Iron-tea polyphenol coordination polymer nanoparticles, and preparation method and application thereof

Abstract

This invention belongs to the field of pharmaceutical technology and relates to an iron-tea polyphenol coordination polymer nanoparticle, its preparation method, and its application. The application of nanotechnology in the medical field is rapidly developing. Nanomaterials, as potential "smart" drugs, can enhance therapeutic effects through targeted drug release. Although research on nanomaterials is promising, the challenge of ineffective drug treatment in the brain, especially in the treatment of radiation-induced brain injury, still needs to be overcome. Therefore, developing novel nanomaterials, particularly those with neuroprotective effects, and further elucidating their neuroprotective effects, will provide new possibilities for the treatment of radiation-induced brain injury. This invention provides a novel iron-tea polyphenol (Fe-TP) coordinated nanomaterial that utilizes the coordination properties of Fe-TP to mitigate adverse reactions caused by radiation damage, thereby addressing the serious health problems caused by radiation-induced brain injury.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a radiotherapy-related preparation, specifically an iron-tea polyphenol (Fe-TP) coordination polymer nanoparticle and its preparation method and application, which can effectively alleviate radiation-induced brain injury. Background Technology

[0002] Radiation therapy is widely used to treat head and neck tumors, primary brain tumors, brain metastases, and arteriovenous malformations. Radiation-induced brain injury (RIBI) is a common complication following cranial radiotherapy. Mild cases may be asymptomatic, while severe cases can cause epilepsy, cognitive and memory impairment, mental abnormalities, and even death. This injury not only causes cell death and neurological dysfunction but can also lead to long-term cognitive and motor impairment, severely impacting patients' quality of life and increasing the burden on families and society. Therefore, radiation-induced brain injury is considered a pressing public health issue that needs to be addressed.

[0003] With the extension of overall survival time for cancer patients and the use of advanced imaging diagnostic and examination methods, the overall diagnosis rate and incidence of radiation-induced brain injury in my country have been increasing year by year. Currently, treatment options for radiation-induced brain injury are limited, mainly relying on conservative treatment based on glucocorticoids and anticoagulants, as well as surgical treatment. In recent years, with advancements in new drug development, novel treatment methods such as anti-VEGF drugs, nerve growth factor (NGF), and hyperbaric oxygen therapy have been increasingly applied to the treatment of radiation-induced brain injury, but the overall treatment effect is not ideal. The Radiation Therapy Oncology Group (RTAO) has confirmed the role of corticosteroids in improving brain injury in radiotherapy patients, but the effect is not ideal for patients with normal baseline neurological function. Donepezil and memantine can improve cognitive impairment and slow disease progression in Alzheimer's disease patients. Furthermore, based on the shared mechanisms between radiation-induced brain injury and neurodegenerative diseases, donepezil and memantine have also been shown to improve cognitive function in patients with radiation-induced brain injury, but they have drawbacks such as poor efficacy and inability to halt the progression of brain injury. In summary, existing drugs show no significant difference in overall efficacy for patients with initially normal neurological function and cannot halt the progression of radiation-induced brain injury. In recent years, anti-angiogenic targeted drugs (bevacizumab, apatinib, etc.) have also been widely used in the treatment of radiation-induced brain injury; however, the current level of evidence is low, the efficacy is unclear, and there are risks such as thrombosis and hemorrhage. Furthermore, recent research has begun to explore the use of nanomedicines for the treatment of radiation-induced brain injury, but clinical application still faces challenges such as uneven release rates and potential toxicity. Therefore, there is an urgent need to develop and explore novel, low-toxicity, and highly effective drugs for the clinical treatment of radiation-induced brain injury. Summary of the Invention

[0004] The application of nanotechnology in the medical field is rapidly developing. Nanomaterials, as potential "smart" drugs, can enhance therapeutic effects through targeted drug release. While research on nanomaterials is promising, the challenge of ineffective drug treatments in the brain, especially for radiation-induced brain injury, remains to be overcome. Therefore, developing novel nanomaterials, particularly those with neuroprotective effects, and further elucidating their neuroprotective role, will provide new possibilities for the treatment of radiation-induced brain injury. This invention provides a novel iron-tea polyphenol (Fe-TP) coordinated nanomaterial that utilizes the properties of Fe-TP coordination to mitigate adverse reactions caused by radiation damage, thereby addressing the serious health problems caused by radiation-induced brain injury.

[0005] The present invention adopts the following technical solution.

[0006] An iron-tea polyphenol coordination polymer nanoparticle comprises tea polyphenols and iron ions coordinated with the tea polyphenols.

[0007] This invention discloses a method for preparing the above-mentioned iron-tea polyphenol coordination polymer nanoparticles, comprising the following steps: mixing tea polyphenols, iron salts, and surfactants in a solvent to obtain iron-tea polyphenol coordination polymer nanoparticles.

[0008] In this invention, the iron salt includes inorganic iron salts, such as ferric chloride; the surfactant includes polyvinylpyrrolidone (PVP), which provides a dispersing effect on the iron-tea polyphenol coordination polymer nanoparticles.

[0009] In this invention, the mass ratio of tea polyphenols, iron salts, and surfactants is (5-15):(10-30):(40-60); for example, the mass ratio of tea polyphenols, iron salts, and surfactants is 10:20:50.

[0010] This invention discloses the application of the above-mentioned iron-tea polyphenol coordination polymer nanoparticles in the preparation of neuroprotective drugs.

[0011] This invention discloses the application of the above-mentioned iron-tea polyphenol coordination polymer nanoparticles in the preparation of neuroprotective drugs.

[0012] This invention discloses the application of the above-mentioned iron-tea polyphenol coordination polymer nanoparticles in the preparation of drugs that reduce radiation-induced neurotoxicity.

[0013] This invention discloses the application of the above-mentioned iron-tea polyphenol coordination polymer nanoparticles in the preparation of drugs to alleviate radiation-induced brain injury.

[0014] This invention discloses the application of the aforementioned iron-tea polyphenol coordination polymer nanoparticles in the preparation of drugs to alleviate brain damage caused by brain treatments. Brain treatments include radiotherapy, drug therapy, etc.

[0015] In this invention, radiation refers to radiotherapy, such as the radiotherapy conventionally used in the treatment of head and neck tumors, primary brain tumors, brain metastases, and arteriovenous malformations.

[0016] This invention provides neuroprotective effects through iron-tea polyphenol coordination polymer nanoparticles. By protecting nerve cells, it reduces radiation-induced neurotoxicity and promotes cell survival and functional recovery. As a drug delivery system, these nanoparticles effectively penetrate the blood-brain barrier, improving drug targeting and biocompatibility, thus achieving better therapeutic effects. This invention promotes new treatment strategies for radiation-induced brain injury, providing a new treatment option for patients, reducing treatment costs, and improving their quality of life. By achieving the above objectives, this invention provides an effective solution for the clinical treatment of radiation-induced brain injury and offers new insights for research in related fields. Attached Figure Description

[0017] Figure 1 (a) Transmission electron microscope image; (b) Optical photograph; (c) Ultraviolet-visible spectrum; (d) Infrared spectrum; (e, f) Raman spectrum.

[0018] Figure 2 It is Fe-TP cytotoxic.

[0019] Figure 3 For the hemolysis test of Fe-TP, (a) optical photographs of hemolysis tests with different concentrations of Fe-TP; (b) hemolysis rate of different concentrations of Fe-TP; (c) morphology of red blood cells under different concentrations of Fe-TP as shown by scanning electron microscopy.

[0020] Figure 4 The toxicity of Fe-TP and TP to sh-sy5y cells (a) and BV2 cells (b) is compared.

[0021] Figure 5 To further confirm that Fe-TP is less toxic than TP using the LIVE / DEAD staining method (PVP and TP concentrations were both 500 μg / mL, SH-SY5Y cells, red: dead cells stained red, green: live cells).

[0022] Figure 6 Under 10 Gy radiation conditions, Fe-TP showed a better ability than TP to alleviate sh-sy5y cell death (red: dead cells stained red, green: live cells).

[0023] Figure 7 Fe-TP was used to alleviate BV2 cell death induced by a high dose of 12 Gy irradiation (red: dead cells stained red, green: live cells).

[0024] Figure 8 Fe-TP was used to alleviate the decrease in mitochondrial membrane potential in BV2 cells induced by 12 Gy irradiation (blue: cell nucleus; red: JC-1 aggregates; green: JC-1 monomers).

[0025] Figure 9 Fe-TP reduces DNA damage in BV2 cells induced by irradiation (red: γH2Ax positive signal, blue: DAPI nuclear staining). Detailed Implementation

[0026] Radiotherapy is widely used to treat head and neck tumors, primary brain tumors, brain metastases, and arteriovenous malformations. Radiation-induced brain injury is a common complication after cranial radiotherapy. This injury not only causes cell death and neurological dysfunction but may also lead to long-term cognitive and motor impairment, severely impacting patients' quality of life and increasing the burden on families and society. Therefore, radiation-induced brain injury is considered a public health problem urgently needing to be addressed. Currently, the pathogenesis and related molecular basis of radiation-induced brain injury are not fully understood, but it is generally believed to be the result of multiple mechanisms. With the development of nanoscience and technology, carbon-based nanomaterials, with their excellent physicochemical properties, have shown great application potential in the field of nanomedicine, demonstrating potential medical value in various neurodegenerative diseases; however, obtaining nanomaterials with good water solubility, good biocompatibility, and the ability to effectively alleviate neurotoxicity at the cellular and animal levels remains a major challenge in this field. This invention discloses an iron-tea polyphenol coordination polymer nanoparticle, its preparation method, and its application. This nanomedicine exhibits excellent neuroprotective properties and shows potential therapeutic effects for radiation-induced brain injury.

[0027] The following specific experiments illustrate the technological advancements of this invention. The raw materials used are existing products, the specific preparation operations and performance tests are conventional techniques, and the data statistics are based on conventional analytical methods. Tea polyphenols (TP) were purchased from Shanghai Yuanye Biotechnology Co., Ltd., in aqueous solution form, with a concentration of 10 mg / ml.

[0028] Example 1: Preparation of Fe-Tea Polyphenol Nanoparticles (Fe-TP) 20 mg FeCl3 was dispersed in 1 ml of ethanol. The FeCl3 ethanol solution was then added to 5 ml of ethanol containing 50 mg polyvinylpyrrolidone (PVP). Next, 1 ml of tea polyphenol aqueous solution was added, and the mixture was stirred for 30 min. After centrifugation, Fe-TP nanoparticles were obtained and dispersed in the solution. During the reaction, the solution changed from yellow to dark black, indicating that Fe... 3+ It successfully coordinated with the phenolic hydroxyl groups of tea polyphenols.

[0029] Figure 1 Characterization of Fe-TP nanopolymers: a) transmission electron microscopy (TEM) image, b) optical photograph, c) UV-Vis spectrum, d) infrared spectrum, e) and f) Raman spectra. The morphology of Fe-TP was characterized by transmission electron microscopy (TEM). Fe-TP consists of ultra-small, uniform nanoparticles with an average particle size of 18 nm. The UV-Vis adsorption wavelength of Fe-TP nanoparticles is around 229 nm. Infrared spectroscopy shows that Fe-tea polyphenols retain the basic structure of tea polyphenols. The phenolic hydroxyl group (-OH) in the tea polyphenol exhibits a broad absorption peak between 3380 cm⁻¹. Compared to pure tea polyphenols, the intensity of this peak in Fe-tea polyphenols is weakened and slightly shifted forward, indicating that the hydroxyl group participates in the coordination reaction with iron ions. The absorption peak at 1633.2 cm⁻¹ is attributed to the stretching vibration of the aromatic ring C=C, suggesting the influence of iron ions on the aromatic ring. The intensity change of the CO stretching vibration peak at 1049.0 cm⁻¹ indicates that the oxygen atom of the phenolic hydroxyl group binds to iron ions through coordination. Raman spectroscopy suggests a coordination interaction between Fe and tea polyphenols. This indicates that iron ions were successfully introduced into the material and formed a stable organometallic complex with tea polyphenols.

[0030] Example 2 BV2 cells (mouse microglia), sh-sy5y cells (human neuroblastoma cell line), and raw264.7 cells (mouse mononuclear macrophage leukemia cells) in logarithmic growth phase were selected, and the cytotoxicity of Fe-TP was detected by the CCK-8 cell counting kit.

[0031] Experimental procedure: Fe-TP nanoparticles at different concentrations were prepared using PBS: 100, 200, 400, and 800 μg / mL.

[0032] BV2, SH-SY5Y, and RAW264.7 were used at 5×10⁻⁶ holes per well. 3Cells were seeded at a density in 96-well plates. After overnight cell adhesion, the old culture medium was discarded, and fresh culture medium containing different concentrations of Fe-TP (100, 200, 400, and 800 μg / mL) was added to the experimental groups for 24 hours. Groups were set up as follows: blank control (wells with only culture medium, no cells, used to correct background absorbance), control group (wells with only cells and complete culture medium), and experimental group (wells with cells and culture medium containing different concentrations of the test drug), with 5 replicates per group. One-tenth of the original culture medium volume of each well was added directly to the plate, and the 96-well plate was returned to a 37°C incubator for 1 to 4 hours in the dark. Using a microplate reader, CCK-8 was used to detect the cells at 450 nm, and the relative cell viability was calculated.

[0033] See Figure 2 Data show that Fe-TP has very low cytotoxicity; even at concentrations as high as 800 μg / mL, the cell survival rate of the three cell types is over 70%.

[0034] To further investigate the biocompatibility of Fe-TP, a hemolysis test was conducted to assess its blood compatibility. The hemolysis test measures the degree of erythrocyte lysis and hemoglobin release caused by in vitro contact between the material / drug and erythrocytes. Materials / drugs with good blood compatibility exhibit a low hemolysis rate. The blood compatibility test used fresh blood from healthy adult mice and was conducted according to the testing requirements of ISO 10993-4:2002. According to ISO evaluation standards: a hemolysis rate of less than or equal to 5% indicates suitability for use as a medical material; a hemolysis rate greater than 5% suggests that the material may cause damage to blood cell membranes and lead to a hemolytic reaction in vivo.

[0035] See Figure 3 The experimental results showed that the hemolysis rate was 2.8% when the Fe-TP concentration was as high as 800 μg / mL, and all experimental concentrations were below 5%. Therefore, it can be considered that Fe-TP has no hemolytic effect and has good blood compatibility.

[0036] See Figure 4 To further evaluate the cytotoxicity of Fe-TP, BV2 cells and sh-sy5y cells were selected, and the cytotoxicity of Fe-TP and tea polyphenols (TP) was compared by CCK-8 assay. The data showed that Fe-TP was less toxic than TP in BV2 cells and sh-sy5y cells (**, P < 0.01).

[0037] Example 3 To further evaluate the cytotoxicity of Fe-TP, the cytotoxicity of Fe-TP and TP was compared under radiation-free and radiation-irradiated conditions.

[0038] Live / dead staining experiments showed that, without radiation, the TP intervention group induced BV2 cell death, and a large number of dead cells stained red were observed in the field of view of this group. Figure 5 However, the number of dead cells in the Fe-TP intervention group was significantly reduced, and a large number of surviving cells (green) were present in the field of view. Figure 5 This indicates that Fe-TP has lower cytotoxicity than TP.

[0039] Under radiation irradiation conditions (10 Gy), the number of dead cells in the TP intervention group was greater than that in the Fe-TP intervention group, and a large number of dead cells (red) were visible in the field of view. Figure 6 This indicates that Fe-TP effectively reduces radiation-induced cell death compared to TP.

[0040] To evaluate the protective effect of Fe-TP against radiation-induced cell death, the live / dead cell viability was determined using a live / dead cell double staining kit (Calcein-AM / PI). BV2 cells were seeded in 6-well plates and treated with 400 μg / mL Fe-TP, followed by X-ray (12 Gy) radiation damage modeling at a dose rate of 3 Gy / min and an irradiation field of 50 × 50 cm. Twenty-four hours after modeling, Calcein AM / PI detection working solution was prepared according to the manufacturer's instructions. 1 ml of Calcein AM / PI detection working solution was added to each well for staining, and the cells were incubated at 37°C in the dark for 30 min. After incubation, the stained cells were imaged using a fluorescence microscope (excitation / emission: calcein AM 488 / 515 nm; PI 535 / 617 nm).

[0041] Irradiation induces abnormal cell death. See also Figure 7 Live / dead staining experiments showed that a 12 Gy irradiation dose induced BV2 cell death, with a large number of dead cells stained red in the field of view. The Fe-TP intervention group showed a significant reduction in the number of dead cells, and a large number of surviving cells (green) were present in the field of view, indicating that Fe-TP effectively reduced irradiation-induced cell death.

[0042] Mitochondrial membrane potential assay: Cell suspension was seeded in 35 mm confocal dishes. When cell confluence reached approximately 70%, a complete culture medium containing 400 μg / mL Fe-TP nanomaterials was prepared. Experimental groups included a control group, an irradiation group (12 Gy), and an intervention group (12 Gy + 400 μg / mL Fe-TP). Six hours after cell irradiation, the mitochondrial membrane potential of BV2 cells was assessed using a JC-1 staining kit (Beyotime). Following the manufacturer's instructions, JC-1 stock solution was diluted with JC-1 staining buffer, and 1 ml of JC-1 working solution was added to each well. The cells were incubated at 37°C in a 5% CO2 incubator for 25 minutes in the dark. After incubation, the JC-1 working solution was carefully aspirated. Cells were washed twice with pre-cooled 1×JC-1 staining buffer and examined using a fluorescence microscope. JC-1 aggregates in healthy mitochondria fluoresce red, while JC-1 monomers in depolarized mitochondria fluoresce green; the nucleus was counterstained with Hoechst 33342 (blue); the ratio of red to green fluorescence was used to quantify mitochondrial membrane potential.

[0043] A decrease in mitochondrial membrane potential is a hallmark event in the early stages of apoptosis. Further fluorescence imaging experiments using a mitochondrial membrane potential detection kit (JC-1) were conducted to verify the reduction of Fe-TP-induced irradiation-induced cell death. At higher mitochondrial membrane potentials, JC-1 aggregates in the mitochondrial matrix, forming J-aggregates that produce red fluorescence; at lower mitochondrial membrane potentials, JC-1 does not aggregate in the mitochondrial matrix and remains a monomer, producing green fluorescence. See also... Figure 8 It can be observed that Fe-TP intervention can significantly reduce the decrease in mitochondrial membrane potential caused by 12Gy irradiation. This trend is consistent with the trend in the live / dead staining experiment, which together indicate that Fe-TP alleviates radiation-induced cell damage and death.

[0044] Example 4 BV2 microglia were seeded onto glass coverslips in 24-well plates and divided into a control group, an irradiation group, and a Fe-TP treatment group (400 μg / mL). After pretreatment, cells were exposed to 12 Gy X-rays and incubated for 24 hours before staining. Cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature, followed by washing three times with PBS. Cells were permeabilized with 0.3% Triton X-100 for 10 minutes and blocked with 5% bovine serum albumin (BSA) for 1 hour at room temperature. Primary antibody against γH2Ax was added, and the cells were incubated overnight at 4°C. The following day, cells were recovered, washed three times with PBS, and then incubated with a fluorophore-conjugated secondary antibody (FITC or Cy3) in the dark at room temperature for 1 hour. Cell nuclei were counterstained with DAPI for 5 minutes. After washing three times with PBS, coverslips were mounted using anti-quenching mounting medium, and images were captured using a fluorescence microscope.

[0045] Microglia are intrinsic immune cells in the brain, playing a crucial role in the pathophysiology of neuroinflammation induced by various central nervous system diseases. In radiation-induced brain injury (RIBI), the functional polarity of microglia undergoes dynamic shifts, which play a key role in the pathogenesis of RIBI. RIBI has become a significant factor affecting the prognosis of patients with head and neck tumors. With the delivery of high-dose radiation to brain tissue, microglia upregulate phagocytic mechanisms and reduce the release of neurotrophic factors, inhibiting cranial neurogenesis and leading to late-stage neurocognitive impairment in RIBI. Based on experimental results showing that Fe-TP initially alleviates BV2 cell death induced by high-dose radiation (12 Gy), this study further investigated whether Fe-TP can regulate microglia activity to inhibit its damaging effects, thereby improving post-radiation brain injury. (See also...) Figure 9 The experimental results showed that irradiation can cause DNA damage in BV2 cells, manifested as increased expression of γH2Ax, while Fe-TP intervention can significantly reduce radioactive DNA damage.

[0046] With the development of nanoscience and technology, carbon-based nanomaterials have shown great promise in the field of nanomedicine due to their excellent physicochemical properties, demonstrating potential medical value in various neurodegenerative diseases. However, obtaining nanomaterials with good water solubility, good biocompatibility, and the ability to effectively alleviate neurotoxicity at the cellular and animal levels remains a major challenge in this field. In particular, as is common knowledge, iron nanoparticles (Fe NPs) belong to a completely different material system, possess strong Fenton reactivity, and can introduce additional toxicity, namely, free Fe... 3+Fe nanoparticles may easily trigger the Fenton reaction, generating highly reactive free radicals such as •OH, leading to enhanced oxidative stress and hindering the repair of radiation-induced brain injury. This invention discloses an iron-tea polyphenol coordination polymer nanoparticle, its preparation method, and its application. This nanomedicine exhibits excellent neuroprotective properties, showing potential therapeutic effects for radiation-induced brain injury. In particular, tea polyphenols themselves possess antioxidant activity. This invention compares Fe-TP with TP, clearly demonstrating that Fe... 3+ The enhanced antioxidant capacity and biocompatibility after coordination.

Claims

1. An iron-tea polyphenol coordination polymer nanoparticle, comprising tea polyphenols and iron ions coordinated with the tea polyphenols.

2. The method for preparing the iron-tea polyphenol coordination polymer nanoparticle of claim 1, characterized in that, The process includes the following steps: mixing tea polyphenols, iron salts, and surfactants in a solvent to obtain iron-tea polyphenol coordination polymer nanoparticles.

3. The method for preparing iron-tea polyphenol coordination polymer nanoparticles according to claim 2, characterized in that, Iron salts include inorganic iron salts; surfactants include polyvinylpyrrolidone.

4. The method for preparing iron-tea polyphenol coordination polymer nanoparticles according to claim 2, characterized in that, The mass ratio of tea polyphenols, iron salts, and surfactants is (5-15):(10-30):(40-60).

5. The application of the iron-tea polyphenol coordination polymer nanoparticles according to claim 1 in the preparation of neuroprotective drugs.

6. The use of the iron-tea polyphenol coordination polymer nanoparticles according to claim 1 in the preparation of neuroprotective drugs.

7. The use of the iron-tea polyphenol coordination polymer nanoparticles according to claim 1 in the preparation of a drug to reduce radiation-induced neurotoxicity.

8. The use of the iron-tea polyphenol coordination polymer nanoparticles according to claim 1 in the preparation of a drug for alleviating radiation-induced brain injury.

9. The use of the iron-tea polyphenol coordination polymer nanoparticles according to claim 1 in the preparation of a medicament for reducing brain damage caused by brain treatment.

10. A drug for alleviating radiation-induced brain injury, characterized in that, The active ingredient of the drug for reducing radiation-induced brain injury includes the iron-tea polyphenol coordination polymer nanoparticles as described in claim 1.

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