Cladding layer for retaining cellular contents, encapsulating method and application

Abstract

The invention discloses a cladding for retaining cell contents, an encapsulation method and application, raw materials of the cladding comprise metal ions and plant polyphenols, the metal ions and the plant polyphenols form metal polyphenol nano-particles under an acidic condition, and after the metal polyphenol nano-particles are combined with cells, the metal polyphenol nano-particles are separated from the cells. And forming a cladding adhered to the cell on the surface of the cell under an alkaline condition. The cladding not only can prevent key substances in cells from leaking and maintain the integrity of the cell structure, but also can prevent harmful substances in the cells from leaking after the cells are broken, so that the risk caused by leakage of contents is reduced.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically relates to a cladding for retaining cell contents, an encapsulation method, and applications thereof. Background Art

[0002] In the fields of biomedicine and biotechnology, the retention of cell contents is crucial for maintaining cell function and ensuring the safety of applications (such as treatment and detection). However, under various conditions, such as in vitro storage, mechanical stress (such as centrifugation, freeze-thaw), pathological states (such as membrane damage), or specific application scenarios (such as the transportation of cell therapy products), cells often experience leakage of intracellular proteins, nucleic acids, metabolites, and organelle components due to the disruption of membrane integrity. Such leakage can cause various negative impacts. For example, in stem cell therapy, the loss of growth factors and cytokines secreted by cells will significantly reduce the therapeutic effect. In single-cell sequencing technology, the leakage of intracellular RNA may lead to cross-contamination between samples, affecting data reliability. In addition, for bacterial cells, the leakage of contents (such as the release of endotoxins) may also pose safety hazards.

[0003] To address the problem of cell content leakage, various encapsulation technologies have emerged in recent years. Among them, the microencapsulation technology based on the alginate system mainly uses ionic crosslinking to form a porous gel layer to wrap cells to protect cells and reduce content leakage. However, studies have shown that the pore size of calcium alginate gel is usually between 50 and 200 nm, which is much larger than the molecular sizes of proteins (molecular weight 10 - 500 kDa) and nucleic acids, resulting in the easy diffusion of these macromolecules into the external environment. The strategy of using lipid bilayer modification to construct a biomimetic cell membrane is to enhance the stability of the cell membrane through liposome fusion or membrane protein insertion to reduce content leakage, but this method faces problems such as low modification efficiency and poor long-term stability in practical applications, and the lipid coating is prone to shedding due to oxidation or mechanical stress. The chemical crosslinking method strengthens the cell membrane structure through crosslinking agents in an attempt to prevent content leakage. This method usually needs to be carried out under severe conditions such as low pH or high temperature, which will irreversibly damage the conformation of cell membrane proteins, resulting in the denaturation or loss of function of the contents. This limits the application of this technology in applications that require retaining the function of the contents.

[0004] Therefore, the development of an encapsulation technology that can effectively prevent cell content leakage remains a key challenge to be solved urgently. Summary of the Invention

[0005] The purpose of the present invention is to provide a cladding for retaining cell contents. The cladding is a metal-polyphenol complex system formed by metal ions and plant polyphenols, which can adhere to the cell surface to effectively prevent cell content leakage, maintain the integrity of the cell structure, and reduce the risks brought about by content leakage.

[0006] The present invention is achieved by the following technical solutions:

[0007] A cladding for retaining cell contents, the raw materials of the cladding including metal ions and plant polyphenols, wherein the metal ions and plant polyphenols form metal polyphenol nanoparticles under acidic conditions, and after the metal polyphenol nanoparticles bind to cells, an adherent cladding is formed on the cell surface under alkaline conditions.

[0008] In this technical solution, the main raw materials of the cladding include metal ions and plant polyphenols. After the metal ions and plant polyphenols are mixed, the pH of the solution is adjusted to acidic. Under acidic conditions, the complexation of plant polyphenols and metal ions is incomplete, and at this time, the size of the formed metal polyphenol nanoparticles is very small, and the connection between the metal polyphenol nanoparticles is still weak; when the small-sized metal polyphenol nanoparticles bind to the cell surface, the pH of the solution is adjusted to alkaline. Under alkaline conditions, the fine metal polyphenol nanoparticles are more closely connected to each other and to the cells, and then are wrapped on the cell surface to form a dense cladding with very small gaps.

[0009] In this technical solution, the gaps between the metal polyphenol nanoparticles in the cladding are small, which can effectively prevent the passage of biological macromolecules such as proteins and nucleic acids. At the same time, the phenolic hydroxyl groups rich in the metal polyphenol nanoparticles can form stable bonds with biological macromolecules such as proteins and nucleic acids through hydrogen bonds, hydrophobic interactions or π-π stacking, so as to adhere these substances inside the cladding to prevent leakage. Therefore, the cladding can not only prevent the leakage of key substances inside the cells and maintain the integrity of the cell structure, but also prevent the leakage of harmful substances inside the cells after the cells rupture, thereby reducing the risk brought by the leakage of the contents. In addition, the small gaps of the cladding can effectively block the proliferation and metabolic pathways of cells physically, achieving the purpose of inactivating cells. After the cladding is formed, it can simultaneously inactivate cells and prevent the leakage of contents.

[0010] In some preferred embodiments, the plant polyphenol can be epigallocatechin gallate (EGCG), procyanidin (PC), tara tannin, tannic acid (TA), etc. The metal ions can be zinc ions, manganese ions, iron ions, etc.

[0011] Furthermore, the raw materials of the cladding further include a stabilizer, and the stabilizer is used to react with the metal ions and plant polyphenols under acidic conditions to form the metal polyphenol nanoparticles.

[0012] In this technical solution, under acidic conditions, a stabilizer is also added during the complexation of metal ions and plant polyphenols to obtain metal polyphenol nanoparticles encapsulated by the stabilizer, thereby improving the stability of the small-sized metal polyphenol nanoparticles and facilitating their subsequent connection with cells. In some preferred embodiments, the stabilizer is hyaluronic acid, chitosan, carboxymethyl cellulose, alginic acid or alginate.

[0013] Furthermore, the pH value under acidic conditions is 3.5 - 5.5, and the pH value under alkaline conditions is 7.0 - 9.0. After the metal ions and plant polyphenols are mixed, the pH value of the solution is adjusted to 3.5 - 5.5 to form incompletely complexed, small-sized metal polyphenol nanoparticles. In one or more embodiments, a buffer solution can be added to the solution to adjust the pH value of the solution. Preferably, the buffer solution can be at least one of acetate buffer solution, citrate buffer solution, phosphate buffer solution, and succinate buffer solution. Subsequently, after the metal polyphenol nanoparticles bind to the cells, the pH value of the solution is adjusted to 7.0 - 9.0 to make the connection between the metal polyphenol nanoparticles and between the particles and the cell surface stable. In one or more embodiments, a buffer solution can be added to the solution to adjust the pH value of the solution. Preferably, the buffer solution can be at least one of phosphate buffer solution, Tris buffer solution, and borate buffer solution.

[0014] Furthermore, the molar ratio of the metal ions to the plant polyphenols is 4:1 - 2:1. In this technical solution, the content of metal ions affects the size and stability of the metal polyphenol nanoparticles. If the content of metal ions is too low, it is not easy to form metal polyphenol nanoparticles, and the formed metal polyphenol nanoparticles have poor stability. The metal polyphenol nanoparticles cannot stably connect to the cells to form a coating. Moreover, fewer metal ions will result in smaller-sized metal polyphenol nanoparticles and an incomplete coating, reducing the anti-leakage effect of the coating. On the contrary, if the content of metal ions is too high, the stability of the metal polyphenol nanoparticles is improved, but the size of the particles increases significantly, leading to an increase in the gap of the coating and also reducing the anti-leakage effect of the coating. Therefore, in this technical solution, the preferred molar ratio of metal ions to plant polyphenols is determined to be 4:1 - 2:1.

[0015] Furthermore, the diameter of the metal polyphenol nanoparticles is 5 - 50 nm. Metal polyphenol nanoparticles with too large a size have larger gaps and cannot effectively prevent the leakage of cell contents, while metal polyphenol nanoparticles with too small a size are difficult to form a stable and complete coating. Therefore, the diameter of the metal polyphenol nanoparticles is preferably controlled within 5 - 50 nm.

[0016] The present invention also provides an encapsulation method for retaining cell contents, comprising the following steps:

[0017] Mix the plant polyphenol solution and the metal ion solution to obtain a first solution;

[0018] Add a stabilizer to the first solution, and at the same time adjust the first solution to be acidic to obtain a suspension of metal polyphenol particles;

[0019] Add the suspension of metal polyphenol particles to the cell suspension containing the cells to be encapsulated to obtain a second solution, adjust the second solution to be alkaline, and form a coating on the surface of the cells to be encapsulated.

[0020] In this technical solution, first mix the plant polyphenol solution and the metal ion solution to obtain a first solution. In some preferred embodiments, first disperse the plant polyphenol solution in physiological saline, and then add the metal ion solution. After mixing evenly, a first solution is obtained. In one or more embodiments, the plant polyphenol solution is obtained by dissolving plant polyphenols in a solvent, and the metal ion solution is obtained by dissolving metal salts in a solvent. Among them, the solvent is preferably deionized water.

[0021] In this technical solution, after obtaining a uniformly mixed first solution, add a stabilizer to the first solution. At the same time, add, for example, a buffer solution to adjust the first solution to be acidic to obtain a suspension of metal polyphenol particles. At this time, the size of the metal polyphenol particles embedded by the stabilizer in the suspension of metal polyphenol particles is small. Subsequently, add the suspension of metal polyphenol particles to the cell suspension to obtain a second solution, and then adjust the second solution to be alkaline to promote the tight connection between metal polyphenol particles and between particles and cells, and form a dense coating on the cell surface to achieve the purpose of preventing the leakage of cell contents.

[0022] Further, the molar ratio of the metal ions in the metal ion solution to the plant polyphenols in the plant polyphenol solution is 4:1 to 2:1.

[0023] In some embodiments, the concentration of the plant polyphenol solution is 100 - 400 mg / mL, and the concentration of the metal ion solution is 40 - 250 mg / L.

[0024] In some embodiments, the concentration of the plant polyphenol solution is preferably 150 - 250 mg / mL. In some embodiments, the concentration of the metal ion solution is preferably 40 - 150 mg / L.

[0025] In some embodiments, the concentration range of the stabilizer is 0.1 - 10 mg / mL, preferably 1 mg / mL, and deionized water is used as the solvent.

[0026] In some embodiments, collect the target cells by centrifugation, and resuspend the pretreated tumor cells in physiological saline (0.9% sodium chloride solution, 1 mL) to make the cell concentration reach 1×10 5~1×10^7 cells / mL. Centrifuge and concentrate the cell suspension (200 - 800 rcf, 3 - 5 min), and resuspend the centrifuged cells in 50 - 400 μL of physiological saline.

[0027] In some embodiments, the encapsulated cells are collected. For mammalian cells, after the reaction, inactivated cells are collected by centrifugation (200 - 800 rcf, 3 - 5 min), and washed 3 - 5 times with phosphate buffer (pH = 7 - 9, 5 - 100 mM, 600 - 1500 μL). The cell suspension is resuspended in 1 mL of physiological saline. The cells are left standing at 4 - 30 °C for 8 - 24 hours to achieve efficient and gentle inactivation of the cells. Among them, mammalian cells can be tumor cells, immune cells, red blood cells, etc. For bacterial cells, after the reaction, inactivated bacterial cells are collected by centrifugation (2000 - 4000 rcf, 3 - 5 min), and washed 3 - 5 times with phosphate buffer (pH = 7 - 9, 5 - 100 mM, 600 - 1500 μL). The bacterial cell suspension is resuspended in 1 mL of physiological saline. The cells are left standing at 4 - 30 °C for 8 - 24 hours to achieve efficient and gentle inactivation of the cells.

[0028] The present invention also provides the application of any one of the foregoing claddings in encapsulating stem cells and maintaining the structural integrity of stem cells.

[0029] Specifically, in stem cell therapy, the retention of growth factors and cytokines (such as TGF-β, IGF-1) is crucial for promoting tissue regeneration and immune regulation. By forming a cladding composed of metal polyphenol nanoparticles on the surface of stem cells, the leakage of these key substances during in vitro manipulation and transplantation can be effectively prevented, thereby maintaining the therapeutic potential of stem cells. For example, in the treatment of myocardial infarction, retaining the vascular growth factor VEGF secreted by stem cells can significantly enhance the angiogenesis effect and improve the treatment success rate. In some embodiments, by centrifuging and analyzing the precipitate after breaking stem cells, it can be known that the stem cells with a cladding still retain the proteome before breaking, and there is no obvious decrease in the protein content and RNA content.

[0030] Similarly, this cladding can also encapsulate tumor cells, prevent the leakage of proteins and RNA in tumor cells, and maintain the structural integrity of tumor cells.

[0031] The present invention also provides the application of any of the foregoing claddings in wrapping bacterial cells and preventing the leakage of biological macromolecules and metabolites inside the bacterial cells. In some embodiments, after disrupting Escherichia coli cells and centrifuging to analyze the precipitate, it can be seen that the bacterial cells with claddings still retain the proteome before disruption. At the same time, in the supernatant, the amount of endotoxin leaked from the bacterial cells with claddings is much less than that of the bacterial cells without claddings. By reducing the leakage of endotoxin, the safety risk can be effectively reduced, providing guarantee for subsequent processes such as vaccine preparation.

[0032] In some embodiments, the bacteria involved include common pathogenic bacteria such as Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Staphylococcus epidermidis (S. epidermidis), Streptococcus pyogenes (S. pyogenes), Pseudomonas aeruginosa (S. aeruginosa), etc.; and common probiotics such as Lactobacillus casei (L. casei), Lactobacillus plantarum (L. plantarum), Bifidobacterium bifidum (B. bifidum), Lactococcus lactis (L. lactis), Streptococcus thermophilus (S. thermophilus), etc.

[0033] In some preferred embodiments, similarly, this application can also be used in single-cell sequencing. The leakage of intracellular RNA may lead to cross-contamination between samples and reduce the data accuracy. The cladding, through its dense barrier and adhesion properties, can effectively prevent the leakage of nucleic acid substances such as RNA, thereby improving the reliability and repeatability of the experiment.

[0034] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0035] 1. The gaps between the metal polyphenol nanoparticles in the cladding of the present invention are small, which can effectively prevent the passage of biological macromolecules such as proteins and nucleic acids. At the same time, the phenolic hydroxyl groups rich in the metal polyphenol nanoparticles can form stable bonds with biological macromolecules such as proteins and nucleic acids through hydrogen bonds, hydrophobic interactions or π-π stacking, thereby adhering these substances inside the cladding to prevent leakage. Therefore, the cladding can not only prevent the leakage of key substances inside the cells and maintain the integrity of the cell structure, but also prevent the leakage of harmful substances inside the cells after cell rupture, thereby reducing the risk brought by the leakage of the contents.

[0036] 2. Through two pH value adjustments in the present invention, during the first adjustment, the complexation of plant polyphenols and metal ions is incomplete under acidic conditions. At this time, the size of the formed metal polyphenol nanoparticles is very small, and the connection between the metal polyphenol nanoparticles is still weak. After the small-sized metal polyphenol nanoparticles bind to the cell surface, the pH of the solution is adjusted to alkaline for the second time. Under alkaline conditions, the small metal polyphenol nanoparticles are more closely connected to each other and to the cells, and then form a dense coating with very small gaps on the cell surface.

[0037] 3. By reasonably setting the molar ratio of metal ions to plant polyphenols in the present invention, more stable and smaller-sized metal polyphenol nanoparticles can be formed on the cell surface.

[0038] 4. The encapsulation method of the present invention does not require complex equipment and cumbersome steps. It can be completed only through simple operations such as preparing solutions, mixing cells and nanoparticle suspensions, etc. This greatly simplifies the operation process, reduces the operation difficulty and cost, and also reduces the risks and errors that may be brought by complex operations, improving safety.

[0039] 5. By preventing the leakage of cell contents, the coating of the present invention can not only maintain the structural integrity of cells such as stem cells and improve their potential functions, but also, for bacterial cells, limit endotoxins within the coating after their rupture, reducing safety risks.

[0040] 6. The present invention is applicable to a variety of cell types, including mammalian cells such as stem cells, immune cells, tumor cells, etc., as well as common pathogenic bacteria such as Escherichia coli and Staphylococcus aureus, and probiotics such as Lactobacillus and Bifidobacterium, providing wide applicability and flexibility for applications in different fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The drawings described herein are used to provide a further understanding of the embodiments of the present invention, form a part of this application, and do not limit the embodiments of the present invention. In the drawings:

[0042] Figure 1 is a flowchart of the encapsulation method in a specific embodiment of the present invention;

[0043] Figure 2 shows the surface of untreated stem cells and the surface of stem cells C1 encapsulated with EGCG-Mn characterized by transmission electron microscopy in a specific embodiment of the present invention;

[0044] Figure 3 shows stem cells C1 labeled with fluorescein-bovine serum albumin in a specific embodiment of the present invention;

[0045] Figure 4Shows the electrophoresis results of the precipitates of untreated stem cells and stem cell C1 after rupture in specific embodiments of the present invention;

[0046] Figure 5 Shows the protein content and RNA content collected from the precipitate of stem cell C1 after rupture in specific embodiments of the present invention;

[0047] Figure 6 Shows Escherichia coli cell C2 labeled with anthocyanin near-infrared fluorescent dye in specific embodiments of the present invention;

[0048] Figure 7 Shows the electrophoresis results of the precipitates of untreated Escherichia coli cells and Escherichia coli cell C2 after rupture in specific embodiments of the present invention;

[0049] Figure 8 Shows the protein content and endotoxin content collected from the supernatant of untreated Escherichia coli cells and Escherichia coli cell C2 after rupture in specific embodiments of the present invention;

[0050] Figure 9 Shows the comparison chart of tumor cells before and after inactivation treated by Calcein / PI live-dead cell staining method in specific embodiments of the present invention. Detailed implementation manners

[0051] To make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with embodiments and drawings. The illustrative embodiments and descriptions of the present invention are only used to explain the present invention and do not limit the present invention.

[0052] There are no special restrictions on the sources of all raw materials of the present invention, and they can be purchased on the market or prepared according to conventional methods well-known to those skilled in the art. There are no special restrictions on the purity of all raw materials of the present invention. The present invention preferably adopts analytical purity or the conventional purity requirements in the field of biotechnology. All raw materials of the present invention, their trademarks and abbreviations all belong to the conventional trademarks and abbreviations in the art, and each trademark and abbreviation is clear and definite in the field of its relevant uses. Those skilled in the art can purchase them from the market or prepare them by conventional methods according to the trademark, abbreviation and corresponding uses.

[0053] The "first", "second", etc. (such as the first solution, the second solution, the first buffer solution, the second buffer solution, etc.) used in the present invention are only used to distinguish the corresponding components for the sake of clear description, and do not aim to limit any order or emphasize importance, etc. In addition, the term "connection" used in this article, without special explanation, can be directly connected or indirectly connected through other groups.

[0054] I. Formation of a coating on the cell surface

[0055] At the present stage, metal polyphenol particles are mainly used for cell delivery, such as probiotic delivery, which requires maintaining the activity and function of cells during the delivery process. Therefore, when preparing metal polyphenol particles, polyphenols need to be connected to cells first, and then metal ions are combined with polyphenols to form a nano-coating. The gap of this nano-coating is very large, and cells can also achieve their functions and maintain activity through this large gap.

[0056] The encapsulation method of the present invention is first based on the formation of loose and fine metal polyphenol nanoparticles by metal polyphenols in a low pH environment. After connecting them with cells, the pH is adjusted to a high value to form a dense coating on the cell surface. The small gap of the coating particles is used to prevent the leakage of the contents in the cells, maintain the integrity of the functions and structures of cells such as stem cells, and at the same time reduce the risk brought by the leakage of the contents of cells such as bacterial cells. Moreover, during the encapsulation, the inventors also found that this coating can physically block the proliferation and metabolic pathways of cells, achieving the effect of inactivating cells, greatly improving the convenience and safety of related cell experiments.

[0057]

Example 1

[0058] In this example, stem cells encapsulated with EGCG-Mn nano-coating were prepared.

[0059] S1: Disperse the epigallocatechin gallate (EGCG) solution (200 μL, 150 mg / mL) in 1 mL of physiological saline, and then add manganese chloride (MnCl2) solution (40 μL, 150 mg / mL), and shake well in a shaker (100 rpm, 5 min) to obtain the first solution;

[0060] S2: Add hyaluronic acid solution (1 mg / mL) and the first phosphate buffer solution (pH = 5.0, 20 mM, 1 mL) to the first solution, and shake well in a shaker (100 rpm, 5 min) to obtain an EGCG-Mn nanoparticle suspension;

[0061] S3: Resuspend the pretreated stem cells (2×10 5 cells) in physiological saline (0.9% sodium chloride solution, 1 mL), add 400 μL of EGCG-Mn nanoparticle suspension to the cell suspension, and shake well in a shaker (200 rpm, 5 min) to obtain the second solution;

[0062] S4: Add the second phosphate buffer solution (pH = 8, 20 mM, 500 μL) to the second solution, and shake well in a shaker (200 rpm, 5 min) to obtain the stem cells C1 encapsulated with the coating composed of HA-embedded EGCG-Mn nanoparticles.

[0063]

Example 2

[0064] In this example, Escherichia coli encapsulated with EGCG-Mn nanolayer was prepared.

[0065] S1: Disperse the epigallocatechin gallate (EGCG) solution (200 μL, 150 mg / mL) in 1 mL of physiological saline, then add manganese chloride (MnCl2) solution (40 μL, 150 mg / mL), and shake well in a shaker (100 rpm, 5 min) to obtain the first solution.

[0066] S2: Add hyaluronic acid solution (1 mg / mL) and the first phosphate buffer solution (pH = 5.0, 20 mM, 1 mL) to the first solution, and shake well in a shaker (100 rpm, 5 min) to obtain the EGCG-Mn nanoparticle suspension.

[0067] S3: Resuspend the pretreated Escherichia coli cells (E. coli, 1×10 7 cells / mL, OD 600 about 0.1) in physiological saline (0.9% sodium chloride solution, 1 mL), add 400 μL of the EGCG-Mn nanoparticle suspension to the cell suspension, and shake well in a shaker (200 rpm, 5 min) to obtain the second solution.

[0068] S4: Add the second phosphate buffer solution (pH = 8, 20 mM, 500 μL) to the second solution, and shake well in a shaker (200 rpm, 5 min) to obtain Escherichia coli cells C2 encapsulated with the nanolayer composed of HA-embedded EGCG-Mn nanoparticles.

[0069]

Example 3

[0070] In this example, tumor cells encapsulated with TA-Fe nanolayer were prepared:

[0071] S1: Disperse the tannic acid (TA) solution (100 μL, 250 mg / mL) in 1 mL of physiological saline, then add iron chloride (FeCl3) solution (50 μL, 100 mg / mL), and shake well in a shaker (100 rpm, 5 min) to obtain the first solution.

[0072] S2: Add chitosan solution (1 mg / mL) and the first phosphate buffer solution (pH = 5.0, 20 mM, 1 mL) to the first solution, and shake well in a shaker (100 rpm, 5 min) to obtain the TA-Fe nanoparticle suspension.

[0073] S3: Resuspend the pretreated B16F10 tumor cells (2×10 5(0) were resuspended in normal saline (0.9% sodium chloride solution, 1 mL), and 400 μL of TA-Fe nanoparticle suspension was added to the cell suspension, and the mixture was shaken well on a shaker (200 rpm, 5 min) to obtain a second solution;

[0074] S4: Add the second phosphate buffer solution (pH = 8, 20 mM, 500 μL) to the second solution, and shake well on a shaker (200 rpm, 5 min) to obtain tumor cells C3 wrapped with a coating composed of TA-Fe nanoparticles.

[0075]

Example 4

[0076] This example prepares tumor cells wrapped with a PC-Zn nanocoating:

[0077] S1: Disperse the procyanidin PC solution (200 μL, 150 mg / mL) in 1 mL of normal saline, and then add zinc chloride ZnCl2 solution (40 μL, 150 mg / mL), and shake well on a shaker (100 rpm, 5 min) to obtain a first solution;

[0078] S2: Add the hyaluronic acid solution (1 mg / mL) and the first phosphate buffer solution (pH = 5.0, 20 mM, 1 mL) to the first solution, and shake well on a shaker (100 rpm, 5 min) to obtain a PC-Zn nanoparticle suspension;

[0079] S3: Resuspend the pretreated B16F10 tumor cells (2×10 5 (0) were resuspended in normal saline (0.9% sodium chloride solution, 1 mL), and 400 μL of PC-Zn nanoparticle suspension was added to the cell suspension, and the mixture was shaken well on a shaker (200 rpm, 5 min) to obtain a second solution;

[0080] S4: Add the second phosphate buffer solution (pH = 8, 20 mM, 500 μL) to the second solution, and shake well on a shaker (200 rpm, 5 min) to obtain tumor cells C4 wrapped with a coating composed of PC-Zn nanoparticles.

[0081]

Example 5

[0082] This example prepares tumor cells wrapped with a PC-Zn nanocoating:

[0083] S1: Disperse the procyanidin PC solution (200 μL, 150 mg / mL) in 1 mL of normal saline, and then add zinc chloride ZnCl2 solution (40 μL, 150 mg / mL), and shake well on a shaker (100 rpm, 5 min) to obtain a first solution;

[0084] S2: Add the first phosphate buffer solution (pH = 5.0, 20 mM, 1 mL) to the first solution, and shake well on a shaker (100 rpm, 5 min) to obtain a PC-Zn nanoparticle suspension;

[0085] S3: Resuspend the pretreated B16F10 tumor cells (2×10 5 cells) in normal saline (0.9% sodium chloride solution, 1 mL). Add 400 μL of the PC-Zn nanoparticle suspension to the cell suspension, and shake well on a shaker (200 rpm, 5 min) to obtain a second solution;

[0086] S4: Add the second phosphate buffer solution (pH = 8, 20 mM, 500 μL) to the second solution, and shake well on a shaker (200 rpm, 5 min) to obtain tumor cells C5 wrapped with a coating composed of PC-Zn nanoparticles.

[0087] II. Performance testing and characterization of cells with the coating

[0088]

Example 6

[0089] In this example, the stem cells C1 wrapped with a coating composed of EGCG-Mn nanoparticles were characterized.

[0090] Specifically, a transmission electron microscope was used to characterize the cell surface, Figure 2 showing TEM images of the cross-sections of the original stem cells and the stem cells C1 wrapped with EGCG-Mn nanoparticles. It can be seen from the figure that the EGCG-Mn nanoparticles embedded in HA formed a dense nano-coating on the cell surface, while the cell membrane of the original stem cells was smooth, indicating that the stem cells were completely encapsulated by the coating formed by using EGCG and manganese ions.

[0091] Furthermore, after encapsulating the stem cells with EGCG-Mn, a fluorescein-bovine serum albumin (FITC-BSA) fluorescent probe was added. The test results are as Figure 3 shown. The polyphenols labeled with fluorescent protein demonstrated the loading of EGCG-Mn on the surface of the stem cells, indicating the presence of the EGCG-Mn coating on the surface of the stem cells.

[0092]

Example 7

[0093] In this example, the changes in the content of the lysed contents of stem cells without metal polyphenol nanoparticles on the surface and stem cells C1 with EGCG-Mn were compared.

[0094] Specifically, the same amount of untreated stem cells and stem cells C1 treated with EGCG-Mn were lysed by hypotonic treatment, centrifuged at 300 g for 3 min, and then the supernatant and precipitate were separated.

[0095] The protein content in the precipitate was analyzed by gel electrophoresis, as Figure 4 shown. Lane 1 is the proteome of the control stem cells, lane 2 is the proteome collected from the untreated stem cells; lane 3 is the proteome collected after the inactivation of stem cell C1 with an EGCG-Mn coating.

[0096] As can be seen from the figure, the protein content collected from the stem cells (mesenchymal stem cells) inactivated by the method of the present invention is basically the same as that of the original cells, indicating that the nanocoating formed by the metal polyphenol nanoparticles can effectively retain the cell contents and avoid the leakage of key contents of stem cells, such as TGF-β and IGF-1, during in vitro operations and transplantation, maintaining the therapeutic potential of stem cells. For example, in the treatment of myocardial infarction, retaining the vascular growth factor VEGF secreted by stem cells can significantly enhance the angiogenesis effect and improve the treatment success rate.

[0097] Furthermore, proteins were extracted from the cells using RIPA lysis and extraction buffer containing protease inhibitors (Beyotime, China). According to the manufacturer's instruction manual, a BCA protein assay kit was used to determine the protein concentration. For the RNA concentration test, Triquick reagent (a Trizol alternative, Solarbio, China) was used to extract the RNA of the cells. The content of RNA was quantified by NanoDrop2000 (Thermo Fisher Scientific, USA).

[0098] Figure 5 The protein content and RNA content of stem cell C1 with an EGCG-Mn coating in the precipitate and the control stem cells are shown. It can be seen that the nanocoating effectively prevents the leakage of stem cell contents and retains a large amount of RNA and almost all proteins in the stem cells. Through calculation, it was found that the protein leakage of stem cells decreased from 50 μg / mL to 5 μg / mL, and the RNA leakage decreased from 100 ng / mL to 10 ng / mL, with the leakage rate reduced by 90%.

[0099]

Example 8

[0100] In this example, Escherichia coli cells C2 encapsulated with an EGCG-Mn nanoparticle coating were characterized.

[0101] Specifically, after encapsulating Escherichia coli cells with EGCG-Mn, cyanine near-infrared fluorescent dye-labeled bovine serum albumin (Cy7-BSA) was added as a fluorescent probe. The test results are as Figure 6 shown. The confocal microscopy results showed fluorescence around the cells, indicating the presence of an EGCG-Mn coating on the cell surface.

[0102]

Example 9

[0103] In this example, the changes in the content of the lysate of Escherichia coli cells without metal polyphenol nanoparticles on the surface were compared with those of Escherichia coli cells C2 with an EGCG-Mn coating.

[0104] First, collect the untreated Escherichia coli and the Escherichia coli broth with an EGCG-Mn coating, and centrifuge (3000 rcf, 5 min) to collect the bacterial precipitate. Resuspend it in phosphate buffer (pH = 7.4, 10 mM), and adjust the OD600 to approximately 1.0, about 1×10 9 CFU / mL.

[0105] The untreated Escherichia coli group and the Escherichia coli group with an EGCG-Mn coating were respectively lysed using a cell disruptor. After centrifugation (3000 rcf, 5 min), the supernatant and precipitate were separated. The endotoxin proteins contained in the Escherichia coli cells in the precipitate were detected by gel electrophoresis. Among them, Laemmli sample buffer (Solarbio Science & Technology Co., Ltd., China) was used to prepare the loading samples, and the protein loading amount per well was 15 μg. After incubation at 95 °C for 5 min, they were loaded onto a 5-20% precast gel. The visualization of protein bands was reflected by immersing the gel in Coomassie Brilliant Blue staining solution.

[0106] As Figure 7 shown, lane 1 is the endotoxin protein group contained in the control Escherichia coli cells, lane 2 is the endotoxin protein group collected after inactivating the Escherichia coli group with an EGCG-Mn coating, and lane 3 is the endotoxin protein group collected by centrifugation after directly irradiating and inactivating the Escherichia coli cells. The results show that the content of the endotoxin proteins collected from the Escherichia coli cells C2 with an EGCG-Mn coating is the same as that of the original control cells, indicating that the coating formed by metal polyphenol nanoparticles can effectively retain the contents of bacterial cells.

[0107] Furthermore, a BCA protein kit and an LAL kit were used to detect the contents of proteins and endotoxins in the supernatants of untreated Escherichia coli and Escherichia coli cells C2, respectively. The test results are as Figure 8 shown. There are a large amount of protein content and endotoxin in the supernatant of the Escherichia coli group without a coating, while the protein and endotoxin contents in the supernatant of the Escherichia coli cells C2 with a coating are much lower than those of the untreated Escherichia coli, indicating that after cell rupture, the coating composed of metal polyphenol nanoparticles can still prevent the leakage of proteins and endotoxins, significantly reducing the safety risk and providing guarantee for subsequent processing (such as vaccine preparation). Through calculation, it is found that the endotoxin leakage of Escherichia coli is reduced from 100 EU / mL to 10 EU / mL, also reducing by 90%.

[0108]

Example 10

[0109] In this embodiment, the Calcein / PI cell viability staining method was used to detect the cell viability after inactivation by the procyanidin PC-Zn ion nano-coating. Specifically, the tumor cells C5 were incubated in a cell culture incubator (37 °C, 5% CO2) with DMEM medium (10% serum, 1% double antibody) for varying lengths of time up to 12 h. Then the cells were collected, washed with PBS, and 195 μL of Annexin V-FITC and 5 μL of PI (per 5 × 10 4 cells) were added. After incubation in the dark at room temperature for 20 min, detection was performed using a laser confocal microscope. The test results are as Figure 9 shown. The tumor cells C5 treated by the inactivation method of the present invention completely lost their proliferative ability within 12 hours, and the cell viability was close to zero, indicating a significant inactivation effect.

[0110] The specific embodiments described above further elaborate on the object, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only for the specific embodiments of the present invention and is not used to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A cladding for retaining cell contents, characterized in that, The raw materials of the cladding include metal ions and plant polyphenols. The metal ions and plant polyphenols form metal polyphenol nanoparticles under acidic conditions. After the metal polyphenol nanoparticles bind to cells, a cladding that adheres to the cells is formed on the cell surface under alkaline conditions.

2. The cladding for retaining cell contents according to claim 1, characterized in that, The raw materials of the cladding further include a stabilizer, and the stabilizer is used to react with metal ions and plant polyphenols together under acidic conditions to form the metal polyphenol nanoparticles.

3. The cladding for retaining cell contents according to claim 2, characterized in that, The stabilizer is hyaluronic acid, chitosan, carboxymethyl cellulose, alginic acid or alginate.

4. The cladding for retaining cell contents according to claim 1, characterized in that, The pH value under the acidic conditions is 3.5 - 5.5, and the pH value under the alkaline conditions is 7.0 - 9.

0.

5. The cladding for retaining cell contents according to claim 1, characterized in that, The molar ratio of the metal ions to the plant polyphenols is 4:1 - 2:

1.

6. The cladding for retaining cell contents according to any one of claims 1 to 5, characterized in that, The diameter of the metal polyphenol nanoparticles is 5 - 50 nm.

7. A method of encapsulation for retaining cell contents, characterized in that, It includes the following steps: Mix the plant polyphenol solution and the metal ion solution to obtain a first solution; Add a stabilizer to the first solution, and at the same time adjust the first solution to be acidic to obtain a metal polyphenol particle suspension; Add the metal polyphenol particle suspension to the cell suspension containing the cells to be encapsulated to obtain a second solution, and adjust the second solution to be alkaline to form a cladding on the surface of the cells to be encapsulated.

8. A method for encapsulating for retaining cell contents according to claim 7, characterized in that, The molar ratio of the metal ions in the metal ion solution to the plant polyphenols in the plant polyphenol solution is 4:1 - 2:

1.

9. Use of the cladding according to any one of claims 1 - 6 in encapsulating stem cells or tumor cells, wherein the cladding is used to prevent the leakage of growth factors and cytokines in the stem cells, or to prevent the leakage of proteins and RNAs in tumor cells, and maintain the structural integrity of the cells.

10. Use of the cladding according to any one of claims 1 - 6 in encapsulating bacterial cells, wherein the cladding is used to prevent the leakage of biomacromolecules and metabolites in the bacterial cells.

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