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Back to Journal »International Journal of Nanomedicine» Volume 16

Curcumin nanoparticles inhibit iron death for enhanced treatment of cerebral hemorrhage

Authors: Yang C, Han Ming, Li Rong, Zhou Li, Zhang Ying, Duan Li, Su Sheng, Li Min, Wang Q, Chen Tao, Mo Yan 

Published on December 14, 2021, the 2021 volume: 16 pages 8049-8065

DOI https://doi.org/10.2147/IJN.S334965

Single anonymous peer review

Editor approved for publication: Professor Dongwoo Khang

Yang Cong,1,*Meng Meng Han,1,2,*Li Ruoyu,1,*Zhou Ligui,1,3 Zhang Ying,1,2 Li Ning Duan,1,4 Su Shiyu,1,4 Li Min,1,4 Qi Wang ,1 Tongkai Chen,1,* Yousheng Mo5,* 1 Science and Technology Innovation Center, Guangzhou University of Chinese Medicine, Guangzhou, 510405; 2 First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510405; 3 Laboratory Animal Center, Guangzhou University of Chinese Medicine, Guangzhou 510405; 4Guangzhou University of Traditional Chinese Medicine Acupuncture and Rehabilitation Clinic, Guangzhou 510405; 5The Second Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine, Guangzhou, 510120 Mail is protected] Background: Intracerebral hemorrhage (ICH) is a severe stroke whose pathology is closely related to a recently discovered form of programmed cell death called iron death. Curcumin (Cur) is a common phenolic compound extracted from turmeric rhizomes, which can produce hematoma volume and related nerve damage in the context of ICH. Despite its therapeutic promise, Cur's efficacy is challenged by its poor water solubility, limited oral bioavailability, and its inability to effectively penetrate physiological barriers. Polymer-based nanoparticles (NPs) are widely used to aid drug delivery due to their ideal biocompatibility and ability to improve the bioavailability and pharmacokinetics of specific target drugs. Methods: In this study, we encapsulated Cur in NPs (Cur-NPs) and explored the role of these Cur-NPs in enhancing Cur delivery in vitro and in vivo. In addition, we evaluated the anti-iron death effect of Cur-NPs in ICH model mice and HT22 mouse hippocampal cells treated with enatine. Results: The obtained Cur-NPs were spherical, with a particle size of 127.31±2.73 nm, a PDI of 0.21±0.01, and a zeta potential of -0.25±0.02 mV. When applied to Madin Darby canine kidney (MDCK) cells in vitro, these Cur-NPs are internalized non-specifically through a variety of endocytic pathways, among which plasma membrane microcapsules and clathrin-mediated uptake are the main mechanisms. Inside the cell, these nanoparticles accumulate in lysosomes, endoplasmic reticulum, and mitochondria. Cur-NPs can cross the physiological barrier in the zebrafish model system. When administered to C57BL/6 mice, they significantly improved the delivery of Cur to the brain. Most notably, when administered to ICH model mice, Cur-NPs achieved better therapeutic effects than other treatments. In the last series of experiments, these Cur-NPs were shown to inhibit the iron death induced by erastin in HT22 mouse hippocampal cells. Conclusion: These Cur-NPs represent a promising method to improve the delivery of Cur to the brain to better treat ICH. Keywords: cerebral hemorrhage, nanoparticles, curcumin, brain delivery, blood-brain barrier, iron death

Intracerebral hemorrhage (ICH) is a serious form of stroke that accounts for 15-25% of total stroke events. 1 The 1-year survival rate of affected patients is less than 40%. 2 Although it is the main cause of human morbidity and mortality, the mechanism by which ICH causes extensive nerve damage is still not fully understood. In the acute phase of ICH just after the beginning of bleeding, hemoglobin is rapidly degraded, producing a large amount of iron in the extracellular space, as well as biliverdin and carbon monoxide. 3 There is evidence that the resulting iron overload can lead to edema around the hematoma, leading to accumulation of peroxides and widespread cell death. 4 Iron death is a recently discovered form of iron-dependent programmed cell death, which can be induced in response to lipid peroxidation. 5 Recent studies have shown that iron death plays an indispensable role in pathology. Therefore, ICH 6 and targeting this cell death pathway may represent an effective treatment for this fatal form of stroke. 7

Surgical hematoma removal can achieve a certain level of therapeutic effect in patients with ICH, but this method is very expensive and risky, which limits its utility for most patients. 8-10 There is currently a lack of effective drug treatments approved for the treatment of ICH, 11 and the development of drugs to treat this disease is hampered by limited oral bioavailability and difficulties associated with drug delivery across the blood-brain barrier (BBB). 12,13 In order to overcome these challenges, many researchers have tried to obtain new medicinal compounds derived from traditional herbs. 14

Curcumin (Cur) is a ubiquitous phenolic compound extracted from the rhizome of turmeric. It is reported to exhibit powerful antioxidant, anti-inflammatory and neuroprotective activities when used in a pharmacological environment. 15-17 When used to treat ICH model mice, Cur can effectively reduce the hematoma volume and related nerve damage in these animals. 18 However, the efficacy of Cur is affected by its undesirable water solubility, poor oral bioavailability, and inability to effectively bridge BBB and other physiological barriers. 19 Previous studies tried to improve the therapeutic effect of Cur by encapsulating Cur in polymer micelles, 20 liposomes 21 and solid lipid nanoparticles. 22 Although these methods can improve the bioavailability of Cur to a certain extent, the related pharmacokinetic characteristics of Cur and the ability of these particles to effectively overcome physiological barriers remain to be clarified. Therefore, it is clear that new treatments need to be developed to improve the oral bioavailability and brain accumulation of Cur as a means to improve the prognosis of ICH patients.

Polymer-based nanoparticles (NPs) have a high degree of biocompatibility and are commonly used as a tool to facilitate drug delivery, and have been approved by the U.S. Food and Drug Administration for medical use. 23-26 NPs have previously been shown to significantly improve solubility, bioavailability, 27,28 Compared with many other drug delivery nanoplatforms, these nanoparticles also have high versatility, toxicity, and immunogenicity. 29 Nanoparticles can also be prepared by an inexpensive and easy-to-use anti-solvent method for precipitation. 30,31

Here, we have prepared Cur polymer-based NPs (Cur-NPs) by anti-solvent precipitation method. We then used Madin Darby Canine Kidney (MDCK) cells as an in vitro model system to study drug absorption because these cells exhibit tight junctions and a polarized mucus layer similar to intestinal epithelial cells. 32 The endothelial BBB of zebrafish is similar to that observed in higher vertebrates, 33 enabling us to use these animals as a model system to monitor the distribution and elimination of Cur-NPs in the body. Then, we explored the pharmacokinetics of Cur-NPs in the brain and plasma of the treated mice, and evaluated the neuroprotective efficacy of these NPs in the mouse ICH model system. Then, HT22 mouse hippocampal cells were used to evaluate the ability of these Cur-NPs to inhibit iron death in vitro.

Cur and PVP-90 (Polyvinylpyrrolidone K90) were purchased from Nantong Feiyu Biotechnology Co., Ltd. (Nantong, China). MPEG-PTMC diblock copolymer, (monomethoxy polyethylene glycol (MPEG), MW, 2000 Da; poly(trimethylene carbonate) (PTMC), MW 16000 Da) purchased from Jinan Daigang Biomaterials Co., Ltd. (Jinan, China). 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), LY294002 (LY), chlorpromazine (CPZ), hypertonic sucrose (HS), MTT solution and methyl-β- Cyclodextrin (MβCD) was purchased from Sigma Aldrich (MO, USA). Coumarin 6 (C6), Lysotracker, ER tracker and Mitotracker were purchased from Molecular Probes Inc. (OR, USA). Erastin was purchased from MCE (NJ, USA). The live/dead detection kit was purchased from KeyGene Biotech (Nanjing, China). Type IV collagenase was purchased from ThermoFisher Scientific (New Jersey, USA). Nissl staining kit was purchased from Beyotime Biotechnology Co. Ltd. (Shanghai, China). The Prussian blue staining kit was purchased from Servicebio (Wuhan, China). H&E staining kit was purchased from Leagene Biotechnology (Beijing, China). The primary anti-GPX4 was obtained from Abcam, Inc. (Cambridge, UK). The secondary anti-rabbit IgG (Alexa Fluor 594) was purchased from Cell Signaling Technology (MA, USA). MDCK cells were purchased from the American Type Culture Collection (MD, USA). Mouse hippocampal neuron cell line HT22 was purchased from Guangzhou Zhenniao Biotechnology Co., Ltd. (Guangzhou, China).

Cur-NP was prepared using the anti-solvent precipitation method. In short, Cur (20mg/mL) and PEG-PTMC (20mg/mL) were dissolved in acetone as the organic phase, and PVP k90 (2mg/mL) was dissolved in water to prepare the aqueous phase. Then, a total of 0.2 mL of the organic phase solution was quickly injected into 10 mL of the aqueous phase solution, and the resulting mixture was stirred at 1000 rpm/min to obtain Cur-NPs. In addition, C6 and mPEG-PTMC were mixed in the organic phase at a ratio of 1:30 to prepare C6-NP. In order to remove residual solvents, the obtained C6-NPs were stirred at room temperature in the dark for 2 hours. The physical and chemical properties of Cur-NPs were analyzed, including transmission electron microscopy (TEM) morphology, dynamic laser scattering (DLS) particle size, PDI and zeta potential, and powder X-ray diffraction (XRD) crystal pattern).

To assess the absorption of Cur-NPs in vitro, MDCK cells were treated with five different endocytic pathway inhibitors (EIPA, LY, CPZ, HS, and MβCD) for 30 minutes. 34 Then treat these cells with C6-NPs for 1 hour, then remove the supernatant and wash the cells three times with PBS. The cells were then fixed with 4% paraformaldehyde (PFA) for 5 minutes and imaged by a confocal laser scanning microscope (CLSM; TCS SPE II, Leica, Germany).

MDCK cells were seeded on coverslips of a 24-well plate (5×104 cells/well) and cultured in complete medium at concentrations of 0.5, 1.0, and 2.0 μg/mL in the presence of C6-NPs. After 10, 30, or 60 minutes, the culture medium was removed from these cells and fixed with 4% PFA before imaging through a fluorescence microscope (DMi8, Leica, Germany) at 488 nm. The accumulation of these nanoparticles in lysosomes, ER and mitochondria was assessed by treating the cells with Lysotracker, ER tracker and Mitotracker dyes for 2 hours. 35 Then wash the sample three times with serum-free medium (wash for 5 minutes) and fix. Then confocal laser scanning microscope (CLSM; TCS SPE II, Leica, Germany) was used to evaluate the localization of C6-NPs to these different organelles.

Zebrafish (Danio rerio) were obtained from the China Zebrafish Resource Center (Wuhan, China) and raised under standard conditions with a 14-hour/10-hour light/dark cycle. After maturation, add male and female zebrafish to a 1 L water tank at a ratio of 1:2, use a sieve overnight, and then collect the fertilized embryos. To prevent the formation of pigments, 1-phenyl-2-thiourea is used. These embryos (7 days after fertilization [dpf]) were incubated in C6-NPs solution at 400 ng/mL C6 for 15 or 60 minutes to assess the biodistribution characteristics of Cur-NP after oral absorption. After treatment, the C6-NP biodistribution was evaluated by fluorescence microscope (DMi8, Leica, Germany).

Male C57BL/6 mice (8 weeks old) are kept under controlled conditions (25°C, 55% relative humidity, 12-hour light/dark cycle), and they can eat and drink freely. Animal oral Cur-NPs or equivalent dose of Cur (5 mg/kg) in 1.5 mg/mL PVP K90 (control). Blood samples were collected from the tail veins of these animals at 1, 2, 4, and 8 hours after treatment (4 points/time point). The blood was centrifuged at 3500 rpm for 10 minutes, and a supernatant serum sample was collected for analysis. At these same time points, the mice were euthanized by anesthesia, and major organs were collected from 4 animals at each time point. Then LC-MS/MS36 was used to assess the level of Cur in mouse plasma and organ samples.

Male C57BL/6 mice (8-10 weeks old) were from the Experimental Animal Center of Guangzhou University of Chinese Medicine (Guangzhou, China). The mouse ICH model was established as described above. 37 In short, the mouse is anesthetized and placed in the prone position, with the head stabilized in the stereotaxic frame. Then use a dental drill to create a 1 mm hole at 2.0 mm to the right of the bregma and 3.5 mm in the brain depth. Next, the acute ICH was induced by slowly injecting 0.1 U of type IV collagenase into the hole. In contrast, control animals were injected with the same volume of saline solution. The Animal Ethics Committee of Guangzhou University of Traditional Chinese Medicine approved these experiments, which were carried out in accordance with the guidelines for the care and use of laboratory animals in China.

For experimental treatment, mice were randomly divided into control, ICH, Cur and Cur-NP groups (n = 6/group). Cur and Cur-NP solutions were orally administered to appropriate animals by gavage 2 hours after the induction of ICH injury. These drugs were administered at a dose of 20 mg/kg/day twice a day after ICH induction for 3 days. Mice in other treatment groups were given an equal volume of saline.

The effect of Cur-NP or Cur treatment on the motor activity of ICH model mice was evaluated using rotating rod and climbing rod tests. For the rotating rod test, place the animal on a rod rotating at 20 rpm for 120 seconds, and record the number of falls during this period and the incubation period of falls. For the pole climbing test, the mouse was placed on a pole (50 cm high, 0.9 cm diameter) and allowed to climb down for 5 minutes without any other interference. Record the time required for the mouse to turn down (T-turn) and the total time to reach the bottom (T-total).

After the behavioral test, the mice were euthanized, and whole brain tissue slices were cut and sliced ​​into 1 mm thick slices after PBS perfusion. Use Epson Perfection V370 photo scanner (Epson China, Beijing, China) to obtain images of these tissue sections, and use ImageJ to measure the percentage of hematoma volume [(hematoma volume/hemispheric brain volume)×100].

After euthanasia, the mice were perfused with PBS through the heart and then perfused with 4% PFA. Then the whole brain was collected from each animal and transferred to 4% PFA solution. After fixation, these brain samples were embedded in paraffin and sectioned to produce 5 μm sections for subsequent analysis.

H&E staining was used to evaluate the effect of Cur-NP treatment on neuronal loss in the area around the hematoma in the brain samples of ICH model mice. In short, tissue sections were deparaffinized with xylene, rehydrated with an ethanol gradient, stained with hematoxylin for 5 minutes, and stained with eosin for 1 minute. Then the sections were dehydrated with ethanol gradient, clarified with xylene, and mounted with neutral gum. Then observe the sample through an optical microscope (DMi8, Leica, Germany).

Nissl staining was used to evaluate the effect of Cur-NP treatment on neuronal degeneration in the area around the hematoma in ICH model mice. In short, tissue sections were deparaffinized with xylene, rehydrated with an ethanol gradient, and stained with 1% toluidine blue for 10 minutes. Then it was dehydrated with ethanol gradient and mounted with neutral gum. Then the Nissl body in these samples was observed by an optical microscope (DMi8, Leica, Germany).

The iron accumulation in the area surrounding the hematoma was assessed by Prussian blue staining. In short, tissue sections were deparaffinized with xylene, rehydrated with an ethanol gradient, stained with Perls Prussian blue for 15 minutes, and treated with hematoxylin for 30 seconds. These samples were then dehydrated using an ethanol gradient, then treated with xylene to be transparent and fixed on a neutral resin cover glass. The slices were then imaged by an optical microscope (DMi8, Leica, Germany).

For GPX4 staining, 30 μm thick frozen brain tissue sections were permeabilized with 0.1% Triton X-100, then blocked in goat serum at room temperature, and then probed with anti-GPX4 overnight at 4°C. After incubating with goat anti-rabbit IgG (Alexa Fluor 594) for another 1 hour in the dark, the nucleus was stained with DAPI, and the cells were imaged by a fluorescence microscope (DMI8, Leica, Germany). The positive cells were stained with 3 per sample. -4 different fields of view.

The effect of Cur-NP treatment on cell viability was evaluated by MTT analysis method. In short, HT22 cells were treated with erastin and Cur-NPs or other experimental treatments for 24 hours. Next, replace the medium in each well with 90 μL of DMEM containing 10 μL of MTT solution, and incubate the cells for another 4 h. Then discard the supernatant in each well and replace with 150 μL DMSO. After shaking with a shaker for 10 minutes, use a microplate reader (Multiskan FC, Thermo Scientific, USA) to measure the absorbance at 490 nm in each well to measure cell viability.

HT22 cells treated with erastin were treated with appropriate concentrations of Cur-NPs or other drugs for 24 hours, and then the supernatant was replaced with 2μM calcein AM and 8μM PI for 30 minutes, while being protected from light. The cells were then imaged by a fluorescence microscope (DMi8, Leica, Germany).

The Annexin V-FITC/PI Apoptosis Detection Kit (KeyGEN Biotech, Nanjing, China) was used to stain HT22 cells treated with Annexin V and PI in different administrations. Flow cytometry was used to detect the apoptosis of HT22 cells. The data was analyzed with CytExpert analysis software.

DCF fluorescence staining was performed to study the intracellular ROS level in the differentially treated HT22 cells, and the fluorescence intensity of the DCF analysis was performed by Image J (NIH software).

DHE is a cell penetrating dye that is oxidized by superoxide to fluorescent ethidium bromide and intercalated with DNA. In this study, it was used to detect ROS levels in HT22 cells after differential treatment. Fluorescence microscope (DMI8, Leica, Germany) was used to identify ROS-positive cells.

RNAiso Plus reagent (Takara, Beijing, China) was used to extract total RNA from the brain tissue around the hematoma of each group of samples. The cDNA template was prepared by reverse transcription using PrimeScript™ RT Master Mix (Takara, Beijing, China). Then the template was diluted in a ratio of 1:3 and amplified on a real-time PCR instrument (ABI, California, USA). Each sample was amplified three times, and the average value was taken to calculate the relative content of the product. GAPDH is used as a housekeeping gene. Measure the relative mRNA concentration by E = 2-ΔΔCt and check the critical threshold cycle (CT) value in each reaction.

Data are expressed as mean ± standard deviation (SD), and evaluated by one-way analysis of variance (ANOVA) and two-tailed Student’s t-test, with P<0.05 as the threshold of significance.

In this study, the anti-solvent precipitation method was used to prepare Cur-NPs (Figure 1A). When evaluated by TEM, the resulting particles are spherical with a diameter of 70-100 nm (Figure 1B). Dynamic light scattering (DLS) analysis showed that the diameter of these particles was 127.31±2.73 nm, the PDI was 0.21±0.01 (Figure 1C), and the zeta potential was -0.25±0.02 mV (Figure 1D). XRD analysis did not show a unique free Cur peak at 17° in Cur-NP, which may be because these peaks were masked by a larger percentage of Cur-NP in the resulting preparation (Figure 1E). In addition, this study also evaluated the size, charge, and surface modification of C6-NPs, which are the main factors affecting the cellular uptake of NPs. 38,39 The results show that C6-NPs have similar size and zeta potential as Cur-NPs. Figure 1 Characterization of Cur-NPs. (A) Preparation of Cur-NPs. (B) TEM imaging result. Scale bar: 100 nm. (C) Size distribution. (D) Zeta potential value. (E) XRD patterns of Cur, PEG-PTMC, PVP K90, Cur-PM (physical mixture of PVP K90 and Cur) and Cur-NPs.

Figure 1 Characterization of Cur-NPs. (A) Preparation of Cur-NPs. (B) TEM imaging result. Scale bar: 100 nm. (C) Size distribution. (D) Zeta potential value. (E) XRD patterns of Cur, PEG-PTMC, PVP K90, Cur-PM (physical mixture of PVP K90 and Cur) and Cur-NPs.

It was found that the uptake of NP by MDCK cells occurred in a time- and dose-dependent manner (Figure 2A and C). In order to clarify the mechanism basis of this uptake, MDCK cells were treated with different endocytosis inhibitors, of which CPZ, HS and MβCD had the most profound inhibitory effects on the endocytosis of NPs. These results strongly indicate that many non-specific endocytosis mechanisms can promote the absorption of NPs (Figure 2B and D). Then we checked the intracellular distribution of C6-NPs in vitro and found that they are mainly distributed in the ER and lysosomes, which can control the bioavailability of the drug (Figure 2E and F). Mitochondria are key regulators of energy metabolism and programmed cell death pathways. Mitochondria are also the main place for the production of reactive oxygen species, making the organelles more susceptible to oxidative damage and mitochondrial function damage. According to reports, curcumin can reduce oxidative damage by increasing reduced glutathione and preventing the permeability transition of isolated brain mitochondrial membranes. 40 It is worth noting that the NPs system can significantly increase the therapeutic index of drugs by promoting the entry of antioxidant drugs into the mitochondria. 41 In this study, NPs were significantly co-localized with mitochondria in the treated cells, indicating that they promoted the entry of Cur into mitochondria 42 (Figure 2G). Figure 2 Cellular uptake and subcellular localization of NPs. (A) C6-NPs showed time and dose-dependent uptake by MDCK cells. Scale bar: 50 μm. (B) Fluorescence image of C6-NPs taken by MDCK cells endocytosis. Scale bar: 50 μm. (C) Quantitative statistics graph of MDCK cell fluorescence intensity at different time points and doses. (D) Quantitative statistical graphs of the fluorescence intensity of MDCK cells treated with different endocytosis uptake inhibitors. (E) The distribution of C6-NPs in cells stained with the designated organelle tracking dye. Scale bar: 25 μm. (F) Quantitative statistical chart of the Pearson value of the distribution of C6-NPs in cells stained with the designated organelle tracking dye. (G) Overview of the uptake process of C6-NP cells. ** means P <0.01.

Figure 2 Cellular uptake and subcellular localization of NPs. (A) C6-NPs showed time and dose-dependent uptake by MDCK cells. Scale bar: 50 μm. (B) Fluorescence image of C6-NPs taken by MDCK cells endocytosis. Scale bar: 50 μm. (C) Quantitative statistics graph of MDCK cell fluorescence intensity at different time points and doses. (D) Quantitative statistical graphs of the fluorescence intensity of MDCK cells treated with different endocytosis uptake inhibitors. (E) The distribution of C6-NPs in cells stained with the designated organelle tracking dye. Scale bar: 25 μm. (F) Quantitative statistical chart of the Pearson value of the distribution of C6-NPs in cells stained with the designated organelle tracking dye. (G) Overview of the uptake process of C6-NP cells. ** means P <0.01.

To explore the biodistribution pattern of NPs in the body after oral administration of these particles, zebrafish larvae were incubated in the presence of free C6 or C6-NPs for 15 or 60 minutes. During this treatment period, a significant increase in fluorescence signal over time was observed in the brain and intestines of zebrafish in the C6-NPs group, while the fluorescence in the free C6 group was negligible. This indicates that these NPs can cross the BBB and are therefore very suitable as a drug delivery method to enhance brain accumulation. These zebrafish also showed accumulation of C6-NPs in the eyes, indicating that these NPs can also cross the blood-retinal barrier. Based on these findings, we concluded that Cur-NPs can enhance the absorption of Cur and the accumulation of this drug in the brain (Figure 3A and B). In the pharmacokinetic analysis, the concentration of Cur-NPs in the plasma of C57BL/6 mice was significantly higher than that of mice given free Cur (Figure 4A). Due to the ideal size and surface characteristics of these particles, these results provide strong evidence for the ability of Cur-loaded NPs to enhance the systemic circulation of the drug. The Cur concentration in the brain of Cur-NPs-treated mice was significantly higher than that in the control animals, and reached a peak 8 hours after treatment, which is consistent with the continuous release of Cur from these NPs in the brain, thereby enhancing its accumulation in it (Figure 4B)) . Compared with control animals, the concentration of Cur-NPs in the heart, liver, spleen, lung and kidney tissue samples of the treated mice also increased, which is consistent with the sustained release of Cur-NP by Cur-NPs and its high plasma exposure rate (Figure 4C-G).43 Figure 3 Assessment of the biodistribution of C6-NPs in vivo. (A) Zebrafish larvae (7 dpf) were treated with C6-NPs (400 ng/mL) for 15 or 60 minutes. Scale bar: 100 μm. (B) Quantitative statistical graph of zebrafish fluorescence intensity. ** means P <0.01. Figure 4 Evaluated Cur-NPs in plasma (A), brain (B), heart (C), liver (D), spleen (E), lung (F), and kidney (G) in treated C57BL/6 The concentration of mice (mean ± SD, n = 4).

Figure 3 Assessment of the biodistribution of C6-NPs in vivo. (A) Zebrafish larvae (7 dpf) were treated with C6-NPs (400 ng/mL) for 15 or 60 minutes. Scale bar: 100 μm. (B) Quantitative statistical graph of zebrafish fluorescence intensity. ** means P <0.01.

Figure 4 Evaluated Cur-NPs in plasma (A), brain (B), heart (C), liver (D), spleen (E), lung (F), and kidney (G) in treated C57BL/6 The concentration of mice (mean ± SD, n = 4).

When the ICH model mice were evaluated by the rotating rod test, they showed a significantly shorter fall latency compared to control animals, which was significantly increased in Cur-NPs-treated mice. Cur-NPs also significantly reduced the number of falls in these ICH model mice (Figure 5A). Similarly, in the pole climbing test, ICH caused a significant increase in T-turn and T-total values, and Cur-NPs treatment was sufficient to reverse these changes (Figure 5B). Importantly, in terms of the observed behavioral improvements in mice, Cur-NPs achieved significantly better therapeutic effects than free Cur. In summary, these results indicate that Cur-NPs treatment can partially overcome the behavioral defects induced by ICH in mice. Figure 5 The effect of Cur-NPs on behavioral defects caused by ICH. (A) The drop latency and number of drops in the rotating rod test of mice in the designated group (mean ± SD, n = 12). (B) T turn and total T time of the pole test of mice in the specified group (mean ± SD, n = 12). * Means P <0.05 and ** means P <0.01.

Figure 5 The effect of Cur-NPs on behavioral defects caused by ICH. (A) The drop latency and number of drops in the rotating rod test of mice in the designated group (mean ± SD, n = 12). (B) T turn and total T time of the pole test of mice in the specified group (mean ± SD, n = 12). * Means P <0.05 and ** means P <0.01.

Compared with the ICH and Cur treatment groups, Cur-NPs treatment was associated with a significant reduction in hematoma volume (Figure 6A and B). H&E staining further showed that ICH is associated with significant neuron loss around the hematoma, and Cur-NPs treatment is sufficient to prevent this neuron damage (Figure 6C). Fig. 6 Cur-NPs treatment is related to the reduction of hematoma volume in the mouse brain. (A) Hematoma image in brain tissue section of ICH model mouse. Scale bar: 10 mm. (B) Hematoma volume percentage of ICH model mice in the designated group (mean ± SD, n = 6). (C) H&E stained brain tissue sections from the specified group. ** means P <0.01.

Fig. 6 Cur-NPs treatment is related to the reduction of hematoma volume in the mouse brain. (A) Hematoma image in brain tissue section of ICH model mouse. Scale bar: 10 mm. (B) Hematoma volume percentage of ICH model mice in the designated group (mean ± SD, n = 6). (C) H&E stained brain tissue sections from the specified group. ** means P <0.01.

ICH model mice showed obvious neuronal degeneration around the hematoma in Nissl-stained brain tissue sections, while in mice treated with Cur-NPs, this neuronal damage was significantly weaker than in Cur-treated mice Observed in is bigger. The accumulation of iron in the cells around the hematoma of these mice was evaluated by Prussian blue staining, which indicated that Cur-NPs and Prussian blue-positive cell frequency were significantly reduced compared with the ICH model group and Cur. The treatment group was related to these Cur-NPs. The ability to inhibit iron deposition in the brain tissue around the hematoma is the same. GPX4 is a glutathione peroxidase that acts by detoxifying lipid peroxides,44 and due to its ability to inhibit intracellular lipid peroxidation, it can serve as a key iron death marker. 45,46 We found that the administration of Cur-NPs was associated with a corresponding increase in the expression of GPX4 around the hematoma in ICH model mice (Figure 7A), thus indicating that Cur-NPs treatment can significantly reduce the induction of iron death in this pathological background. Erastin induces iron death by inhibiting the NRF2/HO-1 pathway. In addition, we studied the effect of Cur-NPs on the NRF2/HO-1 pathway, and the results showed that Cur-NPs significantly regulated the expression of HMOX1 (HO-1) and NFE2L2 (NRF2) in ICH model mice (Figure 7B and C). These findings strongly suggest that Cur-NPs treatment may increase the expression of GPX4 by regulating the NRF2/HO-1 pathway. Figure 7 (A) Representative photomicrographs of mouse brain slices that have received the specified treatment and staining protocol. Scale bar: 250 μm. (BC) Relative mRNA expression of HMOX1 and NFE2L2 in tissues around hematomas in different groups of ICH mice. ** means P <0.01.

Figure 7 (A) Representative photomicrographs of mouse brain slices that have received the specified treatment and staining protocol. Scale bar: 250 μm. (BC) Relative mRNA expression of HMOX1 and NFE2L2 in tissues around hematomas in different groups of ICH mice. ** means P <0.01.

In the MTT analysis, Cur-NPs at a dose of 20 μM or lower did not cause any significant toxicity when used to treat cells (Figure 8A). Then, we used these Cur-NPs together with the known iron death inducer erastin to explore the anti-iron death activity of these Cur-NPs. 47 In the subsequent MTT analysis, we found that Cur-NPs can effectively increase the survival rate of HT22 cells relative to erastin and the Cur-PM treatment group (Figure 8B). This improved survival was further confirmed by live/dead staining analysis. Rate (Figure 8C). In addition, the results of Annexin V/PI staining showed that Cur-NPs significantly reduced the apoptosis induced by enatine in a dose-dependent manner (Figure 8D). In addition, we studied the effects of Cur-NPs on the NRF2/HO-1 pathway and ROS generation. The results showed that the administration of Cur-NPs significantly inhibited the generation of ROS induced by erastin (Figure 9A-D). Importantly, Cur-NPs effectively regulates the expression levels of HMOX1 and NFE2L2, which suggests that it may inhibit the production of ROS by regulating the NRF2/HO-1 pathway (Figure 9E and F). Figure 8 Erastin-induced HT22 cell viability after Cur-NP and Cur-PM treatment. (A) MTT assay is used to evaluate the viability of cells treated with different Cur-NP concentrations. (B) Cells subjected to the designated treatment are used for MTT detection. (C) Live/dead staining is performed on the cells that have received the specified treatment. (D) Flow cytometer dot plot of Annexin V-FITC/PI double staining for apoptosis detection. (Cur-PM, a physical mixture of PVP K90 and Cur). ** means P <0.01. Figure 9 (A) The image of DCF staining in response to the specified treatment. (B) Statistical analysis of the response of DCF fluorescence intensity to the specified treatment. (C) DHE staining responds to the specified processed image. (D) Statistical analysis of DHE fluorescence intensity in response to the specified treatment. (E, F) The relative mRNA expression of HMOX1 and NFE2L2 in cells undergoing the specified treatment. ** means P <0.01.

Figure 8 Erastin-induced HT22 cell viability after Cur-NP and Cur-PM treatment. (A) MTT assay is used to evaluate the viability of cells treated with different Cur-NP concentrations. (B) Cells subjected to the designated treatment are used for MTT detection. (C) Live/dead staining is performed on the cells that have received the specified treatment. (D) Flow cytometer dot plot of Annexin V-FITC/PI double staining for apoptosis detection. (Cur-PM, a physical mixture of PVP K90 and Cur). ** means P <0.01.

Figure 9 (A) The image of DCF staining in response to the specified treatment. (B) Statistical analysis of the response of DCF fluorescence intensity to the specified treatment. (C) DHE staining responds to the specified processed image. (D) Statistical analysis of DHE fluorescence intensity in response to the specified treatment. (E, F) The relative mRNA expression of HMOX1 and NFE2L2 in cells undergoing the specified treatment. ** means P <0.01.

In this study, an anti-solvent precipitation method was used to prepare Cur-NPs, which are easily absorbed by cells through a variety of non-specific endocytosis mechanisms, among which plasma membrane microcapsules and clathrin-mediated absorption are the main internalization methods . These Cur-NPs mainly accumulate in the ER, lysosomes and mitochondria, and are effectively transported across the physiological barrier to enhance the accumulation of Cur in the plasma and brain. What is more noteworthy is that these Cur-NPs have the ability to inhibit iron death and can be used as an effective treatment for ICH. In short, Cur-NPs represent a promising tool that can enhance the delivery of Cur to the brain to achieve excellent therapeutic effects in the treatment of ICH.

All authors have made significant contributions to the work of the report, whether in terms of concept, research design, execution, data acquisition, analysis, and interpretation, or in all these areas; participating in drafting, revising, or critically reviewing articles; final approval requirements Published version; agreed on the journal to which the article was submitted; and agreed to be responsible for all aspects of the work.

This work was funded by the Guangdong Provincial University Key Laboratory Project (2019KSYS005), the Guangdong Provincial Science and Technology Plan International Cooperation Project (2020A0505100052), the Guangdong Basic and Applied Basic Research Fund (2019B1515120043), and the Fundamental Education Key Project Funding Shenzhen Research (JCYJ20200109113603854), Guangdong Provincial Natural Science Foundation (2018A030310623).

The author declares that there is no potential conflict of interest in this work.

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