Nano-Drug Delivery System Targeting the Oxidative Stress Microenvironment: A Prospective Strategy for Intervertebral Disc Degeneration Treatment

Yidian Wang; Pengfei Wen; Bin-Fei Zhang; Shouye Hu; Zhi Yang

doi:10.2147/IJN.S529127

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Review

Nano-Drug Delivery System Targeting the Oxidative Stress Microenvironment: A Prospective Strategy for Intervertebral Disc Degeneration Treatment

Authors Wang Y, Wen P, Zhang BF, Hu S, Yang Z

Received 19 March 2025

Accepted for publication 29 July 2025

Published 25 September 2025 Volume 2025:20 Pages 11717—11780

DOI https://doi.org/10.2147/IJN.S529127

Checked for plagiarism Yes

Review by Single anonymous peer review

Peer reviewer comments 2

Editor who approved publication: Professor Xing Zhang

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Yidian Wang,^* Pengfei Wen,^* Bin-Fei Zhang, Shouye Hu, Zhi Yang

Department of Joint Surgery, Honghui Hospital, Xi’an Jiaotong University, Xi’an, Shaanxi, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Zhi Yang; Yidian Wang, Department of Joint Surgery, Honghui Hospital, Xi’an Jiaotong University, Xi’an, Shaanxi, People’s Republic of China, Email [email protected]; [email protected]

Abstract: Intervertebral disc degeneration (IDD) is the main cause of low back pain (LBP), which imposes a heavy burden on individuals and society. At present, the treatment methods for IDD aim to control clinical symptoms, and it is difficult to fundamentally reverse IDD and reconstruct its mechanical function. Related studies have shown that oxidative stress is a key factor in promoting intervertebral disc (IVD) degeneration and impeding its repair, so antioxidant therapy may be effective in improving or reversing IDD progression. However, considering the non-targeted systemic cytotoxicity and limited bioavailability of antioxidant biopharmaceuticals, it is urgent to establish an efficient drug delivery system. With the development and innovation of nanomedicine and materials science, nano-drug delivery systems (NDDSs) have shown broad application prospects in various diseases. Relying on the characteristics of nanoscale materials, NDDSs can achieve good biodegradability, biocompatibility, targeted drug delivery, and controlled drug release, thereby enhancing the efficacy of antioxidant preparations. This review not only summarizes the research progress of NDDSs in the treatment of IDD, but also evaluates the advantages and challenges faced by various NDDSs. We anticipate that the perspectives articulated in this review will offer valuable insights for the precise management of IDD.

Keywords: intervertebral disc degeneration, oxidative stress, reactive oxygen species, drug delivery system, nanoparticles

Introduction

Low back pain (LBP) is considered as pain or discomfort in the area below the rib margin and above the buttock crease, and it is a common musculoskeletal disorder.¹ With the intensification of global population aging, the incidence rate of LBP is on the rise.² According to relevant studies, the global number of people suffering from LBP was 377.5 million in 1990, which increased to 577 million in 2017.^3,4 In low- and middle-income countries, the prevalence of LBP is even higher. Based on limited data, the annual prevalence of LBP in adults is 57% and 67% in Africa and Latin America respectively,^5,6 with a lifetime prevalence reaching up to 93%.^7,8 LBP is the leading cause of disability, with a 54% increase in disability caused by LBP globally between 1990 and 2015.^3,9 The LBP has also brought about a significant economic burden. According to estimates, the direct medical costs for LBP in the UK and the US are approximately £19.77 billion and $100 billion respectively, while in Japan it is 1.2 trillion yen.^4,10 Although there are many factors contributing to LBP, IDD is the main cause, accounting for approximately 40% of symptomatic LBP.^11,12

Figure 1 The main pathogenic factors of IDD.

Figure 2 Different developmental stages of IDD and their manifestations.

Figure 3 Diagnosis and current IDD treatments.

Figure 4 Redox disorders and their pathogenic mechanisms in degenerative IVD.

Figure 5 Complex signalling networks in degenerating IVD cells and their interaction with the oxidative stress microenvironment.

The intervertebral disc (IVD) is a fibrocartilaginous tissue located between the vertebrae, consisting primarily of the nucleus pulposus (NP), annulus fibrosus (AF), and cartilaginous endplate (CEP), which plays a crucial role in maintaining spinal stability, cushioning spinal pressure, and enhancing spinal mobility.¹³ In the development process of IDD, multiple risk factors are involved in the progression of IDD, including genetic susceptibility, aging, trauma, immune disorders, occupational exposure and abnormal non-physiological mechanical load^14–17 (Figure 1). However, the exact pathogenic mechanism of IDD is unknown. Currently, IDD is considered to be a degenerative process involving molecules, cells, tissues and organs mediated by IVD cells in a specific genetic context, which impairs the normal tissue structure and biomechanical properties of the IVD, ultimately destroying its ability to withstand loading.^8,18 The latest research indicates that the oxidative stress microenvironment plays a crucial role in the occurrence, development, and inhibition of regeneration in IDD.¹⁹ Oxidative stress is caused by an imbalance in intracellular and extracellular redox reactions, which induces cellular senescence, death, and imbalanced metabolism of the extracellular matrix (ECM) through various signaling cascades and intracellular signal transduction pathways, thereby participating in the progression of IDD.¹⁹ Current treatments for IDD are pharmacological and surgical to alleviate symptoms and reduce the incidence of disability, but both have the disadvantages of high complications, high costs and unknown efficacy.²⁰ These methods can only act on the terminal stage of IDD and cannot delay or reverse the progression of IDD by improving the pathological changes of IVD. In addition, some recently emerging preclinical treatment methods, such as small molecule drugs, proteins, and nucleic acids, have shown excellent therapeutic effects in improving the survival and functional impairments of IVD cells.²¹ However, the short half-life and rapid elimination of these drugs limit their treatment efficacy.¹¹ In this regard, approaches based on nano-drug delivery systems (NDDSs) may be promising for the treatment of IDD.²² Currently, NDDSs are mainly involved in the delivery of antioxidant agents through physical encapsulation (liposomes and exosomes, etc.) or chemical binding [inorganic and polymeric nanoparticles (NPs), etc.].²³ Among them, some NDDSs possess excellent antioxidant capacity by themselves (polydopamine NPs, MnO₂ NPs, fullerenes, and fullerols), and their combination with antioxidant agents can exert stronger synergistic antioxidant effects.²⁴ Secondly, NDDSs also have the ability to control release, which mainly depends on physical conformation changes or chemical bond breaks of nanocarriers.²⁵ In particular, some carriers can respond to internal (ROS, pH, enzymes, etc.) or external stimuli (temperature, light, magnetic field, etc.) to achieve precise controlled release of drugs in time and space.^25,26 In addition, NDDSs can increase drug accumulation at the lesion through passive and active targeting capabilities, thereby improving efficacy and reducing side effects.²⁷ These properties make NDDSs an effective method for improving the oxidative stress microenvironment within degenerating IVDs.

The aim of this review is to briefly review the changing pathophysiology of IDD and the limitations of current treatments. In addition, we also emphasized the oxidative stress microenvironment in degenerative IVDs and its role in IDD. Importantly, we demonstrated that biomedical engineering approaches can be used to ameliorate the oxidative stress microenvironment of IDD, thereby promoting regeneration and recovery of IDD, which may help advance the development of NDDSs in the treatment of IDD.

The Pathophysiology of IDD

Normal Structure and Function of IVD

The IVD is an integral part of the spinal tissue and biomechanical composition. The normal spine comprises a minimum of 23 highly hydrated IVDs that connect adjacent vertebrae, playing a crucial role in enhancing spinal mobility and optimizing load distribution.²⁸ The complete IVD comprises NP, AF, and CEP on both sides.

NP is located between the center and posterior of IVD, serving as the core of IVD.²⁹ In general, NP is a soft and elastic gel-like substance. The healthy IVD typically exhibits a relatively high-water content, ranging from approximately 80% to 90%, whereas degenerated IVDs demonstrate a reduction in water content to around 70–75%, or even lower.^30,31 In terms of cellular composition, NP cells are a group of mixed cells at different stages of maturation, used to maintain the metabolic balance of ECM.⁴ Before the age of 10, the mixed cell population consisted of larger vacuolated notochord cells (NC) (mainly expressing CK-8, LGALS3, STMN2, RTN1, PRPH, CXCL12, IGF1, MAP1B, ISL1, CLDN1 and THBS2) and smaller chondrocyte-like cells (mainly expressing HIF1α, GLUT1, proteoglycans, type II collagen, KRT18/19, CA12 and CD24).^31–34 In adulthood, these cells all differentiate into chondrocyte-like cells with reduced metabolic activity.³⁵ Research has shown that NC contributes to the regulation of proteoglycan production and proliferation activity in IVD chondrocytes, suggesting a potential association between NC degradation and IDD.³⁶ In terms of the organization composition of NP, 35–65% is composed of proteoglycans, 5–20% is composed of collagen II (Col II) and elastin protein.³⁷ The high specific gravity and negative charge properties of proteoglycans contribute to the highly hydrated characteristics of NP tissue, while also facilitating the dispersion of loads.^11,38 The mesh composed of Col II and elastic protein fibers is embedded in the gel formed by proteoglycans to maintain the elasticity of ECM.³⁹

The AF is located on the periphery of NP, consisting of well-arranged concentric rings or lamellae composed of collagen fibers and proteoglycans.⁴ The lamellae are inclined approximately 30° from one vertebra to another and cross diagonally at an angle of >60°between adjacent lamellae, which helps to limit rotation and bending between adjacent vertebrae while providing sufficient resistance to axial loads.^37,40 The AF can be further divided into an inner fibrocartilage region and an outer fibrous region, which have different cellular and tissue compositions.²¹ The inner region mainly consists of circular chondrocyte-like cells that produce proteoglycans and Col II, while the outer region contains elongated fibroblast-like cells that primarily produce Col I.^21,41 Correspondingly, the organizational structure of both the internal and external regions has also undergone excessive changes. AF tends to gradually lose proteoglycans, Col II, and water content from the inside out, resulting in a large amount of Col deposition, which effectively restricts NP during axial compression, extension, and bending.^42–44

The endplate is the boundary between the IVD and the adjacent vertebral bodies, including the bony endplate and CEP. The main cell type within the CEP is the chondrocyte, which primarily secretes Col II and proteoglycans.⁴⁵ Compared to NP, the proportion of proteoglycans and Col II in CEP is relatively low, which can provide a solid mechanical barrier between IVD and vertebral body, thereby preventing the protrusion of NP tissue into the trabecular bone of the vertebral body.^46,47 In addition, CEP is also the main channel for IVD to exchange substances with the external environment.⁴⁸

The IVD is the largest avascular tissue in the body.⁴⁹ Compared to the lifelong avascularity in NP, CEP and AF have abundant blood vessels during fetal and infant stages.⁵⁰ However, with age, the blood vessels in CEP will completely regress, while only the outer layer of AF maintains vascularization.⁵¹ The limited distribution of blood vessels in the IVD primarily facilitates substance exchange through diffusion (for small molecular solutes such as glucose, oxygen, and lactate) or convection (for large molecular solutes such as growth factors, hormones, and proteoglycans).^52,53 Therefore, IVD is in a hypoxic microenvironment. According to the study of canine IVD samples, the oxygen tension in the IVD (0.53–1.06 kPa) is lower compared to that in muscle (3.8±0.8 kPa) and brain (3.4±0.2 kPa), and it decreases as it gets closer to the center of the IVD.^54,55 The hypoxic microenvironment makes NP cells partially dependent on anaerobic glycolysis to produce ATP, which contributes to the formation of an acidic microenvironment.^56,57 Regarding the innervation within the IVD, the NP has no innervation, whereas the outer layer of the AF is innervated by branches of the sinus vertebral nerve.⁵⁸

Degenerative IVD: Molecular, Tissue and Mechanical Functions

IDD is an age-related degenerative process that is influenced and accelerated by other unfavourable factors, such as genetic and environmental factors.⁵⁹ The process of degeneration can be divided into three closely connected stages (Figure 2). Firstly, under specific genetic backgrounds, abnormal mechanical load induces functional impairment of IVD cells, leading to an imbalance in ECM metabolism. Subsequently, the microenvironment of IVD continued to deteriorate, leading to further decrease in the number and activity of IVD cells, resulting in a cascade reaction of damage. In the later stage, IVD loses its mechanical function and is accompanied by infiltration of nerves and blood vessels, eventually leading to IVD-related LBP.

Although the traditional view holds that IDD is an age-related disease, it cannot be denied that genetic factors may play a significant role in determining this condition.⁶⁰ The existence of genetic susceptibility to IDD is supported by preliminary evidence from twins and family studies. In 1999, Sambrook et al evaluated the MRI characteristics of cervical and lumbar IVD in 172 identical twins and 154 dizygotic twins.⁶¹ The results indicated a heritability rate of 74% for lumbar IVD degeneration and 73% for cervical IVD degeneration. Another prospective study involving 116 identical twins found that the estimated heritability of IDD within 5 years was between 47–66%.⁶² In addition, in a 10-year longitudinal study involving 234 pairs of twins, Williams et al found that disc herniation has a genetic component across all age groups.⁶³ Recently, genome-wide association studies (GWAS) have made significant contributions to the etiology of complex diseases and have indicated that genetic susceptibility to IDD may be widespread.^64–66 In 2022, Bjornsdottir et al conducted a (58,854 cases, 922,958 controls) GWAS study on IDD and identified 41 genetic variants in 33 loci that were associated with cartilage, bone biology, and inflammatory processes.⁶⁷

In fact, IVD had already undergone degradation in the first decade of life.⁶⁸ The reduction of vacuolar NCs in the NP is considered to be the initiation process of IVD degeneration, because NCs can increase the anabolic and proliferative activity of NP cells.^36,69,70 In all cases, early IDD is mainly characterized by an imbalance between anabolic and catabolic processes in the ECM, which is mainly caused by a phenotypic transition of IVD cells.^71,72 In terms of anabolism, there was a significant reduction in the production of proteoglycans, along with a shift in the composition of their side chains from chondroitin sulphate to keratan sulphate, which led to diminished hydration of NP tissues.^73,74 The balance of collagen production shifts from Col II to Col I, accelerating the process of IVD fibrosis.⁷⁴ In terms of catabolism, the expression of matrix metalloproteinase (MMP)-1, MMP-2, MMP-3, MMP-13, MMP-14, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 significantly increased.^74–76 In contrast, the levels of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 were decreased.⁷⁶ These factors have led to a further decline in the quality of ECM.

Early IVD degeneration lasts for a considerable period of time (10 or 20 years or more), which may be accelerated by other unfavorable factors (genetics, trauma and abnormal loads) and enters the radical phase. During the radical phase, the microenvironment within the IVD is severely deteriorated, accompanied by inflammation and oxidative stress storms.⁷⁷ NP cells secrete various cytokines, including tumor necrosis factor α (TNF-α), interleukin-1 β (IL-1β), IL-6, IL-8, IL-17 and prostaglandin E2 (PGE2).^47,78 Simultaneously, NP cells stimulate the synthesis of chemokines, specifically CCL2, 3, 4, 5, 7, 10 and 13, which activate and recruit immune cells to infiltrate the IVD.^22,32 This further exacerbates the cellular inflammatory response. In addition, degenerating IVDs undergo intense local oxidative stress, with large amounts of reactive oxygen species (ROS) being generated.¹⁹ Excessive ROS can attack the mitochondrial respiratory chain, endoplasmic reticulum, and inhibit the production of reductases, further promoting the accumulation of ROS.^19,79,80 Inflammatory response and oxidative stress promote each other, thus forming a positive feedback loop and acting directly or indirectly on a dynamic signaling network with complex feedback loops composed of different signaling pathways.^72,81,82 Aberrant activation or inhibition of these signaling networks may exacerbate NP dysfunction, including apoptosis, senescence, hypertrophy and calcification, which in turn exacerbate the imbalance of ECM synthesis and degradation.^18,83 In addition, hypertrophy and calcification of CEP cells and sclerosis and occlusion of the bone marrow lumen will lead to loss of CEP permeability, which will result in impaired energy supply and accumulation of metabolic wastes within the IVD, ultimately accelerating IDD.^84–86

When entering the late stage of IDD, IVD will gradually lose its mechanical function and result in IVD-related LBP. At this time, the normally active NP cells are almost completely damaged, and proteoglycan and Col II are replaced by Col I.⁸⁷ As a result, the highly hydrated and elastic jelly-like NP is replaced by stiff fibrous tissue, which results in an inability to effectively distribute the load.⁸⁷ Meanwhile, the imbalance in ECM metabolism leads to structural disorder in AF, resulting in a significant reduction in its ability to resist loads and the occurrence of cracks.⁸⁷ Once the integrity of AF is compromised, internal NP tissue is exposed to the circulatory system.⁸⁸ The immune system will recognize these “foreign” tissues and generate an autoimmune response.⁸⁹ IgG antibodies and antigen-antibody complexes against proteoglycans and collagen were found in human degenerative IVD samples, providing direct evidence for autoimmune reactions.^90,91 In addition, NP cells and immune cells secrete a large amount of IL-1, TNF-α, IL-8, and PGE2, which can stimulate the production of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and vascular endothelial growth factor (VEGF).^92–94 These growth factors induce infiltration of blood vessels and nerves, leading to IVD-induced LBP.^94,95 Moreover, the herniated NP can exert direct compression on the adjacent nerve roots, resulting in significant low back and leg pain.⁹⁶ Meanwhile, the dysfunctional IVD causes spinal instability, leading to degeneration of surrounding small joints, formation of bone spurs, and hypertrophy of surrounding ligaments.^97,98 This in turn results in spondylolisthesis, spinal stenosis, and even degenerative scoliosis.⁹⁸

Diagnosis and Treatment of IDD

The initial diagnosis of IDD is mainly based on the patient’s symptoms and physical signs. Patients with IDD typically present with LBP. This type of pain is often characterized by an unclear location, primarily concentrated in the central region of the lower back or adjacent to the IVD, and worsens after prolonged standing or sitting. When the protruding NP directly compresses or the inflammatory response irritates the nerve root, LBP may worsen and radiate to one or both limbs. In addition to clinical symptoms and signs, the diagnosis of IDD is mainly based on X-ray, CT scan or MRI, and its severity is further assessed by IVD hydration status (Pfirrmann) or CEP Modic changes.^99,100

Currently, the treatment of IDD includes conservative treatment and surgical treatment according to the patient’s symptoms (Figure 3). Conservative treatment includes drug therapy and non-drug therapy, which are mainly suitable for early-stage IDD patients and can alleviate symptoms and improve quality of life. When the degeneration is mild, bed rest, appropriate and regular exercise, weight loss, and physical therapy (such as cold compresses, electrical stimulation, ultrasound therapy, and traction) are simple and effective measures to improve symptoms.^101–103 These methods are designed to reduce stress on the IVD, strengthen the low back muscles and improve spinal stability. When patients experience significant pain in the lower back, medication can be used for treatment, including nonsteroidal anti-inflammatory drugs (NSAIDs), opioid analgesics, muscle relaxants, benzodiazepines, antidepressants, corticosteroids and antiepileptic drugs.¹⁰² For some advanced patients, some invasive methods can be used, such as chemical nucleolysis, radiofrequency ablation, nucleotomy and nerve root blockade.²¹ Once conservative treatment fails, symptoms of nerve root compression are severe, cauda equina syndrome and severe spinal deformity or instability occur, surgical treatment is required. Surgical treatment mainly consists of interbody fusion, partial or total IVD removal and total disc replacement (TDA).¹⁰³ Among them, interbody fusion has always been regarded as the surgical gold standard for symptomatic IVD protrusion and is suitable for the majority of patients with disc herniation.^103,104 TDA has similar clinical results to interbody fusion, but is usually reserved for single-level situations without facet joint disease.^104–106

Oxidative Stress and IDD

Oxidative Stress in IDD

ROS are a class of unstable and highly reactive molecules, including superoxide anion (O₂⁻), hydroxyl radical (OH-), hydrogen peroxide (H₂O₂), and hypochlorite ion (OC1⁻).¹⁰⁷ In addition, some nitrogen-containing compounds (nitric oxide and nitrogen dioxide) are also considered as members of the ROS superfamily due to their similar effects.¹⁰⁸ As a byproduct of aerobic metabolism, the generation of ROS in cells is inevitable Although IVD cells are in a hypoxic environment due to lack of direct blood supply, aerobic metabolism is still an important way for IVD cells to obtain energy and thus generate ROS.⁸⁰ In order to maintain redox balance, the human body has developed a set of ROS scavenging systems, which remove excess ROS to keep them at a level that is harmless to the organism.¹⁰⁹ In normal levels, ROS is an important intracellular signaling molecule that participates in various physiological processes through intracellular signal transduction.¹¹⁰ As IVD degeneration progresses, aging, trauma, abnormal mechanical load, and smoking cause an imbalance between oxidants and antioxidants, resulting in oxidative stress, which in turn leads to damage to molecules, cells, and tissues¹¹¹ (Figure 4). At the molecular level, biomacromolecules such as proteins, nucleic acids, lipids, and carbohydrates in cells are inevitably exposed to ROS and suffer from oxidative damage.¹¹¹ Currently, there are reports indicating a significant elevation of advanced oxidative protein products (AOPPs), 8-oxoguanine (an oxidative product derived from guanine), malondialdehyde (MDA), and advanced glycation end products (AGEs) in degenerative human IVD tissue.^112–115 At the cellular level, large amounts of ROS lead to restricted survival and altered metabolic phenotype of IVD cells by damaging cellular macromolecules, which in turn accelerates IDD progression.⁷⁹ At the tissue level, oxidative stress causes damage to normal IVD tissue structure and biomechanical dysfunction, which in turn leads to corresponding clinical symptoms.¹¹⁶

Redox Imbalance Within the IVD

Increased ROS Production in Degenerative IVD

Mitochondria are the center of oxidative metabolism and the main site of ROS production, because 1–2% O₂ is converted into ROS during oxidative phosphorylation, which depends on the mitochondrial electron transport chain (ETC).^117,118 Indeed, under physiological conditions, 0.2–2% of the electrons do not follow the normal ETC transfer sequence (complex I/III/IV and complex II/III/IV with succinate as substrate), but rather leak directly from the ETC and interact with O₂ to produce O₂ -.¹¹⁹ The complexes I and III are the main sites where electrons transfer to O₂ to generate O₂-.¹²⁰ For complex I, ROS are thought to originate from reduced flavin mononucleotide or N-1a and N-1b iron-sulfur clusters, whereas ROS for complex III are thought to originate from ubiquinone oxidation sites.^121,122 When the activities of complex I and complex III are inhibited or the concentration of succinate is low, complex II becomes the main source of ROS.⁴² Interestingly, under hypoxic conditions, complex II switches its catalytic activity from succinate dehydrogenase to fumarate reductase, and this change is associated with increased ROS generation.^123,124 Therefore, it is possible that complex II has a unique association with increased ROS production in degenerative IVD. In degenerative IVD, the adverse microenvironment can lead to abnormal opening of mitochondrial permeability transition pores (mPTP) in IVD cells, decreased membrane potential, and damage to the respiratory chain, ultimately leading to mitochondrial dysfunction.^125,126 Mitochondrial dysfunction leads to massive electron leakage, which increases ROS production.¹²⁷ A large amount of ROS attacks mitochondrial DNA, proteins, and membrane lipids, which further exacerbates mitochondrial damage, thus forming a vicious cycle.^127,128

The NADPH oxidase (Nox) family comprises seven isoforms (Nox1, Nox2, Nox3, Nox4, Nox5, Duox1, and Duox2), which exhibit distinct expression patterns across various cell types and subcellular compartments.¹²⁹ Nox generates O₂⁻ or H₂O₂ by catalyzing NADPH electron transfer.^130,131 The significant increase in the NADPH/NADP+ ratio in degenerated IVD cells suggests that NOX is involved in the formation of the oxidative microenvironment in IDD.^132,133 In IDD, the most extensively studied are Nox 2 and Nox 4.^134–136 Chang et al found that 15% high cyclic stretch upregulated Nox 2 expression in AF cells, which led to a significant increase in ROS levels.¹³⁷ In addition, 20% O₂ increases the production of ROS by upregulating Nox4 in NP cells, thereby activating the p53-p21-Rb and p16-Rb pathways to induce cellular senescence.¹³⁸ The endoplasmic reticulum (ER) is also an important source of ROS.^139,140 Under the influence of nutritional deficiency, oxidative stress, and genetic mutations, the accumulation of unfolded/misfolded proteins leads to ER stress, which in turn activates the unfolded protein response (UPR).^141,142 This phenomenon is widely observed in IDD.^142,143 During the UPR process, the involvement of protein disulfide isomerase, endoplasmic reticulum oxidoreductin-1 and Nox complex can trigger ROS cascade reactions.⁴² In addition, ER stress leads to Ca²⁺ overload and increases intra-mitochondrial Ca²⁺ levels through the mitochondria-associated membrane (MAM), which in turn leads to mitochondrial dysfunction and increased ROS generation.^139,144 In addition to the above pathways, xanthine oxidase (XO), uncoupled endothelial nitric oxide synthase, cytochrome P-450 monooxygenases, and cyclooxygenases are also sites of ROS generation,^42,120 collectively contributing to endogenous ROS accumulation. In addition to endogenous pathways, external stimuli such as smoking, UV radiation, ionizing radiation, air pollution and drugs can also lead to the accumulation of ROS.^145–147

Reduced Antioxidant Defense in Degenerative IVD

The complete intracellular antioxidant defence system mainly consists of antioxidant enzymes, non-enzymatic small molecules and redox signal transduction.¹¹⁶ Antioxidant enzymes are the first line of defense system, mainly including superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT), and methionine sulfoxide reductase (Msr).^148,149 SOD is an important antioxidant enzyme, which can be classified into Cu/Zn-SOD based on the types of metal ions it binds to.¹⁵⁰ These two enzymes are prevalent in the mitochondria of NP cells and alleviate oxidative stress by efficiently catalysing the conversion of O₂⁻ to H₂O₂ and O2.^151,152 Similarly, GPX and CAT are members of the antioxidant defence within the IVD, both of which can catalyze the decomposition of H₂O₂ into H₂O.¹⁵¹ However, multiple studies have indicated a significant decrease in the expression of SOD, GSH, and CAT in degenerated IVD tissues and cells.^153–155 In addition, Msr is an repair enzyme that can remove ROS by reducing the methionine residues in oxidized proteins.¹⁵⁶ The expression of Msr is significantly reduced in aging AF cells, which makes AF cells vulnerable to oxidative stress damage.¹⁵⁷ Following antioxidant enzymes, non-enzymatic small molecules are a class of compounds that do not rely on the enzyme system in the organism to scavenge free radicals and combat oxidative stress, and mainly include vitamins C and E, β-carotene, lipoic acid, ubiquinone, melatonin (MT), carotenoids, ascorbic acid and uric acid.^97,158 They are widely present in the cytoplasm of IVD cells, and the reduction of their expression levels can also lead to oxidative stress in IVD. In addition to the classical antioxidant system, intracellular redox signaling is also an important way to maintain intracellular redox homeostasis, which mainly includes the Kelch-like ECH-associated protein 1 (Keap1) - Nuclear factor E2- related factor 2 (Nrf2) - Antioxidant response element (ARE) signaling pathway.¹⁵⁹ When the intracellular ROS levels increase, Keap1 dissociates from CUL-E3 ligase, causing a conformational change in Keap1 and releasing Nrf2, which leads to the accumulation of Nrf2 in the cytoplasm and subsequent nuclear translocation.¹⁶⁰ High levels of Nrf2 bind to the ARE, which activates the transcription of antioxidant genes, including heme oxygenase-1 (HO-1), GSH, SOD, and CAT, and thus exerts antioxidant effects.^116,160 I In human degenerated IVD and aged NP cells, Nrf2 expression levels were significantly decreased and correlated with the severity of IDD.^161,162 In addition, high levels of Nrf2 promote mitochondrial biosynthesis by up-regulating the expression of nuclear respiratory factor 1/2 (NRF-1/2), PGC-1α, and mitochondrial transcription factor A, as well as mitochondrial fusion by degrading the mitochondrial fission protein dynamin-related protein 1 (Drp1), which contribute to protection against oxidative stress.^163,164

Role of Oxidative Stress in IDD

ROS is an important signaling molecule within cells, which means it can directly or indirectly interact with dynamic signal networks composed of different signaling pathways and complex feedback loops, thereby regulating the physiological and pathological processes of IDD¹⁵¹ (Figure 5). During this process, there are various phenotypic changes in IVD cells, which accelerate the progression of IDD.

Oxidative Stress and Cell Death

The balance of anabolic and catabolic metabolism within the IVD is dependent on the metabolic capacity conferred by a sufficient number of cells and normal cellular function. When excessive cell death occurs within the IVD, this balance is disrupted and promotes the progression of IDD. ROS are potent inducers of cell death, and here we focus on three cell death types, including apoptosis, pyroptosis, and ferroptosis.^165,166

Apoptosis is a controlled cell death that includes two major pathways: intrinsic and extrinsic,¹⁶⁷ the former of which is often associated with mitochondrial dysfunction. Intrinsic apoptosis depends on the abnormal activation of BCL-2 family proteins on the mitochondrial membrane,¹⁶⁸ which leads to increased mitochondrial outer membrane permeability and leakage of cyt-c, ultimately inducing cell apoptosis by activating a series of caspases.¹⁶⁹ ROS is involved in IVD cell apoptosis through the regulation of multiple signaling pathways, including phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/Akt), NF-κB, mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), Sirtuin (SIRT) protein family, Keap1/Nrf2, and AMP-activated protein kinase (AMPK).^170–173 The activation or inhibition of these signaling pathways leads to the differential expression of pro-apoptotic proteins (such as Bak, Bax, and Bok) and anti-apoptotic proteins (such as Bcl-XL, Bcl-2, Bcl-w, and Mcl-1), thereby inducing cell apoptosis.¹⁶⁸ For example, when IVD cells were co-cultured with oxidants (H₂O₂ and TBHP) in vitro, a decrease in the expression of PI3K/Akt,¹⁷⁴ AMPK,¹⁷⁵ SIRT,¹¹⁴ and Nrf2¹⁶² and an increase in the expression of MAPK/ ERK¹⁷⁶ and NF-κB¹⁷⁷ were observed. This resulted in up-regulation of BAX, Caspase-9 and Caspase-3 expression and down-regulation of Bcl-2 expression, which in turn induced apoptosis. In fact, these signaling pathways are not isolated but intertwined with each other to form positive or negative feedback loops. For example, when the PI3K/Akt signaling pathway is activated, Akt can phosphorylate and inhibit the degradation of IκBα (a member of the IκB protein family and a specific inhibitor of NF-κB), thereby suppressing the activation of NF-κB.¹⁷⁸ In specific circumstances, NF-κB can in turn promote the activation of Akt, forming a positive feedback loop that enhances cell survival capability.¹⁷⁹ In addition, the roles of these signaling pathways in cell apoptosis are diverse and may depend on the cell type, stimulus type, and intracellular environment.¹⁸⁰

Pyroptosis is a type of programmed cell death between apoptosis and necrosis that depends on the activation of inflammasomes.¹⁸¹ Inflammasomes are a class of protein complexes, the most widely studied of which is the NLRP3 inflammasome,¹⁸² which can sense pathogen-associated molecular patterns or damage-associated molecular patterns and then recruit and activate Caspase-1.¹⁸² The activated Caspase-1 can cleave and activate other proteins, such as Gasdermin D (GSDMD), IL-1β, and IL-18.¹⁸³ After being cleaved, the N-terminal fragment of GSDMD forms pores on the cell membrane, leading to leakage of cellular contents and triggering a strong inflammatory response.¹⁸⁴ Compared with non-degenerated cells, high levels of ROS and caspase-1 were observed in degenerated human IVD cells.¹⁸⁵ In vitro, after NP cells were co-cultured with H₂O₂, the expression of ROS, NLRP3 inflammasome and caspase-1 in the cells was significantly upregulated, indicating that oxidative stress can effectively induce cell pyroptosis.¹⁸⁶ In fact, oxidative stress is involved in the induction of cell death through various mechanisms, including activation of inflammasomes, mitochondrial dysfunction, enhanced inflammatory response, and instability of cell membrane.¹⁸⁷ ROS can directly promote the activation of NLRP3 inflammasome, with mitochondrial ROS (mtROS) being the most important signal for NLRP3 inflammasome activation,¹⁸⁸ which has also been demonstrated in cardiovascular disease.¹⁸⁹ In addition, ROS can damage IVD cell mitochondria, leading to abnormal opening of mPTP and leakage of mitochondrial DNA (mtDNA).¹⁹⁰ Released mtDNA promotes cell pyroptosis by activating the Toll-like receptor 9 (TLR9)-NF-κB-NLRP3 axis.¹⁹¹ Recent studies have also shown that thioredoxin-interacting protein (TXNIP) is a crucial bridging molecule for the activation of NLRP3 inflammasome.¹⁹² When ROS is overloaded in IVD cells, TXNIP dissociates from Thioredoxin (Trx) and binds to NLRP3, thereby initiating cell pyroptosis, but this process is inhibited by Nrf2 signaling.^186,193 A possible explanation is that Nrf2 inhibits TXNIP function by up-regulating antioxidant proteins and regulating the balance of Trx/TXNIP complexes.^194–196 In addition, the SIRT protein family is involved in the regulation of cellular pyroptosis.¹⁹⁷ Overexpression of SIRT1 can improve IL-1β-induced mitochondrial dysfunction and NLRP3 inflammasome activation in NP cells by promoting PINK1/Parkin-mediated mitophagy, thereby alleviating NP cell pyroptosis.¹⁸⁸

Ferroptosis is a form of iron-driven cell death characterized by intracellular iron accumulation and oxidative stress, resulting in lipid peroxidation and membrane damage.¹⁹⁸ In fact, there is a vicious cycle of mutual promotion between oxidative stress and iron overload.¹⁹⁹ Iron is a catalyst for many oxidation reactions, especially the Fenton Reaction, in which Fe²⁺ can combine with H₂O₂ to form OH-, accelerating the accumulation of intracellular ROS.²⁰⁰ Furthermore, iron overload can induce the depletion of antioxidant defense systems, rendering cells more vulnerable to oxidative stress-induced damage.^201,202 On the other hand, oxidative stress can exacerbate iron overload. Oxidative stress can lead to the oxidation modification of ferritin, weakening its normal iron storage capacity and exacerbating the accumulation of iron within cells.²⁰³ Compared with the normal control group, the expression of GPX4 and ferritin in degenerated IVD tissues was significantly decreased, accompanied by increased oxidative stress and ferroptosis.²⁰⁴ In vitro, co-culture of oxidants with IVD cells also leads to oxidative stress, lipid peroxidation and iron overload in a dose-dependent manner.^204–206 Multiple molecules and signaling pathways are involved in regulating iron death within the IVD. Ferroportin (FPN) is an iron efflux protein responsible for the translocation of intracellular iron to the extracellular space.²⁰⁷ In TBHP-induced human NP cells, FPN is severely dysfunctional, which leads to intercellular iron overload and ferroptosis.²⁰⁸ Nrf2 is also an important signaling molecule that regulates ferroptosis in IVD cells.¹⁹⁴ In vitro, Nrf2 activation can improve Parkin-mediated mitophagy, which helps to alleviate mitochondrial dysfunction and oxidative stress, thereby inhibiting ferroptosis.²⁰⁹ Similarly, Nrf2 can also upregulate the expression of HO-1 and inhibit the nuclear translocation of NF-κB, thereby alleviating oxidative stress, inflammatory response, and ferroptosis in IVD cells.^84,210,211 In addition, SIRT3 can also alleviate oxidative stress-induced ferroptosis in IVD cells by upregulating the expression of antioxidant genes HO-1, NQO1, SOD2, and GPX4.²¹²

Oxidative Stress and Cellular Senescence

Cellular senescence is the process by which cells gradually lose their function and proliferative capacity during their life cycle, including replicative senescence and stress-induced senescence.²¹³ The replicative senescence is caused by the shortening of telomeres and exacerbated by factors that promote the accumulation or inhibit the breakdown of cellular aging, with ROS being the most important factor inducing cell senescence.¹¹³ In both human and rat degenerated IVD tissues, the proportion of senescence-associated β-galactosidase (SA-β-gal) positive cells significantly increases, which is positively correlated with oxidative stress levels within the IVD.²¹⁴ Cellular senescence not only leads to a decrease in the number of functionally normal IVD cells, but also deteriorates the IVD internal microenvironment through the paracrine effect of senescence-associated secretory phenotype (SASP).¹⁶ These SASP-associated proteins include ECM-degrading enzymes (MMPs and ADAMTS), pro-inflammatory cytokine factors (IL-1β, TNF-α, IL-7, IL-8), chemokines, and other biologically active substances, which can further exacerbate the mitochondrial dysfunction and the production of ROS, thus generating a vicious circle.^39,215 More importantly, high levels of SASP-associated MMPs have been shown to induce autocrine ligand shedding, which renders senescent cells resistant to immune surveillance and clearance.²¹⁶ Multiple signaling molecules are involved in oxidative stress-induced senescence of IVD cells. For example, ROS can promote the ubiquitin-dependent degradation of IκB through ROS-Hsp27-IκB kinase (IKK), leading to increased nuclear translocation of NF-κB (such as p65) and subsequent activation of downstream target genes to regulate cellular functions.^19,217 Compared with normal NP cells, the p65 level in the nucleus of oxidant-induced degenerative NP cells increased, and induced cell senescence by regulating p53-p21-Rb and p16-Rb.^218,219 The PI3K/Akt signaling pathway is an important intracellular signaling pathway that participates in regulating cell apoptosis, aging, growth, and metabolism.²²⁰ When the PI3K/Akt signaling pathway is activated, it activates downstream signaling molecules to alleviate mitochondrial dysfunction and intracellular ROS levels, while downregulating the p53-p21-Rb signaling axis to mitigate cellular senescence in IVD cells.^174,220 Recently, Zhang et al found that high concentrations of lactic acid can induce senescence and oxidative stress in NP cells.²²¹ Mechanistically, lactate can bind to lysine 39 and leucine 52 residues in the Akt PH domain to inhibit Akt kinase activity, thereby inducing NP cell senescence and oxidative stress by regulating p21/p27/cyclinD1 and Nrf2/HO-1 signaling. In addition, the SIRT protein family is also involved in regulating cellular aging in IVD. Activation of SIRT1 has been reported to ameliorate oxidative stress-induced cellular senescence and ECM degradation.^222,223 Similarly, several studies have found that SIRT3 activation can improve mitochondrial function and SOD activity, and alleviate IVD cell senescence by inhibiting p16-Rb signaling.^224,225 In addition, SIRT2 overexpression can also inhibit oxidative stress by upregulating the expression of SOD1/2 and inhibit NP cell senescence by downregulating the level of p53-p21-Rb.²²⁶

Oxidative Stress and Cellular Autophagy

Autophagy is an evolutionarily conserved self-degradation system that degrades and recycles intracellular proteins and damaged organelles to maintain cell homeostasis and function.²²⁷ Compared with healthy IVD tissues, the expression levels of autophagy-related genes (Atg) in degenerated IVD tissues were significantly changed, indicating that autophagy plays an important role in IDD.^228,229 As a stress response system, almost all stress factors that affect cellular homeostasis can induce autophagy. In recent years, an increasing amount of evidence has shown that ROS is a crucial intracellular signaling molecule that affects autophagy.^230,231 However, the relationship between autophagy and oxidative stress is not a simple upstream-downstream signaling regulation relationship, but rather a complex network composed of numerous signaling pathways and crossroads. Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase that responds to nutrient levels and growth signals.⁸² It is a negative regulator of autophagy initiation and its activity is regulated by PI3K-Akt, AMPK and MAPK.^232–234 Excessive ROS down-regulated the ratios of p-PI3K/PI3K, p-Akt/Akt and p-mTOR/mTOR, resulting in the inhibition of the PI3K/Akt/mTOR signaling pathway, which in turn induced autophagy in NP cells.²³⁵ In addition, ROS can also downregulate the level of p-AMPK, leading to the downregulation of ULK1 phosphorylation and upregulation of mTOR phosphorylation, thereby inhibiting NP cell autophagy activation and autophagic flux.²³⁵ Interestingly, under oxidative stress conditions, the upregulation of p38MAPK phosphorylation promotes mTOR phosphorylation, thereby inhibiting autophagy in NP cells.²³⁶ In addition to being regulated by upstream signaling molecules, mTOR can also regulate autophagy through its target genes. Downregulation of mTOR signaling can lead to increased nuclear translocation of EB transcription factor (TFEB) and downregulation of p70S6K levels, thereby enhancing cellular autophagy to combat oxidative stress.^237–239 Mitophagy is a type of cellular autophagy that plays a key role in maintaining mitochondrial quality control.²⁴⁰ PINK1/Parkin signaling pathway is an important signaling axis regulating mitophagy.¹¹⁶ In NP cells, upregulation of the PINK1/Parkin signaling pathway induces mitophagy, thereby delaying the progression of IDD by eliminating damaged mitochondria.^162,188 In addition, the SIRT protein family and Nrf2 can also regulate mitochondrial autophagy in IVD cells.^241,242

Oxidative Stress and Inflammation

The inflammatory response is a defence mechanism against pathogens, damaged cells or specific stimuli, which is induced by a stressor and leads to the expression of inflammatory cytokines through a series of intracellular signal transduction.²⁴³ As a complex immune response, inflammatory response can maintain tissue homeostasis by eliminating pathogens and repairing damaged tissues. However, excessive activation and persistence of inflammatory responses may lead to tissue damage and a variety of diseases. ROS can act as signaling molecules to activate the expression of inflammation-related genes, thereby initiating or exacerbating inflammatory responses.²⁴⁴ In vitro, co-culture of rat NP cells with H2O2 can lead to the expression of pro-inflammatory cytokines, such as IL-1β, IL-6, TNF-α, and nitric oxide synthase (iNOS).²⁴⁵ In addition, ROS can cause damage to intracellular biomacromolecules and organelles (such as mitochondria), which will trigger the activation and assembly of NLRP3, further aggravating the inflammatory response.^246,247 ROS regulates inflammatory responses through a variety of signaling molecules, including PI3K/Akt, MAPK, Nrf2, and NF-κB, among which MAPK and NF-κB pathways play the most important roles.^248,249 ROS can activate MAPK kinase, which in turn phosphorylates and activates downstream MAPK, leading to the expression of pro-inflammatory cytokines in IVD cells.^249,250 In addition, oxidative stress can directly or indirectly activate NF-κB, promoting the expression of inflammatory genes such as IL-1, IL-6, TNF-α in IVD cells and exacerbating the inflammatory response.^251,252 Cell damage caused by oxidative stress can trigger an inflammatory response, while inflammation can also exacerbate oxidative stress, forming a vicious cycle.¹⁹ For example, TNF-α, IL-1β and lipopolysaccharide (LPS) co-cultured with NP cells can lead to oxidative stress by promoting intracellular ROS production and inhibiting the expression of SOD1, SOD2, CAT and GPX3.^248,253,254

Oxidative Stress and ECM Degradation

The complete biomechanical function of IVD depends on the integrity of its tissue structure. With the progression of IVD degeneration, there have been significant changes in the quality and composition of ECM. Due to the death and aging of a large number of IVD cells, the synthesis of ECM is significantly reduced.²⁵⁵ At the same time, the catabolic phenotype of IVD cells is enhanced, which further exacerbates the metabolic imbalance of ECM.¹³⁹ As an important stressor, ROS can regulate cellular behavior through multiple signaling pathways, thereby influencing the expression and secretion of ECM proteins.⁷⁷ According to the current study, excessive ROS can downregulate PI3K/Akt, SIRT, AMPK and Nrf2 and upregulate the expression of NF-κB and MAPK, which in turn promotes the degradation of ECM.^{18,151,225,256} In addition, ROS can disrupt the components of ECM through oxidation, such as causing cross-linking and degradation of collagen protein, affecting its structural stability and functional integrity.²⁵⁷ In conclusion, oxidative stress can lead to abnormal ECM metabolism and oxidative damage of related molecules, promoting the progression of IDD.

Oxidative Stress and Epigenetics

Epigenetics refers to the mechanisms that can influence gene expression without altering the DNA sequence, and these changes can be passed on to future generations.²⁵⁸ Common epigenetic regulations include DNA methylation, histone modification, and non-coding RNA (ncRNA) regulation.²⁵⁹ These changes not only play an important role in the normal physiological processes of cells, but are also involved in driving the pathological processes of various diseases.²⁶⁰ Oxidative stress and epigenetic regulation have a bidirectional control relationship and are involved in the progression of IDD.

DNA methylation is a crucial epigenetic modification involving the addition of a methyl group at specific positions on the DNA molecule, predominantly occurring at the 5′ carbon position of cytosine (C) bases, resulting in the formation of 5-methylcytosine (5-mC).²⁶¹ This process is catalyzed by DNA methyltransferases (DNMTs), especially DNMT1, DNMT3A, and DNMT3B.²⁶² DNA methylation induces gene silencing by interfering with the binding of transcription factors to highly methylated promoter regions, modifying chromatin structure, or reactivating the domains of methyl-binding proteins.^261,263 During oxidative stress, ROS can affect DNA methylation through oxidative DNA damage formed by pyrimidine and 5-methylcytosine (5mC) hydroxylation.²⁶⁴ In addition, ROS can also affect DNA demethylation through DNA oxidation and hydroxymethylation mediated by ten-eleven translocation (TET).²⁶⁴ Meanwhile, DNA methylation can also affect the expression of antioxidant genes, such as SOD, GSH and CAT, affecting the antioxidant defence of cells.^265,266 SLC40A1 is responsible for encoding the FPN, which is the only carrier capable of exporting intracellular Fe²+.^267,268 Upregulated DNMT3B in degenerated NP cells inhibits SLC40A1 expression through methylation, thereby promoting ECM degradation, ferroptosis, and oxidative stress in NP cells.²⁶⁹ DNA methylation also participates in the regulation of various signaling pathways, including MAPK and NF-κB, which are involved in IVD cell survival, aging, inflammatory response, ECM metabolism, and oxidative stress.^18,256 For example, DNMT3A activates the NF-κB pathway by inducing hypermethylation of the peroxisome proliferator-activated receptor γ (PPARγ) promoter, which promotes apoptosis and ECM degradation in NP cells.²⁷⁰ In addition, the hypermethylated genes MAPKAPK5 and PRKCZ, which are associated with the MAPK pathway, have been shown to regulate a wide range of catabolic molecules,⁴² but have not yet been correlated in IDD.

Histone modifications refer to covalent chemical modifications that occur on histone molecules.²⁷¹ These modifications can affect the structure and function of chromatin, thereby regulating gene expression, mainly including acetylation, methylation, phosphorylation and ubiquitination.²⁷² Oxidative stress is strongly associated with histone modification. Evidence suggests that oxidative stress has a direct impact on the function of chromatin remodeling proteins that act as histone modifying enzymes.^273,274 Histone acetyltransferases (HATs) are responsible for adding acetyl groups to the lysine residues of histones, including various subtypes such as Gcn5, Tip60, p300/CBP.^275–277 Among them, the activity of p300 can undergo significant changes after oxidation modification of key cysteine residues.²⁷⁸ Histone demethylases (HDMs) typically require Fe²⁺ as a cofactor, therefore the oxidation state of iron ions (Fe²⁺ or Fe³⁺) is a key factor affecting the activity of this enzyme.^279,280 In this regard, O₂⁻ and OH- can oxidize Fe²⁺ to Fe³⁺ at the catalytic center of HDMs and suppress its activity.^281,282 In addition, the imbalance of non-enzymatic antioxidant GSH oxidation and reduction levels can indirectly affect histone methylation.²⁸³ Histone modification also affects the redox state in IVD. ZRT/IRT-like Protein 4 (ZIP4) is a zinc transporter protein belonging to the ZIP family.²⁸⁴ The upregulation of ZIP4 enhanced inflammation and oxidative stress in NP cells, exacerbating ECM degradation.²⁸⁵ In terms of mechanism, ZIP4 upregulates the expression of histone deacetylase 4 (HDAC4), which in turn exerts its effects by regulating the expression levels of NF-κB, SIRT1, and FoxO3a.

ncRNA is a class of RNA molecules that do not encode proteins, mainly including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs).²⁸⁶ ncRNAs exhibit differential expression in degenerated human IVD tissues and participate in almost all cellular functional processes, including ECM degradation, inflammatory response, cell apoptosis, cellular senescence, oxidative stress, and angiogenesis.^287,288 In addition, N6-methyladenosine (m6A) is the most common RNA modification at the post-transcriptional level and is widely present in IDD.^289,290 Chen et al found that m6A-methylated circGPATCH2L was recognized by the YTHDF2-RPL10-RNaseP/MRP complex and cleaved by endoribonuclease, which was beneficial to alleviate DNA damage accumulation and apoptosis in NP cells.²⁹¹

Advantages of NDDSs for the Treatment of IDD

At present, the administration methods of drugs and bioactive agents for the treatment of IDD mainly include systemic administration and IVD in situ injection, but both have the disadvantages of poor curative effect and many side effects. Due to the non-vascular nature of IVDs, systemic administration makes it difficult to maintain adequate drug concentration and duration of action within the IVD, which results in limited bioavailability in local tissues.²⁹² Systemic administration can also result in altered or impaired active units of the drug, especially as oral drugs are susceptible to degradation by digestive enzymes and intestinal flora as well as first-pass elimination by the liver. In addition, systemic administration may result in widespread distribution of the drug throughout the body, leading to potential off-target effects and toxic side effects on other organs with prolonged excessive exposure.^293,294 For IVD in situ injection, the disadvantage is the poor injection dose and retention of drugs, which leads to limited absorption of drugs by target cells, thus reducing the therapeutic effect. However, increasing the number of injections may lead to intervertebral infection and accelerate the progression of IDD.²⁹⁵ Moreover, the administration of in situ injections may result in drug infiltration into the dural sac, thereby leading to central nervous system infections and other detrimental consequences.²⁹⁶

Nanomedicine refers to the application of nanoscience and technology to solve medical problems, which involves all aspects of diagnosis, prevention and treatment of disease.²⁹⁷ The reduction in size of nanomaterials gives them unique physical and chemical properties, which can be used to enhance existing medical technologies.²⁹⁸ NDDSs are at the forefront of nanomedicine applications and are currently used in the treatment of a wide range of diseases, including IDD.²⁹⁹ Compared with traditional drug delivery methods, NDDSs have the following advantages: (1) Through surface modification, NDDSs can carry specific ligands or antibodies that enable them to recognise and bind to diseased cells or tissues, enhancing drug accumulation at the site of the lesion.³⁷ (2) NDDSs can be designed as controlled drug release systems, allowing drugs to be released at a constant rate or in response to specific stimuli, improving therapeutic efficacy and reducing toxicity.²² (3) With their nanoscale size, certain specific NDDSs can assist drugs in entering the interior of cells through micropores in the cell membrane, which is crucial for drugs that require intracellular action.^37,300 (4) NDDSs generally have good biocompatibility and degradability, which can reduce damage to normal tissues and provide a superior cell microenvironment.²⁹²

Table 1 Common Nano-Drug Delivery Systems in IDD Treatment

Many NDDSs carrying therapeutic agents have been used to ameliorate the oxidative stress microenvironment of degenerating IVDs and to promote IVD repair.³⁷ Table 1 summarises the advantages and limitations of some common NDDSs used to treat IDD. In addition, Table 2 lists several promising nanocarriers that have been used to treat IDD. At the same time, combined with research in other fields, we will explore new NDDSs that are more valuable and effective for the treatment of IDD in the future.

Table 2 Recent Studies on Nano-Drug Delivery Systems for the Treatment of IDD.

Major Types of NDDSs for IDD

Liposomes

Liposome is a kind of nanoscale double-layer spherical structure mainly composed of lipid materials such as phospholipids and cholesterol, which makes liposomes have the ability to transport drugs, genes or other active molecules.³⁵⁷ Compared with other NPs, liposomes have high biocompatibility, easily adjustable physicochemical properties, diverse functionalization modifications and mature preparation methods, making them the first NPs to be converted into clinical applications.^358,359 However, to date, there have been few studies on the use of liposomes in the treatment of IDD. In several limited studies, liposomes have been described as carriers for small molecule drugs and ncRNA to improve cell apoptosis and ECM degradation in degenerative IVD.^360,361 In fact, liposomes also have unique potential in responding to oxidative stress. Liposomes are composed of a hydrophilic core and one or more hydrophobic spaces surrounded by lipid bilayers, which makes liposomes a multifunctional drug delivery platform capable of carrying hydrophobic drugs and hydrophilic drugs at the same time.²² Currently, there is a wide variety of hydrophilic drugs (such as vitamin C, glutathione, water-soluble flavonoids)^362–364and lipophilic drugs (such as vitamin E, retinol analogues, carotenoids, fat-soluble flavonoids and ubiquinone)^365–367 that can be used to improve oxidative stress. In addition, liposomes can carry antioxidant enzymes to directly enhance the cellular antioxidant defense system. For example, Zhang et al developed a liposome encapsulating SOD, which released SOD to improve oxidative damage induced by sodium dextran sulfate and significantly alleviate symptoms of colitis in mice.³⁶⁸ These methods may be used in the future to improve the oxidative stress microenvironment in IDD.

Currently, many challenges remain for liposome-associated drug delivery in IDD, including enhancement of drug encapsulation, tissue targeting, prolonged circulation and reduction of biotoxicity, and these optimisations can help to improve the therapeutic efficacy of liposomal systems.³⁶⁹ Firstly, the ideal size of liposomes is usually between 50 and 200 nm, and excessively large particle sizes can diminish therapeutic efficacy by affecting pharmacokinetic and pharmacodynamic profiles.³⁷⁰ The encapsulation efficiency of drugs in lipid membranes can be influenced by the composition of liposomes, with cholesterol being an example of a component that may decrease this efficiency.³⁷¹ Secondly, the encapsulation strategies for hydrophobic and hydrophilic drugs are also different, as they achieve high encapsulation efficiency by respectively binding with lipid bilayers and aqueous cores.³⁷² In addition, the active loading strategy allows the introduction of a transmembrane pH gradient, which can drive drug molecules into empty vesicles.³⁰¹ For enhanced tissue targeting, bioconjugation strategies can be employed to modify liposomes with small molecule ligands, nucleic acids, peptides, or monoclonal antibodies.³⁷³ In addition, smart stimulus-responsive liposomes can be constructed to target specific pathological environments, tissue sites and subcellular compartments by incorporating microenvironmental features (low pH, oxidative stress, and pro-catabolism) in degenerative IVDs.^374,375 In particular, redox-responsive liposomes can rapidly release encapsulated antioxidants in a high ROS environment and effectively alleviate oxidative stress.^376,377 In addition, liposomes are susceptible to clearance by the mononuclear phagocyte system,³⁷⁸ especially the large amount of immune cell infiltration in the late stage of IDD, which shortens their residence time in the IVD. One of the improvements is the use of biocompatible inert polymers to encapsulate liposomes (such as polyethylene glycol, PEG) to reduce their clearance by phagocytes.³⁷⁹ The degradation and biotoxicity of liposomes are also a concern, especially for some functional components used in the construction of functionalized liposomes.³⁸⁰ For example, cationic lipids can reduce cell mitosis and damage key cellular proteins.^381,382 In summary, developing and improving liposomal carriers is a complex challenge that involves optimizing and balancing multiple parameters to achieve safe and effective drug delivery.

Polymeric Nanoparticles

Polymeric nanoparticles (PNPs) are typically nanoscale homogeneous spherical structures composed of biocompatible and biodegradable polymers, which can encapsulate hydrophilic and hydrophobic pharmaceutical drugs inside the particles or attach to the particle surface.³⁸³ According to the source, PNPs can be divided into natural polymers and synthetic polymers.³⁸⁴

Natural polymers typically come from plants or animals, including polysaccharides, polyphenols, fibroin proteins, gelatin (Gel), collagen protein and albumin.^384,385 Their main advantages as drug delivery carriers are low biotoxicity, biodegradability, high biocompatibility, and unique bioactivity.³⁸⁶ Polysaccharides are common PNPs that naturally possess excellent antioxidant and anti-inflammatory properties.^387,388 It was shown that carboxymethylated chitosan (CMCS) significantly ameliorated H₂O₂-induced mitochondrial dysfunction and ROS production in NP cells, which in turn inhibited ECM degradation and cell apoptosis.³⁸⁹ In addition, fucoidan can upregulate the expression of antioxidant enzymes in NP cells by activating the Nrf2 signaling pathway, thereby alleviating IL-1β-induced oxidative stress damage.³⁹⁰ Gelatin methacrylate (GelMA) microspheres functionalised with fucoidan can effectively preserve the ECM component of IVD and maintain hydration of NP tissues, thus preventing puncture-induced IDD in rats.³⁹⁰ Hyaluronic acid (HA), as an anionic glycosaminoglycan, is the main component of ECM.³⁹¹ As a drug carrier, HA has excellent biocompatibility, biodegradability and mechanical properties.³⁹² Meanwhile, existing evidence also suggests that HA can improve TBHP-induced mitochondrial dysfunction, oxidative stress, cell apoptosis, aging and ECM degradation by activating mitophagy.³⁹³ Importantly, HA can also alleviate IVD-induced pain by altering glycosylation and modulating key inflammatory signaling.³⁹³ Gallic Acid (GA) is a polyphenolic compound with inherent antioxidant properties.³⁹⁴ Recently, Chen et al developed a poly-GA-Mn (PGA-Mn) antioxidant NPs by combining GA with Mn²⁺ via a self-assembly method, and used a mitochondria-targeting peptide (TP04) as a surface modification (PGA-Mn-TP04).³¹² The rapid electron exchange between Mn²⁺ and GA enhances the ROS scavenging ability of PGA-Mn-TP04, effectively alleviating mitochondrial damage. In addition, PGA-Mn-TP04 also promotes mitochondrial fusion by targeting mitochondria to restore mitochondrial function, thereby alleviating NP cell apoptosis and ECM degradation. After in situ injection, PGA-Mn-TP04 can persist in the rat IVD for 13 days and effectively delay the progression of IDD (Figure 6). The natural polymer carrier has great potential in the treatment of IDD, but it lacks batch-to-batch consistency and the diverse functionality possessed by synthetic polymers.³⁹⁵

Figure 6 Metallo phenolic nanoparticles targeting mitochondria for the treatment of IDD. (A) Schematic representation for the synthesis of the PGA-Mn-TP04. (B) OH- and (C) O₂⁻ scavenging efficiency of PGA-Mn with various concentrations. (D) H₂O₂ scavenging efficiency at different concentrations of PGA-Mn over time. (E) Cytosolic ROS (green fluorescence) and mitochondrial ROS (red fluorescence) levels in NP cells under different treatment conditions. (F) Transmission electron microscopy (TEM) images of mitochondria in NP cells under different treatment conditions. (G) Immunofluorescence staining of MMP-13 and COL2A1 in NP cells. (H) Imaging manifestations of PGA-Mn-TP04 in treating IDD in rat acupuncture model. (I) HE staining images of IVD at 4 and 8 weeks after surgery. (J) Safranin-O/Fast Green staining of different groups at 4 and 8 weeks. (*means p value < 0.05, **means p value < 0.01, ***means p value < 0.001, ****means p value < 0.0001). Reprinted with permission from Chen Q, Qian Q, Xu H, et al. Mitochondrial-Targeted Metal-Phenolic Nanoparticles to Attenuate Intervertebral Disc Degeneration: alleviating Oxidative Stress and Mitochondrial Dysfunction. ACS nano. 2024;18(12):8885–8905. Copyright © 2024, American Chemical Society.³¹²

Figure 7 PDA@CNO nanoparticles can mimic the activities of multiple antioxidant enzymes to alleviate oxidative stress in degenerated IVD. (A) Preparation of PDA@CNO. (B) ROS Scavenging Process of PDA@CNO. (C) PDA@CNO attenuates oxidative stress to protect NP cells. (D) Representative Micro-CT and MRI images of the caudal vertebrae of rats at 4 and 8 weeks. (E) Representative images of HE and Safranin O/fast green. Reprinted with permission from Wang J, Wu R, Liu Z, et al. Core-Shell Structured Nanozyme with PDA-Mediated Enhanced Antioxidant Efficiency to Treat Early Intervertebral Disc Degeneration. ACS Appl Mater Interfaces. 2024;16(4):5103–5119. Copyright © 2024, American Chemical Society.³¹⁴

Figure 8 MnO₂ nanoparticles coated with TrkA overexpressing macrophage membranes alleviate oxidative stress and discogenic pain. (A) Preparation process of MnO2@TMNP nanoparticles. (B) Mechanism of MnO2@TMNP in the treatment of IDD. (C) Flowchart of the timing of in vivo experiments to evaluate the effects of MnO₂@TMNP on disc regeneration. (D) Representative T2-weighted MRI images under different conditions. The red arrow marks the modeled section. (E) HE staining and Safranin O solid green staining of target segments of IVDs in different treatment groups. Reproduced from Yang W, Li K, Pan Q, et al. An Engineered Bionic Nanoparticle Sponge as a Cytokine Trap and Reactive Oxygen Species Scavenger to Relieve Disc Degeneration and Discogenic Pain. ACS nano. 2024;18(4):3053–3072. Copyright © 2024, The Authors. Published by American Chemical Society and licensed under CC-BY-NC-ND 4.0. https://creativecommons.org/licenses/by/4.0/.³¹⁵

Figure 9 Mitochondria-targeted carbon dots treat IDD by exerting antioxidant enzyme-like activity. (A) Preparation process of CD-PB-TPP nanoparticles. (B)Mechanism of CD-PB-TPP in the treatment of IDD. (C and D) X-ray and MRI images of rat tail in different treatment groups. (E) DHI of rat caudal IVD in different groups. (F) Mean gray value of the target intervertebral disc. (G) HE and Safranin O/fast green of the rat caudal IVD. (*means p value < 0.05, **means p value < 0.01, ***means p value < 0.001, ****means p value < 0.0001). Reproduced with permission from Shi Y, Bu W, Chu D, et al. Rescuing Nucleus Pulposus Cells from ROS Toxic Microenvironment via Mitochondria-Targeted Carbon Dot-Supported Prussian Blue to Alleviate Intervertebral Disc Degeneration. Adv Healthcare Mater. 2024;13(8):e2303206. Copyright © 2024, Wiley‐VCH.³²³

Figure 10 Injectable mesoporous bioactive glass/sodium alginate hydrogel loaded with melatonin for intervertebral disc regeneration. (A) TEM image of MBG nanoparticles. (B) Scanning electron microscopy (SEM) image of Mel-MBG/SA hydrogel. (C) Photos of the MBG/SA hydrogels before and after crosslinking. (D) Intracellular ROS levels in NP cells were monitored after different treatments using a ROS probe (DCFH-DA). (E) Schematic illustration of the animal experiment and X-ray and MRI images of the caudal vertebrae of rats after different treatments. (F) HE and Safranin O/fast green of the rat caudal IVD. (G) Masson staining of the rat caudal IVD. (H) Immunohistochemical staining images of COL-II and MMP-13. Reproduced with permission from Wu R, Huang L, Xia Q, et al. Injectable mesoporous bioactive glass/sodium alginate hydrogel loaded with melatonin for intervertebral disc regeneration. Mater Today Bio. 2023;22:100731. Under CC-BY-NC-ND 4.0. https://creativecommons.org/licenses/by/4.0/.³²⁷

Figure 11 Injectable decellularized NP hydrogel can be used as a carrier for MSCs to repair degenerated IVD. (A) Schematic overview of the construction of LM. (B) TEM images of poly(allylamine hydrochloride)-coated MnO₂ nanoparticles and LM. (C) Schematic overview of the construction of LMGDNPs.(D) Size distribution and microstructure of microspheres. (E) Cumulative release profile of LOX in LMGDNPs based on the BCA assay. (F) Lactate consumption efficiency of LOX-MnO₂ nanozyme and microspheres. (**means p value < 0.01, ***means p value < 0.001, ****means p value < 0.0001, between LM and CTRL groups; ns, no significance, ^# means p value < 0.05, between LM and LMGDNP groups). (G) Dissolved oxygen profiles of H₂O₂ solution mixed with LOX-MnO₂ nanozyme and microspheres. (**means p value < 0.01, ***means p value < 0.001, between LM and LMGDNP groups). (H) Fluorescence images showing changes in fluorescence intensity after delivery of DiR Iodide-labeled BMSCs via different vectors on days 1, 7, 14 and 21.(**means p value < 0.01, ***means p value < 0.001, GDNPs compared to cell pellets; ^#means p value < 0.05, ^##means p value < 0.01, LMGDNPs compared to cell pellets). (I) Representative images of T2-weighted MRI of rat tails. (J) Histological images based on HE and SO staining. Reproduced with permission from Peng Y, Chen X, Zhang Q, et al. Enzymatically Bioactive Nucleus Pulposus Matrix Hydrogel Microspheres for Exogenous Stem Cells Therapy and Endogenous Repair Strategy to Achieve Disc Regeneration. Advanced Science (Weinheim, Baden-Wurttemberg, Germany). 2024;11(10):e2304761. Copyright © 2023, Wiley‐VCH GmbH. Under CC-BY-NC-ND 4.0. https://creativecommons.org/licenses/by/4.0/.³³²

Synthetic polymers are synthesized by chemical methods, mainly including polyesters, polyamino acids, phosphate-based polymers, vinyl polymers and PEG, etc.³⁰² As drug carriers, they have the advantages of high stability, simple synthesis, strong flexibility and diverse modified structures.³⁹⁶ Polyesters are polymers consisting of repeating ester groups, commonly including poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA) and poly(ε-caprolactone) (PCL).³⁰² PLGA is the most widely studied polyester in IDD.³⁹⁷ Bradykinin (BK) is a nonapeptide hormone that exerts pleiotropic effects by promoting cellular proliferation, attenuating oxidative stress, and inhibiting apoptotic processes.^398,399 Based on this, Qiu et al developed BK/PLGA sustained-release microspheres.⁴⁰⁰ The results indicate that BK/PLGA sustained-release microspheres can inhibit TBHP-induced oxidative stress and mitochondrial dysfunction through the BK receptor 2 and its downstream PI3K/AKT pathway. Unfortunately, the average particle size of such microspheres is about 100 μm, which does not reach the nanoscale. Lim et al improved the process by synthesizing nanoscale PLGA particles and loading the anti-aging drug ABT263 into them (ABT/PLGA).³¹³ After in situ injection of ABT/PLGA into acupuncture-induced IDD of rats for 6 weeks, cell senescence, inflammatory response and ECM degradation in IVD were significantly reduced. In addition, polydopamine (PDA) is a synthetic polymer that possesses the ability to scavenge free radicals and chelate metal ions.⁴⁰¹ Based on this, Wang constructed a core-shell nanozyme (PDA@CNO) with co-doped NiO NPs (CNO) as the core and PDA as the shell³¹⁴ (Figure 7). PDA@CNO can mimic the activities of multiple antioxidant enzymes to effectively scavenge O₂⁻, H₂O₂, and OH−, which can protect NP cells from proliferation arrest, metabolic abnormalities, and inflammation, thereby reestablishing ECM homeostasis. Furthermore, it was shown that PDA@CNO NPs were able to attenuate iron overload through chelation and up-regulation of iron storage protein expression, which in turn attenuated lipid peroxidation-induced ferroptosis in NP cells.²⁰⁵ These results suggest that PDA may serve as an effective drug carrier for the treatment of IDD. Currently, synthetic polymers have broad prospects in drug delivery, but attention should be paid to the degradability and potential biological toxicity of synthetic polymers.⁴⁰²

Inorganic Nanoparticles

Inorganic NPs come from inorganic materials, mainly including metal NPs and non-metal NPs.⁴⁰³ Inorganic NPs have the advantages of adjustable particle size, enhanced stability, and large specific surface area, making them widely used in various industries such as healthcare, cosmetics, composite materials, and energy.⁴⁰⁴ In terms of drug delivery, drug molecules can be combined with NPs through electrostatic interactions, hydrophobic interactions, and covalent bonds to achieve drug delivery.²²

Metal NPs usually include metal-based NPs and their oxides, including silver (Ag), gold (Au), copper (Cu), iron (Fe), zinc (Zn), Mn, cadmium (Cd) and cobalt (Co).⁴⁰⁴ The main purpose of synthesizing metal oxides is to improve their reactivity and efficiency.⁴⁰⁵ MnO₂ is the oxidised form of Mn and its nanoscale form has unique chemical and physical properties of high specific surface area, low toxicity, high adsorption and biocompatibility.⁴⁰⁶ MnO2 can also change the inflammation and oxidative stress microenvironment of IVD cells at the genetic level to achieve IVD repair.^335,407 During the repair process, its metabolite Mn²⁺ is discharged with body fluids, thus reducing the impact on the internal environment.⁴⁰⁸ Therefore, MnO₂ NPs are an excellent drug carrier for the treatment of IDD. In the oxidative microenvironment of degenerating IVDs, excess ROS induces macrophage M1 polarisation, which in turn induces NGF production.⁴⁰⁹ The tropomyosin receptor kinase A (TrkA) belongs to the receptor tyrosine kinase family and is the specific receptor for NGF.⁴¹⁰ Based on the above, Yang et al developed a nanomaterial encapsulating MnO₂ nanoparticles with TrkA overexpressing macrophage cell membrane (TMNP) (MnO2@TMNP)³¹⁵ (Figure 8). MnO2@TMNP can act as a “molecular sponge” to capture NGF, thereby alleviating IVD-derived pain caused by the infiltration of IVD nerve fibers. At the same time, MnO2@TMNP can also target macrophages to deliver MnO2 nanoparticles, which in turn effectively scavenge intracellular ROS and prevent M1 polarisation to improve IDD. In addition, NPs based on Prussian blue (PB) (C₁₈Fe₇N₁₈) can mimic the activities of multiple antioxidant enzymes (CAT and SOD) to effectively remove ROS.⁴¹¹ Zhou et al injected PB NPs into the IVD of a rat IDD model.³¹⁶ The results showed that PB NPs could stabilize SOD1 from ubiquitination-proteasome degradation, thereby improving mitochondrial structure to enhance antioxidant capacity and thus alleviate ROS-induced IDD. Recently, a new type of multifunctional NPs called metallo-phenols have been developed, which can effectively improve the oxidative stress microenvironment of degenerated IVDs.³¹⁷ Such NPs are usually synthesized by self-assembly of metal ions (Cu, Mn, Fe) and phenolic compounds (catechol, gallic acid) in aqueous solution.^317,412 The ortho-hydroxyl groups of phenolic compounds can form stable coordination bonds with metal ions, which promotes the aggregation of metal ions and phenolic molecules in solution and forms NPs.⁴¹³ Metal-phenol NPs can effectively remove ROS in IVD cells by simulating the activities of various antioxidant enzymes through the active sites provided by the metal center.^312,317,318 At the same time, its surface modification molecules can usually accurately target mitochondria to improve mitochondrial function, thereby inhibiting IVD cell death and ECM degradation and effectively delaying the progression of IDD.^312,318

At present, the common inorganic non-metallic NPs in IDD treatment are mainly carbon-based NPs, including fullerenes, carbon nanotubes, graphene and carbon quantum dots.⁴¹⁴ Fullerene is a hollow molecule composed of 60 carbon atoms and is currently the most widely used inorganic NPs.⁴¹⁵ Due to its antioxidant capacity hundreds of times higher than other conventional antioxidants, it is called a “free radical sponge”.⁴¹⁶ Xiao et al combined functionalized nano fullerene with cFlFlF peptide, which can specifically bind to formyl peptide receptor-1 expressed on activated macrophages, thereby effectively reducing oxidative stress, inflammatory response and pain in a mouse model of radiculopathy.⁴¹⁷ Compared with the low water solubility and poor biocompatibility of fullerene, its polyhydroxy derivative fullerol has better biocompatibility and is the most widely studied.⁴¹⁸ In 2014, Jin et al reported that fullerol could significantly improve H₂O₂-induced ROS production in NP cells, thereby alleviating acupuncture-induced IDD in rabbits.³¹⁹ Subsequently, they further verified the antioxidant activity of fullerol in mouse bone marrow stromal cells, and the results showed that fullerol could reduce 23% of basal and 18% of IL-1β-induced intracellular ROS.⁴¹⁹ These studies demonstrate the powerful antioxidant properties of fullerenes and their derivatives, providing a new option for targeted IVD delivery. In addition, carbon nanotubes (CNTs) are considered as new drug and gene delivery carriers due to their high surface-to-volume ratio, high strength, biocompatibility and easy functionalization.^420,421 In 2022, Huang et al developed a cationic polymer-modified CNT for targeted delivery of LINC02569 (a ncRNA synthesized at an active enhancer) siRNA to NP cells.³²⁰ Compared with traditional siRNA delivery systems, CNTs can accumulate in NP cells and significantly reduce the inflammatory response and aging of NP cells by inhibiting p65 phosphorylation. The limitation of this study lies in the absence of in vivo testing for the role of CNT. Recently, carbon dots (CDs) have been widely used in nanomedicine due to their nanoscale size, high water solubility, and strong biocompatibility.^422,423 Related studies have shown that CD has inherent enzyme-like activity and free radical scavenging ability, which can be further enhanced by surface modification and doping elements.⁴²⁴ For example, Wu et al and Sun et al designed NAC-modified CD (NAC-CD)³²¹ and carbonized Mn-containing nanodots (MCD),³²² respectively. The results showed that both NAC-CD and MCD had SOD- and CAT-like activities and strong total antioxidant capacity, which could effectively alleviate H₂O₂-induced mitochondrial dysfunction, cell death and ECM degradation in NP cells, and were verified in the rat IDD model. In addition, surface-modified CDs can easily cross the cell membrane and enter the organelles to exert targeted antioxidant effects.⁴²⁵ For example, Shi et al reported a triphenylphosphine (TPP, a mitochondrial affinity ligand) modified PB-loaded CD (CD-PB-TPP)³²³ (Figure 9). The results showed that CD-PB-TPP could effectively evade lysosomal phagocytosis to achieve effective mitochondrial targeting, thereby significantly reducing ROS in mitochondria and protecting NP cells from senescence, catabolism and inflammatory responses.

Polymer Micelles

Polymer micelle (PM) is a nanostructure formed by self-assembly of amphiphilic polymers in solution.⁴²⁶ The material typically comprises a hydrophobic core and a hydrophilic shell layer, with the former located at the center of the PM and the latter forming an outer layer.⁴²⁷ The shell determines the hydrophilicity, charge, and surface modification of the PM, while the core is used for drug encapsulation.⁴²⁸ PM can be classified as graft or block copolymers, the latter including diblock, triblock and multiblock copolymers.⁴²⁹ Currently, PM drug delivery systems aim to achieve targeted, controlled, and sustained release of hydrophobic drugs.⁴³⁰

In 2021, Yu et al designed a new amphiphilic copolymer PEG-PAPO, which can self-assemble into nanomicelles and load kartogenin (KGN) and apocynin (APO).³²⁴ APO is linked to the polymer chain via a phenolic ester bond, while KGN is physically embedded in the hydrophobic core of the micelle. The results showed that the micelles could achieve precise and controllable delivery of APO and KGN, thereby protecting human adipose-derived stem cells (ADSCs) from oxidative stress and promoting NP-like differentiation of ADSCs. This study provides a potential adjuvant for stem cell transplantation therapy for IDD. In another study, Chang et al synthesized a polyamine-based PM loaded with Runx1 mRNA (a cartilage anabolic factor).⁴³¹ After the PM was injected into the degenerative IVD of rats, the intervertebral height and ECM content were significantly improved. Recently, researchers have used an emulsion-restricted assembly method to encapsulate coenzyme Q10 (CoQ10) (a mitochondria-targeted hydrophobic antioxidant) into lecithin micelles to achieve a water-soluble transformation of CoQ10.³²⁵ The results showed that CoQ10-loaded micelles had better bioactivity and could enhance the antioxidant capacity of rat bone marrow mesenchymal stem cells (BMSCs) by down-regulating NF-κB signaling. Furthermore, co-injection of the micelles with BMSCs maintained IVD height and alleviated IDD in a rat acupuncture model.

Nanofiber

Nanofibres are typically fabricated through phase separation, self-assembly, and electrostatic spinning using biodegradable natural polymers, synthetic polymers, and polymer blends.³⁰⁷ Currently, nanofibers used for drug delivery feature high specific surface area, high porosity, high biocompatibility, and easy modification, thus enabling high drug loading, sustained release properties, improved therapeutic index, and significantly reduced side effects.^432–434 In addition, nanofibers have certain mechanical strength and structural characteristics similar to ECM,^435,436 which help to improve the microenvironment within IVD, promote tissue repair and the interaction between cells and drugs. Therefore, nanofibers are a promising NDDSs for the treatment of IDD.

Initially, nanofibers were mainly used as tissue engineering scaffolds to repair AF.⁴³⁷ In 2011, researchers reported a PCL scaffold produced by electrospinning technology that can mimic the layered structure and mechanical function of AF.⁴³⁸ The scaffold can promote the adhesion, proliferation and ECM production of primary porcine AF cells. Subsequently, Martin et al combined PCL scaffolds with cell-seeded hydrogels to form disc-like angle-ply structures (DAPS).⁴³⁹ They found that only a few cells and ECM were deposited after implanting DAPS into rat IVD, which was significantly improved upon the addition of poly(ethyleneoxide). Although these nanofiber scaffolds demonstrate effective IVD repair, the minimally invasive implantation of the scaffolds remains a problem to be addressed. In 2019, Uysal et al reported an injectable collagen peptide-presenting nanofiber scaffold that can be easily implanted in IVD centers without damaging other healthy tissues.⁴⁴⁰ Injection of this scaffold into degenerated rabbit IVDs significantly induced glycosaminoglycan and collagen deposition. In pursuit of better biocompatibility and biodegradability, Liu et al combined Poly (ether carbonate urethane) urea (PECUU) electrospun nanofibre scaffolds with decellularized AF matrix (DAFM).⁴⁴¹ Compared with PECUU nanofiber scaffolds, DAFM/PECUU electrospun scaffolds can significantly promote ECM production from AF-derived stem cells. In addition, nanofibers can also be surface modified to further endow the scaffold with biological functional properties.^432,442 For example, Yu et al constructed a fucoidan-loaded PECUU nanofiber scaffold (F-PECUU).³²⁶ Compared with PECUU scaffolds, F-PECUU can significantly reduce LPS-induced inflammatory response and oxidative stress in AF cells, while promoting AF cell proliferation and ECM synthesis. In the rat AF defect model, the IVD height and NP hydration after F-PECUU treatment reached 75% and 85% of the sham-operated group, respectively.

Nanohydrogel

Nanohydrogel is a three-dimensional network structure formed by cross-linking hydrogel and nanomaterials through chemical or physical methods.^309,443 Nanohydrogels not only possess the excellent biocompatibility, biodegradability, mechanical strength and adhesion properties of hydrogels, but also the unique properties of each of the different NPs.^309,444 By adjusting the type of nanohydrogel skeleton material, cross-linking degree, type of nanoparticles and functionalization, minimally invasive implantation and in situ curing of hydrogels can be achieved,^445–447 thereby establishing a protective microenvironment that is conducive to cell proliferation and ECM synthesis. Currently, nanohydrogels have been widely studied in IDD. They can be used as sustained-release systems for bioactive substances, templates for ECM deposition, and scaffolds for carrying cells.⁴⁴⁸ According to the source of nanohydrogel skeleton materials, we divide them into natural polymer hydrogels, synthetic polymer hydrogels and composite hydrogels.

The skeleton materials of natural hydrogels are usually composed of natural polymers from animals and plants, including Gel, chondroitin, alginate, HA and chitosan (CS).⁴⁴⁹ Due to their diverse range of material sources, excellent biocompatibility, and degradability,⁴⁵⁰ natural hydrogels have found extensive applications in IDD repair and drug delivery with remarkable efficacy. Alginate is a natural polymer derived from brown algae, and hydrogels based on it have strong biocompatibility, high porosity and water retention capacity.⁴⁵¹ In 2023, Wu et al reported a sodium alginate (SA)-based injectable hydrogel for delivering MT-loaded mesoporous bioactive glass (MBG) NPs (MT-MBG/SA), which can continuously release MT to alleviate IL-1β-induced oxidative stress³²⁷ (Figure 10). Compared with the sham operation group, MT-MBG/SA treatment could significantly improve IVD height and NP water content. To enhance the mechanical capabilities of SA, the researchers added silk fibroin nanofibers to SA hydrogels to provide support for adjacent tissues and promote cell proliferation.³²⁸ In addition to SA, Gel is a widely used natural material, which is obtained by partial hydrolysis of collagen.⁴⁵² In order to enhance the mechanical and biological properties of Gel, it is often combined with other polymers. For example, Gan et al combined dextran with Gel to construct a hydrogel with good biocompatibility and low cytotoxicity for the delivery of PLGA nanoparticles loaded with TGF-β3.³²⁹ The system can stably release TGF-β3 and successfully induce the NP-like differentiation of MSCs. In another study, researchers combined SA with Gel to construct a hydrogel that exhibited good cell adhesion, biocompatibility, and mechanical function.³³⁰ In addition, decellularized ECM (dECM) hydrogel is a new type of biomaterial that retains the natural structure of ECM by removing the cellular components and has injectability, biocompatibility and good mechanical function.⁴⁵³ In 2023, Peng et al reported a decellularized AF matrix hydrogel for the delivery of TrkA-loaded amphiphilic polycarbonate NPs.³³¹ The system can significantly inhibit the expression of neurogenic mediators and nerve axon growth, thereby reducing mechanical hyperalgesia in the rat IVD protrusion model. Related studies have also shown that dECM contains a variety of growth factors and cell signaling molecules that help promote cell proliferation, differentiation and tissue regeneration.⁴⁵⁴ In 2024, in another study by Peng et al, they developed an injectable hydrogel microsphere by incorporating lactate oxidase (LOX)-MnO₂ NPs (LM) into glucose-rich decellularized NP hydrogel (GDNP) through a microfluidic system (LM-GDNP).³³² The results showed that LM-GDNP was able to ameliorate the microenvironment of nutrient deprivation, hypoxia, low pH and oxidative stress in degenerating IVDs and activate autophagy through transforming growth factor β2 overlapping transcript 1 (TGFB2-OT1) and its downstream pathway to promote cell survival and matrix regeneration. After co-injection of LM-GDNP with BMSCs into degenerated IVD in rats, the activity of BMSCs can persist and significantly improve the water content and tissue structure of degenerated IVD (Figure 11). Therefore, LM-GDNP is expected to serve as an ideal delivery system for exogenous stem cell therapy and endogenous repair. In addition, fibrin hydrogel³³³ and DNA hydrogel³³⁴ have also been used for IDD treatment and have shown good stability, controlled release effect and biocompatibility.

Synthetic hydrogels are obtained by processing natural products or other industrial raw materials and are highly customizable biomaterials.⁴⁴⁶ Compared with natural hydrogels, synthetic hydrogels have lower immunogenicity, better mechanical properties, adjustable biodegradability and easy modification.³⁰⁹ GelMA is a photosensitive injectable biohydrogel material formed by polymerization of Gel and methacrylate (MA) in phosphate buffer at 50°C.⁴⁵⁵ Compared with Gel, GelMA has significantly improved degradation rate, immunogenicity and mechanical properties.^447,456 In addition, since GelMA hydrogel is very similar to natural ECM, it can provide excellent mechanical properties and bioactivity, thus providing a suitable microenvironment and corresponding biological matrix for the repair of degenerated IVD.⁴⁵⁷ Currently, GelMA is the most commonly used hydrogel material for repairing IDD.⁴⁵⁶ In a study by Xu et al, they constructed a MnO2/GelMA hydrogel with good biocompatibility and porous structure.³³⁵ The results showed that MnO2/GelMA hydrogel could improve IVD microenvironment by removing intracellular ROS through SIRT1/Nrf2 pathway. However, this study did not verify the effects of the hydrogel in vivo. In another study, Li et al combined CS NPs encapsulating black phosphorus quantum dots (BPQDs) with GelMA through microfluidic technology to construct engineered hydrogel microspheres (GM@CS-BP), which can continuously release highly reducing BPQDs to improve the oxidative and inflammatory microenvironment in IVD.³³⁶ In the rat IDD model, GM@CS-BP microspheres were able to reshape the ECM structure and function of degenerated IVD by providing mechanical support and antioxidant effects. In addition, some researchers combined TGF-β1³³⁷ and aspirin³³⁸ -loaded NPs with GelMA, which significantly improved the NP structure and biomechanical function by reducing the inflammatory response.

Composite hydrogels are hydrogel materials composed of two or more different types of polymers.⁴⁵⁸ This material combines the advantages of multiple polymers, enabling it to achieve performance that is difficult for a single polymer hydrogel to attain.⁴⁵⁹ In 2015, Nair et al reported a composite hydrogel composed of CS and poly(hydroxybutyrate-co-valerate) (CP) for loading chondroitin sulfate NPs.³³⁹ The hydrogel has mechanical properties similar to those of natural tissues and can withstand different stresses under dynamic conditions corresponding to daily activities such as lying down, sitting and standing. The chondroitin sulfate NPs carried by them can enhance the activity of MSCs and promote NP-like differentiation. However, this study did not verify the long-term effects of the hydrogel in vivo. In addition, researchers have constructed a PBNPs-loaded hydrogel (PBNPs@OBG) composed of oxidized hyaluronic acid (OHA), borax and Gel, which exhibits antibacterial activity, antioxidant capacity, biocompatibility, tissue adhesion, self-healing and suitable mechanical properties³⁴⁰ (Figure 12). Compared with the sham operation group, PBNPs@OBG could significantly improve the water content, intervertebral height and tissue integrity of degenerated IVD in rats. In another study, Nezadi et al used laponite (LAP) as a cross-linker to construct a hydrogel composed of quaternized CS (QCS) and Gel.³⁴¹ The results show that the hydrogel-wrapped MgAl layered double hydroxide (LDH) NPs can improve the IVD microenvironment, thereby significantly promoting NP cell proliferation and ECM synthesis. In addition, DNA damage-inducible transcript 4 (DDIT4) is an evolutionarily conserved protein that is overexpressed when cells are subjected to oxidative stress and is associated with a variety of diseases.^438,461 Ma et al constructed a hydrogel loaded with DDIT4 siRNA (siDDIT4) using fifth-generation polyamidoamine (PAMAM) and HA (siDDIT4@ P-HA).¹⁹³ The results showed that siDDIT4@P-HA could alleviate H₂O₂-induced mitochondrial dysfunction and pyroptosis in NP cells by downregulating the ROS-TXNIP-NLRP3 signaling axis. In vivo, siDDIT4@P-HA continuously releases siDDIT4 for up to 35 days, which can significantly alleviate IDD in rats.

Figure 12 Dual-dynamic-bond cross-linked injectable hydrogel of multifunction for intervertebral disc degeneration therapy. (A) Fabrication scheme of the PBNPs@OBG hydrogel. (B) Mitigation of oxidative stress microenvironment within NP cells and notable amelioration of IDD caused by PBNPs-dotted hydrogel.(C and D) X-ray and MRI images of rat tail in different treatment groups. (E) DHI of rat caudal IVD in different groups. (F) Pfirrmann grade of the target intervertebral disc. (G–I) Representative images of HE, Safranin-O/Fast and Alcian Blue staining. (*means p value < 0.05, **means p value < 0.01, ***means p value < 0.001, ****means p value < 0.0001). Reproduced with permission from Yang L, Yu C, Fan X, et al. Dual-dynamic-bond cross-linked injectable hydrogel of multifunction for intervertebral disc degeneration therapy. J Nanobiotechnol. 2022;20(1):433. Copyright © 2022, Springer Nature. Under CC-BY-NC-ND 4.0. https://creativecommons.org/licenses/by/4.0/.³⁴⁰

Figure 13 Exosome-loaded temperature-sensitive decellularised ECM hydrogels for the treatment of IDD. (A) Images of NP tissue decellularization and preparation of hydrogels. (B) TEM analysis of exosomes. (C) SEM analysis of NP, dECM and dECM@exo. (D) Control-release curve of dECM@exo.(E) Fluorescence images of living (green) and dead (red) cells in each group. (F) X-ray images of the intervertebral disc at 0, 4, 8, and 16 weeks. (G) Differences in DHI between different treatment groups at each time period. (H) Images of HE and Safranin O/fast green staining from each group. (* means p value < 0.05). Reproduced with permission from Xing H, Zhang Z, Mao Q, et al. Injectable exosome-functionalized extracellular matrix hydrogel for metabolism balance and pyroptosis regulation in intervertebral disc degeneration. J Nanobiotechnol. 2021;19(1):264. Copyright © 2021, Springer Nature. Under CC-BY-NC-ND 4.0. https://creativecommons.org/licenses/by/4.0/.⁷³

Figure 14 NO-loaded ROS-responsive system for amelioration of oxidative stress microenvironment in degenerating IVDs. (A) NO-loaded ROS-responsive system for amelioration of oxidative stress microenvironment in degenerating IVDs. (B) Cumulative release of NO. (C)Cumulative release of l-Histidine. (D) Cumulative release of zinc ions. (E)X-ray images of rat tail disc in animal experiment. (F) quantitative analysis of DHI. (G) MRI images of rat tail disc in animal experiment. (H and I) The results of quantitative water content in the NP tissue and Modified Pfirrmann grading. (J and K) Representative images of HE, Safranin O/fast green staining, and immunohistochemical staining for Col-I and Col-II. (*means p value < 0.05, **means p value < 0.01, ***means p value < 0.001, ****means p value < 0.0001). Reproduced with permission from Feng Y, Su L, Chen Z, et al. Accurate Spatio-Temporal Delivery of Nitric Oxide Facilitates the Programmable Repair of Avascular Dense Connective Tissues Injury. Adv Healthc Mater. 2024;13(14):e2303740. Copyright © 2024, Wiley‐VCH GmbH.³⁴⁵

Figure 15 pH-responsive NDDS targeting senescent NP cells for the treatment of IDD. (A) The preparation procedures of nanoparticles. (B) Mechanism of SP@NNPm targeting of senescent NP cells. (C) SP@NNPm effectively targets senescent NP cells and activates the PGC1α-NRF2/TFAM pathway to reduce oxidative stress and cellular senescence. (D) Representative X-ray images and T2-MRI images of discs from sham or IDD rats. (E) Statistical analysis of the DHI. (F and G) Statistical analysis of the Pfirmann grades and relative signal intensity of NP tissues. (H) Representative images of HE and Safranin O/fast green staining and statistical analysis. (*means p value < 0.05, **means p value < 0.01, ***means p value < 0.001, ****means p value < 0.0001). Reproduced with permission from Liu S, Li K, He Y, et al. PGC1α-Inducing Senomorphic Nanotherapeutics Functionalized with NKG2D-Overexpressing Cell Membranes for Intervertebral Disc Degeneration. Advanced Science (Weinheim, Baden-Wurttemberg, Germany). 2024;11(22):e2400749. Copyright © 2024, Wiley‐VCH GmbH. Under CC-BY-NC-ND 4.0. https://creativecommons.org/licenses/by/4.0/.³⁴⁷

Figure 16 Hydrogen Ion Capturing Hydrogel Microspheres for the treatment of IDD. (A) Construction of MNP composed of CAT and TGF-β via biomimetic mineralization. (B) Preparation of GMNP loaded with MNP via the microfluidic device. (C) The mechanism by which GMNPs improve the IDD microenvironment, inhibit inflammatory response and promote ECM synthesis. (D) Experimental design of rat IDD model. (E) X-ray and MRI images of rat IDD at different time points. (F) Statistical analysis of the DHI. (G) Quantitative analysis of the gray value of IVDs. (H) Schematic of the material composition in different groups. (I) Images of HE and Safranin O/fast green staining from each group. (*means p value < 0.05, Statistically different from the puncture group; ^#means p value < 0.05, Statistically different from the control group). Reproduced with permission from Zheng D, Chen W, Chen T, et al. Hydrogen Ion Capturing Hydrogel Microspheres for Reversing Inflammaging. Advanced Materials (Deerfield Beach, Fla). 2024;36(5):e2306105. Copyright © 2023, Wiley‐VCH GmbH.³⁴⁸

Figure 17 Sustained and bioresponsive two-stage delivery of therapeutic miRNA by injectable hydrogel loaded with polymeric micelles to inhibit IVD fibrosis. (A) Schematic illustration for formation of miRNA/PGPC polyplex micelles. (B) Encapsulation of miRNA/PGPC polyplexes in PEG hydrogels in an injectable manner and molecular mechanism of MMP-2 silence in nucleus pulposus cells for fibrosis inhibition. (C) Injection sites in the IVDs of rabbits. Reproduced with permission from Feng G, Zha Z, Huang Y, et al.. Sustained and Bioresponsive Two-Stage Delivery of Therapeutic miRNA via Polyplex Micelle-Loaded Injectable Hydrogels for Inhibition of Intervertebral Disc Fibrosis. Adv Healthcare Mater. 2018;7(21):e1800623. Copyright © 2018, WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.³⁴⁹

Figure 18 Multi-responsive spermidine delivery system for improving oxidative stress microenvironment in degenerated IVD. (A) High magnification TEM images of PDA NPs. (B) TEM image of PDA NPs’ responsiveness in simulated media for 12 h. (C) In vitro release of SPD from PDA NPs. (D) Representative fluorescent images of NPCs stained with DCFH-DA (ROS probe). (E) RT-qPCR analyses of the relative expression of the GPX1, SOD1, and CAT genes. (F) Representative images of the general appearance of IVDs in different treatment groups. (G) MRI images of rat IDD at different time points. (H) Representative images of HE staining at different time points. (I) Representative images of Safranin O/fast green staining at different time points. (*means p value < 0.05, ^#means p value < 0.01). Reprinted from Int J Biol Macromol, 274, Wang D, Lu K, Zou G, Wu D, Cheng Y, Sun Y. Attenuating ntervertebral disc degeneration through spermidine-delivery nanoplatform based on polydopamine for persistent regulation of oxidative stress. 132881. Copyright 2024, with permission from Elsevier.³⁵³

Figure 19 MnOx-functionalized temperature-sensitive nanohydrogels contribute to the removal of endogenous ROS and provide a promising stem cell delivery platform for IDD. (A) States of the CS hydrogel and CS@MnOx nanohydrogel at 4 °C and 37 °C. (B) Injectability of the hydrogel. (C) SEM images of the CS hydrogel and the CS@MnOx nanohydrogel. (D) Evaluation of H₂O₂ elimination by the CS hydrogel and CS@MnOx nanohydrogel in vitro. (E) Gross images, X-ray images, and MRI images of IDD repair after treatment. (F) Quantitative analysis of DHI on X-ray images. (G) Quantitative analysis of the relative MRI index in MRI images. (H) Evaluation of Pfirrmann grading on MR images. (I) HE, Safranin-O/Fast Green, and Alcian blue staining of IVDs from different groups. (*means p value < 0.05, **means p value < 0.01, ***means p value < 0.001, ****means p value < 0.0001). Reproduced with permission from Wang X, Yu L, Duan J, et al. Anti-Stress and Anti-ROS Effects of MnOx-Functionalized Thermosensitive Nanohydrogel Protect BMSCs for Intervertebral Disc Degeneration Repair. Adv Healthcare Mater. 2024;13(29):e2400343. Copyright © 2024, Wiley‐VCH GmbH.³⁵⁴

Exosomes

Exosomes (Exos) are tiny membrane vesicles produced by intracellular multivesicular bodies (MVBs), with a diameter of approximately 30–150 nanometers.⁴⁶² Exos typically contain complex biomolecules, including proteins, lipids, DNA and different types of RNA, which can act as messaging molecules playing a key role in intercellular communication.⁴⁶³ In recent years, Exos have been used as carriers of genes and drugs to treat a variety of diseases due to their biocompatibility, low immunogenicity, intrinsic targeting ability, and enhanced intracellular efficiency.^464,465 A large number of studies have explored the role of Exos in IDD, but these results show that Exos may play a “double-edged sword” role in IDD.^466,467 Depending on the cellular source and contents, Exos can both accelerate IDD progression by promoting cellular senescence, inflammatory responses and vascularisation,^468,469 while some Exos can also protect against IVD by promoting NP cell survival, proliferation and ECM synthesis.^470,471 Currently, Exos can be loaded with small molecule drugs, proteins and nucleic acids through loading incubation, electroporation technology, ultrasonic treatment, extrusion and endogenous loading.⁴⁷² However, low drug loading efficiency is a significant drawback.⁴⁷³ Therefore, Exos are often combined with other nanocarriers to enhance their delivery efficiency.^473,474 For example, in 2021, Xing et al combined dECM with Exos derived from ADSCs (dECM@Exo) for the treatment of IDD⁷³ (Figure 13). The hydrogel provides an environment for NP cell growth, while its sustained release of Exos inhibits NP cell pyroptosis and ECM degradation. In vivo experimental results showed that the dECM@Exo hydrogel system maintained the homeostasis of the early IVD microenvironment and improved IDD. Although we have gained some understanding of the role of Exos in IDD, the research on Exos as drug delivery carriers is still focused on the field of tumor research.^475,476 Given the huge differences in pathophysiology between IDD and tumors, further research is needed on the effectiveness and safety of Exos as drug carriers in the future.

Special Stimuli-Responsive Drug Delivery System

Stimuli-responsive nano-drug delivery systems are smart drug carriers that can control drug release based on changes in the internal or external environment.⁴⁷⁷ The advantage of this system lies in its capacity to enhance treatment efficacy and minimize side effects by precisely delivering the drug to the disease site.²⁵

Internal Stimuli-Responsive Drug Delivery System

Redox-Responsive Nano-Drug Delivery System

We have previously described the disturbance of redox state in degenerating IVDs, which can induce the transformation of physicochemical properties, structural changes, functional regulation and degradation of redox-responsive NDDSs, thereby achieving controlled and targeted drug release.⁴⁷⁸ Compared with other carriers, redox-responsive materials remain stable in normal tissues but target lesions, which can significantly reduce the biotoxicity of carriers and drugs and increase drug utilization.⁴⁷⁹ Secondly, it releases cargo under the stimulation of ROS. In particular, redox-responsive materials themselves can alleviate oxidative stress by eliminating the corresponding oxidative species.⁴⁸⁰ In addition, redox-responsive materials can precisely locate subcellular locations, including the cytoplasm, mitochondria, and nucleus.^481,482 Redox-responsive materials include two drug release mechanisms: amphiphilic transition and bond cleavage.⁴⁷⁸ For the former, the transformation of NPs from hydrophobic to hydrophilic properties leads to the dissociation of polyphenylene sulfide, hydrophobic monosulfide, monosilane, or monotellurium polymer structures and subsequent cargo release.⁴⁸³ The functional bonds of the latter (such as diselenide bonds, disulfide bonds, arylboronic acid bonds, succinimide-thioether bonds, and tetrasulfide bonds) are completely broken under the action of ROS, resulting in the release of cargo.⁴⁸⁴

IL-1β is a major component of the inflammatory microenvironment of degenerating IVDs. Under oxidative stress conditions, it is produced by lysosomal-independent secretory autophagy in macrophages, in which tripartite motif containing 16 (TRIM16) plays an important role.³⁴² Based on this, Zheng et al designed a ROS-responsive drug delivery system.³⁴² They loaded water-soluble MR409 (TRIM16/LC3B reducing agent) into ROS-responsive vesicles composed of polyphenylene sulfide-PEG (PPS-PEG) amphiphilic polymers. These vesicles were then embedded in a hydrogel composed of PLGA and PEG for protection and controlled release. The system reaches a stable solid state at around 37°C, and can achieve rapid release of MR409 in the presence of H₂O₂ (more than 80% release in 40 hours), thereby significantly improving oxidative stress and inflammatory microenvironment. However, the degradation products of PLGA (lactic and glycolic acids) can reduce the local pH, which may lead to a deterioration of the IVD microenvironment.⁴⁸⁵ In another study, Yu et al constructed a diselenide block copolymer-based isoginkgetin (IGK)-loaded NPs (IGK@SeNPs), which showed excellent biocompatibility and low cytotoxicity.³⁴³ Compared with the control group, the system showed a cumulative release rate of 63% of IGK in H₂O₂ solution within 36 h, showing sensitive ROS responsiveness. With the combined action of Se-Se bond in diselenide and IGK, intracellular ROS are effectively eliminated. In addition, the researchers developed a rapamycin-loaded ROS-scavenging Gel hydrogel scaffold (Rapa@Gel).³⁴⁴ The hydrogel degraded slowly over 4 weeks without any obvious inflammatory response at the injection site, indicating that the hydrogel has excellent degradability and biocompatibility. In the presence of H2O2, rapamycin can achieve slow and sustained release and inhibit the inflammatory response by promoting M2 polarization of macrophages. Compared with traditional drugs, gas molecules can penetrate dense connective tissues more easily to reach damaged tissues due to their stronger permeability.⁴⁸⁶ Due to its unique biochemical properties, NO has the ability to antioxidant, anti-inflammatory, accelerate tissue regeneration and collagen deposition.^487,488 Based on this, the researchers synthesised NO-loaded biometal-organic frameworks (BioMOFs) and encapsulated them in modified polyvinyl alcohol and PCL composite electrospun fibre membranes with excellent ROS responsiveness.³⁴⁵ The results showed that the system could respond to the high oxidative stress environment in damaged AF tissue to achieve sustained NO release, thereby repairing AF damage by scavenging ROS, inhibiting the expression of inflammatory cytokines, promoting macrophage M2 polarization, and alleviating mitochondrial dysfunction. In a rat model of AF defect, this systemic treatment effectively repaired AF and improved the height and water content of the degenerated IVD (Figure 14).

Although redox-responsive NDDSs show excellent efficacy in alleviating oxidative stress in degenerative IVDs, there are still some issues worth considering. Firstly, it is necessary to systematically detect the redox status of IVDs at different stages of degeneration to distinguish the different ROS levels in normal and degenerated IVDs. Secondly, the sensitivity of the material depends on the specific functional groups and structure of the polymer, which may require individualized design at different disease stages. In addition, redox-responsive NDDSs should have good biocompatibility, degradability, and low cytotoxicity, which can avoid causing additional damage. Finally, while ensuring adequate drug concentration in the IVD, the rate of drug release should be delayed as much as possible through solubility changes and bond cleavage, which can increase efficacy and patient compliance.

pH -Responsive Nano-Drug Delivery System

Compared with the pH of normal IVD (7.1), the pH of mildly, moderately, and severely degraded IVD is 6.8, 6.5, and 6.2, respectively.^489,490 This observed variation in pH serves as the foundation for the application of pH-responsive NDDSs. pH-responsive NDDSs adjust the pH response range by changing the material structure to achieve targeted drug delivery at the lesion site.^491,492 At present, the mainstream response mechanisms of pH-sensitive materials include pH-sensitive bond cleavage and chemical group protonation.⁴⁹³ pH-sensitive chemical bonds (including hydrazone bonds, imine bonds, ester bonds, amide bonds, metal coordination bonds, and non-covalent bonds) act as connectors between drugs and carriers, and undergo conformational changes in response to the pH value of the surrounding environment to achieve controlled release of drugs.⁴⁸³ The latter NPs contain ionizable functional groups (such as carboxylic acids and amines), which can undergo protonation and deprotonation at different pH values, resulting in changes in their physical properties or structures, thereby achieving drug release.⁴⁹³ Based on these properties, researchers loaded antioxidant drugs onto pH-responsive NDDSs to alleviate oxidative stress in degenerated IVDs.

Hydrogen (H₂) is a promising antioxidant and anti-inflammatory substance, but its concentration in tissues is difficult to maintain for a long time.⁴⁹⁴ In 2023, Wang et al developed a pH-responsive hollow polydopamine (HPDA) loaded with ammonia borane (AB) H₂ sustained-release system (AB@HPDA).³⁴⁶ AB releases H₂ efficiently in an acid-responsive manner, while HPDA has the characteristics of large specific surface area, biocompatibility and strong permeability, which can effectively encapsulate AB. Compared with the sham operation group, AB@HPDA can significantly inhibit oxidative stress, inflammatory response and ECM degradation in acupuncture-induced degenerative IVD in rats. Peroxisome proliferator-activated receptor-ɣ coactivator 1α (PGC1α) activates nuclear respiratory factor-1/2 (NRF1/2) to control the expression of mitochondrial transcription factor A (TFAM), which serves as a key regulator of mitochondrial biogenesis and redox homeostasis.^495,496 PGC1α is significantly downregulated in degenerative IVD and is involved in regulating cellular senescence in IVD.⁴⁹⁷ Researchers have developed a pH-responsive and senescent NP cell-targeted PGC1α inducer delivery system³⁴⁷ (Figure 15).They coated mesoporous silica (SiO₂) NPs encapsulating PGC1α inducers (SP) with natural killer cell group 2D (NKG2D, which recognises NKG2D ligands up-regulated by senescent cells) overexpressing NP cell membranes (NNPm) (SP@NNPm), which in turn, through the dual-targeting mechanism of homologous membrane binding and receptor-ligand interactions targeting senescent NP cells. SP@NNPm treatment can significantly improve NP cell mitochondrial dysfunction and mtROS production through the PGC1α –NRF2/TFAM signaling axis, thereby inhibiting SASP-related paracrine effects and delaying the progression of IDD. TGF-β and CAT are important intracellular signalling molecules,^152,498,499 yet their direct delivery to Degenerated IVD tissues results in rapid protein release and degradation. Zheng et al constructed a mineralized nanoparticle (MNP) loaded with GelMA microspheres (GMNP) composed of CAT and TGF-β through biomimetic mineralization and microfluidics technology³⁴⁸ (Figure 16). The calcium carbonate mineralized layer in GMNPs can respond to the acidic environment and release encapsulated TGF-β and CAT. In vitro, GMNPs can inhibit the overactivation of the TXNIP/NLRP3/IL-1β signaling axis, thereby alleviating oxidative stress, inflammatory response, and ECM degradation in NP cells. In the rat IDD model, GMNP treatment could continuously improve intervertebral disc height and water content within 8 weeks, demonstrating its ability to promote the regeneration of degenerated IVD.

The pH-responsive NDDSs utilize the subtle differences in pH between normal and degenerated intervertebral discs, which requires the pH-sensitive functional groups to exhibit sensitive responses to slight changes in acidic microenvironments. The acidic environment in various subcellular organelles is the most commonly used stimulus to trigger the release of drugs from pH-responsive NDDSs. It is important to be aware that some pH-sensitive nanomaterials can induce lysosomal membrane damage and the release of hydrolases, leading to cell autophagy and death.^500,501 In addition, we must consider the biocompatibility and degradation properties of pH-responsive NDDSs, as well as the cytotoxicity of the residues.⁵⁰² For example, the toxic mechanisms of SiO₂ NPs include oxidative stress, inflammation, and cell apoptosis, and these toxic effects involve almost all organs in the human body.⁵⁰³ In addition, nanosized and negatively charged SiO₂ NPs also induced immunosuppression by inhibiting lymphocyte proliferation and NK cell activity.⁵⁰⁴ Therefore, pH-responsive NDDSs are still a long way from the laboratory to clinical treatment.

Enzyme-Responsive Nano-Drug Delivery System

Enzyme-responsive NDDSs are an advanced drug delivery technology that utilizes the activity of specific enzymes to control the release of drugs. It usually consists of two parts, including enzyme-sensitive nanomaterials and therapeutic agents wrapped in biodegradable encapsulation materials.⁵⁰⁵ After being acted upon by specific enzymes, enzyme-sensitive materials release drugs through size shrinkage, surface charge conversion, surface ligand activation, and chemical bond cleavage, which helps increase bioavailability and reduce adverse side effects in the body.⁴⁷⁸ The titers of these enzymes are often positively correlated with the severity of IDD, thus achieving precise controlled release.^83,506 In addition, the target enzymes are localized in different subcellular compartments in IVD cells, which facilitates selective targeted drug delivery.⁵⁰⁷

Protease can catalyze the hydrolysis of peptide bonds within protein molecules, thereby breaking down large molecular proteins into smaller peptides or amino acids.⁵⁰⁸ During IVD degeneration, the expression of multiple proteases is abnormally upregulated (including MMPs, ADAMTS, cathepsins, and hyaluronidase),^76,391,509 which can act as regulatory enzymes of enzyme response NDDSs and mediate the precise release of drugs at the target site. miRNA-29a is an endogenous ncRNA that regulates the expression of multiple ECM components to inhibit abnormal fibrosis and collagen conversion,⁵¹⁰ which may help alleviate NP fibrosis in degenerated IVD. In view of the lack of effective miRNA delivery systems, Feng et al developed a MMPs-responsive nucleic acid delivery system³⁴⁹ (Figure 17). The shell of the encapsulated miRNA-29a carrier is composed of cationic block copolymer micelles and hydrogel. In the presence of MMPs, miRNA-29a achieves a two-stage delivery in a responsive and sustained manner. In a rabbit IDD model, in situ injection of this system significantly inhibited NP fibrosis and increased IVD regeneration capacity. In addition to proteases, esterases can also be used in enzyme-responsive NDDSs.⁵¹¹ NP progenitor cells (NPPCs) derived from NP tissue are an ideal cell source for stem cell therapy of IDD.⁵¹² However, the hypoxic, acidic, and inflammatory microenvironment of IDD severely limits the survival and function of transplanted stem cells.^513,514 To overcome these challenges, Xia et al prepared esterase-responsive ibuprofen copolymer nanomicelles PEG-polyisobutene (PIB), which were internalized by NPPCs to form PEG-PIB-premodified NPPCs.³⁵⁰ Under the stimulation of the acidic microenvironment of the degenerated IVD, the increased esterase activity in the transplanted stem cells led to the sustained release of ibuprofen, which in turn effectively inhibited NPPCs pyroptosis and increased its therapeutic effect through the COX2/NF-κB/Caspase-1 signaling pathway.

At present, a variety of enzyme-responsive NDDSs have been successfully developed for promoting the repair of degenerated IVDs with efficient drug delivery performance and good application prospects. However, no studies have attempted to alleviate oxidative stress in IDD. Prior to undertaking the task, it is imperative to quantitatively assess alterations in enzyme content within IVDs.

Multi-Stimulus-Responsive Nano-Drug Delivery System

There are complex microenvironmental changes in degenerating IVDs, including hypoxia, low pH, oxidative stress, and promotion of catabolism. Therefore, combining the metabolic characteristics of two or more microenvironments can achieve multi-stimulus responsive drug delivery, which will further improve the targeting and side effects of drug delivery.⁵¹⁵ For example, embedding a redox-responsive cross-linked interlayer in pH-sensitive peptide micelles can trigger nanocarrier disassembly and release of encapsulated drugs in a reducing agent-rich environment.⁵⁰¹ In addition, Li et al prepared cross-linked nanogels containing disulfide bonds, which showed pH, temperature and reduction responsiveness.⁵¹⁶

As an important signaling molecule, H₂S plays an important role in various diseases by participating in multiple pathophysiological processes.^517,518 In terms of antioxidation, H₂S has been proven to protect cells from oxidative stress by neutralizing ROS molecules, upregulating the expression of antioxidant enzymes, and modifying cysteine residues on key signaling molecules.^519,520 Existing evidence suggests that H₂S has a protective effect on IDD.⁵²¹ However, traditional H₂S donors (such as NaHS and Na₂S) are difficult to provide continuous and stable H₂S output.⁵²² At the same time, the rapid removal of H₂S donors from the lesion and the volatilization of H₂S gas limit the therapeutic effect of H₂S.⁵²³ Based on this, Zheng et al prepared a pH and enzyme dual-responsive H₂S release system with low cytotoxicity and biocompatibility.³⁵¹ They encapsulated JK1 (a H₂S-releasing agent based on phosphoramidate thioester) into collagen hydrogel (Col-JK1). High levels of MMPs in IDD lead to collagen degradation and release of JK1, which is gradually degraded in a low pH environment to produce H₂S. In the rat IDD model, Col-JK1 treatment can significantly improve the imaging and histological changes of IDD within 16 weeks. The advantages of MnO₂ NPs as drug delivery carriers have been mentioned above, and they are highly sensitive to H₂O₂ and pH.^524,525 Zhu et al reported a smart drug delivery system based on hollow MnO₂ NPs encapsulating TGF-β3 (TGF-β3/MnO₂), which can dissociate and release loaded TGF-β3 under low pH and H₂O₂ environment.³⁵² Compared with simple TGF-β3 and MnO₂ NPs, TGF-β3/MnO₂ was superior in inhibiting H₂O₂-induced ECM degradation, ROS production, and apoptosis of NP cells. In vivo, TGF-β3/MnO₂ ameliorates IDD progression and promotes regeneration in rats. In addition, spermidine (SPD) is a natural biogenic amine that participates in various physiological activities such as DNA synthesis, protein folding, and cell apoptosis.⁵²⁶ Importantly, SPD can reduce excessive accumulation of ROS by protecting mitochondrial function.⁵²⁷ Based on this, the researchers developed a smart drug delivery system that encapsulates SPD with PDA (SPD/PDA).³⁵³ After SPD/PDA was exposed to low pH and H₂O₂ environment, PDANPs dissociated and released SPD, thereby inhibiting ROS production and inducing macrophage M2 polarization, ultimately alleviating the progression of IDD (Figure 18).

Although these systems demonstrate targeted and controlled drug release advantages, their triggering conditions are too complex, which may lead to the failure of encapsulated drug release. To determine the feasibility of these strategies, modulation of responses to each stimulus needs to be demonstrated both in vitro and in vivo.

External Stimulus-Responsive Drug Delivery System

External stimulus-responsive NODDs can control drug release through a variety of external signals (such as light, temperature, magnetism, and ultrasound).⁵²⁸ Compared with internal stimuli that are complex and difficult to control, external stimuli can precisely control drug release in time and space through non-invasive means.^528,529 Therefore, not only can potentially damage to normal tissues be minimized, but also stimulation can be added, removed, provided multiple times and continuously for drug delivery and treatment as needed for disease development. These advantages suggest that external stimuli-responsive drug delivery systems have good prospects in the treatment of IDD.

Thermo-Responsive Nano-Drug Delivery System

Thermo-responsive NDDSs control drug release based on the temperature difference between the lesion site and normal tissue, which requires that the properties of at least one component of the nanocarrier material change dramatically and nonlinearly with temperature.⁵⁰¹ When the temperature is below or above a critical temperature, thermosensitive materials undergo structural changes, leading to the dissolution of drug encapsulation layers or the formation of pores, thereby releasing drugs.⁵³⁰ Currently, common temperature-sensitive materials include functionalized modified liposomes,⁵³¹ poly(N-isopropylacrylamide) (PNIPAM),⁵³² poly(amidoamine) (PAMAM),⁵³³ poly(2-oxazoline) (POxs)⁵³⁴ and poly[2-(2-methoxyethoxy) ethyl methacrylate] (PMEOMA).⁵³⁵

In 2024, Wang et al developed a temperature-sensitive hydrogel composed of CS and glycerophosphate (GP), in which MnOx NPs with excellent water dispersibility were uniformly incorporated (CS@MnOx)³⁵⁴ (Figure 19). The hydrogel is an injectable liquid at 4°C and cross-links into a cavity-rich hydrogel at 37°C. Co-culture of BMSCs with CS@MnOx significantly down-regulated ROS production and increased the expression of SOD and CAT to inhibit oxidative stress, thereby ameliorating apoptosis, senescence and NP-like differentiation of BMSCs. Compared with the sham surgery group, CS@MnOx +BMSCs treatment efficiently restores intervertebral height and IVD water content. In addition, there are a variety of thermo-responsive hydrogels for the treatment of IDD, including PNIPAM-based temperature-sensitive hydrogels,^536–538 dECM temperature-sensitive hydrogels⁷³ and PLGA-PEG-PLGA temperature-sensitive hydrogels.⁵³⁹ Although the size of these materials has not yet reached the nanoscale, the delivery of small molecule drugs, ncRNA and stem cells using them as carriers can effectively improve the microenvironment of degenerative IVDs and promote repair. Similarly, NPs can also be endowed with temperature-sensitive properties. For example, researchers have developed a thermo-responsive nanosphere loaded with diclofenac and cartocinine, whose diameter increases as the temperature decreases (650nm at 4°C and 305nm at 37°C).⁵⁴⁰ Therefore, local cold therapy can promote the release of drugs from nanospheres, thereby improving synovial inflammation and delaying the progression of osteoarthritis in rats. Furthermore, thermosensitive liposomes can achieve temperature-responsive drug release by undergoing phase transitions in their constituent lipids and associated conformational changes in lipid bilayers, rendering them extensively employed in malignancy research.^541,542

Currently, the challenge of thermo-responsive NDDSs is to increase biocompatibility and reduce cytotoxicity while maintaining their thermosensitive properties. Because the thermo-responsive nanocarriers based on the mainstream thermosensitive material PNIPAM are not degradable in the body, which may bring therapeutic side effects.⁵⁴¹

Light-Responsive Nano-Drug Delivery System

Light-responsive NDDSs are typically made of nanoscale carrier materials, which can encapsulate drugs and remain stable in the body.⁵⁰¹ The surface of the nanocarrier is modified with photosensitizers or other photo-responsive components that are sensitive to light in a specific spectral range (UV, visible and near-infrared light).⁵⁴¹ When the photosensitive carrier is stimulated by light of corresponding wavelengths, the photosensitive groups within it can be activated through photoisomerization, photolysis, photocrosslinking, photooxidation-reduction and photothermal triggering, thereby releasing drugs.³⁷ Compared with other carriers, photosensitive carriers can be manipulated with high temporal and spatial precision by adjusting light intensity, emission wavelength, pulse length, exposure time and light direction.^543,544

As early as 2014, Kumar et al constructed a photocurable, biocompatible, degradable and injectable synthetic polymer hydrogel.⁵⁴⁵ The hydrogel solidified under 2.5 minutes of UV irradiation and did not significantly increase the genotoxicity of BMSCs. Under hypoxic conditions, the photocurable hydrogel significantly enhances the NP cell-like differentiation ability of BMSCs. Coincidentally, Chen et al also prepared a photocrosslinked hydrogel that promotes NP-like differentiation of adipose stromal cells (ASC).⁵⁴⁶ This hydrogel uses UV-induced cross-linked GelMA as the backbone, and hyaluronic acid methacrylate (HAMA) is added in a certain proportion to improve its mechanical properties. In vivo, the combination of hydrogel composites and ASCs promoted the repair of degenerated IVD in rats while avoiding osteophyte formation caused by direct injection of stem cells. Recently, researchers have synthesized a biodegradable photocrosslinked hydrogel with collagen and tyramine-modified HA as the backbone and non-toxic riboflavin as the photocrosslinker.⁵⁴⁷ This hydrogel solidified after being exposed to blue light for 3 minutes, and culturing BMSCs in it significantly increased ECM synthesis.

Although these studies have shown that photoresponsive nanomaterials can be used to repair IDD, no studies have yet explored the use of photoresponsive drug carriers to improve the oxidative stress microenvironment of IDD. In addition, compared with the non-nanomaterials mentioned above, nanoscale carriers have the advantages of high specific surface area, excellent biodistribution and strong targeting.⁵⁴⁸ At present, commonly used photoresponsive NDDSs include metal NPs, metal sulfides, metal organic frameworks, carbon nanomaterials, semiconductor NPs, stimuli-responsive micelles, liposome NPs and various organic dyes,^541,544,549 which may be used to repair the oxidative stress microenvironment in degenerative IVD in the future. In addition, for triggering light, UV light has low penetration depth and the risk of cell damage, which limits its clinical application.^550,551 Near-infrared light with a wavelength of 700–1000 nm has strong penetration and low cytotoxicity, which may be more suitable as a trigger light.⁵⁵² However, the lower energy of near-infrared light may not be sufficient to induce photochemical effects, which may lead to decreased drug release efficiency.⁵⁴⁹ Therefore, future attention should be paid to both the update of photoresponsive NDDSs and the optimization of triggering light.

Ultrasound-Responsive Nano-Drug Delivery System

With the development of modern medicine, ultrasound has become an indispensable and important means for the diagnosis and treatment of various diseases. Due to its non-invasive, cost-effective, safe, easy-to-operate, and highly penetrative characteristics, ultrasound has broad prospects in the field of drug delivery.⁵⁵³ Currently, ultrasound-responsive NDDSs induce drug release mainly through cavitation, mechanical effects and local thermal effects.^554,555 Ultrasound also increases the permeability of cell membranes through the double-layer acoustic wave effect.^556,557 In addition, both stable and inertial cavitation can form temporary holes in cell membranes through the “sonoporation” to increase drug delivery and thus improve efficacy.^558,559 Moreover, related studies have shown that low-intensity pulsed ultrasound can promote ECM production in NP cells and reduce inflammatory responses, which shows potential in the treatment of IDD.^560,561

Microbubbles are the most common ultrasound-responsive drug delivery method.⁵⁶² Microbubbles are usually composed of a gas core and a surfactant shell, which can expand, shrink or even rupture under ultrasound pressure, thereby releasing drugs to the desired location.⁵⁵⁶ Common gas cores are usually inert and non-metabolizable high molecular weight perfluorocarbons (PFCs) or sulfur hexafluoride (SF), which prevent dissolution and improve bubble stability.⁵⁵³ In 2018, Shen et al constructed an ultrasound-responsive resveratrol (RES)-loaded PLGA nanobubble with PFC as the gas core and N-cadherin antibody as the surface modification.³⁵⁵ The NPs imploded under external ultrasound induction, which promoted drug delivery through the released microjets and effectively delayed IVD degeneration in rats. In addition to PFC and SF, NO^563,564 and H₂⁵⁶⁵ can also serve as the gas core of microbubbles, and these gases have been shown to effectively alleviate oxidative stress in IDD. In addition to microbubbles, nanodroplets can also be used for ultrasound-responsive NDDSs, which have similar structures and fabrication mechanisms to microbubbles.⁵⁵³ In 2020, Nguyen et al developed nanoscale phase-change PFC droplets loaded with simvastatin.³⁵⁶ Multiple exposures to high-intensity focused ultrasound (HIFU) can lead to a phase transition of nanodroplets into bubbles, thereby inducing the release of simvastatin. The nanodroplets exhibited high stability, low cytotoxicity, and controllable triggered release in an ex vivo rabbit IVD for at least 14 days. In addition to microbubbles and droplets, micelles, liposomes and mesoporous SiO₂ NPs can also be used as ultrasound-responsive nanocarrier materials^562,566 and carry antioxidant drugs for the treatment of IDD in the future.

Although ultrasound-responsive drug carriers have many advantages, there are still many challenges in balancing the sensitivity of ultrasound response and the stability of the carrier in vivo.⁵⁶⁷ In addition, bone has a significant attenuation effect on ultrasound, which may affect its penetration of the targeted IVD. This effect may be ameliorated by changing the frequency, gain compensation or the use of specialised probes.

Magnetic-Responsive Nano Drug-Delivery System

Magnetic-responsive NDDSs are an advanced drug delivery technology that utilizes magnetic NPs as carriers and controls drug release through an external magnetic field.⁵⁶⁸ At present, common magnetic NPs include iron oxide, cobalt oxide, nickel oxide and magnetic SiO2.³⁷ Among them, superparamagnetic iron oxide (SPION) NPs are widely used due to their high level of superparamagnetism, biocompatibility, improved biodistribution, efficient cellular uptake, and minimal toxicity.⁵⁶⁹ In addition, some magnetic polymer composites have also been developed. For example, polysaccharides can be functionalized with other polymers or inorganic NPs to synthesize synergistic smart polysaccharide-based magnetic hydrogels with magnetic responsiveness and mechanical stability.⁵⁷⁰ For the external magnetic field, it mainly includes constant magnetic field and alternating magnetic field (AMF).⁵⁷¹ The constant magnetic field can focus the magnetic field on the biological target, thereby guiding the accumulation of the injectable carrier at the target site.⁵⁷² At the same time, the constant magnetic field can also induce the release of loaded drugs. For example, iron gel consisting of SPION NPs and Pluronic-F127 micelles will undergo aggregation of iron oxide NPs in response to an external magnetic field, which in turn releases the drug by squeezing the micelles.⁴⁸³ When magnetic NPs are in AMFs, they generate thermal effects through hysteresis loss and Néel relaxation,⁵⁷³ which can lead to detachment of the coating material or changes in the nanocarrier structure of magnetic NPs, such as increase in bilayer pores,⁵⁷⁴ disintegration of Fe₃O₄ cores,⁵⁰¹ and deformation of the lattice of single-crystal nanoshells.⁵⁷⁵

Despite the numerous advantages of magnetic responsive NDDSs, their application is limited by the complexity of external magnetic field setup and the potential biotoxicity of magnetic NPs.^570,576 Currently, the research on magnetic responsive NDDSs is mainly focused on the field of tumors,⁵⁷⁷ and there is no application in the treatment of IDD.

Conclusions and Future Perspectives

IDD is a common age-related musculoskeletal disorder, which is characterised by high morbidity, high disability and high healthcare costs. Numerous studies have shown that oxidative stress is an important factor in accelerating IDD. Oxidative stress not only affects the survival and metabolic characteristics of IVD cells by activating downstream signaling pathways, but also interferes with endogenous and exogenous repair by promoting the deterioration of the IVD microenvironment. The current primary objective in the treatment of IDD is to manage clinical symptoms rather than ameliorate pathological changes in degenerative IVD. Meanwhile, these conventional treatment modalities also exhibit limitations including significant invasiveness, numerous systemic adverse reactions, suboptimal patient adherence, and a heightened risk of recurrence. Although some emerging therapies (growth factors, cell therapies, and gene editing technologies) have been tested in preclinical and clinical studies over the past few decades, these approaches have not yet been truly applied in the clinic. The main obstacles leading to repair failure include the avascular structure of IVD, the harsh microenvironment, and the insufficient endogenous repair potential. Fortunately, rapid advances in nanomedicine and NDDSs have brought light to the treatment of IDD. These disruptive and rapidly developing approaches have the potential to address the challenges of current treatment approaches and provide innovative solutions for repairing and reversing IDD.

In this study, we conducted a comprehensive review of NDDSs that have the potential to enhance the oxidative stress microenvironment. We thoroughly analyzed their strengths, weaknesses, and future prospects for development. The ideal NDDSs should possess the advantages of biocompatibility, degradability, high drug encapsulation efficiency, controllable release, robust mechanical performance, low toxicity and facile clinical application. Based on the current study, we believe that nanohydrogels may be relatively more promising NDDSs for IDD repair. As NP repair and drug carrier, nanohydrogels not only have similar mechanical properties to natural tissues, but also provide ideal microenvironment to improve the proliferation, differentiation and signal transduction of endogenous or exogenous cells. In addition, nanohydrogels can achieve multifunctional properties by incorporating functionalized nanoparticles, such as improved controlled drug release, enhanced targeting, and increased cell adhesion. However, nanohydrogels also have limitations for the treatment of IDD and are not a perfect repair medium. For example, nanohydrogels have a better repair effect on NP, but the repair effect on AF is quite limited. In this regard, nanofiber scaffolds can better repair defective AF tissue. In addition, in order to better target lesion tissues to improve the efficacy of the drug and reduce side effects, stimuli-responsive drug delivery systems were introduced into drug delivery. In particular, redox-responsive NDDSs, which target diseased tissues or subcellular regions, can significantly improve the oxidative stress microenvironment in degenerative IVDs. However, stimuli-responsive drug delivery systems also face significant challenges in moving from the laboratory to clinical success. Internal stimuli-responsive NDDSs are dependent on changes in the microenvironment within the IVD and carrier-specific functional groups, demanding conditions that may limit their clinical application. As for external stimuli-responsive NDDSs, they often have difficulty in distinguishing between normal and diseased tissues, and therefore cannot be truly targeted. In summary, these finer vectors often involve limited application conditions or complex synthetic techniques, which make large-scale production and clinical application of vectors more difficult. Therefore, more active targeting strategies for identifying biomarkers in diseased tissues or cells should be introduced in the future. Furthermore, although many potential antioxidants have shown some efficacy in the treatment of IDD. However, it is undeniable that antioxidant therapy alone is not sufficient to halt or reverse the degenerative process. To achieve better clinical outcomes, NDDSs may need to be combined with other more cutting-edge treatments, such as NDDS combined with stem cells or gene therapy. These treatments will help to further enhance targeting and precision while encompassing the multiple pathological processes involved in IVD degeneration.

Although NDDSs have made great progress in treating IDD, they are still a long way from true clinical application, which involves many regulatory and ethical considerations. The progression of IDD is a chronic process, so the efficacy and safety of NDDSs need to be determined by long-term observation. However, the experimental periods of current studies are relatively short. Secondly, the good biocompatibility and biodegradability of nanocarriers is a prerequisite for their wide application, so minimizing immunogenicity and biotoxicity is the focus for further optimization. Currently, regeneration of NP is the current focus of attention, but future treatment should focus on synergistic recovery of NP, AF and CEP. In addition, the model animals currently used to evaluate the therapeutic effects of IDD are mainly rats and rabbits. The anatomical structure and tissue composition of these two animal IVDs differ greatly from humans,⁵⁷⁸ which may affect the accuracy of evaluation results. In comparison, the cell and ECM composition of sheep IVD is similar to that of humans, and may be an ideal experimental animal for studying IDD treatment.^579,580 Finally, the relief of low back and leg pain is the main purpose of patients seeking medical treatment and the main concern of treatment effect. Therefore, it is crucial to establish animal models of IVD-induced pain and reliable evaluation criteria for future clinical applications.

In conclusion, continuous research on the oxidative stress microenvironment of degenerative IVD, development of antioxidant drugs or treatment methods, and optimization of drug delivery strategies may potentially achieve a comprehensive treatment for IDD.

Data Sharing Statement

Data sharing not applicable to this article as no data sets were generated or analysed during the current study.

Acknowledgments

This work was supported by the Cultivation Project of Xi’an Health and Construction Commission (Program No. 2024ms14) and the Innovative Talent Promotion Plan-Technology Innovation Team (Program No. 2024RS-CXTD-86).

Disclosure

The authors report no conflicts of interest in this work.

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