A preparation method and application of lactoferrin crystal gel microspheres

Abstract

The present invention relates to a preparation method and application of lactoferrin crystal colloid microspheres. After lactoferrin and a photoinitiator are prepared into a solution, an emulsification method or a microfluidics method is used to form a sol microsphere emulsion, and then a connecting hole is prefabricated by a gradient freezing method and light-cured to obtain lactoferrin crystal colloid microspheres; lactoferrin is apo-lactoferrin treated with methacrylic acid, and the iron content of apo-lactoferrin before methacrylic acid treatment is less than 0.15 mg / g, and the iron saturation content of apo-lactoferrin after methacrylic acid treatment is more than 10 mg / g; the application is: using a 21G-32G injection needle, the lactoferrin crystal colloid microspheres are injected into the visceral tissue through the meridians for absorbing iron ions and heme. The lactoferrin crystal colloid microspheres prepared by the present invention are physically stuck in the tissue and no longer enter the circulatory system, so as to achieve long-term rapid isolation of heme / iron in situ, and have good application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of hydrogel microspheres and relates to a preparation method and application of lactoferrin crystal gel microspheres. Background Art

[0002] Lactoferrin is a small molecular weight natural milk-derived glycoprotein with a relative molecular mass of about 80KDa. It is widely present in milk, tears and saliva. Lactoferrin has an iron-absorbing capacity 300 times higher than transferrin and can be used as a natural iron chelator. The sequence of lactoferrin has more than 700 amino acids. Structurally, lactoferrin consists of two globular lobes and the structure is stabilized by disulfide bonds. Past theories believed that the center of each of the two globular lobes of lactoferrin can chelate one iron, and the chelation site is supplemented by carbonate provided by the outside world (such as a certain concentration of carbonate ions in body fluids) to stabilize the chelation structure. Lactoferrin will open the iron chelation site and release iron ions in an environment with a pH lower than 4.0; in a neutral environment, apo-lactoferrin can spontaneously re-chelate free iron ions.

[0003] Lactoferrin, as a protein raw material, has the effect of removing excess iron and hemoglobin, but its application is limited to oral administration and solution injection. It is affected by the body's low absorption efficiency, metabolic rate, and targeting ability, and requires multiple doses or injections, which reduces patient compliance. In addition, after the injected protein absorbs toxic hemoglobin and iron in the lesions, it enters the liver, spleen, and kidneys with normal blood circulation, causing protein and hemoglobin metabolism, resulting in iron deposition.

[0004] For example, in CN106511979, apolactoferrin is used to treat Parkinson's disease. The concentration of lactoferrin used is 10 mg / kg. The role of lactoferrin is to reduce iron accumulation in the brain, but the injected protein drug will be rapidly metabolized to the liver and cleared.

[0005] Therefore, it is of great significance to study the preparation method and application of lactoferrin crystal gel microspheres to solve the problem that when protein solution is currently used for injection treatment, the protein easily enters the metabolic organs and is metabolized and eliminated, causing iron deposition. Summary of the invention

[0006] The purpose of the present invention is to solve the problems existing in the prior art and provide a preparation method and application of lactoferrin crystal gel microspheres.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] A method for preparing lactoferrin crystal colloid microspheres, comprising preparing a solution of lactoferrin and a photoinitiator, forming a sol microsphere emulsion by an emulsification method or a microfluidics method, prefabricating interconnecting holes by a gradient freezing method, and performing photocuring to obtain lactoferrin crystal colloid microspheres;

[0009] Lactoferrin is apolactoferrin treated with methacryloylation. The iron content of apolactoferrin before methacryloylation is less than 0.15 mg / g, and the iron saturation content of apolactoferrin after methacryloylation is more than 10 mg / g.

[0010] As the preferred technical solution:

[0011] The preparation method of lactoferrin crystal colloid microspheres as described above, the preparation process of sol microsphere emulsion is: dissolving lactoferrin in 0.01M PBS solution to prepare a mother liquor, adding methacrylic anhydride gelatin to the mother liquor, stirring and dissolving at room temperature, then adding a photoinitiator, stirring and dissolving and filtering to obtain an aqueous phase solution A, under mechanical stirring at 200-500rpm, adding the aqueous phase solution A dropwise at a speed of 20-30uL / s to the oil phase B, and continuously stirring for 10min to form a sol microsphere emulsion; or using a microfluidic method, controlling the injection speed of the aqueous phase solution A to 100-600uL / h, and the injection speed of the oil phase B to 3-6ml / h, to form a sol microsphere emulsion;

[0012] The volume ratio of aqueous solution A to oil phase B is 1:5-60;

[0013] The oil phase B is a mixture of liquid paraffin and Span 80, and the content of Span 80 in the oil phase B is 1-5wt%.

[0014] The lactoferrin used is in the form of freeze-dried powder, and the freeze-drying conditions are: drying at -45 to -55°C for 24 to 72 hours.

[0015] According to the method for preparing lactoferrin crystal gel microspheres as described above, the content of lactoferrin in the mother liquor is 0.05-0.15 g / ml; the grafting degree of methacrylic anhydride gelatin is 80%, and the amount of methacrylic anhydride gelatin added to the mother liquor is 0.03 g / ml; the photoinitiator is lithium phenyl 2,4,6-trimethylbenzoyl phosphinate, and the amount of the photoinitiator added to the mother liquor is 0.3-0.5 g / ml.

[0016] The method for preparing lactoferrin crystal gel microspheres as described above, wherein the wavelength used during light curing is 405 nm and the light curing time is 3 minutes;

[0017] The particle size of lactoferrin crystal gel microspheres is 50 to 400 μm.

[0018] In the method for preparing lactoferrin crystal gel microspheres as described above, the process of prefabricating interconnected pores by the gradient freezing method is as follows: first, the sol microsphere emulsion is stirred in an ice bath environment for 30 minutes to solidify the emulsion droplets therein to obtain a preformed emulsion, and then the preformed emulsion is placed in an environment of -80 to -20°C for 1 to 10 minutes to allow ice crystals to form through pores in the gel network, and then placed in liquid nitrogen to fix the shape.

[0019] The preparation method of lactoferrin crystal gel microspheres as described above further undergoes a cleaning process after light curing and shaping, wherein the cleaning process is: firstly washing with petroleum ether for 3 times, centrifuging for 5 minutes after each washing to remove the supernatant, then washing with -80°C pre-frozen ice acetone for 3 times, centrifuging for 5 minutes after each washing to remove the supernatant, and finally washing with sterile PBS for 3 times;

[0020] The cleaning process is the process of removing liquid paraffin. The use of organic solvents (ice acetone) may destroy the protein hydration layer, so specific operations are required. Improper use will cause protein denaturation, thereby affecting the iron absorption performance of lactoferrin.

[0021] Pre-freezing ice acetone and washing the crystal gel microspheres at low temperature for a short time can reduce its destructive effect on the protein hydration layer;

[0022] Furthermore, the glacial acetone in the cleaning process can be replaced with glacial ether or glacial ethanol.

[0023] The preparation method of lactoferrin crystal gel microspheres as described above, the preparation method of apolactoferrin is: the apolactoferrin solution to be prepared is put into a dialysis bag, the dialysis bag is placed in a PBS solution for dialysis for 48 to 72 hours, and the retentate after dialysis is freeze-dried to obtain apolactoferrin; during the dialysis process, the concentration of the PBS solution gradually decreases, the concentration of the PBS solution gradually decreases from 0.1M to 0.05M within 0 to 24 hours, the concentration of the PBS solution gradually decreases from 0.05M to 0.01M within 24 to 48 hours, and the concentration of the PBS solution gradually decreases from 0.01M to 0M within 48 to 72 hours;

[0024] The molecular weight cut-off of the dialysis bag is 4000-14000Da;

[0025] By gradually reducing the phosphate concentration in the dialysis environment, the iron ions released by lactoferrin in an acidic environment are continuously brought out through osmotic pressure.

[0026] In the method for preparing lactoferrin crystal gel microspheres as described above, the apo-lactoferrin solution is prepared by dissolving apo-lactoferrin powder in a mixed solution at room temperature;

[0027] The apo-lactoferrin is lactoferrin from colostrum, milk and whey of other mammals such as cows, humans and camels, as well as recombinant lactoferrin obtained by fermentation of genetically engineered bacteria and plants; the lactoferrin powder used is freeze-dried powder, and the freeze-drying conditions are: drying at -45 to -55°C for 24 to 72 hours;

[0028] The mixed solution is prepared by adding acetic acid, sodium acetate and EDTA to a PBS solution with a pH of 2.0 to 4.0; the concentration of PBS in the mixed solution is 0.01M, the total concentration of acetic acid and sodium acetate is 0.1M, and the concentration of EDTA is 40 to 500mM;

[0029] The concentration of the apolactoferrin solution is 0.5-10 g / 100 ml.

[0030] In the method for preparing lactoferrin crystal gel microspheres as described above, the process of methacrylating apolactoferrin is as follows:

[0031] (1) dissolving apolactoferrin in a PBS solution at room temperature to prepare an apolactoferrin stock solution with a concentration of 1 to 10 g / 100 ml;

[0032] (2) Add methacrylic anhydride to the apolactoferrin stock solution, then adjust the pH to 6.5-7.5, continue stirring and react at 10-25° C. for 0.5-24 h, dialyze for 24-72 h after the reaction, filter twice with a filter membrane after ultrafiltration concentration, and freeze-dry the filtrate to obtain apolactoferrin treated with methacrylylation.

[0033] In the method for preparing lactoferrin crystal gel microspheres as described above, the pH of the PBS solution in step (1) is 2.0-4.0 (adjusted to 2.0-4.0 by acid, the acid being selected from any one of the following: acetic acid, hydrochloric acid, sulfuric acid, phosphoric acid, citric acid);

[0034] In step (2), the dosage ratio (v / w) of methacrylic anhydride to apolactoferrin is 1 mL / 100 g to 2 mL / 1 g;

[0035] In step (2), methacrylic anhydride is added to the apoptotic lactoferrin stock solution under high-speed stirring at 400 to 1000 rpm;

[0036] The pore size of the filter membrane used for filtration in step (2) is 0.22 μm.

[0037] The present invention also provides the use of lactoferrin crystal colloid microspheres prepared by the method described in any one of the above, using a 21G-32G injection needle to inject the lactoferrin crystal colloid microspheres into visceral tissues (heart, kidney, liver, brain, lung, etc.) through meridians for absorbing iron ions and hemoglobin;

[0038] The particle size of lactoferrin crystal gel microspheres is 50 to 400 μm.

[0039] Principle of the invention:

[0040] Lactoferrin with low iron content exposes the structure with the strongest iron binding ability in the lactoferrin structure, and obtains strong iron absorption ability. After methacrylation treatment, the 3D conformation of lactoferrin with low iron content changes, producing more iron-binding domains (pockets). Due to the increase in protein-binding iron pockets, the protein's ability to absorb iron is greatly improved, and the iron binding saturation is increased to more than 10 mg / g.

[0041] Heme will form hydrogen bonds, salt bridges, π-π conjugation and other interactions with the amino acids (positively charged amino acids) on lactoferrin, thereby chelating inside the protein. After methacryloylation of lactoferrin, the exposure range of the hydrophobic domain of the protein increases (methacryloyl is a hydrophobic group, so the hydrophobic domain of lactoferrin treated with methacryloylation increases), and the electrostatic properties change (methacryloyl is grafted on the amino acid, changing the positively charged group into a neutral group, so the electrostatic properties change); among them, the area of ​​the hydrophobic domain on the protein surface increases, making it easier to bind to the hydrophobic heme; the positive charge of the amino acid surface decreases, and the repulsion with the positively charged iron at the center of the heme decreases, making it easier for heme to enter the protein, thereby significantly improving the heme affinity and binding capacity (i.e., binding sites).

[0042] The prior art generally believes that proteins are molecules, and crystal gels are hydrogel materials (specifically block gels and block porous gels above the millimeter level). The interaction between molecules and ligands (here iron / heme) is instantaneous, and its speed is determined by the molecule-ligand binding constant, which is the property of the protein itself. In addition to the binding of the molecules themselves, the speed of the interaction between proteins and ligands in crystal gels also needs to consider the diffusion speed of the ligands in the hydrogel network. Therefore, after being made into a hydrogel material, due to the diffusion rate limitation, it will have an adverse effect on the protein molecule's absorption of iron / heme.

[0043] The diffusion speed of the ligand in the hydrogel network is caused by the fact that the penetration of the liquid therein is limited by the size of the hydrogel network and the block material itself (the distance of molecular movement from the surface to the inside is long). The lactoferrin crystal gel microspheres of the present invention reduce the osmotic pressure from two aspects: the overall size (reduced from millimeter and centimeter-level blocks to micrometer-level spheres) and the internal structure (connected pore structure). From the test data, the number of iron and hemoglobin absorbed by the lactoferrin crystal gel microspheres is not significantly changed compared with the lactoferrin before being made into crystal gel microspheres.

[0044] The crystal gel microspheres of the present invention have shape memory ability and become a viscous paste after absorbing water (the shape memory ability is due to the fact that the crystal gel microspheres have a connected pore structure, which allows the microspheres to recover quickly after being compressed; the crystal gel microspheres can be stuck together by the tension of the liquid, but when shear force is applied (such as injection), the crystal gel microspheres can slide and look like a paste), and can be implanted into tissues through meridian injection, and have good injection performance;

[0045] Lactoferrin is used as a raw material to prepare crystal colloid microspheres, and combined with meridian minimally invasive surgery, it is possible to achieve the fixed-point delivery of lactoferrin crystal colloid microspheres in tissues (outside blood vessels). The commercial injection needle specifications are 21G~32G. The particle size of the lactoferrin crystal colloid microspheres selected by the present invention during injection is 50~400μm. Since the size of the crystal colloid microspheres is larger than the inner diameter of the capillaries / blood sinuses (6~40μm), the lactoferrin crystal colloid microspheres are physically positioned in the tissue, achieving in situ long-term rapid isolation of heme / iron, and will not enter the liver, spleen, and kidneys through the capillaries / blood sinuses, so that lactoferrin and heme are metabolized in these organs, causing iron deposition. In addition, the interconnected pore structure of the crystal colloid microspheres can quickly absorb iron and heme compared to the dense hydrogel network structure.

[0046] Beneficial effects:

[0047] (1) A method for preparing lactoferrin crystal gel microspheres of the present invention, wherein the lactoferrin crystal gel microspheres are micrometer-level multi-connected pore spherical hydrogels, and the connected pore structure and micrometer-level size enable the crystal gel microspheres to quickly discharge the water in the hydrogel network under the injection pressure and be compressed into a small size, so that they can pass smoothly through the slender injection head, thereby supporting minimally invasive injection and achieving targeted delivery of lactoferrin crystal gel microspheres in tissues;

[0048] (2) A method for preparing lactoferrin crystal gel microspheres of the present invention. Since the size of the crystal gel microspheres is much larger than the inner diameter of capillaries / blood sinuses, the lactoferrin crystal gel microspheres are physically stuck in the tissues and no longer enter the circulatory system, thereby achieving in situ long-term rapid isolation of hemoglobin / iron released from the tissues;

[0049] (3) A method for preparing lactoferrin crystal gel microspheres of the present invention. The interconnected pore structure of the lactoferrin crystal gel microspheres can quickly absorb iron and hemoglobin compared to the dense hydrogel network structure. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 The 3D structural changes of apo-lactoferrin (Apo-Lf) and apo-lactoferrin MA treated with methacrylic acid before and after methacrylic anhydride modification in Example 1 are shown;

[0051] Figure 2Schematic diagram of the specific amino acid sites modified by methacrylic anhydride on Apo-Lf in Example 1;

[0052] Figure 3 The figure is a kinetic simulation diagram of the binding of apo-lactoferrin (Apo-Lf) and apo-lactoferrin treated with methacrylylation (Apo-LfMA) with heme in Example 1, wherein Site1, Site2, ... represent the heme binding sites on the protein;

[0053] Figure 4 The surface plasmon resonance (SPR) spectra of apo-lactoferrin (Apo-Lf) and apo-lactoferrin (Apo-LfMA) treated with methacrylylation and heme (Heme) in Example 1 are shown in Raw, and Fitting is the fitted curve;

[0054] Figure 5 The figure is a comparison of the number of heme-binding apo-lactoferrin (Apo-Lf) and apo-lactoferrin (Apo-LfMA) treated with methacrylylation in Example 1;

[0055] Figure 6 It is a comparison diagram of the iron saturation content of apolactoferrin (Apo-Lf) of Example 1 and apolactoferrin treated with methacrylylation (Apo-LfMA);

[0056] Figure 7 This is a schematic diagram of the lactoferrin crystal gel microspheres of Example 1 injected into the myocardium of myocardial infarction reperfusion 24 hours later; the four myocardial slices in the figure are four equal slices from the upper edge of myocardial infarction to the apex from left to right, and the arrows point to the crystal gel microspheres, which are stuck in the myocardium;

[0057] Figure 8 The change of iron level in the myocardium 24 hours after the lactoferrin crystal gel microspheres of Example 1 were injected into the myocardium of myocardial infarction reperfusion (i.e., the crystal gel microsphere treatment group), and compared with the iron level in the healthy myocardium (i.e., the healthy group) and the myocardium of myocardial infarction reperfusion (i.e., the disease group);

[0058] Fig. 9 The fluorescent staining comparison diagram of the lactoferrin crystal gel microspheres and their solid microspheres in Example 1;

[0059] Fig.10 This is a comparison chart of the porosity of the lactoferrin crystal gel microspheres and their solid microspheres in Example 1;

[0060] Fig.11 The absorption rate of heme by the lactoferrin crystal gel microspheres of Example 1 is compared with the solid microspheres;

[0061] Fig.12The scanning electron microscope (SEM) images of the lactoferrin crystal gel microspheres of Example 1 and Example 3 are shown;

[0062] Fig.13 This is a scanning electron microscope (SEM) image of the lactoferrin crystal gel microspheres of Example 2 compared with the lactoferrin crystal gel microspheres of Example 4. DETAILED DESCRIPTION

[0063] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0064] The test method involved in the present invention is as follows:

[0065] (1) Porosity: Mercury intrusion is a method for determining the pore size distribution of some mesopores and macropores. Mercury intrusion relies on external pressure to make mercury overcome surface tension and enter the pores of the material to determine the pore size and pore distribution of the material. As the external pressure increases, the mercury can enter smaller pores, and the amount of mercury entering the pores of the material will increase. The pore volume of the corresponding pore size can be obtained by measuring the amount of mercury entering the pores under different external pressures. The maximum pressure of the mercury intrusion instrument currently used is about 200MPa, and the pore range that can be measured is: 0.0064~950μm (pore diameter); the mercury intrusion instrument used is model IV-9510 produced by Mack Company in the United States.

[0066] (2) Heme-binding ability: 100 mg of freshly-hydrated wet crystal gel microspheres were placed in 20 mL of 1 mM Heme solution (pH = 7.2-7.4) and stirred for 30 min. The mixture was centrifuged to remove the supernatant and the microspheres were washed with deionized water for 3 times until all unbound heme was removed. 5 mg of freshly-hydrated wet crystal gel microspheres were added with 1 ml of aqua regia and digested at 70 °C for 1 h. After cooling to room temperature, the solution was diluted to 5 ml with deionized water. The diluted solution was sent for inductively coupled plasma (ICP) testing of iron ion concentration. The obtained concentration value was a ug / mL. The heme-binding ability of the crystal gel microspheres was a / 56*652 mg / g.

[0067] (3) Iron absorption capacity: 100 mg of freshly hydrated wet crystal colloid microspheres were placed in 50 mL of 50 μg / mL FeCl3 / NaHCO3 solution (FeCl3:NaHCO3 molar ratio 1:1, pH = 7.2-7.4) and stirred for 30 min. The mixture was centrifuged to remove the supernatant and the microspheres were washed with deionized water for 3 times until all unbound iron was removed. 5 mg of freshly hydrated wet crystal colloid microspheres were added with 1 ml of aqua regia and digested at 70°C for 1 h. After cooling to room temperature, the solution was diluted with deionized water to 5 ml. The diluted solution was sent for inductively coupled plasma (ICP) testing of iron ion concentration. The obtained concentration value a ug / mL was equal to the iron absorption capacity of the crystal colloid microspheres of a mg / g.

[0068] (4) Binding constant K D K value: The technology used is the surface plasmon resonance (SPR) technology of the Biacore instrument. The SPR principle is used to detect the interaction between the ligand (here hemoglobin) and the analyte (here protein) on the biosensor chip. In order to study the interaction between two molecules, one molecule (protein) is fixed to the chip surface, while the other molecule (hemoglobin) flows continuously over the chip surface in the form of a solution. The detector can detect the binding and dissociation process of the molecules in the solution and the molecules on the chip surface in real time, and display it in the form of SPR response value. Calculation formula: K D =kd / ka, ka is the affinity rate of binding between heme and protein, and kd is the dissociation rate.

[0069] Example 1

[0070] A method for preparing lactoferrin crystal gel microspheres, the specific steps are as follows:

[0071] (1) adding acetic acid, sodium acetate (molar ratio of 1:1) and EDTA to a PBS solution with a pH of 2 to obtain a mixed solution, and then dissolving apolactoferrin powder (Bega, Australia) in the mixed solution at room temperature to obtain apolactoferrin solution with a concentration of 10 g / 100 ml;

[0072] The concentration of PBS in the mixed solution was 0.01 M, the total concentration of acetic acid and sodium acetate was 0.1 M, and the concentration of EDTA was 500 mM;

[0073] (2) the apoptotic lactoferrin solution of step (1) is placed in a dialysis bag, the dialysis bag is placed in a PBS solution for dialysis for 72 hours, and the retentate after dialysis is freeze-dried at -55°C for 24 hours to obtain apoptotic lactoferrin with an iron content of 0.0139 mg / g; the concentration of the PBS solution is gradually reduced during the dialysis process, and the concentration of the PBS solution is gradually reduced from 0.1M to 0.05M within the 0th to 24th hour, the concentration of the PBS solution is gradually reduced from 0.05M to 0.01M within the 24th to 48th hour, and the concentration of the PBS solution is gradually reduced from 0.01M to 0M within the 48th to 72th hour;

[0074] Among them, the molecular weight cut-off of the dialysis bag is 7000Da;

[0075] (3) The process of methacrylation of apopactoferrin is as follows:

[0076] (3.1) dissolving apolactoferrin in a PBS solution at pH 2 at room temperature to prepare an apolactoferrin stock solution with a concentration of 10 g / 100 ml;

[0077] (3.2) Under high-speed stirring at 1000 rpm, methacrylic anhydride was added to the apoptotic lactoferrin stock solution, and then the pH was adjusted to 7, and the reaction was continued with stirring at 25° C. for 2 h. After the reaction, the solution was dialyzed for 72 h. After ultrafiltration and concentration, the solution was filtered twice using a filter membrane with a pore size of 0.22 μm. The filtrate was freeze-dried at a temperature of -50° C. and a vacuum degree of 1 Pa to obtain Figure 6 The iron saturation content of apolactoferrin treated with methacryloylation is shown to be 14.43 mg / g;

[0078] The dosage ratio of methacrylic anhydride to lactoferrin is 0.03 mL / 1 g;

[0079] like Figure 1 As shown in Figure 2, the changes in the 3D structure of Apo-Lf and Apo-LfMA before and after methacrylic anhydride grafting (protein morphology constructed by AlphaFold), as shown in Figure 2 Figure 2 Shown is a schematic diagram of the specific amino acid sites modified by methacrylic anhydride on Apo-Lf.

[0080] like Figure 3 As shown, the amount of heme bound to methacryloylated lactoferrin was higher than that of ungrafted lactoferrin.

[0081] like Figure 4 As shown in Figure 2, compared with apolactoferrin, the saturation content of heme binding in apolactoferrin treated with methacrylylation increased, and the affinity also increased (expressed as K D value decreases).

[0082] like Figure 5As shown, the amount of heme bound to apolactoferrin increased after methacryloylation compared with apolactoferrin.

[0083] (4) dissolving the methacrylated apoptotic lactoferrin in a 0.01 M PBS solution to prepare a mother solution, adding methacrylic anhydride gelatin with a grafting degree of 80% to the mother solution, stirring and dissolving at room temperature, then adding lithium phenyl 2,4,6-trimethylbenzoylphosphinate, stirring and dissolving, and filtering to obtain an aqueous solution A;

[0084] The content of lactoferrin in the mother liquor is 0.15 g / mL; the amount of methacrylic anhydride gelatin added relative to the mother liquor is 0.03 g / ml; the amount of phenyl 2,4,6-trimethylbenzoyl phosphinate lithium added relative to the mother liquor is 0.0030 g / ml;

[0085] (5) using a microfluidic method, the injection rate of the aqueous solution A was controlled to be 100 uL / h, and the injection rate of the oil phase B was controlled to be 6 mL / h to form a sol microsphere emulsion;

[0086] Wherein, the oil phase B is a mixture of liquid paraffin and Span 80, and the content of Span 80 in the oil phase B is 5wt%; the volume ratio of the aqueous solution A to the oil phase B is 1:10;

[0087] (6) The process of prefabricating interconnected pores by gradient freezing is as follows:

[0088] First, the sol microsphere emulsion was stirred in an ice bath environment for 30 minutes to obtain a preformed emulsion, and then the preformed emulsion was placed in an environment of -80°C for 1 minute, and then placed in liquid nitrogen to fix the shape;

[0089] (7) The product of step (6) was photocured at a wavelength of 405 nm for 3 min, then washed with petroleum ether for 3 times, centrifuged for 5 min after each wash to remove the supernatant, then washed with -80°C pre-frozen ice acetone for 3 times, centrifuged for 5 min after each wash to remove the supernatant, and finally washed with sterile PBS for 3 times to obtain lactoferrin crystal gel microspheres with a particle size of 123.77±9.39 μm.

[0090] The final lactoferrin crystal gel microspheres have a heme binding capacity of 15.26μg / mg and an iron absorption capacity of 2.165μg / mg.

[0091] Using an injection needle with an inner diameter of 30G, lactoferrin crystal gel microspheres are injected into the visceral tissues through the meridians to absorb iron ions and hemoglobin.

[0092] like Figure 7As shown in the figure, lactoferrin crystal gel microspheres were injected into the pig myocardium through the meridians. After dissecting the myocardium, it was found that the lactoferrin crystal gel microspheres could be retained inside the myocardium without leakage; Figure 8 As shown, lactoferrin crystal gel microspheres were injected into the ischemic-reperfused myocardium. The lactoferrin crystal gel microspheres reduced the iron content in the tissue by absorbing iron, thereby reducing the problem of myocardial ferroptosis in the tissue.

[0093] Comparative Example 1

[0094] A method for preparing lactoferrin solid microspheres is basically the same as that in Example 1, except that the operation of step (6) is omitted, that is, the sol microsphere emulsion formed in step (5) is directly subjected to light curing for 3 minutes at a wavelength of 405 nm, then washed with petroleum ether for 3 times, centrifuged for 5 minutes after each washing to remove the supernatant, then washed with -80°C pre-frozen ice acetone for 3 times, centrifuged for 5 minutes after each washing to remove the supernatant, and finally washed with sterile PBS for 3 times to obtain lactoferrin solid microspheres with a particle size of 123.77±9.39.

[0095] The prepared lactoferrin solid microspheres have a heme-binding capacity of 15.26 μg / mg and an iron-absorbing capacity of 2.165 μg / mg.

[0096] like Figures 9-10 As shown, the present invention obtains cryogel microspheres with interconnected pores by the technology of first freezing and then photo-crosslinking, which have higher porosity than solid microspheres, and the porosity can reach 88.96%. The interconnected pore structure accelerates the iron absorption speed of lactoferrin cryogel microspheres, and can more quickly reduce the iron level in the iron overload microenvironment.

[0097] like Fig.11 As shown, lactoferrin crystal gel microspheres have a faster ability to absorb and isolate hemoglobin than solid microspheres, and lactoferrin crystal gel microspheres maintain good activity and can reach iron saturation within 2 hours.

[0098] Comparative Example 2

[0099] A method for preparing lactoferrin is basically the same as that of Example 1, except that steps (4) to (7) are omitted.

[0100] The number of heme-binding units of the prepared lactoferrin protein was 13, and the iron-absorbing capacity was 14.43 μg / mg.

[0101] By comparing Example 1 with Comparative Example 2, it can be found that the number of iron and hemoglobin absorbed by the lactoferrin crystal colloid microspheres is not significantly changed compared with the lactoferrin before being made into crystal colloid microspheres. This is because the change before and after the protein cross-linking is only the polymerization between the grafted methacryloyl groups, which does not affect the internal structure of the protein.

[0102] Example 2

[0103] A method for preparing lactoferrin crystal gel microspheres, the specific steps are as follows:

[0104] (1) adding acetic acid, sodium acetate (molar ratio of 1:1) and EDTA to a PBS solution with a pH of 2 to obtain a mixed solution, and then dissolving apolactoferrin powder (Bega, Australia) in the mixed solution at room temperature to obtain apolactoferrin solution with a concentration of 0.5 g / 100 ml;

[0105] The concentration of PBS in the mixed solution was 0.01 M, the total concentration of acetic acid and sodium acetate was 0.1 M, and the concentration of EDTA was 500 mM;

[0106] (2) the apoptotic lactoferrin solution of step (1) is placed in a dialysis bag, the dialysis bag is placed in a PBS solution for dialysis for 72 hours, and the retentate after dialysis is freeze-dried at -45°C for 72 hours to obtain apoptotic lactoferrin with an iron content of 0.0139 mg / g; the concentration of the PBS solution is gradually reduced during the dialysis process, the concentration of the PBS solution is gradually reduced from 0.1M to 0.05M within the 0th to 24th hour, the concentration of the PBS solution is gradually reduced from 0.05M to 0.01M within the 24th to 48th hour, and the concentration of the PBS solution is gradually reduced from 0.01M to 0M within the 48th to 72th hour;

[0107] Among them, the molecular weight cut-off of the dialysis bag is 7000Da;

[0108] (3) The process of methacrylation of apopactoferrin is as follows:

[0109] (3.1) dissolving apolactoferrin in a PBS solution at pH 2 at room temperature to prepare an apolactoferrin stock solution with a concentration of 10 g / 100 ml;

[0110] (3.2) adding methacrylic anhydride to the apolactoferrin stock solution under high-speed stirring at 400 rpm, adjusting the pH to 7, stirring and reacting at 25° C. for 2 h, dialyzing for 24 h after the reaction, ultrafiltration and concentration, filtering twice with a filter membrane with a pore size of 0.22 μm, and freeze-drying the filtrate at a temperature of −50° C. and a vacuum degree of 1 Pa to obtain apolactoferrin treated with methacrylic acid with an iron saturation content of 14.43 mg / g;

[0111] The dosage ratio of methacrylic anhydride to lactoferrin is 0.03 mL / 1 g;

[0112] (4) dissolving the methacrylated apoptotic lactoferrin in a 0.01 M PBS solution to prepare a mother solution, adding methacrylic anhydride gelatin with a grafting degree of 80% to the mother solution, stirring and dissolving at room temperature, then adding lithium phenyl 2,4,6-trimethylbenzoylphosphinate, stirring and dissolving, and filtering to obtain an aqueous solution A;

[0113] The content of lactoferrin in the mother liquor is 0.10 g / mL; the amount of methacrylic anhydride gelatin added relative to the mother liquor is 0.03 g / ml; the amount of phenyl 2,4,6-trimethylbenzoyl phosphinate lithium added relative to the mother liquor is 0.0030 g / ml;

[0114] (5) Under mechanical stirring at 200 rpm, the aqueous solution A was added dropwise to the oil phase B at a rate of 30 uL / s and stirring was continued for 10 min to form a sol microsphere emulsion;

[0115] The oil phase B is a mixture of liquid paraffin and Span 80, and the content of Span 80 in the oil phase B is 1 wt %; the volume ratio of the aqueous solution A to the oil phase B is 1:5;

[0116] (6) The process of prefabricating interconnected pores by gradient freezing is as follows:

[0117] First, the sol microsphere emulsion was stirred in an ice bath environment for 30 minutes to obtain a preformed emulsion, and then the preformed emulsion was placed in an environment of -5°C for 10 minutes, and then placed in liquid nitrogen to fix the shape;

[0118] (7) The product of step (6) was photocured at a wavelength of 405 nm for 3 min, then washed with petroleum ether for 3 times, centrifuged for 5 min after each wash to remove the supernatant, then washed with -80°C pre-frozen ice acetone for 3 times, centrifuged for 5 min after each wash to remove the supernatant, and finally washed with sterile PBS for 3 times to obtain lactoferrin crystal gel microspheres with a particle size of 277.50±69.14 μm.

[0119] The final lactoferrin crystal gel microspheres have a heme binding capacity of 10.17 μg / mg and an iron absorption capacity of 1.443 μg / mg.

[0120] Using an injection needle with an inner diameter of 22G, lactoferrin crystal gel microspheres are injected into visceral tissues through the meridians to absorb iron ions and hemoglobin.

[0121] Example 3

[0122] A method for preparing lactoferrin crystal gel microspheres, the specific steps are as follows:

[0123] (1) adding acetic acid, sodium acetate (molar ratio of 1:1) and EDTA to a PBS solution with a pH of 4 to obtain a mixed solution, and then dissolving apolactoferrin powder (Bega, Australia) in the mixed solution at room temperature to obtain apolactoferrin solution with a concentration of 10 g / 100 ml;

[0124] The concentration of PBS in the mixed solution was 0.01 M, the total concentration of acetic acid and sodium acetate was 0.1 M, and the concentration of EDTA was 40 mM;

[0125] (2) the apoptotic lactoferrin solution of step (1) is placed in a dialysis bag, the dialysis bag is placed in a PBS solution for dialysis for 72 hours, and the retentate after dialysis is freeze-dried at -48°C for 24 hours to obtain apoptotic lactoferrin with an iron content of 0.149 mg / g; the concentration of the PBS solution is gradually reduced during the dialysis process, and the concentration of the PBS solution is gradually reduced from 0.1M to 0.05M within the 0th to 24th hour, the concentration of the PBS solution is gradually reduced from 0.05M to 0.01M within the 24th to 48th hour, and the concentration of the PBS solution is gradually reduced from 0.01M to 0M within the 48th to 72th hour;

[0126] Among them, the molecular weight cut-off of the dialysis bag is 7000Da;

[0127] (3) The process of methacrylation of apopactoferrin is as follows:

[0128] (3.1) dissolving apolactoferrin in a PBS solution at pH 4 at room temperature to prepare an apolactoferrin stock solution with a concentration of 10 g / 100 ml;

[0129] (3.2) adding methacrylic anhydride to the apolactoferrin stock solution under high-speed stirring at 600 rpm, adjusting the pH to 7, stirring and reacting at 15° C. for 2 h, dialyzing for 36 h after the reaction, ultrafiltration and concentration, filtering twice with a filter membrane with a pore size of 0.22 μm, and freeze-drying the filtrate at a temperature of −50° C. and a vacuum degree of 1 Pa to obtain apolactoferrin treated with methacrylic acid with an iron saturation content of 14.43 mg / g;

[0130] The dosage ratio of methacrylic anhydride to lactoferrin is 0.03 mL / 1 g;

[0131] (4) dissolving the methacrylated apoptotic lactoferrin in a 0.01 M PBS solution to prepare a mother solution, adding methacrylic anhydride gelatin with a grafting degree of 80% to the mother solution, stirring and dissolving at room temperature, then adding lithium phenyl 2,4,6-trimethylbenzoylphosphinate, stirring and dissolving, and filtering to obtain an aqueous solution A;

[0132] The content of lactoferrin in the mother liquor is 0.12 g / mL; the amount of methacrylic anhydride gelatin added relative to the mother liquor is 0.03 g / ml; the amount of phenyl 2,4,6-trimethylbenzoyl phosphinate lithium added relative to the mother liquor is 0.0030 g / ml;

[0133] (5) using a microfluidic method, the injection rate of the aqueous solution A was controlled to be 300 uL / h, and the injection rate of the oil phase B was controlled to be 3 mL / h to form a sol microsphere emulsion;

[0134] The oil phase B is a mixture of liquid paraffin and Span 80, and the content of Span 80 in the oil phase B is 5wt%; the volume ratio of the aqueous solution A to the oil phase B is 1:60;

[0135] (6) The process of prefabricating interconnected pores by gradient freezing is as follows:

[0136] First, the sol microsphere emulsion was stirred in an ice bath environment for 30 minutes to obtain a preformed emulsion, and then the preformed emulsion was placed in an environment of -20°C for 10 minutes, and then placed in liquid nitrogen to fix the shape;

[0137] (7) The product of step (6) was photocured at a wavelength of 405 nm for 3 min, then washed with petroleum ether for 3 times, centrifuged for 5 min after each wash to remove the supernatant, then washed with -80°C pre-frozen ice acetone for 3 times, centrifuged for 5 min after each wash to remove the supernatant, and finally washed with sterile PBS for 3 times to obtain lactoferrin crystal gel microspheres with a particle size of 203.01±11.34 μm.

[0138] The final lactoferrin crystal gel microspheres have a heme binding capacity of 12.21 μg / mg and an iron absorption capacity of 1.732 μg / mg.

[0139] Using a 26G injection needle, lactoferrin crystal gel microspheres are injected into the visceral tissues through the meridians to absorb iron ions and hemoglobin.

[0140] like Fig.12 As shown, the lactoferrin crystal gel microspheres of Example 1 have smaller pores and sizes than the lactoferrin crystal gel microspheres of Example 3. The small pores give the material a larger specific surface area, and the small size is more conducive to injection in a fine needle and can adapt to more meridian injection systems.

[0141] Example 4

[0142] A method for preparing lactoferrin crystal gel microspheres, the specific steps are as follows:

[0143] (1) adding acetic acid, sodium acetate (molar ratio of 1:1) and EDTA to a PBS solution with a pH of 2 to obtain a mixed solution, and then dissolving apolactoferrin powder (Bega, Australia) in the mixed solution at room temperature to obtain apolactoferrin solution with a concentration of 0.5 g / 100 ml;

[0144] The concentration of PBS in the mixed solution was 0.01 M, the total concentration of acetic acid and sodium acetate was 0.1 M, and the concentration of EDTA was 500 mM;

[0145] (2) the apoptotic lactoferrin solution of step (1) is placed in a dialysis bag, the dialysis bag is placed in a PBS solution for dialysis for 64 hours, and the retentate after dialysis is freeze-dried at -50°C for 72 hours to obtain apoptotic lactoferrin with an iron content of 0.0139 mg / g; during the dialysis process, the concentration of the PBS solution is gradually reduced, from 0.1M to 0.05M within the 0th to 24th hour, from 0.05M to 0.01M within the 24th to 48th hour, and from 0.01M to 0M within the 48th to 72th hour;

[0146] Among them, the molecular weight cut-off of the dialysis bag is 7000Da;

[0147] (3) The process of methacrylation of apopactoferrin is as follows:

[0148] (3.1) dissolving apolactoferrin in a PBS solution at pH 2 at room temperature to prepare an apolactoferrin stock solution with a concentration of 10 g / 100 ml;

[0149] (3.2) adding methacrylic anhydride to the apolactoferrin stock solution under high-speed stirring at 800 rpm, adjusting the pH to 7, stirring and reacting at 20° C. for 5 h, dialyzing for 48 h after the reaction, and filtering twice with a filter membrane with a pore size of 0.22 μm after ultrafiltration concentration, and freeze-drying the filtrate at a temperature of −50° C. and a vacuum degree of 1 Pa to obtain apolactoferrin treated with methacrylic acid with an iron saturation content of 12.46 mg / g;

[0150] The dosage ratio of methacrylic anhydride to lactoferrin is 2 mL / 1 g;

[0151] (4) dissolving the methacrylated apoptotic lactoferrin in a 0.01 M PBS solution to prepare a mother solution, adding methacrylic anhydride gelatin with a grafting degree of 80% to the mother solution, stirring and dissolving at room temperature, then adding lithium phenyl 2,4,6-trimethylbenzoylphosphinate, stirring and dissolving, and filtering to obtain an aqueous solution A;

[0152] The content of lactoferrin in the mother liquor is 0.15 g / mL; the amount of methacrylic anhydride gelatin added relative to the mother liquor is 0.03 g / ml; the amount of phenyl 2,4,6-trimethylbenzoyl phosphinate lithium added relative to the mother liquor is 0.30 g / ml;

[0153] (5) Under mechanical stirring at 500 rpm, the aqueous solution A was added dropwise to the oil phase B at a rate of 30 uL / s and stirring was continued for 10 min to form a sol microsphere emulsion;

[0154] Wherein, the oil phase B is a mixture of liquid paraffin and Span 80, and the content of Span 80 in the oil phase B is 5wt%; the volume ratio of the aqueous solution A to the oil phase B is 1:10;

[0155] (6) The process of prefabricating interconnected pores by gradient freezing is as follows:

[0156] First, the sol microsphere emulsion was stirred in an ice bath environment for 30 minutes to obtain a preformed emulsion, and then the preformed emulsion was placed in an environment of -20°C for 10 minutes, and then placed in liquid nitrogen to fix the shape;

[0157] (7) The product of step (6) was photocured at a wavelength of 405 nm for 3 min, then washed with petroleum ether for 3 times, centrifuged for 5 min after each wash to remove the supernatant, then washed with -80°C pre-frozen ice acetone for 3 times, centrifuged for 5 min after each wash to remove the supernatant, and finally washed with sterile PBS for 3 times to obtain lactoferrin crystal gel microspheres with a particle size of 69.83±28.30 μm.

[0158] The final lactoferrin crystal gel microspheres have a heme binding capacity of 11.74 μg / mg and an iron absorption capacity of 1.869 μg / mg.

[0159] Using an injection needle with an inner diameter of 32G, lactoferrin crystal gel microspheres are injected into the visceral tissues through the meridians to absorb iron ions and hemoglobin.

[0160] like Fig.13 As shown, the lactoferrin crystal gel microspheres of Example 2 have smaller pores and larger sizes than the lactoferrin crystal gel microspheres of Example 4. The small pores give the material a larger specific surface area, and the small size is more conducive to injection in a fine needle and can adapt to more meridian injection systems.

Claims

1. A method for preparing lactoferrin crystal gel microspheres, characterized in that: After lactoferrin and a photoinitiator are prepared into a solution, an emulsification method or a microfluidics method is used to form a sol microsphere emulsion, and then a gradient freezing method is used to preform interconnected holes and light-curing is performed to obtain lactoferrin crystal microspheres; Lactoferrin is apolactoferrin treated with methacrylic acid. The iron content of apolactoferrin before methacrylic acid treatment is less than 0.15 mg / g, and the iron saturation content of apolactoferrin after methacrylic acid treatment is more than 10 mg / g. The preparation process of the sol microsphere emulsion is as follows: lactoferrin is dissolved in a PBS solution to prepare a mother solution, methacrylic anhydride gelatin is added to the mother solution, and the solution is stirred and dissolved at room temperature, and then a photoinitiator is added and stirred and dissolved to obtain an aqueous solution A, and the aqueous solution A is added dropwise to the oil phase B at a speed of 20 to 30 μL / s under mechanical stirring at 200 to 500 rpm, and the solution is stirred continuously for 10 min to form a sol microsphere emulsion; or a microfluidic method is used to control the injection speed of the aqueous solution A to 100 to 600 μL / h and the injection speed of the oil phase B to 3 to 6 mL / h to form a sol microsphere emulsion; The content of lactoferrin in the mother liquor is 0.05-0.15 g / mL; the grafting degree of methacrylic anhydride gelatin is 80%, and the amount of methacrylic anhydride gelatin added relative to the mother liquor is 0.03 g / mL; the photoinitiator is phenyl 2,4,6-trimethylbenzoyl lithium phosphinate, and the amount of the photoinitiator added relative to the mother liquor is 0.3-0.5 g / mL; After light curing, the sample undergoes a cleaning process: first, wash with petroleum ether for 3 times, centrifuge for 5 min after each wash to remove the supernatant, then wash with -80°C pre-frozen ice acetone for 3 times, centrifuge for 5 min after each wash to remove the supernatant, and finally wash with sterile PBS for 3 times.

2. The method for preparing lactoferrin crystal gel microspheres according to claim 1, characterized in that: The volume ratio of aqueous solution A to oil phase B is 1:5~60; The oil phase B is a mixture of liquid paraffin and Span 80, and the content of Span 80 in the oil phase B is 1-5wt%.

3. The method for preparing lactoferrin crystal gel microspheres according to claim 1, characterized in that: The wavelength used for light curing was 405 nm and the light curing time was 3 min.

4. The method for preparing lactoferrin crystal gel microspheres according to claim 1, characterized in that: The process of prefabricating interconnected pores by the gradient freezing method is as follows: first, the sol microsphere emulsion is stirred in an ice bath environment for 30 min to obtain a preformed emulsion, and then the preformed emulsion is placed in an environment of -80~-20℃ for 1~10 min, and then placed in liquid nitrogen to fix the shape.

5. The method for preparing lactoferrin crystal gel microspheres according to claim 1, characterized in that: The preparation method of apolactoferrin comprises the following steps: placing a solution of apolactoferrin into a dialysis bag, placing the dialysis bag in a PBS solution for dialysis for 48-72 hours, and freeze-drying the retentate after dialysis to obtain apolactoferrin; during the dialysis process, the concentration of the PBS solution gradually decreases, from 0.1M to 0.05M within 0-24 hours, from 0.05M to 0.01M within 24-48 hours, and from 0.01M to 0M within 48-72 hours; The molecular weight cut-off of the dialysis bag is 4000~14000 Da.

6. The method for preparing lactoferrin crystal gel microspheres according to claim 5, characterized in that: The apo-lactoferrin solution is prepared by dissolving apo-lactoferrin powder in a mixed solution at room temperature; The mixed solution is prepared by adding acetic acid, sodium acetate and EDTA to a PBS solution with a pH of 2.0 to 4.0; the concentration of PBS in the mixed solution is 0.01M, the total concentration of acetic acid and sodium acetate is 0.1M, and the concentration of EDTA is 40 to 500mM; The concentration of the apolactoferrin solution is 0.5-10 g / 100 mL.

7. The method for preparing lactoferrin crystal gel microspheres according to claim 1, characterized in that: The process of methacrylation of apolactoferrin is as follows: (1) dissolving apolactoferrin in PBS solution at room temperature to prepare an apolactoferrin stock solution with a concentration of 1-10 g / 100 mL; (2) Add methacrylic anhydride to the apopactoferrin stock solution, then adjust the pH to 6.5-7.5, and continue stirring the reaction at 10-25°C for 0.5-24 hours. After the reaction, dialyze for 24-72 hours. After ultrafiltration and concentration, filter twice with a filter membrane, and freeze-dry the filtrate to obtain apopactoferrin treated with methacrylylation.

8. The method for preparing lactoferrin crystal gel microspheres according to claim 7, characterized in that: The pH of the PBS solution in step (1) is 2.0-4.0; In step (2), the ratio of methacrylic anhydride to apolactoferrin is 1 mL / 100 g to 2 mL / 1 g; In step (2), methacrylic anhydride is added to the apoptotic lactoferrin stock solution under high-speed stirring at 400-1000 rpm; The pore size of the filter membrane used in step (2) is 0.22 μm.

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