Alanine-modified fullerene, its preparation method, pharmaceutical composition and application

Abstract

This invention relates to an alanine-modified fullerene, its preparation method, pharmaceutical composition, and applications, belonging to the field of biomedical technology. It addresses the problems in existing treatments for type 1 diabetes, namely, the difficulty in safely and effectively protecting pancreatic β-cells while blocking autoimmune attacks, and the lack of pancreatic tissue-specific targeting ability in existing fullerene derivatives. The alanine-modified fullerene has the general formula shown in Formula I: C 70 - (Ala) n Equation I, where C 70 For fullerenes, Ala is an alanine group, and n is the number of atoms linked to each fullerene C. 70 The average number of alanine groups is taken as an integer from 1 to 20. The alanine-modified fullerene of the present invention has the targeting ability to specifically accumulate in pancreatic islet tissue in vivo, as well as the immunomodulatory function of inhibiting dendritic cell antigen presentation and regulating T cell differentiation, which can achieve efficient intervention on the core pathological links of type 1 diabetes.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an alanine-modified fullerene, its preparation method, pharmaceutical composition, and application. Background Technology

[0002] Type 1 diabetes mellitus (T1DM) is an autoimmune disease driven by a combination of genetic susceptibility and environmental factors. Its core pathological feature is the progressive destruction of pancreatic β-cells and the resulting glucose metabolism disorder. The pathological process of this disease exhibits a clear multi-dimensional cascade reaction: in genetically susceptible individuals, environmental factors such as viral infections can directly or indirectly break the immune tolerance barrier, inducing a large infiltration of autoreactive T cells into pancreatic islet tissue and the formation of "insulitis" lesions. These pathogenic T cells directly attack β-cells by releasing cytotoxic substances such as perforin and granzymes, while simultaneously secreting pro-inflammatory factors such as interferon-gamma (IFN-γ) and tumor necrosis factor-α (TNF-α) to mediate β-cell apoptosis. In addition to cellular immune-mediated damage, humoral immune abnormalities further amplify the damaging effect on β-cells—autoantibodies such as islet cell antibodies (ICA) and insulin autoantibodies (IAA) can activate the complement system to form immune complexes, continuously targeting and damaging β-cells.

[0003] Currently, the clinical treatment of type 1 diabetes mellitus (T1DM) still relies primarily on insulin replacement therapy. While this strategy can temporarily control hyperglycemia symptoms, it cannot block the core pathological processes of islet inflammation progression, irreversible β-cell damage, and autoimmune disorders. Exogenous insulin cannot accurately mimic the physiological pulsatile secretion rhythm, easily leading to adverse reactions such as hypoglycemia or drastic blood glucose fluctuations. Islet transplantation, which can restore physiological insulin secretion function, is limited by donor shortages and post-transplant immune rejection, hindering its widespread adoption. Although immune intervention strategies targeting the core pathological processes of T1DM have been explored with different targets, such as anti-CD3 antibodies reducing immune damage by inhibiting T-cell activation, cyclosporine A reducing pro-inflammatory cytokine secretion by blocking the calcineurin-NFAT pathway, and anti-IL-1β antibodies directly antagonizing inflammatory damage, these approaches all have significant limitations. Systemic immunosuppression easily increases the risk of infection, single-target intervention is difficult to block complex immune regulatory networks, and the lack of islet tissue targeting leads to low treatment efficiency. These problems collectively limit clinical translation and application.

[0004] Fullerenes, as important allotropes of carbon, are spherical cage-like molecular clusters composed of an even number of carbon atoms, whose molecular structure consistently contains 12 five-membered rings and several six-membered rings. Due to the inherent defect of poor water solubility in pure fullerenes, chemical derivatization modification can significantly improve their biocompatibility and aqueous dispersibility. Functionalized fullerene derivatives, while retaining the core activity of the original molecule, can also achieve functional optimization such as targeted enhancement and bioactivity regulation by introducing specific functional groups, providing crucial support for their application in the biomedical field. Due to their unique cage-like structure, excellent free radical scavenging ability, and functionalizability, they show broad application prospects in the biomedical field, such as in antioxidation, neuroprotection, antitumor, and anti-inflammation. Previous studies have reported that water-soluble fullerenes or metallofullerene derivatives can improve insulin resistance and protect pancreatic islet function in animal models of type 2 diabetes mellitus (T2DM) by reducing oxidative stress and inflammatory responses.

[0005] However, the vast majority of existing research focuses on type 2 diabetes models centered on insulin resistance and metabolic disorders. The mechanisms of action of these materials are primarily based on their broad-spectrum antioxidant and anti-inflammatory properties, aiming to improve the function of metabolic organs (such as the liver and muscles) and compensatory damage to the pancreatic islets. Regarding the core immunopathological link specific to type 1 diabetes—the destruction of pancreatic β cells driven by autoreactive T cells—whether fullerene derivatives possess the ability to intervene, and whether they can achieve precise targeting and immunomodulation of the lesion site (the pancreatic islets), systematic research is currently lacking.

[0006] The current treatment of type 1 diabetes mellitus (T1DM) faces two major challenges: First, existing therapies are unable to safely and effectively protect β cells while blocking autoimmune attacks; second, although nanomaterials (such as fullerene derivatives) have been used in diabetes research, their application is limited to the T2DM model, lacks specificity for the immunopathology of T1DM, and generally does not have pancreatic tissue-specific targeting capabilities.

[0007] Therefore, there is an urgent need to develop a novel nanotherapy agent that can overcome the limitations of existing technologies, specifically target the pancreas and islets, precisely regulate local autoimmune responses, and effectively protect β-cell function, providing a completely new solution for the intervention of type 1 diabetes. Summary of the Invention

[0008] In view of the above analysis, the present invention aims to provide an alanine-modified fullerene and its preparation method, pharmaceutical composition and application, in order to solve one of the following technical problems: (1) Existing treatments for type 1 diabetes (insulin replacement therapy) are difficult to safely and effectively protect β cells while blocking autoimmune attacks; (2) Existing modified fullerenes lack specificity for T1DM immunopathology and generally do not have pancreatic tissue-specific targeting ability.

[0009] A first aspect of the present invention provides an alanine-modified fullerene having the general formula shown in Formula I: C 70 -(Ala)n formula I Among them, C 70 For fullerenes, Ala is an alanine group, and n is the number of atoms linked to each fullerene C. 70 The average number of alanine groups on the protein is an integer between 1 and 20.

[0010] Furthermore, n takes the integer range 4-12.

[0011] Furthermore, the alanine-modified fullerene has an average hydrodynamic diameter of 10-100 nm.

[0012] Furthermore, the polydispersity index of the alanine-modified fullerene is 0.259-0.265; Furthermore, the alanine-modified fullerene has a Zeta potential of -44.39 to -47.01 mV.

[0013] A second aspect of the present invention provides a method for preparing the alanine-modified fullerene described in the first aspect, comprising: in an alkaline solution, reacting C... 70 The product was reacted with β-alanine and purified to obtain the alanine-modified fullerene.

[0014] Furthermore, the reaction conditions include: a reaction temperature of 75-80℃ and a reaction time of 8-24h.

[0015] A third aspect of the present invention provides a pharmaceutical composition comprising the alanine-modified fullerene described in the first aspect, and at least one pharmaceutically acceptable carrier.

[0016] Furthermore, the dosage form of the pharmaceutical composition is a targeted formulation.

[0017] Furthermore, the content of the alanine-modified fullerene in each milliliter of the pharmaceutical composition is 12.5 μg-10 mg.

[0018] The fourth aspect of the present invention provides the use of the pharmaceutical composition described in the second aspect in the preparation of a medicament for treating or preventing type 1 diabetes.

[0019] Furthermore, the drug is administered via intraperitoneal injection.

[0020] This invention can achieve at least one of the following beneficial effects: 1. The alanine-modified fullerene (C) of the present invention 70-Ala, through the synergistic effects of targeted enrichment of pancreatic islets and immunomodulation, demonstrates highly effective intervention in the core pathological aspects of type 1 diabetes mellitus (T1DM). Compared to single antioxidant or non-targeted immunosuppressive strategies, this derivative can precisely accumulate at the lesion site (islets) and achieve synergistic effects of targeted delivery and immune homeostasis reconstruction by inhibiting dendritic cell antigen presentation and regulating T cell differentiation (e.g., inhibiting Th1 and promoting Treg). In animal models, this synergistic effect significantly delays disease onset, alleviates islet inflammation, and protects β-cell function, with its overall efficacy superior to a wide range of non-targeted immunosuppressants.

[0021] 2. The present invention C 70 Ala exhibits significant pancreatic islet tissue-specific enrichment. This active targeting characteristic allows the drug to achieve effective concentrations at the lesion site while significantly reducing its distribution in non-target tissues and organs, thereby reducing potential side effects such as infection risk caused by systemic immunosuppression at the source. Therefore, this regimen demonstrates higher expected safety while achieving effective immune intervention.

[0022] 3. The present invention C 70 Ala benefits from its highly efficient pancreatic islet targeting and unique immunomodulatory mechanism. Its proven high islet targeting ability enables effective drug delivery to the lesion site, a characteristic that allows it to achieve high target concentrations with low systemic exposure. This lays the foundation for maintaining efficacy while reducing systemic dosage and potential systemic side effects. Furthermore, by modulating the role of dendritic cells and T cells—an upstream immune hub—it produces a more durable immunomodulatory effect than targeting a single downstream inflammatory factor. This lays the foundation for developing long-acting dosing regimens and improving patient compliance, demonstrating significant potential for dose reduction and enhanced efficacy.

[0023] 4. The C provided by this invention 70 The synthesis of Ala is a mature and simple process, yielding a product with good water solubility and high stability, facilitating subsequent formulation development. It can be further formulated into injections and other dosage forms, fully leveraging its rapid distribution and targeted enrichment characteristics, thus facilitating clinical translation.

[0024] In this invention, the above-described technical solutions can be combined with each other to achieve more preferred combinations. Other features and advantages of this invention will be set forth in the following description, and some advantages may become apparent from the description or be learned by practicing the invention. The objects and other advantages of this invention can be realized and obtained from what is particularly pointed out in the description and drawings. Attached Figure Description

[0025] The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Throughout the drawings, the same reference numerals denote the same parts.

[0026] Figure 1 alanine-modified fullerene C 70 -Ala and C 70 Synthetic process and molecular structure characterization of -Ala-Cy5: A is C 70 -Flowchart of Ala's synthesis; B is C 70 - Ala's infrared spectrum; C is the thermogravimetric and elemental analysis diagram; D is C 70 - Particle size and potential analysis diagram of Ala; E represents C 70 -UV-Vis absorption spectrum of Ala-Cy5.

[0027] Figure 2 Here, A is the infrared spectrum of carboxylated fullerene (TMF); B is the ultraviolet-visible absorption spectrum of carboxylated fullerene-Cy5 (TMF-Cy5).

[0028] Figure 3 This is a comparison of the ability of alanine-modified fullerene and carboxylated fullerene to target and accumulate in pancreatic islets.

[0029] Figure 4 C 70 -Location map of Ala material in immune cells and MIN6 cells.

[0030] Figure 5 C 70 - The regulatory role of Ala on DC differentiation and maturation in an in vitro simulated immune cell model: A is C 70 - Effect of Ala on the expression of CD80 / CD86 double-positive markers of DC maturation; B is C 70 - Effect of Ala on MHCⅡ, a marker of DC maturity.

[0031] Figure 6 C 70 - The regulatory role of Ala on DC antigen presentation and T cell activation in an in vitro simulated immune cell model: A is C 70 -Ala's effect on DC antigen presentation; B is C 70 -Ala T cell activation, i.e., the effect of T cell proliferation.

[0032] Figure 7 C 70 - The regulatory effect of Ala on T cell differentiation in an in vitro simulated immune cell model, where A represents CD8. + T cell activation, B is CD4 + T cell activation.

[0033] Figure 8 C 70 -Results of Ala's effect on T cell subset differentiation: A represents C. 70 - Effects of Ala on Th1 cell subset differentiation; B is C 70 - Effects of Ala on Th2 cell subset differentiation; C is C 70 - Effect of Ala on the differentiation ratio of Th1 / Th2 cell subsets; D is C 70 - Effects of Ala on Th17 cell subset differentiation; E is C 70 - Effect of Ala on Treg cell subset differentiation; F represents C 70 -Ala effect on the differentiation ratio of Th17 / Treg cell subsets.

[0034] Figure 9 C 70 - The protective effect of Ala on pancreatic β-cells and the improvement of glucose-stimulated insulin secretion (GSIS) function. Results of cell protection and restoration of secretory function: A and C represent normal MIN6 cells and normal MIN6 cells + different concentrations of C under 24h and 48h incubation conditions, respectively. 70 - Cell viability graph of Ala; B and D represent normal MIN6 cells, inflammatory cytokine cells, and inflammatory cytokine cells + different concentrations of C under 24h and 48h incubation conditions, respectively. 70 - Cell viability graph of Ala.

[0035] Figure 10 C 70 - The effect of Ala on glucose-stimulated insulin secretion (GSIS) function in MIN6 cells damaged by inflammatory factors is shown in the figure, where A represents serum insulin level and B represents intracellular insulin level.

[0036] Figure 11 The mice included were ICR mice in the T1DM stage, NOD / ShiLtJ model mice, and mice that had undergone C... 70 -Statistical results of disease-related phenotypes and core indicators of glucose metabolism function in ICR mice and NOD / ShiLtJ mice after Ala intervention: A is the statistical results of the cumulative incidence rate of the four groups of mice; B is the statistical results of the organ index of the four groups of mice; C is the statistical results of the oral glucose tolerance test (OGTT) of the four groups of mice.

[0037] Figure 12 The mice included were ICR mice in the T1DM stage, NOD / ShiLtJ model mice, and mice that had undergone C... 70-Statistical results of pancreatitis severity scores and islet morphological parameters (area, number) in ICR mice and NOD / ShiLtJ mice after Ala intervention: A is HE slices of the pancreas of the four groups of mice; B is a statistical graph of pancreatitis scores of the four groups of mice; C is a statistical graph of islet area of ​​the four groups of mice; D is a statistical graph of the number of islets of the four groups of mice. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the present invention clearer, exemplary embodiments of the present invention will be described below in conjunction with the accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. For clarity and brevity, not all features of actual embodiments are described in the specification.

[0039] Type 1 diabetes mellitus (T1DM) is a disease caused by the destruction of pancreatic beta cells due to an attack by the autoimmune system. Current interventions mainly focus on exogenous insulin replacement or the use of broad-spectrum immunosuppressants. However, insulin replacement cannot block the autoimmune process, while systemic immunosuppression has significant drawbacks such as high risk of infection and poor targeting. Existing fullerene derivatives are more focused on treating type 2 diabetes.

[0040] Based on this, the first aspect of the present invention provides an alanine-modified fullerene, wherein the alanine-modified fullerene has the general formula shown in Formula I: C 70 -(Ala) n Formula I Among them, C 70 For fullerenes, Ala is an alanine group, and n is the number of atoms linked to each fullerene C. 70 The average number of alanine groups on the protein is an integer between 1 and 20.

[0041] Within the fullerene family, the molecular configurations and electronic properties of different carbon skeletons differ significantly, directly affecting the biological functions of the derivatives. Through in-depth research, the inventors of this invention discovered that C... 70 With its unique ellipsoidal molecular conformation and electronic configuration, it is irreplaceable in pancreatic islet targeting and immune regulation. The ellipsoidal structure is more conducive to the specific recognition and binding of transport proteins or receptors expressed on the surface of pancreatic islet capillary endothelial cells, thereby mediating efficient transendothelial transport and islet tissue retention; simultaneously, C 70 The electronic structure of the carbon cage enables synergistic interfacial interactions with alanine-modifying groups, providing the optimal molecular basis for subsequent inhibition of dendritic cell antigen presentation and regulation of T cell differentiation, demonstrating superiority over C. 60 Or C 82 The biological activity or stability.

[0042] Furthermore, the inventors discovered that the neutral, moderately hydrophobic side chain properties of alanine have a high affinity for transport proteins or receptors expressed on the surface of pancreatic capillary endothelial cells, thereby mediating C… 70 -Ala exhibits highly efficient transendothelial transport and islet tissue retention. This unique targeting ability is the basis for its subsequent immunomodulatory function at the lesion site.

[0043] According to some embodiments of the present invention, in Formula I, n takes the integer value between 4 and 12.

[0044] The inventors of this invention unexpectedly discovered that the number (n) of alanine modifying groups is the determining factor for C. 70 - The key structural parameters for Ala derivatives to simultaneously achieve the dual core functions of efficient pancreatic islet targeting and precise immune regulation. Preferably, when the n value falls within the specific range of 4-12, the derivative exhibits excellent and unpredictable synergistic biological effects: its molecular surface has an optimal hydrophilic-lipophilic balance and charge distribution, which enables it to not only specifically recognize and accumulate in pancreatic islet tissue with extremely high efficiency, providing a structural basis for the precise accumulation of drugs at the lesion site; but also, after being taken up by antigen-presenting cells and T cells, it can efficiently inhibit excessive immune activation and rebuild immune homeostasis through optimal spatial conformation and interaction with the biological interface. It is worth noting that if the n value is <1, insufficient modification leads to a significant decrease in targeting efficiency and cellular uptake rate; if the n value is >20, excessive modification will completely shield the inherent activity of the fullerene core and may induce non-specific aggregation, thereby weakening its immunomodulatory function and biosafety. For example, the n value can be 1, 4, 5, 6, 8, 10, 12, 15, or 20, preferably 4-12, and more preferably 10.

[0045] According to some embodiments of the present invention, the alanine-modified fullerene has the ability to specifically accumulate in pancreatic islet tissue in vivo, and / or has immunomodulatory functions such as inhibiting dendritic cell antigen presentation and regulating T cell differentiation.

[0046] According to some preferred embodiments of the present invention, the alanine-modified fullerene is a nanoparticle with an average hydrodynamic diameter of 10-100 nm.

[0047] In this invention, the hydrodynamic diameter of the alanine-modified fullerene in aqueous solution meets the above-mentioned range, which facilitates its penetration into tissues through capillary pores (EPR effect) while avoiding excessively small diameters that would lead to rapid filtration and clearance by the kidneys. This promotes circulation in the blood and targets the pancreatic islets. For example, the average hydrodynamic diameter of the alanine-modified fullerene is 10nm, 15nm, 20nm, 30nm, 40nm, 45nm, 48nm, 49nm, 50nm, 80nm, or 100nm, preferably 48-50nm.

[0048] According to some preferred embodiments of the present invention, the polydispersity index of the alanine-modified fullerene is 0.259-0.265. This indicates that the alanine-modified fullerene nanoparticles are highly uniform and do not exhibit severe aggregation or excessively wide distribution, which is of great significance for the stability of drug efficacy. For example, the polydispersity index of the alanine-modified fullerene is 0.259, 0.260, 0.261, 0.262, 0.263, or 0.265.

[0049] According to some preferred embodiments of the present invention, the Zeta potential of the alanine-modified fullerene is -44.39 to 47.01 mV. This indicates that the particles, due to their strong mutual repulsion caused by the same negative charge, are very stable in solution and do not easily aggregate or precipitate. The negative charge here originates from the ionization of the carboxyl group (-COOH) on alanine into -COO in solution. - The high potential also provides a basis for its subsequent interaction with the cell membrane. For example, the Zeta potentials of the alanine-modified fullerenes are -44.39 mV, -44.5 mV, -45.1 mV, -45.7 mV, -46.1 mV, and -47.01 mV.

[0050] A second aspect of the present invention provides a method for preparing the alanine-modified fullerene described in the first aspect, comprising: in an alkaline solution, reacting C... 70 The product was reacted with β-alanine and purified to obtain the alanine-modified fullerene.

[0051] In this invention, alanine-modified fullerene C 70 The synthesis of Ala is a mature and simple process, yielding a product with good water solubility and high stability, facilitating subsequent formulation development. It can be further formulated into injections and other dosage forms, fully leveraging its rapid distribution and targeted enrichment characteristics, thus facilitating clinical translation.

[0052] In this invention, considering the hydrophobic C 70 Fullerene and β-alanine form a covalent bond to achieve water-soluble functionalization. The alkaline solution can be sodium hydroxide solution, potassium hydroxide solution, etc., with a concentration of 8-15wt%, for example, 8wt%, 9wt%, 10wt%, 11wt%, 12wt%, 13wt%, 14wt%, or 15wt%.

[0053] In this invention, the reaction conditions include: a reaction temperature of 75-80℃ and a reaction time of 8-12h.

[0054] In this invention, 75-80℃ is close to the boiling point of ethanol, and the reflux state provides sufficient activation energy to overcome C 70Significant steric hindrance is present, while ensuring the complete dissolution and dispersion of sodium β-alanine in ethanol. The reaction time is controlled at 8-12 hours, ensuring the nucleophilic addition reaction proceeds fully while avoiding prolonged high temperatures that could lead to product decomposition or increased side reactions. These mild and efficient conditions successfully graft hydrophilic β-alanine onto C1. 70 Surface, significantly improved C 70 It has good water solubility and biocompatibility, stable product structure, and high conversion rate.

[0055] Preferably, the purification step includes: (1) Precipitation separation: After the reaction is complete, add 3-5 times the volume of anhydrous ethanol to the reaction solution. C 70 -Ala has extremely low solubility in ethanol and precipitates selectively, while excess β-alanine and its sodium salt, as well as residual base and other impurities, remain in solution. This step achieves preliminary separation and concentration of the product.

[0056] (2) Centrifugal washing: Collect the precipitate and centrifuge and wash it 2-3 times with fresh anhydrous ethanol to thoroughly remove ionic impurities and small molecule organic matter adsorbed on the surface of the precipitate particles.

[0057] (3) Dialysis purification: The washed precipitate is redissolved in ultrapure water and placed in a dialysis bag with a molecular weight cutoff of 1000-3500 Da. The ultrapure water is then dialyzed for 24-48 hours. This step utilizes the principle of membrane separation to efficiently remove any residual salts, tiny organic molecules, and any possible extremely small particulate impurities, ensuring the ionic purity and compositional homogeneity of the final product.

[0058] (4) Freeze-drying the dialyzed solution yields a loose, highly water-soluble C solution with an n value stable in the range of 4-12. 70 -Ala solid powder can be used directly for subsequent formulation research or biological evaluation.

[0059] A third aspect of the present invention provides a pharmaceutical composition comprising the alanine-modified fullerene described in the first aspect, and one or more pharmaceutically acceptable carriers.

[0060] According to some embodiments of the present invention, the dosage form of the pharmaceutical composition is a targeted formulation.

[0061] In some preferred embodiments, the targeted formulation is administered as an injection.

[0062] In this invention, the injectable agent can be a liposome, nanoparticle, or microemulsion, and the liposome, nanoparticle, or microemulsion can be in liquid form or lyophilized powder.

[0063] In this invention, the carrier can be a solvent for injection such as water for injection, physiological saline, or phosphate buffer, or it can be a liposome or nanomicelle, which is prepared from phospholipids and poloxamer.

[0064] In this invention, the content of alanine-modified fullerene in each milliliter of pharmaceutical composition is 12.5 μg-10 mg. For example, it can be 12.5 μg, 25 μg, 50 μg, 100 μg, 1 mg, 5 mg, or 10 mg, preferably 12.5 μg-100 μg.

[0065] The fourth aspect of the present invention provides the use of the pharmaceutical composition described in the third aspect in the preparation of a medicament for the treatment or prevention of type 1 diabetes.

[0066] In this invention, the drug is administered via intraperitoneal injection.

[0067] Specifically, in the application described, the dosage of the pharmaceutical composition is as follows, calculated based on the intraperitoneal injection dose in mice: 1.6-3.3 mg / kg; In some embodiments of the invention, the dosage is 20-40 mg / kg, calculated based on a healthy adult dose.

[0068] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0069] The experimental materials used in the following preparation examples were sourced from the following locations: C 70 Solid powder, purchased from Beijing Funakang Biotechnology Co., Ltd. β-alanine (CAS: 107-95-9, 3.6 g) was purchased from Sigma-Aldrich, a subsidiary of Merck KGaA (Darmstadt, Germany), and was of analytical grade.

[0070] The following preparation examples and comparative examples are used to illustrate the synthesis of modified fullerenes. Preparation Example 1 (1) Preparation of alanine-modified fullerene Take 50 mg C 70Solid powder and 3.6 g of β-alanine were placed in a round-bottom flask, and 50 mL of 14% sodium hydroxide solution was slowly added. After ultrasonic dispersion, the mixture was stirred vigorously at 80 °C for 24 hours. After the reaction was complete, the lower water-soluble liquid was collected using a separatory funnel. A large amount of anhydrous ethanol was added to the water-soluble liquid to precipitate the reactants. The precipitate was collected by centrifugation and washed 2-3 times with anhydrous ethanol. After washing, the product was dissolved in ultrapure water, and the product solution was dialyzed to remove salts using a dialysis bag with a molecular weight cutoff of 1000. After dialysis, the product was lyophilized using a vacuum freeze dryer. The lyophilized product was characterized by infrared spectroscopy, thermogravimetric analysis, elemental analysis, and particle size and potential analysis.

[0071] in, Figure 1 A is alanine-modified fullerene. 70 -A schematic diagram of the synthesis process of Ala Figure 1 B is C 70 -Ala's infrared spectrum, from Figure 1 As can be seen from B, compared to the original C 70 infrared spectrum, C 70 The -Ala product showed new absorption peaks in several characteristic regions, confirming the effect of β-alanine on C. 70 Successful chemical modification. First, at 3400 cm⁻¹ -1 The broad and strong absorption bands observed nearby are attributed to the stretching vibrations of the hydroxyl (-OH) and amino (-NH) groups in the carboxyl group. Secondly, at 1720 cm⁻¹... -1 A sharp, strong absorption peak was observed nearby, with a wavenumber significantly higher than the 1650 cm⁻¹ peak typically observed for amide bonds. -1 This corresponds to the stretching vibration of the C=O double bond in the carboxylic acid structure; the 1580 cm⁻¹ immediately adjacent to it. -1 The absorption peak that appears at C is C 70 The characteristic vibrations of the C=C double bonds in the carbon cage framework indicate that the integrity of the carbon cage structure is preserved, but the symmetry is altered due to modification. Furthermore, the 1380 cm⁻¹ fingerprint region... -1 The absorption peak at 1080 cm⁻¹ corresponds to the stretching vibration of the CO single bond, while the peak at 1080 cm⁻¹ corresponds to the stretching vibration of the CO single bond. -1 The nearby characteristic peaks are attributed to the stretching vibration of the CN single bond, and this key signal directly proves that the amino nitrogen atom has been successfully covalently linked to C. 70 Surface of carbon cage.

[0072] Figure 1 C is C 70- Thermogravimetric and elemental analyses of Ala revealed a distinct multi-stage weight loss pattern in the thermogravimetric (TG) curves. First, a first-stage weight loss of approximately 4.39% was observed in the low-temperature region from room temperature to 109 °C, corresponding to the removal of about six water of crystallization or adsorbed water molecules (~6H₂O). Subsequently, near 200 °C (corresponding to a significant peak in the TG curve), the sample began to experience a second stage of substantial weight loss, attributed to the covalent bonding between C and C. 70 Thermal decomposition and carbonization of the surface organic addition groups (β-alanine moiety). Furthermore, the elemental analysis data below the figure further confirms the occurrence of chemical modification. The number of β-alanine additions, n=10, is calculated based on the percentages of nitrogen (N), hydrogen (H), and carbon (C) in the sample.

[0073] Figure 1 D is C 70 -Ala particle size potential diagram, the figure shows that C 70 Ala exhibits good dispersibility and stability in aqueous solution. Dynamic light scattering (DLS) data show that C 70 -Ala exhibits a unimodal distribution with an average hydrodynamic diameter of 49.18 ± 0.83 nm. Simultaneously, the measured polydispersity index (PDI) is 0.262 (PDI < 0.3 is considered to have a narrow particle size distribution), indicating that the modified C... 70 The derivative was uniformly dispersed in the solvent without significant aggregation. The Zeta potential measurements in the inset show that C... 70 -Ala has a high negative surface charge with a potential of -45.7 mV. This significant negative potential is mainly attributed to the grafting onto C 70 The β-alanine terminal carboxyl group (-COO) on the surface - The high absolute value of the Zeta potential indicates a strong electrostatic repulsion between particles, further confirming the excellent colloidal stability of the system, which is beneficial for its application in the biomedical field.

[0074] (2) Fluorescent labeling synthesis of alanine-modified fullerenes 5 mg of water-soluble near-infrared fluorescent dye (Cy5, sulfo-Cyanine5 carboxylic acid) was dissolved in morpholine ethanesulfonic acid buffer, and 11 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added and stirred for 30 min. Then, 6.5 mg of N-hydroxysuccinimide was added and stirring was continued for 2 h. The pH was adjusted to approximately 7 with 0.1 mol / L sodium hydroxide, and 7 mg of alanine-labeled fullerene was added. The mixture was stirred at room temperature for 48 h. After dialysis to remove impurities using a dialysis bag with a molecular weight cutoff of 3500, the solution was separated using a G-25 dextran gel column. The first green solution to elute was collected, which was the prepared Cy5-labeled alanine-labeled fullerene (Cy5). 70 -Ala-Cy5). Figure 1 E, as confirmed by ultraviolet absorption spectroscopy, indicates the successful preparation of Cy5-labeled alanine-modified fullerene (C). 70 The ultraviolet absorption spectrum of (-Ala-Cy5) can be seen in the figure, and C can be observed in the figure. 70 -Ala-Cy5 was successfully tagged.

[0075] Comparative preparation example (1) Synthesis of carboxylated fullerenes Under nitrogen protection, 4g of sodium hydride was stirred with 40mL of ultra-dry tetrahydrofuran for 10 minutes, and 8mL of diethyl bromomalonate was slowly added dropwise until the solution turned yellow; 0.5g of C 70 Dissolved in 100 mL of ultra-dry o-xylene, the mixture was added to the reaction solution and stirred for 2 h (TLC monitoring for complete reaction). The mixture was filtered to remove impurities, and the filtrate was precipitated with n-hexane. The precipitate was collected by centrifugation, dissolved in toluene, and separated by silica gel column chromatography to obtain the tetraaddition esterification product C. 70 [C(COOCH2CH3)2]4, the tetraaddition esterification product was evaporated to dryness and dissolved in toluene. 6g of sodium hydride was added under nitrogen protection and stirred at 60℃ for 6h. Excess anhydrous ethanol was used to quench and precipitate the product. The precipitate was collected by centrifugation, washed three times with anhydrous ethanol, dissolved in ultrapure water, and dialyzed through a 3500 molecular weight dialysis bag to remove impurities. The product was then freeze-dried to obtain polycarboxylated fullerene TMF.

[0076] from Figure 2 As can be seen from the infrared spectrum, C 70 The characteristic functional group absorption peaks of TMF and its derivatives are clearly visible. At 3400 cm⁻¹... -1 The broad and strong absorption peak at 1630 cm⁻¹ corresponds to the stretching vibration of the OH bond in the carboxyl group, indicating that the hydrolysis reaction successfully introduced a hydrophilic hydroxyl group; -1 The strong absorption peak at 1380 cm⁻¹ is attributed to the characteristic stretching vibration of the C=O double bond (carbonyl group) in the carboxyl group; -1 The absorption peak corresponds to the stretching vibration of the CO single bond. Furthermore, the absorption peak in the 1050-1100 cm⁻¹ range... -1The absorption peaks within the range confirm that C 70 The integrity of the carbon cage framework structure. The presence of these characteristic peaks fully demonstrates the integrity of C. 70 Through the Bingel-Hirsch reaction and subsequent hydrolysis, malonic acid functional groups were successfully grafted and converted into the polycarboxylic acid derivative TMF.

[0077] (2) Fluorescent labeling synthesis of carboxylated fullerenes Weigh out 7 mg of TMF and dissolve it in morpholine ethanesulfonic acid buffer (0.1 mol / L, pH: 6.0). Add 11 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (CAS: 7084-11-9) and stir for 30 minutes. Then add 6.5 mg of N-hydroxysuccinimide (CAS: 6066-82-6) and continue stirring for 2 hours. Adjust the pH of the reaction solution to between 7 and 8 using 0.1 M sodium hydroxide solution. Add 5 mg of sulfo-Cyanine5 NHS (CAS: 2230212-27-6) dissolved in DMSO and stir at room temperature in the dark for 48 hours. After removing excess salt and other substances by dialysis using a dialysis bag with a molecular weight cutoff of 3500, separate the product using a G-25 dextran gel column. The first green solution obtained is TMF-Cy5. Figure 2 B, as confirmed by ultraviolet absorption spectroscopy, indicates the successful preparation of Cy5-labeled alanine-modified fullerene (C). 70 The ultraviolet absorption spectrum of TMF-Cy5 is shown in the figure, which shows that TMF-Cy5 has been successfully labeled.

[0078] The following examples are used to illustrate the application effects of the preparation examples or comparative preparation examples. Example 1 Alanine-modified fullerenes and carboxylated fullerenes target pancreatic islet accumulation. Experimental materials Animals: C57BL / 6J mice; Reagents and Instruments: C 70 -Ala-Cy5, insulin antibody (green fluorescently labeled), PBS buffer; small animal in vivo imaging system, fluorescence microscope, tissue slicer, digital pathology slide scanner, pipettes Experimental methods: Pancreatic tissue fluorescence staining detection: C 70Ala-Cy5 was administered intraperitoneally to C57BL / 6J mice. A blank control group (saline) and a negative control group (Cy5) were set up. Mice were sacrificed at different time points at 8h, 24h, and 48h after administration, and pancreatic tissue was isolated. The pancreatic tissue was fixed, dehydrated, and embedded to prepare tissue sections. After washing with PBS, the sections were incubated with insulin antibody (green fluorescent label) in the dark, washed, and then mounted with a fluorescent anti-quenching agent. The sections were observed under a fluorescence microscope.

[0079] Figure 3 Comparison of the pancreatic islet-targeting enrichment capabilities of alanine-substituted fullerenes and carboxylated fullerenes: from left to right, normal group (CN, i.e., healthy mice that were not given any drugs or dyes), Cy5, TMF-Cy5, and C. 70 -Immunofluorescence labeling images of Ala-Cy5 at different time points after administration: blue labels cell nuclei, green labels insulin (islets of Langerhans), and red labels Cy5 (fullerene drug). Figure 3 As can be seen from this, C 70 In the Ala-Cy5-treated group of mice, a clear Cy5 red fluorescence signal was observed in the islets of Langerhans labeled with insulin green fluorescence in immunofluorescence sections, and this signal gradually increased over time. (The last sentence appears to be incomplete and possibly refers to a different topic.) 70 Pancreatic tissue sections were analyzed at different time points after Ala administration. The islet regions were delineated using Image software, the average fluorescence intensity of the Cy5 channel was read, and the islet-targeting enrichment ratio of each section was calculated. Results showed that the average targeting enrichment ratios at 8h, 24h, and 48h post-administration were 2.2, 4.384, and 6.09, respectively, with the enrichment degree increasing over time. These quantitative results indicate that alanine-modified fullerenes can target and enter pancreatic tissue in vivo, and that administration of alanine-modified fullerenes resulted in greater drug enrichment in the pancreatic islets compared to administration of carboxylated fullerenes with the same fluorescence intensity.

[0080] Table 1

[0081] Example 2 C 70 -Location of Ala material in immune cells and MIN6 cells Experimental materials Cells: DC cells (C57BL / 6J bone marrow mononuclear cells), T cells (OT-1 spleen extract), MIN6 cells (pancreatic β cells); Reagents and instruments: High-glucose DMEM medium (Gibco, C11995500BT) containing 10% fetal bovine serum (Gibco, 10099141C), C 70-Ala-Cy5 (100 μg / mL), Hoechst dye, PBS buffer; confocal dish, laser confocal microscope, pipette.

[0082] Experimental methods: DC cells, T cells, and MIN6 cells were divided into a control group (high glucose DMEM medium containing 10% fetal bovine serum) and an experimental group (containing 100 μg / mL C40, MIN6 ... 70 The cells were divided into groups (using the DMEM medium described above for Ala-Cy5) and inoculated into confocal dishes for normal culture for 6 hours. After culture, Hoechst dye was added and incubated for 10 minutes, followed by washing three times with PBS buffer. The cells were then observed using a confocal microscope (blue Hoechst staining of cell nuclei, red Cy5-labeled fullerenes). Figure 4 C 70 -Location results of Ala material in immune cells and MIN6 cells. As can be seen from the figure, compared with the control group, C 70 The Ala-Cy5 treatment group showed significant red fluorescence in DC cells, T cells, and MIN6 cells. Co-localization (overlay) images showed C 70 -Ala-Cy5 can cross the cell membrane, indicating that this material can be phagocytosed by the aforementioned cells. A distinct red fluorescent signal was observed intracellularly, providing the first cellular-level confirmation of C... 70 -Ala possesses the ability to cross the membrane barriers of immune cells and parenchymal cells. Therefore, C... 70 -Ala can not only reach the lesion, but also enter the key target cells to exert its effects, which constitutes the first key step in achieving its dual therapeutic function.

[0083] Example 3 C 70 - The regulatory role of Ala on DC antigen presentation and T cell activation and differentiation in an in vitro simulated immune cell model Experimental materials Cells: BMM (C57BL / 6J mouse bone marrow mononuclear cells), T cells (OT-1 spleen extract) Reagents and instruments: High-glucose DMEM medium (Gibco, C11995500BT) containing 10% fetal bovine serum (Gibco, 10099141C), C 70- Ala (gradient concentrations: 50 μg / mL, 100 μg / mL, 150 μg / mL), ovalbumin (OVA, model antigen), GM-CSF (DC-induced differentiation factor), IL-4 (DC-induced differentiation factor), H-2Kb-SIINFEKL complex antibody (OVA-specific antigen epitope detection), CD8 antibody, CD4 antibody, Th1 subset marker antibody (e.g., IFN-γ), Th2 subset marker antibody (e.g., IL-4), Treg subset marker antibody (e.g., Foxp3), T cell proliferation assay kit (CFSE kit), PBS buffer, fixative, permeabilization solution, permeabilizing agent; cell culture plates, flow cytometer, multi-functional microplate reader, pipettes, incubator, clean bench, centrifuge.

[0084] Experimental methods Construction of in vitro immune cell model: ① DC cell induction and differentiation: Bone marrow mononuclear cells (BMM) were isolated from the femoral bone marrow of C57BL / 6J mice, seeded into cell culture plates, and cultured in a constant temperature incubator for 8 days until they differentiated into mature dendritic cells (DCs); DC maturation grouping: divided into normal group (complete culture medium) and material control group (complete culture medium + C 10 ... 70 -Ala), positive control group (complete culture medium + LPS (lipopolysaccharide)), experimental group (complete culture medium + LPS + C) 70 -Ala) Incubate for 24 hours. ② DC antigen presentation grouping: divided into normal group (complete culture medium) and material control group (complete culture medium + C). 70 -Ala), positive control group (complete culture medium + OVA antigen), and experimental group (containing different concentrations of C) 70 - Incubate with Ala complete culture medium + OVA antigen for 12 hours. ③ T cell activation and differentiation: Divided into control group (complete culture medium) and material control group (complete culture medium + C). 70 -Ala), positive control group (complete culture medium + OVA incubation of DC cells co-culture) and experimental group (complete culture medium + OVA incubation of DC cells + C 70 -Ala), T cell proliferation was detected after 72 hours of constant temperature culture, and T cell differentiation was detected after 96 hours.

[0085] DC antigen presentation function assay: After culture, DC cells were collected, washed with PBS, and then H-2Kb-SIINFEKL complex antibody (for detecting OVA-specific antigenic epitopes) was added and incubated in the dark for 30 min. After washing, the positive rate of H-2Kb-SIINFEKL complex on the surface of DCs was detected by flow cytometry to assess antigen presentation efficiency.

[0086] T cell activation (proliferation) detection: Before co-culture, T cells were labeled with CFSE dye, incubated in the dark, and then washed with PBS to remove excess dye. The labeled T cells were then co-cultured with mature dendritic cells (DCs) at a predetermined ratio (grouping as before). After culture, cells were collected, and the CFSE fluorescence intensity of T cells was detected by flow cytometry. The degree of fluorescence signal attenuation was used to assess the T cell proliferation level, thereby determining the C... 70 -Ala's intervention on T cell proliferation.

[0087] T cell subset regulation detection: CD8+T and CD4+T cell activation detection: Collect co-cultured T cells, add CD8, CD4 antibodies and activation marker antibodies and incubate in the dark. After washing, analyze the activation level of CD8+ cytotoxic T cells and CD4+ helper T cells by flow cytometry; CD4+T cell subset differentiation detection: Add Th1 (IFN-γ), Th2 (IL-4), Th17 (IL-17A), and Treg (Foxp3) subset-specific antibodies to T cells. After membrane rupture, incubation, and washing, analyze the proportion of each subset and the Th1 / Th2 and Th17 / Treg ratios by flow cytometry.

[0088] Figure 5 C 70 - The regulatory role of Ala on LPS-induced DC maturation in an in vitro simulated immune cell model: Experimental results showed that C 70 -Ala can significantly inhibit LPS-induced DC surface co-stimulatory molecules CD80 / CD86 ( Figure 5 A) and the expression of major histocompatibility complex class II molecules (MHC-II) Figure 5 B). The "+" on the horizontal axis represents adding LPS or C. 70 -Ala, "-" indicates that LPS or C was not added. 70 -Ala.

[0089] Figure 6 C 70 - The regulatory effects of Ala on DC antigen presentation and T cell activation and differentiation in an in vitro simulated immune cell model: These can be seen in the figures: ① Regulation of DC antigen presentation: C 70 -Ala significantly inhibits the OVA antigen presentation efficiency of DCs in a concentration-dependent manner: with increasing C 70 -As Ala concentration increases, the positivity rate of the H-2Kb-SIINFEKL complex on the DC surface decreases in a gradient manner. Figure 6 A); ② T cell activation and proliferation: different concentrations of C 70 Both -Ala and α-Ala can inhibit T cell proliferation, and the inhibitory effect is concentration-dependent. Figure 6 B) Figure 7C 70 -Ala's regulation of T cell subsets in an in vitro simulated immune cell model: C 70 -Ala can significantly inhibit the activation of CD8+ cytotoxic T cells ( Figure 7 A); The activation level of CD4+ helper T cells was regulated to the normal level without OVA stimulation, achieving precise reversal of abnormal activation. Figure 7 B) Figure 8 C 70 - Ala's effect on the balance of CD4+ T subsets in an in vitro simulated immune cell model: C 70 - After Ala treatment, the proportions of Th1 and Th17 subsets decreased significantly, while the proportions of Th2 and Treg subsets returned to normal levels. The Th1 / Th2 and Th17 / Treg ratios were restored to balance, effectively eliminating the pro-inflammatory microenvironment.

[0090] Table 3

[0091] Example 4 C 70 - Protective effect of Ala on pancreatic β cells Experimental materials Cells: MIN6 cells (pancreatic β cells); Reagents and instruments: TNF-α, IL-1β, IFN-γ, high-glucose DMEM medium containing 10% fetal bovine serum (Gibco, C11995500BT), CCK-8 cell viability assay kit, DCFH-DA reactive oxygen species detection probe, Rosup (reactive oxygen species positive control), insulin assay kit, low-glucose glucose buffer solution (2mM), high-glucose glucose buffer solution (16.8mM); cell culture plates, microplate reader, confocal microscope, pipettes, incubator, centrifuge.

[0092] Experimental methods C 70 -Detection of Ala's protective effect on damaged MIN6 cells and restoration of secretory function: ① Grouping and treatment: Inflammatory factors were co-cultured with MIN6 cells for 24h and 48h, and the experimental group was added with different gradient concentrations of C. 70 -Ala (12.5, 25, 50, 100 μg / mL) and a control group (MIN6 cells that have not been treated with any drugs or damaged by inflammatory factors) were set up, and different gradient concentrations of C were added. 70- Ala (12.5, 25, 50, 100 μg / mL) was used to detect cell viability in each group using the CCK-8 assay; ② Insulin secretion function assay: cell culture medium was collected from each group to detect insulin secretion levels; different concentrations of glucose stimulation were set to detect glucose-stimulated insulin secretion (GSIS) response.

[0093] Figure 9 C 70 - Ala's protective effect on pancreatic β cells and the improvement of glucose-stimulated insulin secretion (GSIS) function, resulting in cell protection and restoration of secretory function. Figure 9 A and Figure 9 C represents normal MIN6 cells without inflammatory cytokine damage, and C at different concentrations. 70 The cell viability of -Ala (12.5, 25, 50, 100 μg / mL) at 24h and 48h can be observed, showing C 70 -Ala has no significant toxic side effects on normal MIN6 cells that have not been damaged by inflammatory factors. Figure 9 B and Figure 9 As can be seen from D: different gradient concentrations of C 70 -Ala (0, 12.5, 25, 50, 100 μg / mL) showed protective effects against MIN6 cells damaged by inflammatory factors at 24h and 48h in a concentration-dependent manner, demonstrating excellent repair effects.

[0094] Figure 10 C 70 -Ala can effectively reverse the inflammatory factor-induced abnormal secretory function of pancreatic β cells, restore the insulin secretion level of damaged cells, and make the secretion pattern closer to that of MIN6 cells in a normal physiological state. Figure 10 B).

[0095] Example 5 C 70 -Ala may prevent, treat, or delay the onset of type 1 diabetes in NOD / ShiLtJ mice. Experimental materials Animals: ICR mice, NOD / ShiLtJ mice; Reagents and Instruments: C 70 - Ala, glucose, tissue fixative, dehydrating agent, embedding agent, hematoxylin-eosin (HE) staining solution; blood glucose meter, electronic balance, centrifuge, tissue sectioner, fluorescence microscope, liquid chromatography-mass spectrometry.

[0096] Experimental methods Experimental grouping and intervention: ICR mice and NOD / ShiLtJ mice were randomly divided into four groups: ICR normal mouse group (ICR-C), C 70-Ala intervention followed by ICR mouse group (ICR-F), model group NOD / ShiLtJ mouse group (NOD-M), C 70 - NOD / ShiLtJ mouse group after Ala intervention (NOD-F); mice in the intervention group were given an appropriate dose of C 70 -Ala treatment, the control group was given an equal volume of normal saline, and the intervention continued until the T1DM stage.

[0097] Disease-related phenotypes and glucose metabolism function detection: Incidence statistics: The incidence of T1DM in mice in each group was continuously monitored and recorded, and the incidence was calculated; Organ index detection: After the mice were sacrificed, the relevant organs were separated, weighed, and the organ index (organ weight / mouse body weight × 100%) was calculated; Oral glucose tolerance test (OGTT) detection: After fasting, the mice were given glucose by gavage, and the blood glucose values ​​were measured at different time points to plot the OGTT curve.

[0098] Islet inflammation scoring and islet morphology analysis: Mouse pancreatic tissue was isolated, fixed, dehydrated, embedded, and sectioned for HE staining; islet morphology was analyzed using a pathological section scanner, islet area and number were counted, and the severity of islet inflammation was scored.

[0099] Figure 11 Normal ICR mice, NOD / ShiLtJ model mice, and mice treated with C 70 -Statistical results of disease-related phenotypes and core indicators of glucose metabolism in ICR mice and NOD / ShiLtJ mice after Ala intervention. ICR-C: control group of normal ICR mice, i.e., healthy ICR mice that have not received any drug treatment, serving as a reference for normal physiological state. "ICR" refers to the mouse strain name.

[0100] ICR-F: C 70 -Ala-treated ICR mouse group, i.e., healthy ICR mice receiving C 70 -Ala administration was used to assess whether the drug had adverse effects on normal mice.

[0101] NOD-M: The NOD / ShiLtJ model group mice, i.e., type 1 diabetic model mice that have not received drug treatment, serve as a reference for disease status. "NOD" refers to the mouse strain name (non-obese diabetic mice).

[0102] NOD-F: C 70 - The NOD / ShiLtJ mouse group after Ala intervention, i.e., type 1 diabetes model mice, received C 70 -Ala administration is used to assess the therapeutic effect of the drug.

[0103] The figure shows: ① Disease-related phenotypes and glucose metabolism function results: Figure 11 A. Incidence statistics showed that, compared with the 50% incidence rate in the model group NOD / ShiLtJ mice, C 70 -Ala intervention significantly reduced the incidence of disease in NOD / ShiLtJ mice; Figure 11 B. Organ index statistics showed that the organ index of the intervention group was closer to that of the NOD normal mouse group; C. OGTT test results showed that C 70 - Ala intervention improved glucose tolerance in the model group mice, restoring their glycemic regulation ability. ② Pancreatitis and islet morphology results (see...) Figure 12 A): Analysis of HE slices from the T1DM period. Figure 12 Figure B shows the pancreatitis score, C 70 -After Ala intervention, the severity score of pancreatitis in NOD / ShiLtJ mice was significantly lower than that in the model group; Figure 12 The pancreatic islet area statistics in Figure C and the pancreatic islet number statistics in Figure D show that the results are closer to the normal level, indicating that C 70 -Ala can alleviate pancreatic islet damage. In summary, C 70 -Ala can effectively prevent, treat, or delay the onset of type 1 diabetes in NOD / ShiLtJ mice. Figure 12 The results showed that the islets of Langerhans in the normal ICR mouse control group (ICR-C) were well-formed, with an average islet area of ​​22259 μm. 2 In contrast, the untreated NOD / ShiLtJ model group (NOD-M) experienced severe islet atrophy due to severe islet inflammation and β-cell destruction, with the average islet area decreasing to 5968 μm. 2 The difference was highly significant (P < 0.001). According to C... 70 In the Ala-treated mouse group (NOD-F), the islet area significantly recovered to 21938 μm. 2 The levels were highly significant compared to the model group (P<0.01) and approached those of the normal control group. Furthermore, C... 70 The islet area in the Ala-treated ICR mouse group (ICR-F) was 22227 μm. 2 There was no statistically significant difference between the material and the normal control group (P>0.05), indicating that the material has no significant adverse effects on normal pancreatic islet tissue.

[0104] Figure 12 Results D showed that the number of islets in pancreatic sections further validated the aforementioned protective effect. The average number of islets per section in the ICR-C group was 108, while in the NOD-M group, due to the destruction and disappearance of a large number of islets by the immune system, the number of islets was significantly reduced to 32 (P<0.001). C 70 - After Ala intervention (NOD-F group), the number of islets significantly increased to 75, significantly more than the model group (P<0.01), indicating that C70 -Ala effectively protects the pancreas from complete destruction by the immune system. Similarly, the ICR-F group had 102 pancreatic islets, the same as the normal control group.

[0105] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. An alanine-modified fullerene, characterized in that, The alanine-modified fullerene has the general formula shown in Formula I: C 70 -(Ala) n Formula I Among them, C 70 For fullerenes, Ala is an alanine group, and n is the number of atoms linked to each fullerene C. 70 The average number of alanine groups on the protein is an integer between 1 and 20.

2. The alanine-modified fullerene according to claim 1, characterized in that, n is an integer between 4 and 12.

3. The alanine-modified fullerene according to claim 1, characterized in that, The alanine-modified fullerene has an average hydrodynamic diameter of 10-100 nm. And / or, the polydispersity index of the alanine-modified fullerene is 0.259-0.265; And / or, the alanine-substituted fullerene has a Zeta potential of -44.39 to -47.01 mV.

4. A method for preparing alanine-modified fullerene according to any one of claims 1-3, characterized in that, include: In an alkaline solution, C 70 The product was reacted with β-alanine and purified to obtain the alanine-modified fullerene.

5. The preparation method according to claim 4, characterized in that, The reaction conditions include: a reaction temperature of 75-80℃ and a reaction time of 8-24h.

6. A pharmaceutical composition, characterized in that, It includes alanine-modified fullerene as described in any one of claims 1-3, and at least one pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, characterized in that, The dosage form of the pharmaceutical composition is a targeted formulation.

8. The pharmaceutical composition according to claim 6 or 7, characterized in that, The alanine-modified fullerene content in each milliliter of the pharmaceutical composition is 12.5 μg-10 mg.

9. Use of any one of the pharmaceutical compositions of claims 6-8 in the preparation of a medicament for the treatment or prevention of type 1 diabetes.

10. The application according to claim 9, characterized in that, The drug is administered via intraperitoneal injection.

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