Pharmaceutical composition with the function of homologous recombination repair and its preparation method and application

Abstract

The present application relates to the technical field of medicine, and particularly relates to a drug composition with a sequential synergistic repair effect and a preparation method and application thereof. The drug composition comprises liposome I encapsulating caffeic acid and liposome II encapsulating BH4; the liposome I is a ROS-responsive liposome, can specifically respond in a high ROS microenvironment, and then realize on-demand release of caffeic acid; meanwhile, the liposome II protects BH4 from entering cells to play a role, can effectively overcome the defects of difficult dissolution of caffeic acid and easy oxidation of caffeic acid and BH4, can improve the bioavailability of the two drugs, can realize sequential release of caffeic acid and BH4, and can play a synergistic treatment effect on different mechanisms of occurrence of radioactive skin damage, and provides a sequential synergistic repair strategy for clinic. The liposome I and the liposome II are both prepared by a thin film hydration method, and the operation is simple, the conditions are mild, the lipid film is uniformly formed, and the encapsulation rates of caffeic acid and BH4 are high.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to pharmaceutical compositions with sequential synergistic repair effects, their preparation methods, and applications. Background Technology

[0002] Radiotherapy is a common treatment for tumors. In recent years, with the rapid development of radiotherapy technology, it has shifted from two-dimensional radiotherapy to three-dimensional precision radiotherapy, significantly improving treatment accuracy and effectively reducing non-specific radiation damage to normal tissues. However, as the outermost protective barrier of the human body, the skin is directly exposed to the radiation field and is still inevitably subject to radiation damage. Radiation dermatitis has become a common acute adverse reaction during radiotherapy.

[0003] Studies have confirmed that most cancer patients develop radiation dermatitis during radiotherapy, primarily affecting the skin in folded areas such as the head, neck, and chest. Currently, clinical practice uses the grading criteria developed by the Radiation Therapy Oncology Group (RTOG), classifying radiation dermatitis into five grades (0-IV) based on symptom severity. As radiotherapy progresses and the radiation dose accumulates, skin damage progressively worsens, gradually developing from initial mild symptoms such as redness, swelling, dryness, and desquamation to moist desquamation, erosion, and ulceration. In severe cases, skin necrosis, fibrosis, and pigmentation may occur, not only impacting the patient's quality of life but also potentially leading to radiotherapy interruption, thereby affecting the effectiveness of cancer treatment.

[0004] Clinically, interventions for radiation dermatitis are mostly symptomatic and supportive treatments, primarily including topical skin protectants, growth factors, silver ion dressings, corticosteroids, and physical methods such as laser therapy. These methods only alleviate the surface symptoms of skin damage and cannot target the underlying mechanisms of radiation dermatitis, resulting in poor clinical outcomes and difficulty in effectively preventing skin damage progression and promoting repair. Therefore, developing highly effective intervention strategies targeting the mechanisms of radiation dermatitis is of significant clinical importance.

[0005] The academic community generally believes that radiation dermatitis is the result of the synergistic effects of multiple mechanisms, including DNA damage, oxidative stress, inflammatory response, reduced nitric oxide (NO) synthesis, and altered cellular metabolism. Among these, oxidative stress and abnormal NO synthesis are the core pathogenic factors. Based on this pathogenesis, natural small-molecule drugs with antioxidant activity have become a hot topic in research on the intervention of radiation dermatitis. Caffeic acid, curcumin, astaxanthin, and carotenoids have all been shown to have potential radiation protection and damage repair effects.

[0006] Caffeic acid, as a naturally occurring phenolic acid, has a strong antioxidant capacity. It can effectively remove reactive oxygen species (ROS) generated in cells after radiotherapy, reduce oxidative stress damage, and inhibit the release of inflammatory factors, thus alleviating the inflammatory response.

[0007] Tetrahydrobiopterin (BH4) is a natural nutrient that functions as a cofactor of nitric oxide synthase (NOS) in the body. Due to its unstable chemical properties, BH4 is easily oxidized to its inactive form, dihydrobiopterin (BH2). The large amounts of reactive oxygen species (ROS) generated by radiotherapy accelerate the oxidation of BH4, leading to a significant decrease in the intracellular BH4 / BH2 ratio and subsequently triggering NOS uncoupling. This uncoupling not only reduces NO synthesis but also generates endogenous ROS, further exacerbating oxidative stress damage. Therefore, delivery of exogenous BH4 holds promise for improving post-radiotherapy NOS dysfunction and promoting angiogenesis and skin tissue repair.

[0008] However, both caffeic acid and BH4 are easily oxidized and inactivated. At the same time, caffeic acid is poorly soluble in water, resulting in poor solubility and low delivery efficiency in vivo. Furthermore, the drug distribution in vivo lacks targeting and cannot be accurately concentrated at the site of skin damage. In addition, when caffeic acid or BH4 is delivered alone, it is difficult to achieve the synergistic effects of multiple mechanisms such as anti-oxidation, regulation of NOS function, inhibition of inflammatory response, and improvement of lipid metabolism. As a result, the bioavailability of the two when delivered directly is low, and their radiation protection and skin damage repair efficacy cannot be fully utilized.

[0009] Therefore, improving the physicochemical properties of caffeic acid and BH4, enhancing their bioavailability, and achieving targeted delivery and synergistic effects through multiple mechanisms are pressing technical challenges that need to be addressed. Summary of the Invention

[0010] The present invention aims to at least solve one of the technical problems existing in the related art. Therefore, the first objective of the present invention is to provide a pharmaceutical composition having a sequential synergistic repair effect; the second objective of the present invention is to provide a method for preparing the pharmaceutical composition having a sequential synergistic repair effect; and the third objective of the present invention is to provide the application of the pharmaceutical composition having a sequential synergistic repair effect.

[0011] To achieve the first objective, the technical solution adopted by this invention is as follows: A pharmaceutical composition with sequential synergistic repair function includes liposome I loaded with caffeic acid and liposome II loaded with BH4; The structural formula of caffeic acid is shown below: ; BH4 is tetrahydrobiopterin, and its structural formula is shown below: ; The liposome I is a ROS-responsive liposome containing DHA; DHA is docosahexaenoic acid, with the following structural formula: ; ROS stands for Reactive Oxygen Species.

[0012] Furthermore, the formulation of liposome I includes not only caffeic acid and DHA, but also DSPC and cholesterol; The formulation of liposome II includes BH4, as well as DSPC, cholesterol and HSPC. Among them, DSPC is distearylphosphatidylcholine, and HSPC is hydrogenated soybean phosphatidylcholine.

[0013] Furthermore, the formulation of liposome I includes: The ratio of the mass of caffeic acid to the sum of the masses of DHA, DSPC, and cholesterol was 0.0075:4.05 to 0.06:4.05. The mass ratio of DHA to caffeic acid is 5:6 to 5:0.75. The mass ratio of DHA to the combined mass of DSPC and cholesterol is 0.01:4 to 0.1:4.

[0014] Furthermore, the formulation of liposome II includes: The ratio of the mass of BH4 to the sum of the masses of DSPC, cholesterol, and HSPC is 0.125:4.05 to 1:4.05.

[0015] Furthermore, the mass ratio of caffeic acid to BH4 is 3:400 to 12:25.

[0016] To achieve the second objective, the technical solution adopted by this invention is as follows: A method for preparing a pharmaceutical composition with sequential synergistic repair function, wherein liposome I and liposome II are both prepared by thin-film hydration.

[0017] To achieve the third objective, the technical solution adopted by this invention is as follows: Application of pharmaceutical compositions with sequential synergistic repair effects, and preparation of pharmaceutical formulations for dermatitis using pharmaceutical compositions with sequential synergistic repair effects as described in any of the above-mentioned embodiments.

[0018] Furthermore, the pharmaceutical preparation includes a topical gel preparation.

[0019] Furthermore, the matrix of the topical gel formulation is selected from hydrogel matrices with peptides as the main component, and the structural formula of the peptides is shown below: .

[0020] Furthermore, in the composition of the topical gel formulation: the concentration of caffeic acid is not less than 100 μM, and the concentration of BH4 is not less than 10 μM.

[0021] Furthermore, the dermatitis described is radiation dermatitis.

[0022] The above-described one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects: This invention provides a pharmaceutical composition with sequential synergistic repair effects, comprising liposome I encapsulated with caffeic acid and liposome II encapsulated with BH4. Liposome I is a ROS-responsive liposome, capable of releasing caffeic acid in a high ROS microenvironment. Liposome II protects BH4 from entering cells and exerting its effect, effectively overcoming the drawbacks of poor solubility of caffeic acid and easy oxidation of both caffeic acid and BH4. This pharmaceutical composition improves the bioavailability of both caffeic acid and BH4, achieves sequential release of caffeic acid and BH4, and provides synergistic therapeutic effects targeting different mechanisms of radiation-induced skin damage, offering a sequential synergistic repair strategy for clinical use. Both liposome I and liposome II are prepared using a thin-film hydration method, which is simple to operate, mild, and produces a uniform lipid film formation with high encapsulation efficiency for caffeic acid and BH4. Animal experiments show that the hydrogel preparation obtained using liposomes I and II has significant repair and healing effects on radiation-induced skin damage in mice.

[0023] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0024] Figure 1 This is a TEM image of a sample prepared according to a ROS-responsive liposome formulation with different DHA contents, as provided in Example 1 of this invention.

[0025] Figure 2 This is a graph showing the changes in drug release before and after irradiation of different samples provided in Example 1 of the present invention.

[0026] Figure 3 This is a graph showing the statistical results of encapsulation efficiency and drug loading of different CA@RR-Lipo samples provided in Example 1 of the present invention.

[0027] Figure 4 This is a graph showing the statistical results of encapsulation efficiency and drug loading of different BH4@Lipo samples provided in Embodiment 1 of the present invention.

[0028] Figure 5 These are TEM images of CA@RR-Lipo (sample 1) and BH4@Lipo (sample 3) provided in Embodiment 1 of the present invention.

[0029] Figure 6 This is a particle size distribution curve of CA@RR-Lipo (sample 1) and BH4@Lipo (sample 3) provided in Embodiment 1 of the present invention.

[0030] Figure 7 This is a bar chart showing the results of the particle size stability study of CA@RR-Lipo (sample 1) and BH4@Lipo (sample 3) provided in Example 1 of this invention.

[0031] Figure 8 This is a graph showing the detection results of the free caffeic acid 1,1-diphenyl-2-trinitrophenylhydrazine radical (DPPH) scavenging experiment provided in Example 2 of the present invention.

[0032] Figure 9 This is a graph showing the experimental results of cation scavenging (ABTS) detection of caffeic acid 2,2'-hydrazine-bis(3-ethylbenzothiazoline-6-sulfonic acid) free radicals in CA@RR-Lipo provided in Example 2 of the present invention.

[0033] Figure 10 This is a graph showing the detection results of 2′,7′-Dichlorodihydrofluorescein diacetate (DCFHDA) in different groups provided in Example 2 of the present invention.

[0034] Figure 11 This is a bar chart showing the intracellular BH4 / BH2 ratio of different groups of 3T3 cells after 24 hours of irradiation, as provided in Example 2 of this invention.

[0035] Figure 12 This is a bar chart of intracellular NOS activity in different groups of 3T3 cells after 24 hours of irradiation, provided in Example 2 of this invention.

[0036] Figure 13 These are confocal images of intracellular NO content in different groups of 3T3 cells 24 hours after 6 Gy irradiation, provided in Example 2 of this invention.

[0037] Figure 14 This is a high-resolution mass spectrometry (HRMS) image of a polypeptide provided in Example 3 of the present invention.

[0038] Figure 15This is a circular dichroism chromatogram of the polypeptide provided in Example 3 of the present invention.

[0039] Figure 16 The CA@RR-Lipo provided in Embodiment 3 of this invention Gel BH4@Lipo Gel and BC Gel TEM image.

[0040] Figure 17 The BC provided in Embodiment 3 of the present invention Gel The rheological test results.

[0041] Figure 18 The BC provided in Embodiment 3 of the present invention Gel The results of the biocompatibility test are shown in the figure.

[0042] Figure 19 This is a graph showing the results of skin treatment in different groups of mice provided in Example 3 of the present invention.

[0043] Figure 20 This is a graph showing the change of RTOG scores of different groups of mice over time, as provided in Example 3 of the present invention. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. The following embodiments are used to illustrate this invention, but cannot be used to limit the scope of this invention.

[0045] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available, unless otherwise specified, and are carried out in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions.

[0046] Example 1 I. Preparation of liposome I loaded with caffeic acid (denoted as CA@RR-Lipo).

[0047] First, the formulation composition of ROS-responsive liposomes encapsulating caffeic acid was screened, as follows: DHA is a polyunsaturated fatty acid. When exposed to reactive oxygen species (ROS), the double bonds of DHA are easily oxidized and broken, thus achieving a ROS response effect. Different DHA contents affect the drug release rate after ROS response. This invention aims to enable ROS-responsive liposomes to function in both irradiated tissues and cells. Therefore, the DHA content in the liposomes is optimized, as follows: ROS-responsive blank liposomes were prepared using the thin-film hydration method, and their formulations are shown in Table 1.

[0048] Table 1. Composition of ROS-responsive blank liposome formulations with different DHA contents

[0049] The organic solvents used in Table 1 are a mixture of chloroform and methanol, with a volume ratio of chloroform to methanol of 9:1.

[0050] The process for preparing ROS-responsive blank liposomes using the thin-film hydration method is as follows: Phospholipids, DHA, and cholesterol are dissolved together in an organic solvent according to the above-mentioned formulation. A uniform lipid film is formed on the wall of a flask by rotary evaporation (temperature 40℃, rotation speed 100 rpm). Then, PBS (3 mL) is added for hydration to form ROS-responsive liposomes with uniform particle size. The liposome solution is then imaged using TEM. The results are as follows: Figure 1 As shown; Figure A shows TEM images of ROS-responsive liposomes with different DHA ratios 48 hours after preparation, without X-ray irradiation. From this figure, it can be seen that when the mass ratio of DSPC:cholesterol:DHA is 3:1:0.01 (sample 1), some liposome structures break down, indicating that ROS-responsive liposomes with this ratio have poor stability and cannot be stored for a long time. When the mass ratio of DSPC:cholesterol:DHA is 3:1:0.05 (sample 2) and 3:1:0.1 (sample 3), the liposome structures are intact and the morphology is nearly spherical. ROS-responsive liposomes with these two ratios can be stably stored. Figure B shows TEM images of DHA liposomes irradiated with 6 Gy γ-rays for 24 hours after exposure. When the mass ratio of DSPC:cholesterol:DHA was 3:1:0.05 (sample 2), most of the lipid membrane of the ROS-responsive blank liposomes was ruptured. This result indicates that the formulation of sample 2 can achieve ROS response.

[0051] Subsequently, fluorescein isothiocyanate (FITC) was added during the synthesis of ROS-responsive blank liposomes to determine the drug release levels before and after irradiation. The results were as follows: Figure 2As shown in the figure, it can be seen that when the liposome solution was not irradiated, with a DSPC:cholesterol:DHA mass ratio of 3:1:0.01 (sample 1), after 96 hours of storage, approximately 50% of the drug was released, indicating that the ROS-responsive liposomes at this ratio were unstable. When the DSPC:cholesterol:DHA mass ratio was 3:1:0.05 (sample 2), the release rate of the unirradiated liposome solution was approximately 10%, indicating that the liposomes at this ratio could be stably stored. After the liposome solution was irradiated with 6 Gy rays for 96 hours, the release rate was approximately 50%, indicating that the ROS-responsive liposomes at this ratio could rapidly release some drugs in the high ROS region, and some drugs could still enter the cells to exert their effects. Therefore, the ROS-responsive blank liposomes with a DSPC:cholesterol:DHA mass ratio of 3:1:0.05 (sample 2) were ultimately selected to encapsulate caffeic acid for subsequent processes.

[0052] Secondly, the formulation composition of liposome I (CA@RR-Lipo) was screened using the thin-film hydration method, as shown in Table 2.

[0053] Table 2 Formulation composition of caffeic acid-loaded liposome I (CA@RR-Lipo)

[0054] The organic solvents used in Table 2 are a mixture of chloroform and methanol, with a volume ratio of chloroform to methanol of 9:1.

[0055] The process of preparing CA@RR-Lipo by thin-film hydration is as follows: Caffeic acid, phospholipids, DHA and cholesterol are dissolved together in an organic solvent according to the formula composition. A uniform lipid film is formed on the wall of the eggplant flask by rotary evaporation (temperature 40℃, rotation speed 100rpm). At this time, caffeic acid is embedded in the lipid bilayer. Then, PBS (3mL) is added for hydration to form CA@RR-Lipo with uniform particle size.

[0056] The amount of PBS added depends on the desired final concentration of caffeic acid.

[0057] After demulsification of samples 1, 2, 3, and 4, the supernatant was collected by centrifugation. The absorbance of caffeic acid at 280 nm was measured using a UV spectrophotometer. The encapsulation efficiency and drug loading of caffeic acid were calculated using a standard curve. The encapsulation efficiency and drug loading of CA@RR-Lipo with different formulations were statistically analyzed. The results are as follows: Figure 3 As shown; Figure A shows the encapsulation efficiency of different samples, and Figure B shows the drug loading of different samples. from Figure 3It can be seen that Sample 1 has the best encapsulation efficiency and drug loading. Therefore, CA@RR-Lipo prepared with the formulation of Sample 1 was used for subsequent processes.

[0058] II. Preparation of BH4-encapsulated liposomes II (denoted as BH4@Lipo).

[0059] The formulation composition of BH4@Lipo is shown in Table 3.

[0060] Table 3 Formulation composition of BH4@Lipo

[0061] The organic solvents used in Table 3 are a mixture of chloroform and methanol, with a volume ratio of chloroform to methanol of 9:1.

[0062] The process of preparing BH4@Lipo by thin film hydration is as follows: BH4, DSPC, cholesterol and HSPC are dissolved together in an organic solvent according to the formulation. A uniform lipid film is formed on the wall of the eggplant flask by rotary evaporation (temperature 40℃, rotation speed 100rpm). Then, PBS (3mL) is added for hydration to form BH4@Lipo with uniform particle size. The amount of PBS added depends on the desired final BH4 concentration.

[0063] After demulsification of samples 1, 2, 3, and 4, the supernatant was collected by centrifugation. The absorbance of BH4 at 230 nm was measured using a UV spectrophotometer. The encapsulation efficiency and drug loading of BH4 were calculated using a standard curve. The encapsulation efficiency and drug loading of BH4@Lipo with different formulations were statistically analyzed. The results are as follows: Figure 4 As shown; Figure A shows the encapsulation efficiency of different samples, and Figure B shows the drug loading of different samples. from Figure 4 It can be seen that sample 3 has the best encapsulation efficiency and drug loading. Therefore, BH4@Lipo prepared with the formulation of sample 3 was used for subsequent processes.

[0064] TEM images, particle size distribution curves, and particle size stability assessment results for CA@RR-Lipo (sample 1) and BH4@Lipo (sample 3) are shown below. Figure 5 , Figure 6 and Figure 7 As shown.

[0065] Example 2 The functions of CA@RR-Lipo (sample 1) and BH4@Lipo (sample 3) were investigated using cell experiments.

[0066] I. Investigation of the free radical scavenging efficiency of CA@RR-Lipo.

[0067] Caffeic acid was dissolved in anhydrous ethanol to prepare free caffeic acid at different concentrations (40 μM, 80 μM, 100 μM, 120 μM, 140 μM). The antioxidant activity of the free caffeic acid was detected by a DPPH scavenging assay. Figure 8 As shown in the figure, it can be seen that when the concentration of free caffeic acid solution is 100 μM, the free radical scavenging efficiency is approximately 70%. The antioxidant activity of CA@RR-Lipo at different caffeic acid concentrations (40 μM, 80 μM, 100 μM, 120 μM, and 140 μM) was detected by ABTS scavenging assay. The results are as follows: Figure 9 As shown in the figure, when the caffeic acid concentration is 100 μM, the free radical scavenging efficiency is approximately 70%.

[0068] Free radicals are normal byproducts of cellular metabolism. Appropriate amounts of free radicals participate in physiological processes such as immune defense and cell signaling. They also participate in various intracellular pathways. At low concentrations, free radicals have regulatory effects on the immune system and cell proliferation. Therefore, excessive scavenging of free radicals can interfere with normal intracellular pathways, affect immune cell activity, increase the risk of infection, or trigger autoimmune diseases. Furthermore, caffeic acid at high concentrations (approximately 500 μM) is cytotoxic; higher concentrations are not necessarily better. Scavenging approximately 70% of the free radicals generated after radiation can reduce oxidative stress. Therefore, a caffeic acid concentration of 100 μM was ultimately chosen for the experiment. The relevant reference documents are as follows: 1. Sies H, Jones D P. Reactive oxygen species (ROS) as pleiotropicphysiological signaling agents[J]. Nature Reviews Molecular Cell Biology, 2020, 21(7): 363-383.

[0069] 2. Lennicke C, Cochemé H M. Redox metabolism: ROS as specificmolecular regulators of cell signaling and function[J]. Molecular Cell, 2021,81(18): 3691-3707.

[0070] To investigate the antioxidant properties of different substances in cells, they were divided into the following groups: Blank: The blank control group that was not treated with probes or other substances and was not irradiated; NRT: Control group with added probes, no other treatments, and no irradiation; CA@RR-Lipo: The treatment group that has been treated with probe, CA@RR-Lipo solution (caffeic acid concentration of 100 μM) and irradiation; Blank liposome group (Lipo): The treatment group that has been treated with probes, blank liposome (CA@RR-Lipo without caffeic acid) solution and irradiation.

[0071] Different groups were treated with cells for 24 hours, followed by 6 Gy of gamma ray irradiation. The intracellular ROS positivity rate was then detected by flow cytometry using the ROS-sensitive probe DCFHDA. The results are as follows: Figure 10 As shown; Figure A shows the flow cytometry histogram of intracellular fluorescence intensity distribution, and Figure B shows the quantitative statistical analysis of the flow cytometry data. from Figure 10 It can be seen that the intracellular ROS in the CA@RR-Lipo treatment group was significantly reduced.

[0072] II. To investigate whether BH4@Lipo has a protective effect and whether simultaneous delivery with caffeic acid (CA@RR-Lipo) can protect BH4.

[0073] The grouping is as follows: NRT: Control group with added probes, no other treatments, and no irradiation; BH4@Lipo: The treatment group that has been treated with probe, BH4@Lipo solution (BH4 concentration of 10 μM) and irradiation; BC: The treatment group was treated with probe, BH4@Lipo and CA@RR-Lipo mixed solution (caffeic acid concentration of 100 μM and BH4 concentration of 10 μM) and irradiated.

[0074] Lipo: The treatment group that has had probes added, blank liposomes (BH4@Lipo without BH4) added, and has been irradiated.

[0075] In 3T3 cells, cells were treated according to groups for 24 hours; subsequently, the cells were irradiated with 6 Gy of gamma rays and incubated for another 24 hours. The levels of BH4 and BH2 in the cells were then detected using an ELISA assay, and the BH4 to BH2 ratio was calculated. The results are as follows: Figure 11As shown in the figure, it can be seen that: delivery of exogenous BH4 alone (BH4@Lipo group) can increase the intracellular BH4 / BH2 ratio, but the effect is limited. The intracellular BH4 / BH2 ratio of the group that delivers caffeic acid and BH4 simultaneously (BC group) is significantly increased and close to that of the unirradiated group. This result indicates that caffeic acid has a protective effect on BH4.

[0076] Cells were treated using the same methods as described in the previous experiment, and intracellular NOS activity was measured. Cell groups were the same as above, and the results are as follows: Figure 12 As shown in the figure, exogenous delivery of BH4 (BH4@Lipo group) can improve NOS activity, but there is still a difference compared with the unirradiated group. After simultaneous delivery of caffeic acid and BH4 (BC group), the NOS activity is not different from the unirradiated group. This result indicates that simultaneous delivery of caffeic acid and BH4 is more helpful in improving the intracellular NOS activity after irradiation.

[0077] After treating the cells using the same methods as in the experiments described above, nitric oxide (NO), the terminal substance of NOS in the cells, was detected. The grouping is the same as the previous grouping except that NRT adds probes to the grouping. Intracellular NO expression was detected using the nitric oxide-sensitive probe 2',7'-dichlorodihydrofluorescein diacetate (DCF-FM) via confocal microscopy. The results are as follows: Figure 13 As shown in the figure, it can be seen that the intracellular NO content in the BH4 group (BH4@Lipo group) was slightly increased compared with the untreated group after irradiation, while the intracellular NO content in the group that was delivered with both caffeic acid and BH4 (BC group) was significantly increased.

[0078] The above experimental results collectively indicate that simultaneous delivery of caffeic acid can protect BH4 by scavenging ROS, thereby increasing the intracellular BH4 / BH2 ratio, enhancing intracellular NOS activity and NO content. Caffeic acid and BH4 can synergistically improve post-radiation damage through multiple functions.

[0079] Example 3 To facilitate the application of the synthesized CA@RR-Lipo and BH4@Lipo liposomes to the skin and achieve long-term sustained drug release, hydrogels loaded with CA@RR-Lipo and BH4@Lipo were prepared.

[0080] I. A polypeptide was synthesized using a traditional solid-phase synthesis method. The polypeptide structure is shown below: ; The synthesis process, taking the synthesized polypeptide (0.5 mmol) as an example, involves the following specific steps: Step 1, Resin Swelling: Weigh 0.5g of resin and add it to a solid-phase synthesis tube, then add 5mL of dichloromethane (DCM) and place it on a shaker to swell for 5min; Step 2, First amino acid linkage: Weigh 234 mg (0.5 mmol) of fluorenylmethoxycarbonyl-arginine (2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl)-hydroxy (Fmoc-Arg(Pbf)-OH) into a vial, add DCM (10 mL) to dissolve it, then add N,N-diisopropylethylamine (DIEA) (200 μL, 1 mmol), mix thoroughly to obtain a mixed solution; squeeze out the swollen liquid from the solid-phase synthesis tube, add the mixed solution to the solid-phase synthesis tube, and react at room temperature for 2 h; Step 3, sealing: After the reaction is complete, squeeze out the reaction solution from the solid synthesis tube, wash the resin with DCM 5 times, and then add DCM-CH3OH-DIEA mixed sealing solution (the volume ratio of DCM-CH3OH-DIEA is 17:2:1) (10mL) to the solid synthesis tube. Seal at room temperature (about 25℃) for 30min. Step 4, Deprotection: After the blocking reaction is complete, squeeze out the blocking solution, wash the resin 5 times with DCM, and then wash the resin 5 times with N,N-dimethylformamide (DMF); then, add 5 mL of 20% piperidine DMF solution and deprotect at room temperature for 30 min to remove the fluorene methoxycarbonyl (Fmoc) protecting group. Step 5, Second Amino Acid Ligation: After the deprotection reaction is complete, squeeze out the deprotection solution and wash the resin 5 times with DMF; dissolve fluorenemethoxycarbonyl-glycine-hydroxy (Fmoc-Gly-OH) (2 mmol), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate (HBTU) (2 mmol), and N,N-diisopropylethylamine (DIEA) (4 mmol) in DMF (10 mL) to obtain the reaction solution; add the reaction solution to a solid-phase synthesis tube and react at room temperature for 2 h; Step 6, Subsequent Amino Acid Ligation: After the second amino acid reaction is completed, squeeze out the reaction solution and repeat the operations of Steps 3, 4 and 5 above, adding the required amino acids in sequence; until the last step, use 2-naphthaleneacetic acid as the peptide capping reagent and complete the capping reaction according to the above amino acid ligation process. Step 7, Peptide chain cleavage: After the end-capping reaction is completed, squeeze out the reaction solution, rinse the resin 5 times with DMF, and then wash the resin 5 times with DCM. Add a cleavage solution composed of trifluoroacetic acid (TFA), tris(hydroxymethyl)aminomethane (Tris), and deionized water (H2O) (the volume ratio of TFA, Tris, and H2O is 95:2.5:2.5) to the solid-phase synthesis tube, and react at room temperature (about 25°C) for 30 minutes to cleave the peptide chain from the resin. Step 8: Preparation of crude product: Collect the cleavage reaction solution in the solid-phase synthesis tube and concentrate it using a rotary evaporator; rinse the concentration container with DCM 3-4 times, add the rinsing solution to the concentrate, continue rotary evaporation until nearly dry, add ice-cold ether to the concentrate, stir evenly, and a white solid will precipitate; then, use a vacuum pump to dry it to obtain crude peptide product; Step 9: Peptide Purification: After dissolving the crude peptide in methanol, filter the solution through a 0.22 μm organic phase filter membrane. Purify the filtered sample using high-performance liquid chromatography (HPLC) at an injection concentration of approximately 100 mg / mL, with 80 μL injected each time. Detect the target peak in the HPLC chromatogram using high-resolution mass spectrometry (HRMS). Collect the eluent corresponding to the target peak, concentrate the eluent by rotary evaporation, and freeze-dry to obtain a white flocculent lyophilized peptide, i.e., the target peptide. Its HRMS chromatogram is shown below. Figure 14 As shown; its circular dichroism chromatogram is as follows. Figure 15 As shown.

[0081] II. Preparation of liposome-hydrogel.

[0082] The peptide was prepared into a 5 mg / mL solution using PBS, and then alkaline phosphatase aqueous solution (1 U / μL, 5 μL), CA@RR-Lipo and / or BH4@Lipo liposomes were added. After thorough mixing, the mixture was allowed to stand for about 1 h to obtain a liposome-hydrogel. Four types of hydrogels were prepared using the above method: Gel: Hydrogel unloaded with CA@RR-Lipo and BH4@Lipo liposomes.

[0083] CA@RR-Lipo Gel Hydrogel loaded with CA@RR-Lipo liposomes, with a concentration of 100 μM in the hydrogel; BH4@Lipo Gel Hydrogel loaded with BH4@Lipo liposomes, with a BH4@Lipo concentration of 10 μM in the hydrogel; BC Gel A hydrogel simultaneously loaded with CA@RR-Lipo and BH4@Lipo liposomes, with CA@RR-Lipo concentration of 100 μM and BH4@Lipo concentration of 10 μM in the hydrogel; CA@RR-Lipo Gel BH4@Lipo Gel and BC Gel TEM images, such as Figure 16 As shown in the figure, a liposome-hydrogel delivery system was successfully constructed using this peptide. BC Gel The rheological test results, such as Figure 17 As shown; Figure A is the amplitude scan diagram, and Figure B is the frequency scan diagram. from Figure 17 It can be concluded that: BC Gel The structurally stable elastic hydrogel possesses good mechanical strength and structural integrity, providing a reliable mechanical basis for subsequent applications.

[0084] The viability of cells after treatment with different concentrations of samples was determined using the CCK-8 assay to evaluate BC. Gel Biocompatibility, results as follows Figure 18 As shown, within the test concentration range of 1.5825–200 μM, the cell viability of each group remained above 90%, with no significant concentration-dependent changes. This result indicates that BC… Gel It exhibits excellent biocompatibility within the concentration range of 1.5825–200 μM, with a wide safety concentration window, which can meet the concentration requirements of subsequent cell function experiments and biomedical applications, laying a safety foundation for its further in vivo studies.

[0085] III. Pharmacodynamic experimental results of a mouse skin radiation injury model.

[0086] A mouse skin radiation injury model was established by irradiating the right hind leg of Balb / c mice with a single 40 Gy X-ray. Immediately after irradiation, hydrogels loaded with different drugs were applied to the irradiated area (specifically, the groups were: ①Gel, gel alone; ②BH4@Lipo Gel ③CA@RR-Lipo Gel ④BC Gel The hydrogels co-loaded with CA@RR-Lipo and BH4@Lipo were applied once daily for 21 days, and the results were as follows: Figure 19 As shown in the figure, we can see that: BC Gel Compared with other groups, the mice in this group showed symptoms in the irradiated areas later and the symptoms were milder 21 days after irradiation. The change of RTOG score in mice over time, such as Figure 20 As shown in the figure, we can see that: BC Gel The group of mice had lower scores; The above experimental results show that the simultaneous application of caffeic acid and BH4 is more helpful in reducing skin radiation damage and protecting the integrity of the skin.

[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A pharmaceutical composition having a sequential synergistic repair effect, characterized in that, This includes liposome I loaded with caffeic acid and liposome II loaded with BH4; The structural formula of caffeic acid is shown below: ； BH4 is tetrahydrobiopterin, and its structural formula is shown below: ； The liposome I is a ROS-responsive liposome containing DHA; DHA is docosahexaenoic acid, with the following structural formula: ； ROS stands for Reactive Oxygen Species.

2. The pharmaceutical composition with sequential synergistic repair effect as described in claim 1, characterized in that, The formulation of liposome I includes caffeic acid and DHA, as well as DSPC and cholesterol; The formulation of liposome II includes BH4, as well as DSPC, cholesterol and HSPC. Among them, DSPC is distearylphosphatidylcholine, and HSPC is hydrogenated soybean phosphatidylcholine.

3. The pharmaceutical composition with sequential synergistic repair effect as described in claim 2, characterized in that, The formulation of liposome I includes: The ratio of the mass of caffeic acid to the sum of the masses of DHA, DSPC, and cholesterol was 0.0075:4.05 to 0.06:4.

05. The mass ratio of DHA to caffeic acid is 5:6 to 5:0.

75. The mass ratio of DHA to the combined mass of DSPC and cholesterol is 0.01:4 to 0.1:

4.

4. The pharmaceutical composition with sequential synergistic repair effect as described in claim 2, characterized in that, The formulation of liposome II includes: The ratio of the mass of BH4 to the sum of the masses of DSPC, cholesterol, and HSPC is 0.125:4.05 to 1:4.

05.

5. The pharmaceutical composition with sequential synergistic repair effect as described in claim 2, characterized in that, The mass ratio of caffeic acid to BH4 is 3:400 to 12:

25.

6. A method for preparing a pharmaceutical composition with sequential synergistic repair effects, characterized in that, For preparing a pharmaceutical composition with sequential synergistic repair effects as described in any one of claims 1 to 5, both liposome I and liposome II are prepared by thin-film hydration.

7. The application of a pharmaceutical composition with sequential synergistic repair effects, characterized in that, A pharmaceutical preparation for dermatitis is prepared using the pharmaceutical composition having a sequential synergistic repair effect as described in any one of claims 1 to 5.

8. The application of the pharmaceutical composition with sequential synergistic repair effect as described in claim 7, characterized in that, The pharmaceutical preparations include topical gel preparations.

9. The application of the pharmaceutical composition with sequential synergistic repair effect as described in claim 8, characterized in that, The matrix of the topical gel formulation is selected from a hydrogel matrix with peptides as the main component, and the structural formula of the peptides is shown below: 。 10. The application of the pharmaceutical composition with sequential synergistic repair effect as described in claim 7, characterized in that, The dermatitis mentioned is radiation dermatitis.

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