Engineering modified tumor cell-derived microparticle drug-carrying preparation and preparation method thereof

Abstract

The present invention belongs to the field of anti-tumor treatment technology, and specifically discloses an engineered tumor cell-derived microparticle drug-loaded preparation and a preparation method thereof, wherein the drug-loaded preparation comprises tumor cell-derived microparticles modified by a pH-responsive hydrophilic-hydrophobic transition polymer and a drug encapsulated in the tumor cell-derived microparticles; the pH-responsive hydrophilic-hydrophobic transition polymer is hydrophilic in the blood environment and hydrophobic in the acidic tumor microenvironment. The present invention enhances the ability of drug-loaded microparticles to circulate for a long time in the blood, improves their targeted enrichment and deep penetration in the tumor site, promotes tumor cells and tumor stem cells to efficiently absorb drug-loaded microparticles and release drugs, thereby improving the anti-tumor efficacy.

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-tumor treatment, and more specifically, relates to an engineered tumor cell-derived microparticle drug-loaded preparation and a preparation method thereof. Background Art

[0002] Malignant tumors are one of the diseases that seriously threaten human life. When traditional chemotherapy drugs act on tumor tissues, tumor stem cells with extremely strong DNA repair ability and high expression of ABC transporters use their unique properties to reduce the effect of chemotherapy drugs and make them resistant. The surviving tumor stem cells will differentiate into a large number of tumor cells, causing the tumor to grow violently and significantly reducing the patient's survival rate.

[0003] Nano drug delivery systems have obvious advantages in anti-tumor treatment. They can not only effectively solve the solubility and in vivo degradation problems of anti-tumor drugs, but also actively target loaded drugs to the site of action through their enhanced permeability and retention effect (EPR). On the other hand, smart responsive nano drug delivery systems can selectively release loaded drugs according to changes in different factors in the tumor microenvironment (pH, temperature, bioenzymes, ultrasound, etc.), effectively reducing drug toxicity and thus improving the therapeutic effect of chemotherapy drugs. Based on this, nano drug delivery systems are very beneficial for targeted treatment of tumor stem cells.

[0004] Microparticles (MPs) are cell membrane vesicle structures secreted by cells, with a particle size between 100 and 1000 nm. Different types of cells can produce a large number of microparticles under physical and physiological conditions. MPs contain a large number of messenger molecules, enzymes, RNA, and DNA, and can transport the contents between different cells. Therefore, MPs can also be used as a transport carrier to transport chemotherapy drugs in the body. Tumor cell-derived microparticles can be used as a good anti-tumor drug carrier for tumor treatment. They not only have good biosafety, but can also be enriched in the tumor area through homologous targeting, improve the therapeutic effect, and open up new ideas for anti-tumor treatment. However, traditional tumor cell-derived drug-loaded microparticles have poor long-term circulation performance in the blood, cannot selectively release chemotherapy drugs in the tumor area and effectively kill tumor stem cells, which greatly limits their application in anti-tumor treatment. Summary of the invention

[0005] In view of the defects of the prior art, the purpose of the present invention is to provide an engineered tumor cell-derived microparticle drug-loaded preparation and a preparation method thereof, aiming to solve the problems that traditional tumor cell-derived drug-loaded microparticles have poor long circulation performance in the blood, less targeted enrichment at the tumor site, weak deep penetration ability, and cannot selectively release chemotherapy drugs in the tumor area and effectively kill tumor stem cells.

[0006] To achieve the above-mentioned purpose, the present invention provides an engineered tumor cell-derived microparticle drug-loaded preparation, which includes tumor cell-derived microparticles modified with a pH-responsive hydrophilic-hydrophobic transition polymer and a drug encapsulated inside the tumor cell-derived microparticles; the pH-responsive hydrophilic-hydrophobic transition polymer is hydrophilic in the blood environment and hydrophobic in the acidic tumor microenvironment.

[0007] Preferably, the pH-responsive hydrophilic-hydrophobic transition polymer is N - a chain polymer formed by polymerization of isopropyl acrylamide, an acrylic acid monomer and octadecyl acrylate, wherein the acrylic acid monomer is acrylic acid, methacrylic acid, propyl acrylate or N -Methallylamine.

[0008] More preferably, the N The molar ratio of isopropyl acrylamide, the acrylic monomer and the octadecyl acrylate is 100:(2-3):1.

[0009] Preferably, the tumor cells include common tumor cells of liver cancer, breast cancer or melanoma, or tumor stem cells of liver cancer, breast cancer or melanoma.

[0010] Preferably, the drug is one or more of doxorubicin, methotrexate, 5-fluorouracil, cisplatin, paclitaxel and dextran.

[0011] Preferably, the particle size of the engineered tumor cell-derived microparticle drug-loaded preparation is 200 nm to 600 nm.

[0012] According to another aspect of the present invention, a method for preparing the above-mentioned engineered tumor cell-derived microparticle drug-loaded preparation is also provided, which comprises the following steps:

[0013] S1, using ultraviolet rays to irradiate cultured tumor cells to induce apoptosis, then incubating the tumor cells with high concentrations of drugs so that the drugs are taken up by the cells, and finally collecting the released drug-loaded microparticles;

[0014] S2. Incubate the drug-loaded microparticles with a pH-responsive hydrophilic-hydrophobic transition polymer, so that the pH-responsive hydrophilic-hydrophobic transition polymer is modified on the surface of the drug-loaded microparticles to obtain an engineered tumor cell-derived microparticle drug-loaded preparation.

[0015] Preferably, in step S1, the drug-loaded microparticles are collected at 4°C to 6°C and at a centrifugal force of 400g to 20000g.

[0016] Preferably, in step S2, the mass ratio of the protein in the drug-loaded microparticles to the pH-responsive hydrophilic-hydrophobic transition polymer is 1:(0.5-1).

[0017] Preferably, the specific operation of step S2 is: during the incubation process of the drug-loaded microparticles and the pH-responsive hydrophilic-hydrophobic transition polymer, the long-chain alkane in the pH-responsive hydrophilic-hydrophobic transition polymer is inserted into the hydrophobic layer of the phospholipid bilayer of the drug-loaded microparticles through hydrophobic action, so that the pH-responsive hydrophilic-hydrophobic transition polymer is adsorbed and fixed on the surface of the drug-loaded microparticles, thereby obtaining the drug-loaded microparticles modified with the pH-responsive hydrophilic-hydrophobic transition polymer.

[0018] Preferably, in step S2, the drug-loaded microparticles and the pH-responsive hydrophilic-hydrophobic transition polymer are incubated at 4°C to 6°C for 10 h to 16 h.

[0019] In general, the above technical solution conceived by the present invention has the following beneficial effects compared with the prior art:

[0020] (1) The present invention provides a pH-regulated tumor cell microparticle drug-loaded nanoformulation, which modifies a specific polymer on the surface of the tumor cell microparticles so that the polymer can undergo a hydrophilic and hydrophobic transformation in response to pH changes. The polymer is in a hydrophilic and stretched state under the condition of blood pH 7.4, which can effectively prevent protein adsorption and conditioning and clearance of the reticuloendothelial system during blood circulation, thereby enabling the drug-loaded microparticles to have the ability of long blood circulation; when the drug-loaded microparticle preparation enters the acidic tumor microenvironment, the polymer is converted into a hydrophobic state, which promotes the enrichment of the drug-loaded microparticles in tumor tissues, deep penetration of tumors, and uptake of the preparation by tumor cells and tumor stem cells. The pH 4.5 environment in the tumor cell lysosome further promotes the drastic contraction of the polymer, resulting in the collapse of the phospholipid bilayer of the microparticles and the release of the loaded drug, thereby exerting a better killing effect.

[0021] (2) The polymer modified on the surface of the nanoparticle drug-carrying preparation of the present invention is N-Isopropyl acrylamide (NIPAM), acrylic monomers and octadecyl acrylate (ODA) are polymerized. The volume phase transition temperature of the polymer under pH 7.4 is greater than 37°C, and it is in a hydrophilic and fluffy state in the blood environment in the body. However, under the slightly acidic conditions of tumors at pH 6.5, its volume phase transition temperature is slightly less than 37°C, showing a hydrophobic state; the polymer will quickly transform into a contracted state under pH 4.5, so when the nano drug-loaded microparticles enter the lysosomes of tumor cells, the polymer on the surface of the microparticles shrinks violently, destroying the membrane structure of the microparticles and promoting the release of the loaded drugs.

[0022] (3) After the nanoparticles of the present invention are loaded with anti-tumor drugs, a nano-drug delivery system responsive to the tumor microenvironment can be prepared, which can not only increase the circulation time of the loaded drugs in the blood, but also enhance their enrichment in the tumor site, deep penetration ability and uptake by tumor cells and tumor stem cells; and can further promote the responsive release of loaded drugs from the drug-loaded microparticles in tumor cells, thereby improving the anti-tumor effect of chemotherapy drugs.

[0023] (4) The synthesis process of the nanoparticle drug-loaded preparation of the present invention is simple and can rapidly achieve hydrophilic-hydrophobic conversion under the slightly acidic conditions of the tumor, thereby promoting the controlled release of the drug-loaded microparticles. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1A FITR detection spectrum of polymers P1 and P2 synthesized in Example 1 of the present invention;

[0025] Figure 1B is the transmittance of polymer P1 in Example 1 of the present invention under different temperature and pH conditions;

[0026] Figure 1C is the transmittance of polymer P2 in Example 1 of the present invention under different temperature and pH conditions;

[0027] Figure 2A is the hydrated particle size of the drug-carrying nanoparticles in Example 2 of the present invention;

[0028] Figure 2B is the surface potential of the drug-carrying nanoparticles in Example 2 of the present invention;

[0029] Figure 3 The drug release results of the nano drug-loaded microparticles in Example 3 of the present invention under different pH conditions;

[0030] Figure 4 The uptake of drug-loaded microparticles by macrophages RAW264.7 under different pH conditions in Example 4 of the present invention;

[0031] Figure 5The accumulation of drug-loaded nanoparticles in tumor tissue in Example 5 of the present invention;

[0032] Fig. 6A The penetration behavior of the drug-loaded nanoparticles in the tumor cell cluster in Example 6 of the present invention;

[0033] Figure 6B The fluorescence quantification of the penetration behavior of the drug-carrying nanoparticles in the tumor cell clusters in Example 6 of the present invention;

[0034] Fig. 7A The uptake of the drug-carrying nanoparticles by mouse H22 tumor cells under different pH conditions in Example 7 of the present invention;

[0035] Figure 7B The killing ability of the drug-loaded nanoparticles in Example 7 of the present invention on mouse H22 tumor cells under different pH conditions;

[0036] Fig. 8A The uptake of the drug-loaded nanoparticles by H22 CSCs under different pH conditions in Example 8 of the present invention;

[0037] Figure 8B The effect of the drug-loaded nanoparticles on the number of H22CSCs tumor clones under different pH conditions in Example 8 of the present invention;

[0038] Figure 8C The effect of the drug-loaded nanoparticles on the size of H22CSCs tumor clones under different pH conditions in Example 8 of the present invention;

[0039] Fig. 9 The results of the doxorubicin-loaded nanoparticles in Example 9 of the present invention inhibiting the growth of H22 subcutaneous tumor mice liver cancer;

[0040] Fig.10 The results of the methotrexate-loaded nanoparticles in Example 10 of the present invention inhibiting the growth of H22 subcutaneous tumor liver cancer in mice;

[0041] Fig.11 The results of the 5-fluorouracil-loaded nanoparticles in Example 11 of the present invention inhibiting the growth of H22 subcutaneous tumor liver cancer in mice;

[0042] Fig.12 The results of the nano drug-loaded microparticles derived from mouse H22 liver cancer stem cells in Example 12 of the present invention inhibiting the growth of H22 subcutaneous tumor mice liver cancer;

[0043] Fig.13 This is the result of the mouse 4T1 breast cancer cell-derived nanoparticles in Example 13 of the present invention inhibiting the growth of 4T1 orthotopic tumor mice with breast cancer. DETAILED DESCRIPTION

[0044] In order to make the purpose, technical solution 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 used to limit the present invention.

[0045] The present invention provides an engineered tumor cell-derived microparticle drug-loaded preparation, which includes tumor cell-derived microparticles modified with a pH-responsive hydrophilic-hydrophobic transition polymer and a drug encapsulated in the tumor cell-derived microparticles; the pH-responsive hydrophilic-hydrophobic transition polymer is hydrophilic in a blood environment and hydrophobic in an acidic tumor microenvironment.

[0046] The pH-responsive hydrophilic-hydrophobic transition polymer modified on the surface of the drug-loaded microparticles of the present invention is in a hydrophilic and stretched state under the condition of blood pH of about 7.4, which can effectively avoid protein adsorption and conditioning and removal of the reticuloendothelial system during blood circulation, thus having the ability of long blood circulation; when the nano preparation enters the acidic tumor microenvironment, the polymer on the surface of the microparticles is converted into a hydrophobic state, which promotes the enrichment of the drug-loaded microparticles in tumor tissues, deep penetration of tumors, and uptake of the preparation by tumor cells and tumor stem cells. The pH 4.5 environment in the lysosomes of tumor cells further promotes the drastic contraction of the polymer, causing the phospholipid bilayer of the microparticles to collapse and release the loaded drug, thereby exerting a better killing effect.

[0047] In some embodiments, the pH-responsive hydrophilic-hydrophobic transition polymer is N-A chain polymer formed by polymerization of poly(N-isopropylacrylamide) (NIPAM), acrylic monomers and octadecyl acrylate (ODA), wherein the acrylic monomers are acrylic acid (AA), methacrylic acid, propyl acrylate or N-methylallylamine. Generally, polymers containing NIPAM have the property of pH-responsive hydrophilic-hydrophobic transition. NIPAM is a temperature-sensitive monomer with a lower critical solution temperature (LCST) of 33°C. When poly(N-isopropylacrylamide) (PNIPAM) is in an environment above the LCST, the PNIPAM polymer chain exists in a compressed coil-like conformation; when it is below the LCST, PNIPAM exists in an extended conformation. Its phase change mechanism is as follows: PNIPAM contains hydrophilic amide groups, hydrophobic ethylene backbone chains and isopropyl side chains. At low temperatures, the favorable free energy generated by the hydrogen bonding between the hydrophilic group and water is greater than the unfavorable free energy generated by the hydrophobic group being exposed to water, making the polymer more soluble in water; when the temperature rises, the hydrogen bonding effect weakens, and the hydrophobic effect between the hydrophobic side groups increases. When the temperature exceeds the LCST, the effect between the hydrophobic groups becomes dominant, causing the polymer stretched conformation to collapse and produce phase separation. The addition of these acrylic monomers will change the LCST of the polymer because the acrylic monomers contain hydrophilic carboxyl groups. The LCST of the polymer is very sensitive to changes in the ratio of monomers. During the preparation of the polymer, the phase transition temperature of the synthetic polymer can be adjusted by adjusting the feed ratio of the monomers, so that it has the ability to transform between hydrophilic and hydrophobic under different pH conditions. The present invention has experimentally found that the LCST of the polymer is the ratio of monomers required at a human body temperature of 37°C, that is, N The molar ratio of isopropyl acrylamide, acrylic monomer and octadecyl acrylate is 100:(2-3):1. The polymer-modified drug-loaded microparticles have the same morphology and potential as the unmodified tumor cell-derived drug-loaded microparticles and still have good biocompatibility.

[0048] The tumor cell-derived microparticles of the present invention can be of various cancer cell types, including but not limited to liver cancer cells, breast cancer cells, melanoma cells, etc. The tumor cells can be either ordinary tumor cells or corresponding tumor stem cells, preferably tumor stem cells, and the curative effect of the drug-loaded microparticles derived from engineered tumor stem cells is stronger. The drug loaded in the drug-loaded microparticles of the present invention can be any small molecule chemical drug, preferably an anti-tumor drug, including but not limited to doxorubicin, methotrexate, 5-fluorouracil, cisplatin, paclitaxel, dextran, etc.

[0049] In some embodiments, the particle size of the engineered tumor cell-derived microparticle drug-loaded preparation is 200nm to 600nm, preferably about 400nm. The drug-loaded preparation of the present invention is nano-sized, which is more conducive to its enrichment at the tumor site, deep penetration of tumor tissue, more effective targeting and effective killing of tumor cells and tumor stem cells, and avoiding the use of exogenous materials as carriers to produce toxic side effects on the body.

[0050] On the other hand, the method for preparing the engineered tumor cell-derived microparticle drug-loaded preparation provided by the present invention comprises the following steps:

[0051] S1. Using ultraviolet rays to irradiate cultured tumor cells or tumor stem cells to induce apoptosis, then incubating the tumor cells or tumor stem cells with high concentrations of drugs to allow the drugs to be taken up by the cells, and finally collecting the released drug-loaded cell microparticles;

[0052] S2. Incubate the drug-loaded microparticles with a pH-responsive hydrophilic-hydrophobic transition polymer, so that the pH-responsive hydrophilic-hydrophobic transition polymer is modified on the surface of the drug-loaded microparticles to obtain an engineered tumor cell-derived microparticle drug-loaded preparation.

[0053] In step S2 of some embodiments, the mass ratio of the protein in the drug-loaded microparticles to the pH-responsive hydrophilic-hydrophobic transition polymer is 1:(0.5-1). The protein content in the microparticles can be detected using a special commercial kit. Furthermore, the drug-loaded microparticles and the pH-responsive hydrophilic-hydrophobic transition polymer are incubated at 4°C for 10h-16h.

[0054] The above technical solution is described in detail below in conjunction with specific embodiments.

[0055] The various tumor cells, drugs and experimental animals used in the following examples: H22 mouse hepatoma cells and mouse macrophage cell line RAW264.7, both of which can be purchased from ATCC in the United States or China Type Collection Center CCTCC; BALB / C mice, 5 weeks old, purchased from Alpha Biotech (Wuhan) Biotechnology Co., Ltd. N-isopropylacrylamide (NIPAM), octadecyl acrylate (ODA), acrylic acid (AA) and azobisisobutyronitrile (AIBN) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0056] Example 1 Characterization of the physicochemical properties of polymers P1 and P2

[0057] 1. Experimental Materials and Reagents

[0058] N -Isopropyl acrylamide, octadecyl acrylate, acrylic acid and azobisisobutyronitrile

[0059] 2. Experimental steps

[0060] (1) Accurately weigh 0.565g N -Isopropyl acrylamide (NIPAM), 0.017 g of octadecyl acrylate (ODA), 0.009 g of acrylic acid (AA), and 0.012 g of azobisisobutyronitrile (AIBN) were dissolved in a 50 mL three-necked flask, dissolved with 9 mL of 1,4-dioxane, and mixed by ultrasonication.

[0061] (2) Nitrogen was introduced into the above reaction system to remove oxygen in the reaction vessel. The reaction was heated in a water bath under magnetic stirring, and the temperature was slowly raised to 68°C and the reaction was continued for 12 hours.

[0062] (3) After the reaction product is cooled to room temperature, an appropriate amount of ether is added for extraction. After filtering, the product is dissolved in ultrapure water and transferred to a dialysis bag for dialysis for one week to remove unreacted monomers and solvents. The molecular weight cutoff of the dialysis bag is 3000Da. Finally, the purified product is freeze-dried and stored. The obtained polymer is called polymer P2 and serves as the experimental group. In a similar treatment method, when the amount of acrylic acid added is increased to 0.018g, the product obtained is polymer P1, which serves as the control group.

[0063] (4) First, add 10% FBS to phosphate buffered saline (PBS), that is, add 1 mL of FBS to 9 mL of PBS, and mix the polymer at a concentration of 5 mg / mL. -1 Dissolved in it. Adjust the pH of the above solution to 4.5, 6.5, and 7.4 with NaOH and HCl, and detect the change of the transmittance of the polymer under different pH and temperature conditions. The temperature is set to 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 and 50°C. Incubate for 10 minutes under each temperature condition. Use a spectrophotometer to detect the transmittance of the nanogel aqueous solution at 560nm.

[0064] 3. Experimental results

[0065] FITR was used to detect the polymerization of monomers in polymers P1 and P2. Figure 1A As shown, both polymers P1 and P2 appear N -The characteristic peak of isopropyl acrylamide - the stretching vibration peak of amide carbonyl (1627cm -1 ) and the overtone peak of amide II band (3070 cm -1 ), this FITR preliminarily verifies the presence of N-isopropylacrylamide in the polymer. Figure 1B and 1CAs shown in the figure, with the increase of temperature, the permeability of polymers P1 and P2 at different pH values ​​decreased significantly. The data obtained by Boltzmann fitting and derivation were used to obtain the volume phase transition temperatures at different pH values ​​as follows: the LCST of polymer P2 at pH 7.4 was 41.7°C, which was greater than the normal physiological temperature of 37°C, indicating that polymer P2 was in a hydrophilic stretch state under physiological conditions (pH 7.4, 37°C); the LCST at pH 6.5 was 36.3°C, which was less than the physiological temperature, indicating that polymer P2 was in a hydrophobic contraction state in tumor tissue (pH 6.5, 37°C); the LCST at pH 4.5 was 33.7°C, and polymer P2 contracted more violently. The volume phase transition temperatures of polymer P1 under different pH conditions were all greater than 37°C, indicating that polymer P1 was in a hydrophilic stretch state in the above three physiological environments.

[0066] Example 2 Drug-loaded microparticles produced by inducing tumor cell apoptosis using ultraviolet light are incubated with small molecule drugs and modified with synthetic polymers to obtain nano drug-loaded microparticles

[0067] 1. Experimental Materials and Reagents

[0068] The mouse H22 liver cancer cells, synthetic polymers P1 and P2 used, and the ultraviolet device were all from a conventional cell clean bench.

[0069] 2. Experimental steps

[0070] (1) Take 10 7 H22 tumor cells were cultured in a sterile culture dish with a diameter of 10 cm. After overnight culture, the culture medium was discarded, washed with PBS, irradiated with ultraviolet light for 2 hours, and then doxorubicin (DOX) was added to make the final concentration of DOX in the culture medium 200 μg / mL.

[0071] (2) 24 hours after the administration of doxorubicin, the tumor was separated. First, the supernatant was taken for step-by-step separation. After centrifugation at 200g and 600g for 10 minutes each, it was centrifuged at 14000g for 1 minute to remove cells and debris. The supernatant after centrifugation was further centrifuged at 20000g for 1 hour to obtain drug-loaded microparticles produced by apoptosis of H22 mouse liver cancer cells.

[0072] (3) The drug-loaded microparticles were incubated with polymer P2 at a ratio of 1 mg protein: 500 μg at 4°C for 12 h, and the supernatant was removed by centrifugation at 20,000 g to obtain polymer P2-modified drug-loaded microparticles (DOX@P2-MPs) as the experimental group. In a similar manner, polymer P1-modified drug-loaded microparticles (DOX@P1-MPs) were obtained, and unmodified drug-loaded microparticles (DOX@MPs) were used as the control.

[0073] (4) The obtained microparticles were dissolved in PBS, and the particle size and zeta potential distribution were measured using a nanoparticle size and zeta potential instrument with a 633 nm He-Ne laser. The specific setting conditions were: temperature of 25 °C and equilibrium time of 120 s.

[0074] 3. Experimental results

[0075] Laser particle size analyzer was used to detect the hydrated particle size and zeta potential of drug-loaded nanoparticles. Figure 2A As shown in Figure 2, the hydrated particle size of the two nanocarriers is about 450 nm, indicating that the nanoparticles can be passively targeted to the tumor area through the EPR effect. Figure 2B As shown, the surface potential of the drug-loaded nanoparticles is about -10MV, indicating that they can remain stable in a physiological environment.

[0076] Example 3 Drug Release from Drug-Loaded Microparticles

[0077] 1. Experimental materials and reagents

[0078] Dialysis bag (molecular weight cut-off: 14000Da)

[0079] 2. Experimental steps

[0080] Take 30 mL of PBS solution and adjust the pH to 7.4, 6.5 or 4.5 with HCl or NaOH, respectively. Add 1 mL of drug solution (150 μg mL -1 ) was placed in a dialysis bag (14000Da), sealed, and the dialysis bag containing the drug solution was immersed in 30mL of the release solution. Finally, it was placed in a shaker at 37°C and 250 rpm. 1mL of the release solution was taken out at 5min, 10min, 15min, 30min, 1h, 2h, 4h, 8h, 12h and 24h, and 1mL of the release solution of the corresponding pH was added. The drug release experiment was carried out under light-proof conditions throughout the process. The drug concentration of the released solution was determined by fluorescence and the cumulative release amount was calculated.

[0081] 3. Experimental results

[0082] From the data, it can be seen that the drug-loaded microparticles modified with polymer P2 have a certain pH responsiveness. As the pH decreases, the drug release in 24 hours increases from 26.0% at pH 7.4 to 40.9% at pH 6.5, and the release at pH 4.5 is 57.9%. This shows that polymer P2 further promotes the release of the drug-loaded microparticles at pH 4.5.

[0083] Example 4 Phagocytosis of drug-loaded microparticles by macrophages

[0084] 1. Experimental Materials and Reagents

[0085] Mouse macrophage cell line RAW264.7

[0086] 2. Experimental steps

[0087] RAW264.7 cells were plated into 12-well plates at a cell density of 2.0 × 10 per well. 4 After culturing overnight, the pH of the culture medium was adjusted to 7.5 or 6.5 with lactic acid, and then polymer P2-modified nanoparticles (experimental group) were added. In a similar manner, polymer P1-modified drug-loaded microparticles (DOX@P1-MPs) and unmodified drug-loaded microparticles (DOX@MPs) were obtained as controls. The final concentration of DOX added was 1 μg / mL. After incubation at 37°C for 1 hour, the cells were collected and detected by flow cytometry.

[0088] 3. Experimental results

[0089] Since polymer P2 is hydrophilic at pH 7.4 and undergoes a hydrophobic transition at pH 6.5, the drug-loaded nanoparticles are more phagocytosed by macrophages at pH 6.5; while polymer P1 does not have the ability to regulate the hydrophilic-hydrophobic transition by pH, so there is no statistical difference in the amount of macrophage uptake of its modified drug-loaded microparticles under the two pH conditions.

[0090] Example 5 Accumulation of drug-loaded nanoparticles in tumor tissue

[0091] 1. Experimental Materials and Reagents

[0092] Mouse H22 hepatoma cells and BALB / C mice were purchased from Alvacell (Wuhan) Biotechnology Co., Ltd.

[0093] 2. Experimental steps

[0094] (1) Establishment of mouse H22 liver cancer subcutaneous tumor model: 100 μL of H22 liver cancer cell suspension (cell number 2×10 6 ) to establish a mouse hepatocellular carcinoma H22 subcutaneous tumor model.

[0095] (2) When the volume of mouse liver cancer H22 subcutaneous tumor is 0.2cm 3At the same time, the tumor-bearing mice were randomly divided into 4 groups, with 4 mice in each group. The prepared polymer P2-modified tumor cell-derived drug-loaded microparticles were injected into 4 BALB / C mice via the tail vein at a dose of 0.75 mg (DOX equivalent / kg, 200 μL) as the experimental group (DOX@P2-MPs). In a similar manner, 4 BALB / C mice were injected with the same dose of free DOX (FreeDOX); the prepared drug-loaded microparticles without polymer modification were injected into 4 BALB / C mice via the tail vein at a dose of 0.75 mg (DOX equivalent) / kg, 200 μL as (DOX@MPs); at the same time, the prepared polymer P1-modified drug-loaded microparticles were injected into 4 BALB / C mice via the tail vein at a dose of 0.75 mg (DOX equivalent) / kg, 200 μL as the control (DOX@P1-MPs).

[0096] (3) The mice were killed 24 hours after the tail vein injection, and the tumors were removed and prepared into tissue homogenates, and the drug content was detected by an ELISA instrument.

[0097] 3. Experimental results

[0098] like Figure 5 As shown, 24 hours after tail vein injection of polymer P2-modified drug-loaded microparticles, the concentration of DOX in the tumor was 3.33 μg / g tissue, which was significantly higher than that of polymer P1-modified drug-loaded microparticles (2.20 μg / g) and drug-loaded microparticles without polymer modification (2.36 μg / g), and there was no significant difference between the latter two groups, indicating that the pH-regulated hydrophilic-hydrophobic transition property of the polymer can more effectively increase the enrichment of drug-loaded microparticles in the tumor area.

[0099] Example 6 Penetration of drug-loaded nanoparticles into tumor cell clusters

[0100] 1. Experimental Materials and Reagents

[0101] H22 mouse liver cancer cells, chemotherapy drugs doxorubicin, and Fibrinogen were purchased from Reagent proteins.

[0102] 2. Experimental steps

[0103] (1) H22 cells were inoculated in 3D soft fibrin gel to obtain H22 CSCs. The specific operation was as follows: H22 mouse liver cancer cells were cultured in RPMI1640 cell culture medium. H22 cells were pre-diluted to 1×10 4 / mL to obtain a cell suspension, dilute the 3D soft fibrin glue with T7 buffer to a solution of 2 mg / mL, mix the solution with the H22 cell suspension at a volume ratio of 1:1 to obtain a mixture; add 1 μL of thrombin (0.1 U / μL) to a pre-cooled 96-well plate, and mix with 50 μL of the above mixture by pipetting to obtain a mixed solution, incubate at 37°C for 15 minutes, add the mixed solution to 200 μL of RPMI 1640 complete culture medium, and culture for 6 days to obtain tumor stem cell clones.

[0104] (2) The pH of the culture medium was adjusted to 7.5 or 6.5 using lactic acid, and then polymer P2-modified drug-loaded microparticles (DOX@P2-MPs) were added as the experimental group. Free doxorubicin (FreeDox), unmodified drug-loaded microparticles (DOX@MPs), and polymer P1-modified drug-loaded microparticles (DOX@P1-MPs) were obtained in a similar manner as the control group. The cells were incubated for 24 h, and the final concentration of DOX was 1 μg / mL. After washing three times with PBS, the cells were fixed with 4% paraformaldehyde for 30 min and imaged using a laser confocal microscope. Doxorubicin was observed using filters of Ex = 488 nm and Em = 560 nm.

[0105] 3. Experimental results

[0106] As shown in Figure 6 (the gray area in the figure is the fluorescence distribution area of ​​doxorubicin), under the condition of pH 6.5, the fluorescence intensity of doxorubicin at different levels of H22 tumor cell clusters of the drug-loaded microparticles modified with polymer P2 was significantly higher than that under the condition of pH 7.4. In addition, there was no significant difference in the fluorescence intensity of other control groups under different pH conditions, indicating that the hydrophilic and hydrophobic reversal of polymer P2 on the surface of the drug-loaded microparticles under pH 6.5 promoted the penetration of nano drug-loaded microparticles in tumor cell clusters.

[0107] Example 7 pH-regulated hydrophilicity conversion promotes uptake and killing of drug-loaded microparticles by H22 cells

[0108] 1. Experimental Materials and Reagents

[0109] Mouse H22 hepatoma cells

[0110] 2. Experimental steps

[0111] (1) H22 cells were plated into a 12-well plate at a cell density of 2.0 × 10 cells per well. 4After culturing overnight, the pH of the culture medium was adjusted to 7.5 or 6.5 with lactic acid, and then polymer P2 modified drug-loaded microparticles (DOX@P2-MPs) were added as the experimental group. Free doxorubicin (FreeDox), unmodified drug-loaded microparticles (DOX@MPs) and polymer P1 modified drug-loaded microparticles (DOX@P1-MPs) were obtained in a similar manner as controls. The final concentration of DOX was 1μg / mL. After continuing to incubate at 37℃ for 4 hours, the cells were collected and the content of drug uptake in the cells was detected by flow cytometry.

[0112] (2) H22 cells were cultured at 8×10 3 The cells were inoculated in a 96-well plate at a density of 100 μg / mL. After overnight incubation, the serum-free medium was replaced, and the pH of the medium was adjusted to 7.5 or 6.5 with lactic acid. Then, polymer P2-modified drug-loaded microparticles (DOX@P2-MPs) were added as the experimental group. Free doxorubicin (FreeDox), unmodified drug-loaded microparticles (DOX@MPs) and polymer P1-modified drug-loaded microparticles (DOX@P1-MPs) were obtained in a similar manner as controls. The final concentration of DOX was 1 μg / mL. Finally, after incubation for 24 hours at 37°C and 5% CO2, 10 μL of CCK-8 solution was added to each well and incubated for 4 hours. The absorbance of each well at 450 nm was detected using an ELISA reader.

[0113] 3. Experimental results

[0114] like Fig. 7A As shown in the figure, the uptake of polymer P2 modified drug-loaded microparticles by H22 cells at pH 6.5 was significantly higher than that at pH 7.4, while there was no significant difference in the uptake of polymer P1 modified drug-loaded microparticles under the two pH conditions, indicating that the pH-regulated hydrophilic-hydrophobic transition can effectively increase the uptake of polymer P2 modified drug-loaded microparticles by tumor cells. Figure 7B As shown, the experimental results of in vitro tumor cell toxicity showed that compared with other groups, polymer P2 modified drug-loaded microparticles had stronger tumor cell killing ability under pH 6.5 conditions.

[0115] Example 8 pH-regulated hydrophilicity conversion promotes uptake and killing of drug-loaded microparticles by H22 CSCs

[0116] 1. Experimental Materials and Reagents

[0117] H22 mouse liver cancer cells, 3D soft fibrin glue

[0118] 2. Experimental steps

[0119] (1) The culture method of H22 mouse liver cancer stem cells is the same as that in Example 6. H22 mouse liver cancer cells are cultured using RPMI1640.

[0120] (2) Use lactic acid to adjust the pH of the culture medium to 6.5 or 7.4, and then add free DOX (FreeDOX), drug-loaded microparticles (DOX@MPs), polymer P1 modified drug-loaded microparticles (DOX@P1-MPs), and polymer P2 modified drug-loaded microparticles (DOX@P2-MPs) with a final DOX concentration of 1 μg / mL. Incubate at 37°C and 5% CO2 for 4 h, collect the cells, wash them three times with PBS, resuspend the cells in 500 μL pre-cooled PBS, pass through a 200-mesh sieve, and use a flow cytometer to detect intracellular DOX fluorescence.

[0121] (3) 400 H22 cells / well were seeded in a 96-well plate covered with 1 mg / mL 3D soft fibrin glue and cultured at 37°C and 5% CO2. After 5 days, the culture medium was discarded, and free DOX (FreeDOX) with a final concentration of 1 μg / mL, drug-loaded microparticles (DOX@MPs), polymer P1-modified drug-loaded microparticles (DOX@P1-MPs), and polymer P2-modified drug-loaded microparticles (DOX@P2-MPs) were added to the plates for 24 hours. Finally, the cells were observed and photographed under a microscope to count the number and size of live tumor cell spheres.

[0122] 3. Experimental results

[0123] As shown in Figure 8, in H22 CSCs, under pH 6.5 conditions, the amount of drug-loaded microparticles treated with polymer P2 modified with the drug-loaded microparticles was significantly higher than that in other groups, indicating that the pH-regulated hydrophilicity and hydrophobicity transition of polymer P2 effectively promoted the uptake of tumor cell drug-loaded microparticles by CSCs, and therefore had a stronger CSCs killing effect.

[0124] Example 9 H22 tumor inhibition effect of drug-loaded DOX nanoparticles

[0125] 1. Experimental Materials and Reagents

[0126] Mouse H22 hepatoma cells and BALB / C mice were purchased from Alvacell (Wuhan) Biotechnology Co., Ltd.

[0127] 2. Experimental steps

[0128] (1) A mouse subcutaneous liver cancer tumor model was established according to the method of Example 5.

[0129] (2) When the mouse tumor volume reaches 100mm 3At the same time, the tumor-bearing mice were randomly divided into 5 groups, with 6 mice in each group. The drug-loaded nanoparticles were injected into the mice through the tail vein at a dose of 0.5 mg / kg and a dose of 200 μL on days 1, 3, 5, 7, 9 and 11. BALB / C mice treated with polymer P2-modified drug-loaded microparticles were the experimental group (DOX@P2-MPs); similar treatment methods, PBS treatment (PBS), free DOX-treated BALB / C mice (FreeDOX); BALB / C mice treated with drug-loaded microparticles without polymer modification (DOX@MPs); BALB / C mice treated with P1-modified drug-loaded microparticles (DOX@P1-MPs) were used as controls. The longest (L) and widest (W) parts of the tumor were measured with a vernier caliper every day, and the tumor volume V = L × W was calculated. 2 / 2.

[0130] 3. Experimental results

[0131] From Fig. 9 It can be seen that compared with the control group PBS and FreeDOX, the tumor volume of each drug-loaded microparticle group was significantly reduced. At the same time, after the administration, the relative tumor volume of the experimental group DOX@P2-MPs was significantly lower than that of DOX@MPs and DOX@P1-MPs. This shows that polymer P2 modified tumor cell microparticles loaded with DOX have a better therapeutic effect on mouse H22 liver cancer subcutaneous tumors.

[0132] Example 10 H22 tumor inhibition effect of methotrexate-loaded nanoparticles

[0133] 1. Experimental Materials and Reagents

[0134] Mouse H22 hepatoma cells and BALB / C mice were purchased from Alvacell (Wuhan) Biotechnology Co., Ltd.

[0135] 2. Experimental steps

[0136] (1) A mouse subcutaneous liver cancer tumor model was established according to the method of Example 5.

[0137] (2) When the mouse tumor volume reaches 100mm 3The tumor-bearing mice were randomly divided into 5 groups, with 6 mice in each group. The drug-loaded nanoparticles were injected into the mice through the tail vein at a dose of 0.5 mg / kg and 200 μL on days 1, 3, 5, 7, 9 and 11. BALB / C mice treated with polymer P2-modified drug-loaded microparticles were the experimental group (MTX@P2-MPs); similar treatments were used for PBS-treated BALB / C mice (PBS), BALB / C mice treated with microparticles without polymer modification and drug loading (MPs), BALB / C mice treated with P1-modified non-drug-loaded microparticles (P1-MPs), BALB / C mice treated with polymer P2-modified non-drug-loaded microparticles (P2-MPs), BALB / C mice treated with free methotrexate (FreeMTX), BALB / C mice treated with drug-loaded microparticles without polymer modification (MTX@MPs), and BALB / C mice treated with P1-modified drug-loaded microparticles (MTX@P1-MPs) as controls. The longest part (L) and the widest part (W) of the tumor were measured with a vernier caliper every day, and the tumor volume V = L × W was calculated. 2 / 2.

[0138] 3. Experimental results

[0139] from Fig.10 It can be seen that compared with PBS and FreeMTX, the tumor volume of each drug-loaded microparticle group was significantly reduced. At the same time, after the administration, the relative tumor volume of the experimental group was significantly lower than that of MTX@MPs and MTX@P1-MPs. This shows that polymer P2 modified drug-loaded microparticles loaded with methotrexate still have a good therapeutic effect on H22 liver cancer subcutaneous tumors in mice.

[0140] Example 11 H22 tumor inhibition effect of drug-loaded 5-fluorouracil (5-FU) nanoparticles

[0141] 1. Experimental Materials and Reagents

[0142] Mouse H22 hepatoma cells and BALB / C mice were purchased from Alvacell (Wuhan) Biotechnology Co., Ltd.

[0143] 2. Experimental steps

[0144] (1) A mouse subcutaneous liver cancer tumor model was established according to the method of Example 5.

[0145] (2) When the mouse tumor volume reaches 100mm 3The tumor-bearing mice were randomly divided into 5 groups, with 6 mice in each group. The drug-loaded nanoparticles were injected into the mice through the tail vein at a dose of 0.5 mg / kg and 200 μL on days 1, 3, 5, 7, 9 and 11. BALB / C mice treated with polymer P2-modified drug-loaded microparticles (5-FU@P2-MPs) were the experimental groups; similar treatments were used for PBS-treated BALB / C mice (PBS), BALB / C mice treated with microparticles without polymer modification and drug loading (MPs), BALB / C mice treated with P1-modified non-drug-loaded microparticles (P1-MPs), BALB / C mice treated with polymer P2-modified non-drug-loaded microparticles (P2-MPs), BALB / C mice treated with free 5-fluorouracil (Free 5-FU), BALB / C mice treated with drug-loaded microparticles without polymer modification (5-FU@MPs), and BALB / C mice treated with P1-modified drug-loaded microparticles (5-FU@P1-MPs). The longest part (L) and the widest part (W) of the tumor were measured with a vernier caliper every day, and the tumor volume V = L × W was calculated. 2 / 2.

[0146] 3. Experimental results

[0147] Fig.11 The results showed that polymer P2-modified tumor cell microparticles loaded with 5-fluorouracil had a stronger tumor inhibitory effect compared with the other groups.

[0148] Example 12 Inhibitory effect of engineered stem cell-derived DOX-loaded microparticles on H22 tumors

[0149] 1. Experimental Materials and Reagents

[0150] Mouse H22 hepatoma cells and BALB / C mice were purchased from Alvacell (Wuhan) Biotechnology Co., Ltd.

[0151] 2. Experimental steps

[0152] (1) The culture method of H22 mouse liver cancer stem cells is the same as that of Example 6. Then, referring to Example 2, polymer-modified tumor stem cell-derived drug-loaded microparticles are prepared.

[0153] (2) When the mouse tumor volume reaches 100 mm 3At the same time, the tumor-bearing mice were randomly divided into 5 groups, with 6 mice in each group. The drug-loaded nanoparticles were injected into the mice through the tail vein at a dosage of 0.5 mg / kg and a dose of 200 μL on days 1, 3, 5, 7, 9 and 11. BALB / C mice treated with polymer P2-modified tumor stem cell-derived drug-loaded microparticles (DOX@P2-3D-MPs) were the experimental group; the same treatment method was used for BALB / C mice treated with PBS (PBS), BALB / C mice treated with common tumor cell-derived microparticles without polymer modification and drug loading (2D-MPs); BALB / C mice treated with P1-modified common tumor cell-derived microparticles without drug loading (P1-2D-MPs); BALB / C mice treated with polymer P2-modified common tumor cell-derived non-drug-loaded microparticles (P2-2D-MPs); BALB / C mice treated with polymer P2-modified tumor stem cell-derived microparticles without polymer modification and drug loading (3D-MPs); BALB / C mice treated with P1-modified tumor stem cell-derived microparticles without drug loading (P1-3D-MPs); polymer P2-modified tumor stem cell-derived microparticles without drug loading (P1-3D-MPs); BALB / C mice treated with particles (P2-3D-MPs); BALB / C mice treated with free doxorubicin (FreeDOX); BALB / C mice treated with common tumor cell-derived drug-loaded microparticles without polymer modification (DOX@2D-MPs); BALB / C mice treated with common tumor cell-derived drug-loaded microparticles modified with P1 (DOX@P1-2D-MPs); BALB / C mice treated with common tumor cell-derived drug-loaded microparticles modified with polymer P2 (DOX@P2-2D-MPs); BALB / C mice treated with tumor stem cell-derived drug-loaded microparticles without polymer modification (DOX@3D-MPs); BALB / C mice treated with P1-modified tumor stem cell-derived drug-loaded microparticles (DOX@P1-3D-MPs); BALB / C mice treated with commercial liposome-encapsulated doxorubicin preparation (Doxil) as control. The longest part (L) and the widest part (W) of the tumor were measured with a vernier caliper every day, and the tumor volume V = L × W was calculated. 2 / 2.

[0154] 3. Experimental results

[0155] like Fig.12 As shown, compared with other groups, the cancer stem cell-derived drug-loaded microparticles modified with polymer P2 had better tumor inhibition effects.

[0156] Example 13 4T1 tumor inhibition effect of DOX-loaded nanoparticles

[0157] 1. Experimental Materials and Reagents

[0158] Mouse 4T1 breast cancer cells and BALB / C mice were purchased from Alva (Wuhan) Biotechnology Co., Ltd.

[0159] 2. Experimental steps

[0160] (1) Establish a mouse breast cancer orthotopic tumor model.

[0161] (2) When the mouse tumor volume reaches 100mm 3 At the same time, the tumor-bearing mice were randomly divided into 5 groups, with 6 mice in each group. The drug-loaded nanoparticles were injected into the mice through the tail vein at a dosage of 0.5 mg / kg and a dose of 200 μL on days 1, 3, 5, 7, 9 and 11. BALB / C mice treated with polymer P2-modified drug-loaded microparticles (DOX@P2-MPs) were used as the experimental group; similar treatment methods were used for PBS-treated BALB / C mice (PBS), BALB / C mice treated with microparticles that were not polymer-modified and not drug-loaded (MPs), BALB / C mice treated with P1-modified non-drug-loaded microparticles (P1-MPs), BALB / C mice treated with polymer P2-modified non-drug-loaded microparticles (P2-MPs), BALB / C mice treated with free doxorubicin (FreeDOX), BALB / C mice treated with drug-loaded microparticles that were not polymer-modified (DOX@MPs), BALB / C mice treated with P1-modified drug-loaded microparticles (DOX@P1-MPs), and BALB / C mice treated with commercial liposome-encapsulated doxorubicin preparations (Doxil). The longest (L) and widest (W) parts of the tumor were measured with a vernier caliper every day, and the tumor volume V = L × W was calculated. 2 / 2.

[0162] 3. Experimental results

[0163] from Fig.13 It can be seen that the polymer P2 modified drug-loaded microparticles loaded with doxorubicin also have a good therapeutic effect on mouse 4T1 breast cancer in situ tumors, indicating that the method of engineering modified microparticles provided in the present invention has universal applicability.

[0164] 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. An engineered tumor cell-derived microparticle drug-loaded preparation, characterized in that: It comprises tumor cell-derived microparticles modified by a pH-responsive hydrophilic-hydrophobic transition polymer and drugs encapsulated in the tumor cell-derived microparticles; the pH-responsive hydrophilic-hydrophobic transition polymer is hydrophilic in a blood environment and is hydrophobic in an acidic tumor microenvironment; The pH-responsive hydrophilic-hydrophobic transition polymer is N - Chain polymer formed by polymerization of isopropyl acrylamide, acrylic acid monomer and octadecyl acrylate; Said N - the molar ratio of isopropyl acrylamide, the acrylic monomer and the octadecyl acrylate is 100:(2-3):1; The tumor cells are common tumor cells of liver cancer or breast cancer, or tumor stem cells of liver cancer or breast cancer; The drug is one or more of doxorubicin, methotrexate, 5-fluorouracil, cisplatin, paclitaxel and dextran.

2. The engineered tumor cell-derived microparticle drug-loaded preparation according to claim 1, characterized in that: The acrylic monomer is acrylic acid, methacrylic acid, propyl acrylate or N -Methallylamine.

3. The engineered tumor cell-derived microparticle drug-loaded preparation according to claim 1, characterized in that: The particle size of the engineered tumor cell-derived microparticle drug-loaded preparation is 200nm-600nm.

4. A method for preparing the engineered tumor cell-derived microparticle drug-loaded preparation according to any one of claims 1 to 3, characterized in that: The steps include: S1. Using ultraviolet rays to irradiate cultured tumor cells to induce apoptosis, then incubating the tumor cells with high concentrations of drugs so that the drugs are taken up by the cells, and finally collecting the released drug-loaded microparticles; S2. Incubate the drug-loaded microparticles with a pH-responsive hydrophilic-hydrophobic transition polymer, so that the pH-responsive hydrophilic-hydrophobic transition polymer is modified on the surface of the drug-loaded microparticles to obtain an engineered tumor cell-derived microparticle drug-loaded preparation.

5. The method for preparing the engineered tumor cell-derived microparticle drug-loaded preparation according to claim 4, characterized in that: In step S1, the drug-loaded microparticles are collected at 4°C to 6°C and at a centrifugal force of 400g to 20000g.

6. The method for preparing the engineered tumor cell-derived microparticle drug-loaded preparation according to claim 4, characterized in that: In step S2, the mass ratio of the protein in the drug-loaded microparticles to the pH-responsive hydrophilic-hydrophobic transition polymer is 1:(0.5-1).

7. The method for preparing the engineered tumor cell-derived microparticle drug-loaded preparation according to claim 4, characterized in that: The specific operation of step S2 is: during the incubation process of the drug-loaded microparticles and the pH-responsive hydrophilic-hydrophobic transition polymer, the long-chain alkane in the pH-responsive hydrophilic-hydrophobic transition polymer is inserted into the hydrophobic layer of the phospholipid bilayer of the drug-loaded microparticles through hydrophobic action, so that the pH-responsive hydrophilic-hydrophobic transition polymer is adsorbed and fixed on the surface of the drug-loaded microparticles, thereby obtaining the drug-loaded microparticles modified with the pH-responsive hydrophilic-hydrophobic transition polymer.

8. The method for preparing the engineered tumor cell-derived microparticle drug-loaded preparation according to claim 4, characterized in that: In step S2, the drug-loaded microparticles and the pH-responsive hydrophilic-hydrophobic transition polymer are incubated at 4° C. to 6° C. for 10 h to 16 h.

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