of particular genes. It is comprised of the Cas9 protein and a single-guide RNA (sgRNA) (RNA-guided endonuclease) [66]. To be effective in gene editing, the CRISPR/Cas9 system must be supplied to receptor cells in a certain way. The researchers employed a Liposome-exosome hybrid nano system to introduce the CRISPR/Cas9 system within MSCs. Ultracentrifugation was used to separate exosomes from HEK293FT cells. Exosomes were incubated with liposomes to form hybrid exosomes; before being delivered to MSCs, significant DNAs were enclosed within exosomes. They were capable of transporting hybrid nano-systems into MSCs and controlling the expression of targeted genes [67]. Exosomes have been discovered to be ideal carriers for cancer treatment due to cell tropism-induced selective accumulation. Exosomes containing CRISPR/Cas9 reduced the formation of poly (ADP-ribose) polymerase-1 (PARP-1), causing ovarian cancer cells to die. CRISPR/Cas9 genetic material transformation improved cisplatin chemosensitivity, resulting in synergistic cytotoxicity [64].

This entry is adapted from the peer-reviewed paper 10.3390/molecules27217289


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