The New Approach to Treating Erectile Dysfunction: Stem Cell-Derived Extracellular Vesicles

Abstract

Erectile dysfunction refers to the inability of men to achieve or maintain sufficient erection during sexual activity, which significantly impacts their quality of life and mental health. The causes of erectile dysfunction are complex, involving multiple systems such as vascular, neural, endocrine, and psychological factors. Current treatment methods for erectile dysfunction have significant limitations in addressing organic lesions, particularly penile fibrosis and hypoxic microenvironments. However, the latest stem cell-derived nanovesicle technology has been found to significantly reverse cellular fibrosis and alter the cellular microenvironment, potentially offering a new therapeutic approach for erectile dysfunction. Additionally, extracellular vesicles derived from stem cells can exert stem cell-like regenerative effects while overcoming challenges associated with cell therapy, making them excellent tools for regenerative repair. Therefore, this paper summarizes the potential of extracellular vesicles from different stem cell sources in the treatment of erectile dysfunction and analyzes their engineered preclinical studies to facilitate their translation from the laboratory to clinical applications.

Introduction

Background

Erectile dysfunction (ED) has been documented since ancient times, with descriptions found in medical literature dating back over 5000 years.¹ ED, also known as penile erectile dysfunction, refers to the inability of men to achieve or maintain an erection sufficient for sexual intercourse under sexual stimulation.² According to the latest report from the International Consultation Committee for Sexual Medicine on Definitions/Epidemiology/Risk Factors for Sexual Dysfunction, ED is widespread among men over 40, with its prevalence gradually increasing with age.^3,4 In particular, among men over 70, the prevalence of ED ranges from 50% to 100%.^5–9 It is estimated that by 2025, the global number of ED patients will rise to 322 million.^10,11 The high incidence of ED makes it an increasingly prominent health issue in aging societies.

Although current treatment methods such as lifestyle adjustments, psychotherapy, and pharmacological interventions have achieved certain effects, ED treatment still faces many challenges. Studies have shown that broad improvements in lifestyle, such as dietary changes and increased physical activity, can effectively alleviate ED symptoms in some patients.^12–17 Psychotherapy can help reduce anxiety and stress, especially for those with psychological ED.¹⁸ However, for many patients with comorbid chronic diseases such as diabetes or cardiovascular disease, the effect of lifestyle adjustments is limited.^19–21 While dietary regulation or the use of statins to lower blood lipid levels and cholesterol concentrations may improve symptoms in some patients,²² these approaches do not fundamentally address ED caused by organic factors. In terms of pharmacological treatment, selective phosphodiesterase type 5 inhibitors (PDE5i), such as sildenafil, are currently the most commonly used drugs in clinical practice.^23–27 These drugs improve erectile function by enhancing the relaxation effect of nitric oxide (NO) on penile smooth muscle^19,28–30 with their effectiveness depending on the integrity of the patient’s own neurological and vascular functions. Therefore, for patients with severe vascular or neurological damage, such as those with vascular complications from diabetes or nerve injury after prostate surgery, the efficacy of PDE5i is limited. Moreover, although PDE5i shows significant short-term effects, the issue of drug resistance and dependence with long-term treatment limits its effectiveness, and it is not suitable for some patients with cardiovascular diseases.^31,32

With the in-depth study of the pathophysiological mechanisms of ED, SCs-EVs have emerged as a novel biological therapeutic approach, showing significant potential for treating ED. SCs-EVs possess multiple biological functions, including anti-inflammatory, pro-angiogenic, and tissue repair properties,^33–36 which specifically target the core causes of organic ED, such as nerve damage and vascular lesions. Particularly in neurogenic and vascular ED, SCs-EVs can promote the regeneration and functional recovery of damaged tissues, filling the gap left by existing therapeutic methods. Furthermore, as natural intercellular communication vehicles, EVs can circumvent common issues such as immune rejection and ethical concerns in cell-based therapies, demonstrating good safety and feasibility.³⁷ Therefore, although current lifestyle interventions and pharmacological treatments can improve ED to some extent, the efficacy remains unsatisfactory, especially for patients with complex, organic ED.^38,39 SCs-EVs, as a promising therapeutic approach, offer new possibilities, particularly due to their unique advantages in repairing nerve and vascular damage, suggesting their potential in future clinical applications.

Etiology and Pathogenesis of ED

Sexual activity is an indispensable part of many men’s lives, with sexual function often closely tied to self-esteem and male identity. ED not only undermines self-esteem and confidence but also significantly affects overall quality of life.^40,41 The causes of ED can be broadly classified into psychological and organic categories. Psychological factors include anxiety, depression, low self-esteem, psychological stress, and relationship issues with a partner,^38,42–46 while organic causes encompass abnormalities in the neurological, endocrine, vascular, and anatomical systems. Specifically, organic ED can be further subdivided into neurogenic, endocrine, arterial, cavernous, and drug-induced types.^19,38

The specific causes of organic ED are extensive. Neurogenic causes include multiple sclerosis, Parkinson’s disease, spinal cord injuries, and prostate surgery,^19,47–51 while endocrine factors primarily involve low testosterone and hyperprolactinemia.^22,52 Vascular issues, such as diabetes, hypertension, hyperlipidemia, and metabolic syndrome, are also common causes of ED, as these conditions negatively impact vascular function, obstructing normal blood flow.^53–61 Additionally, conditions like obstructive sleep apnea and lower urinary tract symptoms are closely associated with ED.^53–61 It is important to note that ED is not only a reproductive system disorder but has also been identified as an early warning sign or associated symptom of cardiovascular diseases,^58,60,62 with independent associations to coronary heart disease, stroke, and all-cause mortality.^63,64 Besides organic factors, environmental and lifestyle factors also play a significant role in ED. Smoking, obesity, and lack of physical activity have been confirmed as risk factors for ED.^63,65,66 Moreover, certain medications, particularly antihypertensive and antidepressant drugs, may trigger or worsen ED symptoms.⁶⁷

Normal sexual function is a complex physiological process coordinated by the psychological, endocrine, vascular, and nervous systems.⁶⁸ During this process, penile erection is regulated by multiple mechanisms, with smooth muscle relaxation and contraction playing a key role (Figure 1). Adrenergic nerves, endogenous muscle factors, and endothelial factors together regulate smooth muscle contraction.^69–71 Upon sexual stimulation, non-adrenergic, non-cholinergic nerve fibers release NO, while parasympathetic cholinergic nerve fibers release acetylcholine, promoting smooth muscle relaxation.^71,72 This process is achieved by increasing cyclic guanosine monophosphate (cGMP) levels and reducing intracellular calcium ion concentration.^71,72 After smooth muscle relaxation, blood fills the cavernous bodies, compressing the veins and preventing blood from flowing back, thus achieving an erection. When phosphodiesterase type 5 (PDE5) breaks down cGMP, the erection process is reversed^71,72 (Figure 2). Therefore, any disruption in these mechanisms can lead to the occurrence of ED.

Figure 1.

Flaccidity State. a. Intracellular Ca²⁺ influx is regulated by norepinephrine signaling and the level of inositol-1,4,5-trisphosphate (Ins(1,4,5)P₃), which is generated by phospholipase C (PLC) from phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P₂). Elevated intracellular Ca²⁺ binds to calmodulin, facilitating the formation of the calmodulin-myosin light-chain kinase (MLCK) complex. This results in phosphorylation of myosin light chains (MLC), leading to smooth muscle contraction and penile flaccidity. Norepinephrine signaling also inhibits adenylate cyclase and modulates the Rho-associated protein kinase (ROCK) pathway, thereby increasing MLC sensitivity to Ca²⁺. Testosterone negatively regulates this process. Additionally, endothelin and prostaglandins from the endothelium further elevate intracellular Ca²⁺, promoting smooth muscle contraction. (b) During smooth muscle contraction, minimal blood enters through the cavernosal artery, allowing free outflow via the subtunical venous plexus. inhibitory effect; promoting effect.

Abbreviations: ER, endoplasmic reticulum; MLCP, myosin light-chain phosphatase.

Figure 2.

Erection State. (a) Upon sexual stimulation, nitric oxide (NO) released from non-adrenergic non-cholinergic (NANC) nerve fibers activates guanylate cyclase, increasing cyclic guanosine monophosphate (cGMP) levels. Concurrently, acetylcholine released from parasympathetic cholinergic nerve fibers activates adenylate cyclase, elevating cyclic adenosine monophosphate (cAMP) levels. (b) Smooth muscle relaxation enables blood to fill the cavernosal sinusoids, compressing the subtunical veins and blocking venous outflow. This process reverses when phosphodiesterase type 5 (PDE5) hydrolyzes cGMP. inhibitory effect; promoting effect.

Abbreviations: ER, endoplasmic reticulum; InsP₃, inositol trisphosphate; NOS, nitric oxide synthase; PtdIns(4,5)P₂, phosphatidylinositol-4,5-bisphosphate.

Stem Cells-Derived Extracellular Vesicles

Since Ernst Haeckel first proposed the concept of “stem cells” in 1868, the exploration of stem cells in scientific research and clinical applications has become one of the key fields of study.⁷³ In fact, stem cells have significant therapeutic potential for various diseases and have greatly advanced the development of regenerative medicine.⁷⁴ Stem cells not only possess self-renewal ability but can also differentiate both in vitro and in vivo into tissues derived from the mesoderm and non-mesodermal layers, ultimately differentiating into mature cells.⁷⁵ Based on their differentiation potential, stem cells can be classified into totipotent stem cells (eg, zygotes), pluripotent stem cells (eg, embryonic stem cells), multipotent stem cells (eg, mesenchymal stem cells), oligopotent stem cells, and unipotent stem cells.^76,77 Among them, mesenchymal stem cells (MSCs) are the most commonly used and easily accessible type, considered multipotent adult stem cells. They are nearly non-tumorigenic and free from ethical concerns.⁷⁸

Extracellular particles (EPs) is the preferred overarching term for cell‐derived multimolecular assemblies in the nanometre to micron size range, including both EVs and Non‐vesicular extracellular particles (NVEPs).⁷⁹ EVs is the term for particles that are delimited by a lipid bilayer and cannot replicate on their own (vesicular component of extracellular particles). In addition to naturally released EVs, there are also EV mimetics, artificial cell-derived vesicles (ACDVs), and synthetic vesicles.⁷⁹ Other terms, such as exosomes, ectosomes, and microvesicles, are generally used in specific contexts as the research on EVs is finer grained.⁷⁹ EVs contain various molecules from within the cell and act as a novel tool for intercellular communication. Based on their size and biological origin, EVs are derived from a wide range of sources, with nearly all living cells capable of releasing EVs. These EVs are rich in RNA, bioactive lipids, and proteins associated with various biological processes.^80–82 Through membrane fusion or endocytosis, they transfer soluble factors, such as growth factors, cytokines, and genetic material, to target cells,^83–86 thereby regulating their biological activity.^87,88 Moreover, EVs can regulate gene expression by transporting miRNAs and can program the function of cells involved in tissue repair,^89,90 playing roles in physiological and pathological processes within the body. Due to their broad biological effects, EVs play a key role in regulating the tissue microenvironment, promoting the repair and regeneration of damaged or diseased tissues, and helping to clear unnecessary proteins and toxic substances.^85,91,92 These unique properties make EVs particularly important in the study of the pathophysiological mechanisms of various diseases, including cancer, cardiovascular diseases, infectious diseases, tissue repair and regeneration, and neurodegenerative diseases.^{33,85,92–94}

EVs play a significant synergistic role with stem cells in activating immune and inflammatory pathways.^33,34,95 After immune cells reach the site of tissue damage, SCs-EVs regulate the immune process by reducing inflammation and enhancing the local immune response.⁸² Specifically, EVs can modulate both innate and adaptive immune responses.^33,34 Broadly, SCs-EVs have been found to perform the following functions: (i) promote the survival of hematopoietic stem cells; (ii) activate monocytes, macrophages, and B cells; (iii) enhance the function of CD4+ regulatory T cells; (iv) inhibit the activity of natural killer cells and CD8+ cytotoxic T cells. Furthermore, studies have reported that EVs can recruit various stem cells from the microenvironment to the site of tissue damage, coordinate their functions, and promote tissue repair.^90,92 Once mobilized, stem cells reach the damaged tissue, where they secrete growth factors and cytokines through paracrine signaling, promoting tissue repair and wound healing.^35,36 Evidence suggests that stem cells can migrate to the injury site via chemotactic gradients and form connections with damaged cells, generating replacement and supporting cells to facilitate tissue regeneration.^96,97 Studies also show that SCs-EVs have broad biological functions, such as promoting the self-renewal of bone marrow cells,^98,99 aiding the repair and regeneration of nerve damage,^100,101 accelerating skin regeneration after burns,^102,103 inducing angiogenesis in quiescent endothelial cells,^104,105 and influencing the phenotypic differentiation of downstream recruited stem cells.

Since the discovery of the immune-regulating and regenerative properties of EVs, numerous animal models have demonstrated the important role of stem cells and their derived EVs in treating various diseases, including but not limited to skin injury, cardiac injury, spinal cord nerve injury, kidney damage, retinal diseases, arthritis, skeletal deformities, muscle degeneration, liver injury, brain injury, and cancer immunotherapy.^106,107 Importantly, increasing research shows that SCs-EVs can effectively increase smooth muscle content, promote angiogenesis, reduce fibrosis, and thereby restore tissue function.^108–110 Additionally, EVs can inhibit cell apoptosis, reduce neuronal damage, and promote the recovery of neurological functions.^111–113 These findings highlight the tremendous potential of SCs-EVs in restoring the function of tissues and organs.

Given the role of EVs in stem cell recruitment, inflammatory response modulation, and tissue repair, EVs are regarded as a promising new therapeutic tool. They not only serve as carriers for therapeutic molecules but also act as adjunctive drugs to enhance the effects of stem cell transplantation therapy at injury sites (Table 1).¹⁰³

Table 1.

The Comparison Between Stem Cell Therapy and Stem Cell-Derived Exosome Therapy

Treatment Modality	Advantages	Limitations
Stem cells therapy	Multilineage differentiation potential	Short survival time after injection, low transplantation rate
	Suitable for the treatment of various diseases	Strict storage and transportation requirements
	Accumulation of extensive laboratory and clinical data	Potential tumorigenicity
	Easy to isolate, suitable for large-scale production	Infusion toxicity
	Less pain and fewer complications	Immunogenicity
	Well-established regulatory guidelines	Some stem cells may have ethical issues (eg, embryonic stem cells)
		Preparation process needs improvement
		Variability in therapeutic outcomes
Stem cells-derived extracellular vesicles therapy	Comparable therapeutic effects to stem cells but with a smaller size	Batch-to-batch inconsistency
	More concentrated functional carriers (eg, cytokines)	Lack of standardized purification and storage protocols
	Can be modified on their surface and within the carriers	Relatively low yield in large-scale production
	Various delivery methods	Higher costs
	Long-term storage and high transport efficiency	No industry-standard quality specifications
	EVs derived from non-tumorigenic cells pose minimal tumorigenic risk.	Insufficient regulatory oversight
	Lack of ethical issues

Exploration of Stem Cells-Derived EVs in the Treatment of ED

Stem cells applications in the treatment of ED began relatively late, with relevant studies only emerging in the 21st century.¹¹⁴ However, significant progress has been made in Stem cells therapy (SCT) for treating ED.^115,116 A wealth of basic research and clinical trials have demonstrated that SCT can partially restore erectile dysfunction caused by various factors and significantly improve patients’ quality of life.^117–119 More importantly, stem cells not only alleviate symptoms but also exert therapeutic effects by repairing the underlying pathological mechanisms. These mechanisms include differentiation into specific functional cells, secretion of a large number of growth factors and extracellular vesicles (Table 2), thereby improving the local pathological environment and promoting tissue repair and regeneration (Figure 3).^{114,120–122}

Table 2.

Summary of Key Studies on Stem Cell-Derived EVs for ED Treatment and Their Mechanisms

Stem Cell EV Source	ED Model	Key Findings	Study Reference
BM-MSC-EVs	Rat (Cavernous nerve injury)	↑ Smooth muscle content, ↑ nNOS; ↓ Caspase-3; Improved erectile function.	[123]
MSC-EVs	Rat (Iliac artery injury)	↑ Endothelialization, ↓ Oxidative stress; Comparable efficacy to whole MSCs.	[124]
CCSMC-EXOs	Rat (Diabetic ED)	Superior uptake and retention; ↑ NO/cGMP pathway; ↓ Fibrosis.	[125]
MSC-EXOs	Rat (Aged ED)	Regulated PTEN/PI3K/AKT via miR-296-5p/miR-337-3p; ↓ Apoptosis.	[126]
ADSC-EVs	Rat (Diabetic ED)	Restored erectile function via ↑ Angiogenesis, ↓ Fibrosis.	[127]
ADSC-EXOs	Rat (Diabetic ED)	↑ Corin gene activity; Improved neurovascular parameters.	[128]
ADSC-EVs	Rat (Type 2 Diabetic ED)	↑ ICP/MAP ratio; ↑ Bcl-2, ↓ Caspase-3; ↓ Endothelial apoptosis.	[129]
ADSC-EVs vs BMSC-EVs	Rat (Bilateral nerve injury)	Both ↑ Cavernous pressure; Enhanced endothelial internalization.	[130]
ADSC-EVs (miR-301a-3p)	Rat (OSA-related ED)	Targeted PTEN/TLR4; ↑ Autophagy, ↓ Apoptosis in smooth muscle.	[131]
Engineered ADSC-Exos (circPIP5K1C)	Rat (CIH-induced ED)	miR-153-3p/SMURF1 axis ↓ Glycolysis; Restored erectile function.	[132]
USC-EVs	Rat (Peyronie’s disease)	↓ Fibroblast transformation; Balanced MMP/TIMP; ↓ Tunica albuginea fibrosis.	[133]
USC-EVs	Rat (Diabetic ED)	Pro-angiogenic miRNAs (miR-21-5p, let-7); ↑ CD31/eNOS; ↑ ICP/MAP ratio.	[134]
ESC-NVs	Mouse (Diabetic ED)	↑ Angiogenesis (HGF, Angiopoietin-1) and neuroregeneration (NGF); 96% functional recovery.	[135]
PC-NVs	Mouse (Nerve injury)	Activated Akt/eNOS; ↑ Endothelial tube formation and neuronal sprouting.	[136]
MCP-EVs (miR-148a-3p)	Mouse (Diabetic ED)	↓ PDK4; ↑ Endothelial migration/proliferation; ↓ Apoptosis.	[137]

Figure 3.

SC-EVs ameliorate ED through comprehensive multi-target mechanisms involving endothelial repair, smooth muscle modulation, antifibrotic processes, and neural regeneration. In endothelial repair, MSC-EVs enhance NO production by upregulating eNOS/nNOS expression and activating the PTEN/PI3K/AKT pathway via miR-296-5p, while USC-EVs promote CD31+/eNOS+ endothelial proliferation and improve erectile function (ICP/MAP ratio) through miR-21-5p and let-7 family-mediated mechanisms. For smooth muscle regulation, MSC-EVs restore contractile function by increasing α-SMA expression and inhibiting caspase-3-dependent apoptosis, whereas ADSC-EVs counteract fibrosis and metabolic dysfunction via miR-301a-3p-mediated PTEN/TLR4 suppression and the circPIP5K1C-miR-153-3p-SMURF1 axis. In antifibrotic processes, USC-EVs reduce collagen deposition by modulating MMP/TIMP balance, while ADSC-EVs enhance endothelial function through corin gene activity. Neural regeneration is facilitated by ESC/PC-EVs, which promote axonal growth through neurotrophin-3 and Schwann cell migration via Akt/eNOS signaling. Additionally, MSC-EVs mitigate oxidative stress by suppressing NOX4 through miR-337-3p, and ADSC-EVs exert antiapoptotic effects by upregulating Bcl-2 and reducing caspase-3 activity, collectively restoring erectile function through synergistic pathways..

MSCs-EVs

Mesenchymal stem cells-derived extracellular vesicles (MSCs-EVs) are vesicles secreted by mesenchymal stem cells, rich in a variety of bioactive molecules, including microRNAs, proteins, and lipids. They play an important role in promoting tissue repair, reducing inflammation, and regulating immune responses, and have been widely applied in the treatment of cardiovascular diseases, nerve injuries, and other conditions.

Ouyang et al isolated MSCs-EVs from rat bone marrow and applied them to corpus cavernosum smooth muscle cells (CCSMCs). The study found that treatment with MSCs-EVs significantly increased the smooth muscle content in the corpus cavernosum and the level of neuronal nitric oxide synthase, improved the ratio of smooth muscle to collagen, enhanced CCSMCs viability, and reduced caspase-3, effectively improving erectile dysfunction in a rat model of cavernous nerve injury.¹²³ Similarly, Liu et al injected MSCs-EVs into the corpus cavernosum of a rat model of ED induced by iliac artery injury. The results indicated that MSCs-EVs could promote endothelial formation in the cavernous sinus, alleviate oxidative stress damage, and increase nitric oxide synthase and smooth muscle content in the corpus cavernosum. Compared with MSCs treatment alone, MSCs-EVs showed similar therapeutic effects but also provided unique advantages, making them an effective means of treating erectile dysfunction caused by arterial injury.¹²⁴

However, the experiments by Ouyang and Song did not further investigate the composition and mechanisms of MSCs-EVs, which is an important aspect for future applications of MSCs-EVs in ED treatment. As research continued, Song et al further validated the effects of MSCs-EVs in a diabetic rat model, confirming that MSCs-EVs could improve erectile function in diabetic erectile dysfunction (DMED) rats. Interestingly, they compared the effects of corpus cavernosum smooth muscle cell-derived exosomes (CCSMCs-EXOs) and MSC-derived exosomes (MSCs-EXOs). The results showed that CCSMCs-EXOs were more readily absorbed by CCSMCs and had a higher peak concentration and longer retention time in the corpus cavernosum. By inhibiting fibrosis and regulating the NO/cGMP signaling pathway (including increasing smooth muscle content, reducing collagen deposition, and elevating NO and cGMP levels), CCSMCs-EXOs significantly improved erectile function in DMED rats.¹²⁵ Additionally, Li et al found that injecting MSCs-EXOs into elderly rats improved age-related erectile dysfunction (AED). These EVs regulated the PTEN/PI3K/AKT signaling pathway through miR-296-5p and miR-337-3p, improving the histological structure of the corpus cavernosum and inhibiting cell apoptosis.¹²⁶

MSCs-EVs have effectively alleviated ED caused by nerve injury, diabetes, and arterial injury by improving smooth muscle content in the corpus cavernosum, reducing fibrosis, and influencing key signaling pathways.

ADSCs-EVs

Adipose-derived mesenchymal stem cells-derived extracellular vesicles (ADSCs-EVs) are vesicles secreted by ADSCs that are rich in angiogenic and anti-fibrotic components. These extracellular vesicles show promising potential in improving wound healing, promoting angiogenesis, and alleviating fibrosis, especially in the context of diabetes and ED.

Zhu et al utilized ADSCs-EVs to treat diabetic ED in rats and found that these EVs, containing angiogenic and anti-fibrotic microRNAs, could induce endothelial cell proliferation, restore erectile function, and reduce cavernous fibrosis.¹²⁷ Wang et al also applied ADSCs-EXOs to a rat model of diabetic ED. Their results showed that ADSCs-EXOs could restore erectile function in diabetic rats, enhance neurovascular function, and inhibit the expression of inflammatory factors. Furthermore, they identified that the content of the corin gene in EVs was a critical factor in their therapeutic effect.¹²⁸ Additionally, Chen et al found that ADSCs-EVs injection significantly improved the ratio of cavernous pressure to mean arterial pressure, increased the smooth muscle to collagen ratio, and promoted the expression of the anti-apoptotic protein Bcl-2 while reducing the apoptotic protein caspase-3, leading to reduced apoptosis of endothelial and smooth muscle cells.¹²⁹

In a bilateral cavernous nerve injury (BCNI) model, Li et al compared ADSCs-EVs with bone marrow mesenchymal stem cells-derived extracellular vesicles (BMSCs-EVs) and found that both types of EVs could effectively internalize into human umbilical vein endothelial cells and improve cavernous function. After treatment, the BCNI rats showed a significant increase in the cavernous pressure/mean arterial pressure ratio, with noticeable improvement in erectile function.¹³⁰ To further elucidate the specific components of ADSCs-EVs involved in improving ED, Li et al extracted ADSCs-EVs and used them to treat obstructive sleep apnea (OSA)-related ED in rats. The study revealed that ADSCs-EVs, enriched with miR-301a-3p, targeted phosphatase and tensin homolog (PTEN) and Toll-like receptor 4 (TLR4), promoting autophagy and inhibiting apoptosis, which successfully restored erectile function in corpus cavernosum smooth muscle cells. Treatment also significantly elevated α-smooth muscle actin (α-SMA) levels.¹³¹

Furthermore, Gu et al injected ADSCs-Exos-circ (ADSCs-derived exosomes overexpressing circPIP5K1C) into the corpus cavernosum of OSA-related ED rats. Their findings indicated that ADSCs-Exos-circ improved erectile function induced by chronic intermittent hypoxia (CIH) exposure. The mechanism involved the adsorption of miR-153-3p in the corpus cavernosum, which promoted the expression of the downstream target gene SMURF1, leading to the binding and ubiquitination of SMURF1 and PFKFB3, inhibiting smooth muscle glycolysis, and ultimately restoring smooth muscle function.¹³²

ADSCs-EVs show significant therapeutic effects in treating ED associated with diabetes, OSA, and aging by regulating key signaling pathways, reducing fibrosis, and promoting angiogenesis. These findings suggest that ADSCs-derived EVs hold great potential as a therapeutic modality in ED treatment.

USCs-EVs

Urine-derived stem cells-derived extracellular vesicles (USCs-EVs) are extracellular vesicles extracted from urine, rich in various biomolecules such as microRNAs and proteins. These EVs have shown potential in the study of renal diseases, urinary tract infections, and other urological disorders due to their non-invasive nature, serving as biomarkers and therapeutic carriers.

Yang et al injected EVs derived from human urine-derived stem cells (USCs-EVs) into the testes of a Peyronie’s disease (PD) rat model. Their findings revealed that USCs-EVs could inhibit the transformation of fibroblasts into myofibroblasts, reduce the expression of tissue inhibitors of metalloproteinases (TIMPs) (−1, −2, and −3), and enhance the activity of matrix metalloproteinases (MMPs) (−1, −3, and −9). These effects significantly improved testicular tunica fibrosis and enhanced erectile function in the rats.¹³³

To explore the specific components in USCs-EVs that improve ED, Ouyang et al extracted USCs-EVs and applied them to diabetic ED rat models. The USCs-EVs were found to be rich in angiogenesis-promoting microRNAs (miR-21-5p, let-7 family, miR-10 family, miR-30 family, and miR-148a-3p), which improved the intracavernous pressure (ICP) and the ICP/mean arterial pressure (MAP) ratio. The study demonstrated that USCs-EVs significantly improved the cavernous pressure/mean arterial pressure ratio in rats, increased the expression of CD31, eNOS, phospho-eNOS, and nNOS in the corpus cavernosum, significantly enhanced the smooth muscle to collagen ratio, and improved erectile function in diabetic rats.¹³⁴

These findings highlight the significant role of USCs-EVs in promoting angiogenesis, improving erectile function, and reducing testicular fibrosis induced by diabetes and Peyronie’s disease. USCs-EVs hold promise as a potential therapeutic approach for ED, offering non-invasive treatment options with beneficial effects on vascular regeneration and tissue repair.

ESCs-EVs

ESCs-EVs (Embryonic stem cells-derived EVs) are vesicles secreted by embryonic stem cells, which have the ability to promote cell proliferation and differentiation. These EVs hold significant value in regenerative medicine, particularly due to their potential to modulate immune responses and promote tissue repair. They are especially promising in the fields of tissue engineering and regenerative therapies.

Kwon et al extracted embryonic stem cells-derived nanovesicles (ESCs-NVs) and applied them to diabetic ED mice models. The study found that ESC-NVs enhanced the expression of angiogenic factors and neurotrophic factors, such as hepatocyte growth factor (HGF), angiopoietin-1, nerve growth factor (NGF), and neurotrophin-3. Additionally, ESCs-NVs activated cell survival and proliferation pathways, including the Akt and ERK pathways. In high glucose conditions, ESCs-NVs promoted the formation of endothelial and pericyte tubes in primary cultures of mouse cavernous endothelial cells and pericytes, and accelerated the sprouting of microvessels and neurons in the aortic ring and major pelvic ganglia. By enhancing angiogenesis and nerve regeneration in vivo, ESC-NVs completely restored erectile function to approximately 96% of control values, while ESC treatment alone only partially restored erectile function to about 77% of control values.¹³⁵

These results highlight the potential of ESCs-NVs in significantly improving erectile dysfunction caused by diabetes and nerve damage by enhancing vascular regeneration and nerve repair. ESCs-NVs, through their regenerative properties, could provide a promising therapeutic strategy for ED treatment, particularly in cases where both vascular and neural damage contribute to the dysfunction.

PC-EVs and MCP-EVs

Pericyte-derived EVs (PC-EVs) are vesicles secreted by pericytes, which play crucial roles in regulating vascular function, supporting cell survival, and promoting tissue repair. These EVs show potential therapeutic applications in cardiovascular diseases, neurodegenerative disorders, and the tumor microenvironment.

Yin et al demonstrated the positive effects of PC-NVs (pericyte-derived nanovesicles) in a mouse model of cavernous nerve injury. Their research showed that PC-NVs promoted endothelial cell tube formation, Schwann cell migration, and neuronal sprouting. Additionally, PC-NVs significantly enhanced cell survival signaling pathways (such as Akt and eNOS) and induced the expression of neurotrophic factors, ultimately improving erectile function.¹³⁶

Mouse corpus cavernous pericyte-derived extracellular vesicle (MCP-EVs) also reflect the physiological state and functional characteristics of the originating cells. These EVs contain various bioactive molecules and play a key role in studying intercellular interactions, disease mechanisms, and potential therapeutic approaches. Ock et al injected MCP-EVs into diabetic ED mice, and the study revealed that miR-148a-3p in MCP-EVs inhibited PDK4 expression, promoting the migration, proliferation, and tube formation of mouse cavernous endothelial cells (MCECs). Additionally, MCP-EVs reduced MCECs apoptosis under high-glucose conditions, thereby improving erectile function in diabetic mice.¹³⁷ PC-NVs and MCP-EVs can effectively improve erectile function in diabetic ED mice by enhancing angiogenesis, promoting nerve regeneration, and reducing cell apoptosis.

The therapeutic potential of stem cell-derived EVs (MSCs-EVs, ADSCs-EVs, USCs-EVs, ESCs-EVs, etc) has been demonstrated across different types of ED, not limited to neurogenic or age-related ED, but also extending to diabetes and other endocrine-related ED.^125,138,139 These EVs improve cavernous body function through mechanisms such as promoting angiogenesis, inhibiting fibrosis, and enhancing cell survival. EVs-based therapies present a promising supplement to standard ED treatments, particularly in cases where conventional methods are less effective or not applicable.

However, there remains a lack of detailed research into the content and specific mechanisms of EVs, which limits our understanding of their mode of action. Future studies should focus on identifying specific biomarkers and functional components in EVs derived from different sources of stem cells, to better elucidate their roles in promoting angiogenesis, antifibrosis, and cell protection.

Engineered SCs-EVs: Strategies and Applications in ED

Tissue engineering approaches that integrate biotechnology, materials science, and engineering methodologies have provided innovative solutions for ED treatment. These therapeutic strategies offer distinct advantages including precise targeting capability, minimal immunogenicity, and significant tissue regenerative potential. Engineered stem cells, through genetic editing and bioengineering techniques, enable precise modulation of cellular functions to substantially enhance tissue repair capacity. Engineered EVs, serving as biomimetic nanocarriers derived from natural EVs, demonstrate tremendous promise in precision medicine following genetic or molecular modifications.^140,141 Research has shown that EVs exhibit excellent tissue tropism in various disease models. Local administration in myocardial infarction,¹⁴² ischemic limb,¹⁴³ or murine wound healing models¹⁴⁴ results in rapid accumulation within target tissues within hours, providing a rationale for sustained-release strategies to optimize tissue regeneration.

Current engineering strategies for EVs primarily fall into two categories: First, parental cell modification through vectors that induce progenitor cells to express specific proteins or non-coding RNAs,¹⁴⁵ enabling packaging of these therapeutic molecules into EVs for release in specific microenvironments.¹⁴⁶ Second, direct EV modification approaches including genetic engineering, metabolic engineering, and surface molecular engineering.^147,148 For instance, membrane permeabilization techniques allow small molecule drug loading into EVs, while biochemical modifications enable covalent conjugation of chemotherapeutic agents or siRNA to EVs surfaces for tissue-specific targeted therapy.^147,149

In the field of ED treatment, engineered stem cells and EVs have achieved remarkable progress. For example, Liu et al generated OE-miR-145-BMSCs by transfecting bone marrow mesenchymal stem cells (BMSCs) with lentivirus overexpressing miR-145, which significantly increased smooth muscle content and elevated expression of α-SMA, desmin, and SM-MHC in penile tissues of ED rats.¹⁵⁰ Kim’s team seeded mesenchymal stem cells onto nanofiber scaffolds and transplanted them around injured cavernous nerves, successfully promoting regeneration of neurons, endothelial cells, and smooth muscle.¹⁵¹ Fu et al developed a black phosphorus nanosheet-SDF1α complex (BP@SDF1-α) that effectively recruits endogenous/exogenous stem cells, markedly improving ED caused by nerve injury.¹⁵² Chen et al discovered that melatonin-pretreated MSC-EVs (MT-EVs) improved corpus cavernosum smooth muscle function via the miR-10a-3p/PKIA/RhoA/ROCK pathway.¹⁵³ Zhang’s team demonstrated that MSC-EVs enriched with miR-200a-3p alleviated oxidative stress damage in diabetic ED through the Keap1/Nrf2 pathway.¹⁵⁴ Huo et al transfected miR-21-5p into MSCs and isolated modified exosomes (miR-21-5p-MSC-Exos) that reduced apoptosis and increased proliferation of corpus cavernosum smooth muscle cells (CCSMCs), improving diabetes-induced ED and smooth muscle density.¹⁵⁵ Additionally, miR-126-modified muscle-derived stem cell EVs targeted IRS1/KLF10 to promote cavernosal repair,¹⁵⁶ while MCP-derived EVs delivering Hebp1 enhanced neurovascular regeneration.¹⁵⁷

However, direct injection of exosomes into injured areas may yield suboptimal results, as free exosomes in aqueous solutions struggle to remain localized in target regions, leading to washout or rapid clearance that prevents full biological functionality.¹⁵⁸ Therefore, effective tissue repair requires an exosome delivery vehicle with dose- and time-dependent controlled release properties, minimal adverse effects on exosome internalization, and appropriate degradation rates.¹⁵⁹ The combination of exosomes with biomaterials has partially addressed these challenges, with hydrogels being the most widely utilized. Materials such as hyaluronic acid,^144,160,161 alginate,^162,163 chitosan,^164,165 and collagen^166,167 can form stable delivery carriers through physical or chemical conjugation with EVs, significantly prolonging EV retention in target tissues.^{144,161,163,165,167,168} These biomaterial-based EV delivery systems have markedly enhanced their potential for tissue repair applications.

The development of intelligent delivery systems has further improved therapeutic outcomes. Liu et al’s injectable thermosensitive hydroxyethyl chitosan hydrogel (HG@Exo) substantially extended ADSC-Exos retention time and improved ED following nerve injury.¹⁶⁹ Zhuang’s team combined USC-EVs with hyaluronic acid (USC-EVs-HA) to effectively enhance endothelial cell function.¹⁷⁰ Liang et al designed a polydopamine nanoparticle composite hydrogel (PDNPs-PELA) that combines photoacoustic navigation with controlled release capabilities, promoting endothelial cell and neuronal regeneration while increasing intracavernosal pressure to restore erectile function.¹⁷¹

SC-EVs play a crucial role in promoting tissue regeneration, while engineered EVs strategies offer additional advantages including diversified administration routes, enhanced drug-loading capacity, reduced side effects, improved targeting precision, controllable release mechanisms, and fewer ethical concerns, demonstrating excellent clinical translation potential. Future research should further explore combinatorial applications of multiple engineered EVs platforms or their integration with other therapeutic modalities such as pharmacotherapy, physical therapy, or biomaterials to optimize treatment efficacy.

Prospects and Challenges

SC-EVs demonstrate unique therapeutic potential for ED, particularly in addressing the multifactorial pathogenesis involving vascular insufficiency, neural damage, and smooth muscle dysfunction. The primary bottleneck lies in EVs heterogeneity, where variations in cellular sources and isolation methods lead to divergent biological properties, compromising treatment predictability.^140,172 Scaling up production remains problematic, with clinical-grade EVs requiring optimization in both yield and purity.^141,148 The primary challenge lies in penile tissue-specific delivery, as the unique anatomical structure of corporal tissue presents distinct barriers for EVs homing. Current studies show only partial accumulation of intravenously administered EVs in the penile tissue, with significant off-target distribution to other organs.^173–175 The lack of definitive surface markers for corpus cavernosum endothelial cells and smooth muscle cells hampers targeted delivery design.^176,177 Moreover, current research predominantly focuses on final therapeutic outcomes, with most studies examining single-factor triggers or isolated biomarkers, failing to replicate the intricate penile microenvironment.¹⁷⁸ In addition, the unique hemodynamic environment during erection affects EVs retention, requiring specially engineered formulations.¹⁷⁸

Production challenges are particularly acute for ED applications, where clinical-grade EVs demand: Advanced biomanufacturing platforms including microfluidic systems and bioreactors show promise for scaling up clinical-grade EVs production while maintaining quality standards, higher purity standardsto avoid pro-inflammatory responses in sensitive penile tissue, specialized cryopreservation protocols to maintain bioactivity after intracavernosal injection, scalable manufacturing for chronic administration needs in ED patients.^179–181 Nanotechnology and novel biomaterials are being leveraged to improve targeted delivery precision, ensuring focused action on damaged penile tissues.^182–184 Furthermore, by optimizing electroporation, membrane fusion, 3D printing and other technical means, the loading efficiency of therapeutic molecules can be improved, immune response can be reduced and safety can be enhanced, thus enhancing the overall therapeutic efficacy and safety for ED.^185–187 Ethical considerations are being addressed through stringent review frameworks ensuring compliant cell sourcing, transparent informed consent protocols, and multi-stakeholder engagement involving scientists, ethicists, and public representatives to balance scientific progress with social responsibility.

The transition from animal models to human clinical trials represents a pivotal phase in EVs therapeutics development. While translational pathways are becoming clearer.¹⁸⁸ The clinical trial, which starts on September 18, 2024, focuses on evaluating the role of SC-EVs in ED. Participants will receive an injection directly into the penis and blood flow will be checked through a specific scoring system and ultrasound testing. Participants will undergo multiple assessments at the start and then be followed up at 3, 6, 9 and 12 months to track their progress and any side effects (ClinicalTrials.gov identifier (NCT number): NCT06605508). Large-scale clinical validation remains essential for determining optimal treatment parameters including dosage, administration routes, and long-term efficacy. These systematic investigations will facilitate the translation of SC-EVs therapies from bench to bedside, ultimately providing ED patients with safer and more effective treatment options. Future directions should emphasize standardized production protocols, comprehensive safety profiling, and development of combinatorial approaches to address ED’s multifactorial pathophysiology.

Summary

SC-EVs have demonstrated remarkable potential in the treatment of ED, particularly as an effective alternative to stem cell therapy in the fields of regenerative medicine, anti-inflammation, anti-apoptosis, and immunomodulation. By modulating multiple signaling pathways (eg, NO/cGMP, miR-10a-3p/PKIA/RhoA/ROCK, and PTEN/PI3K/Akt), SC-EVs can ameliorate various ED-related pathophysiological conditions, including cavernous nerve injury, oxidative stress-induced tissue fibrosis, and vascular endothelial dysfunction. Notably, engineered extracellular vesicles with enhanced targeting capabilities have shown improved therapeutic efficacy.

It is important to recognize that the therapeutic effects of SC-EVs vary depending on the etiology of ED. For diabetic ED, MSC-EVs and ADSC-EVs primarily exert their benefits by improving endothelial dysfunction and exerting antifibrotic effects, thereby restoring the smooth muscle/collagen ratio and NO signaling. Among these, CCSMC-EVs exhibit superior cellular uptake and longer-lasting therapeutic effects. In contrast, neurogenic ED benefits more significantly from ESC-EVs and MSC-EVs, which promote neural repair through neurotrophic factor delivery and vascular-nerve crosstalk. These differences highlight the importance of selecting EV subtypes based on the underlying pathology—antifibrotic EVs for diabetic ED and proneurogenic EVs for nerve injury-related ED—to optimize treatment outcomes. This tailored approach underscores the potential of SC-EVs as a precision medicine strategy for ED subtypes.

Despite these advances, specific challenges related to ED remain. First, the consequences of penile fibrosis often indicate that cells are processing chronic inflammatory states, and the high inflammatory state of the extracellular environment may prematurely disrupt the phospholipid bilayer of extracellular vesicles, leading to their premature lysis. Second, a hypoxic microenvironment can lead to excessive lactic acid secretion, and the extracellular environment is often acidic, which may also damage extracellular vesicles. This is similar to the limited efficacy of many oral extracellular vesicle therapies. Third, the targeting of extracellular vesicles remains inadequate. Even engineered extracellular vesicles require specific targeting molecules, but current research on penile cell surface-specific molecules is insufficient. In summary, these specificity challenges limit the biodistribution and targeted therapeutic efficacy of extracellular vesicles. Therefore, innovative technological development is needed, such as considering collagenase-activated extracellular vesicles resistant to external interference or hypoxia-targeted delivery systems, especially appropriate administration methods such as local injection or topical application. Future research should prioritize extracellular vesicles customized according to the etiology of ED, as well as multimodal treatment regimens combining extracellular vesicles with existing therapies. The focus should be on integrating engineered innovations with the complex pathophysiology of ED to redefine the treatment standards for this debilitating condition using stem cell-derived extracellular vesicles (SCs-EVs).

Funding Statement

There is no funding to report.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors declare that they have no conflict of interest.