Polyphenol condensation liquid drop based on hydrogen bond interaction and preparation and application thereof

Abstract

The invention relates to a polyphenol condensation liquid drop based on hydrogen bond interaction and preparation and application thereof, and belongs to the technical field of medical bioengineering. The method comprises the following steps: firstly, carrying out preliminary screening from various natural polyphenol and high-molecular polymer libraries according to comprehensive investigation of factors such as molecular structure, solubility, source, hydrogen bond interaction, clinical accessibility and the like, then pairing different polyphenols and high-molecular polymers to form a condensation body, observing the morphological structure of the condensation body, and finally obtaining the polyphenols and high-molecular polymers. Finally, screening out a formula for forming the condensed liquid drops; various properties of the polyphenol condensation liquid drops are represented, physicochemical properties, drug entrapment performance and surface modification strategies of the polyphenol condensation liquid drops are researched, the feasibility of the polyphenol condensation liquid drops as liquid preparations and dry powder preparations is further explored, and the application of the polyphenol condensation liquid drops in drug delivery or tissue engineering is expanded.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical bioengineering, and more specifically, relates to polyphenol aggregate droplets based on hydrogen bond interactions and their preparation and application. Background Art

[0002] The biomolecules inside the cell aggregate due to multivalent weak interactions and undergo phase transition, forming substances similar to "droplets", which are called biomolecular condensates. The formation of biomolecular condensates is a common biological phenomenon. The principle of this process is liquid-liquid phase separation (LLPS). Cells form droplets by concentrating specific reaction components in an area inside the cell to form membraneless reaction compartments. Droplets can accelerate biochemical processes and isolate other non-related components to prevent unnecessary interference. Biomolecular condensates are generally considered to be the basis for the formation of membraneless compartments inside cells. During the droplet formation process, various molecules can be spontaneously enriched in the droplets.

[0003] Droplets refer to spherical liquids that can settle under static conditions and remain suspended under turbulent conditions. The particle size range is mainly in the micrometer and below size range. They are commonly found in various organisms in nature and play an important role in the life process. As a kind of liquid droplet with fluidity, flexibility and deformability, condensate droplets can exchange substances, deform, expand and contract, merge and split. They are a unique material form, different from condensate particles, fibers and gels (patent document CN114191568B), and have broad application prospects.

[0004] Studies have found that a variety of small molecules, ions, peptides, proteins, nucleic acids, polymers, etc. can undergo LLPS. The current research hotspot is to construct artificial condensate droplets by simulating the process of biomolecule condensation through various chemical substances. During the formation process of condensate droplets, specific small molecules and macromolecular drugs can be spontaneously enriched inside. The process is simple and gentle, and is especially suitable for biological drugs that need to maintain activity; at the same time, the unique size, density, flexibility, fluidity and other properties of condensate droplets themselves make them have the potential to become a new drug delivery system. Condensation is driven by the intermolecular interaction force (electrostatic interaction, hydrogen bond interaction, π-π interaction, etc.), and the molecules undergo liquid-liquid phase separation and spontaneously transform from the continuous phase to the condensed phase, which can present droplet state, fiber state, gel state, etc., and the morphology after condensation is related to the strength of their interaction. The intermolecular interaction force is determined by the chemical structure of the molecule, so the key to constructing artificial condensate droplets is to select paired molecules with appropriate interaction forces.

[0005] Polyphenols are natural active compounds widely found in nature and have the function of promoting health. Polyphenols are mainly derived from vegetables, fruits, tea, cocoa, nuts, soybeans, etc. They have antioxidant, anti-tumor, antibacterial, antiviral and other activities, and play an important role in the treatment and prevention of diseases. Polyphenols are a general term for polyhydroxy compounds present in plants, mainly simple phenolic acids, flavonoids, tannins, anthocyanins, etc. Experts from the World Health Organization believe that plant polyphenols are the well-deserved seventh nutrient for humans and a green, environmentally friendly, and renewable century resource treasure house. Polyphenols contain multiple phenolic hydroxyl groups in their structure, which can serve as hydrogen bond donors. Polymers are functional materials synthesized by artificial chemistry. Many polymers have been approved for use as pharmaceutical excipients. Polymers contain a large number of oxygen, nitrogen, and fluorine atoms, which can serve as hydrogen bond acceptors. Therefore, polyphenols and polymers condense through hydrogen bond interactions. It is a frontier exploratory field to explore polyphenols and polymers with different structures and properties and pair them to form condensate droplets. Summary of the invention

[0006] In order to expand the formula of existing condensate droplets and overcome the shortcomings of existing drug delivery carriers such as complex preparation processes, the purpose of the present invention is to develop new polyphenol condensate droplets: first, various natural polyphenols are selected from nature, and preliminary screening is carried out based on structural characteristics, solubility and other factors, and then condensates are formed by pairing with different high molecular polymers, and the morphological structure of the condensates is observed. Finally, the formula for forming condensate droplets is screened out, its various properties are characterized, and the performance of condensate droplets and their application potential as drug delivery systems are explored.

[0007] According to a first aspect of the present invention, a method for preparing polyphenol aggregate droplets based on hydrogen bond interactions is provided, comprising the following steps:

[0008] (1) preparing a polyphenol into a polyphenol aqueous solution with a concentration of 0.1-50 mg / mL, wherein the number of phenolic hydroxyl groups of the polyphenol is greater than 8; preparing a high molecular polymer into a high molecular polymer aqueous solution with a concentration of 0.1-50 mg / mL, wherein the high molecular polymer contains oxygen, nitrogen or fluorine atoms;

[0009] (2) Mixing the polyphenol aqueous solution obtained in step (1) with the polymer aqueous solution, wherein the mass ratio of the polyphenols in the polyphenol aqueous solution to the polymer in the polymer aqueous solution is 0.5-10, thereby obtaining polyphenol aggregate droplets.

[0010] Preferably, the polyphenol is tannic acid or epigallocatechin gallate.

[0011] Preferably, the high molecular polymer is Pluronic F-68, Pluronic F-127, polyethylene glycol, four-arm polyethylene glycol, polyvinyl alcohol, Tween 20, Tween 40 or Tween 80.

[0012] Preferably, in step (2), before the polyphenol aqueous solution and the high molecular polymer aqueous solution are mixed, the high molecular polymer aqueous solution is first mixed with the drug solution and then mixed with the polyphenol aqueous solution to prepare drug-loaded polyphenol aggregate droplets.

[0013] Preferably, the drug is a hydrophilic small molecule, a hydrophobic small molecule or an amphiphilic small molecule drug.

[0014] Preferably, the drug is a polypeptide, a protein, a nucleic acid or a nanodrug.

[0015] Preferably, in step (1), the polyphenol aqueous solution and the high molecular polymer aqueous solution are both disaccharide aqueous solutions; after step (2), the polyphenol aggregate droplets are freeze-dried to obtain dry powder.

[0016] Preferably, after step (2), the polyphenol aggregate droplets are mixed with proteins, lipid membranes, and extracellular vesicles to obtain surface-modified polyphenol aggregate droplets.

[0017] According to another aspect of the present invention, provided are polyphenol aggregate droplets based on hydrogen bond interactions prepared by any one of the methods of the present invention.

[0018] According to another aspect of the present invention, there is provided a use of the polyphenol aggregate droplets based on hydrogen bond interactions in the preparation of drug delivery carriers or artificial tissues.

[0019] In general, the above technical solutions conceived by the present invention mainly have the following technical advantages:

[0020] (1) The primary characteristics of polyphenol aggregate droplets are spherical or vesicular aqueous liquid droplets, which have fluidity, flexibility and deformability, and can undergo material exchange, deformation, expansion, fusion and splitting, which is different from material forms such as particles, fibers and gels. A series of formulations after pairing candidate polyphenols with high molecular polymers are used to screen droplets based on microscopic morphology and internal fluidity, which can be selected as needed for practical applications.

[0021] (2) The preparation method of polyphenol aggregate droplets has mild conditions, simple operation, strong repeatability, does not require complicated instruments and equipment and preparation processes, and is easy to scale up production and quality control.

[0022] (3) Polyphenol aggregate droplets can quickly and efficiently encapsulate drugs with different physical and chemical properties and are universally applicable to the encapsulation of drugs, including hydrophilic small molecules, hydrophobic small molecules, amphiphilic small molecules, peptides, proteins, nucleic acids, nanomedicines, etc., and can be used as drug delivery carriers.

[0023] (4) Polyphenol condensate droplets have a variety of surface modification methods, including proteins, lipid membranes, extracellular vesicles, etc., which not only improve the stability of polyphenol condensate droplets, but also give them additional biological functions, and can be used as artificial cells or tissues.

[0024] (5) After freeze-drying and re-dissolving, the polyphenol condensate droplets maintain their original morphology, structure and function and can be used in liquid preparations, freeze-dried powders, dry powder inhalation preparations, high-end biological preparations, etc., providing more options for disease treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Workflow diagram for the construction and application of polyphenol condensate droplets.

[0026] Figure 2 The results of formula screening of polyphenols and high molecular polymers of Examples 1 and 2 are shown.

[0027] Figure 3 The tool materials and methods required for preparing the liquid sample observation device prepared in Example 3 are demonstrated.

[0028] Figure 4 The microscopic morphology of the polyphenol aggregate droplet prepared in Example 4 is displayed under a microscope.

[0029] Figure 5 The microscopic morphology of the polyphenol aggregate droplet prepared by Tween in Example 4 is shown under a microscope.

[0030] Figure 6 This is the situation of whether the two components of the polyphenol agglomerate droplets of different concentrations and ratios prepared in Example 5 can coagulate.

[0031] Figure 7 This is a fluorescent image of the polyphenol aggregate droplets containing different molecules prepared in Example 6.

[0032] Figure 8 This is a fluorescent image of the polyphenol aggregate droplets encapsulating nucleic acids and nanoparticles prepared in Example 6.

[0033] Fig. 9 This is the fluorescence emission spectrum of the polyphenol aggregate droplets encapsulating two FRET pairing molecules prepared in Example 7.

[0034] Fig.10The FRAP experimental results of the polyphenol aggregate droplets in Example 8 are shown.

[0035] Fig.11 The results of polyphenol agglomerate dry powder prepared by freeze-drying polyphenol agglomerate droplets in different dispersion media in Example 9 are presented.

[0036] Fig.12 The microscopic morphology of the polyphenol aggregate droplets in Example 9 before and after freeze-drying is shown.

[0037] Fig.13 The surface modification strategy and results of the polyphenol aggregate droplets in Example 10 are presented. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0039] The purpose of the present invention is to artificially simulate the process of biological liquid-liquid phase separation with chemical substances and develop new polyphenol condensate droplets. Droplets refer to spherical liquid material forms that can settle under static conditions and remain suspended under turbulent conditions. The particle size range is mainly in the micrometer and below size range. They are commonly found in various organisms in nature and play an important role in the life process. As a liquid droplet with fluidity, flexibility and deformability, condensate droplets can undergo material exchange, deformation, expansion and contraction, fusion and splitting. They are a unique material form, different from condensate particles, fibers and gels, and have broad application prospects.

[0040] One purpose of the present invention is to screen out polyphenols suitable for coacervate pairing from natural plant polyphenols based on comprehensive consideration of factors such as solubility, molecular structure, source, etc.; comprehensively consider factors such as the solubility, molecular structure, hydrogen bond interaction, clinical accessibility, etc. of the polymer to screen out a polymer suitable for coacervate pairing; preliminarily screen out the formula of the coacervate based on the phenomenon after the polyphenol and the polymer are mixed and the ultraviolet absorbance; confirm the microscopic morphology of the coacervate, and finally obtain a series of formulas of polyphenol coacervate droplets.

[0041] Another object of the present invention is to provide a platform and method for encapsulating drugs in polyphenol droplets. The polyphenol droplets are universal for encapsulating different molecules, have high encapsulation efficiency, and a gentle and rapid drug loading process, and can be used as a drug delivery system.

[0042] The third object of the present invention is to provide polyphenol aggregate droplets suitable for freeze-drying, which have the potential to be used as freeze-dried powders for high-end biological preparations, dry powder inhalation preparations, etc.

[0043] The fourth purpose of the present invention is to provide surface modification strategies such as proteins, lipid membranes, and extracellular vesicles to improve the stability of polyphenol aggregate droplets, enhance their storage and preservation capabilities, and expand their applications in the fields of drug delivery, tissue engineering, and disease treatment.

[0044] The formula of the polyphenol condensate droplets based on hydrogen bond interaction and the preparation process thereof include the following steps:

[0045] (1) The primary characteristics of polyphenol aggregate droplets are spherical or vesicular aqueous liquid droplets, which have fluidity, flexibility and deformability, and can undergo material exchange, deformation, expansion, fusion and splitting. They are different from particles, fibers, gels and other material forms, and their components are polyphenols and high molecular polymers.

[0046] (2) Polyphenols are characterized by having more than 8 phenolic hydroxyl groups in their structure and a water solubility greater than 5 mg / mL, including but not limited to tannic acid, epigallocatechin gallate, etc.;

[0047] (3) High molecular weight polymers are characterized by containing oxygen, nitrogen or fluorine atoms in their structure and having a water solubility greater than 5 mg / mL, including but not limited to: Pluronic F-68, Pluronic F-127, polyethylene glycol, four-arm polyethylene glycol, polyvinyl alcohol, Tween 20, Tween 40, Tween 80, etc.;

[0048] (4) a series of polyphenol aggregate droplet formulas are obtained by pairing polyphenols with high molecular weight polymers;

[0049] (5) The preparation method of polyphenol condensate droplets is simple and can be prepared in one step. The polyphenol and the high molecular polymer are dissolved in ultrapure water to prepare their own aqueous solutions, and then the polyphenol solution and the high molecular polymer solution are mixed to obtain condensate droplets.

[0050] (6) The concentration and ratio of polyphenols and high molecular weight polymers to form polyphenol aggregate droplets have a critical range. The mass ratio of polyphenols to high molecular weight polymers is 0.5-10, and the concentration range is 0.1-50 mg / mL.

[0051] In some embodiments, polyphenols and high molecular weight polymers are dissolved in ultrapure water respectively to prepare their own aqueous solutions. The high molecular weight polymer solution is first mixed with the working solution of the drug, and then mixed with the polyphenol solution to prepare drug-loaded condensate droplets; the polyphenol condensate droplets are universal for encapsulating different drugs, including but not limited to: hydrophilic small molecules, hydrophobic small molecules, amphiphilic small molecules, polypeptides, proteins, nucleic acids, nanomedicines, etc.

[0052] In some embodiments, the freeze-drying and reconstitution method of polyphenol aggregate droplets and the freeze-drying protective agent formulation are

[0053] (1) polyphenol and high molecular polymer are respectively prepared in the following dispersion media: 10% sucrose aqueous solution, 5% sucrose + 5% lactose aqueous solution and 10% lactose aqueous solution, and then the polyphenol solution and the high molecular polymer solution are mixed to prepare coacervate droplets;

[0054] (2) grinding the solid powder obtained by freeze-drying the condensate droplets into a fine and loose dry powder;

[0055] (3) The condensate dry powder is redissolved in the same volume of ultrapure water to obtain condensate droplets with the same morphological structure.

[0056] In some embodiments, the surface modification method of polyphenol aggregate droplets is to prepare the aggregate droplets and after the aggregate droplets have been grown stably, mix them with coating materials such as proteins, lipid membranes, and extracellular vesicles to obtain surface-coated polyphenol aggregate droplets.

[0057] In some embodiments, polyphenol aggregate droplets are used in the fields of drug delivery, artificial tissue, etc.

[0058] In some embodiments, the polyphenol aggregate droplets are used in liquid preparations, lyophilized powder injections, dry powder inhalation preparations, etc.

[0059] The present invention relates to the formulation screening, preparation method, drug encapsulation and surface modification of polyphenol aggregate droplets. The working process is as follows: Figure 1 As shown, the following steps are included:

[0060] Step 1, selecting polyphenol molecules suitable for coacervate pairing from natural plant polyphenols based on solubility, molecular structure, source and other factors;

[0061] Step 2, based on comprehensive consideration of factors such as the solubility, molecular structure, hydrogen bonding interaction, and clinical accessibility of the polymer, a polymer suitable for coacervate pairing is selected;

[0062] Step 3, preliminarily screening out the formula of the coacervate according to the phenomenon and ultraviolet absorbance after the polyphenol and the high molecular polymer aqueous solution are mixed;

[0063] Step 4, confirming the microscopic morphology of the polyphenol aggregate droplets;

[0064] Step 5: Study the physicochemical properties and drug loading performance of polyphenol aggregate droplets:

[0065] Step 6: Study the effect of freeze-drying process on polyphenol condensate droplets, confirm the potential of polyphenol condensate droplets as freeze-dried powder, and further apply them to high-end biological preparations, dry powder inhalation preparations, etc.

[0066] Step 7: Propose surface modification strategies such as proteins, lipid membranes, and extracellular vesicles to improve the stability of polyphenol aggregate droplets.

[0067] The invention discloses an application of polyphenol aggregate droplets based on hydrogen bond interactions in the fields of drug delivery, tissue engineering and disease treatment.

[0068] The following are specific embodiments.

[0069] Example 1

[0070] In this example, polyphenols were preliminarily screened based on solubility. The necessary requirement for the components constituting the coacervate droplets is good water solubility, so fat-soluble flavonoid polyphenols were first excluded. Water-soluble simple phenolic acid, tannin, and anthocyanin polyphenols entered the next round of screening (as shown in Table 1).

[0071] Table 1 Classification, solubility and representative compounds of natural plant polyphenols

[0072]

[0073] Example 2

[0074] This embodiment performs subsequent screening on water-soluble polyphenols. The structure of simple phenolic acid polyphenols only contains a few phenolic hydroxyl groups, the number of hydrogen bond donors is too small, and the intermolecular force is weak enough to form agglomerates. The composition of anthocyanidin polyphenols is relatively complex. Anthocyanidins in the natural state exist in the form of glycosides, called anthocyanins. There are very few free anthocyanidins, which are not convenient for research. Therefore, tannin polyphenols with clear structure, single composition and wide sources are the best candidate agglomerate composition materials. Tannin polyphenols include tannic acid (TA), epigallocatechin gallate (EGCG), epicatechin gallate (ECG), epigallocatechin gallate (GCG), epigallocatechin (EGC), epicatechin (EC), gallic acid (GA), ellagic acid (EA), etc. The number of phenolic hydroxyl groups in each structure is different, and the water solubility is also different. Four polyphenol molecules were representatively selected for subsequent research, namely TA, EGCG, EGC and EC (as shown in Table 2).

[0075] Table 2 Comparison of tannin polyphenol molecules

[0076]

[0077] Therefore, the above four polyphenol molecules (as hydrogen bond donors) were selected to pair with high molecular polymers (as hydrogen bond acceptors) to screen the formula of coacervates. TA, EGCG, EGC and EC were dissolved in ultrapure water to prepare a solution with a concentration of 1 mg / mL. At the same time, Pluronic F-68 (F68), Pluronic F-127 (F127), polyethylene glycol (PEG), four-arm polyethylene glycol (4-arm-PEG), and polyvinyl alcohol (PVA) were dissolved in ultrapure water to prepare a solution with a concentration of 1 mg / mL. Mix the polyphenol solution with the high molecular polymer solution, observe whether the solution becomes turbid, and use a multifunctional microplate reader to detect the ultraviolet absorbance of the solution to preliminarily determine whether coacervates are formed. Figure 2 As shown, EGC and EC cannot condense due to the small number of phenolic hydroxyl groups in their structures and the weak intermolecular interaction. When the characteristics of polyphenols are that the number of phenolic hydroxyl groups in the structure is greater than 8 and the water solubility is greater than 5 mg / mL, and the characteristics of high molecular polymers are that the structure contains hydroxyl groups, ether bonds, and amino groups and the water solubility is greater than 5 mg / mL, the materials that meet these characteristics can form condensates through hydrogen bond interactions, and the condensate formula is preliminarily screened out through macroscopic phenomena and ultraviolet absorbance.

[0078] Example 3

[0079] Droplets refer to small liquid particles that can settle under static conditions and remain suspended under turbulent conditions. The particle size range is mainly in the micron size and below. Whether the microscopic morphology of the agglomerate is a droplet can only be determined under a microscope. Therefore, it is necessary to prepare a suitable liquid sample observation device for laser confocal microscopy observation. The tools and materials for preparing the liquid sample observation device in this embodiment are a glass slide, a glue stick, and a hot melt gun. The specific steps are to use a hot melt gun to heat the glue stick and evenly coat it on the glass slide to form a liquid sample loading chamber. After it cools down, the liquid sample observation device (such as Figure 3 shown).

[0080] Example 4

[0081] TA and EGCG were dissolved in ultrapure water to prepare a solution with a concentration of 10 mg / mL. Meanwhile, Pluronic F-68 (F68), Pluronic F-127 (F127), polyethylene glycol (PEG), four-arm polyethylene glycol (4-arm-PEG), polyvinyl alcohol (PVA), Tween 20 (T20), Tween 40 (T40), and Tween 80 (T80) were dissolved in ultrapure water to prepare a solution with a concentration of 10 mg / mL. The polyphenol solution and the high molecular polymer solution were mixed to prepare a coacervate, and the prepared coacervate was placed in the liquid sample observation device prepared in Example 3 to observe its microscopic morphology under a white light microscope. Figure 4 and Figure 5As shown, the microscopic morphology of the condensates of different formulations are all transparent bubbly droplets, proving that a series of formulations of these materials are paired respectively are all condensate droplets, and their respective sizes are determined by the properties of the constituent materials themselves.

[0082] Example 5

[0083] This example and the following examples use the condensate droplets of TA / F127 formula as an example to carry out research. TA was dissolved in ultrapure water to prepare solutions with concentrations of 1, 2, 4, 6, 8, and 10 mg / mL. At the same time, F127 was dissolved in ultrapure water to prepare solutions with concentrations of 1, 2, 4, 6, 8, and 10 mg / mL. TA solutions of different concentrations were successively mixed with F127 solutions of different concentrations to prepare condensates. The ultraviolet absorbance of the solution was detected using a multifunctional microplate reader. The contour map was drawn using the Origin software, and the critical curve for the formation of condensates was fitted. At the same time, the condensate solution was centrifuged at 12000g for 10 minutes to observe whether solid condensates were collected to further determine whether a phase change occurred. Figure 6 As shown, the coagulation effect gradually increases with the increase of the concentration of the two components, but if the proportion of F127 is too high, coagulation cannot occur. TA plays a dominant role in the formation of coagulated droplets, proving that the concentration and proportion on the right side of the critical line can form coagulation bodies, while the concentration and proportion on the left side cannot. The concentration and proportion of polyphenols and polymers to form polyphenol coagulation droplets have a critical range. The mass ratio of polyphenols to polymers is 0.5-10, and the concentration range of the two raw materials is 0.1-50 mg / mL.

[0084] Example 6

[0085] This example investigates the encapsulation performance of condensate droplets for molecules with different physicochemical properties, biomacromolecules, and nanomedicines, and selects lipophilic molecules: green long-chain carbocyanine dye (DiO), red long-chain carbocyanine dye (DiD), coumarin-6 (C6), desalted doxorubicin (DOX), 5-carboxyfluorescein (5-FAM), hydrophilic molecules: rhodamine B (RB), methylene blue (MB), fluorescein isothiocyanate (FITC), blue DNA dye (DAPI), propidium iodide (PI), small interfering RNA, and nanoparticles (PLGANPs) as representative molecules. TA was dissolved in ultrapure water to prepare a solution with a concentration of 10 mg / mL, and F127 was dissolved in ultrapure water to prepare a solution with a concentration of 10 mg / mL. First, the F127 solution was mixed with the working solution of each fluorescent molecule, and then mixed with the TA solution to prepare condensate droplets, and the encapsulation efficiency of the condensate droplets for the representative fluorescent molecules was observed by laser confocal microscopy. Figure 7 and Figure 8 As shown, the polyphenol aggregate droplets have excellent encapsulation capacity for molecules with different physicochemical properties.

[0086] Example 7

[0087] This example investigates the spatial confinement effect of condensate droplets on the molecules encapsulated inside, and conducts a fluorescence resonance energy transfer (FRET) experiment. The spatial distance of the FRET paired fluorescent molecules is determined by the fluorescence migration of the FRET paired fluorescent molecules. The FRET fluorescent molecule pairs are selected: hydrophilic FITC (FRET donor) and RB (FRET acceptor) and lipophilic DiI (FRET donor) and DiD (FRET acceptor). According to the method of Example 6, condensate droplets (herein the condensate droplets are named Droplet) that simultaneously encapsulate the FRET fluorescent molecule pairs are prepared, and the fluorescence emission spectrum of the sample is scanned with a multifunctional microplate reader. Fig. 9 As shown in the figure, the distance between fluorescent molecules in aqueous solution is far, and FRET cannot occur. Due to the spatial confinement effect of the condensate droplets on the molecules, independent compartments of the droplets are formed, which limits the movement area of ​​the molecules, brings the distance between the molecules closer, and FRET occurs in the FRET paired fluorescent molecules, proving the ability of polyphenol condensate droplets to enrich and concentrate molecules.

[0088] Example 8

[0089] According to Example 6, FITC-encapsulated coacervate droplets were prepared, and a fluorescence recovery after photobleaching (FRAP) experiment was performed using a laser confocal microscope. A pulsed high-intensity laser was used to bleach a selected area of ​​the microscope, and then the fluorescence recovery process of the area was observed over time. Fig.10 As shown, a strong laser irradiates a pre-set area for a short time to quench the fluorescent molecules in this area, and then the fluorescence intensity of this area is observed to gradually recover over time, proving that the droplets have fluidity and the fluorescent molecules diffuse and migrate in the droplets and then redistribute evenly.

[0090] Example 9

[0091] TA and F127 were prepared in the following dispersion media: ultrapure water and 10% sucrose, 5% sucrose + 5% lactose and 10% lactose aqueous solution, TA and F127 concentration was 10mg / mL solution, and then the TA solution and F127 solution were mixed to obtain condensate droplets. The solid powder obtained by freeze-drying the condensate droplets was ground into fine and loose dry powder, and the hygroscopicity and stability of the dry powder obtained after freeze-drying in different dispersion media were investigated. Fig.11 As shown, the coacervate dry powder prepared by dissolving TA and F127 in a 10% lactose aqueous solution is a fluffy and uniform powder, and its shape can still remain stable after being exposed to air for 30 minutes, and has good anti-hygroscopicity.

[0092] The above-mentioned condensate dry powder was redissolved in the same volume of ultrapure water, and the microscopic morphology of the condensate droplets was observed by laser confocal microscopy. Fig.12As shown, the freeze-drying process has no effect on the morphological structure of the polyphenol aggregate droplets, and the polyphenol aggregate droplets have the potential to be used as freeze-dried powder.

[0093] Example 10

[0094] The condensate droplets have a tendency to merge and grow, and have characteristics such as salt, pH, and temperature sensitivity, so surface modification is required to improve the stability of the condensate droplets. This embodiment explores different coating layers to achieve surface modification of the condensate droplets. The structure of polyphenols is rich in phenolic hydroxyl groups, so proteins and extracellular vesicles can be adsorbed on the surface of condensate droplets through hydrogen bonding interactions. The surface of polyphenol condensate droplets is negatively charged, so positively charged lipid membranes or polysaccharides can be adsorbed on the surface of condensate droplets through electrostatic interactions.

[0095] In this example, albumin (content of 10-100%), lipids (DDAB:DPPC=1:1, lipid content of 0.5-5%), and extracellular vesicles (isolated from the cell culture supernatant by differential centrifugation, protein content of 1-10%) are selected as representative coatings to modify the surface of the coacervate. TA is dissolved in ultrapure water to prepare a solution with a concentration of 10 mg / mL. At the same time, F127 is dissolved in ultrapure water to prepare a solution with a concentration of 10 mg / mL. The F127 solution is mixed with the TA solution to prepare coacervate droplets. After standing for half an hour to allow the coacervate droplets to grow to a stable state, a fluorescently labeled coating is added, and the microscopic morphology of the coacervate droplets is observed using a laser confocal microscope. Fig.13 As shown, the corresponding fluorescence is distributed on the periphery of the condensate droplets to form a circle structure, proving that the surface of the condensate droplets was successfully modified.

[0096] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing polyphenol aggregate droplets based on hydrogen bond interactions, characterized in that: The following steps are involved: (1) preparing a polyphenol into a polyphenol aqueous solution with a concentration of 0.1-50 mg / mL, wherein the number of phenolic hydroxyl groups of the polyphenol is greater than 8; preparing a high molecular polymer into a high molecular polymer aqueous solution with a concentration of 0.1-50 mg / mL, wherein the high molecular polymer contains oxygen, nitrogen or fluorine atoms; (2) The polyphenol aqueous solution obtained in step (1) and the high molecular polymer aqueous solution are mixed, wherein the mass ratio of the polyphenols in the polyphenol aqueous solution to the high molecular polymer in the high molecular polymer aqueous solution is (0.5-10):1, thereby obtaining polyphenol aggregate droplets.

2. The method for preparing polyphenol aggregate droplets based on hydrogen bond interaction according to claim 1, characterized in that: The polyphenol is tannic acid or epigallocatechin gallate.

3. The method for preparing polyphenol aggregate droplets based on hydrogen bond interaction according to claim 1, characterized in that: The high molecular polymer is Pluronic F-68, Pluronic F-127, polyethylene glycol, four-arm polyethylene glycol, polyvinyl alcohol, Tween 20, Tween 40 or Tween 80.

4. The method for preparing polyphenol aggregate droplets based on hydrogen bond interaction according to claim 1, characterized in that: In step (2), before the polyphenol aqueous solution and the high molecular polymer aqueous solution are mixed, the high molecular polymer aqueous solution is first mixed with the drug solution, and then mixed with the polyphenol aqueous solution to prepare drug-loaded polyphenol aggregate droplets.

5. The method for preparing polyphenol aggregate droplets based on hydrogen bond interaction according to claim 4, characterized in that: The drug is a hydrophilic small molecule, a hydrophobic small molecule or an amphiphilic small molecule drug.

6. The method for preparing polyphenol aggregate droplets based on hydrogen bond interaction according to claim 4, characterized in that: The drug is a polypeptide, a protein, a nucleic acid or a nano drug.

7. The method for preparing polyphenol aggregate droplets based on hydrogen bond interaction according to claim 1, characterized in that: In step (1), the polyphenol aqueous solution and the high molecular polymer aqueous solution are both disaccharide aqueous solutions; after step (2), the polyphenol aggregate droplets are freeze-dried to obtain dry powder.

8. The method for preparing polyphenol aggregate droplets based on hydrogen bond interaction according to claim 1, characterized in that: After step (2), the polyphenol aggregate droplets are mixed with proteins, lipid membranes, and extracellular vesicles to obtain surface-modified polyphenol aggregate droplets.

9. Polyphenol aggregate droplets based on hydrogen bond interaction prepared by the method according to any one of claims 1 to 8.

10. Use of the polyphenol aggregate droplets based on hydrogen bond interaction as claimed in claim 9 in preparing drug delivery carriers or artificial tissues.

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