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Drug Delivery to the Bone Microenvironment Mediated by Exosomes: An Axiom or Enigma

Authors Samal S, Dash P, Dash M 

Received 22 February 2021

Accepted for publication 30 March 2021

Published 21 May 2021 Volume 2021:16 Pages 3509—3540

DOI https://doi.org/10.2147/IJN.S307843

Checked for plagiarism Yes

Review by Single anonymous peer review

Peer reviewer comments 2

Editor who approved publication: Professor Israel (Rudi) Rubinstein



Sasmita Samal,1,2 Pratigyan Dash,1,2 Mamoni Dash1

1Institute of Life Sciences, Nalco Square, Bhubaneswar, Odisha, 751023, India; 2School of Biotechnology, Kalinga Institute of Industrial Technology (KIIT) University, Bhubaneswar, Odisha, 751024, India

Correspondence: Mamoni Dash
Institute of Life Sciences, Bhubaneswar, Odisha, India
Email [email protected]

Abstract: The increasing incidence of bone-related disorders is causing a burden on the clinical scenario. Even though bone is one of the tissues that possess tremendous regenerative potential, certain bone anomalies need therapeutic intervention through appropriate delivery of a drug. Among several nanosystems and biologics that offer the potential to contribute towards bone healing, the exosomes from the class of extracellular vesicles are outstanding. Exosomes are extracellular nanovesicles that, apart from the various advantages, are standing out of the crowd for their ability to conduct cellular communication. The internal cargo of the exosomes is leading to its potential use in therapeutics. Exosomes are being unraveled in terms of the mechanism as well as application in targeting various diseases and tissues. Through this review, we have tried to understand and review all that is already established and the gap areas that still exist in utilizing them as drug delivery vehicles targeting the bone. The review highlights the potential of the exosomes towards their contribution to the drug delivery scenario in the bone microenvironment. A comparison of the pros and cons of exosomes with other prevalent drug delivery systems is also done. A section on the patents that have been generated so far from this field is included.

Keywords: drug delivery, bone diseases, exosomes, nanosystems, RANK-RANKL pathway, exosome-liposome hybrid

Introduction

Bone is a complex rigid organ of the human body which provides the basic skeletal framework, stores calcium, and produces blood cells.1,2 The adult bone comprises 50–70% mineral as hydroxyapatite [Ca10(PO4)6(OH)2], about 20–40% organic matrix, consisting mostly of type I collagen.3,4 A well-balanced homeostasis requires active communication between osteoblasts and osteoclasts mediated by various cytokines and growth factors.5–8 However, this delicate balance tends to get lost with time due to aging,9 hormonal imbalance,10,11 impaired signaling pathways,12,13 external factors, etc. leading to excess osteoclastic bone resorption as compared to bone formation. Drugs that inhibit the osteoclastic activity or induce bone formation are important for treating osteoporosis,14,15 bone metastases,16 osteosarcoma,17 arthritis,18 and Paget’s disease.19

The currently available drug treatments are based on the principle of either suppressing osteoclast formation (e.g; estrogens, SERMs) or suppressing osteoclast activity (e.g; Bisphosphonates).20 However, besides the anabolic effects of most of the drugs on bone, they have certain limitations which include affecting the normal bone cells as well as other tissues including uterus,21 breasts.22–24 Also, the intravenous25 or oral administration26 of high doses of drugs for a prolonged period can cause secondary adverse effects.27 Thus, it is clear that bone-drug delivery application is yet to meet perfections in terms of targeting and eliminating the side effects. Therefore, it is important to understand the disease-specific pathways for more accurate tailored targeting mechanism and a controlled release at the target site. The research area of controlled drug delivery to bone has experienced tremendous progress owing to the emergence of nanotechnology.28,29 The novel features of these nanosystems have enabled site-specific drug delivery,30 high loading efficiency,31 controlled release,32 prolonged blood circulation time,33 etc. Moreover, with the emergence of BPs, tetracycline, and acidic oligopeptides (AO), having the highest affinity for bone hydroxyapatite,34–36 the bone-specific targeting has boosted up. Several polymer-drug conjugates37 and multifunctional hybrid nanoparticles (HNPs) for enhanced drug activity has received great impetus in recent years.38–40 In spite of several progresses, some challenges and obstacles are involved in the translation of these nanomaterials from laboratory to clinical therapy. High cost to benefit ratio, rapid clearance of bare nanoparticles by mononuclear phagocyte system (MPS), bioaccumulation, and cytotoxicity are some of the major limitations.41–43

In this context, exosomes, the nano-sized (30–120 nm) lipid bilayer vesicles secreted by almost all eukaryotic cells44,45 have come up as an important tool for diagnostic and therapeutic purposes.46–48 Right from the discovery of exosomes in 1987, by Johnstone et al from sheep reticulocytes,49 these were thought to be the cell garbage cans, which remove wastes or byproducts from cells.50 Thanks to the advancement of electron microscopic technology, the vital role of exosomes as the cellular messenger has been revealed.51–53 Compared with the synthetic nano-delivery systems, exosomes have gained much attention because of some of their vital characteristic features. As exosomes are released by budding off from the plasma membrane, they tend to contain surface adhesion proteins and carry various cargos like protein, lipids and nucleic acids,54,55 which also shows their strong cargo loading and cargo protection ability. They show tremendous inherent capacity to interact with recipient cells.56,57 They can be internalized by the host cell and transfer their payload, thereby modulating the host cell machinery,58,59 which proves their inherent cargo delivery properties. Exosomes qualify all the criteria to become an effective and safe delivery vehicle such as high biocompatibility,60 strong cargo loading61 and cargo releasing ability,62 ability to cross difficult biological barriers,63,64 easy surface modification and drug loading,65,66 etc. Recent studies have revealed the pivotal roles played by bone cell-derived exosomes in maintaining normal bone homeostasis by transferring biologically active molecules to target cells.67–69 In a study, exosomal miR-214 released from osteoclasts significantly suppressed osteoblast activity and the inhibition of exosome secretion via Rab27a siRNA potentially prevented osteoblast dysfunction.70 In another study, osteoblast-derived exosomes mediated intracellular communication was seen to play important role in osteoclast differentiation.71 Furthermore, bone marrow mesenchymal stem cell (BM-MSC) derived exosomes have been seen to regulate osteoblastic activity and differentiation72 to promote fracture healing73 and osteoporosis improvement74 via important paracrine signaling. BM-MSC-derived exosomal miR-150-3p could effectively promote osteoblast proliferation and differentiation in the ovariectomized (OVX) rat model of osteoporosis.75 These findings provide new insights and motivation to exploit the naturally occurring exosomes for finding novel treatments of existing bone diseases.

In this review, the major highlight is the utilization of exosomes as a potential drug delivery vehicle to treat bone diseases as compared to conventional nanosystems. A brief idea of the biogenesis and importance of exosomes in bone homeostasis regulation is provided. Then, the potential advantages and limitations of each of these nano-drug transporters are compared with naturally occurring exosomes. Furthermore, the existing patents on the therapeutic applications of exosomes as a drug carrier device targeted to the bone have been listed out. Lastly, the improved translational potential of exosomes by using innovative engineering techniques to enhance the targeting efficiency and prolonged stability in the blood has been discussed.

The Clinical Need for Drug Delivery Systems to Bone

Drugs and therapeutic strategies are currently available for common bone disorders occurring due to increased bone resorption like osteoporosis, bone metastasis, osteosarcoma, osteoarthritis, and Paget’s disease.76 These broadly include bone-targeted anti-resorptive drugs like bisphosphonates (BPs),77,78 antibody treatment,79 combination anti-resorptive therapy,80,81 antibiotics like tetracyclines,82 chemotherapeutic agents83,84 and hormonal therapy.85 The Food and Drug Administration (FDA) approved anti-resorptive drugs BPs,86 such as alendronate, risedronate, ibandronate and zoledronic acid, are used to treat osteoporosis,87,88 bone metastases89,90 and osteosarcoma91,92 by inhibiting bone demineralization93,94 and tumor growth.95,96 Denosumab, a RANKL-inhibitor,97,98 is a human monoclonal antibody-based treatment,99 which has been strongly discussed for its superior effects on skeletal-related events (SREs),100,101 osteoporotic prevention in postmenopausal women102 over BPs and treatment of bone metastases.103 Tetracyclines having a high affinity for bone hydroxyapatite (HA)104 inhibit matrix metalloproteinases (MMPs)82,105 involved in bone metastasis. Standard chemotherapeutic drugs, such as doxorubicin,106,107 cisplatin,108 ifosfamide,109 cyclophosphamide,110 and high-dose methotrexate111 with leucovorin rescue,112 are being used singly or in combination with osteosarcoma chemotherapeutic regimen107,113,114. Selective estrogen receptor modulators (SERMs) such as raloxifene,115 bazedoxifene116,117 and lasofoxifene118 are FDA approved drugs used for the prevention and treatment of postmenopausal osteoporosis.119

Despite the significant innovation of pharmaceutical drugs for various diseases, these are not able to achieve the proposed goal in most of the cases due to the various disadvantages associated with the drugs. For example, nitrogen-containing bisphosphonates have a short plasma half-life, low drug absorption, slow-release effect of higher affinity BPs, some side effects like osteonecrosis of the jaw (ONJ), atrial fibrillation (AF), esophageal cancer, musculoskeletal pain etc120 are associated. Moreover, lingered anti-fracture benefits are observed after stopping the treatment therapy, for which drug holidays are recommended.121 Although zoledronate has come up as an advanced drug, the optimal dosing interval has to be established for different patient populations.122 In the case of antibody treatment with denosumab, hypocalcemia is observed in severe cases of renal dysfunction. Osteoclastic inhibitors (BPs and denosumab) have been shown to adversely affect advanced breast cancer.123 In some cases, the treatment requires oral administration of high doses of the drug to achieve effective drug concentrations in the target tissue, which results in drug accumulation in normal tissues.25 Moreover, some of the drugs may have poor biocompatibility and rapid clearance by body metabolism. Also, the higher hydrophobicity of some drugs results in poor penetration ability to cells.23,124 Therefore, there is an alarming need for the development of novel carriers for efficient drug delivery.

Potential drug delivery systems can control the rate of drug release at the targeted site and provide excellent stealth upon coating and drug protection from cellular degradation.28,29 Recent decades have witnessed huge studies being done on bone-targeted delivery using nanotechnology as a potential solution to the current limitations (Figure 1). Nanomaterials provide the advantage of enlarged drug-loading capacity with a large surface area: volume ratio due to the smaller size (30–200nm), which also allows them to traverse biological barriers for more efficient delivery.125 Furthermore, the combination of conjugates, nanoparticles, and drugs has shown increased targeting ability and efficacy.77 In a study, magnetic nanoparticles (̴ 20nm) coated with calcium phosphates (CaP) were used to treat osteoporosis and bacterial infection. The study also demonstrated that the optimum dose of magnetic nanoparticles has the potential to promote osteoblast density and inhibit bacterial growth.126 For successful bone-targeted drug delivery, site-specificity, or active targeting by engineering the nanosystem surface with specific targeting moieties is essential. Wang D et al, synthesized polymeric bone-targeting conjugates based on PEG and poly[N-(2-hydroxypropyl)methacrylamide] (pHPMA) conjugated to well-known bone-targeting compounds, alendronate, and aspartic acid peptide as bone-targeting moieties. When tested for bone-targeting potential in vitro and mice (Balb/c), they found that these conjugates could specifically accumulate in the bone tissue and hydroxyapatite (HA) proving to be promising candidates for bone-targeted delivery of therapeutic agents.127 In a recent study, Sun et al came up with a novel bone-targeted nano-platform using mesoporous silica nanoparticles loaded with gold nanorods (Au@MSNs) which were then conjugated with zoledronic acid (ZOL) forming a novel nanosystem (Au@MSNs-ZOL). They demonstrated targeted bone delivery in vivo as well as an inhibited formation of osteoclast-like cells and enhanced osteoblast differentiation in vitro.128 With a limited number of such materials, Peng Z et al synthesized carbon dots (1.5 to 5.0 nm) (C-dots: CD1) from carbon nanopowder with non-toxic and unique affinity and specificity towards calcified bones in live zebrafish larvae. These novel nanodots can potentially be used as bioimaging agents for the early detection of bone diseases as well as drug carriers for targeted drug delivery.129 Although the improved nanotechnology methods are often successful for specifically treating osteogenic disorders, these also come up with environmental and health concerns due to toxic by-products and lack of biocompatibility as well as biodegradation issues. Medina-Cruz D et al have given an overview of green nanotechnology-based approaches to treat osteogenic disorders. These latest advancements have emerged as potential tools for efficient drug delivery to the bone without showing any side-effects and toxicity, unlike the traditionally synthesized nanomaterials. An informative discussion has been done on the use of various green nanotechnologies, such as polysaccharide-based, protein-based, calcium-based, and silica-based in bone regeneration and controlled delivery.130 Wang F et al synthesized a liposomal drug delivery system conjugated with cyclic arginine-glycine-aspartic acid-tyrosine-lysine peptide (cRGDyk) as the ligand for αvβ3 integrin. They loaded it with hydrophilic cisplatin (CIS) and hydrophobic PbS quantum dots (PbS QD) to form RGD-CIS-liposomes and RGD-PbS-liposomes, respectively (Figure 2) to target bone metastasis from prostate cancer. The resultant product, RGD-CIS-liposomes, showed improved therapeutic efficacy with selective accumulation at the tumor site, enhanced EPR effects, and synergistic anti-tumor activities in mice.131

Figure 1 Examples of common nanomaterials used for targeted bone delivery. Titanium nanotubes, gold nanoparticles, calcium phosphate nanoparticles, and mesoporous silica nanoparticles (MSNs) constitute the inorganic nanomaterials. The organic nanomaterials include chitosan nanoparticles, poly(L-lactide-co-glycolide) PLGA nanoparticles, and liposomes. MSC: mesenchymal stem cell. Adapted with permission from Drug Discovery Today, Vol /edition number 22(9), Cheng H, Chawla A, Yang Y, et al, Development of nanomaterials for bone-targeted drug delivery, Pages No. 1336–1350, Copyright (2017), with permission from Elsevier.125

Figure 2 Schematic diagrams for the structure of RGD-CIS-liposomes and RGD-PbS-liposomes. Adapted with permission from Journal of Controlled Release, Vol /edition number 196, Wang F, Chen L, Zhang R, Chen Z, Zhu L, RGD peptide conjugated liposomal drug delivery system for enhance therapeutic efficacy in treating bone metastasis from prostate cancer, Pages No. 222–233, Copyright (2014), with permission from Elsevier.131

A novel approach was attempted by Chaudhari et al to target bone metastasis. They prepared zoledronate conjugated PLGA nanoparticles (ZOL-PLGA) for specific bone targeting with higher affinity and utilized the synergistic effects of ZOL along with Docetaxel (DTX). Their results showed a remarkable increase in cellular uptake, facilitated delivery of DTX inhibiting cancer cell proliferation and increased apoptosis, prolonged blood circulation time, and higher tumor retention ability.37 A comparative analysis was done by Marra M between ZOL-containing self-assembly PEGylated NPs and ZOL-encapsulated stealth liposomes towards anti-cancer effect which suggested the development of ZOL-based NPs for the treatment of human cancer.132 Carmona et al developed a novel multifunctional nanodevice for bone cancer treatment. They functionalized the surface of mesoporous silica nanoparticles (MSNs) with carboxylic acid groups (MSN-COOH) and conjugated with a targeting ligand, lectin concanavalin A (ConA) (Figure 3). This novel multifunctional nanosystem showed a higher degree of internalization into human osteosarcoma cells (HOS) as compared to healthy pre-osteoblast cells (MC3T3-E1). Moreover, a lower dose of DOX loading (2.5 µg/mL) could kill almost all the HOS cells.133

Figure 3 Schematic representation of modified multifunctional MSNs loaded with anti-tumor drug DOX for targeted bone cancer treatment. Adapted with permission from Acta Biomaterialia, Vol 65, Martínez-Carmona M, Lozano D, Colilla, M, Vallet-Regí M, Lectin-conjugated pH-responsive mesoporous silica nanoparticles for targeted bone cancer treatment, Pages No. 393–404, Copyright (2018), with permission from Elsevier.133

From all the above examples it can be evidently inferred that as compared to the therapeutic drugs used alone, their loading into potential nano-delivery vehicles has better therapeutic efficacy in terms of enhanced drug stability, protection from lysosomal degradation, and prolonged blood circulation time. Also, it is translucent that enhanced targeting ability can be achieved by surface conjugation with molecules having a higher affinity to bone HA.

Existing Drug Delivery Systems for Bone Therapeutics

Drug delivery vehicles are diverse and have received enormous popularity due to the versatile applications they possess. Engineered and natural nanomaterials have the properties to deliver drugs or fluorescent molecules for therapeutic advantage. Different systems have different criteria for delivering a molecule of interest. Fabrication of nanoparticles to augment therapeutic dosage plays a key role in translational science. In this section, we have presented the most commonly used nanoparticles as well as the naturally occurring nanovesicles for drug carriers in bone therapeutics.

Nanoparticles

Polymeric nanoparticles

Polymeric nanoparticles prepared either from natural or synthetic polymers, have a wide array of biomedical applications due to the fact that they are biodegradable and biocompatible.134 Most of the synthesized polymeric nanoparticles such as PLA (polylactic acid) and PLGA (polylactic glycolic acid) have even been approved for human consumption by the US FDA. Various polymers such as polycyanoacrylate and poly(lactide-co-glycolide) PLA or poly(lactic acid) have been used in targeted drug delivery, gene silencing, and siRNA delivery.135 Besides synthetic ones, natural polymers such as chitosan, dextran, albumin, and gelatin have also been used for encapsulating drugs and therapeutics. In addition to the effective cargo loading criteria, the smaller size (increased surface area-to-volume ratio) facilitates facile conjugation for attaching several ligand and functional groups to specifically target the desired site.136–138 Inert polymers such as polyethylene glycol are now being used as stealth coats to avoid the RES (Reticulo-endothelial system) and successfully evade the immune system to make the targeted drug delivery even more fruitful.139 Wang and co-workers studied effective combinational chemotherapy via encapsulation of both the drugs inside PEGylated PLGA nanoparticles directed to MG63 and Saos-2 osteosarcoma cell lines. PEG surface modification to PLGA NPs was successful in evading macrophage and thus the RES. The co-encapsulated drugs in PLGA NPs exhibited dose and time-dependent cytotoxicity and exerted higher cytotoxicity, apoptosis, and cell cycle arrest with evasion of RES as compared to free drug formulations.140

However, these polymeric coatings are also now being questioned as they are starting to become immunogenic. According to Garay et al, repeated infusion of PEGylated therapeutics can elicit a hypersensitive reaction and evoke anti-PEG antibodies against the nanomaterial.141 Apart from all these advantages, polymeric nanoparticles suffer from batch to batch variability and are prone to faster degradation.

Polymeric Micelles

Micelles comprise a polar hydrophilic head and a lipophilic tail that aggregate into an amphiphilic structure in solution. These compact structures are formed between a strong covalent bond that holds the molecules together and the intermolecular forces. Due to the peculiar size and morphology of these nanovesicles, they have gained much attention in targeted drug delivery system approaches. Amphiphilic block copolymers self-assemble into stable structures in aqueous media that can contain hydrophobic drugs.142 They are not mechanically cleared from the spleen and also possess minimal cytotoxicity with potent stability inside the body system.143 They can be made stimuli or thermo-sensitive based on the release of the drug at the required site. Alendronate (ALN), as a bone homing agent decorated with polymeric micelles encapsulated with docetaxel, has been developed for treating breast cancer bone metastases with better pharmacokinetics and sustained release.144

However, the major setbacks that micelles possess are the high cost for the preparation of micelles at a large scale and difficulty in loading hydrophilic drugs.145

Dendrimers

Dendrimers are nano-sized hyperbranched macromolecules with radial symmetry, containing an outer and an inner shell. They are defined by a highly intricate structure containing protruding functional groups resembling branches of a tree, thus the name dendrimer.146 Dendrimers can be attached to the periphery or can be engaged in their interior space.147 In most cases, dendrimers are employed because the functional groups are free for conjugation to various targeting moieties, such as monoclonal antibodies or folate that facilitate entry into the tumor or say destined cell. Janus peptide-based dendrimer comprising of RGD dimer and 5-FU dimer have been synthesized with the ability to carry a chemotherapeutic agent for the treatment of bone tumor.148 Drugs attached to polymeric dendrimers are highly soluble with enhanced stability.149 The limitation is the controlled multistep synthesis parameter.

Mesoporous Silica Nanoparticles (MSNs)

Mesoporous silica nanoparticles (MSNs) are widely used in targeted delivery approaches due to their hydrophobic core that enables loading of drug and hydrophilic surface that prevents opsonization as such.150 The amorphous silica matrix contains numerous uniform pores ranging from 2 to 50 nm, each being tuned to the size of the drug to be loaded. MSNs have a higher potential in loading multiple drugs at a time and release of the drugs to the desired site. The large surface area is amenable to attaching multiple functionalities.151 Ma and team fabricated hollow mesoporous silica nanoparticles to deliver doxorubicin (Dox) and siRNA against the Bcl‐2 protein (a protein that regulates apoptosis) that was decorated with folic acid conjugated polyethyleneimine (PEI‐FA).152 Recently, mesoporous silica NPs have been fabricated with alendronate for bone-specific targeting and loaded with ibuprofen as the model drug. Alendronate was electrostatically bonded to the carboxyl end of MSNs. A high affinity between alendronate and hydroxyapatite was observed. The nanosystem proved to be a successful bone targeting entity with good biocompatibility towards normal human fibroblast cells (BJ-hTERT c).153

The major disadvantage of porous silica nanoparticles is due to the interaction of silanol groups with the surface of the phospholipids of the RBC membranes that can gradually lead to hemolysis.154

Magnetic Nanoparticles

For biomedical applications, magnetic nanoparticles are generally employed in detection, separation, and magnetorelaxometry with some extended advantages, such as hyperthermia, drug-targeting, nuclear magnetic resonance, and imaging.155 In targeted drug delivery approaches, the use of magnetic nanoparticles has exceeded rapidly in recent times. External magnetic field guides the iron oxide nanoparticles to the desired target area. To render the magnetic particles more biocompatible organic polymers are usually functionalized over them.156 Several examples can be cited for magnetic nanoparticles employed in drug delivery. For instance, Hu et al fabricated tamoxifen encapsulated magnetite/poly(l-lactic acid) composite nanoparticles for the in vitro anticancer activity against MCF-7 breast cancer cells. The nanofabricated system had enhanced cytotoxicity compared to free tamoxifen.157 Also, efforts have been made for preparing biocompatible Fe3O4 nanoparticles for human bone osteosarcoma cell (MG63). The doxorubicin-loaded magnetic nanoparticle exhibited better colloidal stability and biocompatibility with sustained release profile at low pH conditions. The self-healing attribute under an external AC magnetic field made the nanocarrier suitable for magnetic hyperthermia.158

However, this area requires intense in vivo studies which will lead to improved biocompatibility and negate the side effects due to the injection of magnetic nanoparticles.159

Liposomes

Liposomes are made up of lipids and cholesterol and mimic lipid membranes. They can encapsulate both hydrophobic and hydrophilic molecules. Among all other nanoformulations, liposomes are a popular choice when it comes to being a drug delivery vehicle. A wide number of therapeutics (peptides, RNA moieties, drugs) can be encapsulated inside the aqueous phase of liposomes to increase the therapeutic advantage. Usually, liposomes consist of non-immunogenic lipids and have slower degradation kinetics.160 However, liposomes can also be decorated with various targeting ligands for facilitating active targeting.161 Liposomes modified with TAT peptide enhanced the permeation of the liposomes facilitating the transfollicular pathway. Treatment of mannose modified liposomes encapsulating p-coumaric acid (ML-CA) in synovial macrophages isolated from AIA rats could result in reduced differentiation of osteoclast by downregulating the master transcription factor, NFATc1 that subsequently resulted in lower production of cytokines like Interleukins and TNF-α.162,163

Although various such advantages can be listed, the major limiting step for liposomes in DDS is the physical and chemical instability. Leakage of drugs out of the bilayer has also been reported in some cases.164 Other than this, heterogeneity in size distribution is also a crucial drawback.

Cell Membrane Coated Nanoparticles

Another promising area, which is rapidly evolving and is a newer niche in biomaterial, is using cell membrane-coated nanoparticles. It acts as a bridge between synthetic nanocarriers and natural membranes.165 Synthetic nanoparticles confer long-term stability and natural membranes cloaked onto them evade the immune system. The presence of membrane proteins has the ability to bind with specific cells. The membrane proteins are isolated from the desired cell (for targeting) and then coated with a synthetic nanomaterial (PLGA, chitosan, MSNs, liposomes, gold nanocages) by different approaches like sonication and extrusion.166 Until now, various cells have been taken into consideration due to the unique properties of each cell type. RBCs evade the immune system, decreased uptake by macrophages,167 WBCs and platelets home to injured and inflammation prone areas,168 cancer cells for homotypic targeting to the desired cancer cell abiding by the principle of hemophilic adhesion between cell adhesion molecules in the cancer cell membrane.169 Although there is no such advancement being made for bone-related disorders with cell membrane-coated NPs, still some efforts are on the way to reach progress in this field. RBC coated PLGA NPs have been rehydrated into macroporous alginate scaffolds and it was observed that there are reduced infiltrating neutrophils in the case of RBC-PLGA-alginate scaffold treated cells isolated from C57BL/6J mice.170

Cell membrane coated nanoparticles hold a promising future in translational science for mimicking the natural cell entities with synthetic nanocarriers,; however, this field is still in its pilot stage and the cost for manufacturing processes is usually higher than the synthetic nanomaterials.165

Along with the wide range of advantages, these nanosystems come with several challenges and major issues like bioaccumulation, cytotoxicity, and high cost-to-benefit ratio which make their translational use a difficult task. In this respect, modern-day clinical research is slowly shifting its gear in search of a more natural delivery system to meet the standards along with the added advantage of desired surface modification.

Exosomes as a Powerful Tool for Drug Delivery to Bone

Biogenesis and Host Cell Interaction

Based on the size and pathway of biogenesis, extracellular vesicles are categorized into three classes; apoptotic bodies (1–4 μm), microvesicles (100–1000 nm) and exosomes (30–150 nm). Apoptotic bodies are generally released from the dying cells by cell membrane blebbing and programmed cell death; microvesicles are shed directly by outward budding of the plasma membrane;44 and exosomes, the smallest of all, are actively released to the extracellular environment after the fusion of late endosomes or multivesicular bodies (MVBs) carrying intraluminal vesicles (ILVs) with the plasma membrane.

Since exosomes are of endocytic origin, their biogenesis starts with the formation of an endosome, which is a membrane-bound component inside a eukaryotic cell. While early endosomes mature into late endosomes, ILVs (intraluminal vesicles) are also generated by the invagination of the endosomal membrane.171 These late endosomes with ILVs inside are known as MVBs (multivesicular bodies). Afterward, MVBs may either fuse with lysosomes and become degraded by hydrolysis or may fuse with the plasma membrane to release its ILVs into extracellular space.172 These vesicles once come out of the cell are known as exosomes (Figure 4A).

Figure 4 Representative image of exosome biogenesis and interaction with the recipient cell. (A) Early endosomes bud into MVBs (multivesicular bodies) as ILVs (intraluminal vesicles) from which exosomes are formed by merging with the plasma membrane. (B) Released exosomes carry a variety of components mainly proteins, lipids, and nucleic acids which can modulate the function once entered into the target cell. (C) Exosomes in the extracellular space can enter into the recipient cells by different mechanisms including (i) receptor binding, (ii) phagocytosis, and (iii) membrane fusion. Adapted with permission from Gulei D, Irimie AI, Cojocneanu-Petric R, Schultze JL, Berindan-Neagoe I. Exosomes-Small Players, Big Sound. Bioconjugate Chemistry. 2018; 29(3):635–648. Copyright © 2018 American Chemical Society.173

The function of exosomes in target cells is determined by the specific cargo they contain. Exosomes contain a variety of molecules of which proteins, lipids, and nucleic acids are the major cargos that regulate their specificity (Figure 4B).173 A large number of exosomal cargos, identified in exosomes, are compiled into a database named ExoCarta174 which has been subsequently integrated into a broader database Vesiclepedia,175,176 and a more recent one named EVpedia.177 According to ExoCarta datasets among the top 20 most identified proteins in exosomes, transmembrane proteins, such as CD81, CD9, CD63, Tspn8, and so on, are abundant.178 These marker proteins are generally involved in cargo and exosome biogenesis. Heat shock proteins like Hsp 70, Hsp 90, Hsp 27, etc., and MVB biogenesis proteins are also involved in the exosome biogenesis process.179 Membrane receptor proteins like transferrin receptors are involved in iron transportation. Lipid contents of exosomes do not necessarily represent parent cells, rather they play an important role in maintaining the exosomal structure, cargo sorting, and their biological activity.173 Inhibition of these lipids in a controlled cell culture environment leads to reduced secretion of exosomes. Sphingolipids are involved in Ca2+ influx and exosomal membrane construction.56 Loading of RNA species into exosomes is a tightly regulated mechanism. Exosomes do not come out with all the mRNAs present in the parent cell. mRNAs present in exosomes are involved in the intracellular transfer of genetic information as these could be translated into proteins in the target cell or could also regulate post-transcriptional modification.180 miRNAs are present in the most abundant form of nucleic acid in exosomes. miRNA-19b-3p released from tubular epithelial cells have been seen to promote macrophage activation in case of kidney injury.181 Recent genomic research suggests that long non-coding RNAs and more stable circular RNAs are also present in exosomes, which regulate the host cellular machinery.182 Due to the enriched forms of RNAs in specific disease conditions, circulating exosomes are being used as disease biomarkers and diagnostic purposes.

Exosomes can send vital signals to the recipient cells by unloading their payload. The fusion of exosomes can transfer cell surface receptors, transcription factors, genetic materials, oncogenes, and infectious particles into host cells thereby performing biological functions.183 Broadly, there are three proposed mechanisms of interaction between exosomes and host cells: (a) specific receptor-ligand binding, (b) membrane fusion of exosomes with the plasma membrane of the host cell, (c) receptor-mediated endocytosis/phagocytosis (Figure 4C). Studies suggest that intracellular communication via exosomes is organ-specific and depends upon the recipient cells. However, this specific phenomenon is still not very clear and further studies should be done on understanding the exosomal interaction process.

Exosomes in Maintaining Bone Homeostasis

The process of bone regeneration is a complex but well-defined physiological and continuous remodeling process of bone formation.184 The entire adult life witnesses, bone as a tissue which possesses a tremendous inherent capacity for regeneration in response to either injury or during skeletal development. The cellular communication and crosstalk between osteoblasts and osteoclasts are responsible for the maintenance of bone homeostasis.185 Exosomes are one such nano-communicator that helps to maintain bone homeostasis through the exchange of vital cellular information. The last decade has evidenced a considerable amount of research results in the field of exosomes and specifically its role in the bone microenvironment. The regulated differentiation of osteoblasts and osteoclasts happens by the exchange of biologically active molecules (proteins, mRNAs, miRNAs) with target cells (Figure 5).67 Matrix vesicles, which are the family of extracellular vesicles have been shown to contain miR-125b, which targets the osteoblast precursors in the bone marrow microenvironment thereby increasing expression of anti-osteoclastogenic factors, and bone mass in mice.186 MC3T3-E1 cell-derived exosomes promoted the differentiation of bone marrow stromal cells (ST2) to osteoblasts. These exosomes significantly influenced the miRNA profiles in the stromal cells, through the activation of the Wnt signaling pathway by inhibiting Axin1 expression and increasing β-catenin expression.187 Recently, in a mCherry transgenic zebrafish model, engulfment of osteoblast-derived EVs by osteoclasts could be seen by live-imaging. This promoted osteoclast differentiation via RANKL signaling, suggesting the EV-mediated intercellular communication to promote osteoclastogenesis.71 Osteoclast-derived exosomal miR-214-3p inhibits osteoblastic bone formation, indicating that the inhibition of miR-214-3p in osteoclasts could be used for treating low bone formation skeletal disorders.15 These research findings give a strong and positive indication of the matrix formation and bone tissue regeneration via mineralizing osteoblast- and stem cell-derived exosomes.

Figure 5 Illustration of regulation of bone homeostasis by the transfer of various biologically active molecules via bone-derived exosomes. Adapted with permission from Xie et al (2017). Copyright © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cell and Molecular Medicine. This article is an open access article under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits use, distribution, and reproduction in any medium, provided the original work is properly cited.67

Researchers have demonstrated that receptor activator of the nuclear factor κB (RANK)/RANK ligand (RANKL) system, which plays an important role in osteoclast formation, greatly contributes to maintaining normal bone physiology.13,188 Osteoblasts express RANKL which, when interacts with RANK on the surface of monocyte precursor cells, promotes osteoclastogenesis by osteoclast maturation.189,190 It is now well known that exosomes play a very crucial role in bone cell-cell communication by stimulating the RANKL-RANK pathway.69 Exosomes from mature osteoclasts are enriched with RANK and it has been observed that their depletion inhibits osteoclast maturation and bone resorption.191 A very interesting feedback mechanism is observed in the regulation of bone homeostasis. RANKL-enriched exosomes secreted from osteoblasts are found to activate the osteoclastic function, whereas osteoclasts derived RANK-enriched exosomes competitively inhibit this process. Osteoclasts are also seen to inhibit osteoblastic differentiation by exosomal delivery of miR-214-3p (Figure 6).192,193

Figure 6 Schematic representation of RANK-RANKL mediated network of interaction between bone cells via the exosomal transfer of miRNA-214-3p. Adapted with permission from Gao et al Copyright © 2018, The Author(s). This is an open access article distributed under the terms of the Creative Commons CC BY license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.192

In a study conducted by Cappariello et al, exosomes isolated from Rankl−/- osteoblasts showed the loss of function to regulate osteoclastic maturation, which proved the importance of Rankl in this process. Also, they demonstrated the internalization and shuttling of anti-resorptive drugs like zoledronate and dasatinib that inhibited osteoclastic activity in vitro and in vivo.194

Exosome as a Potential Drug Delivery Vehicle to Bone Diseases

An ideal drug delivery vehicle demands, targeted drug delivery of smaller doses to the target site in order to reduce toxicity and other unwanted side effects, biocompatibility, non-immunogenicity, and biodegradability.195 To fulfill these demands, a lot of attempts have been done over the past few decades to develop some innovative drug delivery systems, such as liposomes,196–198 micelles,199,200 polymeric and synthetic nanoparticles,201,202 extracellular vesicles,203–205 dendrimers,206,207 and so on. However, due to some of their limitations, the naturally occurring exosomes are gaining much attention in this aspect, thanks to their vital characteristic features. The advantages of using exosomes as drug delivery vehicles are as follows (Figure 7);208 a) direct communication with target cells due to exposed surface adhesion proteins,56,209 b) high cargo carrying capacity210,211 c) cargo protection and enhanced targeting ability,212,213 d) passive targeting (EPR)214 and prolonged blood circulation,215 e) easy surface modification216,217 and cargo loading,65,218 f) ability to cross difficult biological barriers,219,220 g) low immunogenicity and biodegradability.60,221

Figure 7 Various advantages associated with opting for exosomes as a drug delivery system. Reproduced from Peng, H; Ji, W; Zhao, R; Yang, J; Lu, Z; Li, Y; Zhang, X, Exosome: a significant nano-scale drug delivery carrier. Journal of Materials Chemistry B. 2020;8(34):7591–7608 with permission from Royal Society of Chemistry.208

The native form of exosomes, released from cells, shows the intrinsic ability to transfer their internal cargo content to host cells. Song H et al found that the naturally occurring endothelial cell-secreted exosomes (EC-Exos) can deliver osteogenic regulating miRNA-155 specifically to bone tissue, thereby inhibiting osteoclastogenesis in vitro and reducing bone resorption in an ovariectomized mouse model. Sequencing of exosomal miRNA revealed the abundance of miR-125 in endothelial cell-derived exosomes, which showed a higher bone targeting efficiency when tracked using DiI labeling, as compared to other bone cell-derived exosomes (Figure 8). They demonstrated that EC-Exos are promising nanomaterial with excellent bone targeting capacity which can be utilized in the therapeutics for bone resorption disorders.222

Figure 8 Bone targeting efficiency of EC-Exos in vivo. Biophotonic images showing tissue distribution of DiI labeled exosomes in mice injected with A) PBS, B) DiL reagent, C) DiI-labeled BMSCs-Exos, D) DiI-labeled MC3T3-Exos, and E) DiI-labeled EC-Exos. Adapted with permission from Song H, Li X, Zhao Z et al. Reversal of Osteoporotic Activity by Endothelial Cell-Secreted Bone Targeting and Biocompatible Exosomes. Nano Letters. 2019;19(5):3040–3048. Copyright © 2019 American Chemical Society.222

One of the key factors that are expected from a nanoscale delivery platform is the advantage of high payload and co-delivery of therapeutic agents.61 Mostly, the cargo molecules are loaded into exosomes in an ex vivo manner, after being purified from the cell of origin.223 The common approaches are simple incubation, freeze-thaw cycles, sonication, extrusion, and electroporation (Figure 9A). Out of these methods, Haney et al found the highest catalase loading efficiency as well as prolonged and sustained release in case of sonication (Figure 9B).213

Figure 9 (A) Different approaches for drug incorporation into macrophage-derived exosomes in the presence of catalase. The novel exosomal-based catalase formulation (ExoCAT) efficiently crossed the blood-brain barrier showing the antioxidant effect in the neuronal cells of the PD mouse model. (B) The different loading formulations were examined by I) western blot, II) catalytic enzymatic activity, and III) catalase release.213 All data were represented as mean±S.E.M (n=4); data were analysed using t-test; *p < 0.05, **p < 0.05. Adapted with permission from Journal of Controlled Release, Vol /edition number 207, Haney MJ, Klyachko NL, Zhao Y, et al, Exosomes as drug delivery vehicles for Parkinson’s disease therapy, Pages No. 18–30, Copyright (2015), with permission from Elsevier.213

In a study, Wei H and group developed a nano-drug formulation by loading chemotherapeutic drug doxorubicin (Dox) into exosomes (Exo-Dox) derived from mesenchymal stem cells, which showed enhanced cellular uptake and anti-tumor activity in osteosarcoma cell-line MG63 as compared to free doxorubicin (Figure 10A I, II). They also showed a much weaker uptake of Exo-Dox by myocardial H9C2 cells and reduced cytotoxicity (IC50), as compared to free Dox (Figure 10B I, II), thereby reducing the chances of cardiac toxicity, the major side effect induced by free Dox.224

Figure 10 (A) I) Quantification of internalized Dox, Exo-Dox in MG63 cells for 1h, 4h, and 24h in terms of fluorescence intensity, II) cell viability of MG63 cells exposed to different concentrations of Exo-Dox and free Dox. (B) I) Quantification of internalized Dox, Exo-Dox in H9C2 cells for 1h, 4h and 24h in terms of fluorescence intensity, II) cell viability of H9C2 cells exposed to different concentrations of Exo-Dox and free Dox. Bars represent mean±standard deviation (n=3); data were analysed with two-way ANOVA; n.s.: no significant difference, ****P < 0.0001. Adapted with permission from Wei H, Chen J, Wang S et al Nanodrug Consisting Of Doxorubicin And Exosome Derived From Mesenchymal Stem Cells For Osteosarcoma Treatment In Vitro. Int J Nanomedicine. 2019;14:8603–8610. Dove Medical Press (DMP) publishes many of its articles under a Creative Commons Attribution Non-Commercial license (CC-BY-NC). This allows for the non-commercial reuse of the published paper so long as the published paper is fully attributed.224

Furthermore, the easy surface chemical modification makes exosomes most suitable for targeted drug delivery. In a recent study conducted by Luo et al, mesenchymal stem cell-derived exosomes (STExos) were modified by specific recognizable ligands to avoid rapid metabolism and clearance (Figure 11A). Bone marrow mesenchymal stem cell-specific aptamer (5′-ACGACGGTGATATGTCAAGGTCGTATGCACGAGTCAGAGG-3′) was designed to be a stable structure with an aldehyde group modification at the 5ʹ end, which could react with the amino group of exosomal membrane proteins forming a stable Schiff base (Figure 11B). This modified aptamer conjugated to exosomes by incubation (STExo-Apt), facilitated internalization of exosomes in BMSCs with significantly higher distribution in the bone region (91.8% positive) (Figure 11C). While investigating the bone regeneration capability of STExo-Apt in postmenopausal osteoporosis mouse model after bilateral ovariectomy (OVX), once per week after two months of intravenous injection, higher trabecular volume, trabecular number, and trabecular thickness were observed as compared to vehicle or STExo treated mice. An increase in the bone mass was also observed in OVX mice by increased osteogenesis together with increased callus tissue formation and bone mineralization.225 This study provides a novel promising approach for the targeted therapeutic drug treatment of osteoporosis.

Figure 11 (A) Schematic illustration of aptamer functionalized exosomes to promote bone regeneration. (B) Schematic of conjugation procedure between the aptamer and STExos. (C) Representative FMT images of FL signals showing significantly more accumulation of STExo or STExo-Aptamer in the bone as compared to spleen, lungs, liver, and kidney. Reproduced from Luo ZW, Li FXZ, Liu YW et al Aptamer-functionalized exosomes from bone marrow stromal cells target bone to promote bone regeneration. Nanoscale. 2019;11(43):20884–20892 with permission from Royal Society of Chemistry.225Abbreviations: Lu, lungs; Li, liver; Sp, spleens; Ki, kidneys.

The newest enigma of using exosome-based biomimetic nano-platforms in drug delivery has been proved to be successfully loading various drugs to many disease target sites. However, their application in the treatment of bone diseases is rarely studied.226,227 Recently, Yan et al in 2020 constructed a novel biomimetic-exosome (Exo) nanoparticle formulation (Exo/Dex), by electrophoretic loading of dexamethasone sodium phosphate (Dex) with folic acid (FA)-polyethylene glycol (PEG)-cholesterol (Chol) compound (FPC)– surface modification to attain FPC-Exo/Dex for achieving active targeting drug delivery system to treat rheumatoid arthritis (RA) (Figure 12A). Keeping in mind the abundant presence of folic acid receptor (FRβ) on matured macrophages in the inflamed areas of RA, the authors decided to derive exosomes from RAW264.7 macrophages, so as to get maximum exosome internalization. They found that this novel formulation FPC-Exo/Dex inhibited the secretion of pro-inflammatory cytokines TNF-α and IL-1β while significantly up-regulating the level of anti-inflammatory cytokine IL-10. Greater accumulation into joints was observed after intravenous injection of collagen induced arthritis (CIA) mice with FPC-Exo/Dex as compared to other formulations, proving the superior targeting ability as well as longer systemic circulation (Figure 12B). Micro CT analysis showed significantly lower ankle bone erosion with ROI values similar to that of the healthy control. Based on H&E staining and SO staining, the histopathology scores of ankle joints were found to be lowest in FPC-Exo/Dex treated CIA mice (Figure 12C). The reduced hepatotoxicity and all the above advantages such as better therapeutic efficacy, better stability, longer persistence suggested the usefulness of this drug delivery system for glucocorticoids to treat RA.228

Figure 12 (A) Schematic illustration showing the preparation of novel biomimetic nanoparticles FPC-Exo/Dex, for enhanced targeting based on folic acid (FA) receptor–ligand interaction and better treatment of RA. (B) I) Real-time fluorescence imaging of CIA mice after i.v. injection with free DiD, Lip/Dex, Exo/Dex, and FPC-Exo/Dex, II) ex-vivo imaging of plasma and organs after 24 hr of i.v. injection, III) semi-quantitative fluorescence intensity in joints and plasma. Data were expressed as mean±SD (n=3) and analysed using two-way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs mice treated with DID/FPC-Exo/Dex, #p < 0.05, ##p < 0.01, ###p < 0.001 are mice treated with DID/Lip/Dex compared to DID/Exo/Dex. (C) Histopathology analysis of ankle joints I) representative H&E staining, II) representative SO staining, III) histopathology score of SO staining. Data were expressed as mean±SD (n=3) and analysed using one-way ANOVA with Dunnett’s multiple comparisons test; **p < 0.01, ***p < 0.001 vs mice treated with saline; #p < 0.05 is mice treated with Lip/Dex compared to with Exo/dex. Adapted with permission from Yan et al (2020). This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. (http://creativecommons.org/licenses/by/4.0/).228

Translational Potential of Exosomes in the Field of Bone Therapeutics: Recent Patents

Scientists are always trying to press forward to find out new possibilities of exosome engineering for therapeutic applications in a smarter way. Pertaining to this, some novel techniques for exosome-based bone disease diagnostics and treatment methods have been patented. The utilization of exosomes in the area of bone therapeutics holds strong translational potential as is evident from the patents available on exosomes. Based on some studies from Google patents and Espacenet (data obtained in 22/11/2020), a compiled list of certain recent patents that have been filed or granted on the use of exosomes in diagnostic and therapeutic applications towards bone diseases is provided in this section (Table 1). It encloses exosome-based delivery of the therapeutic drug, cargos like miRNAs to treat painful bone diseases, such as osteosarcoma, rheumatoid arthritis, and osteoporosis. The utilization of exosomes for early disease diagnosis and detection based on measuring the level of some marker proteins and mRNAs is also included in the list. In most of the cases, the exosomes are isolated from either osteoblast lineage cells, or stem cells (bone marrow, umbilical cord, and adipose-derived), or monocytes and macrophages to avoid any chances of immunogenicity. From the given list of filed patents, it can be observed that apart from the exosomes loaded with therapeutic drugs or cargoes, naturally occurring exosomes with their inherent cargo are also important in the therapeutic applications. This proves the unique advantage of opting for exosomes as drug delivery systems.

Table 1 Patents Related to the Diagnostic and Therapeutic Application of Exosomes Towards Bone Disorders

Current Limitations of Using Exosomes as Drug Delivery Vehicles

Despite the considerable advancements in the exosome research area, its therapeutic application is still in a premature phase due to some key issues to be addressed in future studies. The lack of proper isolation methods for getting a better yield of pure exosomes stands as a major obstacle in their therapeutic usage.229–231 The relatively low release of exosomes from some mammalian cells is one of the primary reasons behind this.44,232 The widely used gold standard ultracentrifugation method usually fails to provide a pure form of exosomal yield.233,234 To address this issue, attempts have been made to develop advanced isolation techniques, such as density gradient centrifugation,235 ultrafiltration,165 immunoisolation based on antigen–antibody interaction,236 precipitation method using commercially available kits,237 size-exclusion chromatography,238 and the newest chip-based microfluidics technology.239,240 However, none of these techniques qualify to be the most superior of all due to some or the other limitations like high equipment cost, the requirement of skilled manpower, lower yield, impure exosome, filter plugging, high reagent cost, laboratory standardization, etc.229,230,241 Furthermore, exosome loss during drug encapsulation method and less net output of drug-loaded naturally occurring exosomes make the use of native exosomes very tricky.61,242 Again, out of the drug-loaded exosomes, not all bind to the targeted site, and some are cleared by excretion.61 Considering the presence of MHC class I and II molecules on the surface,243,244 exosomes may trigger immunogenic reactions, resulting in rapid clearance.245,246

Utilization of exosomes secreted from immune cells like dendritic cells and macrophages should be more emphasized for addressing this issue.247 Optimization of methods to cargo encapsulation and surface engineering without corrupting the intrinsic properties of exosomes is also very important for the best utility of this delivery vehicle. Many researchers are optimistically focused on developing appropriate methods to modify exosomes and proper loading of drugs/genes to get the best out of it. Modern clinical research is coming up with smartly engineered exosomes with innovative methods as an attempt to combat these issues.66,218,248 With a proper understanding of the biology and continued efforts towards resolving the limitations, we envision that an exosome-based drug delivery system will lead to the emergence of a novel therapeutic strategy.

Perspective on Innovative Systems Utilizing Exosomes for Drug Delivery

To improve the drug loading efficiency and half-life in blood as well as for achieving a decreased immunogenic profile, there have been several attempts. Some examples of the use of innovative methods of exosome modifications for improved and targeted drug delivery have been enlisted in Table 2. One such example is the recently trending technique of synthesizing liposome-exosome hybrid delivery systems as the smarter approach.249 This can be achieved by some of the very commonly used methods such as freeze-thaw, incubation, and sonication (Figure 13A). One of the most widely applied methodologies is the freeze-thaw method that can retain the targeting moieties of exosomal membrane and cargo loading potential of exosomes, to release both the hydrophobic and hydrophilic drug agents.250 Sato and coworkers worked on exosomes isolated from RAW264.7 cells and fused it with liposomes constituting 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC; zwitterionic), 1,2-dioleoyl-sn-glycero3-phospho-l-serine; (DOPS, anionic), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP; cationic) or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N–[methoxy(polyethylene glycol)-2000] (PEG-DSPE)251 (Figure 13B). Basically, a proteoliposome (liposome carrying connexin) was prepared and was made to fuse with exosomal vesicle by a simple freeze-thaw technique. The hybrid system was effectively uptaken by HeLa cells and the fate of the exosomes was decided by the lipids or PEG-lipids anchored onto them after the fusion process.

Table 2 Innovative Methods of Exosome Modifications for Improved and Targeted Drug Delivery

Figure 13 Synthesis of liposome-exosome hybrid nanosystem. (A) Schematic illustration of the three mainly used methods: freeze-thaw, incubation, and sonication for the synthesis. Adapted with permission from Elkhoury et al (2020). Copyright © 2020 by the authors. License MDPI, Basel, Switzerland. This is an open access distributed under the terms of the Creative Commons (CC BY) license (http://creativecommons.org/licenses/by/4.0/).249 (B) Fusion of RAW 264.7 cell-derived exosomes with liposomes by using freeze-thaw technique. Adapted with permission from Sato et al Copyright © 2016, The Author(s). This is an open access article distributed under the terms of the Creative Commons CC BY license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.251

Another approach is the surface chemical modification of the isolated exosomes for improved targeting efficiency. Tian and team in 2018 studied modified exosomes for specifically targeting cerebral ischemia. The cyclo(Arg-Gly-Asp-D-Tyr-Lys) peptide [c(RGDyK)], which has the highest affinity for integrin αvβ3 on active cerebral vascular endothelial cells, was conjugated to mesenchymal stromal cell (MSCs)-derived exosomes by a facile technique called bio-orthogonal chemistry. The cellular uptake pattern of MSCs-derived exosomes was thoroughly studied with HeLa and U87 glioblastoma cells, as shown in Figure 14. Similarly, the surface-modified exosomes were found to enter into microglia, astrocytes, and the region of ischemic junction. To augment the activity of surface-modified exosomes, curcumin was loaded onto it which decreased the inflammation in the lesion area without compromising toxicity in mice.216

Figure 14 Representative results showing cellular tropism of cRGD-Exosomes (surface-modified exosomes) in vitro. (A) Fluorescence microscopic images of native and modified exosomes, green color indicates DiI-stained lipid membranes (of only native exosomes) and red is Cy5.5 that indicates the peptide-functionalized cy5.5 labeled exosomes, scale bar: 5 µm. The arrows indicate the colocalization between both the cy5.5 and DiI. (B) Western blotting of integrin subunits αv, β3. β5, α5, β1, αIIb from two cell lines U87 and HeLa cells that represent different integrin expression patterns, the results indicate that HeLa does not express αvβ3 while U87 cells express high levels of αvβ3. (C) Flow cytometry results indicate that the uptake level as measured by the fluorescence intensity of cRGD-Exo by αvβ3-positive U87 cells is about 3-times higher than that of exosomes. (D) The relative percentage of Cy5.5-positive HeLa or U87 cells compared to Cy5.5-Exo group, c(RGDyK) peptide acts dose-dependently. Data were represented as mean±SEM (n=5); n.s., not significant; ***P < 0.001 by Student’s t-test. (E) Fluorescence images of Cy5.5-labeled exosomes or Scr-Exo (exosomes with functionalized scrambled peptide) and cRGD-Exo post 30 minutes incubation with U87-GFP and HeLa cells. Green indicates U87-GFP cells, magenta color represents Cy5.5-labeled exosomes, Scr-Exo, or cRGD-Exo and blue color indicates nuclei stained with Hoechst. cRGD-Exo entered more efficiently into U87-GFP cells as compared rest groups. The lower panel figure shows magnified images; arrows indicate internalized exosomes; scale bar, 30 µm. Adapted with permission from Biomaterials, Vol /edition number 150, Tian T, Zhang H-X, He C-P, et al, Surface functionalized exosomes as targeted drug delivery vehicles for cerebral ischemia therapy, Pages No. 137–149, Copyright (2018), with permission from Elsevier.216

In a similar study, Zhuang et al fabricated a synergistic system, i.e. SPION-conjugated exosomes with TNF-α anchored in the membrane. Recombinant plasmids carrying the sequence of cell-penetrating peptide (CPP) and TNF- α were synthesized and were established stably in MSC cell lines. The isolated exosomes, decorated with TNF-α were then conjugated with SPIONs to produce CTNFα-exosome-SPIONs and then the anticancer activity was examined to validate the TNF-α mediated apoptosis. It was found that in the presence of an external magnetic field (MF) applied to the tumor site, CTNF-α-exosome-SPION/MF exhibited higher cytotoxicity to tumor cells with less harm caused to normal cells. There was a marked increase in apoptotic cell populations, evident by the large G1 population (71.48% by the combination of CTNF-α-exosome-SPIONs and MF) in the case of human melanoma cells A375 after being exposed for 24 h along with an increased expression of cleaved-caspases 2, 3, and 8. The in vivo biodistribution profile was gauged in A375 tumor-bearing mice by a non-invasive NIRF (near-infrared fluorescence) technique. In comparison to the absence of MF, increased Cy5.5 signal of the CTNF-α-exosome-SPION was observed with strong MF which caused a significant reduction of tumor volume.252

Considering the relatively low release of exosomes from mammalian cells affecting their yield and purity, and the strenuous purification methods, researchers are now coming up with the idea of developing bioinspired exosome-mimetics. They display similar characteristics to exosomes in terms of membrane proteins, and at the same time, they offer advantages of high scalability and low production cost.253–255 Recently, Pisano et al developed Immune Derived Exosome Mimetics (IDEM) as a promising approach for the effective treatment of ovarian cancer. They synthesized IDEM by subjecting ThP-1 monocytic cells to serial extrusion through filters with decreasing pore size (10 µm and 8 µm), followed by purifications using size-exclusion chromatography. Exosomes were isolated from the same number of ThP-1 cells using ultrafiltration followed by kit-based precipitation method. As per nanoparticle tracking analysis (NTA), IDEMe were found to be 2.8 times more concentrated than exosomes. Both were found to be positive for the exosomal marker proteins CD63 and CD81 with a rounded morphology (Figure 15A). When loaded with the drug doxorubicin (DOXO), IDEMs showed 28% encapsulation efficiency, whereas exosomes retained 17% of the drug. IDEM-DOXO showed reduced cell viability of ovarian cancer cells (SKOV-3) with increasing concentration of DOXO with 85% cell death at 1µg/mL concentration after 24h, whereas 90% cell death was observed at 96h with EXO-DOXO treatment. A similar trend was observed for the level of apoptosis marker Caspase 3. In a 3D spheroid system that better mimics the in vivo environment, 5 µg/mL of both IDEM-DOXO and EXO-DOXO showed a loss of integrity with 20% reduced proliferation after 24 h due to increased necrosis (Figure 15B). This biomimetic system showed the potential to revolutionize the treatment of cancers due to stable drug delivery with reduced immunogenicity.256

Figure 15 (A) Characterization of EXO and IDEM. I) Size (nm) and concentration (particles/mL) as per NTA, II) the number of CD63 positive EXO and IDEM obtained from ELISA (*P < 0.05), III) positive signal in flow cytometry for APC-CD81 antibody stained EXO and IDEM, IV) particle morphology revealed from scanning electron microscopy, V) presence of tsg-101 marker obtained from Western blotting. (B) I) Effect on 3D spheroid ovarian cancer model at 24h, 48h, 72h and 96h of treatment with 5µg/mL of EXO and IDEM. Red arrows showing loss of integrity, change in color due to necrosis, II) percentage of cell viability showing greater effectiveness of IDEM-DOXO than only DOXO at 24 h and 96h (*P < 0.05). Data indicating EXO-DOXO to be most effective at all-time points (***P < 0.001 at 72h and 96h). Data were analysed by a two-tailed Student’s t-test and represented as mean±standard deviation; *P < 0.05, **P < 0.01, ***P < 0.001. Adapted with permission from Pisano S, Pierini, I, Gu Jet al Immune (Cell) Derived Exosome Mimetics (IDEM) as a Treatment for Ovarian Cancer. Front Cell Dev Biol. 2020;8: 553576.Copyright © 2020 Pisano, Perini, Gu, Gazze, Francis, Gonzalez, Conlan and Corradetti. This is an open access distributed under the terms of the Creative Commons Attribution License (CC BY).256

A similar study was conducted by Kalimuthu et al in 2018, demonstrating an alternative to paclitaxel (PTX) drug delivery by synthetically prepared exosome-mimetics (EMs). To isolate EMs, human mesenchymal stem cells (MSCs) were treated with PTX and serially extruded using polycarbonate membrane filters in a mini-extruder similar to the previously discussed example. This was followed by filtration and ultracentrifugation to get PTX-MSC-EMs. These synthesized EMs were successfully internalized into triple-negative breast cancer cell-line MDA-MB-231 where they showed cytotoxicity with increasing concentration of PTX from 25 to 50µg/mL. When injected intratumorally into a xenograft MDA-MB-231 cancer mouse model, PTX-MSC-EMs inhibited tumor growth and a significant reduction of tumor weight was observed after 5 days of treatment. Their study revealed the therapeutic efficiency of the innovative exosome mimetic system, against breast cancer cells, which can be used as novel drug delivery vehicles for cancer treatment.257

Exosomes exert valuable biological properties that endow advantages for a suitable delivery vehicle. Its intrinsic cargo loading capacity, along with its high targeting potential due to the presence of membrane proteins, makes it an advanced nanosystem.258 However, due to issues like low plasma half-life and rapid clearance, it has been on demand to make innovative and hybrid exosomes to bring overall stability in the nanosystem. Although in the pilot stage, this area is proving to be one of the hot topics in exosome research. Bone defects are hard to cure and the regimens currently available for skeletal-related events and other bone diseases like osteoarthritis and osteosarcoma are ineffective.30 To minimize the possible off-target effects that are mainly seen with signaling pathway crosstalk in poor drug delivery systems, it is essential to develop nanosystems that will enhance the drug deposition in the bone niche with a better biodistribution profile.259 For these valid reasons, effective and advanced nanosystems are of keen interest in the treatment of bone diseases. Synthetic nanosystems that are highly developing still lack some features and often fail in the mice model being recognized as foreign material.258 It will not be futile to state the fact that innovative ideas as discussed in this section will be interesting to make the naturally occurring exosomes a stable nanosystem, which will be more in demand in the long run.

Conclusion

In line with the title of the review, a lot of information on exosomes has been already deciphered while a long way is still to go before we can evidence bench to bedside results. Based on the results that have been obtained so far, exosomes are definitely promising candidates for therapeutics and at the same time, the scalability and a non-expensive user-friendly technique for isolation is the need of the hour. Other features like biocompatibility and efficient cellular internalization together with high therapeutic loading capacity are inherently present. Another advantage of using exosome mediation is the cell-free approach that it offers which eliminates a lot of drawbacks. The lipid bilayer also looks promising in terms of chemistries that can be taken up to further functionalize this system towards effective targeting. However, looking at the number of patents that have been generated so far, it appears that the field is still open with ample scopes that must be explored to fully utilize the exosome-based systems for effectively acting as drug delivery vehicles to the bone. The review of the literature and presentation of our perspective is an effort towards that direction.

Acknowledgments

Mamoni Dash acknowledges the Ramalingaswami Fellowship 2016-17 (D.O.NO.BT/HRD/35/02/2006), Department of Biotechnology, Government of India, Institute of Life Sciences (ILS), Sasmita Samal acknowledges the Council of Scientific and Industrial Research (CSIR), New Delhi, Government of India, Institute of Life Sciences, Pratigyan Dash acknowledges UGC, Government of India. Dr Sandra Van Vlierberghe, Lana Van Damme, are highly acknowledged for valuable discussion on the topic.

Disclosure

The authors report no conflicts of interest in this work.

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