Research Advances and Application Progress on miRNAs in Exosomes Derived From M2 Macrophage for Tissue Injury Repairing

Introduction

Tissue injury, which may arise from various causes such as infections, autoimmune disorders, mechanical trauma, or toxic agents, triggers a complex and dynamic reparative process. In the acute phase following an injury to tissue or organs, there is a rapid recruitment of neutrophils, monocytes (precursors to macrophages), and other immune cells to the affected area. These immune cells play a dual role; they are involved in the clearance of necrotic tissue and the elimination of pathogens, while also actively participating in the subsequent repair of tissue.¹ Monocytes and macrophages are pivotal components of the human immune system, significantly influencing tissue repair and regeneration.^2,3 They secrete matrix metalloproteinases (MMPs) that degrade the basement membrane and produce chemokines, which facilitate the migration of inflammation-associated cells to the injury site. Additionally, these cells engage in the phagocytosis of cellular debris and pathogenic microorganisms, aiding in the removal of damaged cells.⁴ The recruitment and activation of macrophages at the injury site are essential; any limitation in this process can lead to an extended inflammatory phase, thereby delaying the repair of injuries. Moreover, the phenotypic transformation of macrophages is crucial for regulating cell proliferation and regeneration, and any disruption in this transformation may result in incomplete tissue regeneration.^5,6 Consequently, the orchestrated recruitment and activation of macrophages, along with their functional transformations at various stages, are imperative for the effective repair of tissue injury.⁷

Macrophages play a crucial role in the regulation of tissue repair following injury by secreting signaling molecules that facilitate communication with adjacent cells. Among the various macrophage subtypes, M2-type macrophages are particularly proficient in enhancing tissue repair processes. Their communication with surrounding cells is usually mediated through exosome-dependent mechanisms, which are significantly influenced by microRNAs (miRNAs) contained within these exosomes.^8–10 These miRNAs are derived from diverse genomic loci and undergo post-transcriptional processing and modification within the cell, ultimately maturing into single-stranded RNA molecules approximately 22 nucleotides in length. The mature miRNAs are associated with RNA-induced silencing complexes (RISCs), which act as regulatory entities by modulating the degradation or translation of target message RNAs (mRNAs).¹¹ The miRNAs derived from M2-type macrophage exosomes (M2-Exos) are closely linked to tissue injury and are involved in various biological processes, including cell proliferation, granulation tissue formation, angiogenesis, and inflammation regulation. Through the regulation of these activities, M2-Exos contributes to the therapeutic management of respiratory, neurological, circulatory, systemic, and chronic inflammatory diseases.^12–15 Therefore, M2-Exos and their associated miRNAs provide a robust theoretical framework for the synergistic application of exosome nanotechnology and macrophage immunotherapy.^13,16,17

Considering the crucial regulatory function of miRNAs in M2-Exos concerning tissue repair mechanisms and their prospective clinical implications, we have undertaken an extensive examination and literature review focused on miRNAs in M2-Exos. This review aims to clarify existing challenges while providing insights into the utilization of intracellular miRNAs within M2-Exos, as well as identifying potential directions for future research in this field (Figure 1).

Figure 1 A scheme of miRNAs in exosomes derived from M2 macrophage for tissue injury repairing.

Biogenesis and Function of M2-Exos

Exosomes have gained significant attention in the field of research as crucial mediators of communication between M2 macrophages and their surrounding microenvironment. These nanoscale extracellular vesicles are derived from the endomembrane system. Their formation involves a process characterized by the double invagination of the plasma membrane, which leads to the development of intracellular multivesicular bodies (MVBs). Within these MVBs are intraluminal vesicles (ILVs), which are subsequently processed into exosomes with diameters ranging from 40 to 160 nm. The fate of MVBs can vary; they may either merge with lysosomes or autophagosomes for degradation, or they may fuse with the plasma membrane, facilitating the release of exosomes into the extracellular space via a process known as exocytosis (Figure 2).¹⁸ The process of exosome release plays a crucial role in cellular communication and the transmission of biological signals. Numerous regulatory factors involved in the synthesis and secretion of exosomes have been identified, such as the Endosomal Sorting Complex Required for Transport (ESCRT), Rab proteins (GTPases), Sytenin1, and tumor susceptibility genes (TSG101). However, the precise functions of these factors have yet to be elucidated.¹⁹

Figure 2 A visual illustration depicting the process of exosome formation:1) The first invagination of the plasma membrane forms a cup-shaped structure that includes cell surface proteins and soluble proteins associated with the extracellular environment through the formation of early sorting endosomes (ESEs).20,21 2) Subsequently, a double-layered MVB is formed through the inward invagination of the plasma membrane of the ESE. The ESE matures into a late-sorting endosome (LSE), which also includes intraluminal vesicles (ILVs; future exosomes) that are about 100 nm in diameter. 3) Finally, the MVB may fuse with lysosomes or autophagosomes and undergo degradation, or it may fuse with the plasma membrane to release the stored ILVs as exosomes.19,20,22

The established mechanisms by which exosomes interact with their target cells encompass three primary processes: membrane fusion, direct receptor interaction, and internalization.²³ Variations in these mechanisms and the pathways involved in the uptake of exosomes by recipient cells, along with the metabolic state of these cells and the chemotactic specificity of exosomes for particular cell types, may result in alterations to the composition of exosomes. Such variations complicate the investigation of exosomal functions in intercellular communication.^22,24,25 Current research predominantly emphasizes the induction of phenotypic changes and the biological alterations in recipient cells. Most experimental investigations of exosomes are conducted using exosomes derived from in vitro cultures; however, it remains uncertain whether these in vitro-generated exosomes maintain the same properties as those produced in vivo when utilized in functional studies.^26–28 Additionally, discrepancies in the conditions under which exosomes are extracted may lead to differential gene expression within the exosomes, potentially resulting in variations in their regulatory functions.^29,30 Therefore, further elucidation of the pathways through which exosomes exert their effects on recipient cells is warranted. It is also essential to explore whether the regulatory functions of exosomes cultured in vitro are physiologically comparable to those derived from in vivo sources.

Exosomal miRNAs Biology of Macrophages

The synthesis and maturation of miRNAs is a complex, multistep process, as illustrated in Figure 3. ^31,32 Following their synthesis and maturation, miRNAs perform their regulatory functions through interactions with mRNA. A single miRNA can modulate the expression of multiple target mRNAs, while a single mRNA may be subject to regulation by various miRNAs.³³ miRNAs can engage in several other mechanisms of action: 1) Inhibition of translation: miRNAs primarily exert their effects post-transcriptionally by base pairing with the 3′ untranslated region (3′ UTR) of mRNAs. miRNAs can impede translation at the initiation phase by targeting the cap recognition process or by obstructing the assembly of the ribosomal 80S complex, as well as at the post-initiation stage.³⁴ In a non-classical mechanism, after binding to mRNAs, miRNAs may also attach to the 5′ UTR of mRNAs to inhibit translation.³⁵ 2) Under specific conditions, miRNAs may enhance translation rather than inhibit it. Research by Vasudevan et al indicates that miRNAs suppress translation in proliferating cells but promote it in quiescent cells that are arrested at the G0/G1 checkpoint.³⁶ Furthermore, Orom et al demonstrated that miR-10a interacts with the 5′ UTRs of various mRNAs encoding ribosomal proteins, thereby activating their translation.^34,36,37 3) Deadenylation and decay of mRNAs: miRNAs can destabilize target mRNAs through mechanisms such as deadenylation, subsequent decapping, and 5′-3′ nucleic acid exonuclease activity.³⁴ Notably, some mature miRNAs have been identified in the nucleus, where they can induce the degradation of nuclear mRNAs.³⁸ 4) Activation of transcription: In addition to repressing the expression of target genes, certain miRNAs can activate the transcription of target genes following their binding to mRNAs.²⁹

Figure 3 The formation of miRNA and its main mechanism of action: First, in the nucleus, RNA polymerase II catalyzes the formation of double-stranded RNAs with a stem-loop structure called primary miRNAs (pri-miRNAs). Subsequently, the pri-miRNAs are cleaved into 60–70 nucleotide precursor miRNAs (pre-miRNAs) by RNA polymerase III (RNase III), Drosha and its cofactor, DGCR8, are then, transferred to the cytoplasm via the transport protein Exportin 5. In the cytoplasm, the pre-miRNAs are sheared and processed by another RNase III, known as Dicer to generate a segment of complementary miRNA duplex. Finally, with the help of the Argonaute protein, the miRNA duplex removes the non-functional coding single strand and dissociates into a functionally active “leading strand”, which forms a miRNA-induced silencing complex (miRISC) with RISC protein. The RISC-coated miRNA is complementarily paired with the 3′-UTR of the target mRNA through its “seed region” (2–8 nucleotides at the 5′ end). This binding leads to the inhibition of gene expression or promotion of mRNA degradation, resulting in the regulation of the expression of target genes. 31,39,40

While the specific mechanisms by which miRNAs regulate gene transcription or translation through classical or non-classical pathways are not yet fully elucidated, these processes highlight the intricate nature and importance of miRNAs in the functional regulation of diverse biological processes.

Isolation Methods of M2-Exos

Exosomes play a crucial role in intercellular communication, and their regulatory functions are of considerable importance, leading to an annual increase in research focused on these entities. However, current extraction methods do not consistently yield the desired levels of purity and concentration. To address the challenges of reducing extraction costs and enhancing the purity of isolated exosomes to fulfill research requirements, there is a growing diversification and targeting of extraction techniques. In this context, we provide a comprehensive summary of the most recent methodologies for the extraction of macrophage-derived exosomes, as detailed in Table 1.

Table 1 Methods Used to Extract Macrophage-Derived Exosomes

Ultracentrifugation is the predominant technique employed for the extraction of macrophage-derived exosomes and is regarded as the “gold standard” for this purpose.^52,53 The methodology is predicated on the differential size and density of exosomes compared to other cellular components, rendering it particularly effective for isolating exosomes from samples characterized by significant variations in size and density.⁴¹ The extraction process encompasses several sequential steps. Initially, the cell supernatant undergoes centrifugation at low speeds (500–10,000 g) at 4 °C, followed by filtration to eliminate debris from the samples. Subsequently, exosomes present in the culture medium or tissue fluid are precipitated through ultracentrifugation. The morphological features of the isolated exosomes are then evaluated using transmission electron microscopy (TEM), while their sizes are quantified via nanoparticle tracking analysis (NTA).^41,48 This method is recognized for its ability to yield exosomes of high purity and activity. However, it is important to note that the process is labor-intensive and necessitates costly equipment. Furthermore, various factors, including centrifugation duration and force, can significantly influence the extraction efficiency and purity of the target exosomes.⁵⁴

Given the labor-intensive and equipment-dependent characteristics of ultracentrifugation, researchers have devised alternative methods for exosome extraction that can be executed using standard centrifuges or even in the absence of centrifugation. For instance, conventional centrifugation techniques such as polymer precipitation and ultrafiltration can effectively isolate and purify exosomes.⁵⁵ Additionally, methods such as Tangential Flow Filtration (TFF), Size Exclusion Chromatography (SEC), and Anion Exchange Chromatography (AEC) do not necessitate the use of a centrifuge.^41,56,57 However, the purity and recovery rates of exosomes obtained through these methods tend to be suboptimal, which may hinder subsequent functional analyses.

In response to these challenges, recent technological advancements have led to the development of numerous commercial kits designed to streamline the exosome extraction process. These kits facilitate the rapid acquisition of significant quantities of exosomes while preserving their structural integrity, thereby offering considerable potential for various applications. Nonetheless, these kits frequently do not achieve optimal separation efficiency, and their high cost, coupled with the inadequate purity of the extracted exosomes, presents notable limitations. To address these shortcomings, employing a combination of different exosome extraction techniques or integrating multiple methods tailored to specific research objectives may enhance both the purity and yield of exosomes.⁴¹

M2-Exos miRNAs in Diagnosis and Prognosis of Tissue Injury

Exosomes are present in a variety of tissue and body fluids, including blood, urine, and bile,⁵⁸ and their surface proteins, combined with their inherent stability, render them promising candidates for diagnostic and prognostic biomarkers in numerous injury-related diseases.^59–61 Concurrently, macrophages, which play pivotal roles in various tissue injury diseases and exhibit complex regulatory functions, release exosomes that may be more valuable for diagnostic and preventive applications than exosomes derived from other sources (Figure 4). The miRNA within exosomes derived from macrophages is influenced by their phenotype, with exosomes from M2 macrophages potentially exhibiting higher levels of specific miRNAs compared to those from M1 macrophages.⁶² Furthermore, it has been proposed that the detection of exosomes can facilitate longitudinal sampling to monitor disease progression and may be incorporated into minimally invasive liquid biopsy methodologies in clinical settings.^18,63 This is particularly relevant as macrophage-derived exosomes represent a substantial fraction of blood-borne exosomes, and analyzing their content and dynamic release provides a novel strategy for tracking disease progression.⁶⁴

Figure 4 M2-Exos for disease prevention and diagnosis.

Current detection methodologies primarily focus on capturing the complex extracellular and intracellular “molecular cargo” and conducting multiparameter diagnostic assays. Historically, exosomes have been utilized mainly as diagnostic tools for diseases associated with cardiovascular and central nervous system (CNS) injuries. However, recent investigations have broadened the focus to encompass injuries affecting other tissues and organs, such as the liver, kidneys, and lungs.^48,65,66 In a review, Yang et al highlighted the role of miRNAs in exosomes from various cell types, including hepatic macrophages, where these miRNAs significantly contribute to the inhibition of fibrosis, inflammatory responses, and lipogenesis, thereby facilitating the diagnosis of non-alcoholic fatty liver disease (NAFLD). They further posited that miRNAs in macrophage-derived exosomes hold considerable promise for the diagnosis and prognosis of NAFLD.⁶⁷ Nevertheless, it is important to note that macrophage-derived exosomes may differ based on the characteristics of their parent cells. Therefore, identifying the specific exosomes secreted by distinct macrophage phenotypes may enhance efforts in the prevention, diagnosis, and treatment of related diseases.⁶⁴

M2-Exosomal miRNAs for Tissue Injury Repairing

Numerous studies on M2-Exos miRNAs have demonstrated their ability to either promote or hinder tissue injury repair in both cellular and animal models. In this section, we present a detailed overview of the known functions of miRNAs in M2-Exos and the mechanisms that regulate these functions in relation to tissue repair, systematically arranged based on the anatomical structures of the human body (Figure 5).

Figure 5 The regulatory role of M2-Exos miRNAs in tissue injury repair: (A) Repairing for respiratory system injury. (B) Repairing for central nervous system injury. (C) Repairing for circulatory system injury. (D) Repairing for urinary system injury. (E) Repairing for skin injury. (F) Repairing for locomotor system injury.

Repairing for Respiratory System Injury

Pulmonary Fibrosis Therapy

Pulmonary fibrosis (PF) is a permanent lung condition marked by the growth of fibroblasts, significant accumulation of extracellular matrix (ECM), inflammation, and damage to lung tissue structure. Research by Yao et al indicated that miRNA-328 from M2-Exos can stimulate the growth of lung interstitial fibroblasts and enhance the expression of proliferation and differentiation markers, such as collagen, by inhibiting FAM13A. Increased collagen levels lead to the release of inflammatory factors from macrophages, worsening fibrosis after tissue injury and accelerating the progression of PF.⁶⁸ However, Guiot et al reported that macrophage-derived exosomes can alleviate the progression of fibrosis in alveolar epithelial cells and lung fibroblasts by delivering miR-142-3p, which inhibits transforming growth factor beta receptor 1 (TGFβ-R1).^69,70 Conversely, Guiot et al found that exosomes derived from macrophages can slow down fibrosis in alveolar epithelial cells and lung fibroblasts by delivering miR-142-3p, which blocks the transforming growth factor beta receptor 1 (TGFβ-R1).⁶⁹ Considering the opposing roles of miRNAs within M2-Exos, we propose that miRNA-328, which promotes PF progression, may serve as a future diagnostic biomarker for PF, whereas miR-142-3p, which inhibits PF progression, could be used as a therapeutic miRNA in clinical settings.

Asthma Therapy

Many studies have identified the therapeutic significance of miRNAs of M2-Exos in the progression of asthma. Li et al discovered that miR-370 from M2-Exos can reduce the growth of mouse airway smooth muscle cells (ASMCs) and airway remodeling by decreasing the levels of fibroblast growth factor 1 (FGF1) and the MAPK/STAT1 signaling pathway, thereby mitigating the progression of asthma.⁷¹ In a similar study, Tang et al demonstrated that a powder made from Chinese medicine, specifically scorpion and centipede, encourages M2 polarization and boosts the release of M2-Exos. This process helps to reduce inflammation in asthmatic mice by delivering miR-30b-5p, which lowers the levels of NLRP3, caspase-1, and IL-1β, as well as mitochondrial swelling, contributing to asthma treatment.⁷² Consequently, miR-370 and miR-30b-5p found in M2-Exos can help reduce asthma symptoms by preventing fibrosis in airway epithelial cells and inhibiting pyroptosis in these cells, respectively, which offered a more effective and innovative approach to treating the progression of asthma.

Repairing for Central Nervous System Injury

Cerebral Ischemia-Reperfusion Therapy

Following cerebral ischemia-reperfusion injury, intracranial macrophages serve as the initial defense against tissue injury. As the disease progresses, intracerebral macrophages change from the M2 type to the M1 type, which worsens the injury to brain tissue. Luckily, in the central nervous system, microglia polarize to the M2 phenotype and exert neuroprotective effects during cerebral ischemic injury.⁷³ Furthermore, M2 microglia not only promote angiogenesis but also attenuate brain damage.⁷⁴ Song et al showed that miR-124 in M2 microglia-derived exosomes would enter the damaged neurons and downregulate the expression of ubiquitin-specific protease 14 (USP14), thereby attenuating ischemic brain injury and promoting the survival of neurons.⁷⁵ Additionally, Zhang et al found that microglial exosomes inhibit the expression of Notch by transporting miR-137, which reduces neuronal apoptosis and attenuates ischemia-reperfusion brain injury.⁷⁶ Thus, the protective effect of M2-Exos on neurons and the reduction of brain damage, mediated by miRNAs, offer new therapeutic solutions for cerebral ischemia-reperfusion injury.

Multiple Sclerosis Therapy

Multiple sclerosis (MS) is a central nervous system (CNS) demyelinating disease of unknown origin, marked by damage to myelin, difficulties in repair, loss of neurons, and worsening neurological symptoms. The lesion areas contain numerous oligodendrocyte precursor cells (OPCs) as well as activated resident microglia and infiltrating macrophages. Exosomes derived from activated microglia carry miR-615-5p to OPCs, where they bind to the myelin regulatory factor (MYRF), an important transcription factor for myelin production, and hinder the maturation of OPCs. Reducing the expression of miR-615-5p in microglia can slow the progression of the disease, enhance the maturation of oligodendrocytes (OLGs), and encourage the regeneration of myelin.⁷⁷ Consequently, targeting miR-615-5p in exosomes derived from microglia could be a viable approach for treating multiple sclerosis (MS) by enhancing myelin regeneration and acting as a diagnostic marker for the disease. While research on miRNA in M2-Exos related to MS is still scarce, we are optimistic that the delivery and therapeutic properties of exosomes indicate a strong potential for future use.

Repairing for Circulatory System Injury

Myocardial Injury Therapy

Myocardial ischemic injury typically manifests as either ischemic necrosis or temporary dysfunction of heart muscle cells, accompanied by the infiltration of macrophages. In a study using a rabbit model of myocardial ischemia, Dai et al discovered that miR-148 delivered via M2-Exos reduced the levels of thioredoxin-interacting protein (TXNIP) and inhibited the activation of the TLR4/NF-κB/NLRP3 pathway, thereby protecting heart tissue from damage caused by ischemia.⁷⁸ In cases of acute myocardial infarction (AMI), Long et al found that M2-Exos delivered miR-1271-5p, which downregulated the expression of SOX6, a direct target, leading to a decrease in hypoxia-induced cell death and lessening cardiac injury in AMI.⁷⁹ Additionally, Wei et al demonstrated that M2-Exos containing miR-145-5p prevented the death of heart muscle cells caused by hypoxia-reoxygenation (H/R) by lowering TLR4 expression.⁸⁰ Regarding post-sepsis myocardial injury, Sun et al reported that miR-24-3p within M2-Exos can impart cardioprotection by decreasing the expression of Tnfsf10.⁸¹ Recent research highlights that the regenerative ability of adult cardiomyocytes is very limited. However, miRNAs in M2-Exos can safeguard cardiac muscle by minimizing cardiomyocyte injury, inhibiting apoptosis and pyroptosis in these cells, and curbing fibroblast proliferation, which presents opportunities for developing biologically-based therapies to repair myocardial damage.

Atherosclerosis Therapy

Current therapeutic strategies for AS aim to decrease inflammation and prevent plaque rupture by modulating the M2 macrophage polarization in the arterial wall.^82,83 The exosomes isolated from the supernatant of bone marrow-derived macrophages (BMDMs) polarize into M2-type, containing anti-inflammatory microRNA-99a/146b/378a, inhibit inflammation by targeting NF-κB and TNF-α signaling and also promoted M2 polarization in recipient macrophages.⁸⁴ Cheng et al demonstrated that overexpressing miR-221-3p in M2-Exos can attenuate the injury-induced inflammatory response and apoptosis of endothelial cells.⁸⁵ Additionally, Xie et al developed P-M2EV (platelet membrane-modified M2EV), which contains miR-99a-5p, the most highly expressed miRNA, capable of targeting the mRNA of Homeobox A1 (HOXA1).⁸⁶ This targeting effectively suppresses the formation of foam cells in vitro and successfully inhibits the progression of AS. This targeting effectively inhibits foam cell formation in vitro and slows the progression of AS. To summarize, the miRNAs from M2-Exos have potential therapeutic benefits for mitigating AS and preventing its advancement through various mechanisms, including reducing inflammation in arterial plaques, decreasing the inflammatory response and apoptosis in injured endothelial cells, and inhibiting foam cell formation.

Repairing for Urinary System Injury

Diabetic Nephropathy Therapy

Diabetic nephropathy (DN) is a frequent complication of diabetes mellitus, characterized by elevated glucose levels that can trigger pro-inflammatory M1-type macrophages, resulting in podocyte apoptosis due to the release of inflammatory mediators. Wang et al discovered that M2-Exos can reduce lipopolysaccharide (LPS)-induced podocyte apoptosis by modulating the miR-93-5p/TLR4 pathway.⁸⁷ Similarly, Huang et al showed that miR-25-3p found in M2-Exos promotes cellular autophagy, which decreases DUSP1 expression and mitigates podocyte damage caused by high glucose (HG) levels.⁵⁵ Zhuang et al reported that γ-aminobutyric acid (GABA) can reverse the polarization of M1/M2 macrophages in HG conditions, reducing podocyte injury through the miR-21a-5p-Tnpo1/miR-25-3p-ATXN3 signaling pathway in M2-Exos.⁸⁸ The miRNAs present in M2-Exos are effective in minimizing podocyte damage and slowing the progression of DN. These results offer new approaches for the prevention and treatment of DN, although additional research is necessary to explore the underlying mechanisms and pathways.

Post-Traumatic Urethral Stricture Therapy

Post-traumatic urethral stricture poses a significant challenge for both patients and healthcare providers. Focusing on glutamine metabolism to curb the excessive activation of urethral fibroblasts (UFBs) offers a promising and effective method to avert urethral scarring and stricture. Research has indicated that miR-381 found in M2-Exos can diminish the formation of myofibroblasts in UFBs by inhibiting YAP/GLS1-dependent glutamine breakdown, thereby preventing urethral scarring and stricture.⁸⁹ Therefore, this strategy may be a viable therapeutic option for preventing urethral stricture.

Repairing for Skin Injury

Research has demonstrated that M2-Exos have a strong ability to promote angiogenesis during the healing of skin injuries.^90–92 Lyu et al found that M2-Exos can enhance the vascularization of endothelial cells by transferring miR-21, which suppresses PTEN expression in these cells and activates the AKT/mTOR signaling pathway.¹⁵ Additionally, the researchers utilized a mouse model with complete skin defects and discovered that M2-Exos significantly expedited the healing of traumatic wounds due to their remarkable angiogenic properties, making them a promising clinical treatment for skin injuries. However, there is still a significant lack of understanding regarding the miRNAs present in these exosomes and the regulatory mechanisms involved.

Repairing for Locomotor System Injury

Bone Injury Therapy

Approximately 90% of fractures generally heal well with timely treatment and rest, but a small number of patients may face complications such as nonunion or delayed healing.⁹³ Osteoblast differentiation plays an important role in fracture healing, with macrophages serving as crucial mediators between the inflammatory response and bone regeneration. M2-Exos can promote tissue repair by reducing inflammation and promoting osteoblast differentiation.^94,95 Research by Xiong et al revealed that miR-5106, which is prevalent in M2-Exos, is transferred to bone mesenchymal stem cells (BMSCs) and targets the SIK2 and SIK3 genes to facilitate osteoblast differentiation.⁹⁶ Furthermore, the equilibrium between osteogenesis and lipogenesis in BMSCs is vital for bone formation. It has been reported that M2-Exos can enhance osteogenesis while inhibiting adipogenesis via the miR-690/IRS-1/TAZ pathway, presenting a promising therapeutic approach for bone loss conditions.⁹⁷ Additionally, Zhang et al found that miRNA-26a-5p found in M2-Exos can also promote osteogenesis and suppress lipogenesis in BMSCs.⁹⁸

Radiation therapy is a successful method for treating cancer, but it can harm the healthy tissues nearby. Bone tissue is particularly vulnerable to radiation. Bone marrow mesenchymal stem cells (BMMSCs) are also affected by radiation, which may link them to radiation-related bone damage. Huang et al demonstrated that miR-142-3p from M2-Exos can help restore the normal differentiation balance of irradiated BMMSCs by targeting TGF-β1. This discovery highlights a potential pathway for a cell-free strategy to address radiation-induced bone injuries.⁹⁹

Rheumatoid arthritis (RA) is an autoimmune condition that leads to inflammation and pain in the joints, eventually resulting in bone damage. Pascual-García et al reviewed the role of miRNAs found in the exosomes of osteoclasts, which are derived from the blood’s monocyte-macrophage system, to inform treatment strategies for bone damage caused by RA.¹⁰⁰ They found that blocking miR-23a, miR-29b, and miR-214 in osteoclast exosomes promotes the growth of osteoblasts, while increasing levels of miR-22-3p, miR-26a, miR-27a, miR-29a, miR-125b, and miR-146a can inhibit osteoblast activity. Additionally, they explored potential therapeutic targets for these exosomal miRNAs and conducted tests in mice and in vitro RA models. Their findings indicate that these approaches could be significant in restoring the balance of osteoclast and osteoblast differentiation, aiding in the treatment of bone damage associated with RA.

Articular Cartilage and Muscle Injuries Therapy

Osteoarthritis (OA) is a prevalent age-related condition that impacts the entire joint structure, including the articular cartilage and subchondral bone.¹⁰¹ Qian et al found that miR-26b-5p from M2-Exos targets the TLR3 signaling pathway in vitro, which enhances the polarization of M2 macrophages and inhibits the hypertrophy of chondrocytes induced by M1 macrophage-conditioned medium by targeting COL10A1.¹⁰² In an OA animal model, miR-26b-5p improved gait abnormalities and alleviated pain, while also reducing synovitis and cartilage degeneration, thereby slowing the progression of OA. Additionally, Zhou et al demonstrated that miR-501, which is abundant in M2-Exos, can promote the differentiation of C2C12 myoblasts by targeting YY1.¹⁰³ Their results offer new insights into how M2 macrophages can enhance myogenesis and indicate that delivering miR-501 through M2-Exos could be a promising treatment for muscle injury-related conditions.

In summary, the miRNAs from M2-Exos can facilitate healing and hinder the advancement of damage in the respiratory, circulatory, urinary, central nervous, and motor systems, as well as other systemic organ injuries by influencing various pathways. Research into the mechanisms and therapeutic benefits of these miRNAs offers a promising approach for addressing related health issues and establishes a basis for utilizing M2-Exos in the treatment of different diseases. Nonetheless, more comprehensive research and confirmation of the mechanisms involved are necessary, along with improvements in the biosafety of their use.

Application Methods of M2-Exos in Tissue Injury Repair

Direct Exertion of M2-Exos in Tissue Injury Repair

Numerous significant therapeutic outcomes have been documented through the direct utilization of M2-Exos in the repair of tissue injuries (Figure 6).^75,104 For instance, exosomes derived from macrophages can be administered at the site of tendon injuries to stimulate the intrinsic reparative capabilities of the tissues, thereby facilitating effective tendon repair.¹⁰⁵ Additionally, M2-Exos has demonstrated anti-inflammatory properties by inhibiting the release of pro-inflammatory enzymes and cytokines, while also promoting angiogenesis and re-epithelialization in diabetic wounds, which accelerates the processes of wound healing and flap survival.^14,106,107 Despite the significant therapeutic potential of M2-Exos, their advancement is impeded by challenges such as low yield and high production costs. Consequently, researchers typically pursue several strategies to enhance their application: 1) Modifying the culture conditions of exosome-producing cells to increase exosome secretion.¹⁰⁸ 2) Employing the most suitable extraction method tailored to the specific characteristics of the exosomes. 3) Enhancing therapeutic efficacy through pretreatment aimed at improving exosome uptake by the donor.¹⁰⁹ 4) Achieving synergistic effects by co-delivering additional drug components.¹¹⁰ 5) The integration of multiple distinct exosomes may facilitate the attainment of synergistic therapeutic outcomes.¹¹¹

Figure 6 Application methods of M2-Exos in tissue injury repair.

M2-Exos as Drug Delivery for Tissue Injury Repair

Under physiological conditions, extracellular vesicles, including exosomes, serve as mediators of intercellular communication, as they transport various bioactive components that can influence recipient cells.^112,113 In the context of atherosclerosis (AS), macrophages transform into foam cells, leading to the release of substantial amounts of pro-inflammatory factors. Exosomes engineered from modified M2 macrophages, in conjunction with the FDA-approved compound 5-aminolevulinic acid hexyl ester hydrochloride (HAL), exhibit the anti-inflammatory properties characteristic of M2 macrophages, with HAL further augmenting their anti-inflammatory effects.¹¹⁴ However, the therapeutic applications of exosomes have often been limited in effectiveness. To address this issue, Li et al developed an integrated hydrogel system incorporating functionalized gold nanorods (AuNRs) and M2-derived exosomes. This system facilitates wound angiogenesis through the sustained release of M2 exosomes, while simultaneously scavenging reactive oxygen species, inhibiting inflammation, promoting angiogenesis, and exhibiting antimicrobial properties, thereby accelerating the wound healing process.¹⁰⁷

Several factors underscore the potential of exosome-based drug delivery systems. Firstly, the exosomal membrane is composed of a relatively impermeable lipid bilayer that regulates and prolongs the release of encapsulated substances, including therapeutic agents. Moreover, the surface of exosomes is adorned with various adhesion proteins that exhibit low immunogenicity and cytotoxicity, particularly in the case of autologous exosomes. This characteristic not only facilitates their efficient cellular uptake and the transport of therapeutic drugs but also protects against immune rejection.¹¹⁵ Additionally, exosomes can enhance the stability of certain drug monomers by increasing their resistance to enzymatic degradation and reducing their solubility, thereby preventing rapid degradation in the bloodstream. Consequently, exosomes represent an ideal and distinctive platform for the encapsulation and delivery of drug molecules.

Engineered M2-Exos in Tissue Injury Repair

Exosomes possess the capacity to elicit both adaptive and innate immune responses, rendering them advantageous for the development of therapeutic agents. Nonetheless, challenges persist in the utilization of exosomes, including inadequate delivery efficiency and suboptimal targeting, which impose certain constraints on their application. Various strategies exist that may enhance their therapeutic efficacy, particularly in the context of engineered exosomes.^115–117

Genetic engineering techniques enable the modification of ligands on the exosomal membrane, thereby enhancing the specificity and prolonging the in vivo action of exosomes.^54,118–120 Numerous studies have employed these techniques to create engineered exosomes with specific functionalities. For instance, Liu utilized CD47 and human antigen R (HuR) to alter the exosomal membrane, transforming it into an effective drug delivery vehicle.¹²¹ Additionally, the modulation of gene expression within exosomes through genetic engineering can influence their functional capabilities to achieve targeted therapeutic outcomes. Huang et al exemplified this by developing genetically engineered M2-Exos that were designed to silence casein kinase 2 interacting protein-1 (Ckip-1), which subsequently rescued the mineralization and cementogenesis suppressed by Pg, thus promoting the regeneration of cementum.¹²² To further improve the targeted delivery efficacy of exosomes, researchers have increasingly focused on integrating exosomes with synthetic nano-microspheres for targeted therapeutic applications. For example, Zeng created a dual-layer microneedle-based wound dressing system (MEs@PMN) that encapsulates micelles in the needle tip and polydopamine (PDA) nanoparticles in the backing layer, which collectively enhances pro-angiogenic effects by elevating the expression of CD31 and von Willebrand factor (vWF).¹²³

Limitations and Future Perspectives

Current methodologies for the isolation of exosomes yield satisfactory levels of both quantity and purity; however, the absence of standardized protocols for their extraction and characterization poses significant challenges for subsequent applications.¹²⁴ The properties of exosomes, such as size and surface charge, vary depending on the extraction technique employed, which subsequently influences the drug loading capacity and encapsulation efficiency for future drug delivery systems. For exosomes to be effectively utilized in clinical settings, it is imperative to develop standardized extraction and validation protocols. We advocate for researchers to take into account various factors that could enhance the efficacy of exosome-based therapies during their investigations. This includes the establishment of criteria for the selection of appropriate exosomes and the identification of optimal drug delivery timing to maximize therapeutic outcomes.

Additionally, there exists a substantial disparity between the actual therapeutic effectiveness and the levels required for clinical implementation.¹²⁵ Numerous researchers have suggested strategies to enhance therapeutic efficacy through the synergistic systemic effects of multiple active components, as well as by modifying the exosomal membrane to improve the uptake rate by receptor cells. Furthermore, the integration of exosomes with nanoengineering techniques, such as the incorporation of iron oxide and nanogold, has augmented the translational capacity of exosomes in clinical settings, indicating substantial research potential within the realm of interdisciplinary medicine. We contend that utilizing exosomes as vehicles for therapeutic drug delivery presents considerable promise, particularly in terms of enabling controlled release and minimizing the frequency of drug administration.

Exosomes are usually kept at −80°C for experiments to be used later. However, research has shown that multiple freeze-thaw cycles can alter the size, structure, and function of exosomes, which can affect future studies. Thus, preserving the activity of the bilayer lipid membrane is a matter that needs more exploration. For clinical applications, ensuring their safety is crucial, as is confirming their stable activity (therapeutic effectiveness). If exosomes are to be utilized as treatments for humans, their safety must be guaranteed. In China, exosomes are currently in Phase III clinical trials involving human participants; if they successfully pass further evaluations, they could be approved as drugs. Nonetheless, a few reports have indicated that significant allergic reactions can occur after facial injections of a standard dose of exosomes. Such severe allergic reactions can pose risks to human health and even life, highlighting the need for thorough testing and protocols.

Conclusion

The process of repairing tissue damage is complex and dynamic, involving various cell types and molecular signals, with macrophages being particularly important. Exosomes have garnered significant research interest, both nationally and internationally, due to their wide range of miRNAs. This review examines the role of miRNAs from M2-Exos in diagnosing, preventing, and treating diseases related to tissue injury. Although there has been extensive research on miRNAs from M2 macrophages, challenges remain, especially in improving yield and therapeutic effectiveness. Current studies are focused on creating drug-loaded and engineered exosomes to enhance their efficacy, showing promising results. In conclusion, miRNAs found in M2-Exos hold great potential for aiding tissue repair and could serve as novel targets for disease diagnosis and treatment, offering significant therapeutic opportunities for the future.