Category: 3. Business

Oslo/Cairo, 08 December: Scatec ASA, a leading renewable energy solutions provider, has signed shareholder agreements with Norfund, the Norwegian investment fund for developing countries and EDF power solutions, an international energy player which develops, builds and operates renewable and low-carbon energy production facilities, for an equity partnership in Scatec’s 1.1GW solar and 100MW/200MWh battery storage hybrid project, Obelisk in Egypt.

“We are very pleased to continue our valuable collaborations with Norfund and EDF power solutions through these partnerships. Obelisk is Scatec’s largest project to start construction to date and combines solar and batteries to deliver stable and cost competitive renewable energy to support Egypt’s growing power demand and energy transition,” says Scatec CEO Terje Pilskog.

Following the transaction, Norfund will own 25% of the Obelisk holding company with Scatec owning the remaining 75%. EDF power solutions will own 20% of the operating company (SPV), bringing Scatec’s and Norfund’s total economic interest to 60% and 20% respectively with Scatec retaining economic control of the power plant.

Inviting equity partners at several ownership levels is part of Scatec’s strategy to enhance capital efficiency and increase value creation, while retaining control of the power producing entities. Scatec is in advanced discussions with additional equity partners, aiming to reduce Scatec’s economic interest in the project further.

For further information, please contact:
For analysts and investors:
Andreas Austrell, SVP IR
andreas.austrell@scatec.com
+47 974 38 686

For media:
Meera Bhatia, SVP External Affairs & Communications
meera.bhatia@scatec.com
+47 468 44 959

About Scatec 
Scatec is a leading renewable energy solutions provider, accelerating access to reliable and affordable clean energy in emerging markets. As a long-term player, we develop, build, own, and operate renewable energy plants, with 6.2 GW in operation and under construction across five continents today. We are committed to growing our renewable energy capacity, delivered by our passionate employees and partners who are driven by a common vision of ‘Improving our Future’. Scatec is headquartered in Oslo, Norway and listed on the Oslo Stock Exchange under the ticker symbol ‘SCATC’. To learn more, visit www.scatec.com or connect with us on LinkedIn.

This information is subject to the disclosure requirements pursuant to Section 5-12 the Norwegian Securities Trading Act

Introduction

When mitochondria are mentioned, their name comes to mind naturally as “powerhouse”. As is well known, they are semi-autonomous organelles enclosed by two membranes, possessing own genetic material and found in most eukaryotic cells. Mitochondria provide adenosine triphosphate (ATP) to cells and serve as the primary site for aerobic respiration, with a diameter of approximately 0.5 to 1 micrometer.^1,2 ATP production occurs through oxidative phosphorylation (OXPHOS) and the tricarboxylic acid (TCA) cycle. In the inner mitochondrial membrane, the electron transport chain (ETC) transfers electrons from reduced coenzymes (NADH and FADH₂) to oxygen, driving proton pumps to form a transmembrane proton gradient, which ultimately synthesizes ATP through ATP synthase.^3,4 Mitochondria are not only the centers of energy metabolism but also participate in various cellular processes such as calcium ion homeostasis, apoptosis, and reactive oxygen species (ROS) regulation.^5,6 They play a dual role in maintaining physiological homeostasis and mediating pathological damage, thereby determining the survival and death of cells. Mitochondrial dysfunction, characterized by oxidative stress, abnormal energy metabolism, and impaired apoptosis, is a key pathological mechanism in neurodegenerative diseases, cancer, and cardiovascular and kidney disorders. Under pathological conditions, ETC dysfunction leads to energy crises and ROS surges, while mitochondrial dynamics imbalance (abnormal fusion/fission) and autophagy defects contribute to neurodegeneration. Studies have shown that the frequency and duration of the mitochondrial permeability transition pore (mPTP) opening are correlated with increased ROS production, electron leakage, and a reduction in mitochondrial membrane potential (MMP), collectively precipitating cellular apoptosis.⁷ Zhou et al discovered that the transcription factor Nynrin is highly expressed in hematopoietic stem cells, with further upregulation under stress conditions such as radiation. Nynrin serves as a key regulator in maintaining HSCs by modulating mitochondrial function. Deficiency of Nynrin leads to a reduction in HSC numbers, loss of quiescence, impaired self-renewal capacity, and mitochondrial dysfunction characterized by increased mPTP opening, mitochondrial swelling, and elevated ROS levels.⁸ Strategies targeting MMP restoration, mtDNA protection, regulate the opening of mPTP, and quality control pathway activation offer novel therapeutic avenues.⁹

Central nervous system (CNS) diseases, including neurodegenerative diseases, brain tumors, and cerebrovascular lesions, pose significant global public health challenges due to their high disability and mortality rates.^10–12 The common pathological features of these diseases are closely related to mitochondrial dysfunction. For instance, mitochondria in the brain of patients with Alzheimer’s disease (AD) differ significantly in number and morphology from those in healthy individuals.¹³ Amyloid-β (Aβ) oligomers in the brains of AD patients induce abnormal opening of the mPTP, leading to calcium overload and neuronal apoptosis.^14,15 In Parkinson’s disease (PD), abnormal α-synuclein aggregation disrupts mitophagy, leading to accumulated damaged mitochondria in dopaminergic neurons.^16,17 Post-stroke ischemia-reperfusion generates excessive ROS that directly damages mtDNA, exacerbating neuronal death.^18,19 These findings collectively underscore mitochondria as central targets for treating CNS diseases.

Nano-drug delivery systems have emerged as key tools for CNS therapy, enhancing drug bioavailability and retention while enabling site-specific delivery via modifications with peptides, membranes, or microenvironment-responsive ligands.^20,21 Notably, due to the presence of blood-brain barrier (BBB), over 98% of small-molecule drugs and nearly all large-molecule therapeutics struggle to effectively reach brain lesions. Therefore, precise mitochondrial interventions in CNS diseases face dual challenges, including overcoming the BBB’s biological bottleneck, and further achieving subcellular-level precision delivery.²² Most previous studies have reviewed the strategies for nanomaterials crossing the BBB, which could be achieved via carrier, receptor or cell-mediated transcytosis, as well as some external modulation strategies.^23–25 Mitochondria-targeted nanosystems are emerging as promising therapeutic approaches for mitochondria-related disorders treatment.^26–28 These nanosystems have the potential to directly deliver drugs to mitochondria, reduce oxidative stress of mitochondria, and restore energy homeostasis.²⁹

This review discusses the design principles and applications of mitochondria-targeted nanosystems, with a focus on their use in CNS diseases (Figure 1). We aim to provide insights for researchers in mitochondrial-targeted therapy and inspire future designs of precision delivery platforms.

Figure 1 Schematic illustration of mitochondria-targeted nanosystems in CNS disorders. (A) Mitochondrial function and mitochondria-targeted approaches. (B) The microenvironment of mitochondria-related CNS diseases. (C) The role of mitochondria-targeted nanosystems in regulating the mitochondria function. Created with BioRender.com.

Design Principles and Applications of Mitochondria-Targeted Nanosystems

Mitochondria have a double-membrane structure consisting of the outer mitochondrial membrane (OMM) and inner mitochondrial membrane (IMM). Both membranes are primarily composed of proteins and lipids. The space between them is termed the inter-membrane space (IMS), while the IMM is subdivided into the inner boundary membrane (IBM), cristae junctions (CJs), and cristae.³⁰ Porins (eg, VDAC) are abundant on the OMM, allowing free diffusion of substances <5 kDa. In contrast, the IMM exhibits extremely low permeability and high tortuosity with only specific molecules (such as O₂, CO₂) passively diffusing while other substances rely on transport proteins. Located in the IMM, the mPTP is a non-selective channel formed by large protein complexes. It critically regulates apoptosis—aberrant opening mediates leakage of pro-apoptotic factors (eg, Cytochrome C/Cyt C) from the IMS into the cytoplasm, activating apoptotic cascades. The IMM also houses oxidative phosphorylation machinery and is enriched with cardiolipin, a phospholipid essential for OXPHOS complex stability.³¹

Mitochondria-targeted nanosystems usually employ four key strategies, namely (1) small-molecule ligands (eg, triphenylphosphonium/TPP), which exploit the IMM’s negative membrane potential to accumulate drugs in the matrix; (2) peptide ligands that leverage lipophilicity/cationic properties; (3) mitochondrial membrane-derived vesicles; and (4) biomimetic membrane systems. The latter two utilize natural membrane components (eg, proteins, lipids) for intrinsic homing to mitochondria. Herein, we discuss the design principles of these nanosystems and summarize their applications (Table 1).

Table 1 Representative Mitochondria-Targeted Groups

Small Molecules

TPP

Owing to the transmembrane potential (Δψm) of −150 to −180 mV (matrix negative relative to cytoplasm) across the IMM, lipophilic cations accumulate in the mitochondrial matrix via electrophoresis. Their delocalized surface charge enables lipid bilayer penetration, driving selective enrichment via the electrochemical gradient.^32,60

Meanwhile, the unique electrochemical properties of mitochondria provide feasibility for the targeted delivery of antioxidant molecules. This is achieved by chemically coupling therapeutic molecules with lipophilic cations, which enables selective accumulation through the membrane potential of mitochondria, thereby establishing a specific protective mechanism against mitochondrial oxidative damage.^32,61 TPP, as a targeting moiety, delivers various antioxidants into mitochondria and accumulates within mitochondrial organelles. As one of the most common mitochondria-targeted molecules, TPP is an organophosphate compound with a lipophilic cation that can effectively target mitochondria through electrostatic interactions. The key mechanism behind its targeting strategy stems from the electrochemical characteristics of the IMM.⁶² So far, research on using TPP as a mitochondria-targeted modification has been extensive. TPP can be modified on various nanocarriers such as liposomes, inorganic nanoparticles, micelles, and other polymer nanoparticles. A common strategy for modifying liposomes with TPP involves synthesizing TPP-PEG-PE or TPP-PEG-DSPE first to improve the stability and targeting efficiency of the carrier by coupling it with other groups. When inorganic nanoparticles are used as carriers, the modification methods usually include direct coupling and indirect modification techniques. Inorganic nanoparticles featuring -COOH, -NH₂, or -SH groups on their surface are suitable for direct coupling. For example, Xia et al³³ utilized TPP coupled with DSPE-TK-PEG2000 to achieve mitochondrial targeting and demonstrated ROS-responsive effects via the thioketal (TK) bond. They developed a drug delivery system using nanoliposomes to deliver tripterygium glycoside to mitochondria for treating hepatocellular carcinoma. It was shown that modifying the liposome surface with TPP-PEG2000-TK-DSPE could significantly enhance the aggregation ability of these liposomes in mitochondria. Li et al synthesized TPP-modified micelles loaded with puerarin for the treatment of acute myocardial infarction, they likewise began by synthesizing the mitochondriotropic polymer TPP-PEG-PE. Compared to free puerarin, these micelles exhibited enhanced cellular uptake, improved mitochondrial targeting, and increased lysosomal escape.³⁴ However, this method requires multi-step organic synthesis, more complex preparation processes and higher costs. In Gu et al’s study³⁵ bimetallic cluster nanozymes were employed to reduce neuropathic pain. TPP was pre-modified to TPP-COOH and then covalently linked to the surface of bimetallic cluster nanozymes. Covalent bonds give it high stability and prevent TPP from detaching in the internal environment. Nevertheless, it is important to note that TPP is inherently a cationic species. High-density TPP modification also carries potential toxicity, as it may disrupt mitochondrial membrane integrity, thereby impairing the respiratory chain and ATP synthesis. This cascade of events can ultimately lead to cellular dysfunction or apoptosis. Furthermore, TPP may exhibit metabolic instability in vivo and has a limited ability to cross the blood-brain barrier, posing a significant challenge for treating brain disorders.^63,64

Besides TPP, other antioxidants, including ubiquinol, alpha-tocopherol, vitamin C, metformin, and tempol, have also been investigated.^32,65,66 The combination of lipophilic cations and antioxidants is applied in the treatment of various diseases, such as chronic obstructive pulmonary disease, traumatic brain injury, inflammatory bowel disease, cardiovascular diseases, renal ischemia-reperfusion injury, PD, AD.^67–73

Other Cationic Targeting Molecules

Rhodamine, initially discovered as a dye in 1887, was later utilized as a probe.⁷⁴ Rhodamine 123 belongs to the rhodamine dye family and is a lipophilic cationic compound that accumulates in mitochondria in a mitochondrial membrane potential-dependent manner, serving as a fluorescent probe for monitoring membrane potential in living cells. Later, it was also gradually used for mitochondria-targeted modifications. Similar to TPP, it can be linked to drugs through chemical coupling or modified liposomes, polymer nanoparticles, and other carriers in a similar fashion.^36,37,75,76

Another derivative of rhodamine, tetramethyl rhodamine methyl ester (TMRM), functions similarly to Rhodamine 123. Its structure includes a tetramethyl-substituted rhodamine skeleton with methyl ester groups. As a cationic dye, TMRM enriches in the mitochondrial matrix with a higher negative potential through electrochemical gradients, and its fluorescence signal can quantitatively reflect changes in MMP.^40,77–79 TMRM can be conjugated to other molecules, such as polymers and drugs, through functional group reactions to form functional probes or drug delivery systems. Additionally, F16 and its analogs are used for mitochondrial tracing and serve as mitochondria-targeted anticancer agents, primarily effective against cancer.^80–82 F16 is mainly conjugated to drug molecules through chemical synthesis, thereby enhancing its mitochondrial targeting and antitumor activity.^41,83

Moreover, dequalinium (DQA) is another cationic mitochondria-targeted molecule—a bis-quaternary ammonium compound with two positively charged quaternary ammonium groups and a hydrophobic carbon chain, combining hydrophilicity with hydrophobicity. DQA can be used in nanoemulsions or liposome assemblies.^84–86 A distinctive application of DQA is its ability to self-assemble in aqueous solution to form vesicular structures known as DQAsomes, which are used for the delivery of genes or small-molecule drugs.⁸⁷ DQA can be covalently linked directly with lipids, such as DSPE-PEG, modified on the surface of liposomes, and form mitochondria-targeted carriers. In polymer nanoparticles, such as polylactic acid-glycolic acid copolymer, DQA binds to the carriers through electrostatic interactions or chemical coupling.^43,88 Although these small molecules exhibit good mitochondria-targeting abilities, their potential toxicity due to their cationic properties limits their further applications.⁸⁹

Mitochondria-Targeted Peptides

SS Peptides (with SS-31 as a Representative)

Mitochondria-targeted peptides (MTPs) are short peptides engineered to deliver therapeutics or modulate mitochondrial functions. Their design leverages mitochondrial membrane properties or natural targeting signals. Szeto-Schiller (SS) peptides, exemplified by tetrapeptides with alternating aromatic (eg, phenylalanine and tyrosine) and basic (eg, lysine and arginine) residues, exhibit potent molecular recognition and membrane penetration.⁹⁰ They specifically target the IMM via lipophilic-cationic structures and confer mitochondrial protection through dual mechanisms.⁹¹ On the one hand, they can directly eliminate mitochondrial ROS and inhibit their excessive generation. On the other hand, SS peptides can maintain mitochondrial membrane integrity by blocking pathological mPTP opening. Critically, SS peptides mitigate apoptosis and necrosis triggered by oxidative stress or ETC dysfunction,demonstrating efficacy in ischemia-reperfusion, neurodegeneration (AD/PD), cognitive deficits, and renal fibrosis models with high biocompatibility.^91,92 Szeto-Schiller Peptide 31 (SS-31, also known as MTP-131, elamipretide), a representative aromatic cationic peptide, selectively binds cardiolipin on the IMM via electrostatic interactions driven by its alternating cationic/aromatic structure.^58,93–96 This targeting mechanism operates independently of strict membrane potential dependence. SS-31 is frequently conjugated to nanocarriers (eg, liposomes, micelles, or polymer nanoparticles) to enhance mitochondrial targeting efficiency through the synergistic effect of passive targeting of nanocarriers (eg, enhanced permeability and retention effect) and active targeting of SS-31.^97–99 Methods for conjugating it to nanocarriers primarily include covalent connection and hydrophobic insertion. For example, in polymer nanoparticles, SS-31 can be connected to the particle surface through chemical coupling of amino or carboxyl groups (such as EDC/NHS reaction). It is also possible to covalently link the cysteine residues on SS-31 with DSPE-PEG₂₀₀₀-Mal (maleimide group) in the liposome formulation through thioether bonds.^{48,49,100,101} SS-31 has a favorable safety profile and is considered a promising strategy for the treatment of diabetic cardiomyopathy. Xiong et al revealed a novel mechanism of mitochondria-targeted peptide SS-31 for the treatment of diabetic cardiomyopathy (DCM): SS-31 inhibits mitochondrial lipid peroxidation and attenuates mitochondria-dependent ferroptosis through specific activation of mitochondrial glutathione peroxidase 4 (mitoGPX4). In a diabetic cardiomyopathy model, high glucose and metabolic disorders lead to enhanced mitochondrial oxidative stress, imbalanced iron metabolism, and reduced mitoGPX4 activity, which in turn triggers ferroptosis in cardiomyocytes. SS-31 restored the antioxidant function of mitoGPX4 by targeting mitochondria and stabilizing the structure of cardiolipin, reduced mitochondrial ROS and lipid peroxidation accumulation, as well as inhibited the expression of key markers of ferroptosis, thereby protecting cardiomyocytes from ferroptosis-driven injury. The experimental results showed that SS-31 significantly improved cardiac function, attenuated myocardial fibrosis and mitochondrial structural disruption in diabetic mice, and provided a novel strategy for targeting mitochondrial ferroptosis in the treatment of DCM.¹⁰² In addition, Zhu et al summarized that, as a mitochondria-targeted antioxidant, SS-31 alleviates oxidative stress by inhibiting the overproduction of mitochondrial ROS, repairing the function of the mitochondrial electron transport chain to enhance ATP synthesis, inhibiting mitochondrial membrane permeability transition pore opening and down-regulating inflammatory factors expression, thereby reducing oxidative stress damage to renal tubular epithelial cells and podocytes, as well as attenuating renal inflammatory responses, suggesting that SS-31 may be a candidate for the treatment of mitochondria-associated nephropathy, providing a new direction in the treatment of nephropathy.¹⁰³ Currently, SS-31 has been widely used in the treatment of mitochondrial dysfunction-related diseases in various systems, such as the nervous and cardiovascular systems. Du et al systematically explored the mechanism of action and efficacy of the novel mitochondria-targeted antioxidant peptide SS-31 in alleviating mitochondrial dysfunction, highlighting the broad prospects of SS-31 as a therapeutic tool for precisely regulating mitochondrial function in the treatment of mitochondrial-related diseases.¹⁰⁴ Furthermore, many nano-delivery systems utilize the mitochondria-targeting ability of SS-31 to deliver drugs, genes or probes to the mitochondria in a targeted manner, which synergistically enhances the repairing effect on mitochondrial dysfunction with the delivered agents, reduces systemic toxicity and improves the bioavailability.^{49,50,105–107}

XJB-5-131

XJB-5-131 is also a synthetic peptide and a mitochondrial-targeted antioxidant. Its structural design combines mitochondrial targeting and free radical scavenging functions. The molecular structure of XJB-5-131 is composed of two functional modules linked by chemical bonds. The mitochondrial-targeting moiety is a cationic oligopeptide structurally inspired by gramicidin S. It targets the negatively charged regions of the mitochondrial inner membrane via positively charged amino acid residues and binds to phosphatidylserine in the mitochondrial inner membrane through electrostatic interactions. The antioxidant active part is composed of nitroxide radicals. Usually, the targeted peptide is coupled with the antioxidant group through ester bonds or amide bonds to ensure the structural stability and functional synergy.^{54,55,108,109} It targets mitochondria more rapidly and does not require a carrier, showing low toxicity and little interference with MMP. Besides, it is mainly utilized in relation to oxidative stress diseases, with its application in drug delivery being less explored and requiring further development.¹¹⁰ It has been shown, XJB-5-131 is a novel mild oxidative phosphorylation uncoupler that partially disrupts the proton gradient in the IMM, thereby reducing ATP synthesis efficiency and increasing heat production, thus regulating mitochondrial energy metabolism.⁵⁴ Unlike potent uncoupling agents, XJB-5-131 exhibits controllable uncoupling effects at low doses, reducing ROS accumulation and alleviating oxidative stress damage while avoiding side effects such as excessive heat production and energy depletion. Experiments have shown that this compound selectively acts on mitochondrial membrane structures to improve cellular energy metabolism disorders and has demonstrated protective potential in models of neurodegenerative diseases and ischemic reperfusion injury. Its “mildness” makes it safer for treating metabolic syndrome and age-related diseases, but further research is needed on its long-term effects, tissue specificity, and clinical translation potential. Sun et al found that the mitochondria-targeting compound XJB-5-131 restored Pebp1 protein expression in chondrocytes, inhibiting ferroptosis (reducing lipid peroxidation products and upregulating GPX4 activity), thus protecting chondrocytes and alleviating osteoarthritis (OA) progression; in vivo experiments showed that XJB-5-131 joint injection significantly reduced cartilage degeneration and inflammation in mouse OA models with a favorable safety profile, providing a new strategy for OA treatment targeting ferroptosis.¹¹¹

Mitochondrial-Penetrating Peptides, MPPs

Mitochondrial-penetrating peptides (MPPs) have been extensively studied as a means of mitochondrial uptake. MPPs are synthetic mitochondria-targeted peptides whose typical structure consists of 4–16 amino acid residues, which typically contain positively charged basic and hydrophobic amino acids. Studies have shown that the mitochondrial targeting efficiency of MPPs is closely related to their surface positive charge properties and hydrophobicity. MPPs, as a type of short peptides with the ability to penetrate both cellular and mitochondrial membranes, present diversified strategies in their mechanism of action and structural design. Innovative strategies based on the structural similarity between mitochondria and bacterial membranes have attracted attention, such as semi-Grammycin analogues designed to simulate Grammycin S. MPPs relies on the characteristics of positively charged amino acids and achieves delivery through interaction with negatively charged mitochondrial membranes, which can enhance the precise accumulation of drugs in mitochondria and reduce off-target effects.^27,32,112 Zhou et al successfully developed a polymer-MPP-doxorubicin (P-M-Dox) coupling that achieved specific mitochondrial delivery through mitochondria-targeting properties and significantly improved doxorubicin delivery efficiency by circumventing the P-gp efflux pump effect.¹¹³

Advantages and Challenges of MTPs

Peptides targeting mitochondria have advantages such as high targeting efficiency, biocompatibility and stability, and avoiding the risk of genomic integration.^114–116 However, several concerns should be addressed issues. The mitochondrial targeting of some peptides mainly depends on the electrochemical potential of the electron transport chain, which may fail under pathological conditions (such as reduced membrane potential). Additionally, peptides tend to dissociate after binding to mitochondria, affecting sustained action. Off-target effects may also occur, as peptides may non-specifically bind to other negatively charged cellular structures or interfere with normal mitochondrial function. Furthermore, large-scale production is challenging.^112,115,117 There is still room for improvement in terms of structure, joint delivery systems, and dynamic response design.^96,118,119

Mitochondria-Targeted Vesicles and Mitochondrial Membrane Mimetic Systems

MITO-Porter is a liposome-based mitochondrial targeting delivery system that encapsulates drugs in double-layer liposomes and is surface-modified with mitochondrial targeting ligands (such as mitochondrial signaling peptides or membrane fusion-related lipids), to achieve mitochondrial delivery.⁵⁷ The MITO-Porter system has been widely utilized in the field of mitochondrial targeting.¹²⁰ The core principle behind MITO-Porter’s mitochondrial targeting lies in achieving specific delivery through the membrane fusion mechanism of lipid nanoparticles. It adopts a bilayer liposome structure, where surface-modified ligands (eg, octaarginine/R8) mediate binding to the outer mitochondrial membrane (OMM), followed by liposome-OMM fusion, which directly releases the cargo into the intermembrane space.^121–123 This mechanism overcomes the physical barrier posed by the mitochondrial bilayer membrane structure and avoids lysosomal degradation via the endocytosis pathway.²⁶ Yuma Yamada et al demonstrated mitochondrial gene therapy using MITO-Porter-encapsulated tRNA nanoparticles composed of DOPE, sphingomyelin, and stearylated R8 at a molar ratio of 9:2:1.¹²⁴ However, its poor stability, limited drug loading capacity, and difficulty in delivering hydrophilic drugs restrict further applications.⁵⁸

Biomimetic cell membrane encapsulation technology, which leverages the integration of natural membrane components and targeting mechanisms, has emerged as a promising approach to address these limitations.^125–127 Specifically, biomimetic cell membranes can confer immune evasion, prolonged circulation, and precise targeting capabilities on nanoparticles, thereby significantly enhancing delivery efficiency and reducing off-target effects.¹²⁸ This biomimetic design not only retains the characteristics of natural membranes but also overcomes the spatial resolution limitations of traditional delivery systems through biocompatible transport pathways.^129–132 Compared to whole cell membranes, mitochondria-derived membrane-coated nanosystems can deliver therapeutic molecules to mitochondria more efficiently, enabling precise subcellular-level drug delivery through membrane fusion-mediated intracellular transport mechanisms.⁵⁸ The extraction of mitochondrial membranes from cells typically involves two steps: first, cells are homogenized using a glass homogenizer; then, mitochondria are isolated via gradient centrifugation. Finally, mitochondrial membranes are obtained by ultracentrifugation at low temperature.^59,133 By leveraging the natural properties of mitochondrial membranes to achieve homologous targeting, existing research has explored methods for directly wrapping nanoparticles with membranes to enhance targeting, including co-extrusion and sonication.^134–137 Chen et al reported a mitochondrial-mimicking therapeutic strategy capable of specifically targeting dilated cardiomyopathy and effectively regulating mitochondrial homeostasis.¹³⁸ They synthesized nano-micelles (TPTN) and achieved mitochondrial targeting in cardiomyocytes by encapsulating the micelles with OMM-derived vesicles. The surface of MTPTN is functionalized with OMM-specific proteins, which enable direct homophilic binding to their counterparts on the mitochondrial membrane of cardiomyocytes. This membrane-membrane recognition mechanism significantly enhances targeting specificity. In addition, by combining two types of membranes through methods such as sonication, extrusion, or a combination of both, multifunctional hybrid membranes can be constructed that integrate the inherent properties of both components.^139,140 Shi et al developed hybrid membranes using cancer cell membranes and mitochondrial membranes to achieve dual targeting.⁵⁹ Mitochondrial membrane proteins integrated into the hybrid membranes—such as the outer membrane protein TOM20 and mitochondrial fusion-related proteins—mimic the natural surface features of mitochondria, promoting either membrane fusion or endogenous uptake between nanoparticles and mitochondria, thereby enabling subcellular organelle-level targeting. The inner and outer mitochondrial membranes contain various proteins and lipids that can be used for homologous targeting and lipid fusion. Targeted delivery using membrane-coated nanocarriers or membrane-fused liposomes can facilitate homologous targeting via antibody-antigen interactions.

Moreover, in addition to effectively deliver the therapeutics to mitochondria, some vesicles also have the property to rescue the mitochondrial dysfunction. Deng et al have proposed that the extracellular vesicles collected from mesenchymal stem cells (MSCs) with 3D dynamic culture could improve the proliferation ability and cell viability of aging-gingival MSCs, as well as ameliorate senescence. This effects was attributed to the up-regulated genes which were related to the mitochondrial dynamics, cell cycle and DNA repair functions.¹⁴¹

Mitochondria-Targeted Nanosystems for CNS Disorders

Given the central role of mitochondria in cellular energy homeostasis, mitochondria-targeted nanosystems show therapeutic potential for mitochondrial-related pathologies such as cancer, cardiovascular diseases, and brain disorders.^142,143 The substantial accumulation of nanoparticles in a subset of mitochondria disrupts mitochondrial network connectivity by inducing fragmentation, impairs calcium buffering capacity through persistent mPTP opening, and activates retrograde signaling to the nucleus via released ROS and Mitochondrial DNA (mtDNA). This mechanism can be therapeutically harnessed to either trigger apoptosis in cancer cells or protect neurons in neurodegenerative diseases by modulating mitochondrial function.^144,145 These nanosystems exert therapeutic effects by modulating energy metabolism, targeting mitochondrial DNA, regulating ROS, directing mitochondrial proteins, exerting anti-inflammatory effect, or enhancing mitophagy.^146,147 Compared with other organs, the brain is more sensitive and reliant on energy produced by mitochondria. Additionally, nearly 15% of global deaths are closely related to mitochondrial damage in brain diseases.¹⁴⁸ This section discusses applications of mitochondrial-targeted nanosystems in CNS disorders, including neurodegenerative diseases, brain tumors, and ischemic stroke (Table 2).

Table 2 Mitochondria-Targeted Nanosystems in CNS Disorders

Before achieving the mitochondria of CNS-related cells, these nanosystems should bypass the first barrier of BBB. Recently, a variety of transport therapeutics have been developed for enhancing the BBB crossing ability of nanosystems, including adsorptive-mediated transcytosis, carrier-mediated transcytosis, receptor-mediated transcytosis, cell-mediated transcytosis, and modulation of tight junction.^180,181 However, the specific diseased BBB-based brain-targeting strategies should be designed based on the BBB evolutions of CNS diseases, which owing to that the diseased BBB of various CNS disorders is different and complex.

Neurodegenerative Diseases

Neurodegenerative diseases, including AD, PD, Huntington’s disease (HD), and Amyotrophic Lateral Sclerosis (ALS), are a group of heterogeneous disorders caused by the progressive degeneration of neurons.¹⁸² Mitochondrial dysfunction is closely associated with the progression of neurodegenerative diseases, leading to oxidative stress and impaired neuronal metabolic activity.¹⁸³ Consequently, mitochondria represent a promising target for therapies aimed at mitigating neurodegeneration. In recent years, various mitochondria-targeted nanosystems have been developed to modulate mitochondrial functions in neurodegenerative diseases, such as regulating calcium ion transport, delivering antioxidants, chelating metals, and modifying proteins.¹⁸⁴

AD

As the most common neurodegenerative disease, AD is characterized by pathological aggregation of Aβ plaques and hyperphosphorylated Tau neurofibrillary tangles, as well as abnormally elevated ROS, which are primarily produced in mitochondria.¹⁸⁵ Notably, hyperphosphorylated Tau exacerbates mitochondrial damage by generating excessive ROS and promoting mitochondrial fission. Conversely, mitochondrial oxidative stress also promotes abnormal phosphorylation and aggregation of Tau protein.¹⁸⁶ Hence, targeting mitochondria and alleviating mitochondrial dysfunction are critical strategies for treating AD.

Various mitochondria-targeted nanosystems have been employed in AD treatment, designed with mitochondria-targeting peptides (such as TPP and SS-31) and loaded with therapeutic agents (eg, curcumin, resveratrol, methylene blue, etc).^{149–153,155,186} Ceria (CeO₂) nanoparticles, widely studied for their potential ROS-scavenging activity through shuttling between Ce³⁺ and Ce⁴⁺ oxidation states, have been modified to target mitochondria. Kwon et al designed TPP-conjugated CeO₂ nanoparticles (TPP-ceria NPs) that could specifically target mitochondria and suppress neuronal death in an AD mouse model.¹⁵⁶ After TPP modification, significantly higher mitochondrial localization was observed in SH-SY5Y cells treated with FITC-TPP-ceria NPs compared to FITC-ceria NPs. Moreover, TPP-ceria NPs markedly inhibited Aβ-induced mitochondrial ROS production and reduced reactive gliosis and morphological mitochondrial damage, demonstrating that mitochondrial therapeutics hold promise for AD treatment. Li et al have also utilized TPP to modify tetrahedral DNA framework-based nanoparticles (TDFNs) for mitochondria-targeting, as well as cholesterol for BBB crossing and antisense oligonucleotide (ASO) for diagnosis and gene silencing therapy of AD.¹⁸⁷ The results showed the efficient colocalization of TDFNs and MitoTracker Red in neuron cells, demonstrating the superior mitochondia-targeted ability of TDFNs.

In contrast to TPP, the SS-31 peptide not only targets neurons and mitochondria but also directly alleviates mitochondrial dysfunction and oxidative damage.¹⁸⁸ Specifically, SS-31 has shown beneficial effects in treating AD^189–191 and has been conjugated with cyclosporin A (CsA) via a TK bond (Figure 2).⁹⁸ The resulting self-assembled micelles (CsA-TK-SS-31, abbreviated as CTS) efficiently targeted the mitochondria of damaged neurons and microglia driven by SS-31. Upon exposure to high levels of ROS within mitochondria, the TK bond breaks, releasing CsA and SS-31. The CTS micelles demonstrated enhanced mitochondrial targeting ability and alleviated neuronal mitochondrial dysfunction and neuroinflammation in 5×FAD mice by restoring mitochondrial function and increasing anti-inflammatory cytokines.

Figure 2 CTS micelle-based mitochondria-targeted nanosystems for AD. (A) Schematic illustration of the construction, targeting, and therapeutic mechanisms of CTS micelles for AD treatment. (B) Responsive release profile of CsA from CTS micelles. (C) Intracellular mitochondrial superoxide levels and (D) mitochondrial membrane potential following various treatments in Aβ1-42-damaged primary mouse neurons. Cellular uptake of FITC-labeled CTS (FITC@CTS) in both normal and Aβ1-42-damaged (E) BV2 or (F) SH-SY5Y cells as analyzed by flow cytometry. Confocal laser scanning microscopy (CLSM) images showing the colocalization of FITC@CTS with mitochondria in (G) BV2 or (H) SH-SY5Y cells. Reproduced with permission.98 Copyright 2024, Wiley-VCH.

Furthermore, mitochondrial targeted fluorescence probe (Mito-DDP) was also designed by Li et al.¹⁵⁴ The results of confocal images have demonstrated that Mito-DDP could effectively penetrate the membrane and target to mitochondria. Besides, this probe has the effect of viscosity sensing, including mitochondrial malfunction, cellular inflammation and AD models, providing a novel strategy for exploring the pathological role of viscosity in AD.

PD

PD, the second most common neurodegenerative disorder after AD, involves dopaminergic neuron degeneration and α-synuclein (α-syn) aggregation.¹⁶¹ Mitochondrial dysfunction drives pathogenesis through impaired mitophagy and ROS overproduction.¹⁹² Critically, α-syn accumulation induces cardiolipin exposure on the OMM, disrupting respiratory chain complexes, while mtROS promote α-syn oligomerization, forming a self-amplifying pathological loop.¹⁹³

Based on the promising mitochondria-targeting properties of the TPP peptide in AD therapy, several studies have explored its use for PD treatment due to its efficient targeting capability. For example, Zhang et al constructed ursodeoxycholic acid (UDCA)-loaded macrophage-derived nanovesicles (NVs) modified with TPP (UDCA-NVs-TPP) to combat PD.¹⁵⁷ UDCA-NVs-TPP could efficiently target damaged neuronal mitochondria and restore mitochondrial dysfunction by mitigating oxidative stress, providing a promising therapeutic strategy for PD treatment. Additionally, TPP was utilized to confer mitochondria-targeting properties to an iron (Fe)-isolated single-atom nanozyme (Fe-ISAzyme) liposome (Mito@Fe-ISAzyme, MFeI).¹⁶² Cells treated with MFeI showed higher enrichment in mitochondria compared to Fe-ISAzyme, increasing mitochondrial numbers and improving therapeutic outcomes. Furthermore, sequence-targeted nanodots (TPP-rHuHF-LYC) were created by loading lycopene into recombinant human H-ferritin (rHuHF) and coupling it with TPP as the mitochondria-targeting peptide.¹⁵⁸ TPP-rHuHF-LYC efficiently targeted neuronal mitochondria via receptor-mediated transcytosis at the BBB facilitated by rHuHF and the mitochondria-targeting ability of TPP. Results indicated that TPP-rHuHF-LYC could repair mitochondrial function by enhancing mitophagy and alleviate pathological deterioration, significantly improving the therapeutic efficiency for PD. Similarly, Zheng et al designed ultrasmall Cu_2-xSe nanoparticles carrying curcumin, encapsulated within DSPE-PEG₂₀₀₀-TPP-modified macrophage membranes (CSCCT NPs).¹⁵⁹ With focused ultrasound assistance and the targeting role of TPP, CSCCT NPs specifically targeted the mitochondria of inflammatory neurons, effectively scavenging mitochondrial ROS and promoting mitochondrial biogenesis, demonstrating a promising PD therapy strategy. Moreover, nanosystems without mitochondria-targeting peptide modifications have also shown effective mitochondrial targeting. Single-atom catalysts Pt/CeO₂ were developed for PD treatment by inducing autophagy.¹⁶⁰ After 4 h of Pt/CeO₂ treatment, mitochondria in mouse midbrain dopaminergic neuronal cells (MN9D) exhibited the highest colocalization coefficient with the nanomaterials, displaying a time-dependent increase. Interestingly, this colocalization decreased significantly from 0.692 to 0.347 when MN9D cells were pre-incubated with carbonyl cyanide m-chlorophenylhydrazone (CCCP), which reduces mitochondrial membrane potential. These findings indicate that the mitochondria-targeting property of Pt/CeO₂ is mainly due to electrostatic attraction between the positively charged surface of Pt/CeO₂ and the negatively charged mitochondria. Furthermore, coating with a neutrophil membrane and modifying with RVG29 enabled RVG29@AHM@Pt/CeO₂ to cross the BBB effectively, break down mitochondrial ROS, and induce mitophagy for PD treatment, as well as improve the locomotor ability of PD mice (Figure 3).

Figure 3 RVG29@AHM@Pt/CeO2-based mitochondria-targeted nanosystems for PD. (A) Schematic illustration of the construction, targeting, and therapeutic mechanisms of RVG29@AHM@Pt/CeO2 for PD treatment. (B) TEM image of cells after Pt/CeO2 coincubation for 4 h. (C) Mechanism of Pt/CeO2 targeting of mitochondria. (D) Mitochondria colocalization images of Pt/CeO2 cultured with MN9D cells for 0, 1, 2 and 4 h. (E) ROS levels of cells. (F) Fluorescent images of the head of PD mice. (G) Latency to fall during the rotarod test of mice. (H) Delayed time to climb down during the pole test. Reproduced with permission.160 Copyright 2023, American Chemical Society.

In summary, mitochondria-targeted nanosystems represent promising strategies for treating neurodegenerative diseases through either mitochondria-targeting peptide modifications or the positive surface charge of nanosystems. Although positively charged nanosystems facilitate cellular uptake and permeability, their cytotoxicity and short blood circulation times must be considered.^194,195 Further efforts should focus on enhancing the targeting capabilities and therapeutic outcomes of these nanosystems.

Brain Tumor

Due to the critical role of mitochondria in energy supply, cell differentiation, and growth, numerous therapeutic approaches have been designed to target mitochondria for precise tumor treatment by inducing mitochondrial dysfunction, such as decreasing ATP level, disrupting redox balance, depolarizing the mitochondrial membrane potential, interfering with ion homeostasis, and dysregulating mitophagy.¹⁴² Hence, developing mitochondria-targeted nanosystems represents an effective strategy for cancer treatment by causing intrinsic apoptotic cell death.¹⁹⁶ Glioma, especially glioblastoma multiforme (GBM), is a malignant brain tumor characterized by rapid proliferation, high recurrence rates, and high mortality. Recently, mitochondria-targeted nanosystems have also been developed for the treatment of glioma or GBM, which can be achieved through mitochondria-targeted peptides or modifications of the mitochondrial cell membrane, as well as charge conversion.

Similar to their use in neurodegenerative diseases, TPP and SS-31 are commonly employed to endow nanosystems with mitochondria-targeting capabilities for brain tumor treatment. For instance, Kang et al constructed mitochondria-targeted photosensitizer-loaded albumin nanoparticles by conjugating (4-carboxy-butyl)-TPP with pheophorbide-a (PS@chol-BSA NPs) for targeting mitochondria in the treatment of malignant brain tumors.¹⁶³ TPP has also been linked to the photosensitizer Chlorin e6 (Ce6), self-assembled with drugs and ions, or encapsulated into brain endothelial cell-derived extracellular vesicles for enhanced brain penetration and mitochondria targeting, ultimately inducing mitochondrial impairment.^164,171 In addition, researchers have constructed nanosystems modified with mitochondria-targeted peptides for glioma/GBM treatment. Cascade-targeted liposomes (Lip-TPGS) were developed using glucose and TPP as targeting peptides.¹⁶⁵ Similarly, SS-31 was used to modify doxorubicin-loaded liposomes (LS-DOX) for BBB penetration and mitochondria targeting.⁴⁹

As natural biomaterials, biomimetic nanomaterials based on nanovesicles and cell membranes have emerged as promising platforms for treating brain disorders due to their excellent biocompatibility and homologous-targeting ability.^197,198 Shi et al utilized GBM-cell membranes to camouflage nanoparticles for targeting homologous cancer cells, achieving remarkable targeting efficiency and anti-GBM efficacy.¹⁹⁹ Based on the unique advantages of GBM-cell membranes, Shi et al further developed a cancer cell-mitochondria hybrid membrane strategy for GBM treatment (Figure 4).⁵⁹ Compared to single cancer cell membranes, subcellular organelle-derived membranes can enhance the immune evasion capability of nanomaterials and be tailored for specific subcellular targeting. The results showed that hybrid membrane nanosystems (HM-NPs) had the highest overlap with mitochondria, indicating their superior mitochondria-targeting properties. After hybrid membrane coating, Gboxin could be effectively delivered to the mitochondria of cancer cells, interrupting ATP synthase function, leading to electron transport and energy metabolism disruption, ultimately causing mitochondria-mediated apoptosis.

Figure 4 HM-NPs@G-based mitochondria-targeted nanosystems for brain tumor. (A) Schematic illustration of the preparation of HM-NPs@G. (B) CLSM images and (C) colocalization analysis of U87 cells after various nanosystems incubation. (D) ATP content of U87 cells after various treatments. (E) The fluorescence intensity of JC-1 monomer of U87 cells. (F) Schematic illustration of the changes of JC-1 structures with MMP. (G) TEM images of mitochondria after various treatments. (H) in vivo fluorescence images of U87-tumor bearing mice after Cy5-labeled nanosystems injection. (I) Ex vivo fluorescence images of Cy5 in major organs and brains, and the colocalization with mitochondria in GBM sites. (J) The Gboxin accumulation in major organs, tumors and brains. Reproduced with permission.59 Copyright 2023, Springer Nature.

Tumor microenvironment-triggered charge-convertible nanosystems have also been designed to enhance mitochondria uptake while reducing toxicity to normal cells. Jin et al reported pH-triggered charge-convertible micelles (GPTD) for glioma therapy.¹⁶⁶ By conjugating 2,3-dimethylmaleic anhydride to TPP-modified chitosan-poly (lactic-co-glycolic acid) polymers to form dimethyl maleic amides, these compounds convert from cationic to anionic via the transformation of amino groups to carboxyl groups in acidic microenvironments. Under the low pH conditions found in tumors, GPTD could efficiently accumulate in mitochondria following charge-mediated cellular internalization, subsequently releasing drugs to increase the anti-tumor effect. Additionally, Xiang et al designed a mitochondrion-disturbing nanosystem (GBEPPT) capable of enzyme-mediated charge conversion.¹⁶⁷ Once delivered to the tumor site where γ-glutamyl transpeptidase (GGT) is overexpressed, GBEPPT would reverse its surface charge from negative to positive. With the assistance of this positive charge, GBEPPT could easily target mitochondria to disrupt their homeostasis, resulting in enhanced deep penetration within GBM treatments.

Furthermore, some mitochondria-targeting molecules without additional targeting equipment, such as hypericin, pheophorbide a-quinolinium conjugate (PQC), and carbon dots modified with metformin and gallic acid (MGA-CDs), inherently localize to the mitochondria to trigger oxidative stress, induce severe mitochondrial dysfunction, or promote ferroptosis for GBM.^168,172,173 Recently, a novel mitochondria-targeting strategy was demonstrated by Gao et al.¹⁶⁹ They found that tertiary amine modification could confer triterpene nanoparticles (eg, oleanolic acid, lupeol, and glycyrrhetinic acid) with mitochondria-targeting properties, enabling effective GBM treatment through pyroptosis after interaction with the translocase of the outer mitochondrial membrane 70 (TOM70).

Collectively, mitochondria-targeting strategies represent a promising avenue for brain tumor treatment by generating excessive ROS, triggering oxidative stress, and exacerbating mitochondrial dysfunction. Through the use of mitochondria-targeted peptides or membrane modifications, microenvironment-responsive charge conversion, and leveraging their inherent natural properties, nanosystems can effectively target the mitochondria of tumor cells and exert their functions therein.

Ischemic Stroke

Ischemic stroke has become a prevalent neurological disorder globally, characterized by high mortality and disability rates. Following ischemic injury, mitochondrial function is compromised under hypoxic or ischemic conditions, leading to oxidative stress, microglial activation, inflammation, and ultimately neuronal cell death.²⁰⁰ Targeting mitochondria can directly modulate cellular metabolism, thereby maximizing therapeutic outcomes while minimizing toxicity. The overproduction of ROS by damaged mitochondria plays a critical role in ischemic stroke injury. Therefore, strategies aimed at targeting mitochondria represent promising approaches for mitigating ischemic stroke damage.

Similar to neurodegenerative diseases and brain tumors, TPP and SS-31 are common mitochondria-targeted modification strategies used in ischemic stroke.^50,107 For instance, Wang et al proposed a stepwise targeting nanoplatform by modified pH-responsive micelles with cRGD and TPP peptide (cRGD/TPP@Res) for brain-mitochondria targeting.¹⁷⁴ Upon entering ischemic tissues with an acidic environment, the PEG shell detaches from the micelles due to the breakage of acetal bonds, followed by exposing TPP for mitochondria targeting. Resveratrol accumulates within the mitochondria, effectively scavenging ROS and alleviating oxidative stress and inflammation.

Ceria nanoenzymes (CeNZs) have garnered attention due to their excellent ROS scavenging capabilities and reversible conversion properties.^201,202 Liao et al developed a mitochondria-targeted nanosystem based on CeNZs and incorporated roflumilast for synergistic treatment of ischemic stroke (Figure 5A–5D).¹⁷⁵ After TPP-targeted modification, TPP@(CeO₂+ROF) nanosystems were rapidly taken up by neuronal cells within 1.5 h, exhibiting higher co-localization with mitochondria compared to non-targeting nanosystems. Additionally, TPP@(CeO₂+ROF) exhibited effective ROS scavenging, mitochondrial function regulation, and anti-inflammatory properties, demonstrating comprehensive treatment of ischemic stroke. To improve the brain-targeting ability of nanosystems, they further developed a magnetic field-driven and mitochondria-targeting ceria nanosystem (MMTCe) by anchoring TPP-Ce nanoparticles coated with BV2 cell membranes to maleimide-functionalized superparamagnetic iron oxide nanoparticles (Figure 5E–5H).¹⁷⁶ Under external magnetic field guidance, MMTCe efficiently reached the brain and targeted damaged mitochondria with the assistance of TPP. Subsequently, MMTCe exerted its therapeutic efficiency by scavenging ROS, reducing oxidative stress, thereby rebalancing the ischemic microenvironment. This study presented a magnetically driven, mitochondria-targeted nanosystem, providing a multifaceted approach for ischemic stroke treatment.

Figure 5 Ce-based mitochondria-targeted nanosystems for ischemic stroke. (A) Schematic illustration of the construction, mitochondria-targeting, and therapeutic effects of TPP@(CeO2+ROF). (B) Co-localization of nanosystems with mitochondria in PC12 cells. (C) TEM images of TPP@(CeO2+ROF) in mitochondria of PC12 cells. (D) JC-1 fluorescence analyzed by flow cytometry. Reproduced with permission.175 Copyright 2024, American Chemical Society. (E) Schematic representation of the preparation of MMTCe nanosystems and their neuroprotective effects. (F) CLSM images of mitochondria-targeted ability of DiR-labeled MMCe and MMTCe. (G) Ex vivo fluorescence images of brain and section from rats after various treatments. (H) The cerebral infarct volume using TTC staining. Reproduced with permission.176 Copyright 2025, Wiley-VCH.

Mitochondrial dysfunction plays a significant role in the pathogenesis of ischemic stroke, impairing energy supply, exacerbating oxidative stress, and causing neuronal death. Hence, restoring mitochondrial functions is essential in treating ischemic stroke. Researchers have recently employed mitochondria-targeted peptides, including TPP and SS-31, to modify nanosystems for scavenging ROS, alleviating inflammation, and promoting mitophagy. Further investigations should focus on mitochondrial biology and specific molecular mechanisms, which will facilitate the development of novel mitochondria-targeted approaches in ischemic stroke treatment.

Others

Similar to acute central nervous system injuries and neurodegenerative diseases, mitochondrial dysfunction also plays an essential role in other brain disorders, such as traumatic brain injury (TBI), cognitive deficits, and neuropathy.^203,204 Neurological functions are impaired by changes in mitochondria resulting from increased ROS production and decreased energy supply. Therefore, proposing potential therapeutic approaches for reversing mitochondrial dysfunction is crucial for the treatment of brain diseases.

TBI is a complex and biphasic condition. The initial phase involves direct tissue damage, while the secondary phase is characterized by calcium overload, excessive ROS production, and inflammation.²⁰⁵ Mitochondrial dysfunction frequently occurs during this secondary phase. Consequently, mitochondria represent a promising target for intervention in TBI. Researchers have developed mitochondria-targeted nanosystems for managing TBI. For instance, Sharma et al utilized polyamidoamine dendrimers to encapsulate N-acetyl cysteine (NAC), which acts as an antioxidant.¹⁷⁷ After being modified with TPP, the NAC was specifically targeted to the mitochondria in injured glial cells. This approach significantly reduced oxidative stress both in vitro using cell models and in vivo using rabbit TBI models, demonstrating that mitochondria-targeted strategies could enhance therapeutic efficacy in TBI.

Furthermore, Sun et al introduced a biomimetic nanosystem co-modified with C3 peptides and SS31 (C3/SS31-RBCNLCs) for targeting neuronal mitochondria in the brain.¹⁷⁸ Both in vitro and in vivo experiments showed that the encapsulated drug olaparib could be efficiently delivered into brain mitochondria, thereby improving mitochondrial function, reducing neuronal death, and delaying the pathological progression of TBI. Besides, various fluorescence probes were also constructed by researchers to realize mitochondria-targeting and brain imaging. For instance, Pan et al proposed a ligand engineering of luminescent AuAg nanoclusters conjugated with TPP on the surface.¹⁷⁹ In vitro cell experiments have revealed that AuAg NCs@TPP probe could effectively target to the mitochondria of 4T1 and BV2 cells, exhibiting outstanding mitochondria-targeted property.

Conclusion and Perspective

Mitochondria, as central orchestrators of cellular energetics, calcium homeostasis, and apoptotic signaling, represent pivotal therapeutic targets for CNS disorders. Mounting evidence implicates mitochondrial dysfunction as a convergent pathological mechanism underpinning neurodegenerative diseases (eg, AD, PD), brain tumors, ischemic stroke, and TBI. Consequently, strategies aimed at restoring mitochondrial homeostasis hold transformative potential for CNS therapeutics. Mitochondria-targeted nanosystems have emerged as a paradigm-shifting approach, enabling spatiotemporally precise delivery of therapeutic cargo to rescue mitochondrial function and amplify treatment efficacy.

This review has delineated the rational design principles and expanding therapeutic applications of mitochondria-targeted nanosystems. Key targeting modalities—including cationic molecules, mitochondrial-penetrating peptides, and bioengineered mitochondrial membranes—demonstrate markedly enhanced mitochondrial accumulation and intervention capacity. We further synthesized recent advances in deploying these nanosystems across diverse CNS pathologies (including AD, PD, glioma, ischemic stroke, and TBI etc)., highlighting their roles in mitigating oxidative stress, modulating mitophagy, and promoting mitochondrial biogenesis to achieve superior therapeutic outcomes. Despite these promising advances, critical challenges demand resolution before clinical translation (Figure 6).

Figure 6 Current challenges and future prospects of mitochondria-targeted nanosystems. Created with BioRender.com.

To date, mitochondria-targeted nanosystems have yet to be employed in clinical studies, potentially due to challenges related to their complex preparation, reproducibility, and metabolism. Consequently, greater emphasis should be placed on the thorough investigation of the physicochemical characterization of these nanosystems. Moreover, before these systems can advance to preclinical trials, it is essential to evaluate their in vivo stability, long-term effects, and metabolic pathways. Addressing these aspects will be crucial for translating mitochondria-targeted nanosystems from experimental models to potential clinical applications.

Size, shape, and charge play essential roles in determining the biological fate of nanosystems, which influence their in vivo cellular uptake and circulation lifetime.^206,207 For example, nanosystems with over 200 nm size have poor penetration and cell interaction properties. In contrast, nanosystems with small size can enhance the penetration, but are rapidly cleared from the systems by kidneys during the in vivo circulation.²⁰⁸ As for the charge of nanosystems, the cationic charge of nanosystems improves cellular uptake efficiency, which is mainly attributed to the negative charge of cell membrane, but it is also associated with cytotoxicity. For instance, the inherent cationic charge of common mitochondrial ligands (eg, TPP) raises concerns regarding membrane perturbation and off-target cytotoxicity. Hence, future research must prioritize rigorous toxicological profiling of nanosystems across relevant cell types and disease models, coupled with the development of less cytotoxic strategies such as coating with biomimetic mitochondrial membranes and engineering delivery systems with targeted ligands.

Effective CNS delivery necessitates sequential navigation of formidable obstacles: the BBB, the complex brain parenchyma, and ultimately, the mitochondrial double membranes. Furthermore, in order to enhance the accumulation of nanosystems in the mitochondria of specific cell types, various sub-organelle targeting strategies have been developed in combination with mitochondria-targeted approaches. For instance, T807 and TPP peptides were co-functionalized to red blood cell-modified nanosystems by Gao et al, which could not only penetrate the BBB but also target neuronal mitochondria.¹⁴⁹ The similar functions could also be observed by RVG29 peptide modification.¹⁵² While dual-targeting strategies (eg, conjugating BBB-penetrating ligands like transferrin or angiopep-2 with mitochondrial homing signals like SS-31) show promise, their efficiency remains limited. A concerted effort is required to engineer next-generation ligands with enhanced specificity, avidity, and stimuli-responsiveness for both brain entry and mitochondrial localization, thereby minimizing systemic exposure and maximizing the therapeutic index.

A fundamental limitation of current platforms is their inability to distinguish pathologically damaged mitochondria from functional counterparts. Cellular homeostasis relies on mitochondrial quality control mechanisms: damaged organelles (exhibiting fragmentation, depolarized membrane potential, and proteotoxic/oxidative stress) are segregated via fission and targeted for mitophagy, while healthy units sustain essential functions. Indiscriminate nanocarrier accumulation in all mitochondria poses potential risks. Delivery of cytotoxic payloads or nanomaterial-induced stress to healthy mitochondria could irreversibly compromise cellular viability. Next-generation systems demand molecular intelligence for damaged mitochondrial targeting, such as energy-state-responsive delivery, damage marker-guided binding, or ROS/protease-activatable systems.

In summary, subcellular targeting strategies, including but not limited to mitochondrial-targeted nanosystems, offer promising approaches for treating CNS diseases. Future success hinges on prioritizing safety by design, enhancing targeting sophistication, and demonstrating scalable manufacturability, paving the way for these potent nanoscale interventions to transform the treatment landscape for devastating neurological diseases.

Data Sharing Statement

No data was used for the research described in the article.

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. Conceptualization: Y.Z., X.C. and X.Z.; literature curation: X.Z., J.C. and B.W., writing – original draft: X.Z., J.C., B.W. and X.C.; writing—review and editing: Y.Z. and X.C.; supervision: Y.Z. All authors read and approved the final manuscript.

Funding

This work was supported by grants from the National Natural Science Foundation of China (82473900, 82273903), Zhejiang Provincial Natural Science Foundation (LD25H090002), and Research Project of Zhejiang Chinese Medical University (2025JKZKTS33).

Disclosure

The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper.

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DUBAI, United Arab Emirates, December 8, 2025 — Bybit, the world’s second-largest cryptocurrency exchange by trading volume, today announced a strategic partnership with an affiliate of Circle Internet Group, Inc. (‘Circle’) (NYSE:CRCL), a global financial technology firm and the issuer of USDC through its regulated entities. The partnership aims to expand USDC access across Bybit’s global ecosystem, strengthen liquidity of the world’s largest regulated stablecoin, and reinforce Bybit’s position as a regulatory-compliant platform committed to transparency and trust.

As part of this partnership, Bybit will enhance USDC liquidity across spot and derivatives markets,  enabling a more efficient trading environment for retail and institutional users. The two companies will also roll out a series of campaigns and initiatives to increase the utility of USDC across Bybit’s products and services. 

The partnership will further extend to fiat on- and off-ramp solutions, combining Circle’s infrastructure and network of partners with Bybit’s global reach to simplify deposits and withdrawals across key markets. Bybit users will then benefit from greater speed, transparency, and efficiency when converting between local currencies and digital assets.

Bybit also plans to expand USDC integration across its ecosystem,  including Bybit Earn for savings, Bybit Card for cashback rewards, and Bybit Pay for everyday transactions — reinforcing its commitment to delivering secure, compliant, and accessible technology-enabled financial solutions. 

In addition, Bybit was among the first wave of more than one hundred companies to join the public testnet of Circle’s Arc network, a new layer-1 blockchain purpose-built for stablecoin-native finance. Arc’s public testnet launched in October 2025, with broad-based engagement and collaboration from across the financial and economic system, deep infrastructure support, and global participation. 

 
Regulatory Compliance as a Cornerstone of Innovation

Bybit recently secured a full Virtual Asset Platform Operator License from the UAE’s Securities and Commodities Authority (SCA), marking a major milestone in its expansion in the Middle East  and positioning it as one of the first global exchanges to secure this level of regulatory approval in the region.

In addition, Bybit has expanded its regulatory oversight across the European Economic Area (EEA), Turkey, and other jurisdictions around the world. These developments demonstrate Bybit’s long-term commitment to aligning with global regulatory standards and bridging traditional finance with the digital asset economy. 

USDC is a digital asset fully backed by highly liquid cash and cash-equivalent assets, and is redeemable 1:1 with the U.S. dollar. The reserve assets are held with trusted financial institutions, and monthly attestation by independent third parties provide a high degree of transparency.

Ben Zhou, Co-founder and CEO of Bybit, said: “Bybit’s partnership with Circle represents a major milestone in our mission to offer a fully compliant, liquid, and user-friendly ecosystem. From trading to payments to savings, we are integrating USDC to power the next phase of our platform’s growth and stability.”

Jeremy Allaire, Chairman, Co-founder, and CEO of Circle, added: “At Circle, we’re powering the future of internet activity with enterprise-grade infrastructure and stablecoins built for scale. Together, Circle and Bybit are making it easier for users to access and use USDC with the confidence, transparency, and speed they expect.”

Looking ahead, Bybit and Circle continue to explore deeper integrations to unlock new opportunities for cross-chain liquidity and institutional-grade financial solutions.

About Bybit

Bybit is the world’s second-largest cryptocurrency exchange by trading volume, serving a global community of over 60 million users. Founded in 2018, Bybit is redefining openness in the decentralized world by creating a simpler, open and equal ecosystem for everyone. With a strong focus on Web3, Bybit partners strategically with leading blockchain protocols to provide robust infrastructure and drive on-chain innovation. Renowned for its secure custody, diverse marketplaces, intuitive user experience, and advanced blockchain tools, Bybit bridges the gap between TradFi and DeFi, empowering builders, creators, and enthusiasts to unlock the full potential of Web3. Discover the future of decentralized finance at Bybit.com.

For more details about Bybit, please visit Bybit Press
For media inquiries, please contact: [email protected]
For updates, please follow: Bybit’s Communities and Social Media

‍

About Circle Internet Group, Inc.

Circle (NYSE: CRCL) is one of the world’s leading internet financial platform companies, building the foundation of a more open, global economy through digital assets, payment applications, and programmable blockchain infrastructure. Circle’s platform includes the world’s largest regulated stablecoin network anchored by USDC, Circle Payments Network for global money movement, and Arc, an enterprise-grade blockchain designed to become the Economic OS for the internet. Enterprises, financial institutions, and developers use Circle to power trusted, internet-scale financial innovation. Learn more at circle.com. 

Introduction

Regulatory T cells (Tregs) play an essential role in the immune system and FoxP3 is considered a specific marker for them.¹ Tregs are a special subset of CD4⁺ T cells and function in peripheral tissues.^2,3 In immune responses, Tregs can directly contact and inhibit effector T cells, suppress the activity of antigen-presenting cells (APCs), and exert their effects by secreting immunosuppressive cytokines. These functions make Treg cells of great significance in preventing autoimmune reactions, regulating immune responses, promoting tissue repair, and maintaining immune tolerance.² However, such functions may be utilized in certain pathological states, especially in tumor progression, to form adverse outcomes. In the tumor microenvironment (TME), the function of Treg cells becomes more complex and crucial.³ Many tumor cells recruit and maintain Treg cells by releasing specific cytokines and chemical factors (such as CCL22, TGF-β, etc)., causing them to aggregate in tumor tissue.^4,5 These Treg cells form a suppressive tumor immune microenvironment that benefits tumor cells in immune evasion, thereby accelerating tumor progression. Due to the immunosuppressive effect of Treg cells in the TME, their function has become one of the crucial targets in cancer immunotherapy. Regulating Treg cells to enhance the efficiency of immunotherapy has become a novel treatment regimen for cancers. However, Treg cells have a double-edged sword role in cancers. They also contribute to good survival in cancer patients. A high rate of FOXP3+ tumour-infiltrating lymphocytes positively correlated with CD8+ T cells in oestrogen receptor-negative breast cancer.⁶ Similar results were also found in colorectal cancer⁷ and ovarian cancers,⁸ which indicated Treg cells are associated with better prognosis. The reasons for these contradictory phenomena include the heterogeneous functions and phenotypes of Treg subpopulations and the complex cell communications in TME.⁹

Extracellular vesicles (EVs) are nanoscale particles secreted by all cells that play important roles in multiple biological processes, including intercellular communication, immune responses, and disease development.¹⁰ EVs include multiple subtypes, mainly including exosomes, microvesicles, and apoptotic bodies, which vary in diameter, origin, and function. EVs usually carry various biological molecules, including RNA (both mRNA and non-coding RNAs, such as miRNAs, lncRNAs, and circRNAs), proteins, lipids, metabolites, etc. EVs are considered essential communication mediators due to their ability to transmit biomolecules across different cells.^10,11 The tremendous amount of EV research in tumors during the past years has demonstrated their wide range of effects on tumor progression, from tumor proliferation, metastasis, immune evasion, metabolism reprogramming, treatment resistance, angiogenesis, etc.^10,12 More importantly, EVs are considered ideal drug carriers because of their natural biocompatibility and low immunogenicity. Researchers can engineer EVs to carry specific drugs or gene therapy agents and deliver them precisely to both malignant and non-malignant cells.¹¹ Therefore, exploring the roles of EVs in tumor progression and taking advantage of EVs as drug delivers become a hotspot.

Recently, the effects of EVs on Tregs were uncovered rapidly, and repressing Treg-beneficial EVs seems helpful in tumor treatment. Studies on targeting Treg via EVs to improve antitumor immunity are merging gradually, especially the important roles of non-coding RNAs in EVs. For example, miR-196b-5p and miR-3200-3p from non-small-cell lung cancer (NSCLC) cells can induce the proliferation and senescence of Treg cells;^13,14 miR-320c, miR-27a-3p and miR-30a-5p from colon cancer cells can promote the immunosuppressive function of Treg.¹⁵ To provide an overall understanding of these advances in the relationships between EVs and Tregs in tumors, we systematically reviewed the expanding functions of Tregs in TME, the effects of EVs on Treg in the context of various kinds of tumors, and the clinical potential of Treg-targeting EVs in cancer treatment.

Tregs in Tumor

Treg-Related Immunity in Tumor

Tregs are highly associated with various malignant behaviors in tumors, such as tumor growth and metastasis, immune evasion, metabolism reprogramming, therapy resistance, and so on. Among these, the ability of Tregs to reshape tumor immune microenvironment is outstanding. First, Tregs could repress immune function by direct intercellular contact via some well-known immune inhibitory receptors, including cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), Lymphocyte-activation gene 3 (LAG-3), etc. CTLA-4 is an essential immune inhibitory receptor on the Tregs surface. On the one hand, CTLA-4 on Tregs could bind to the CD80/CD86 on the surface of antigen-presenting cells (APCs), such as dendritic cells, to block their co-stimulatory signals for effector T cells and further inhibit the activation and proliferation of effector T cells.¹⁶ On the other hand, CTLA-4 on Tregs could remove CD80/CD86 on the APC surface via trogocytosis to prevent T cell activation signaling and increase the release of PD-L1, another famous immune inhibitory protein.¹⁷ Besides CD80/CD86, CTLA-4 also removes other immune-stimulating factors. For example, Tregs deletes B7 on APC in a similar way that trogocytosis and CTLA4-mediated cis-endocytosis function cooperatively to clear B7 on APCs.¹⁸ The high-affinity binding of LAG-3 to MHC Class II, the main ligand of LAG-3, can directly inhibit the function of APCs, thereby indirectly inhibiting the activation and proliferation of effector T cells.¹⁹ The interaction between LAG-3 and MHC II not only acts on APCs but also inhibits the ability of other immune cells (for example, macrophages).²⁰ Apart from direct interaction, Tregs can also exert immune suppression by releasing immunosuppressive cytokines including IL-10, TGF-β, and IL-35.^21,22 TGF-β has multiple immunosuppressive functions, including inhibiting the activation and biofunction of effector T cells, NK cells, macrophages, and other immune cells. Besides, TGF-β also enhances the differentiation and expansion of Tregs.²³ Therefore, TGF-β secretion by Tregs may further promote their differentiation and expansion while inhibiting other immune cells, thus forming a vicious cycle in the TME. IL-10 can induce the expression of PD-L1 in monocytes, thereby reducing T-cell infiltration.²⁴ IL-35 can exert inhibitory effects on anti-tumor immune responses by promoting the expression of various immunosuppressive molecules such as PD-1, TIM-3, and LAG-3, limiting T cell recruitment, and inhibiting anti-tumor immune memory. Tregs can also secrete EVs containing IL-35, thereby exerting immunosuppressive effects.²⁵ The different factors secreted by Tregs can cooperate to jointly shape a tumor immune suppressive microenvironment. For instance, IL-10 and IL-35 are secreted by different subgroups of Tregs. Still, the inhibitory molecules IL-10 and IL-35 released by these Tregs work together to exert immunosuppressive function and promote the depletion of T cells in tumors.²² Both IL-10, IL-35, and TGF-β can alter the polarity of macrophages, transforming them into M2-type immunosuppressive phenotypes, which have the effects of promoting tumor growth and immune suppression.^26–28 Interestingly, in addition to secreting immunosuppressive factors, IL-2 receptor CD25, a marker highly expressed on Treg, can also absorb the immunostimulatory molecule IL-2, thereby reducing the promoting effect of IL-2 on other immune cells, preventing them from fully proliferating and exerting their function of killing tumors.^29,30 In addition, Tregs can also alter the function and vitality of immune cells through metabolism. On the one hand, in solid tumors, as the tumor progresses rapidly, aerobic glycolysis (ie Warburg effect) often occurs, resulting in a large accumulation of lactate. In this microenvironment, tumor-killing immune cells such as cytotoxic T cells are significantly inhibited, while Tregs can adapt well to lactate. Even worse, studies have shown that lactate can further promote the survival and function of Tregs.^31,32 On the other hand, Tregs can consume a large amount of ATP through CD39 and CD73, metabolize ATP into the immunosuppressive adenosine, and further shape the tumor-suppressive immune microenvironment.³³

Other Functions of Tregs in Tumor

Tregs can indirectly facilitate tumor growth, metastasis, and treatment tolerance by shaping the aforementioned immunosuppressive microenvironment. However, some studies suggest that Tregs can directly participate in the malignant biological behavior of these tumors. Tregs can promote angiogenesis. When recruited into tumors in a hypoxic microenvironment, Tregs can release vascular endothelial growth factor A (VEGFA), which is beneficial for cancer angiogenesis.³⁴ Tregs and vascular endothelial cells interact tightly with each other. Endothelial cells can promote the differentiation, expansion, migration, and aggregation of Tregs in various ways.^35–37 Endothelial cells exposed to prostaglandin E2 (PGE2), hypoxia, and Treg-induced VEGF and IL-10 express Fas ligand (FasL), which promotes T cell apoptosis. However, Tregs can evade FasL-mediated apoptosis by expressing the anti-apoptotic gene FADD like IL-1β-converting enzyme.³⁸ Considering the pro-angiogenic and immunosuppressive properties of Tregs, the interaction between endothelial cells and Tregs may form a feedback interaction, potentially creating a favorable microenvironment for angiogenesis and immunosuppression. In terms of promoting tumor metastasis, Tregs can promote metastasis not only via enhancing angiogenesis but also through directly affecting the invasion and metastasis ability of tumors. The pro-metastatic effect of TGF-β has been widely confirmed.^39–41 Meanwhile, Oh et al confirmed that TGF-β secreted by Tregs can induce EMT transformation in melanoma cells. Cells undergoing EMT transformation have strong mobility, which is a crucial step in the cascade of tumor invasion and metastasis.⁴² The formation of novel blood vessels is beneficial for the further growth and proliferation of tumor cells.⁴³ In addition, considering the promoting effect of VEGF on tumor proliferation,^44–46 VEGF secreted by Tregs may have a direct promoting effect on the proliferation and growth of tumor cells, which is similar to the release of TGF-β by Tregs to enhance cancer cell invasion ability.

Based on the multifarious roles of Tregs in tumors mentioned above (Figure 1), Tregs are often closely associated with adverse clinical outcomes of tumors. The level of Tregs in peripheral blood increases with tumor staging, with the highest Tregs level observed in patients with metastases.⁴⁷ An increasing number of infiltrating Tregs in tumor tissues indicates a poorer prognosis and a higher risk of recurrence.^48–50 Tregs may lead to resistance to various treatments, including traditional radiotherapy, chemotherapy, target therapies, and immune-related therapies.^51–54 However, it should be noted that a higher number of Tregs does not always indicate a poor prognosis, and some studies even suggest that a higher number of Tregs may indicate a better tumor prognosis. Two independent studies from Salama and Frey et al showed that a higher number of FOXP3⁺ Tregs in colorectal tumors suggests a better prognosis.^7,55 This may be related to the anti-inflammatory effect of Tregs. Inflammation is considered an important factor in promoting tumor progression, but the immunosuppressive environment formed by Tregs may counteract this effect.^56–58 The different prognostic roles of Tregs in tumors indicate that our understanding of the complex functions of Tregs in tumors is not yet perfect, and more research is needed to reveal them.

Figure 1 Roles of Treg in tumor. (A) Treg could directly inhibit other immune cells, especially antigen-presenting cells, via LAG-3 and CTLA-4. (B) On the one hand, Treg could secret immune inhibitory factors, such as IL-10, IL-35, and TGF-β, to inhibit other immune cells. On the other hand, Treg could inhibit immune cells via metabolism reprogramming, such as metabolizing ATP into adenosine via CD39 and CD73. (C) Tumor microenvironment (TME) benefits Treg but impairs tumor-inhibiting immune cells. Lactate in TME could promote Treg function but inhibit other immune cells, such as CD8+ T cells. CD25 on the Treg surface could consume IL-2 to active Treg to avoid IL-2-mediated activation in other immune cells. (D) Treg could secret VEGFA, TGF-β, and other factors to induce angiogenesis, tumor proliferation, and metastasis.

The Types of EVs

The first category is exosomes, which originate from the intracellular endosomal system. Specifically, the plasma membrane invaginates to form early endosomes, which undergo further sorting and maturation into late endosomes (referred to as multivesicular bodies, MVBs). Intraluminal vesicles (ILVs) within MVBs then fuse with the plasma membrane and are released into the extracellular space, giving rise to exosomes.⁵⁹ As one of the most extensively studied EV subtypes, exosomes are characterized by a small diameter, typically ranging from 30 to 150 nm. Their surface is enriched with specific marker proteins, including members of the tetraspanin family (CD9, CD63, CD81), Alix, and TSG101.⁶⁰ Exosomes encapsulate bioactive molecules derived from their parental cells, such as nucleic acids (mRNA, miRNA, lncRNA) as well as proteins and lipids, enabling them to participate in long-range intercellular signaling.⁶¹

The second category is microvesicles (MVs), also referred to as shedding vesicles. Unlike exosomes, MVs are formed directly through budding and shedding from the plasma membrane without involving the endosomal pathway. Their biogenesis is closely linked to the disruption of the asymmetric distribution of phospholipids in the cell membrane. With a larger diameter than exosomes, MVs typically range from 100 to 1000 nm and exhibit a broad size distribution.⁶² They retain surface markers characteristic of their parental cell membrane, including integrins, selectins, and adhesion molecules.⁶³ Internally, MVs carry nucleic acids and proteins, enabling them to participate in local intercellular signaling. Additionally, they are involved in physiological and pathological processes such as coagulation and inflammatory responses.⁶⁴

The third category is apoptotic bodies (ABs), which are exclusively generated during cellular apoptosis (programmed cell death). Specifically, the cell membrane of apoptotic cells undergoes shrinkage and budding (forming apoptotic blebs) before the cell ultimately fragments to release large vesicles containing nuclear fragments, organelles (eg, mitochondria, ribosomes), and other cytoplasmic contents—these are apoptotic bodies. As the largest subtype of EVs, ABs typically have a diameter ranging from 1 to 5 μm (with some reaching up to 10 μm).^65,66 They characteristically expose phosphatidylserine on their surface, which acts as a “phagocytic signal” to recruit phagocytes such as macrophages for their clearance. Internally, ABs contain apoptosis-related proteins (eg, caspase family members) and nuclear DNA fragments. Their primary function is to facilitate the clearance of apoptotic cellular debris, thereby preventing the leakage of cellular content that could induce inflammation. Additionally, ABs may modulate the functions of neighboring cells through the transmission of genetic material.⁶⁷

In addition to these three mainstream types of EVs, there are also some rare types, such as oncosomes—large-sized vesicles (0.5 to 10 μm) released by tumor cells, rich in oncogenic proteins and nucleic acids, and closely associated with tumor invasion and metastasis—and exosome-like vesicles, which are vesicles of a size similar to exosomes that some cells (eg, platelets, nerve cells) may release through non-endosomal pathways, have functions overlapping with those of exosomes, and are temporarily classified as a special subtype.⁶⁸

Effects of EVs on Treg

With the gradual deepening of research on EVs, it is now clear that various cells can influence the proliferation, differentiation, expansion, metabolism, and other biofunctions of Tregs by releasing EVs (Figure 2). Next, based on relevant research on different tumors, we will systematically summarize the effects of EVs from different cell sources on Tregs under different tumor backgrounds (Table 1).

Table 1 Effects of EVs on Treg in Different Tumors

Figure 2 Effects of different EVs on Treg in tumor. In the context of cancer, EVs from various cells (including tumor cells, adipocytes, endothelial cells, macrophages, and stem cells) and patients’ body liquid (including plasma and effusion) could promote the expansion and function of Treg in tumors.

Head and Neck Cancer

In the field of head and neck cancer (HNC), most research explored the effects of tumor-derived EVs or EVs from patient’s plasma on Treg. In 2010, Whiteside’s team reported that tumor-derived EVs containing TGF-β1 and IL-10 could induce the differentiation of CD4⁺CD25⁺ Tregs from CD4⁺CD25⁻ T cells, promote the proliferation of Treg, and augment the immunosuppression of Tregs.⁶⁹ In 2014, Whiteside’s team showed that both exosomes from normal control or head and neck squamous cell carcinoma (HNSCC) patients carried enzymatically active CD39 and CD73 and could hydrolyze eATP to 5’-AMP and to adenosine.⁷⁰ When CD4⁺CD39⁺ Tregs contact with CD73⁺ exosomes, they could produce immunosuppressive adenosine.⁷⁰ In 2016, Whiteside’s team examined the response of different T cell subsets to tumor-derived EVs via mRNA profiles. They found that Tregs were more sensitive to exosomes from HNSCC cell line PCI-13 than other T cell subsets and the PCI-13-derived EVs downregulated the expression of the adenosine pathway gene and enhanced the production of adenosine in CD4⁺CD39⁺ Tregs.⁷¹ In 2017, Whiteside’s team demonstrated that PCI-13-derived EVs also enhanced inosine production in Tregs and the effects of tumor-derived EVs on Tregs depend on cell surface signaling rather than uptake mechanisms.⁷² Besides, Whiteside’s team re-emphasized exosomes from HNSCC patients’ plasma induce the CD39 expression and adenosine production in CD4⁺CD39⁺ regulatory T cells and the effects of exosomes from patients with active disease were more effective than that from patients with active disease no evident disease after oncologic therapies.⁷³ In 2018, Whiteside’s team noticed that CD3⁻ exosomes from HNSCC patients’ plasma were more effective than CD3⁺ exosomes in reshaping the metabolism of Tregs that induces the generation of 5’-AMP and purines in Tregs and inducing the differentiation of CD4⁺CD39⁺ Tregs from CD4⁺ T cells.^74,75 In 2020, Beccard et al, who are Whiteside’s et al, discovered CD45⁻ exosomes from high-stage HNSCC patients’ plasma were more effective than other exosomes in inducing the differentiation of CD4⁺CD39⁺ Tregs from CD4⁺ T cells.⁷⁶ Since this year, researchers from other institutes also reported the impacts of EVs on Tregs in the context of HNSCC. Lopatina et al found that exosomes from HNSCC endothelial cells also stimulated CD4⁺CD25⁺FoxP3⁺ Tregs from peripheral blood mononuclear cells (PBMCs).⁷⁷ Wei et al showed that cancer cell-derived exosomal PD-L1 could induce the differentiation of activated Treg and M2 tumor-associated macrophages (TAM).⁷⁸ Besides, cancer cell-derived exosomal PD-L1 also enhances the positive feedback loop between aTreg-M2 that aTregs promotes the M2 polarization of M0 macrophages and M2 macrophages also promote the differentiation of aTregs from CD4⁺CD25⁻ T cells.⁷⁸ In nasopharyngeal carcinoma (NPC), a subtype of HNC, exosomes from NPC cells reshape tumor immunity by inhibiting the proliferation and expansion of T cells, blocking the differentiation of Th1 and Th17, and boosting Tregs.⁸¹ Mrizak et al discovered similar phenomena: NPC-derived exosomes recruited conventional CD4⁺CD25⁻ T cells and mediated their conversion into CD4⁺CD25⁺ Treg; NPC-derived exosomes enhanced Tregs expansion via inducing the expression of CD25 and FOXP3 in Tim3⁻ Tregs.⁷⁹ Later, Ye et al further demonstrated that miR-24-3p in NPC-derived exosomes targets FGF11 to mediate inhibitory changes in tumor immunity above.⁸⁰ Two studies focused on oral squamous cell carcinomas (OSCC), another subtype of HNC. Chen et al found that OSCC cells could maintain the expression of Foxp3 in Tregs via inhibiting miR-325-3p-mediated Foxp3 degradation due to the transmission of exosomal has_circ_0069313 from cancer cells to Tregs.⁸² Ko et al reported that arecoline-induced cytosolic mtDNA D-loop leakage and PD-L1 expression in OSCC cells were packaged into EVs in arecoline-treated OSCC cells to promote the number of Tregs.⁸³

Digestive System Neoplasms

In esophageal cancer, cancer-derived EV carried PD-L1 and promoted the expansion and immunosuppression of circulating follicular Tregs. In gastric cancer, exosomes from the peripheral blood of gastric cancer patients contain TGF-β1 to induce the differentiation of Tregs from naïve T cells and increase Tregs in lymph nodes. In hepatocellular carcinoma (HCC), 14-3-3 protein zeta (14-3-3ζ), miR-500a-3p, and circGSE1 in exosomes from HCC cells were reported to increase the expansion of Tregs.^86–88 Similarly, exosomal miRNA-425-5p from chemoresistant HCC cells can be absorbed by CD4+ T cells to promote the increase of Tregs through regulating the translation of PTEN.⁸⁹ In pancreatic cancer, EVs from BxPC-3, a pancreatic ductal adenocarcinoma cell line, promote the regulatory phenotype of T lymphocytes via inducing the upregulation of immune checkpoint proteins represented by PD-1, PD-L1, CTLA4, and Tim-3 and the expansion of FOXP3⁺ Tregs.⁹⁰ The studies about the influences of EVs on Tregs in the context of colorectal cancer (CRC) were relatively more than other digestive system neoplasms. EVs from CRC carried TGF-β which induced the phenotype conversion of T cells into Treg-like cells via TGF-β/Smad signaling.⁹³ Wang et al demonstrated that exosomes from colon cancer cells transmit miR-27a-3p, miR-30a-5p, and miR-320c into Tregs to increase Tregs via inhibiting the expression of IRF4, which is an inhibitory factor for the number of Tregs in colon cancer tissue.¹⁵ Another study also showed similar results. Exosomal miR-208b is related to oxaliplatin resistance in CRC patients, and exosomal miR-208b from CRC cells could induce the expansion of Tregs via targeting PDCD4.⁹² However, some research showed the opposite effects of EVs on Treg. Ganji et al showed that CT26-derived exosomes can block cancer progression in vivo by decreasing Tregs and upregulating IFN-γ.⁹¹ Kobayashi et al used another CRC cell line Colon-26 and found similar inhibitory impacts of CRC-derived EVs on Treg. Colon-26-derived EVs significantly inhibit the number of CD4⁺FoxP3⁺ Tregs in the mice model of CRC lung metastasis via upregulating PDCD1, GITR, and CD69.⁹⁴ The anti-Treg roles of cancer-derived EVs may be attributed to the heterogeneity properties of EVs. The heterogeneity of EVs does not exist randomly; instead, it is co-regulated by factors such as the type of source cell, cell activation state, and microenvironmental signals. It can be mainly categorized into the following four dimensions, each of which is closely coupled with function. Due to the presence of heterogeneity, the molecular composition of EVs also exhibits high heterogeneity. The molecular components of EVs (proteins, nucleic acids, lipids) are the direct executors of their functions—even EVs derived from the same source may exhibit completely different functions due to differences in their molecular cargo. Heterogeneities across different dimensions can act synergistically to co-regulate complex biological processes.^120,121

Lung Cancer

In lung cancer, EVs from tumor cells could directly or indirectly induce the differentiation of Treg. Exosomal CD39 from non-small-cell lung cancer (NSCLC) cells also results in the insufficiency of ATP and the hyperactivation of AMPK to directly induce the mal-differentiation of T cells and increase the differentiation of Tregs from CD4⁺ T cells.⁹⁷ Exosomal epidermal growth factor receptor (EGFR) could indirectly affect the differentiation of Tregs via inducing tolerogenic dendritic cells which can promote the differentiation of Tregs from Th0 cells. Similarly, Ning et al reported that PD-L1-containing exosomes released by lung LLC Lewis lung carcinoma inhibit the differentiation of CD11c⁺ dendritic cells from myeloid precursor cells and promote the immunosuppressive function of dendritic cells, which indirectly induces the increase of Tregs.⁹⁸ Wang reported that lung cancer-derived exosomes indirectly increase CD4⁺ FoxP3⁺ Tregs in the lung by enhancing the contact between fibroblasts and Treg. In detail, lung cancer-derived exosomes increase the secretion of CCL1 from fibroblasts, which promotes Tregs differentiation by activating CCR8.⁹⁵ Besides, exosomal miR-196b-5p from lung cancer cells could promote pyroptosis in T cells and the proliferation of Tregs via regulating the expression of ING5.¹³ Interestingly, exosomal miR-3200-3p from lung cancer cells could induce the senescence of Treg. However, VEGFR2, which usually are overexpressed in lung cancer cells, reduces miR-3200-3p in cancer-derived exosomes. As a result, the senescence of Tregs was inhibited in the lung cancer TME to accelerate disease progression.¹⁴

Female Cancers

Zhu et al explored the influence of EVs released from adipose-derived stem cells (ASCs) on tumor immune microenvironment in the background of breast cancer in vitro. They showed that EVs released from ASCs could not only block the M1 polarization and enhance the M2 polarization of CD14⁺ monocytes but also promote CD4⁺ T cells to differentiate into Tregs, thus forming the immunosuppressive microenvironment of breast cancer.⁹⁹ Fathollahi et al studied the effect of EVs released by either adipose-derived MSCs or breast cancer cells on the recall-antigen-specific immune responses. The results showed that EVs released by either adipose-derived MSCs or breast cancer cells can upregulate Foxp3 mRNA, the main regulatory factor of Tregs, in splenocytes, and reduce the expression of Tbx21 and Gata3 mRNA, the main mediators of T helper (TH) 1 and TH2 responses. However, only EVs from breast cancer cells can increase the secretion of IL-10 and TGF-βin splenocytes.¹⁰³ Exosomes from breast cancer stem cells carried Foxp3 to generate Foxp3⁺ Tregs from anti-CD3/anti-CD28-treated (antigen-activated) CD4⁺ T cells at an early time-point of 24 h.¹⁰¹ These studies strongly suggest that EVs can increase the number and/or enhance the function of Tregs. Ni et al further explored the influence of breast cancer-derived EVs on Tregs. They found that exosomes secreted by breast cancer cells can transmit lncRNA SNHG16 which induces γδT1 cells to express CD73 by regulating the miR-16-5p/SMAD5 axis, ultimately promoting the emergence of CD73⁺γδT1 Tregs and forming the immunosuppressive microenvironment of breast cancer.¹⁰² Cancer-derived EVs also carry TGF-β1 to induce Tregs differentiation in breast cancer.¹⁰⁴ Interestingly, PD-L1 in EVs released from breast cancer cell line 4T1 induced DCs which can promote CD4⁺ T cells to differentiate into CD4⁺Foxp3⁺ Tregs and inhibit the differentiation of CD4⁺IFN-γ⁺ Th1 cells.⁹⁸ This result indicates that tumor-derived EVs can indirectly affect the differentiation of Tregs, thereby forming an immunosuppressive microenvironment. However, a study showed that EVs from breast cancer cells reduce the number of Tregs. Santoro et al explored the influence of EVs released by breast cancer cell lines BT474 and HS578T under 2D and 3D conditions on various immune cells from peripheral blood through in vitro and found that EVs derived from BT474 significantly reduce CD39⁺ Tregs.¹⁰⁰

In cervical cancer, Ni et al demonstrated that exosomal TGF-β induces the expansion of Tregs from naïve CD4⁺ T cells via activating STING signaling which induces FOXP3 transcription through TBK1-IRF3-mediated SMAD3 and STAT5 phosphorylation independent of interferon-β.¹⁰⁵ In ovarian cancer, three studies reported the effects of EVs from different origin cells on Treg. Szajnik proved that exosomal TGF-β could induce the proliferation of Treg, promote the differentiation of CD4⁺CD25⁺ Tregs from CD4⁺CD25⁻ T cells, and enhance the immunosuppressive function of Tregs.⁶⁹ Zheng et al reported that cancer-associated adipocyte-derived EVs (CAA-EVs) delivered SIRT1 to form a suppressive immune microenvironment via inhibiting tumor-inhibitory M1 macrophages and increasing tumor-promoting M2 macrophages and Tregs.¹⁰⁷ Zhou et al reported that compared to exosomes from untreated monocytes or M1 macrophages, exosomes from M2 macrophages significantly increased the ratio of Treg/Th17 when CD4⁺ T cells were treated with these exosomes from different macrophages.¹⁰⁶

Hematological System Tumors

As the effects of EVs on Tregs in the context of hematological system tumors, present most research, if not all, reported tumor-derived EVs benefit Treg. In paediatric pre-B acute lymphoblastic leukaemia, tumor-derived exosomes induce apoptosis of T cells and enhance the regulatory phenotype of T cells via increasing FOXP3 expression and the secretion of Tregs-related cytokines, such as TGF-β and IL-10.¹¹³ In B-cell lymphoma, exosomal CD19 from tumor cells benefit the initial activation of CD19-CAR T-cells but subsequently induce the apoptosis of CAR-T cells and the differentiation into Tregs phenotype.¹⁰⁹ In myeloid leukemias, 4–1BBL/CD137L in EVs from leukemia cells enhance the immunosuppressive function Tregs via regulating the expression of CD30 and TNFR2¹¹¹ and exosomal miR-24-3p reduces the apoptosis of Tregs and stimulate Tregs proliferation via activating JAK3/STAT5 signaling and increasing the expression of p-NF-κB and p-ERK protein.¹⁰⁸ Exosomes from Kasumi-1, acute leukemia cell line expressing high TP53, increase Tregs and decrease CD8⁺ T cells.¹¹² In multiple myeloma (MM), exosomes from MM cell lines OPM2 and U266B1 inhibited the apoptosis of Tregs from healthy people and elevated the viability of Tregs from healthy people.¹¹⁰ However, U266B1-derived exosomes increase the apoptosis of Tregs from MM patients and both U266B1-derived and OPM2-derived exosomes could reduce the viability of Tregs from MM patients.¹¹⁰

Melanoma, Glioma, Mesothelioma and Malignant-Effusion

Zhu et al systematically explored the effects of melanoma-derived sEVs on systemic immunity and found that melanoma-derived sEVs increased Tregs in lymph nodes.¹¹⁷ Nakazawa et al reported that B16 melanoma-derived EVs could indirectly promote Tregs proliferation via dendritic cells.¹¹⁸ In detail, B16 melanoma-derived EVs were internalized into endosomes in dendritic cells to activate TLR3-TRIF signaling to generate IFN-β which induces the proliferation of Treg. However, when dendritic cells express CD300a, CD300a could be internalized into endosomes as well to prevent EV-mediated IFN-β secretion and following Tregs proliferation.¹¹⁸ Cytokine-free and PD-1-containing exosomes from glioblastoma multiforme (GBM) cells could increase the infiltration and expansion of Tregs, which is inhibited by LRRC4, a tumor suppressor for GBM.¹¹⁹ Another study on glioma reported that exosomes from glioma stem cells form an immunosuppressive microenvironment via myeloid-derived suppressor cells rather than Tregs.¹²² Mesothelioma-derived exosomes augment the immunosuppressive function of CD4⁺CD25⁺ Tregs.¹¹⁴ In the patients with malignant effusion, TGF-β1 in exosomes collected from cancer malignant effusions help maintain the number of Tregs and the expression of FOXP3.¹¹⁶ Besides, macrophages in malignant effusion exhibit the M2 phenotype and secrete more exosomes than macrophages in the blood, and macrophage-derived exosomal miR-4443 also induces the differentiation of Tregs.¹¹⁵

In conclusion, within TME and physiological as well as pathological processes, the functions of EVs are highly dependent on their cellular sources. EVs secreted by different cells exhibit significant differences in the proportion of vesicle subtypes, the composition of molecular cargo, and the effects on target cells (eg, regulatory T cells, Treg).¹²³ Such differences are the core manifestation of EV heterogeneity and determine their diverse roles in tumor immune regulation.¹²⁴ From the perspective of regulatory effects on Treg, EVs from different cellular sources show functional differentiation into “pro-suppression” or “pro-activation”: most tumor cell-derived EVs (eg, miR-196b-5p-containin EVs from NSCLC cells, TGF-β⁺ EVs from ovarian cancer cells) focus on “enhancing Treg function”.¹³ They create a TME conducive to tumor escape by inducing Treg proliferation, inhibiting Treg senescence, and increasing the secretion of immunosuppressive factors (eg, IL-35, adenosine); a small number of tumor cell-derived EVs (eg, EVs from CT26 colon cancer cells and Colon-26 colon cancer cells), however, due to cargo heterogeneity (eg, high expression of PDCD4 and IFN-γ-related RNA), can instead reduce the number of Treg, downregulate the expression of FoxP3 in Treg, and exert anti-tumor immune effects; the regulatory effects of immune cell-derived EVs are strongly associated with the polarization state of the source cells.¹²⁵ EVs secreted by non-activated dendritic cells (DCs) can promote Treg differentiation by transmitting MHC-II/LAG-3 signals. In contrast, EVs secreted by DCs stimulated with tumor antigens (eg, DCs loaded with Hepa1-6 cell lysates) can reduce the number of CD25⁺FoxP3⁺ Treg and reverse immune suppression; mesenchymal stem cell-derived EVs generally exhibit the property of “promoting Treg generation”, but the abundance of their cargo is regulated by the microenvironment.⁷⁷ For instance, EVs from adipose-derived stem cells (ASCs) can significantly promote Treg differentiation in breast cancer, while in inflammatory models, they can alleviate tissue damage by regulating Treg metabolism.

Treg-Derived EVs

Tregs are well-known for their unique immunosuppressive properties, and the EVs they release may exhibit similar effects. Studies have shown that Treg-derived EVs carry the immunosuppressive molecule CD73, which contributes to the formation of a Treg cell-mediated immunosuppressive microenvironment.¹²⁶ Similarly, miRNAs carried by EVs from Tregs can also inhibit the functions of other immune cells, such as pathogenic T helper 1 cells and macrophages.^127,128 Consequently, in TME, Treg cell-derived EVs may be important contributors to the formation of the tumor immunosuppressive microenvironment. A study showed that in head and neck cancer patients, CD3+CD15s+ exosomes (from Tregs) increased in patients with cancer recurrence, which indicates a significant role of Treg-derived EVs in cancer progression.¹²⁹ Another two studies provided more direct evidence and showed exosomes from CD8+25+ Tregs contain CD8, CD25, GITR, Foxp3, pMHC-II and pMHC-I, and can inhibit dendritic cell-induced CD8+ T responses and immunity against B16 melanoma and lung cancer.^130,131 The immunosuppressive property of Treg cell-derived EVs provides a new approach for the treatment of various diseases characterized by excessive immune responses, such as transplantation rejection, tissue repair, acute myocardial infarction, inflammatory bowel disease, etc.^132,133 However, the application of natural Treg-EVs in cancers is limited mainly because re-activating immunity rather than inhibiting immunity is the common strategy to treat cancer.

EV-Related Therapy to Target Treg for Cancer Treatment

The therapeutic potential of EVs in cancer mainly includes targeting EVs and taking advantage of EVs for delivery tools, which are the most important clinical implications of EVs on Treg.¹²¹ In this section, we discuss how to treat cancer via these two ways to interfere with Treg.

Targeting EVs

As mentioned above, the promoting effects of EVs on Tregs are one main reason for cancer progression. Therefore, reducing these Treg-beneficial EVs represents a helpful strategy for cancer treatment. Some popular methods include RAB27A knockdown, GW4869, as well as novel AH-D peptide.¹²¹ These methods may also work well in targeting Treg-beneficial EVs. For example, the aforementioned 4–1BBL-containing EVs could result in the expansion of Treg, and these EVs are secreted from leukemic cells in a Rab27-dependent way.¹¹¹ Therefore, knockout Rab27 in leukemic cells reversed the immunosuppressive function of Tregs.¹¹¹ AH-D peptide could result in the rupture of EV membrane via sensing EV membrane from cancer cells with high curvature to reduce the number of EVs in vivo.¹³⁴ The usage of AH-D peptide greatly decreases the percentage of CD4⁺CD25⁺ Tregs in B16F10 tumor-bearing C57BL/6 mice.¹³⁴ Interestingly, the emergence of Treg-beneficial EVs is due to the function loss or down-expression of some tumor suppressor genes. The recovery of these genes also is an effective way to target Treg-beneficial EVs. The best example is LRRC4 mentioned above.¹¹⁹ Besides these, other drugs or biomaterials also decrease Treg-beneficial EVs. In contrast to tumor suppressor genes, some tumor-promoting genes could increase Treg-beneficial EVs. For example, PKC-ζ is related to radiotherapy resistance. In detail, irradiation-induced the phosphorylation level of PKC-ζ in breast cancer 4T1 cells, which results in the secretion of TGF-β1-containing EVs to form immunosuppressive TME via promoting the differentiation of Tregs from CD4⁺ T cells.¹⁰⁴ Inhibition of PKC-ζ via siRNA or naringenin, a natural flavonoid, could revere the secretion of TGF-β1-containing EVs and immune suppression to overcome radiotherapy resistance.¹⁰⁴ Some known drugs also repress tumors by reducing Treg-beneficial EVs. Macitentan, an oral drug approved by the FDA, can decrease the number of PD-L1-containing EVs. The combination of macitentan and anti-PD-L1 antibody can boost the number and activity of CD8⁺ T cells but reduce Tregs number in tumors and lymph nodes in the tumor model of triple-negative breast cancer (TNBC), colon cancer, and lung cancer.¹³⁵ Similarly, the combination of botulinum neurotoxin type A1 and anti-PD-L1 therapy also reduces blood exosomes and reverses the increase of Tregs in the B16-F10 syngeneic mouse tumor model.¹³⁶ Estrogen, a well-known hormone therapy for some cancers, could reduce the level of TGF-β1 in EVs from colon cancer MC38 cells thus blocking the ability of MC38-derived EVs to induce Tregs.¹³⁷ These research proved the feasibility of targeting Treg-beneficial EVs for cancer treatment (Table 2).

Table 2 Targeting EVs to Inhibit Tregs for Cancer Treatment

EV Delivery

Harnessing EVs, including native EVs, pre-generation modified, post-generation modified, and hybrid EVs, as drugs or tools to deliver drugs/biomaterials is another strategy for cancer treatment (Figure 3 and Table 3).

Table 3 Treg-Targeting EVs in Cancer Treatment

Figure 3 Therapeutic EVs for targeting Treg in cancer treatment. Therapeutic EVs could be generated via pre-generation modification, post-generation modification, and hybridization.

In the field of drug delivery, EVs exhibit unique and irreplaceable advantages over traditional drug delivery (such as CTLA-4 antibody therapy, etc) and artificially synthesized nanoparticles (such as liposomes, polymeric nanoparticles, and metallic nanoparticles). The core of these advantages lies in the high alignment between their “natural biological properties” and “precise delivery capabilities”.^150–152 First, EVs possess excellent biocompatibility and low immunogenicity. As naturally released vesicles from cells, their membrane structure is fully compatible with the components of biological membranes (eg, phospholipids, cholesterol, membrane proteins), which allows them to avoid foreign body rejection reactions commonly induced by traditional drug or artificial nanoparticles (such as complement system activation and excessive phagocytosis by macrophages).^152,153 Autologous EVs can achieve long-term circulation in the body, significantly reducing toxic and side effects. In contrast, even after surface modification (eg, PEGylation), traditional drug or artificial nanoparticles still struggle to completely eliminate the risk of immune recognition.^154,155 Second, EVs have precisely targeted delivery capabilities. The surface of their membrane carries cell-specific molecules from their source cells (eg, integrins, adhesion proteins, glycosylation modifications), which can act like a “biological navigator” to recognize specific receptors on the surface of target cells (eg, EGFR highly expressed on tumor cells, CD31 on vascular endothelial cells), enabling active targeted accumulation of drugs at lesion sites.^153,156 By comparison, the targeting ability of artificial nanoparticles or traditional drug mostly relies on the chemical conjugation of exogenous ligands (eg, antibodies, peptides).¹⁵¹ This modification process is not only complex and prone to affecting particle stability but may also lead to reduced targeting efficiency due to ligand detachment. Furthermore, EVs feature efficient cargo loading and release mechanisms. Their natural compartmental structure is compatible with various types of drugs (eg, small-molecule chemotherapeutic drugs, nucleic acid drugs, protein drugs). Moreover, they can directly deliver drugs into the cytoplasm of target cells through membrane fusion or endocytosis, preventing drugs from being degraded in lysosomes.^157,158 On the other hand, traditional drug or artificial nanoparticles often face issues such as low drug encapsulation efficiency, easy leakage, or drug inactivation due to insufficient lysosomal escape efficiency after entering cells.¹⁵⁹ Finally, EVs also demonstrate excellent biological barrier penetration. Thanks to their nanoscale size (30–1000 nm) and natural membrane structure, EVs can penetrate physiological barriers that are difficult for artificial nanoparticles to cross, such as the blood-brain barrier and blood-ocular barrier. This provides a new pathway for drug delivery in refractory diseases like central nervous system diseases and ocular diseases.^153,156 These natural advantages make EVs a core candidate for the next generation of drug delivery systems. Especially in terms of precise therapy and reducing drug toxic and side effects, their application potential significantly surpasses that of traditional drug or artificially synthesized nanoparticles.

Native EVs with inhibitory effects on Tregs have been mentioned above^91,94,100 (Figure 4). Pre-generation modified EVs include EVs acquired via endogenous loading and EVs from cells with/without certain stimulation. The endogenous loading mainly involves gene editing. Gao et al acquired Treg-targeting EVs from miR-124-3p transfected bone marrow mesenchymal stromal cells (BM-MSCs).¹³⁸ Exosomes from genetically engineered BM-MSCs carried large amounts of miR-124-3p, which directly targets monocarboxylate transporter 1 (MCT1) to reduce the lactate uptake in Treg, finally repressing the immunosuppressive function of Tregs.¹³⁸ These genetically engineered exosomes greatly improve the efficiency of anti-PD-1 therapy in ovarian cancer-bearing mice via reversing Treg-mediated immunosuppression.¹³⁸ Lu et al used genetically engineered DC2.4 cells to obtain therapeutic EVs.¹³⁹ They transfected DC2.4 cells with lentivirus expressing α-fetoprotein (AFP) gene and then collected DEX_AFP (dendritic cell-derived exosomes expressing AFP) from these modified DC2.4 cells.¹³⁹ DEX_AFP stimulated more IFN-γ-expressing CD8⁺ T cells and caused a decrease in CD25⁺Foxp3⁺ Tregs, which induced effective tumor suppression in vivo.¹³⁹ Ji et al also developed Exo^smart (exosomes with high expression of CD62L and OX40L) via infecting donor DC2.4 cells with lentivirus overexpressing CD62L and OX40L.¹⁴⁰ CD62L on the surface of Exo^smart provides target T cells with a high affinity to lymph nodes while OX40L on the surface of Exo^smart promotes the expansion of T cells and inhibits Tregs.¹⁴⁰ In 4T1 breast tumor-bearing mice, Exo^smart greatly inhibits tumor growth and metastasis in lymph nodes via activating immunity in tumor-draining lymph nodes.¹⁴⁰ However, some research also demonstrated the validity of EVs from donor cells treated with certain stimulation for cancer treatment via targeting Treg. Zhong et al treated dendritic cells with Hepa1-6 cell lysates and then collected exosomes (mDC-derived exosomes).¹⁴¹ Compared with microwave ablation (MWA) monotherapy, the combination of MWA and mDC-derived exosomes greatly decreased the number of CD25⁺Foxp3⁺ Tregs to improve the immune microenvironment and inhibit tumor growth.¹⁴¹ More interestingly, besides these non-malignant cells, tumor-derived EVs also gained much attention in the field of therapeutic EVs. Guo et al discovered that heat-stressed MC38 colon cancer-derived exosomes contain HSP70 and could induce the conversion of Tregs into Th17 cells in MC38-bearing mice.¹⁴² Semionatto et al generated genetically engineered tumor cells via transfecting B16F10 melanoma cells with retroviral vectors encoding OX40L and then harvested OX40-EVs for cancer treatment.¹⁴³ OX40-EVs exhibited a great ability to inhibit the expression of FoxP3 to block the immunosuppressive phenotype of Tregs.¹⁴³ The exploration of the application of post-generation modified EVs is another hotspot in cancer treatment and the way to load drugs or biomaterials into EVs could be divided into endogenous and exogenous loading,¹⁶⁰ thus some drugs or biomaterials could be loaded into EVs after generation. Coincidentally, Rezaei et al also acquired miR-124-3p-expressing exosomes, but they adapted post-generation modification rather than genetically engineering as Gao et al did.^125,138 Rezaei et al cocultured exosomes from CT-26 cancer cells with miR-124-3p via a modified calcium chloride method.¹²⁵ These post-generation modified miR-124-3p-expressing exosomes also function well in inhibiting CD4⁺CD25⁺Foxp3⁺ Tregs.¹²⁵ Wang et al designed RSL3-loading exosomes via coculturing RSL3 with exosomes but they treated primary born-derived macrophage with lipopolysaccharide in advance before collecting exosomes.¹⁴⁴ These RSL3-loading exosomes stimulated anti-cancer immunity, including reducing the number of CD4⁺CD3⁺FOXP3⁺ Tregs in tumor-beating mice.¹⁴⁴ RSL-loading exosomes are the product of the combination of pre-generation and post-generation modification in essence. Phung et al also applied a similar combination to acquire EXO-OVA-mAb, which increased the ratio of CTLs/Tregs (cytotoxic T lymphocytes/Treg) in tumor sites. They cocultured exosomes from bone marrow cells, which are treated with OVA in advance, with anti-CTLA-4 antibody.¹⁴⁵ The combination is not limited to this. Zhou et al combined cell stimulation, electroporation, and incubation.¹⁴⁶ They treated PANC-02 cells with mitoxantrone to acquire exosomes (MEXO), then loaded CCL22 siRNA into MEXO via electroporation, and finally obtained spMEXO via incubating CCL22 siRNA-loading MEXO with CM peptide, the conjugation of CP05 peptide and MART-1 peptide.¹⁴⁶ spMEXO can indirectly suppress Tregs via inhibiting the CCR4/CCL22 axis between dendritic cells and Tregs in vivo, and the combination of spMEXO with chemotherapy achieved a synergistic effect, including boosting anti-tumor immunity, suppressing tumor growth, and improving overall survival rate.¹⁴⁶ Hybrid exosomes include mixing exosomes with nano-biomaterials and mixing exosomes from different cells. GT-exos (exosomes from genetically engineered CD47-overexpressed cancer cells) and M1-exos (exosomes from M1 macrophages) were hybridized to generate genetically engineered hybrid exosomes (gHE). Then, SN38 and MnO₂ were successively added into gHE via sonication and extrusion respectively to acquire SN/Mn@gHE.¹⁴⁷ SN/Mn@gHE decreased CD4⁺Foxp3⁺ Tregs and myeloid-derived suppression cells but increased central CD44⁺CD62L⁻ and effector memory T cells.¹⁴⁷ Liposome is another popular drug delivery nanoparticle and also is a good partner for exosomes. Yang et al designed hybrid exosomes (HE) based on exosomes from M1 macrophages and liposomes via simple thin film hydration followed by a membrane extrusion.¹⁴⁸ Thalidomide (THD) was loaded into HE to form HE-THD via a physical encapsulation technique. In vivo, HE-THD inhibited CD4⁺Foxp3⁺ Tregs expansion and proliferation for anti-cancer immunity.¹⁴⁸ Another study generated hybrid exosomes loaded with paclitaxel (ELP) via fusing exosomes from mesenchymal stem cells and folate-targeted liposomes based on repeated freeze-thaw cycles with the existence of paclitaxel.¹⁴⁹ In CT26 tumor-bearing mice, ELP significantly decreased Tregs and raised the proportion of activated CD8⁺ cells and CD4⁺ T cells.¹⁴⁹

Figure 4 Schematic illustration of native, engineered, and hybrid extracellular vesicles (EVs) for therapeutic applications. Native EVs can be engineered to load various therapeutic cargos—including proteins, nucleic acids, lipids, drugs, peptides, and liposomes—through multiple modification strategies such as biological interaction, freeze–thaw cycles, electroporation, sonication, incubation, hybridization, transfection, and extrusion. These hybrid or engineered EVs can be tailored to deliver specific therapeutic molecules in vivo, enabling precision applications such as regulatory T cell (Treg)-targeted EV therapy for cancer treatment.

Conclusion and Perspective

A great deal of research emphasized the fundamental effects of EVs on Tregs. In TME, EVs form a communication network thus EVs from various cells including tumor cells, macrophages, adipocytes, etc., patients’ plasma, and effusion could regulate the expansion, proliferation, differentiation, and death of Tregs. The function molecules in Treg-regulating EVs include PD-L1, PD-1, TGF-β, non-coding RNA, CD19, CD39, CD73, IL-10, SIRT1, 4–1BBL, CD137L, EGFR, and other biological molecules, most of which are well-known immunosuppressive factors. Generally speaking, most studies proved the promoting effects of EVs on Treg in the context of tumors which reflects the fact that tumors often harness EVs to benefit tumor-promoting cells again.¹²¹ More interestingly, the emergence of Treg-beneficial EVs is associated with the downregulation of tumor-inhibiting genes or upregulation of tumor-promoting genes in some research, which is consistent with the natural tumor progression. However, a few reports discovered the inhibitory effects of EVs on Treg, which reflect EV heterogeneity and our imperfect understanding of the interaction between EVs and Treg in cancer.

The wide promoting effects emphasized the importance of targeting tumor-promoting EVs again. Some methods function well in preventing the release of Treg-promoting EVs or deleting them in cell experiments or tumor-bearing models, which showed clinical potential in cancer treatment. Besides, the engineering EVs inhibiting Tregs also provide a novel selection for tumor treatment. However, there remain some challenges before EV-related therapy enters clinical practice. Which kinds of cells are best for EV origin? Tumor-derived EVs provide a high affinity to tumor cells but could inherit some tumor-promoting properties; immune cell-derived EVs could activate anti-tumor immunity, but their target ability and yield may not be perfect. The isolation method and EV generation also need further improvement. The batch effect is another problem in generating therapeutic EVs. In addition, present research usually uses cell lines or mouse models, which indicates the development of EV-related therapy still is in the laboratory stage. Clinical trials and other 3D models will upgrade the current understanding of them. Anyway, the emerging work focusing on the influences of EVs on Tregs and EV-related therapy for targeting Treg shows our deepening comprehension of tumors and promises a better treatment strategy in the future.

Consent for Publication

The authors have consented to publish this article.

Acknowledgments

The figures in this review were created by Figdraw.

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.

Funding

This research was funded by the Department of Education of Liaoning Province (JYTJCZR2020059) and the Department of Education of Liaoning Province (LJ212410159099).

Disclosure

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Introduction

Ovarian cancer is the third most common gynecological malignancy globally, with a five-year survival rate of 40–45%.^1–3 Cytoreductive surgery serves as the first-line treatment for ovarian cancer and requires multi-organ resection.⁴ The surgical trauma associated with this operation results in various types of pain, including incision, inflammatory, and visceral pain, leading to severe postoperative pain for patients. Therefore, pain management following primary cytoreductive surgery remains a major challenge. Improved opioid analgesics should be prioritized in the analgesic strategy for cytoreductive surgery, aligning with the requirements of Enhanced Recovery After Surgery protocols.⁵

Butorphanol, a synthetic opioid receptor agonist-antagonist, exhibits a potency ratio of 25:4:1 against κ, μ, and δ receptors, respectively. Varying affinities for these receptors results in distinct clinical effects.⁶ Compared to pure μ receptor agonists, butorphanol is more effective at suppressing visceral pain while reducing opioid-associated adverse effects and opioid dependence risk. A study by Du et al⁷ on postoperative analgesia in patients undergoing laparoscopic hysterectomy showed that dexmedetomidine combined with butorphanol patient-controlled intravenous analgesia (PCIA) significantly decreased postoperative visual analog scale (VAS) scores and postoperative nausea and vomiting, thus improving patient satisfaction. Similarly, butorphanol has been shown to reduce postoperative visceral pain in patients undergoing microwave ablation for liver tumors.⁸ However, its application in large open abdominal surgeries, such as ovarian cancer surgery has not been reported. Considering the existing literature, pharmacological characteristics of butorphanol, and lack of reported effective perioperative analgesia for ovarian cancer, we anticipate that butorphanol PCIA will provide effective analgesia with fewer adverse reactions in postoperative ovarian cancer. However, the optimal dosage remains to be determined.

Furthermore, patients with tumors often experience a suppressed immune function, making them susceptible to immune dysfunction during the perioperative period. Surgical trauma, perioperative stress, inflammatory responses, and anesthetic agents facilitate dissemination of tumor cells.⁹ In vitro studies have demonstrated that butorphanol protects PC12 cells from inflammation and apoptosis induced by oxygen-glucose deprivation/reperfusion.¹⁰ Additionally, butorphanol may inhibit malignant biological behavior of ovarian cancer cells by downregulating the expression of TMEFF1 (Tomoregulin-1, a transmembrane protein with an epidermal growth factor-like domain and two follistatin-like structural domains).¹¹ Consequently, we hypothesized that butorphanol PCIA as an opioid agonist-antagonist in postoperative patients undergoing primary cytoreductive surgery for ovarian cancer may stabilize the immune status of these patients.

Materials and Methods

Patients

This was a prospective, double-blind, randomized controlled trial. This study was approved by the Medical Research Ethics Committee of the First Affiliated Hospital of the University of Science and Technology of China (approval number: 2021KY [258]). This study was registered in the Chinese Clinical Trial Registry (Registration Number: ChiCTR 2300069879). Compliance with the Declaration of Helsinki and CONSORT standards was ensured during the trial. All patients signed an informed consent form. We enrolled patients who underwent primary cytoreductive surgery for ovarian cancer at the First Affiliated Hospital of the University of Science and Technology of China (Anhui Provincial Hospital) between May 2023 and March 2025. Four groups were formed by dividing the patients according to the postoperative use of sufentanil or different doses of butorphanol PCIA: S (sufentanil 0.04 μg·kg⁻¹·h⁻¹), B1 (butorphanol 3.0 μg·kg⁻¹·h⁻¹), B2 (butorphanol 3.5 μg·kg⁻¹·h⁻¹), and B3 (butorphanol 4.0 μg·kg⁻¹·h⁻¹).

The following inclusion criteria were applied: American Association of Anesthesiologists (ASA) classification I, II, or III; ovarian cancer confirmed by surgical and pathological diagnosis; no history of allergies to the study drugs; International Federation of Gynecology and Obstetrics classification I, II, or III; and age 18–65 years. Patients who had multiple chronic diseases, were unable to tolerate surgical anesthesia, had severe cardiopulmonary or renal insufficiency, had coagulation dysfunction, were allergic to sufentanil or butorphanol, had severe sequelae of neurological disorders, had advanced ovarian cancer with cachexia, had a recent history of opioid use, had recent infections or immune system diseases, or were pregnant or lactating were excluded from the study. The removal criteria were as follows: intraoperative bleeding volume more than 2000 mL, surgical duration >6 h, or need for a second procedure within 48 h postoperatively.

Anesthesia and Analgesia

All patients fasted for 8 h before surgery and had no oral intake for 4 h. General anesthesia was administered via endotracheal intubation. Upon entering the operating room, patient information was verified and intravenous access was established. Heart rate, electrocardiography, oxygen saturation, and end-tidal carbon dioxide were continuously monitored. Under local anesthesia, radial artery catheterization was performed to monitor arterial pressure and internal jugular vein catheterization was performed to monitor central venous pressure. All groups of patients were induced with intravenous administration of midazolam (0.05 mg/kg), sufentanil (0.5 μg/kg), etomidate (0.3 mg/kg), and rocuronium (0.8 mg/kg). Following satisfactory muscle relaxation, endotracheal intubation was performed and intermittent positive pressure ventilation was initiated. Target-controlled infusion was used to maintain a target plasma concentration of propofol between 2.0~4.0 μg/mL and remifentanil between 2.0~6.0 ng/mL during the surgery, with inhalation of 1–2% sevoflurane and intermittent bolus doses of cisatracurium (0.1 mg/kg) to maintain a Bispectral Index value between 40 and 60. Intraoperative monitoring included nasal temperature measurements and temperature management was implemented for thermal protection. The acid–base balance, electrolyte levels, blood glucose levels, and other parameters were adjusted according to the arterial blood gas results.

After completion of surgery, all patients underwent bilateral, four-point transversus abdominis plane (TAP) block under ultrasound guidance, and 15 mL of 0.25% ropivacaine was injected at each injection site through different puncture pathways.^12,13

Postoperatively, all the patients received PCIA. The study protocols were sealed in envelopes and were randomly distributed to each participant. The study drugs for the PCIA pump were prepared by an independent anesthetic nurse, with an anesthesiologist handling the recording of study outcomes. The PCIA formulations were as follows: sufentanil 0.04 μg·kg⁻¹·h⁻¹ for group S, low-dose butorphanol (3.0 μg·kg⁻¹·h⁻¹) for group B1, medium-dose butorphanol (3.5 μg·kg⁻¹·h⁻¹) for group B2, and high-dose butorphanol (4.0 μg·kg⁻¹·h⁻¹) for group B3, with background infusion of 2 mL/h, a bolus dose dose of 1 mL and a lockout interval of 30 minutes. When the patients’ VAS scores exceeded 4, rescue analgesia was administered via intravenous injection of 50 mg flurbiprofen axetil as analgesic rescue.

Data Collection

The patients’ general characteristics were recorded, including age, height, weight, body mass index (BMI), and ASA classification. Surgical parameters, such as operative time, intraoperative blood loss, urine output, fluid intake, extubation time, and surgical complexity scores were also documented. Additionally, postoperative pain VAS scores at T1 (2 h), T2 (6 h), T3 (12 h), T4 (24 h), and T5 (48 h), the curve of VAS time (AUC_VAS-time) over 48 hours, the number of PCA button presses, the frequency of rescue analgesia, the occurrence of adverse reactions, postoperative recovery indicators and preoperative and postoperative inflammatory indicators, including neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and lymphocyte-to-monocyte ratio (LMR) were recorded for each group.

Levels of T lymphocytes (CD3+), B lymphocytes (CD19+), helper/inducer T lymphocytes (CD3+CD4+), suppressor/cytotoxic T lymphocytes (CD3+CD8+), and natural killer cells (CD3-CD16+ and CD56+), as well as the CD4+/CD8+ ratio were determined at baseline and 48 h postoperatively by flow cytometry using an Attune NxT flow cytometer. A schematic diagram of the gating strategy for the flow cytometry of various lymphocyte subsets and representative data of different parameters for each group are shown in Figure 1. To minimize the potential confounding effect of neoadjuvant chemotherapy on hematological parameters, patients who had received preoperative chemotherapy were excluded from this study. Furthermore, all postoperative blood samples were collected prior to administering any postoperative chemotherapy.

Figure 1 Flow cytometry workflow and representative plots of each group. (A) Gating strategy for lymphocyte subpopulation analysis by flow cytometry. (B) Representative flow cytometry plots showing the percentages of CD3+ T cells, CD19+ B cells, CD4+ T cells, CD8+ T cells, CD4+/CD8+ ratio, and NK cells across four groups at different time points.

Statistical Analysis

A preliminary trial was conducted with 10 patients in each group, followed by recording postoperative VAS scores at 12 h (B1: 3.5 ± 0.85; B2: 2.80 ± 0.63; B3: 2.60 ± 0.70; S: 2.9 ± 0.74). Sample size was calculated using PASS 15.0 statistical software, with 90% power and a two-sided α value of 0.05. The required sample sizes for the four qualifying groups were computed using one-way analysis of variance (ANOVA) and averaged, resulting in a required sample size of 69. Considering a 20% loss to follow-up rate, a total sample size was set at 88 patients, with 22 patients allocated to each group.

Data analysis was performed using SPSS 25.0 statistical software. Results for normally distributed parameters are expressed as mean ± standard deviation (), while non-normally distributed parameters are presented as median and interquartile range. ANOVA was used for multigroup measurements to compare means among the three groups, with Bonferroni post-hoc tests for multiple comparisons. Categorical variables were analyzed using appropriate non-parametric tests based on data distribution. Ordered categorical data were analyzed using rank-sum tests, whereas unordered categorical data were analyzed using chi-square tests. The Fisher’s exact test was used when the cell frequency was less than 5. The significance level was set at a two-sided α = 0.05.

Results

Patient Characteristics

Among the 119 patients who underwent ovarian cancer cytoreductive surgery under general anesthesia between May 2023 and March 2025, 88 were included in this study and randomized into four groups (Figure 2). As shown in Table 1, no statistically significant differences in age, BMI, ASA scores, comorbidities, blood loss, operative time, extubation time, surgical complexity scores, intraoperative fluid volume, or bleeding volume were observed among the four groups (P > 0.05).

Table 1 Demographic and Clinical Characteristics of the Patients Included

Figure 2 Flow diagram of participants in the study.

Postoperative Pain Score

At 2 and 6 h postoperatively, no statistically significant differences in pain VAS scores were observed among the groups (P = 0.789 and P = 0.154, respectively). At 24 h postoperatively, the VAS score in group B3 was significantly lower than that in group S (P = 0.042). Additionally, the VAS scores at 12 and 24 h were lower in groups B2 (P = 0.007 and P < 0.001, respectively) and B3 (P = 0.005 and P < 0.001, respectively) than in group B1. However, no statistically significant differences in VAS scores were observed between groups B2 and B3 at the time points of 2, 6, 12, 24, and 48 h (P = 1.000, P = 1.000, P = 1.000, P = 0.663, and P = 1.000, respectively) (Figure 3). Compared with that in group S, the AUC_VAS-time was higher in group B1 and lower in group B3 (P = 0.010 and P = 0.004, respectively). Furthermore, the AUC_VAS-time was lower in groups B2 and B3 than in group B1 (P < 0.001), as shown in Figure 4.

Figure 3 Postoperative VAS scores were measured at 2, 6, 12, 24, and 48 hours after surgery. The VAS score in group B3 compared with group S, aP<0.05. The VAS score in group B2 and group B3 compared with group B1, bP<0.05.

Figure 4 The area under the VAS-time curve (AUCVAS-time) over 48 hours. AUCVAS-time: The area under the VAS-time curve (AUCVAS-time) over 48 hours was calculated using the trapezoidal rule, which represents the cumulative pain intensity. Compared with group S, aP<0.05, Compared with group B1, bP<0.05.

PCIA and Rescue Analgesia

The number of effective presses for PCIA was lower in group B3 than in group S (P = 0.045). Group B3 also exhibited a lower number of effective presses on the analgesic pump compared with group B1 (P = 0.005). However, no statistically significant differences in the total number of presses on the analgesic pump were observed among the four groups (P = 0.075). Additionally, the proportion of patients requiring rescue analgesia was lower in group B3 than in group B1 (P = 0.002), as shown in Table 2.

Table 2 PCIA and Rescue Analgesia Among the Four Groups

Comparison of Adverse Effects and Postoperative Rehabilitation Indexes Among the Four Groups

No statistically significant differences in postoperative nausea and vomiting, dizziness, drowsiness, or respiratory depression were observed among the four groups (P > 0.05). Additionally, no statistically significant differences in the incidence of complications, such as enteric fistula, pulmonary complications, and wound healing were observed among the four groups (P > 0.05), as shown in Table 3. The differences in time to first flatus, time to first ambulation, and length of hospital stay among the four groups were not statistically significant (P > 0.05). However, the time to ambulation was significantly shorter in group B3 than in group S (P = 0.031) (Table 3).

Table 3 Adverse Effects and Postoperative Rehabilitation Indexes Among Four Groups

Comparison of Inflammatory Biomarkers Among the Four Groups

Within each group, patients exhibited a significant postoperative increase in PLR and NLR compared to preoperative values, whereas LMR showed a significant postoperative decrease. No statistically significant differences were observed among the four groups, either preoperatively or postoperatively (Figure 5).

Figure 5 Box plots of NLR (a), PLR (b) and LMR (c) for the four groups of patients at different time points. T0: preoperative, T5: 48 hours postoperatively. Abbreviations: NLR, neutrophil-to-lymphocyte ratio; PLR, platelet-to-lymphocyte ratio; LMR, lymphocyte-to-monocyte ratio.

Expression of CD3+, CD4+, CD8+, CD4+/CD8+, and CD3‐CD16+CD56+

Intergroup comparisons at T0 (preoperative) and T5 showed no significant differences in immune markers among the four groups (P > 0.05), as illustrated in Figure 6. Group S exhibited a lower NK cell count (P = 0.007) and a significantly higher CD19+ B cell count (P = 0.005) at T5 than at T0. Group B1 showed an increase in CD19+ B cells (P = 0.012), CD4+ T cell levels (P = 0.007), and the CD4+/CD8+ ratio (P = 0.014) at T5 compared to T0, whereas CD8+ T cell count decreased (P = 0.011). No statistically significant differences were observed in T cell subsets and NK cell levels between groups B2 and B3 at T5 compared with those at T0 (P > 0.05) (Figure 6).

Figure 6 The level of lymphocyte subsets and NK cells in the four groups at different time points. The percentages of CD3+T cells (a), CD19+B cells (b), CD4+T cells (c), CD8+T cells (d) T lymphocytes, CD4+/CD8+ ratio (e), NK cells (f) in the four groups at different time points.

Discussion

In the present study, we evaluated the clinical effects of butorphanol for PCIA on pain relief and immune function in patients undergoing cytoreductive surgery for ovarian cancer. Our results suggest that butorphanol PCIA at a high dose of 4.0 μg·kg⁻¹·h⁻¹ in patients undergoing intubated general anesthesia for ovarian surgery results in optimal analgesic outcomes without increasing the incidence of adverse events, and does not significantly impact patient immune markers, demonstrating its safe application for postoperative analgesia.

Several studies have indicated that¹⁴ the postoperative analgesic efficacy of sufentanil is approximately 1000 times greater than that of morphine, whereas the analgesic potency of butorphanol is 5–8 times that of morphine. This indicates that 1 mg sufentanil may be equivalent to the analgesic effect of 200 mg butorphanol. A study investigating the postoperative analgesic effects of dexmedetomidine combined with sufentanil or butorphanol in patients undergoing laparoscopic gastrointestinal tumor resection has demonstrated that sufentanil (2.0 μg/kg) or butorphanol (0.15 mg/kg) combined with dexmedetomidine can be safely used for postoperative pain management in these patients.¹⁵ In light of the increasing adverse reactions associated with higher doses, our study compared butorphanol dosages of 3.0 μg·kg⁻¹·h⁻¹, 3.5 μg·kg⁻¹·h⁻¹, and 4.0 μg·kg⁻¹·h⁻¹ with sufentanil at 0.04 μg·kg⁻¹·h⁻¹.

Butorphanol, with its unique mechanism of action and specific efficacy against visceral pain, can enhance analgesic effects of opioids while reducing the occurrence of common opioid-related adverse reactions.⁷ The TAP block is recognized for its efficacy in managing somatic or incisional pain; however, it does not provide analgesia for visceral pain caused by intra-abdominal surgeries. Studies have shown that the analgesic duration of TAP block typically ranges from 12 to 24 h, depending on the type of injected medication and individual patient differences.^16,17 Our study showed that the VAS scores for four groups peaked at 12 h postoperative (mean ± standard deviation): B1 (3.27 ± 0.77), B2 (3.73 ± 0.83), B3 (3.00 ± 0.62), and B4 (2.73 ± 0.63). Some patients used PCIA or rescue analgesics when their VAS score exceeded 4, with a subsequent decline in VAS scores observed after the 12-h mark. We interpret this to be due to the gradual metabolism of local anesthetic agents from the TAP block at 12 h postoperatively, resulting in diminished analgesic effects from TAP and the need to incrementally increase the intensity of PCIA analgesia to provide supplementary pain relief. Consequently, the high (4.0 μg·kg⁻¹·h⁻¹) and medium (3.5 μg·kg⁻¹·h⁻¹) dose butorphanol groups demonstrated superior analgesic benefits beyond the 12-h period. Statistically significant differences in VAS scores were observed at the 12-h and 24-h time points (P < 0.001). At 12 h postoperative, the VAS scores of groups B2 (P = 0.007 and P < 0.001) and B3 (P = 0.005 and P < 0.001) were significantly lower than that of group B1, indicating that high (4.0 μg·kg⁻¹·h⁻¹) and medium doses (3.5 μg·kg⁻¹·h⁻¹) of butorphanol PCIA provided stronger analgesic efficacy compared to low dose butorphanol (3.0 μg·kg⁻¹·h⁻¹) PCIA. This suggests that low-dose butorphanol PCIA was insufficient for pain management. At 24 h postoperative, group B3 had lower VAS scores compared to group S (P = 0.042), thus indicating that postoperative analgesia with 4.0 μg·kg⁻¹·h⁻¹ butorphanol provides better pain relief for ovarian cancer patients. At the time points of 2, 6, 12, 24, and 48 h, no statistically significant differences in analgesic efficacy were observed between the medium-dose butorphanol (3.5 μg·kg⁻¹·h⁻¹) and the sufentanil groups (0.04 μg·kg⁻¹·h⁻¹) (P > 0.05), suggesting similar analgesic efficacy and intensity between the two groups.

The area under the VAS-time curve (AUC_VAS-time) was calculated for 48 h postoperatively to compare cumulative pain intensity.¹⁸ Compared to group S, the AUC_VAS-time for group B1 increased (P = 0.010), while the AUC_VAS-time for group B3 decreased (P = 0.004). Compared to group B1, the AUC_VAS-time for groups B2 and B3 decreased (P < 0.001). These results suggest that PCIA with butorphanol or sufentanil combined with TAP can efficiently relieve postoperative pain in the short term. Importantly, the butorphanol dose in group B1 was inadequate to address postoperative pain in patients with ovarian cancer. Group B3 demonstrated superior analgesic efficacy compared to the S group. Additionally, the effective press counts on the analgesic pump and the need for rescue analgesia in group B3 were significantly lower than those in groups B1 and S. The present study revealed that butorphanol PCIA at a dose of 4.0 μg·kg⁻¹·h⁻¹ resulted in optimal analgesic effects.

The results on adverse reaction incidence demonstrate no statistically significant differences across the groups. A meta-analysis indicated that butorphanol in PCA significantly reduces adverse events such as nausea, vomiting, pruritus, and dizziness.¹⁹ Although six patients in group B3 experienced postoperative drowsiness (a common butorphanol-associated adverse reaction), this was not statistically significant compared to group S.²⁰ All six patients were mildly drowsy, easily awakened, and did not require special intervention. No instances of respiratory depression were observed in any group. Additionally, group B3 ambulated earlier than postoperatively than group S, likely due to better pain relief, conditions favoring early mobilization, and enhanced recovery. This finding may be influenced by the study’s single-center design and modest sample size. Therefore, future research should plan to adopt a multicenter approach to increase sample size and explore the occurrence of adverse reactions among groups.

Various inflammatory markers have been widely used in cancer patients, such as neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and lymphocyte-to-monocyte ratio (LMR).^21,22 NLR might be a reliable indicator of opioid-related immunosuppression after after thoracoscopic Surgery.²³ This study demonstrated a significant increase in postoperative PLR and NLR, along with a decrease in LMR, indicating that surgical stress induces inflammation and promotes a hypercoagulable state, predisposing patients to postoperative thrombotic events. Intergroup comparisons revealed no statistically significant differences in these hematological indices, at either preoperative or postoperative timepoints (P > 0.05), suggesting uniform inflammatory dynamics across all groups. Postoperative inflammatory marker changes were consistent across groups, indicating that butorphanol PCIA may exert similar effects to sufentanil in modulating these responses, with no observed intergroup differences.

NK cells are a major component of innate immunity, which can kill cells, promote the production of proinflammatory cytokines, and enhance the cytotoxicity and persistence of NK cells in vivo. NK cells are one of the targeted therapies for cancer and play an important role in tumor defense.²⁴ Group S exhibited a significant reduction in NK cell levels at 48 h postoperatively compared to preoperative levels (P = 0.007). It indicated that sufentanil suppresses NK cell activity in patients with ovarian cancer. This was consistent with previous studies.²⁵ In contrast, NK cell levels in the three butorphanol groups showed no significant differences at 48 h postoperatively compared to the preoperative levels (P > 0.05), suggesting that butorphanol does not adversely affect NK cell function in patients with ovarian cancer.

Opioids can not only directly affect immune cells, such as neutrophils, macrophages and NK cells, but also affect immune function through the hypothalamic pituitary-adrenal (HPA) axis. In addition, opioids can also regulate sympathetic nerve activity to affect immune function. In this study, group B1 showed significantly higher CD19+ B lymphocyte levels (P = 0.012), CD4+ T lymphocyte levels (P = 0.007), and CD4+/CD8+ ratio (P = 0.014) at 48 h postoperatively (T5) compared with preoperative (T0), along with a lower CD8+ T cell levels (P = 0.011). In contrast, no significant differences were observed between groups B2 and B3. In the vast majority of physiological and pathological processes, CD4+ T cells serve as the initiators and regulators of immune responses, while CD8+ T cells act as the key effector executors—both are indispensable. In this study, patients in the low-dose group exhibited elevated CD4+ T lymphocyte levels and decreased CD8+ T lymphocyte levels postoperatively, suggesting that their immune function remained somewhat compromised. In contrast, no significant differences were observed in any parameters between groups B2 and B3. Moderate to high doses of butorphanol could partially alleviate surgery-induced stress and pain-related immunosuppression in ovarian cancer patients, with minimal impact on their postoperative immune status. Previous research has shown that μ-receptor agonists can inhibit T cell proliferation and macrophage activity, thereby affecting immune response.²⁶ Studies have shown that opioids enhance the proliferation and metastatic potential of tumor cells through μ-receptor expression while inhibiting immune cell function.²⁷ Research conducted by Gupta et al showed that morphine suppresses macrophage and T cell activity by activating the μ-opioid receptor, which helps to reduce the antitumor immune response.²⁸ Butorphanol enhances analgesia for visceral pain through κ-receptor activation while mitigating tumor-associated immunosuppression in recurrence and metastasis processes through reduction of μ-opioid receptor overexpression. In vivo experiments have shown that butorphanol provides protective effects against ischemia-reperfusion injury in rat myocardial tissue,²⁹ which enhances macrophage phagocytic activity and balances cytokine production. This indicates that butorphanol may enhance immune factor activity and bolster overall immune function, which is consistent with the results of this study.

In this study, the analgesic efficacy in group B1 was insufficient, allowing groups B2 and B3 to achieve better pain relief without adversely affecting immune function. Although butorphanol did not demonstrate significant immunoenhancement effects in this study, moderate to high doses partially alleviated surgery-induced immunosuppression. Importantly, higher doses of butorphanol may play a crucial role in preserving perioperative immune function and potentially improving clinical outcomes in ovarian cancer patients. Therefore, the use of butorphanol in Ovarian cancer patients needs to weigh the pros and cons. High-dose butorphanol (4.0 μg·kg⁻¹·h⁻¹) PCIA may also be an option for postoperative analgesia in patients undergoing cytoreductive surgery for ovarian cancer, although it can not Up-regulation immune function.³⁰ This study has several limitations. First, its single-center design, despite an adequate calculated sample size, limits broader applicability due to the relatively small number of ovarian cancer patients included. Future studies should adopt a multicenter design to increase the sample size for a more comprehensive clinical assessment. Second, our study assessed outcomes only up to 48 h postoperatively; a longer follow-up period may be required to fully understand subsequent outcomes and prognosis. Third, while our assessment of immune cell functional status focuses on overall immune cell quantity and function, interpreting individual lymphocyte subsets may lack strong clinical guidance; therefore, future investigation should explore additional laboratory markers for comprehensive diagnostic evaluations. In addition, this study are applicable to the studied population of ovarian cancer patients aged 18–65 and that dose adjustments may be necessary for other groups, such as the elderly or pediatric patients, which should be determined by future dedicated studies.

Conclusion

High-dose butorphanol (4.0 μg·kg⁻¹·h⁻¹) PCIA effectively relieves postoperative pain and reduces the time to early ambulation, without affecting immune indicators within 48 h postoperatively. Therefore, high-dose butorphanol (4.0 μg·kg⁻¹·h⁻¹) PCIA is recommended for postoperative analgesia in adults patients undergoing primary cytoreductive surgery for ovarian cancer.

Data Sharing Statement

The individual deidentified participant data will not be made available for public sharing. The data contain sensitive clinical information of participants. The informed consent documents signed by participants do not include provisions for public data sharing. Researchers may submit a methodologically sound proposal to the corresponding author (Wei Zhang, [email protected]) for access to the deidentified data. Data requests will be reviewed by the study’s steering committee. Approval will be granted subject to a signed data access agreement.

Funding

This work was supported by Rui Research Special Funds of Chen xiao-ping foundation for the development of science and technology of Hubei province.

Disclosure

The authors report no conflicts of interest in this work.

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Applied Materials Inc. (NASDAQ:AMAT) is one of the most profitable tech stocks to buy. On December 4, TD Cowen analyst Krish Sankar raised the firm’s price target on Applied Materials to $315 from $260 and maintained a Buy rating on the shares. The firm believes the company is currently at the intersection of two major market uptrends: one in the DRAM sector and another in leading-edge Foundry.

Earlier in its Q4 2025 earnings report, the company disclosed achieving a revenue of $28.4 billion for the full year 2025, representing a 4% increase from 2024. The Non-GAAP EPS increased by 9% year-over-year. The revenue growth was broad-based across the company’s segments. Semiconductor Systems revenue was up 4%, setting a record for both foundry systems revenue and DRAM sales outside China. Applied Global Services revenue grew 3% to a record $6.4 billion. Display revenue rose by 20%.

For FQ1 2026, Applied Materials anticipates a revenue of ~$6.85 billion and Non-GAAP EPS of ~$2.18. The company is positioned to benefit from the AI computing boom, which is driving investment in advanced semiconductors and wafer fab equipment/WFE. Applied Materials is confident in its ability to capture more than 50% of its served market in these segments, supported by deep co-innovation relationships that provide visibility into future technology nodes, sometimes extending to over 2 years.

Applied Materials Inc. (NASDAQ:AMAT) provides manufacturing equipment, services, and software to the semiconductor, display, and related industries. The company operates through three segments: Semiconductor Systems, Applied Global Services, and Display.

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Disclosure: None. This article is originally published at Insider Monkey.

Key stat: The US accounts for just 10% of TikTok users but generates 41% of the platform’s ad revenue worldwide, according to a July forecast from EMARKETER.

Beyond the chart:

• TikTok’s US audience is highly responsive to advertising. In fact, 83% of weekly US TikTok users ages 13 and older have taken action after seeing an ad on the platform, including making a purchase (43%), according to a December Edison Research report.
• Meanwhile, the platform’s US user base is expanding beyond its Gen Z core. TikTok adoption among users 45 and older has grown 1,200% between 2019 and 2025, according to an August survey from CivicScience.

Use this chart: Drop this into any deck questioning TikTok’s role in your media mix. Even with the app’s future uncertain, this chart makes the case for why the US market still commands attention and budget. Use it to defend domestic TikTok investment despite regulatory headwinds.

Related EMARKETER reports:

Methodology: Estimates are based on the analysis of various elements related to the ad spending market, including macro-level economic conditions, historical trends of the advertising market, historical trends of each medium in relation to other media, reported revenues of major ad publishers, estimates from other research firms, data from benchmark sources, consumer media consumption trends, consumer device usage trends, and EMARKETER interviews with executives at ad agencies, brands, media publishers, and other industry leaders.