Hyaluronic acid modified flavin adenine dinucleotide and synthesis method thereof

Abstract

The invention discloses hyaluronic acid modified flavin adenine dinucleotide and a synthesis method thereof, relates to the field of biological medicine, in particular to the field of tumor immunotherapy, and provides a synthesis method of hyaluronic acid (HA) modified flavin adenine dinucleotide (FAD), which is characterized in that amidation reaction of HA and FAD is mediated by DAST (diethylamino sulfur trifluoride), and the FAD is converted into the FAD. Aiming at the reaction which is sensitive to reaction conditions and low in byproduct tolerance, the invention provides a solution for effectively ensuring the biological activity of the product. The obtained hyaluronic acid modified flavin adenine dinucleotide product shows biological activity in related experiments of anti-tumor performance, targeting performance and the like.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the field of tumor immunotherapy, and particularly to a hyaluronic acid-modified flavin adenine dinucleotide and its synthesis method. Background Technology

[0002] Cancer is a malignant disease caused by abnormal cell proliferation, seriously endangering life and health. Current radiotherapy and chemotherapy treatments for cancer both significantly attack healthy cells. While killing tumor cells, existing radiotherapy and chemotherapy methods lack precise targeting, easily causing significant damage to rapidly dividing healthy cells (such as bone marrow, digestive tract mucosa, and hair follicle cells), leading to serious adverse reactions such as bone marrow suppression, nausea and vomiting, and hair loss. These adverse reactions affect patients' quality of life and treatment adherence, necessitating improvements in treatment selectivity to reduce toxic side effects.

[0003] Flavin adenine dinucleotide (FAD) is a key redox coenzyme in the body, participating in a variety of metabolic reactions and mitochondrial function regulation.

[0004] This invention provides a specific method for synthesizing specially modified flavin adenine dinucleotide (FAD), which enables the obtained reaction product to exhibit biological activity in performance characterization tests such as antitumor or targeted therapy, and has the potential for application in anticancer treatment. Currently, there are no related reports on this synthesis method. Summary of the Invention

[0005] The main objective of this invention is to propose a method for synthesizing hyaluronic acid-modified flavin adenine dinucleotide. The synthesized hyaluronic acid-modified flavin adenine dinucleotide product has improved anti-tumor or targeted effects in the treatment of cancer.

[0006] A secondary objective of this invention is to provide a hyaluronic acid-modified flavin adenine dinucleotide, synthesized by the aforementioned synthetic method.

[0007] To achieve the above objectives, this invention proposes a method for synthesizing hyaluronic acid-modified flavin adenine dinucleotide, comprising the following preparation steps: S0: Hyaluronic acid solution and flavin adenine dinucleotide solution were prepared using hyaluronic acid and flavin adenine dinucleotide as substrates, respectively. S1: Add DAST dropwise to the hyaluronic acid solution at a rate of 0.05~0.10 mL / min under ice bath conditions. After the addition is complete, remove the ice bath and heat the hyaluronic acid solution to 20~30℃ for stirring and activation. S2: Add the flavin adenine dinucleotide solution dropwise into the hyaluronic acid solution, and an amidation reaction occurs at 22~28℃ to obtain the reaction solution; S3: Add pre-cooled organic solvent (-20~0℃) to the reaction solution until a precipitate forms. Collect the precipitate by centrifugation to obtain the crude product.

[0008] Preferably, the molecular weight of hyaluronic acid is 5-200 kDa.

[0009] Preferably, the organic solvent in S3 is one of diethyl ether, n-hexane, petroleum ether, chilled pentane, or dichloromethane. More preferably, the organic solvent in S3 is pre-cooled diethyl ether at -20°C.

[0010] The present invention provides a method for synthesizing hyaluronic acid (HA)-modified flavin adenine dinucleotide (FAD). The resulting product has been experimentally found to possess antitumor and targeted properties, i.e., it exhibits biological activity. The synthesis method of the present invention is simple, using hyaluronic acid (HA) and flavin adenine dinucleotide (FAD) as reaction substrates. A slow, low-temperature addition of DAST (diethylaminotrifluoride) is employed to avoid the decomposition of the flavin ring in FAD. The amidation reaction is carried out at room temperature to avoid the destruction of FAD by high temperatures. After the reaction, a pre-cooled organic solvent is used to precipitate the product, preventing unreacted raw materials from affecting the final product. In existing studies, such as the paper "Synthesis of Novel 2,2'-Glycidyl-L-Threonylpyrimidine Nucleoside" published by the Institute of Pharmaceutical Sciences of Zhengzhou University in 2023, DAST was used to participate in the condensation reaction and the yield was improved. However, the process is complex and requires nine steps. Moreover, the condensed product obtained did not show any biological activity in in vitro antiviral and antitumor studies. Traditional condensing agents (such as EDC / NHS) form active esters (O-acylisourea / NHS esters) through carboxyl activation, which then react with amino groups. This usually requires room temperature or slightly higher temperatures, and sometimes heating. The reaction usually takes a long time (12-24 hours). The urea byproducts derived from EDC are difficult to remove, affecting the purity of the product and resulting in a low yield (30-60%). The operation usually requires two steps, including EDC and NHS activation of carboxyl groups and amidation. The long reaction time and potential high temperature requirements increase the risk of FAD degradation and affect its biological activity. This invention utilizes DAST to mediate the amidation reaction between HA and FAD. The reaction involves the activation of the carboxyl group to form an acyl fluoride intermediate, which then reacts with the amino group. This reaction can be carried out at room temperature with a significantly shortened reaction time of approximately 8 hours, significantly improving reaction efficiency and yield, reaching over 60%. The main byproducts are volatile gases such as HF, Et₂NH, and SO₂, which are easy to handle and have minimal impact on product purity. In terms of operation, DAST simultaneously activates the carboxyl group and mediates the amidation reaction, achieving a one-step reaction. The short reaction time under mild room temperature conditions is more conducive to maintaining the biological activity and structural integrity of FAD.

[0011] In the specific synthesis, hyaluronic acid-modified flavin adenine dinucleotide (HA-FAD) is formed by an amidation reaction between the carboxyl group (-COOH) on HA and the hydroxyl group (-NH2) on FAD under specific conditions, creating a stable covalent bond and avoiding premature release caused by physical mixing. However, high molecular weight HA (>1000 kDa) may lead to steric hindrance, reducing the binding efficiency of FAD; while low molecular weight HA (<5 kDa), although easily bound, may affect targeting. Therefore, medium molecular weight HA (5-200 kDa) is preferred for synthesizing the HA-FAD complex, achieving a balance between binding efficiency and targeting performance.

[0012] Preferably, the molecular weight of hyaluronic acid is 8000 Da. In step S0, the hyaluronic acid and flavin adenine dinucleotide (FAD) used as substrates are pretreated and activated during solution preparation. The activation step for hyaluronic acid is as follows: hyaluronic acid is dissolved in anhydrous DMSO / TEA mixture at a volume ratio of 3:1 to 5:1, and stirred at 30 to 40°C until transparent, resulting in a hyaluronic acid solution with a solute mass concentration of 17 g / L to 25 g / L. The activation step for flavin adenine dinucleotide (FAD) is as follows: flavin adenine dinucleotide (FAD) is dissolved in anhydrous DMSO, and then TEA is added at a volume ratio of 65:1 to 75:1. The mixture is stirred at 22 to 28°C to obtain a flavin adenine dinucleotide (FAD) solution with a solute mass concentration of 30.5 g / L to 38.5 g / L. This substrate pretreatment and activation step addresses the low reactivity of the polysaccharide carboxyl group, and combined with the low-temperature dropwise addition of DAST, achieves a highly efficient reaction at room temperature.

[0013] Preferably, after step S3, step S4 is included, in which the crude product is dissolved in PBS buffer at pH 7.3-7.5 to obtain a mixture. This mixture is then transferred to a dialysis bag and dialyzed in the dark, with the buffer changed 4-6 times. The resulting residue is freeze-dried to obtain purified hyaluronic acid-modified flavin adenine dinucleotide (FAD) product. The molecular weight cutoff of the dialysis bag is greater than or equal to the maximum molecular weight of the remaining substrate material in the reaction, but less than the molecular weight of the hyaluronic acid-modified FAD product. Step S4 effectively improves the dialysis efficiency, yielding a high-purity hyaluronic acid-modified FAD product with a purity exceeding 60%. More preferably, the molecular weight cutoff of the dialysis bag is 3500-5000 Da. Adding excess flavin adenine dinucleotide (FAD) to the reaction helps to completely consume the hyaluronic acid (HA). Using a dialysis bag with a molecular weight cutoff of 3500-5000 Da effectively improves the dialysis efficiency and product purity.

[0014] Preferably, the amidation reaction occurring in S2 includes the following two steps: 1) HA-COOH + Et2N-SF3 (DAST) → HA-COF + Et2NSOF2+ HF; 2) HA-COF + FAD-NH2→ HA-CO-NH-FAD + HF; The overall reaction is: HA-COOH + FAD-NH2 + Et2N-SF3 → HA-CO-NH-FAD + Et2NSOF2 + 2HF.

[0015] This invention also proposes a hyaluronic acid-modified flavin adenine dinucleotide, prepared by the synthetic method for hyaluronic acid-modified flavin adenine dinucleotide described in any of the aforementioned schemes. It possesses all the beneficial effects of any of the aforementioned preparation schemes, which will not be elaborated upon here.

[0016] Unlike existing technologies, the main beneficial effects of this invention are: 1. This invention obtains a bioactive hyaluronic acid-modified flavin adenine dinucleotide product by DAST-mediated amidation reaction of HA and FAD. For this type of reaction which is sensitive to reaction conditions and has low tolerance for byproducts, this invention provides a solution that effectively ensures the bioactivity of the product. The obtained hyaluronic acid-modified flavin adenine dinucleotide product shows bioactivity in antitumor and targeting performance experiments.

[0017] 2. This invention provides a highly efficient, rapid, mild, and easily removable byproduct synthesis reaction for hyaluronic acid-modified flavin adenine dinucleotide, which is beneficial for obtaining products with high yield and high purity. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The above is the 1H NMR spectrum of the reaction product HA-FAD (hyaluronic acid-modified flavin adenine dinucleotide) in the embodiments of the present invention; Figure 2 The above is the 1H NMR spectrum of the reaction substrate HA (hyaluronic acid) in this embodiment of the invention. Figure 3 This is the 1H NMR spectrum of the reaction substrate FAD (flavin adenine dinucleotide) in the embodiments of the present invention; Figure 4This invention employs a dialysis method and a UV spectrophotometer to detect the drug release behavior of the reaction product HA-FAD in a simulated tumor microenvironment (pH 5.0).

[0020] Figure 5 This is a diagram showing the uptake of FAD by THP1 cells after co-culturing FAD (flavin adenine dinucleotide) and HA-FAD (hyaluronic acid modified flavin adenine dinucleotide) with THP1 cells for 24 h using flow cytometry, according to the present invention. Figures 6-A to 6-C This is a comparison of mouse tumor growth in an in vivo experiment using a mouse orthotopic liver cancer model to verify the efficacy of HA-FAD for HCC immunotherapy. Figure 7 This is a graph showing the changes in FAD content in orthotopic liver cancer tissue after HA-FAD treatment, detected by LC-MS method according to the present invention. Figure 8 This is a graph showing the changes in the proportion of macrophage M1 and M2 phenotypes in orthotopic liver cancer tissue after different drug treatments using flow cytometry, according to the present invention. Figure 9 This is a graph showing the changes in the proportion of CD4+ and CD8+ T cells in orthotopic liver cancer tissue after different drug treatments using flow cytometry, as described in this invention. Figure 10 This invention utilizes flow cytometry to detect changes in the proportion of memory T cells in the spleen. Figure 11 This is a graph showing the change in the proportion of Tregs cells in orthotopic liver cancer tissue detected by flow cytometry according to the present invention.

[0021] The realization of the purpose, functional features and advantages of this application will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. In addition, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0023] This application provides a method for synthesizing hyaluronic acid-modified flavin adenine dinucleotide, comprising the following preparation steps: S0: Hyaluronic acid solution and flavin adenine dinucleotide solution were prepared using hyaluronic acid and flavin adenine dinucleotide as substrates, respectively. S1: Add DAST dropwise to the hyaluronic acid solution at a rate of 0.05~0.10 mL / min under ice bath conditions. After the addition is complete, remove the ice bath and heat the hyaluronic acid solution to 20~30℃ for stirring and activation. S2: Add the flavin adenine dinucleotide solution dropwise into the hyaluronic acid solution, and an amidation reaction occurs at 22~28℃ to obtain the reaction solution; S3: Add pre-cooled organic solvent (-20~0℃) to the reaction solution until a precipitate forms. Collect the precipitate by centrifugation to obtain the crude product.

[0024] In a preferred embodiment, the molecular weight of hyaluronic acid is 5-200 kDa.

[0025] In a further preferred embodiment, the molecular weight of hyaluronic acid is 8000 Da; in step S0, the hyaluronic acid and flavin adenine dinucleotide, which are used as substrates, are pretreated and activated during solution preparation. The activation step of hyaluronic acid is as follows: hyaluronic acid is dissolved in anhydrous DMSO / TEA mixture with a volume ratio of 3:1 to 5:1, and stirred at 30 to 40°C until transparent, to obtain a hyaluronic acid solution with a solute mass concentration of 17 g / L to 25 g / L; the activation step of flavin adenine dinucleotide is as follows: flavin adenine dinucleotide is dissolved in anhydrous DMSO, and then TEA is added, with an anhydrous DMSO:TEA volume ratio of 65:1 to 75:1, and stirred at 22 to 28°C to obtain a flavin adenine dinucleotide solution with a solute mass concentration of 30.5 g / L to 38.5 g / L.

[0026] In a preferred embodiment, after S3, S4 is further included, in which the crude product is dissolved in PBS buffer at pH 7.3-7.5 to obtain a mixture. The mixture is transferred to a dialysis bag and dialyzed in the dark for 4-6 times, changing the buffer each time. The resulting retention solution is freeze-dried to obtain purified hyaluronic acid-modified flavin adenine dinucleotide product. The molecular weight cutoff of the dialysis bag is greater than or equal to the maximum molecular weight of the remaining substrate raw material in the reaction, and less than the molecular weight of the hyaluronic acid-modified flavin adenine dinucleotide product. More preferably, the molecular weight cutoff of the dialysis bag is 3500-5000 Da.

[0027] In a preferred embodiment, the organic solvent in S3 is one of diethyl ether, n-hexane, petroleum ether, cold pentane, or dichloromethane.

[0028] In a preferred embodiment, the organic solvent in S3 is pre-cooled diethyl ether at -20°C.

[0029] In a preferred embodiment, the amidation reaction that occurs in S2 includes the following two steps: 1) HA-COOH + Et2N-SF3 (DAST) → HA-COF + Et2NSOF2+ HF; 2) HA-COF + FAD-NH2→ HA-CO-NH-FAD + HF; The overall reaction is: HA-COOH + FAD-NH2 + Et2N-SF3 → HA-CO-NH-FAD + Et2NSOF2 + 2HF.

[0030] This application also provides a hyaluronic acid-modified flavin adenine dinucleotide, prepared by the synthetic method of hyaluronic acid-modified flavin adenine dinucleotide as described in any of the preceding schemes.

[0031] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. In the following embodiments, raw materials and reagents, unless otherwise specified, are obtained commercially available, and preparation methods without special conditions are prepared using conventional and well-known preparation methods.

[0032] Example 1 A method for synthesizing hyaluronic acid-modified flavin adenine dinucleotide is provided, comprising the following steps: 1. Substrate pretreatment 1) HA activation: Dissolve HA (100mg, 8000Da) in 5 mL of anhydrous DMSO / TEA mixture (V:V=4:1) and stir at 40℃ for 1 h until a clear solution is obtained.

[0033] 2) FAD activation: Dissolve FAD (230.8 mg) in 7 mL of anhydrous DMSO, add 0.1 mL of TEA, and stir at 25°C for 30 min.

[0034] 2. Cool the activated HA solution to 0℃ and slowly add DAST solution (1 mL / min). 3. Remove the cold bath, raise the temperature to 25℃, and stir to activate for 30 minutes.

[0035] 4. Add FAD solution dropwise (0.5 mL / min) and maintain the reaction at 28±1℃ for 8 h to obtain the reaction solution (TLC monitoring: EtOAc / MeOH / H2O=5:4:1, FAD Rf=0.3→product Rf=0.1).

[0036] 1) Inject the reaction solution into pre-cooled diethyl ether (100 mL, -20℃) to precipitate. 2) Centrifuge (8000 rpm, 5 min) to collect the solid, and wash with cold ether (3 × 5 mL). 3) Dissolve the crude product in 10 mL of pH 7.4 PBS buffer, transfer the mixture to a 3500 Da dialysis bag, and dialyze for 24 h in the dark, changing the buffer 5 times. 4) Freeze-drying yielded a pale yellow, spongy HA-FAD compound.

[0037] Results: The yield of the obtained product was 80%, the purity was 95%, and it showed biological activity in the characterization and detection of antitumor and targeted properties.

[0038] Example 2 Compared with Example 1, all other conditions were the same, except that the molecular weight of hyaluronic acid (HA) in the substrate was lower, at 5000 Da, and other process parameters were adjusted accordingly.

[0039] Results: The yield of the obtained product was 75%, the purity was 85%, and it showed biological activity in the characterization and detection of antitumor and targeted properties.

[0040] Example 3 Compared with Example 1, all other conditions were the same, except that the molecular weight of hyaluronic acid (HA) in the substrate was higher, at 200 kDa, and other process parameters were adjusted accordingly.

[0041] Results: The yield of the obtained product was 70%, the purity was 90%, and it showed biological activity in the characterization and detection of antitumor and targeted properties.

[0042] Comparative Example 1 A method for synthesizing hyaluronic acid-modified flavin adenine dinucleotide is provided, comprising the following steps: HA-FAD is synthesized by covalently linking the carboxyl group on HA and the hydroxyl group on FAD through an esterification reaction. 100 mg HA (8000 Da) was added to 5 ml of anhydrous formamide, and the mixture was slowly heated and stirred until completely dissolved. It was then cooled to room temperature. 96 mg EDC and 58 mg NHS were weighed and added to the HA solution, and stirred at room temperature (25°C) for 2 hours. 230.8 mg FAD was added to 7 ml of dimethylformamide, heated to 80°C to dissolve, and then slowly added to the HA mixture while hot. The mixture was stirred at 60°C at 300 rpm in the dark for 12 hours, and then stirred in the dark at room temperature for 24 hours. The mixture was transferred to a 3500 Da dialysis bag and dialyzed sequentially with deionized water / ethanol (volume ratios 1:3, 1:2, 1:1), for a total dialysis time of 48 hours. It was then dialyzed with deionized water for 24 hours, filtered through a 0.45 μm filter membrane, and dried in a vacuum dryer for 24 hours to produce a dry powder.

[0043] Results: The yield of the obtained product was 30%, and the purity was 60%. The total reaction time was too long, the temperature was too high, and the urea byproducts were difficult to remove. The yield and purity were worse than those of Example 1.

[0044] Comparative Example 2 Compared with Example 1, all other conditions were the same, except that: no pretreatment activation of the two substrates HA and FAD was performed, and the reaction time in step 4 reached 72h.

[0045] Results: The yield of the obtained product was 20%, its purity was 70%, the total reaction time was too long, and no biological activity was shown in the characterization of antitumor and targeted properties.

[0046] As can be seen from Examples 1 to 3, in the synthesis method of this application, the substrate used is hyaluronic acid with a molecular weight in the range of 5k to 200Da, and the reaction products all show biological activity. At the same time, the yield reaches 70-80%, the purity is greater than 85%, and the bioactivity is shown in the characterization and detection of anti-tumor and targeted performance.

[0047] Comparing Example 1 with Comparative Examples 1 and 2, it is evident that the synthesis method of this application significantly improves the purity and yield of the reaction product compared to the conventional synthesis reaction using the traditional condensing agent EDC. Example 1 exhibits a significantly shorter reaction time than Comparative Examples 1 and 2, while also demonstrating biological activity (not shown in Comparative Example 2). It is clear that the amidation reaction of HA and FAD is sensitive to reaction conditions and has low tolerance for byproducts. This application, through a DAST-mediated HA-FAD amidation reaction, strictly controls experimental conditions to obtain a hyaluronic acid-modified flavin adenine dinucleotide product that demonstrates biological activity in characterization and detection of antitumor and targeted properties.

[0048] The reaction product HA-FAD obtained in Example 1 was characterized and measured to confirm its biological activity.

[0049] in: I. The method for determining the content of FAD in tumor tissue is by liquid chromatography-mass spectrometry (LC-MS), and the steps are as follows: (1) Weigh 300 mg of sample and add 1100 μL of 75% methanol, vortex for 60 seconds. (2) Add steel balls, put them in a grinder, and grind 6 times until uniform. (3) Place the sample in liquid nitrogen for 1 minute, thaw at room temperature, and sonicate for 10 minutes. (4) Repeat step 3 twice. (5) Centrifuge 17000g for 15 minutes and take the supernatant for later use.

[0050] (6) Prepare a series of standard solutions of different concentrations using 75% methanol. (7) Using MultiQuant 3.0.3 analysis software, the sample concentration was calculated by using the external standard method to plot a standard curve with the standard response and corresponding concentration.

[0051] II. The effect of the reaction product HA-FAD on the phenotype of immune cells in tumor tissue was detected by flow cytometry, as follows: (1) Extraction of immune cells from tumor tissue. Fresh tumor tissue was placed in pre-cooled PBS and cut into 1 mm³ pieces using sterile surgical scissors. The tissue was then digested in prepared enzymatic digestion buffer (RPMI-1640 medium containing 1 mg / mL collagenase IV, 0.2 mg / mL DNase I, and 0.2 mg / mL hyaluronidase) at 37°C with shaking for 30 min (200 rpm). After digestion, the cells were passed through a 40 μm cell sieve and centrifuged (300×g, 5 min, 4°C). 1 mL of erythrocyte lysis buffer was added, and the cells were incubated at room temperature for 5 min. After centrifugation, the cells were resuspended in 1 mL of sample diluent and slowly stacked onto 4 mL of separation medium. The cells were centrifuged at 400×g density gradient for 25 min (without brakes), and the white membrane layer cells were aspirated for target marker labeling and screening.

[0052] (2) Extraction of immune cells from spleen. Fresh spleen was placed in a cold RPMI-1640 containing 2% FBS. The capsule and blood vessels were removed. Cells were ground on a 70 μm cell sieve using a sterile syringe plunger, and the cells were washed and collected. After centrifugation (300×g, 5 min, 4℃), the supernatant was discarded, 1 mL of erythrocyte lysis buffer was added, and the mixture was incubated at room temperature for 5 min. After centrifugation, the cells were resuspended in 1 mL of sample diluent and slowly stacked onto 4 mL of separation medium. The mixture was centrifuged at a density gradient of 400×g for 25 min (without brakes), and the white membrane layer cells were aspirated for target marker labeling and screening.

[0053] 1. Characterization of HA-FAD The structure of HA-FAD was confirmed using nuclear magnetic resonance (NMR) spectroscopy. 10-20 mg of the reaction product HA-FAD, substrate HA, and substrate FAD were dissolved in 0.6 mL of deuterated reagent, then injected into a syringe. The proton NMR spectra were recorded using a magnetic resonance spectrometer. The structure of HA-CUR was confirmed using the characteristic peaks of HA and FAD. Figure 1 ~Appendix Figure 3 .

[0054] 2. Determination of in vitro HA-FAD release Objective: To investigate the drug release behavior of the reaction product HA-FAD in a simulated tumor microenvironment (pH 5.0) using dialysis.

[0055] Methods: 2 mL of HA-FAD solution was added to a dialysis bag (molecular weight cutoff 3500 Da), and then the dialysis bag was immersed in 50 mL of PBS (pH 5.0) release medium. The sample was shaken at 200 rpm in a 37°C constant-temperature shaker. At 0, 1, 2, 4, 6, 8, 10, 12, 24, 48, and 72 h, 2 mL of sample was removed from the release medium, and an equal volume of fresh medium was added. The absorbance of the removed sample was measured using a UV spectrophotometer, and the concentration was calculated by substituting the values ​​into the FAD standard curve. Finally, the amount of FAD released into PBS was calculated using the following formula.

[0056] Results: The release rate of FAD was detected using a UV spectrophotometer. The release rate was 48.6% at 10 hours and 77.6% at 72 hours. (See attached image) Figure 4 As shown.

[0057] 3. Determination of in vitro HA-FAD uptake Objective: To detect the uptake of FAD by cells using flow cytometry by utilizing the autofluorescence of the reaction product FAD at 450 nm.

[0058] Methods: In vitro cell uptake experiments were conducted using THP1 cells as the experimental subjects. THP1 cells were treated with HA-FAD and the same dose of FAD for 24 h, respectively. The intracellular FAD content was detected by flow cytometry.

[0059] Results: The rate of FAD uptake in THP1 cells was 5.48%, and the rate of HA-FAD uptake was 21.91%, as shown in the attached figure. Figure 5 As shown.

[0060] 4. Antitumor effect of the reaction product HA-FAD Based on the therapeutic need for specific targeting of the tumor microenvironment, this study innovatively constructed a HA-functionalized FAD (HA-FAD) and verified its efficacy in in vivo for HCC immunotherapy. A mouse orthotopic hepatocellular carcinoma model was established using luc-Hepa1-6 cells, with separate control groups (administered with saline), FAD-treated groups, and HA-FAD-treated groups. After one week of continuous administration, tumor growth was monitored using a small animal in vivo imaging system on day 7, and the size of intrahepatic tumors was compared. The results showed that, compared with the control group, FAD slowed tumor cell growth; and compared with the FAD-treated group, the HA-FAD-treated group inhibited tumor cell growth (Figure 6 AC).

[0061] Figure 6 shows the experimental treatment pattern: C57BL / 6J mice were orally implanted with Hepa1-6-luc cells in their livers for 7 days before drug treatment. From day 0 to day 6, control mice were intraperitoneally injected with saline, while experimental mice were treated with FAD (flavin adenine dinucleotide) and HA-FAD (hyaluronic acid-modified flavin adenine dinucleotide). Samples were collected on day 7. Figure B shows hematoxylin and eosin (H&E) staining of mouse liver tumor tissue. Figure C shows changes in the tumor detected by a small animal in vivo imaging system.

[0062] 5. Verification of the targeting of HA-FAD in the reaction products To verify the targeting of HA-FAD, the content of FAD in tumor tissues of each group after treatment in a luc-Hepa1-6 cell mouse orthotopic liver cancer model was detected by LC-MS.

[0063] The results showed that, compared with the FAD monotherapy group, the concentration of FAD in tumor tissue increased after HA-FAD treatment, as shown in the appendix. Figure 7 As shown.

[0064] 6. Effects of the reaction product HA-FAD on tumor-associated macrophages To investigate the effect of FAD on the polarization phenotype of TAMs in HCC tissues, this study used flow cytometry to detect changes in the proportion of immune cell populations in tumor and spleen tissues of mice in each group.

[0065] A C57BL / 6J mouse Hepa1-6-luc cell hepatocellular carcinoma orthotopic model was constructed. Control mice were injected intraperitoneally with saline, while experimental mice were treated with FAD (flavin adenine dinucleotide) and HA-FAD (hyaluronic acid-modified flavin adenine dinucleotide) for 7 days. Flow cytometry was used to detect the changes in the proportion of macrophage M1 and M2 phenotypes in tumor tissue.

[0066] The results showed that the percentage of M1 phenotype macrophages (CD80, CD86) in tumor tissue was 13.46% in the control group, 23.29% after FAD treatment, and significantly higher after HA-FAD treatment (32.49%) compared to the FAD treatment group. Furthermore, after FAD treatment, the percentage of M2 phenotype macrophages (CD163, CD206) infiltrating tumor tissue was 9.7%, while after HA-FAD treatment, the percentage decreased to 4.31%. These results suggest that compared to FAD alone, HA-FAD can further promote macrophage phenotype remodeling and improve the tumor immunosuppressive microenvironment, as shown in the appendix. Figure 8 As shown 7. Effects of the reaction product HA-FAD on tumor-associated T cells Flow cytometry was used to detect changes in the proportions of CD4+ T cells and CD8+ T cells in mouse liver cancer tissue after treatment with FAD (flavin adenine dinucleotide) and HA-FAD (hyaluronic acid-modified flavin adenine dinucleotide). The results are shown in the attached figure. Figure 9 As shown.

[0067] The changes in the proportion of memory T cells in the spleen of mice after treatment with FAD (flavin adenine dinucleotide) and HA-FAD (hyaluronic acid-modified flavin adenine dinucleotide) were detected by flow cytometry. The results are shown in the attached figure. Figure 10 As shown.

[0068] The changes in the proportion of Treg cells in mouse hepatocellular carcinoma tissues after treatment with FAD (flavin adenine dinucleotide) and HA-FAD (hyaluronic acid-modified flavin adenine dinucleotide) were detected by flow cytometry. The results are shown in the attached figure. Figure 11 As shown.

[0069] From the appendix Figures 9-11 The results showed that FAD treatment increased the number of CD4+ T cells resident in liver cancer tissue by 6.22%, and HA-FAD treatment further increased this to 11.09%; at the same time, HA-FAD treatment significantly increased the proportion of CD8+ T cells (18.54%). Figure 9 The above indicates that, compared with FAD, HA-FAD can effectively enhance the recruitment of cytotoxic effector T cells in tumor tissue. Detection of changes in memory T cell populations in the spleen of mice revealed that, compared with the FAD treatment group, HA-FAD effectively enhanced the level of memory T cells in mice with hepatocellular carcinoma (19.93%). Figure 10 Studies have shown that the accumulation of regulatory T cells (Tregs) can inhibit the activation of effector T cells and promote tumor progression and spread. In this study, we comprehensively evaluated the role of FAD in regulating the tumor immune microenvironment by assessing the levels of Tregs in different treatment groups. The results showed that compared with the FAD-treated group, HA-FAD significantly inhibited the infiltration of Tregs in hepatocellular carcinoma tissue (11.07%). Figure 11 The above results collectively demonstrate that HA-FAD can effectively activate the immune response in mice with liver cancer and exert a pharmacological effect against liver cancer.

[0070] The above description is merely a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural transformations made using the contents of the present invention under the inventive concept of the present invention, or direct / indirect applications in other related technical fields, are included within the patent protection scope of the present invention.

Claims

1. A method for synthesizing hyaluronic acid-modified flavin adenine dinucleotide, characterized in that, The preparation steps include the following: S0: Hyaluronic acid solution and flavin adenine dinucleotide solution were prepared using hyaluronic acid and flavin adenine dinucleotide as substrates, respectively. S1: Under ice bath conditions, DAST is added dropwise to the hyaluronic acid solution at a rate of 0.05~0.10 mL / min. After the addition is completed, the ice bath is removed and the hyaluronic acid solution is heated to 20~30℃ and stirred for activation. S2: The flavin adenine dinucleotide solution is added dropwise to the hyaluronic acid solution, and an amidation reaction occurs at 22~28℃ to obtain a reaction solution; S3: Add an organic solvent that has been pre-cooled to -20~0℃ to the reaction solution until a precipitate forms. Collect the precipitate by centrifugation to obtain the crude product.

2. The method for synthesizing hyaluronic acid-modified flavin adenine dinucleotide as described in claim 1, characterized in that, The molecular weight of the hyaluronic acid is 5-200 kDa.

3. The method for synthesizing hyaluronic acid-modified flavin adenine dinucleotide as described in claim 2, characterized in that, The molecular weight of the hyaluronic acid is 8000 Da; In step S0, hyaluronic acid and flavin adenine dinucleotide, which are used as substrates, are pretreated and activated during solution preparation. The activation step of the hyaluronic acid is as follows: dissolve the hyaluronic acid in an anhydrous DMSO / TEA mixture with a volume ratio of 3:1 to 5:1, stir at 30 to 40°C until transparent, and obtain a hyaluronic acid solution with a solute mass concentration of 17 g / L to 25 g / L. The steps for activating the flavin adenine dinucleotide are as follows: dissolve the flavin adenine dinucleotide in anhydrous DMSO, then add TEA, wherein the volume ratio of anhydrous DMSO to TEA is 65:1 to 75:1, and stir at 22 to 28°C to obtain a flavin adenine dinucleotide solution with a solute mass concentration of 30.5 g / L to 38.5 g / L.

4. The method for synthesizing hyaluronic acid-modified flavin adenine dinucleotide as described in claim 1 or 3, characterized in that, Following S3, S4 is also included, in which the crude product is dissolved in PBS buffer at pH 7.3-7.5 to obtain a mixture. The mixture is then transferred to a dialysis bag and dialyzed in the dark for 4-6 times. The resulting retention solution is freeze-dried to obtain a purified hyaluronic acid-modified flavin adenine dinucleotide product. The molecular weight cutoff of the dialysis bag is greater than or equal to the maximum molecular weight of the remaining substrate raw material in the reaction, and less than the molecular weight of the hyaluronic acid-modified flavin adenine dinucleotide product.

5. The method for synthesizing hyaluronic acid-modified flavin adenine dinucleotide as described in claim 4, characterized in that, The molecular weight cutoff of the dialysis bag is 3500~5000 Da.

6. The method for synthesizing hyaluronic acid-modified flavin adenine dinucleotide as described in claim 1, characterized in that, The organic solvent mentioned in S3 is one of diethyl ether, n-hexane, petroleum ether, cold pentane, or dichloromethane.

7. The method for synthesizing hyaluronic acid-modified flavin adenine dinucleotide as described in claim 6, characterized in that, The organic solvent mentioned in S3 is pre-cooled diethyl ether at -20°C.

8. The method for synthesizing hyaluronic acid-modified flavin adenine dinucleotide as described in claim 1, characterized in that, The amidation reaction that occurs in S2 includes the following two steps: 1) HA-COOH + Et2N-SF3 (DAST) → HA-COF + Et2NSOF2 + HF; 2) HA-COF + FAD-NH2 → HA-CO-NH-FAD + HF; The overall reaction is: HA-COOH + FAD-NH2 + Et2N-SF3 → HA-CO-NH-FAD + Et2NSOF2 + 2HF.

9. Hyaluronic acid-modified flavin adenine dinucleotide, characterized in that, It is prepared by the method for synthesizing hyaluronic acid-modified flavin adenine dinucleotide as described in any one of claims 1 to 8.

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