CN114053420B - Preparation method of calcium carbonate nano-drug - Google Patents
Preparation method of calcium carbonate nano-drug Download PDFInfo
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- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 title claims abstract description 144
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- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 238000000502 dialysis Methods 0.000 claims abstract description 100
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims abstract description 50
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 35
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims abstract description 32
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- 108091003079 Bovine Serum Albumin Proteins 0.000 description 18
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- 238000010586 diagram Methods 0.000 description 4
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 4
- 230000002194 synthesizing effect Effects 0.000 description 4
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- 239000003937 drug carrier Substances 0.000 description 3
- 238000004627 transmission electron microscopy Methods 0.000 description 3
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- UQSXHKLRYXJYBZ-UHFFFAOYSA-N Iron oxide Chemical compound [Fe]=O UQSXHKLRYXJYBZ-UHFFFAOYSA-N 0.000 description 2
- 206010057249 Phagocytosis Diseases 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
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- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000002411 thermogravimetry Methods 0.000 description 2
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
- 102000011727 Caspases Human genes 0.000 description 1
- 108010076667 Caspases Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
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- 101001045444 Proteus vulgaris Endoribonuclease HigB Proteins 0.000 description 1
- 101001100822 Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) Pyocin-S2 Proteins 0.000 description 1
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- 229910001424 calcium ion Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 description 1
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- 235000011837 pasties Nutrition 0.000 description 1
- 208000024981 pyrosis Diseases 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
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- 239000000758 substrate Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/02—Inorganic compounds
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/38—Albumins
- A61K38/385—Serum albumin
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
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Abstract
The invention provides a preparation method of a calcium carbonate nano-drug, which comprises the following steps: uniformly mixing a calcium chloride aqueous solution and a protein medicine, pouring the obtained mixed solution into a dialysis bag A, then filling the dialysis bag A into a dialysis bag B which is larger than the dialysis bag A, filling deionized water between the dialysis bag A and the dialysis bag B, sealing, placing the dialysis bag A and the dialysis bag B into a sodium carbonate aqueous solution, stirring and dialyzing, and performing aftertreatment on the obtained reaction solution to obtain the calcium carbonate nano medicine; the method for preparing the calcium carbonate nano particles by using the double-layer dialysis bag to control ion diffusion has the advantages of simple preparation method, uniform particle size, no use of surfactant, polymer and other substances for controlling the growth of calcium carbonate crystal grains, and ensures the biocompatibility of the nano calcium carbonate to the greatest extent.
Description
Technical Field
The invention relates to a preparation method of a calcium carbonate nano-drug.
Background
Among inorganic materials, materials such as calcium carbonate, calcium phosphate, silica, and iron oxide are widely used in the medical field as drug carriers due to their excellent properties. CaCO, in comparison with other inorganic materials 3 Nanoparticles have a variety of sizes, porosities, crystals and morphologies, and thus are of particular advantage as drug carriers. In addition, calcium carbonate has pH sensitivity, which is stable in a neutral environment, but generates certain structural micro-dissolution and destruction in a weak acid environment, and the pH of the tumor tissue micro-environment is lower than that of normal tissues, so that the drug carrier constructed based on the calcium carbonate has certain low pH responsiveness. However, calcium carbonate is easy to agglomerate in the conventional chemical synthesis, so that the particle size of the calcium carbonate exceeds the nano level, and the calcium carbonate is unfavorable for phagocytosis of cells. For synthesizing small-particle-size calcium carbonate particles, a surfactant or a polymer template is usually required, but polymer residues are remained on the generated calcium carbonate nanoparticles, so that the biocompatibility of the calcium carbonate nanoparticles is greatly reduced. Meanwhile, the addition of the surfactant or the polymer also easily leads to the denaturation and inactivation of the carried biomacromolecule, so that the research on a method capable of stably synthesizing the high-activity calcium carbonate nano-particles is urgent. The invention provides a method for preparing calcium carbonate nano particles with uniform particle size and high biological activityThe method is as follows.
With the intensive exploration of the mechanism of cell death, recent studies have demonstrated: there is another way of programmed death of cells, namely pyrosis (pyroptis). The occurrence of many diseases in humans, especially inflammatory diseases, has been reported to be closely linked to the apoptosis of normal cells. Although focal death is detrimental to normal cells and tissues, it is a potential cancer treatment strategy that can address the problem of natural or acquired anti-apoptosis of certain cancer cells if used in cancer treatment. Researchers found that the substrate GSDMD protein of inflammatory caspases is a key "killer protein" that mediates cell apoptosis. However, due to the drawbacks of easy degradation, instability, limited bioavailability, etc., the application of GSDMD proteins in the field of cancer treatment has only made limited progress, so it is urgent to construct a nano-delivery system capable of stabilizing GSDMD proteins.
Disclosure of Invention
Aiming at the defect that the calcium carbonate nano-drug is easy to agglomerate in the prior art, the invention provides a preparation method; the preparation method of the calcium carbonate nano-drug is simple, uniform in size and good in stability.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the invention provides a preparation method of a calcium carbonate nano-drug, which comprises the following steps:
mixing 1.0-5.0mg/mL (preferably 3 mg/mL) of calcium chloride aqueous solution with protein medicines (the amount is small, the mixed solution can be added in the form of aqueous solution for convenience of quantification), pouring the obtained mixed solution into a dialysis bag A, then filling the dialysis bag A into a dialysis bag B which is larger than the dialysis bag A, filling deionized water between the dialysis bag A and the dialysis bag B, sealing the two dialysis bags (keeping the deionized water in a cavity between the dialysis bag A and the dialysis bag B independently of the deionized water), placing the two dialysis bags into 0.1-1mg/mL of sodium carbonate aqueous solution (preferably 0.5 mg/mL), dialyzing for 8-12h (preferably 12 h) under stirring, and carrying out post-treatment on the obtained reaction solution in the dialysis bag A to obtain the calcium carbonate nano-medicine; the mass of the protein medicine is 5-20 mug/mL (preferably 5 mug/mL) based on the volume of the calcium chloride aqueous solution, and excessive amount can cause waste); the volume of the aqueous sodium carbonate solution is at least 40 times the volume of the aqueous calcium chloride solution.
Further, the post-treatment is as follows: and (3) putting the dialysis bag B into deionized water for continuous dialysis for 12-48h, changing the deionized water (free calcium chloride, sodium carbonate and GSDMD protein are completely removed) at least once every 2h, taking out the solution in the dialysis bag A, and freeze-drying at-80 ℃ for more than 24h to obtain the calcium carbonate nano-medicament.
Further, the protein medicine is added in the form of protein medicine water solution with the concentration of 50-200 mug/mL.
Preferably, the proteinaceous drug is a negatively charged protein, particularly preferably GSDMD protein (pyro-protein).
Preferably, the dialysis bag A or the dialysis bag B has a molecular weight cut-off that is greater than the molecular weight of the proteinaceous drug (facilitating subsequent removal of the non-nanoparticle-supported protein by dialysis).
Further preferably, the molecular weight cut-off of the dialysis bag A or the dialysis bag B is 100kDa, and the molecular weight of the protein medicine is 60-70kDa.
When the aqueous solution of calcium chloride is mixed with the protein medicines, the mixture can be stirred for 5 to 20 minutes at room temperature, so that negatively charged proteins are fully combined with positively charged calcium ions.
In order to maintain the osmotic pressure and the reaction rate, the volume of the aqueous sodium carbonate solution is at least 40 times that of the aqueous calcium chloride solution.
Compared with the prior art, the invention has the beneficial effects that:
when the single-layer dialysis bag is used for preparing the calcium carbonate particles, pasty large particles can obviously be generated on the inner wall of the dialysis bag, and the nano calcium carbonate particles cannot be obtained. The preparation method of the calcium carbonate nano-particles controls ion diffusion by using a double-layer dialysis bag, has the advantages of simple preparation method, uniform particle size, no use of surfactant or polymer and the like, and ensures the biocompatibility of the calcium carbonate nano-drug to the greatest extent. The smaller nanometer size facilitates the cell phagocytosis of the calcium carbonate nanometer medicine and provides more possibility for the development of the calcium carbonate nanometer medicine.
Drawings
FIG. 1 is a schematic diagram of a double-layer dialysis bag method for preparing nano calcium carbonate particles in examples 1 and 2;
FIG. 2 is a schematic diagram of a two-layer dialysis bag method for preparing calcium carbonate scorch nano-drug particles in examples 3 and 4;
FIG. 3 is a transmission electron microscopy image of calcium carbonate nanoparticles at 12h of dialysis of example 1;
FIG. 4 is a transmission electron microscopy image of calcium carbonate nanoparticles at 24h dialysis in example 2;
FIG. 5 is a transmission electron microscope image of bovine serum albumin-loaded calcium carbonate nano-drug of example 3;
FIG. 6 is a thermogravimetric analysis of bovine serum albumin-loaded calcium carbonate nano-drug of example 3;
FIG. 7 is an ultraviolet absorption diagram of bovine serum albumin-loaded calcium carbonate nano-drug of example 3.
Detailed Description
The technical scheme of the invention is described below through specific embodiments. It is to be understood that the mention of one or more method steps of the present invention does not exclude the presence of other method steps before and after the combination step or that other method steps may be interposed between these explicitly mentioned steps; the nanoparticles of the present invention may not only carry the proteins described herein, including but not limited to antibodies, enzymes, cytokines, etc., but biological macromolecules capable of carrying the protein structure are within the scope of the present invention. It should also be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
The yields in the examples below were calculated from the theoretical yield obtained when calcium chloride and sodium carbonate were fully reacted. The amount of calcium carbonate nanoparticles collected was used to obtain a value (small protein, not calculated) that was higher than the theoretical yield.
Example 1: the preparation of the nano calcium carbonate particles without carrying the medicine comprises the following steps.
(1) 60mg of calcium chloride is dissolved in 20mL of deionized water to prepare 3mg/mL of calcium chloride solution; 200mg of sodium carbonate was dissolved in 1L of deionized water to prepare a sodium carbonate solution of 0.2 mg/mL.
(2) First, a dialysis bag (us spec labs, cat 131417) having a cut-off molecular weight of 100kda, md24 was set in a dialysis bag (us spec labs, cat 131420) having a cut-off molecular weight of 100kda, md31, then 10mL of deionized water was added between the two dialysis bags to fill the space between the two dialysis bags, and finally the solution of calcium chloride was transferred to the inner dialysis bag. The double dialysis system was placed in the above prepared sodium carbonate solution for dialysis for 12 hours.
(3) To remove free calcium chloride, sodium carbonate, the crude product after dialysis in step (2) was placed in a 1L beaker filled with deionized water along with a dialysis bag for continued dialysis, during which water was changed every 2 hours for a total of 6 times. Unreacted ions are removed. After the dialysis is completed, 0.5mL of the solution is taken out from the inner dialysis bag, the nano particles are characterized by utilizing a transmission electron microscope, and the calcium carbonate nano particle solution in the dialysis bag is freeze-dried to finally obtain 2mg of calcium carbonate nano particles, wherein the yield is 3.3%.
As shown in figure 3, the nano calcium carbonate particles synthesized by the method of controlling ion diffusion by using the double-layer dialysis bag have uniform particle size and particle size of 100-200nm, which indicates that the nano calcium carbonate particles with uniform particle size and proper size can be obtained by controlling the method of controlling ion diffusion by using the double-layer dialysis bag and controlling the reaction time to be 12h.
Example 2: the preparation of the nano calcium carbonate particles without carrying the medicine comprises the following steps.
(1) 60mg of calcium chloride is dissolved in 20mL of deionized water to prepare 3mg/mL of calcium chloride solution; 200mg of sodium carbonate was dissolved in 1L of deionized water to prepare a sodium carbonate solution of 0.2 mg/mL.
(2) First, a dialysis bag (us spec labs, cat 131417) having a molecular weight cut-off of 100kda, md24 was set into a dialysis bag (us spec labs, cat 131420) having a molecular weight cut-off of 100kda, md31, then 10mL of deionized water 3 was added between the two dialysis bags, and finally the solution of calcium chloride was transferred to the inner dialysis bag. The double dialysis system was placed in the above prepared sodium carbonate solution for dialysis for 24 hours.
(3) To remove free calcium chloride, sodium carbonate, the crude product after dialysis in step (2) was continued to be dialyzed in deionized water, during which time water was exchanged every 2 hours for a total of 6 times. Sampling and shooting a transmission electron microscope. After the dialysis is completed, the calcium carbonate nanoparticle solution in the dialysis bag is freeze-dried, and finally 3mg of calcium carbonate nanoparticles are obtained, and the yield is 5.7%.
As shown in fig. 4, the synthesis of larger calcium carbonate particles occurred at 24 hours of reaction by controlling ion diffusion with a double-layered dialysis bag. In comparison with example 1, as the reaction time increases, small particles start to adsorb and accumulate to form large particles, so the control time is preferably about 12 hours.
Example 3: the preparation of a calcium-loaded bovine serum albumin (bovine serum albumin produced from Allatin) calcium carbonate medicament comprises the following steps (pre-experiments were performed using calcium carbonate loaded bovine serum albumin prior to loading with the pyro-protein because the pyro-protein has a similar molecular weight and similar structure to bovine serum albumin). The protein is subjected to fluorescent labeling by FITC, and the labeling process comprises the following steps: first, 2g of bovine serum albumin was dissolved in 10mL of deionized water, 10mg of FITC was dissolved in 1mL of deionized water, and then the FITC solution was slowly added to the bovine serum albumin solution and stirred for 8 hours in the dark. After the reaction was completed, the mixed solution was transferred to a dialysis bag having a molecular weight cut-off of 1W, and dialyzed in a beaker to remove free FITC, and dialyzed for 12 hours or more. The liquid in the beaker was taken for UV absorption testing, as no absorption peak at 495nm indicates that dialysis was complete.
(1) 60mg of calcium chloride is dissolved in 20mL of deionized water to prepare 3mg/mL of calcium chloride solution; 200mg of sodium carbonate is dissolved in 1L of deionized water to prepare 0.2mg/mL of sodium carbonate solution; 10mg of bovine serum albumin was dissolved in 10mL of deionized water to prepare a 1mg/mL bovine serum albumin solution. (2) 20mL of the calcium chloride solution prepared in (1) and 100. Mu.L of the bovine serum albumin solution were sequentially added to a 50mL round bottom flask, and then stirred at room temperature for 10min.
(3) First, a dialysis bag (U.S. specrum labs, cat. No. 131417) having a molecular weight cut-off of 100kDa, MD24 is set in a dialysis bag (U.S. specrum labs, cat. No. 131420) having a molecular weight cut-off of 100kDa, then 10mL of deionized water is added between the two dialysis bags, and finally 20mL of a mixed solution of bovine serum albumin and calcium chloride is transferred to the inner dialysis bag. The double dialysis system was placed in the above prepared sodium carbonate solution for dialysis for 12 hours.
(4) To remove free calcium chloride, sodium carbonate and bovine serum albumin, the crude product after dialysis in step (3) was continued to be dialyzed in deionized water, with 6 water exchanges every 2 hours. After the dialysis is completed, the calcium carbonate nanoparticle solution in the dialysis bag is freeze-dried, and finally 2.9mg of calcium carbonate nanoparticle loaded with bovine serum albumin is obtained, and the yield is 5.3%.
Example 4: the preparation of the calcium carbonate nano-drug loaded with the pyro-protein (the pyro-protein GSDMD is produced by Ai Bokang) comprises the following steps.
(1) 60mg of calcium chloride is dissolved in 20mL of deionized water to prepare 3mg/mL of calcium chloride solution; 200mg of sodium carbonate is dissolved in 1L of deionized water to prepare 0.2mg/mL of sodium carbonate solution; 1mg GSDMD protein was dissolved in 1mL deionized water to prepare a 1mg/mL GSDMD protein solution.
(2) 20mL of the calcium chloride solution prepared in (1) and 100. Mu.L of GSDMD protein solution were sequentially added to a 50mL round bottom flask, and then stirred at room temperature for 10min.
(3) First, a dialysis bag (US speclomlabs, cat. No. 131417) having a molecular weight cut-off of 100kDa, MD24 was set in a dialysis bag (US speclomlabs, cat. No. 131420) having a molecular weight cut-off of 100kDa, MD31, followed by adding 10mL of deionized water between the two dialysis bags, and finally transferring 20mL of a mixed solution of GSDMD protein and calcium chloride to an inner dialysis bag. The double dialysis system was placed in the above prepared sodium carbonate solution for dialysis for 12 hours.
(4) To remove free calcium chloride, sodium carbonate and GSDMD protein, the crude product after dialysis in step (3) was continued to be dialyzed in deionized water, with 6 water exchanges every 2 hours. After the dialysis is completed, the calcium carbonate nanoparticle solution in the dialysis bag is freeze-dried for 24 hours, and finally 2.7mg of the calcium carbonate nanoparticle loaded with GSDMD pyroprotein is obtained, and the yield is 4.9%. The yield is the mass of carbonic acid produced by adding 100% of calcium chloride in terms of the amount of nanoparticles collected.
The morphology of the nano particles formed by the calcium is changed, and perfect crystals and regular shapes are formed when the protein is not added. The morphology of the particles formed after the addition of the protein changes, and the insertion of protein molecules results in imperfect crystallization.
FIGS. 5 and 6 are transmission electron microscopy and thermogravimetric analysis diagrams of bovine serum albumin-loaded calcium carbonate nano-drug synthesized by a method of controlling ion diffusion using a double-layer dialysis bag; FIG. 7 is a graph of ultraviolet absorption of a fluorescence-labeled bovine serum albumin-loaded calcium carbonate nanomedicine having ultraviolet absorption at 495nm, demonstrating that fluorescence-labeled bovine serum albumin is loaded onto the nanomedicine. The method of combining nano calcium carbonate loaded medicines by using a double-layer dialysis bag method is proved to be feasible.
The invention uses the example to demonstrate that the mode of synthesizing the calcium carbonate nano-particles by controlling ion diffusion by a double-layer dialysis bag method is feasible, not only proves that the calcium carbonate nano-particles with high biological activity can be synthesized, but also proves that the method can be used for well synthesizing the calcium carbonate nano-particles loaded with medicines.
Claims (10)
1. The preparation method of the calcium carbonate nano-drug is characterized by comprising the following steps:
uniformly mixing 1.0-5.0mg/mL of calcium chloride aqueous solution and protein medicines, pouring the obtained mixed solution into a dialysis bag A, then filling the dialysis bag A into a dialysis bag B which is larger than the dialysis bag A, filling deionized water between the dialysis bag A and the dialysis bag B, sealing the two dialysis bags, placing the two dialysis bags into 0.1-1mg/mL of sodium carbonate aqueous solution, dialyzing 8-12h under stirring, and performing post-treatment on the reaction solution obtained in the dialysis bag A to obtain the calcium carbonate nano medicine; the mass of the protein medicine is 5-20 mug/mL calculated by the volume of the calcium chloride aqueous solution; the volume of the sodium carbonate aqueous solution is at least 40 times that of the calcium chloride aqueous solution; the protein medicine is added in the form of protein medicine water solution; the protein medicine is negatively charged protein with molecular weight of 60-70kDa.
2. The method for preparing the calcium carbonate nano-drug according to claim 1, wherein the post-treatment is as follows: and (3) putting the dialysis bag B into deionized water for continuing dialysis for 12-48h, changing the deionized water at least once every 2h, taking out the solution in the dialysis bag A, and freeze-drying at the temperature of-80 ℃ for more than 24h to obtain the calcium carbonate nano-medicament.
3. The method for preparing the calcium carbonate nano-drug according to claim 1, wherein: the concentration of the protein medicine aqueous solution is 50-200 mug/mL.
4. The method for preparing the calcium carbonate nano-drug according to claim 1, wherein: the protein medicine is GSDMD protein.
5. The method for preparing the calcium carbonate nano-drug according to claim 1, wherein: the molecular weight cut-off of the dialysis bag A or the dialysis bag B is larger than the molecular weight of the protein medicine.
6. The method for preparing the calcium carbonate nano-drug according to claim 5, wherein: the molecular weight cut-off of the dialysis bag A or the dialysis bag B is 100 kDa.
7. The method for preparing the calcium carbonate nano-drug according to claim 1, wherein: the concentration of the calcium chloride aqueous solution was 3 mg/mL.
8. The method for preparing the calcium carbonate nano-drug according to claim 1, wherein: the concentration of the sodium carbonate aqueous solution was 0.5 mg/mL.
9. The method for preparing the calcium carbonate nano-drug according to claim 1, wherein: the mass of the protein medicine is 5 mug/mL calculated by the volume of the calcium chloride aqueous solution.
10. The method for preparing the calcium carbonate nano-drug according to claim 1, wherein: the dialysis time was 12h.
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| Synthesis of Precipitated Calcium Carbonate Nanoparticles Using a Two-Membrane System;Zeshan Hu等;Colloid Journal;第66卷(第6期);第829–834页 * |
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