A tea polyphenol ester-rna self-assembled hybrid vesicle with probiotic outer membrane vesicle and a preparation method thereof
By regulating the hybridization of tea polyphenol ester-RNA nanovesicles and probiotic outer membrane vesicles, the degradation and barrier crossing problems of RNA in the gastrointestinal environment were solved, achieving efficient oral delivery and stabilization effects.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHEJIANG UNIV
- Filing Date
- 2026-01-23
- Publication Date
- 2026-06-05
AI Technical Summary
In existing technologies, RNA is easily degraded by enzymes in the gastrointestinal environment and has difficulty crossing the intestinal barrier, resulting in low oral bioavailability. The self-assembly performance of tea polyphenols is difficult to control, and the loading efficiency of probiotic outer membrane vesicles for hydrophilic macromolecules is low, which limits their application as RNA drug carriers.
By modifying the hydrophilicity and hydrophobicity of the tea polyphenol component EGC through molecular modification strategies, tea polyphenol ester-RNA nanovesicles were prepared and fused with probiotic outer membrane vesicles to form hybrid vesicles. By integrating the advantages of both, efficient encapsulation of RNA and intestinal targeted delivery were achieved.
This technology enables efficient encapsulation and stable delivery of RNA, significantly improving RNA stability in the gastrointestinal tract and intestinal cell uptake, thus enhancing the effectiveness of oral delivery.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of pharmaceutical biotechnology, specifically relating to a hybrid vesicle of tea polyphenol ester-RNA self-assembly and probiotic outer membrane vesicles and its preparation method. Background Technology
[0002] Nucleic acid drugs, such as small interfering RNA (RNA), messenger RNA (mRNA), and antisense oligonucleotides (ASO), have become a key next-generation therapeutic approach, greatly expanding the range of drug candidates. Oral administration is highly favored due to its superior convenience, high patient compliance, and lower risk of systemic side effects, showing great clinical promise. However, the active RNA components face severe challenges in the complex gastrointestinal environment; they are not only easily degraded by enzymes but also struggle to effectively cross the intestinal barrier, resulting in extremely low oral bioavailability and significantly reduced efficacy. Therefore, developing oral technologies that can effectively protect RNA and achieve efficient intestinal-targeted delivery is crucial.
[0003] Tea polyphenols are the main active components of tea. In addition to their excellent antioxidant activity, their abundant phenolic hydroxyl structures endow them with the unique advantage of binding and polymerizing with nucleic acids through multiple non-covalent bonds (such as hydrogen bonds and hydrophobic interactions), making them highly promising nucleic acid carrier materials. However, the strong polarity and hydrophilicity of natural tea polyphenols make it difficult to precisely control their self-assembly properties, often making it difficult to spontaneously form a uniform and stable nanoscale delivery system.
[0004] Outer membrane vesicles (OMVs) secreted by Gram-negative probiotics are lipid bilayer nanostructures ranging in size from 20 to 400 nanometers. Their natural components endow them with gastrointestinal stability, low cytotoxicity, and good biocompatibility, making them an ideal platform for oral delivery. However, OMVs have low efficiency in actively loading hydrophilic macromolecules, resulting in typically low RNA encapsulation rates, which limits their widespread application as RNA drug carriers. Summary of the Invention
[0005] To address the problems existing in the prior art, this invention provides a method for preparing hybrid nanovesicles based on the self-assembly of tea polyphenol esters and RNA and probiotic outer membrane vesicles, as well as the resulting product. This method first modifies the hydrophilicity and hydrophobicity of the tea polyphenol component EGC (epigallocatechin gallate) through a molecular modification strategy. The EGC palmitate obtained through this modification can directly self-assemble with RNA to form tea polyphenol ester-RNA nanovesicles (self-assembled vesicles), exhibiting superior drug loading capacity. Based on this, OMVs are further introduced as intestinal-targeted delivery carriers and fused with tea polyphenol ester-RNA vesicles to form structurally stable hybrid vesicles. This hybrid vesicle integrates the advantages of both: the tea polyphenol ester-RNA self-assembled vesicles ensure efficient RNA encapsulation, while OMVs provide natural protection for its passage through the gastrointestinal tract and promote intestinal cell uptake through their inherent bioadhesion. This strategy effectively promotes the oral delivery, encapsulation, and stabilization of RNA.
[0006] A method for preparing hybrid vesicles of tea polyphenol ester-RNA self-assembly and probiotic outer membrane vesicles includes the following steps:
[0007] (1) EGC, propionic anhydride and triethylamine undergo ortho-phenol hydroxylation reaction in solvent. After the reaction is completed, a protected product is obtained.
[0008] (2) Dissolve the protected product in the reaction solvent, and add palmitoyl chloride and pyridine to carry out the fatty alcohol hydroxyl esterification reaction;
[0009] (3) Hydroxylamine was added to the system after esterification to remove the propionyl protecting group. After the reaction was completed, EGC palmitate was obtained.
[0010] (4) Disperse RNA into an ethanol solution under ultrasonic conditions, add EGC palmitate, and stir the reaction after ultrasonication to obtain tea polyphenol ester-RNA self-assembled vesicles;
[0011] (5) The tea polyphenol ester-RNA self-assembled vesicles and the outer membrane vesicles of probiotics were fused by ultrasound and then subjected to a shaking reaction to obtain the hybrid vesicles.
[0012] In step (1) above,
[0013] Preferably, the molar ratio of EGC to propionic anhydride is 1:(2~30). More preferably, it is 1:(5~10), which helps to achieve sufficient acylation of the ortho-phenolic hydroxyl groups.
[0014] Preferably, the molar ratio of EGC to triethylamine is 1:(2~30). More preferably, it is 1:(5~10), which can fully neutralize the byproduct propionic acid and at the same time fully activate the phenolic hydroxyl groups of EGC to increase the reaction rate.
[0015] Preferably, the solvent is anhydrous acetonitrile.
[0016] Preferably, the molar volume ratio of EGC to solvent is 0.05~0.2 mol / L. More preferably, it is 0.08~0.12 mol / L.
[0017] Preferably, the reaction temperature is room temperature and the reaction time is 2-6 h. More preferably, it is 4 h.
[0018] As a preferred method, after the reaction is completed, the reaction mixture is extracted with ethyl acetate, washed twice with deionized water, dehydrated and dried with anhydrous magnesium sulfate, filtered, and then the ethyl acetate is removed by rotary evaporation to obtain the EGC protected product.
[0019] In step (2) above,
[0020] Preferably, the molar ratio of the protected product to palmitoyl chloride is 1:(1~10). More preferably, it is 1:(2~4), which helps to achieve full esterification of the fatty alcohol hydroxyl groups.
[0021] Preferably, the molar ratio of the protected product to pyridine is 1:(1~10). More preferably, it is 1:(1~4), which can greatly improve the efficiency of the esterification reaction.
[0022] Preferably, the reaction solvent is anhydrous dichloromethane.
[0023] Preferably, the molar volume ratio of the protected product to the reaction solvent is 0.05~0.2 mol / L. More preferably, it is 0.08~0.12 mol / L.
[0024] Preferably, the reaction temperature is room temperature and the reaction time is 0.5 to 2 hours. More preferably, it is 0.5 hours.
[0025] Preferably, after the esterification reaction is completed, 95% ethanol is added to the system to react and remove excess palmitoyl chloride, and the system after removing excess palmitoyl chloride is used in step (3).
[0026] As a further preferred embodiment, the volume ratio of added ethanol to the reaction solvent is 1:(0.5~2). A further preferred ratio is 1:1.
[0027] As a further preferred option, the reaction temperature after adding ethanol is room temperature, and the reaction time is 1–4 h. A further preferred option is 2 h, which allows for the complete removal of excess palmitoyl chloride.
[0028] Preferably, pyridine and palmitoyl chloride are added dropwise to the solution of the protected product.
[0029] In step (3) above,
[0030] Preferably, hydroxylamine is added in the form of an aqueous solution with a concentration of 50 wt.%.
[0031] Preferably, the molar ratio of the protected product to hydroxylamine is 1:(10~20). More preferably, it is 1:(10~15), which can greatly improve the removal efficiency of the propionyl protecting group.
[0032] Preferably, the reaction temperature for removing the propionyl protecting group is room temperature, and the reaction time is 1 to 4 hours. More preferably, it is 2 hours.
[0033] As a preferred option, the following post-treatment is performed after the deprotection reaction is completed:
[0034] After extraction with ethyl acetate, the reaction solution was washed twice each with 1 mol / L hydrochloric acid solution and deionized water, and then dehydrated and dried with anhydrous magnesium sulfate. The organic phase was then collected by filtration and rotary evaporation. The resulting solid was washed with petroleum ether, filtered, and dried to obtain EGC palmitate.
[0035] In step (4) above,
[0036] Preferably, the mass ratio of RNA to EGC palmitate is 1:(20~100). More preferably, it is 1:(50~70). Even more preferably, it is 1:60.
[0037] Preferably, the ethanol concentration used to disperse the RNA is 10-50%. More preferably, it is 30-40%. Even more preferably, it is 35%.
[0038] Preferably, the concentration of RNA dispersed in ethanol is 1-2 μg / mL. More preferably, it is 1.5 μg / mL.
[0039] Preferably, EGC palmitate is added in the form of an ethanol solution, wherein the ethanol concentration is 60-100%. More preferably, it is 60-80%. Even more preferably, it is 70%.
[0040] As a further preferred embodiment, the concentration of EGC palmitate in the ethanol solution is 2-6 mg / mL. Even more preferably, it is 4 mg / mL.
[0041] As a further preferred option, the ethanol solution of EGC palmitate is added dropwise.
[0042] As a preferred option, after adding EGC palmitate, the water bath ultrasonic time is 2 min and the ultrasonic power is 200 W.
[0043] Preferably, the stirring reaction temperature is room temperature, and the reaction time is 0.5 to 2 hours. More preferably, it is 1 hour.
[0044] Preferably, after the stirring reaction is complete, the following post-treatment is performed:
[0045] The reaction system was added to a 50 kDa ultrafiltration centrifuge tube and centrifuged at 4°C. The retentate containing tea polyphenol ester-RNA self-assembled vesicles was washed with nuclease-free water and subjected to three repeated ultrafiltration cycles to remove residues, yielding purified tea polyphenol ester-RNA self-assembled vesicles. The purified tea polyphenol ester-RNA self-assembled vesicles were then resuspended in nuclease-free water and stored at 4°C for later use.
[0046] In step (5) above,
[0047] Preferably, the probiotic is a Gram-negative bacillus, such as Escherichia coli (EcN).
[0048] Furthermore, taking *Escherichia coli* (EcN) as an example, the extraction method for outer membrane vesicles (OMVs) is as follows:
[0049] Single colonies of EcN, preserved by streaking, were inoculated into LB medium and cultured overnight at 37°C with shaking. 200 μL of the bacterial culture was transferred to 1600 mL of fresh LB medium and cultured overnight at 37°C with shaking. Subsequently, the bacterial cells were removed by centrifugation at 4°C. The resulting supernatant was filtered through a 0.45 μm sterile filter and concentrated using a 100 kDa ultrafiltration centrifuge tube. Finally, OMVs were collected by ultracentrifugation at 4°C, washed with PBS, resuspended in PBS, and stored at -80°C for later use.
[0050] As a further preferred option, the ultracentrifugation speed for collecting OMVs is 180,000 × g, and the centrifugation time is 30 min.
[0051] Preferably, tea polyphenol ester-RNA self-assembled vesicles and outer membrane vesicles (OMVs) are mixed at a particle number concentration ratio of 1:(0.5~10). More preferably, the ratio is 1:1.
[0052] As a preferred method, the parameters for ultrasound-induced fusion are set as follows: amplitude 30%, ultrasound for 5 seconds, interval for 5 seconds, and cycle for 24 times.
[0053] Preferably, ultrasound-assisted fusion is performed under ice bath conditions with an ice bath temperature of 0°C.
[0054] Preferably, the shaking reaction temperature is 37°C, and the reaction time is 0.5~2h. More preferably, it is 1h. The shaking reaction in this step can reconstruct the membrane structure of the vesicles, obtaining hybrid vesicles.
[0055] As a preferred embodiment, a method for preparing hybrid nanovesicles of tea polyphenol ester-RNA self-assembly and probiotic outer membrane vesicles includes the following steps:
[0056] (1) Dissolve EGC in anhydrous acetonitrile, then add propionic anhydride and triethylamine dropwise to carry out acylation reaction. After the reaction is completed, a protected product is obtained.
[0057] (2) Dissolve the protected product in anhydrous dichloromethane, then add pyridine dropwise to obtain a mixed solution, and add palmitoyl chloride dropwise to the mixed solution to carry out the fatty alcohol hydroxyl esterification reaction. After the reaction is completed, a reaction solution containing intermediate esterification product is obtained.
[0058] (3) Add excess 95% ethanol to the reaction solution containing the intermediate esterification product to remove excess palmitoyl chloride, and then add hydroxylamine dropwise to selectively remove the propionyl protecting group. After the reaction is completed, EGC palmitate is obtained.
[0059] (4) Add RNA to an ethanol solution, disperse it by sonication, and then add EGC palmitate dropwise to the mixture. Sonicate and stir the mixture to obtain tea polyphenol ester-RNA self-assembled nanovesicles.
[0060] (5) The tea polyphenol ester-RNA self-assembled nanovesicles were fused with probiotic OMVs by ultrasonication and then subjected to a shaking reaction to obtain hybrid vesicles.
[0061] This invention discloses a method for preparing hybrid nanovesicles based on tea polyphenol ester-RNA self-assembly and probiotic outer membrane vesicles, for achieving efficient oral delivery of RNA. The method first hydrophobically modifies natural tea polyphenol EGC, resulting in an amphiphilic derivative, EGC palmitate, which can autonomously load RNA and self-assemble into nanovesicles, exhibiting excellent RNA encapsulation efficiency. Based on this, the above nanovesicles are further constructed by membrane fusion with probiotic-derived OMVs to create hybrid vesicles. This hybrid system integrates the characteristics of both components: tea polyphenol ester-RNA nanovesicles achieve effective loading of nucleic acids (RNA); while OMVs significantly enhance the resistance of RNA to gastric acid and enzymatic degradation. The delivery system (hybrid nanovesicles) established by the preparation method of this invention successfully integrates the synergistic advantages of synthetic vesicles and natural biological vesicles, effectively promoting the encapsulation and stabilization of RNA for oral delivery.
[0062] This invention provides a hybrid vesicle of tea polyphenol ester-RNA self-assembly and probiotic outer membrane vesicles, which is prepared by any of the preparation methods described above.
[0063] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0064] (1) The preparation method of the present invention first uses propionic anhydride to protect the ortho-phenolic hydroxyl groups of EGC, and then uses palmitoyl chloride to perform a fatty alcohol hydroxyl esterification reaction on the protected product to obtain an intermediate esterified product. The protecting group is then removed by hydroxylamine to obtain EGC palmitate. This strategy can regulate the hydrophilicity and hydrophobicity of EGC and achieve controllable molecular assembly. It can directly encapsulate RNA and self-assemble into tea polyphenol ester-RNA nanovesicles without the need for additional lipid assistance.
[0065] (2) Compared with the low encapsulation efficiency of traditional liposomes for hydrophilic macromolecules, the tea polyphenol ester-RNA nanovesicles of the present invention achieve superior RNA encapsulation efficiency.
[0066] (3) In this invention, probiotic-derived OMVs are further fused with tea polyphenol ester-RNA nanovesicles to form structurally stable hybrid vesicles. The lipid bilayer structure of OMVs can provide additional protection for RNA, significantly enhancing its stability in the complex gastrointestinal environment.
[0067] (4) The present invention uses an ultrasonic method to prepare hybrid vesicles. This method is mild and the process is controllable. It can efficiently guide different membrane systems to reconstruct into structurally stable and uniform hybrid vesicles, while effectively preserving their biological activity. Attached Figure Description
[0068] Figure 1 The 1H NMR spectrum of the EGC palmitate obtained in Example 1;
[0069] Figure 2 This is the mass spectrum of the EGC palmitate obtained in Example 1. Detailed Implementation
[0070] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto.
[0071] The TNF-α siRNA sequences used in the following examples are: sense strand: 5'-CACAACCAACUAGUGGUGCUU-3'; antisense strand: 5'-AAGCACCACUAGUUGGUUGUG-3'. The sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd.
[0072] Example 1: Preparation of hybrid vesicles of tea polyphenol ester-RNA self-assembly and OMVs
[0073] (1) Synthesis of EGC palmitate
[0074] Propionic anhydride (131.5 μL, 1.0 mmol) and triethylamine (138.6 μL, 1.0 mmol) were added dropwise to anhydrous acetonitrile (2 mL) containing dissolved EGC (61.2 mg, 0.2 mmol). The resulting mixture was stirred at room temperature for 4 h. After the reaction was complete, the reaction mixture was extracted with ethyl acetate, washed twice with deionized water, and then dehydrated and dried over anhydrous magnesium sulfate. After filtration, ethyl acetate was removed by rotary evaporation to obtain the protected EGC product.
[0075] Pyridine (24.2 μL, 0.3 mmol) was added dropwise to anhydrous dichloromethane (2 mL) containing the EGC protected product (117.2 mg, 0.2 mmol), followed by slow dropwise addition of palmitoyl chloride (125.8 μL, 0.4 mmol). The resulting mixture was stirred at room temperature for 30 min to obtain the intermediate esterified product. To remove excess palmitoyl chloride, 95% ethanol (2 mL) was added to the reaction system, and the reaction was continued for 2 h.
[0076] Hydroxylamine aqueous solution (50 wt.%, 122.5 μL, 2.0 mmol) was slowly added to the above reaction system, and the reaction was stirred at room temperature for 2 h. The reaction solution was extracted with ethyl acetate, washed twice each with 1 mol / L hydrochloric acid solution and deionized water, and then dehydrated and dried with anhydrous magnesium sulfate. The organic phase was collected by filtration and rotary evaporation. The resulting solid product was suspended in petroleum ether, filtered, and dried to obtain the directed esterification product of EGC palmitate (yield 89%).
[0077] (2) Preparation of tea polyphenol ester-RNA self-assembled nanovesicles
[0078] Before preparing EGC palmitate-RNA self-assembled vesicles (sEPVs), 4 mg of EGC palmitate was dissolved in 1 mL of 70% ethanol as a stock solution. TNF-α siRNA (1.5 μg) was dispersed in 1 mL of 35% ethanol and sonicated in a water bath to ensure uniform RNA dispersion. Under gentle stirring, 22.5 μL of the stock solution (containing 90 μg of EGC palmitate) was added dropwise to the RNA ethanol solution. The mixture was sonicated in a water bath at 25°C (200 W, 2 min) and then stirred continuously at room temperature for 1 h to form sEPVs. To remove unencapsulated RNA and free EGC palmitate, the reaction mixture was added to a 50 kDa ultrafiltration centrifuge tube and centrifuged at 4°C. The vesicle-containing retentate was washed with nuclease-free water and subjected to three repeated ultrafiltration cycles to remove residues. The purified vesicles were resuspended in nuclease-free water and stored at 4°C for later use.
[0079] (3) Extraction of OMVs
[0080] Single colonies of EcN, preserved by streaking, were inoculated into LB medium and cultured overnight at 37°C with shaking. 200 μL of this bacterial culture was transferred to 1600 mL of fresh LB medium and cultured overnight at 37°C with shaking. Subsequently, the bacterial cells were removed by centrifugation at 4°C. The resulting supernatant was filtered through a 0.45 μm sterile filter and concentrated using a 100 kDa ultrafiltration centrifuge tube. Finally, OMVs were collected by ultracentrifugation (180,000 × g, 30 min) at 4°C, washed with PBS, resuspended in PBS, and stored at -80°C for later use.
[0081] (4) Preparation of hybrid vesicles of tea polyphenol ester-RNA self-assembly and OMVs
[0082] sEPVs and OMVs were mixed in equal volumes (200 μL each), with a concentration of 2.0 × 10⁻⁶ for both. 9 The mixture was sonicated using an integrated ultrasonic cell disruptor with the following parameters: amplitude 30%, sonication for 5 seconds, interval for 5 seconds, and 24 cycles. Ultrasonic fusion was performed in an ice bath (0°C) to avoid damage to the membrane components caused by ultrasonic heat. The sonicated reaction mixture was then agitated at 37°C for 1 h to allow for vesicle membrane structure reconstruction, yielding hybrid vesicles (HVs).
[0083] Characterization tests of hybrid vesicles:
[0084] (1) Structural characterization of EGC palmitate
[0085] The 1H NMR spectrum of EGC palmitate (see...) Figure 1 A significant saturated fatty chain signal was observed in the 0.8–1.4 ppm range, confirming the successful attachment of the palmitoyl chain to the EGC molecule. (Mass spectrometry [see image]) Figure 2 This further confirms the structural characteristics ([2M-H]). - 1087.18).
[0086] (2) RNA encapsulation efficiency determination
[0087] The prepared sEPVs were purified by ultrafiltration to remove unencapsulated RNA, excess EGC palmitate, and ethanol. RNA bands in the retentate were obtained by 2% agarose gel electrophoresis. Encapsulation efficiency = band intensity of RNA in the retentate / band intensity of the initial RNA feed. After preparing HVs, the unencapsulated RNA was separated from the permeate by ultrafiltration and centrifugation as described above. Encapsulation efficiency = 1 - (band intensity of RNA in the permeate / band intensity of the initial RNA feed).
[0088] The results showed that under the condition of RNA to EGC palmitate mass ratio of 1:60, sEPVs achieved an encapsulation efficiency of up to 85% for RNA. In addition, 67% of the initial RNA feed was successfully encapsulated inside HVs, while when RNA was directly loaded into OMVs by ultrasound without pre-encapsulation by sEPVs, its drug loading efficiency was only 27%, demonstrating the superior drug loading capacity of sEPVs and HVs.
[0089] (3) RNA tolerance test
[0090] To assess the resistance of RNA to ribonuclease (RNase) degradation, RNA, sEPVs, and HVs were co-incubated with RNase (2 ng / µL) for 5 min, followed by the addition of an RNase inhibitor to terminate the reaction. Electrophoretic analysis showed that unencapsulated RNA was completely degraded. In contrast, sEPVs and HVs effectively prevented RNA degradation by the enzyme, with RNA retention rates as high as 73% and 88%, respectively. Furthermore, the stability of nanovesicles in simulated gastric fluid at 37°C for 2 h was evaluated. The results showed that the OMV membrane structure provided effective protection for the encapsulated RNA, reducing the RNA degradation rate in HVs to 58%, significantly lower than the 97% degradation rate in sEPVs. These results confirm that HVs can significantly improve the stability of encapsulated RNA and effectively resist the disruptive effects of the gastrointestinal environment.
Claims
1. A method for preparing hybrid vesicles of tea polyphenol ester-RNA self-assembly and probiotic outer membrane vesicles, characterized in that, Includes the following steps: (1) EGC, propionic anhydride and triethylamine undergo ortho-phenol hydroxylation reaction in solvent. After the reaction is completed, a protected product is obtained. (2) Dissolve the protected product in the reaction solvent, and add palmitoyl chloride and pyridine to carry out the fatty alcohol hydroxyl esterification reaction; (3) Hydroxylamine was added to the system after esterification to remove the propionyl protecting group. After the reaction was completed, EGC palmitate was obtained. (4) Disperse RNA into an ethanol solution under ultrasonic conditions, add EGC palmitate, sonicate and stir to react, and obtain tea polyphenol ester-RNA self-assembled vesicles; (5) The tea polyphenol ester-RNA self-assembled vesicles and the outer membrane vesicles of probiotics were fused by ultrasound and then subjected to a shaking reaction to obtain the hybrid vesicles.
2. The method for preparing hybrid vesicles of tea polyphenol ester-RNA self-assembly and probiotic outer membrane vesicles according to claim 1, characterized in that, In step (1), the molar ratio of EGC to propionic anhydride is 1:(2~30); The molar ratio of EGC to triethylamine is 1:(2~30).
3. The method for preparing hybrid vesicles of tea polyphenol ester-RNA self-assembly and probiotic outer membrane vesicles according to claim 1, characterized in that, In step (2), the molar ratio of the protected product to palmitoyl chloride is 1:(1~10); The molar ratio of the protected product to pyridine is 1:(1~10).
4. The method for preparing hybrid vesicles of tea polyphenol ester-RNA self-assembly and probiotic outer membrane vesicles according to claim 1, characterized in that, The molar ratio of the protected product to hydroxylamine is 1:(10~20).
5. The method for preparing hybrid vesicles of tea polyphenol ester-RNA self-assembly and probiotic outer membrane vesicles according to claim 1, characterized in that, The mass ratio of RNA to EGC palmitate is 1:(20~100).
6. The method for preparing hybrid vesicles of tea polyphenol ester-RNA self-assembly and probiotic outer membrane vesicles according to claim 1, characterized in that, Tea polyphenol ester-RNA self-assembled vesicles and outer membrane vesicles were mixed according to the particle number concentration ratio of 1:(0.5~10).
7. The method for preparing hybrid vesicles of tea polyphenol ester-RNA self-assembly and probiotic outer membrane vesicles according to claim 1, characterized in that, After the esterification reaction in step (2) is completed, 95% ethanol is added to the system to react and remove excess palmitoyl chloride. The system after removing excess palmitoyl chloride is then used in step (3). The volume ratio of added ethanol to reaction solvent is 1:(0.5~2). The reaction temperature after adding ethanol was room temperature, and the reaction time was 1-4 h.
8. The method for preparing hybrid vesicles of tea polyphenol ester-RNA self-assembly and probiotic outer membrane vesicles according to claim 1, characterized in that, In step (1), the solvent is anhydrous acetonitrile; The reaction temperature was room temperature, and the reaction time was 2-6 hours.
9. The method for preparing hybrid vesicles of tea polyphenol ester-RNA self-assembly and probiotic outer membrane vesicles according to claim 1, characterized in that, In step (2), the reaction solvent is anhydrous dichloromethane; The reaction temperature was room temperature, and the reaction time was 0.5 to 2 hours.
10. A hybrid vesicle of tea polyphenol ester-RNA self-assembly and probiotic outer membrane vesicles, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 9.