CN111994935A - Preparation method of porous hollow calcium carbonate drug-loaded microspheres - Google Patents
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Abstract
A preparation method of porous hollow calcium carbonate drug-loaded microspheres comprises the following steps: (1) culturing of microbial cells: adding the strain into liquid culture mediumTaking the precipitate, and placing the precipitate in a NaCl solution for re-suspension for later use; (2) preparing a layer-by-layer self-assembly coating on the surface of a strain: and (3) alternately adding polydiene dimethyl ammonium chloride solution and polystyrene sulfonic acid solution into the bacteria to obtain the bacterial cells coated with surface polyelectrolyte. (3) Calcium salt pretreatment: adding Ca to strain cells2+The solution was magnetically stirred and mixed well. (4) Preparing a calcium carbonate shell: adding the same amount of CO dropwise into the solution3 2‑The solution was magnetically stirred for 16 h. The resulting mixture was repeatedly washed with pure water, and then dried and ground. Thus obtaining the nano calcium carbonate shell on the surface of the strain cell. (5) Preparing porous hollow calcium carbonate microspheres: and (3) putting the nano calcium carbonate shell particles into a tube furnace, and removing strain cells in the inner layer to obtain the hollow porous calcium carbonate microspheres. The invention has low cost and simple preparation process.
Description
Technical Field
The invention belongs to a drug sustained release carrier, relates to a preparation method of porous hollow calcium carbonate drug-loaded microspheres, belongs to the field of biomedical engineering, and can be used as a drug sustained release carrier material in a biological material. Can endow various oral drug carriers with pH sensitivity and biodegradability.
Background
Drug carriers, such as microcapsules, nanotubes and hollow nanoparticles, have attracted considerable attention in tumor therapy for decades because they can passively spill over into tumor tissue and accumulate, thereby effectively reducing side effects, enhancing the pharmacokinetic properties of the drug, extending the circulating half-life, and improving the availability, safety and efficacy of the drug. To date, such as liposomes, dendrimers, polymeric nanoparticles, calcium phosphate, silicon oxide, zinc oxide and calcium carbonate (CaCO)3) Have been developed for drug delivery. Among these nanocarriers, CaCO3Nanoparticles or nanospheres have high drug delivery potential due to CaCO3Is the main component of human skeleton or animal shell in nature, and has good biocompatibility and biodegradability. In addition, the preparation process of the calcium carbonate is simple and mild. Overall, CaCO3The base nanocarriers have several potential advantages in drug delivery: (1) has good biocompatibility and no obvious toxicity or immune response; (2) is sensitive to low pH value and is particularly suitable for tumor environment; (3) Biodegradability in a biological environment; (4) simple preparation and low cost. Heretofore, there have been many methods for synthesizing porous calcium carbonate nanoparticles or microparticles, including a template method, an emulsion liquid membrane method, a coprecipitation method, a solvent/hydrothermal method, a gel crystallization method, a salting-out method, and the like. Among these methods, the template method is a commonly used one. The main principle is that a layer of calcium carbonate is coated on the surface of a selected template agent to form a core-shell structure, and then the template is removed by solvent dissolution, high-temperature calcination or chemical reaction and the like, so that particles form a hollow structure. The templates used at present are polyethylene glycol (PEG) and carbon dioxide (CO)2) Sodium Dodecyl Sulfate (SDS), sodium polystyrene sulfonate (PSS), Polyacrylamide (PAM), soluble starch, lotus root and the like. The shape and size of the template has a significant effect on the structure and size of the drug carrier. The template method is simple, no special solvent is needed, and the preparation conditions are mild. However, CaCO prepared using the above template3Poor structure controllability, too small cavities and difficult access of some protein drugs with large molecular weight into the porous CaCO3In (1). Therefore, the search for innovative templates to obtain the hollow microspheres with larger holes has important significance, and CaCO can be further expanded3The application range of the material in the aspect of drug carriers.
Disclosure of Invention
The invention provides a preparation method of porous hollow calcium carbonate drug-loaded microspheres, which is low in cost, simple in preparation process and free of chemical pore-forming agents.
Under mild and controllable conditions, the invention utilizes yeast, escherichia coli and staphylococcus aureus cells as biological templates by a safe and simple method, utilizes layer-by-layer self-assembly technology (LbL) to encapsulate 4-6 layers of (PDDA/PSS) membranes on the cell surfaces, combines with calcium, and prepares [ cell @ (PPDA/PSS) n ] by a precipitation method] @CaCO3Mixing the microspheres, and sintering at high temperature to remove the cell template to obtain the porous hollow calcium carbonate microspheres.
The preparation method of the porous hollow calcium carbonate drug-loaded microsphere comprises the following steps:
(1) microbial cells (Yeast, Escherichia coli, Staphylococcus aureus)) The culture of (2): adding the strain into 5 mL of liquid culture medium, placing into a constant temperature shaking table with the temperature of 180 rpm and 37 ℃ for culturing for 16 h until the strain grows to 10%6 centrifuging at 3000 rpm after cfu/mL for 5min, removing supernatant, taking precipitate, and placing in 0.9% NaCl solution for re-suspension;
(2) preparing a layer-by-layer self-assembly coating on the surface of a strain: adding 20 mL of polydiene dimethyl ammonium chloride (PDDA) solution with the concentration of 1.0 g/mL into the bacterial liquid, shaking for 15 min at 180 rpm in a constant-temperature shaking table at 37 ℃, then centrifuging for 5min at 6000 rpm, then removing the supernatant, adding 20 mL of poly (styrene sulfonic acid) PSS solution with the concentration of 1.0 g/mL, shaking uniformly and scattering, shaking for 15 min at 180 rpm in a constant-temperature shaking table at 37 ℃, then centrifuging for 5min at 6000 rpm, and repeating the step for 4-6 times.
(3) Calcium salt pretreatment: the treated seed cells were transferred to a beaker and 100 mL of 0.33M Ca was added2+The solution is placed in a water bath kettle, stirred for 2 hours under the magnetic force of 37 ℃ and fully mixed.
(4) Preparing a calcium carbonate shell: 150 mL of 0.33M CO was added dropwise to the above solution3 2-The solution was stirred magnetically for 16 h after addition. The resulting mixture was repeatedly washed with pure water, and then dried and ground. Thus obtaining the nano calcium carbonate shell on the surface of the strain cell.
(5) Preparing porous hollow calcium carbonate microspheres: and (3) putting the nano calcium carbonate shell particles into a tube furnace, and sintering for 4 hours at 500 ℃ to remove strain cells in the inner layer to obtain the hollow porous calcium carbonate microspheres.
The yeast cell in the step (1) is saccharomyces cerevisiae, the main components of a culture medium of the saccharomyces cerevisiae are tryptone 10 g, yeast extract 5 g and glucose 10 g, then the saccharomyces cerevisiae and saccharomyces cerevisiae are dissolved in 1000 mL of deionized water, sterilized by a sterilization pot at 115 ℃ for half an hour, cooled and then put into a refrigerator at 4 ℃ for standby; the main components of the culture medium for the escherichia coli and the staphylococcus aureus are tryptone 10 g, yeast extract 5 g and NaCl 8g, then the culture medium is sterilized in 1000 mL of deionized water at 121 ℃ for half an hour by a sterilizing pot, and the sterilized culture medium is placed in a refrigerator at 4 ℃ for standby after being cooled.
The weight average molecular weight of the PDDA in the step (2) is 100000, and the weight average molecular weight of the PSS is 70000.
Ca described in step (3)2+Suitable concentration is 0.33M, soluble calcium salt can be selected, but is not limited to CaCl2,Ca(NO3)2。
CO described in step (4)3 2-Suitably at a concentration of 0.33M, soluble carbonate may be used (e.g. Na)2CO3, (NH4)2CO3Etc.).
Through the process steps of the preparation method, the prepared porous hollow calcium carbonate microspheres are spherical and regular in shape, and the average particle size is 3 microns.
The invention firstly cultures the bacteria cells with proper concentration (10)6cfu/mL), alternately depositing PDDA and PSS on the cell surface by a layer-by-layer assembly technology, increasing the charge on the cell surface, increasing calcium ion binding sites, adding carbonate particles after coaction with calcium ions, performing coprecipitation to prepare cell polyelectrolyte calcium carbonate composite particles, and sintering in a tube furnace to remove cells and polymers to obtain the porous hollow calcium carbonate microspheres.
The calcium carbonate drug-loaded microsphere with high drug-loading capacity, pH sensitivity and degradability and good biocompatibility is obtained by taking the saccharomycetes, the escherichia coli and the staphylococcus aureus cells as the templates under mild and stable conditions, the cost is low, the preparation process is simple, and safe, efficient and large-scale production can be realized. The invention provides a new idea for preparing the porous hollow inorganic drug-loaded material, and has wide application value in the aspect of drug carriers.
The foregoing and other objects, features, and advantages of the invention will be apparent from the following more particular description of the embodiments of the invention, as illustrated in the accompanying drawings. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. Various substitutions and alterations according to the general knowledge and conventional practice in the art are intended to be included within the scope of the present invention without departing from the technical spirit of the present invention as described above.
Drawings
FIG. 1 is an SEM image of cells and sintered calcium carbonate microspheres; electron micrographs of CaCO3 microspheres at different sintering temperatures. Unsintered CaCO3Cellular microspheres, a) unsintered CaCO3Cell microspheres;
FIG. 2 is 600 ℃ sintered CaCO3Electron microscopy of microspheres.
FIG. 3 is 700 ℃ sintered CaCO3Electron microscopy of microspheres.
FIG. 4 is a 750 ℃ sintered CaCO3Electron microscopy of microspheres.
FIG. 5 is 800 ℃ sintered CaCO3Electron microscopy of microspheres.
FIG. 6 is CaCO sintered at 900 deg.C3Electron microscopy of microspheres.
FIG. 7 is a calcium carbonate microsphere XRD pattern; wherein: a) sintered CaCO3Microspheres; b) unsintered CaCO3Microspheres; c) CaCO3XRD pattern of the powder.
FIG. 8 is a slow release curve of calcium carbonate microspheres loaded with doxorubicin; the cumulative release profile of doxorubicin under different circumstances (a) pH =4.8, (b) pH = 7.
Fig. 9 is the degradation performance of calcium carbonate microspheres. a) -e) scanning electron microscopy of CaCO3-HNPs degraded for different times in a PBS environment at pH = 4.8; f) -j) at pH =7 PBS environment.
Detailed Description
Example 1
Adding yeast into 5 mL liquid culture medium, placing into constant temperature shaking table with 180 rpm and 37 deg.C, culturing for 16 h until the strain grows to 10%6centrifuging at 3000 rpm after cfu/mL for 5min, removing supernatant, taking precipitate, and placing in 0.9% NaCl solution for re-suspension; adding 20 mL of polydiene dimethyl ammonium chloride (PDDA) solution with the concentration of 1.0 g/mL into the bacterial liquid, shaking for 15 min at 180 rpm in a constant-temperature shaking table at 37 ℃, then centrifuging for 5min at 6000 rpm, then removing the supernatant, adding 20 mL of polystyrene sulfonic acid solution with the concentration of 1.0 g/mL, shaking uniformly and scattering, shaking for 15 min at 180 rpm in a constant-temperature shaking table at 37 ℃, then centrifuging for 5min at 6000 rpm, and repeating the step for 4 times. The treated seed cells were transferred to a beaker and 100 mL of 0.33M Ca was added2+The solution is placed in a water bath kettle, stirred for 2 hours under the magnetic force of 37 ℃ and fully mixed. 150 mL of 0.33M CO was added dropwise to the above solution3 2-The solution was stirred magnetically for 16 h after addition. The resulting mixture was repeatedly washed with pure water, and then dried and ground. Thus obtaining the nano calcium carbonate shell on the surface of the strain cell. And (2) placing the nano calcium carbonate shell particles into a tube furnace, sintering for 4 hours at 500 ℃ to remove strain cells in the inner layer to obtain hollow porous calcium carbonate microspheres, and observing the surface appearance of the microspheres by using SEM (shown in figure 1). Then, carrying out crystal form test on the microspheres by an X-ray diffractometer to obtain an XRD (X-ray diffraction) pattern (attached figure 2) of the microspheres; 10 mL DOX solution was taken and 5mg CaCO was added thereto3And (3) vibrating the microspheres at room temperature at 100 rpm for 24 h, centrifuging to obtain a proper amount of supernatant, testing the absorbance at 490nm by using an enzyme-labeling instrument, substituting the absorbance into a standard curve, calculating the drug loading rate by using the difference, and arranging three groups of parallel samples for each sample. Weighing 1 mg DOX-loaded CaCO3The microspheres were immersed in 5 mL of PBS solution (pH =4.8 or 7), shaken at 60 rpm at room temperature, and 0.2 mL of the solution was taken out at regular intervals to measure absorbance at 490nm with a microplate reader while supplementing the same amount of fresh PBS solution. Origin software was used to count cumulative drug release. Three sets of replicates were set up for each sample (fig. 3). Weighing hollow CaCO with same mass3The microspheres (0.2 g) were placed in a centrifuge tube containing 5 mL of PBS solution (pH =4.8 or 7) and continuously shaken at 60 rpm in a constant temperature shaker set at 37 ℃. One sample was taken every week and dried after centrifugation, weighed and compared to the original 0.2 g to obtain the degradation of the microspheres over this time period, which often lasted 7 weeks, and then dried and SEM obtained to obtain SEM pictures (fig. 4).
Example 2
Adding the above yeast into 5 mL liquid culture medium, placing into constant temperature shaking table set at 180 rpm and 37 deg.C, culturing for 16 times until the bacterial growth reaches 106centrifuging at 3000 rpm after cfu/mL for 5min, removing supernatant, taking precipitate, and placing in 0.9% NaCl solution for re-suspension; adding 20 mL polydiene dimethyl ammonium chloride (PDDA) solution with concentration of 2.0 g/mL into the above bacterial solution, and maintaining at 37 deg.CShaking at 180 rpm in a warm shaker for 15 min, centrifuging at 6000 rpm for 5min, removing supernatant, adding 20 mL of PSS solution with concentration of 2.0 g/mL, shaking up and scattering, shaking at 180 rpm in a warm shaker at 37 ℃ for 15 min, centrifuging at 6000 rpm for 5min, and repeating the steps for 4-6 times. Transferring the treated strain cells into a beaker, adding 100 mL of 0.5M Ca2+The solution is placed in a water bath kettle, stirred for 2 hours under the magnetic force of 37 ℃ and fully mixed. 150 mL of 0.5M CO was added dropwise to the above solution3 2-The solution was stirred magnetically for 16 h after addition. The resulting mixture was repeatedly washed with pure water, and then dried and ground. Thus obtaining the nano calcium carbonate shell on the surface of the strain cell. And (3) placing the nano calcium carbonate shell particles into a tube furnace, sintering for 4 hours at 550 ℃ to remove strain cells in the inner layer to obtain hollow porous calcium carbonate microspheres, and observing the surface appearance of the microspheres by using SEM. Then, carrying out crystal form test on the microspheres by an X-ray diffractometer to obtain an XRD (X-ray diffraction) pattern of the microspheres; 10 mL DOX solution was taken and 5mg CaCO was added thereto3And (3) vibrating the microspheres at room temperature at 100 rpm for 24 h, centrifuging to obtain a proper amount of supernatant, testing the absorbance at 490nm by using an enzyme-labeling instrument, substituting the absorbance into a standard curve, calculating the drug loading rate by using the difference, and arranging three groups of parallel samples for each sample. Weighing 1 mg DOX-loaded CaCO3The microspheres were immersed in 5 mL of PBS solution (pH =4.8 or 7), shaken at 60 rpm at room temperature, and 0.2 mL of the solution was taken out at regular intervals to measure absorbance at 490nm with a microplate reader while supplementing the same amount of fresh PBS solution. Origin software was used to count cumulative drug release. Three replicates were set up for each sample. Weighing hollow CaCO with same mass3The microspheres (0.2 g) were placed in a centrifuge tube containing 5 mL of PBS solution (pH =4.8 or 7) and continuously shaken at 60 rpm in a constant temperature shaker set at 37 ℃. One sample was taken every week and dried after centrifugation, weighed and compared to the original 0.2 g to obtain the degradation of the microspheres over this time period, which often lasted 7 weeks, and then dried and SEM taken to obtain SEM pictures.
Example 3
Adding the Escherichia coli into 5 mL of liquid culture medium, and placingCulturing in constant temperature shaking table set at 180 rpm and 37 deg.C for 16 h until the strain grows to 10 deg.C6centrifuging at 3000 rpm after cfu/mL for 5min, removing supernatant, taking precipitate, and placing in 0.9% NaCl solution for re-suspension; adding 20 mL of polydiene dimethyl ammonium chloride (PDDA) solution with the concentration of 1.0 g/mL into the bacterial liquid, shaking for 15 min at 180 rpm in a constant-temperature shaking table at 37 ℃, then centrifuging for 5min at 6000 rpm, then removing the supernatant, adding 20 mL of polystyrene sulfonic acid (PSS) solution with the concentration of 1.0 g/mL, shaking uniformly and scattering, shaking for 15 min at 180 rpm in a constant-temperature shaking table at 37 ℃, then centrifuging for 5min at 6000 rpm, and repeating the step for 4-6 times. The treated seed cells were transferred to a beaker and 100 mL of 0.33M Ca was added2+The solution is placed in a water bath kettle, stirred for 2 hours under the magnetic force of 37 ℃ and fully mixed. 150 mL of 0.33M CO was added dropwise to the above solution3 2-The solution was stirred magnetically for 16 h after addition. The resulting mixture was repeatedly washed with pure water, and then dried and ground. Thus obtaining the nano calcium carbonate shell on the surface of the strain cell. And (3) putting the nano calcium carbonate shell particles into a tube furnace, sintering for 4 hours at 500 ℃ to remove strain cells in the inner layer to obtain hollow porous calcium carbonate microspheres, and observing the surface appearance of the microspheres by using SEM. Then, carrying out crystal form test on the microspheres by an X-ray diffractometer to obtain an XRD (X-ray diffraction) pattern of the microspheres; 10 mL DOX solution was taken and 5mg CaCO was added thereto3And (3) vibrating the microspheres at room temperature at 100 rpm for 24 h, centrifuging to obtain a proper amount of supernatant, testing the absorbance at 490nm by using an enzyme-labeling instrument, substituting the absorbance into a standard curve, calculating the drug loading rate by using the difference, and arranging three groups of parallel samples for each sample. Weighing 1 mg DOX-loaded CaCO3The microspheres were immersed in 5 mL of PBS solution (pH =4.8 or 7), shaken at 60 rpm at room temperature, and 0.2 mL of the solution was taken out at regular intervals to measure absorbance at 490nm with a microplate reader while supplementing the same amount of fresh PBS solution. Origin software was used to count cumulative drug release. Three replicates were set up for each sample. Weighing hollow CaCO with same mass3The microspheres (0.2 g) were placed in a centrifuge tube containing 5 mL of PBS solution (pH =4.8 or 7) and continuously shaken at 60 rpm in a constant temperature shaker set at 37 ℃. Taking out a sample every other week, centrifuging and dryingThe microspheres were dried, weighed and compared to the original 0.2 g to determine the degradation of the microspheres over this time period, which often lasted 7 weeks, and then dried and SEM-scanned to obtain SEM images.
Example 4
Adding the staphylococcus aureus into 5 mL of liquid culture medium, putting into a constant-temperature shaking table with the temperature of 180 rpm and 37 ℃ for culturing for 16 h until the bacterial growth reaches 10%6centrifuging at 3000 rpm after cfu/mL for 5min, removing supernatant, taking precipitate, and placing in 0.9% NaCl solution for re-suspension; adding 20 mL of polydiene dimethyl ammonium chloride (PDDA) solution with the concentration of 1.0 g/mL into the bacterial liquid, shaking for 15 min at 180 rpm in a constant-temperature shaking table at 37 ℃, then centrifuging for 5min at 6000 rpm, then removing the supernatant, adding 20 mL of polystyrene sulfonic acid solution with the concentration of 1.0 g/mL, shaking uniformly and scattering, shaking for 15 min at 180 rpm in a constant-temperature shaking table at 37 ℃, then centrifuging for 5min at 6000 rpm, and repeating the step for 4-6 times. The treated seed cells were transferred to a beaker and 100 mL of 0.33M Ca was added2+The solution is placed in a water bath kettle, stirred for 2 hours under the magnetic force of 37 ℃ and fully mixed. 150 mL of 0.33M CO was added dropwise to the above solution3 2-The solution was stirred magnetically for 16 h after addition. The resulting mixture was repeatedly washed with pure water, and then dried and ground. Thus obtaining the nano calcium carbonate shell on the surface of the strain cell. And (3) putting the nano calcium carbonate shell particles into a tube furnace, sintering for 4 hours at 500 ℃ to remove strain cells in the inner layer to obtain hollow porous calcium carbonate microspheres, and observing the surface appearance of the microspheres by using SEM. Then, carrying out crystal form test on the microspheres by an X-ray diffractometer to obtain an XRD (X-ray diffraction) pattern of the microspheres; 10 mL DOX solution was taken and 5mg CaCO was added thereto3And (3) vibrating the microspheres at room temperature at 100 rpm for 24 h, centrifuging to obtain a proper amount of supernatant, testing the absorbance at 490nm by using an enzyme-labeling instrument, substituting the absorbance into a standard curve, calculating the drug loading rate by using the difference, and arranging three groups of parallel samples for each sample. Weighing 1 mg DOX-loaded CaCO3The microspheres were immersed in 5 mL of PBS solution (pH =4.8 or 7), shaken at 60 rpm at room temperature, and 0.2 mL of the solution was taken out at regular intervals to measure absorbance at 490nm with a microplate reader while supplementing the same amount of fresh PBS solution. Using origin softwareAnd (5) counting the cumulative release amount of the medicine. Three replicates were set up for each sample. Weighing hollow CaCO with same mass3The microspheres (0.2 g) were placed in a centrifuge tube containing 5 mL of PBS solution (pH =4.8 or 7) and continuously shaken at 60 rpm in a constant temperature shaker set at 37 ℃. One sample was taken every week and dried after centrifugation, weighed and compared to the original 0.2 g to obtain the degradation of the microspheres over this time period, which often lasted 7 weeks, and then dried and SEM taken to obtain SEM pictures.
Claims (3)
1. A preparation method of porous hollow calcium carbonate drug-loaded microspheres is characterized by comprising the following steps:
(1) culturing of microbial cells: adding yeast into 5 mL liquid culture medium, placing into constant temperature shaking table set at 180 rpm and 37 deg.C, culturing for 16 h until yeast grows to 10%6 centrifuging at 3000 rpm after cfu/mL for 5min, removing supernatant, taking precipitate, and placing in 0.9% NaCl solution for re-suspension;
(2) preparing a layer-by-layer self-assembly coating on the surface of a yeast strain: adding 20 mL of polydiene dimethyl ammonium chloride solution with the concentration of 1.0 g/mL into the bacterial liquid, shaking for 15 min at 180 rpm in a constant-temperature shaking table at 37 ℃, then centrifuging for 5min at 6000 rpm, then removing supernatant, adding 20 mL of polystyrene sulfonic acid solution with the concentration of 1.0 g/mL, shaking uniformly and scattering, shaking for 15 min at 180 rpm in a constant-temperature shaking table at 37 ℃, then centrifuging for 5min at 6000 rpm, and repeating the step for 4-6 times;
(3) calcium salt pretreatment: transferring the strain cells treated in the step 2 into a beaker, and adding 100 mL of 0.33M Ca2+The solution is placed in a water bath kettle, stirred for 2 hours under the magnetic force of 37 ℃ and fully mixed;
(4) preparing a calcium carbonate shell: 150 mL of 0.33M CO was added dropwise to the step 3 solution3 2-After the solution is added, magnetically stirring for 16 hours, repeatedly cleaning with pure water, and then drying and grinding to obtain a nano calcium carbonate shell on the surface of the strain cell;
(5) preparing porous hollow calcium carbonate microspheres: and (3) placing the shell of the nano calcium carbonate into a tube furnace, and sintering for 4 hours at 500 ℃ to remove strain cells in the inner layer to obtain the hollow porous calcium carbonate microspheres.
2. The preparation method of the porous hollow calcium carbonate drug-loaded microspheres according to claim 1, wherein the crystal form of the porous hollow calcium carbonate microspheres is calcite, the average particle size is 3 μm, and the dispersibility is good.
3. The preparation method of the porous hollow calcium carbonate drug-loaded microspheres of claim 1, wherein the porous hollow calcium carbonate microspheres loaded with doxorubicin hydrochloride have a 99% sustained release effect of 120 days at pH =4.8, and have degradability under acidic conditions.
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