Ischemic stroke is an acute cerebrovascular disease characterized by cerebral artery stenosis or embolism leading to blood flow interruption, resulting in ischemic and hypoxic necrosis of brain tissue [1]. Actively rescuing the ischemic penumbra and implementing various measures for treatment during the acute phase of ischemic stroke, including thrombosis, anticoagulation, and the use of neuroprotective drugs, may still result in poor efficacy and residual neurological deficits due to cerebral ischemia-reperfusion injury (CI/RI) [2,3]. At present, there is still no effective method in clinical practice to treat secondary injury caused by cerebral ischemia reperfusion. Therefore, it is particularly urgent and important to develop safe and effective drugs for intervention targets.

A growing body of evidence suggests that immune system inflammation, especially neuroinflammation, is involved in the progression and repair of CI/RI [4,5]. Exploring the mechanism of polarization of microglia/macrophages in CI/RI plays an important role. Microglia are one of the types of macrophages in the brain, serving as the first line of defense for the immune system. They maintain the homeostasis of the internal environment of brain neurons, monitor the microenvironment, and initiate immune response responses [6].

After ischemia-reperfusion injury, microglia are activated as early as 2 h, with the acute phase predominantly characterized by M2-type microglia. M2 microglia will suppress local inflammatory responses and exhibit protective effects on post-stroke brain tissue. After stroke 24 h, M1-type microglia become dominant, and result in migration, aggregation, activation of microglia, and a cascade of inflammatory cytokines [7]. Microglia play a dual role in CI/RI to maintain the homeostasis of the central nervous system microenvironment after stroke through two opposing pathophysiological effects. M1 phenotype activation releases harmful inflammatory factors such as nitric oxide (NO) and interleukin 1-beta (IL-1β), promoting inflammation and causing neuronal damage [8]. M1 microglia release pro-inflammatory factors to mediate neuroinflammation and lead to the death of pathogenic microorganisms [9]. Moreover, M2 phenotype activation secrete anti-inflammatory factors such as transforming growth factor-beta (TGF-β) and interleukin 10 (IL-10), which alleviates the inflammatory response [10]. Therefore, by inhibiting M1 microglia polarization and promoting M2 microglia polarization, will reduce inflammation, excessive immune responses and neuronal damage. Therefore, regulating the polarization balance of M1/M2 microglia is of great significance in mitigating CI/RI. Targeting specific inflammatory pathways, promoting the transition from M1 phenotype to M2 phenotype, and regulating the polarization balance of M1/M2 microglia are currently hot research topics in alleviating CI/RI.

Metformin is a potent drug that primarily acts on oxidative stress and inflammatory responses [11]. For decades, it has been used as a drug for clinical treatment of diabetes. However, metformin currently is becoming a drug for treating central nervous system damage. On the one hand, metformin can stimulate adult hippocampal dentate gyrus neurogenesis [12], and on the other hand, metformin has effective anti-inflammatory properties [13,14]. In fact, recent studies have shown that metformin has neuroprotective effects in different central nervous system injury models [15,16], and metformin treatment can restore cognitive and emotional deficits in mice with traumatic brain injury. Metformin treatment can induce differentiation of microglia, indicating a reduction in neuroinflammation [17]. However, most studies on the effect of metformin have not discussed the activation of microglia, particularly inflammation mediated by NLRP3 inflammasome. Here, we aim to investigate the anti-inflammatory effect of metformin and its regulatory effect on NLRP3 inflammasome, thereby inhibiting microglial cell activation.

Nearly 90% of the absorbed drugs of metformin are excreted through the kidneys within 24 h. Its plasma elimination half-life is about 6.2 h, so metformin needs to be taken two to three times a day [18]. Repeated administration of metformin can lead to increased toxic side effects, such as severe lactic acidosis, symptoms including tremors, dizziness, muscle pain, severe drowsiness, fatigue, skin cyanosis/cold, shortness of breath/difficulty, slow/irregular heartbeat, nausea or vomiting, and stomach pain and abdominal diarrhea [19,20]. Multiple drug delivery systems, especially nanodelivery systems, have been studied with the aim of reducing the side effects and frequency of drug use of metformin, while improving its efficacy.

As a cage like protein, ferritin is the storage protein for most organisms. It is a natural nanoparticle widely present in organisms, with good biocompatibility, specific active targeting, and easy preparation and self-assembly properties. Apoferritin nanocages has a large molecular “nanocage” structure, and its inner cavity can carry drugs. In addition, the particle size of apoferritin nanocages is relatively small, about 12 nm, making it easy to escape the phagocytosis of the reticuloendothelial system and thus achieve long-term circulation in the body [21,22]. Importantly, apoferritin nanocages can pass through the blood-brain barrier and achieve brain targeted drug delivery. Apoferritin can specifically bind to cells expressing transferrin receptor 1 (TfR1) [23]. Due to the high expression of TfR1 in brain endothelial cells, apoferritin can cross the blood-brain barrier and deliver drugs to the brain [24], achieving brain targeted drug delivery.

In this research, we constructed brain targeted apoferritin nanocages encapsulated with metformin, named APO@Metformin nanoparticles. APO@Metformin not only could target the CI/RI brain through TfR1 expression in microglia, but also improve the efficiency of metformin and reduce toxic side effects. Additionally, APO@Metformin released metformin, inhibited NLRP3 activity and M1 polarization activation of microglia, thereby inhibiting inflammatory response, protecting neurons, and improving CI/RI.