PH-sensitive liposome for entrapment of cell culture additive and preparation method of pH-sensitive liposome

Abstract

The invention discloses a pH-sensitive liposome for entrapment of a cell culture additive and a preparation method of the pH-sensitive liposome, and relates to the technical field of pH-sensitive liposomes. According to the invention, nutrient substances such as glucose, amino acid and glutamine and pH regulators such as NaOH and NaHCO3 required in the middle and later periods of cell growth are prepared into pH-sensitive lipidosome, the substances are respectively wrapped in cavities of the lipidosome, and after the pH of a cell growth solution system is reduced, the substances can be respectively released to be beneficial to improving the growth state of cells, improving the cell growth state and improving the cell growth efficiency. The pH balance of the cell growth liquid can be realized, and the cell growth liquid is kept neutral. The prepared pH sensitive liposome is uniform in size, good in dispersity, stable in structure in a neutral solution and capable of being effectively split in an acid solution, and the growth state of cells can be improved after the pH sensitive liposome is loaded with nutrient substances and a pH regulator. The pH-sensitive lipidosome can be added with substances required by cell culture in a manner of not opening a cell culture container, so that the risk of cell pollution is reduced.

Description

Technical Field

[0001] The invention relates to the technical field of pH sensitive liposomes, in particular to a pH sensitive liposome for encapsulating cell culture additives and a preparation method thereof. Background Art

[0002] One of the matrices for the preparation of inactivated vaccines is cells. During the culture process, cells consume nutrients such as glucose, amino acids, and glutamine, and the carbon dioxide and other substances produced increase the acidity in the growth medium. The reduction of nutrients and the increase in acidity are both detrimental to cell growth. Therefore, in the middle and late stages of cell growth, it is necessary to add nutrients such as glucose, amino acids, glutamine, or pH regulators such as NaOH and NaHCO3 that can increase the pH of the solution.

[0003] The containers for cell growth, from culture flasks, shake flasks, cell factories to fermenters of different sizes, are all closed and sterile environments. During the cell growth process, if new substances need to be added, the cell culture container needs to be opened, which will inevitably increase the risk of cell contamination. Therefore, a method is needed to reduce the risk of cell contamination without opening the container.

[0004] Liposomes are artificial membranes made of heptadecanoic acid phospholipids and ceramide, and have a bilayer structure similar to the cell membrane structure. The diameter of liposomes is generally between 25 and 1000 nm. The drugs and substances that can be encapsulated by liposomes include not only large molecular bioactive substances (glucose, amino acids, glutamine, etc.), but also small molecular active substances, which indicates that liposomes have the potential to encapsulate various types of substances. In particular, the reverse evaporation method in liposome preparation is particularly suitable for encapsulating water-soluble and large molecular bioactive substances, which further proves that liposomes can encapsulate a variety of substances including pH regulators (NaOH / NaHCO3).

[0005] The principle of pH-sensitive liposomes is that low pH can lead to the protonation of the carboxyl groups of fatty esters and cause the formation of hexagonal crystal phases, which is the main mechanism of membrane fusion. Under acidic conditions, when the pH decreases from 7.4 to 6.3-5.3, the pH-liposome membrane undergoes structural changes, causing the liposome membrane to dissolve and rupture, releasing the encapsulated substances in the liposomes. Chinese patent CN201810297334.8 discloses a method for preparing co-loaded liposomes by thin film hydration. The preparation method can provide a reference for the preparation of the liposomes of the present invention, but the direction of adding nutrients for cell culture is currently lacking in research on liposome controlled release methods. Summary of the invention

[0006] The purpose of the present invention is to provide a pH-sensitive liposome for encapsulating cell culture additives and a preparation method thereof, wherein the liposome meets the following requirements: in the early stage of cell growth, the pH of the growth medium is neutral, and the structure of the pH-sensitive liposome is stable and unchanged; in the late stage of cell growth, the pH liposome membrane structure changes under the acidic environment of the growth medium, which causes the liposome membrane to be destroyed, thereby slowly releasing nutrients in the liposome for cell growth, or slowly releasing NaOH, NaHCO3 and the like to neutralize the acidity in the growth medium, thereby creating a pH environment more suitable for cell growth; the pH-sensitive liposome is applied to cell culture to solve the problems raised in the above-mentioned background technology.

[0007] The invention discloses a pH-sensitive liposome respectively encapsulating one or more nutrients such as glucose, amino acid, glutamine, etc. or a pH regulator such as NaOH, NaHCO3, etc. which can increase the pH of a solution.

[0008] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0009] A pH-sensitive liposome for encapsulating cell culture additives, wherein each milliliter of the pH-sensitive liposome comprises: a pH-sensitive phospholipid modified with polyethylene glycol or polyacrylamide, cholesterol, an antioxidant, a nutrient or a pH regulator;

[0010] The pH sensitive phospholipids are one or more of dipalmitoyl phospholipids, heptadecanoyl phospholipids and disulfide phosphatidyl phospholipids.

[0011] According to the more optimized solution, the specific components of each milliliter of pH-sensitive liposomes include: 1-2 mg / mL of pH-sensitive phospholipids modified with polyethylene glycol or polyacrylamide, 0.05-0.15 mg / mL of cholesterol, 0.06-0.1 mg / mL of antioxidants, and 0.4-1 mg / mL of nutrients or pH regulators.

[0012] In the more optimized solution, the specific components of each milliliter of pH-sensitive liposomes include: 2 mg / mL of pH-sensitive phospholipids modified with polyethylene glycol or polyacrylamide, 0.1 mg / mL of cholesterol, 0.1 mg / mL of antioxidant, and 1 mg / mL of pH regulator.

[0013] According to the more optimized solution, the specific components of each milliliter of pH-sensitive liposomes include: 2 mg / mL pH-sensitive phospholipids modified with polyethylene glycol or polyacrylamide, 0.1 mg / mL cholesterol, 0.1 mg / mL antioxidant, and 0.8 mg / mL nutrients; when adding nutrients, 8 to 10 mL of buffer solution is also added.

[0014] In a more optimized solution, the antioxidant is any one of coenzyme Q10 and vitamin E; the nutrients are one or more of glucose, amino acids, and glutamine; the pH adjuster is any one of NaOH and NaHCO3; and the buffer solution is any one of citrate buffer, phosphate buffer, and carbonate buffer.

[0015] In a more optimized solution, the buffer solution is a phosphate buffer solution with a pH of 7.30-7.50, and the specific formula ingredients include: 8g / LNaCl, 0.2g / LKCl, 1.44g / LNa2HPO4, and 0.24g / LKH2PO4.

[0016] The more optimized scheme, the specific preparation method of the pH-sensitive phospholipid modified with polyethylene glycol is as follows: dissolve polyethylene glycol in chloroform, add acetic acid propionic anhydride as a catalyst, then add an activator, activate the reaction at 25°C for 1 to 2 hours, then add pH-sensitive phospholipid, react at 55 to 65°C for 24 hours, add the reactant to ethanol, slowly stir magnetically, centrifuge, and obtain the pH-sensitive phospholipid modified with polyethylene glycol. The molar ratio of polyethylene glycol: acetic acid propionic anhydride: activator: disulfide phosphatidyl amide is 16:2:1:1.

[0017] The more optimized scheme, the specific preparation method of polyacrylamide-modified pH-sensitive phospholipid is as follows: dissolve the pH-sensitive phospholipid in acetone, add an activator, react at 25°C for 1 to 2 hours, add the reactant to an ethanol solution of polyacrylamide, react at 50 to 60°C for 24 to 36 hours, slowly stir magnetically, and centrifuge to obtain the polyacrylamide-modified pH-sensitive phospholipid.

[0018] The activator is any one of DCC·HCL (1,3-dicyclohexylcarbodiimide hydrochloride), DIC·HCL (N,N'-diisopropylcarbodiimide hydrochloride), and EDC·HCL (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride); the molecular weight of the polyethylene glycol is any one of 2000, 3400, and 5000.

[0019] A more optimized solution is a method for preparing a pH-sensitive liposome for encapsulating a cell culture additive according to any one of the above items, wherein the specific steps are:

[0020] Step 1: Adding polyethylene glycol or polyacrylamide-modified pH-sensitive phospholipids, antioxidants and cholesterol to an organic solvent, mixing them thoroughly, and evaporating the organic solvent by rotary evaporation to form a lipid film;

[0021] Step 2: Add an aqueous solution of nutrients or pH regulators to the lipid film obtained in step 1, and ultrasonicate in an ice-water bath for 6 to 8 minutes to obtain a uniform liposome solution; when adding nutrients, a buffer solution must be added.

[0022] Step 3: The liposome solution obtained in step 2 is purified by gel filtration column to obtain pH-sensitive liposomes.

[0023] In a more optimized solution, in step 1, the organic solvent is any one of chloroform, methanol and acetone; in step 2, the frequency of ice-water bath ultrasound is 250-550 Hz, preferably 450 Hz.

[0024] A more optimized solution is the use of a pH-sensitive liposome for encapsulating cell culture additives according to any of the above items in cell culture.

[0025] The invention discloses a pH-sensitive liposome for encapsulating a cell culture additive, which can be added to a culture bottle, a shaking bottle, a cell factory and fermenters of different specifications when used for cell culture.

[0026] Compared with the prior art, the beneficial effects achieved by the present invention are:

[0027] In this scheme, the pH-sensitive phospholipid is one or more of dipalmitoyl phospholipids, heptadecanoyl phospholipids, and disulfide phosphatidyl amide. After being modified with polyethylene glycol or polyacrylamide, it can reduce the leakage rate of liposomes, increase the stability of liposome solutions, reduce their aggregation and fusion, and extend the shelf life; the antioxidant is any one of coenzyme Q10 and vitamin E, wherein coenzyme Q10 is a fat-soluble antioxidant that is essential for cell growth and health. It can improve the utilization rate of oxygen by cells, produce the energy required for cell growth and health, and at the same time, it can also resist oxidation and protect cells from free radicals. Vitamin E is also called vitamin E, also known as tocopherol or progesterone. It is a general term for fat-soluble vitamins such as α, β, γ and δ-tocopherol and α, β, γ and δ-tocotrienol. It is a nutrient that cannot be synthesized or is insufficiently supplied in animal bodies and is one of the most important antioxidants. It is soluble in organic solvents such as fat and ethanol, insoluble in water, stable to heat and acid, unstable to alkali, sensitive to oxygen, insensitive to heat, and particularly outstanding in blocking lipid oxidation. The addition of oil-soluble antioxidants (coenzyme Q10, vitamin E) can enhance the antioxidant properties of liposomes and improve the utilization rate of oxygen by cells.

[0028] The present application prepares the nutrients such as glucose, amino acids, glutamine and pH regulators such as NaOH and NaHCO3 required for the middle and late stages of cell growth into pH-sensitive liposomes, and encapsulates these substances in the liposome cavity respectively. After the pH of the cell growth solution system is reduced, they can be released respectively to improve the growth state of the cells, and the pH balance of the cell growth solution can be achieved and maintained at neutral. The pH-sensitive liposomes prepared by the present invention are uniform in size, good in dispersibility, stable in structure in neutral solutions, and can be effectively lysed in acidic solutions. After being loaded with nutrients and pH regulators, they are beneficial to improving the growth state of cells.

[0029] The application of pH-sensitive liposomes in the present application can add substances required for cell culture without opening the cell culture container, thereby reducing the risk of cell contamination compared to the traditional method of directly opening the lid. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Transmission electron microscopy images of pH-sensitive liposomes prepared in Examples 3 to 5;

[0031] Figure 2 This is the particle size distribution diagram of the empty pH-sensitive liposomes prepared in Example 3;

[0032] Figure 3 This is the particle size distribution diagram of the glucose pH sensitive liposomes prepared in Example 4;

[0033] Figure 4 This is the particle size distribution diagram of the NaHCO3 pH sensitive liposomes prepared in Example 5;

[0034] Figure 5 The transmission electron micrographs of the empty pH-sensitive liposomes prepared in Example 3 after being treated with hydrochloric acid solutions of different pH values;

[0035] Figure 6 This is a statistical diagram of the MTT results of the pH-sensitive liposomes prepared in Examples 3 to 5;

[0036] Figure 7 The MTT method was used to detect the effects of the pH sensitive liposomes prepared in Examples 3 to 5 on the viability of Vero cells; wherein A was a cell control group, B was an empty pH sensitive liposome group, C was a glucose pH sensitive liposome group, and D was a NaHCO3 pH sensitive liposome group. DETAILED DESCRIPTION

[0037] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] In the following examples, the molecular weight of polyethylene glycol is 2000; the molecular weight of cationic polyacrylamide is 8 million.

[0039] Embodiment 1:

[0040] (1) The specific preparation method of polyethylene glycol-modified pH-sensitive phospholipid (disulfide phosphatidyl-polyethylene glycol) is as follows:

[0041] 2 g of polyethylene glycol was dissolved in 300 mL of chloroform, acetic anhydride was added as a catalyst, and then 1,3-dicyclohexylcarbodiimide hydrochloride was added, and the reaction was activated at 25°C for 1 hour. Then, disulfide phosphatidylcholine was added and the reaction was carried out at 55°C for 24 hours. The reactants were added to ethanol, slowly stirred with magnetic stirring, and centrifuged at 3000 g for 10 minutes. The precipitate was taken to obtain disulfide phosphatidylcholine-polyethylene glycol.

[0042] The molar ratio of polyethylene glycol, acetic anhydride, 1,3-dicyclohexylcarbodiimide hydrochloride and disulfide phosphatidyl amide is 16:2:1:1.

[0043] (2) The specific preparation method of polyethylene glycol-modified pH-sensitive phospholipid (dipalmitoyl phospholipid-polyethylene glycol) is as follows:

[0044] 2 g of polyethylene glycol was dissolved in 300 mL of chloroform, acetic anhydride was added as a catalyst, and then 1,3-dicyclohexylcarbodiimide hydrochloride was added, and the reaction was activated at 25°C for 1 hour. Then, dipalmitoyl phospholipid was added and the reaction was carried out at 55°C for 24 hours. The reactants were added to ethanol, slowly stirred with magnetic stirring, and centrifuged at 3000 g for 10 minutes. The precipitate was taken to obtain dipalmitoyl phospholipid-polyethylene glycol.

[0045] The molar ratio of polyethylene glycol, acetic anhydride, 1,3-dicyclohexylcarbodiimide hydrochloride and dipalmitoyl phospholipid is 16:2:1:1.

[0046] Example 2: The specific preparation method of the pH-sensitive phospholipid modified with polyacrylamide (heptadecanoic acid phospholipid-polyacrylamide) is as follows:

[0047] 3 g of lecithin heptadecanoate was dissolved in 500 mL of acetone, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added, and the mixture was reacted at 25° C. for 2 hours. The reactant was added to an ethanol solution of polyacrylamide and the mixture was reacted at 55° C. for 24 hours. The mixture was slowly stirred with a magnetic stirrer and centrifuged at 3000 g for 10 minutes. The precipitate was collected to obtain lecithin heptadecanoate-polyacrylamide.

[0048] The molar ratio of heptadecanoic acid phospholipid: polyacrylamide (PPA): 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 2:16:1.

[0049] Example 3: The specific preparation method of empty pH sensitive liposomes is:

[0050] Step 1: 20 mg of disulfide phosphatidyl-polyethylene glycol obtained in Example 1, 1 mg of antioxidant and 1 mg of cholesterol were added to 6 mL of chloroform solution, and the mixture was thoroughly mixed. The chloroform was evaporated to dryness by a rotary evaporator to form a lipid film;

[0051] Step 2: Add 10 mL of phosphate buffer solution (pH 7.40, specific formula ingredients include: 8 g / L NaCl, 0.2 g / L KCl, 1.44 g / L Na2HPO4, 0.24 g / L KH2PO4) to the lipid film obtained in step 1, and perform ice-water bath ultrasound for 8 minutes to obtain a uniform liposome solution; the frequency of ice-water bath ultrasound is 450 Hz.

[0052] Step 3: The liposome solution obtained in step 2 is purified by gel filtration column to obtain empty pH-sensitive liposomes.

[0053] Example 4: The specific preparation method of glucose pH sensitive liposomes is:

[0054] Step 1: 20 mg of disulfide phosphatidyl-polyethylene glycol obtained in Example 1, 1 mg of antioxidant and 1 mg of cholesterol were added to 6 mL of chloroform solution, and the mixture was thoroughly mixed. The chloroform was evaporated to dryness by a rotary evaporator to form a lipid film;

[0055] Step 2: Add 2 mL of glucose aqueous solution (concentration of 4 mg / mL) and 8 mL of phosphate buffer solution (pH 7.40, specific formula ingredients include: 8 g / L NaCl, 0.2 g / L KCl, 1.44 g / L Na2HPO4, 0.24 g / L KH2PO4) to the lipid film obtained in step 1, and perform ice-water bath ultrasound for 8 minutes to obtain a uniform liposome solution; the frequency of ice-water bath ultrasound is 450 Hz.

[0056] Step 3: The liposome solution obtained in step 2 is purified by gel filtration column to obtain pH-sensitive liposomes.

[0057] Example 5: The specific preparation method of NaHCO3 pH sensitive liposomes is:

[0058] Step 1: 20 mg of disulfide phosphatidyl-polyethylene glycol obtained in Example 1, 1 mg of antioxidant and 1 mg of cholesterol were added to 6 mL of chloroform solution, and the mixture was thoroughly mixed. The chloroform was evaporated to dryness by a rotary evaporator to form a lipid film;

[0059] Step 2: Add 10 mL of NaHCO3 aqueous solution (concentration of 1 mg / mL) to the lipid film obtained in step 1, and perform ice-water bath ultrasound for 8 minutes to obtain a uniform liposome solution; the frequency of the ice-water bath ultrasound is 450 Hz.

[0060] Step 3: The liposome solution obtained in step 2 is purified by gel filtration column to obtain pH-sensitive liposomes.

[0061] Example 6: The process is the same as that of Example 4, except that the amount of glucose is adjusted. The specific preparation steps are:

[0062] Step 1: 20 mg of disulfide phosphatidyl-polyethylene glycol obtained in Example 1, 1 mg of antioxidant and 1 mg of cholesterol were added to 6 mL of chloroform solution, and the mixture was thoroughly mixed. The chloroform was evaporated to dryness by a rotary evaporator to form a lipid film;

[0063] Step 2: Add 2 mL of glucose aqueous solution (concentration of 3 mg / mL) and 8 mL of phosphate buffer solution (pH 7.40, specific formula ingredients include: 8 g / L NaCl, 0.2 g / L KCl, 1.44 g / L Na2HPO4, 0.24 g / L KH2PO4) to the lipid film obtained in step 1, and perform ice-water bath ultrasound for 8 minutes to obtain a uniform liposome solution; the frequency of ice-water bath ultrasound is 450 Hz.

[0064] Step 3: The liposome solution obtained in step 2 is purified by gel filtration column to obtain pH-sensitive liposomes.

[0065] Example 7: The process is the same as that of Example 5, except that the amount of NaHCO3 is adjusted. The specific preparation steps are:

[0066] Step 1: 20 mg of disulfide phosphatidyl-polyethylene glycol obtained in Example 1, 1 mg of antioxidant and 1 mg of cholesterol were added to 6 mL of chloroform solution, and the mixture was thoroughly mixed. The chloroform was evaporated to dryness by a rotary evaporator to form a lipid film;

[0067] Step 2: Add 10 mL of NaHCO3 aqueous solution (concentration of 0.6 mg / mL) to the lipid film obtained in step 1, and perform ice-water bath ultrasound for 8 minutes to obtain a uniform liposome solution; the frequency of the ice-water bath ultrasound is 450 Hz.

[0068] Step 3: The liposome solution obtained in step 2 is purified by gel filtration column to obtain pH-sensitive liposomes.

[0069] Example 8: Preparation of glutamine pH sensitive liposomes;

[0070] Step 1: 20 mg of disulfide phosphatidyl-polyethylene glycol obtained in Example 1, 1 mg of antioxidant and 1 mg of cholesterol were added to 6 mL of chloroform solution, and the mixture was thoroughly mixed. The chloroform was evaporated to dryness by a rotary evaporator to form a lipid film;

[0071] Step 2: Add 2 mL of glutamine aqueous solution (concentration of 5 mg / mL) and 8 mL of phosphate buffer solution (pH 7.40, specific formula ingredients include: 8 g / L NaCl, 0.2 g / L KCl, 1.44 g / L Na2HPO4, 0.24 g / L KH2PO4) to the lipid film obtained in step 1, and perform ice-water bath ultrasound for 8 minutes to obtain a uniform liposome solution; the frequency of ice-water bath ultrasound is 450 Hz.

[0072] Step 3: The liposome solution obtained in step 2 is purified by gel filtration column to obtain pH-sensitive liposomes.

[0073] Example 9: Preparation of NaOH pH-sensitive liposomes;

[0074] Step 1: 20 mg of disulfide phosphatidyl-polyethylene glycol obtained in Example 1, 1 mg of antioxidant and 1 mg of cholesterol were added to 6 mL of chloroform solution, and the mixture was thoroughly mixed. The chloroform was evaporated to dryness by a rotary evaporator to form a lipid film;

[0075] Step 2: Add 10 mL of NaHCO3 aqueous solution (concentration of 0.4 mg / mL) to the lipid film obtained in step 1, and perform ice-water bath ultrasound for 8 minutes to obtain a uniform liposome solution; the frequency of the ice-water bath ultrasound is 450 Hz.

[0076] Step 3: The liposome solution obtained in step 2 is purified by gel filtration column to obtain pH-sensitive liposomes.

[0077] Detection experiment:

[0078] Test experiment 1: The liposomes prepared in Examples 3 to 5 were imaged by transmission electron microscopy. Figure 1 a is the empty pH-sensitive liposome prepared in Example 3, Figure 1 b is the glucose pH sensitive liposome prepared in Example 4, Figure 1 c is the NaHCO3 pH-sensitive liposome prepared in Example 5. As can be seen from the image, the three liposomes are spheres with uniform size and stable structure, and the particle size is 150nm to 200nm.

[0079] Test experiment 2: The three liposomes prepared in Examples 3 to 5 were tested for their hydrated particle size, dispersion coefficient PDI and surface membrane potential by a particle size analyzer. The results are shown in Table 1. The particle size distribution diagram is shown in Figures 2 to 4The results showed that the hydrated particle sizes of the three pH-sensitive liposomes were between 160nm and 200nm, and the liposomes had good dispersibility in the solution.

[0080] Table 1

[0081] Liposome Type Hydrated particle size (nm) PDI Surface membrane potential (mV) Example 3 (empty pH sensitive liposomes) 168.2±5.093 0.292±0.071 -8.9±0.44 Example 4 (Glucose pH-sensitive liposomes) 203.0±7.351 0.472±0.086 -9.5±0.38 <![CDATA[Example 5 (NaHCO3 pH-sensitive liposome)]]> 191.1±6.093 0.410±0.038 -8.3±0.62

[0082] Test experiment 3, determination of glucose pH-sensitive liposome encapsulation efficiency (EE%) and drug loading efficiency (LE%):

[0083] Take the glucose pH sensitive liposomes prepared in Example 4 and Example 6, add dilute hydrochloric acid to destroy the lipid bilayer and release glucose. Transfer to a centrifuge and centrifuge at 12000rpm for 10min, separate the liquid, and precipitate the liposomes at the bottom. Take the supernatant, which is the solution containing glucose. Using the UV-HPLC method, the concentration of glucose is calculated from the peak area-glucose quantitative analysis curve, and then the mass of the encapsulated glucose is calculated. The calculation method of EE% and LE% is as follows; the test is performed 3 to 5 times, and the average value is calculated. The specific test results are shown in Table 2.

[0084] EE% = mass of encapsulated glucose / mass of total glucose input × 100%;

[0085] LE% = mass of encapsulated glucose / mass of input pH-sensitive phospholipid and cholesterol × 100%.

[0086] Test experiment 4, determination of NaHCO3 pH sensitive liposome encapsulation efficiency (EE%) and drug loading efficiency (LE%):

[0087] Take the NaHCO3 pH sensitive liposomes prepared in Example 5 and Example 7, add enough chloroform to destroy the lipid bilayer and release NaHCO3, mix thoroughly with a magnetic stirrer, and use a pH meter to detect the pH of the solution after the liposomes are added. According to the conversion of the ion product constant, that is, c(H+)*c(OH-)=Kw;

[0088] Taking the negative logarithm of both sides of the above formula, we can obtain PH+POH=14POH=-lg[c(OH-)]PH=-lg[c(H+)].

[0089] Note: The ion product constant increases with the increase of temperature. At 25 degrees Celsius, it is Kw=10^-14. The concentration of NaHCO3 is calculated, and the mass of NaHCO3 is further calculated. The calculation method of EE% and LE% is as follows; the test is performed 3 to 5 times and the average value is calculated. The specific test results are shown in Table 2.

[0090] EE% = mass of loaded NaHCO3 / mass of total NaHCO3 input × 100%;

[0091] LE% = mass of encapsulated NaHCO3 / mass of input pH-sensitive phospholipids and cholesterol × 100%.

[0092] Table 2

[0093]

[0094] Test experiment 5:

[0095] The empty pH-sensitive liposomes prepared in Example 3 were placed in three hydrochloric acid solutions with pH values ​​of 7.4, 6.0, and 5.0, respectively, and reacted slowly for 12 hours with magnetic stirring. After 12 hours, the liposomes were photographed by scanning with a transmission electron microscope.

[0096] The results are as follows Figure 5 As shown, after 12 h, the morphology of the empty pH-sensitive liposomes in pH 7.4 solution was intact, the membrane structure of the empty pH-sensitive liposomes in pH 6.0 hydrochloric acid solution was broken, and the membrane of the empty pH-sensitive liposomes in pH 6.0 hydrochloric acid solution was completely disintegrated into heterogeneous fragments.

[0097] Test experiment 6: MTT method to detect the effect of glucose pH-sensitive liposomes on Vero cell viability:

[0098] Four experimental groups were set up: a cell control group, an empty pH-sensitive liposome prepared in Example 3, a glucose pH-sensitive liposome prepared in Example 4, and a NaHCO3 pH-sensitive liposome prepared in Example 5; the following experiments were performed respectively, and each experimental group was tested 3 to 5 times, and the average value was taken:

[0099] (1) Digest the Vero cells in the logarithmic growth phase, resuspend and count. Inoculate 2000 cells per well in a 96-well plate, with 6 replicate wells for each experimental group, and the volume of cell culture fluid in each well is 100 μL. Inoculate the cells in a cell culture incubator at 37°C and 5% CO2 for 36 hours. After 36 hours of cell culture, observe the growth status of the cells and add the corresponding liposomes to each treatment group.

[0100] (2) Preparation of MTT solution: Weigh an appropriate amount of MTT powder and dissolve it in a certain amount of PBS to obtain a 0.5 mg / mL MTT solution. After filtering through a 0.22 μm filter membrane, dilute the MTT solution to 0.5 mg / mL using cell growth medium.

[0101] (3) After 36 h of cell culture, the liquid in the wells was aspirated, rinsed once with PBS, and 100 μL of MTT solution was added to each well. The cells were incubated in a dark 37°C, 5% CO2 cell culture incubator for 4 h.

[0102] (4) Add 100 μL of 15% SDS lysis buffer to each well and place the cells back into the 37°C cell culture incubator and continue incubating in the dark for 12 h;

[0103] (5) Take out the 96-well plate and place it in an ELISA reader to detect the OD value at a wavelength of 570 nm.

[0104] (6) After 36 h of cell culture and before adding MTT, take photos under a microscope to record cell morphology.

[0105] The test results are shown in Table 3. Figure 6 , Figure 7 As shown, there was no significant difference in cell viability between the empty pH-sensitive liposome group and the cell control group, indicating that the empty pH-sensitive liposomes were not toxic to cells because liposomes have the advantages of biocompatibility, cell affinity and low toxicity; the cell viability of the glucose pH-sensitive liposome group was better than that of the cell control group and the empty pH-sensitive liposome group because the addition of glucose provided nutrition for cell growth. The cell viability of the NaHCO3 pH-sensitive liposome group was better than that of the cell control group compared with the empty pH-sensitive liposome group because the NaHCO3 released by the NaHCO3 pH-sensitive liposomes neutralized the acidity in the growth medium and maintained the pH balance.

[0106] Table 3 MTT results of different treatment groups

[0107]

[0108]

[0109] Conclusion: This application prepares the nutrients such as glucose, amino acids, glutamine and pH regulators such as NaOH and NaHCO3 required for the middle and late stages of cell growth into pH-sensitive liposomes, and encapsulates these substances in the liposome cavity respectively. After the pH of the cell growth solution system is reduced, they can be released respectively to improve the growth state of the cells, and the pH balance of the cell growth solution can be achieved and maintained at neutral. The pH-sensitive liposomes prepared by the present invention are uniform in size, have good dispersibility, are structurally stable in neutral solutions, can be effectively lysed in acidic solutions, and are beneficial to improving the growth state of cells after being loaded with nutrients and pH regulators.

[0110] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

Claims

1. A pH-sensitive liposome for encapsulating cell culture additives, characterized in that: The specific components of each milliliter of pH-sensitive liposomes include: pH-sensitive phospholipids modified with polyethylene glycol or polyacrylamide, cholesterol, antioxidants, nutrients or pH regulators; The pH sensitive phospholipids are one or more of dipalmitoyl phospholipids, heptadecanoyl phospholipids and disulfide phosphatidyl phospholipids.

2. The pH-sensitive liposome for encapsulating cell culture additives according to claim 1, characterized in that: The specific components of each milliliter of pH sensitive liposomes include: 1-2 mg / mL of pH sensitive phospholipids modified with polyethylene glycol or polyacrylamide, 0.05-0.15 mg / mL of cholesterol, 0.06-0.1 mg / mL of antioxidant, and 0.4-1 mg / mL of nutrients or pH regulators.

3. The pH-sensitive liposome for encapsulating cell culture additives according to claim 2, characterized in that: The specific components of each milliliter of pH-sensitive liposomes include: 2 mg / mL of pH-sensitive phospholipids modified with polyethylene glycol or polyacrylamide, 0.1 mg / mL of cholesterol, 0.1 mg / mL of antioxidant, and 1 mg / mL of pH regulator.

4. The pH-sensitive liposome for encapsulating cell culture additives according to claim 2, characterized in that: The specific components of each milliliter of pH-sensitive liposomes include: 2 mg / mL of pH-sensitive phospholipids modified with polyethylene glycol or polyacrylamide, 0.1 mg / mL of cholesterol, 0.1 mg / mL of antioxidants, and 0.8 mg / mL of nutrients; a buffer solution is also added when the nutrients are added.

5. The pH-sensitive liposome for encapsulating cell culture additives according to claim 4, characterized in that: The antioxidant is any one of coenzyme Q10 and vitamin E; the nutrients are one or more of glucose, amino acids, and glutamine; the pH regulator is any one of NaOH and NaHCO3; and the buffer solution is any one of citrate buffer, phosphate buffer, and carbonate buffer.

6. The pH-sensitive liposome for encapsulating cell culture additives according to claim 1, characterized in that: The specific preparation method of polyethylene glycol-modified pH-sensitive phospholipids is as follows: polyethylene glycol is dissolved in chloroform, acetic acid and propionic anhydride are added as a catalyst, and then an activator is added, and the activation reaction is carried out at 25°C for 1 to 2 hours, and then pH-sensitive phospholipids are added, and the reaction is carried out at 55 to 65°C for 24 hours. The reactants are added to ethanol, slowly magnetically stirred, and centrifuged to obtain polyethylene glycol-modified pH-sensitive phospholipids.

7. The pH-sensitive liposome for encapsulating cell culture additives according to claim 1, characterized in that: The specific preparation method of the pH-sensitive phospholipid modified with polyacrylamide is as follows: dissolve the pH-sensitive phospholipid in acetone, add an activator, react at 25°C for 1 to 2 hours, add the reactant to an ethanol solution of polyacrylamide, react at 50 to 60°C for 24 to 36 hours, slowly stir magnetically, and centrifuge to obtain the pH-sensitive phospholipid modified with polyacrylamide.

8. A method for preparing pH-sensitive liposomes for encapsulating cell culture additives according to any one of claims 1 to 7, characterized in that: The specific steps are: Step 1: Adding polyethylene glycol or polyacrylamide-modified pH-sensitive phospholipids, antioxidants and cholesterol to an organic solvent, mixing them thoroughly, and evaporating the organic solvent by rotary evaporation to form a lipid film; Step 2: adding an aqueous solution of a nutrient or a pH regulator to the lipid film obtained in step 1, and ultrasonicating in an ice-water bath for 6 to 8 minutes to obtain a uniform liposome solution; Step 3: The liposome solution obtained in step 2 is purified by gel filtration column to obtain pH sensitive liposomes.

9. The method for preparing a pH-sensitive liposome for encapsulating cell culture additives according to claim 8, characterized in that: In step 1, the organic solvent is any one of chloroform, methanol and acetone; in step 2, the frequency of ice-water bath ultrasound is 250-550 Hz.

10. Use of the pH-sensitive liposome for encapsulating cell culture additives according to any one of claims 1 to 7 in cell culture.

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