CN110898034A - Serum albumin nanoparticle loaded with anti-tumor active drug and preparation method thereof - Google Patents
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Abstract
The invention relates to a serum albumin nanoparticle loaded with an anti-tumor active drug and a preparation method thereof, wherein the preparation method specifically comprises the following steps: (1) weighing shikonin oxime derivative DMAKO-201-3 parts, and dissolving in an organic solvent to obtain an organic phase; (2) weighing 7-9 parts of serum albumin, and dissolving in deionized water to obtain a water phase; (3) adding the organic phase into the water phase under the stirring action, and stirring to obtain an emulsion; (4) then homogenizing the emulsion under high pressure to obtain a homogenized product; (5) and then separating the obtained homogeneous product, adding 0-1600 parts of freeze-drying protective agent into the obtained supernatant, and continuously freezing and drying to obtain the target product. Compared with the prior art, the invention effectively improves the solubility and stability of the alkannin oxime derivative in aqueous solution, can realize the passive targeting drug delivery of tumor parts by utilizing EPR effect, and has safe and reliable whole preparation process, simple and controllable working procedures and the like.
Description
Technical Field
The invention belongs to the technical field of nano-drugs, and relates to a serum albumin nanoparticle loaded with an anti-tumor active drug and a preparation method thereof.
Background
Lithospermum erythrorhizon is a perennial herb of Boraginaceae, and is derived from dried root of Sinkiang Lithospermum erythrorhizon (Arnebia euchroma Johnst) or Lithospermum erythrorhizon (Arnebia guttata Bunge) according to pharmacopoeia of the people's republic of China (2015 edition). The traditional Chinese medicine considers that the lithospermum has bitter taste and cold property, has the effects of cooling blood, activating blood, detoxifying, promoting eruption and the like, and can be used for treating pyocutaneous disease, stranguria with turbid urine, heat disease and the like for a long time. Modern medical research shows that a plurality of chemical components in the lithospermum have various biological activities of resisting inflammation, promoting wound healing, resisting bacteria, viruses, thrombus and hyperthyroidism, enhancing the immune function, reducing blood sugar, protecting liver, resisting tumors and the like.
Shikonin and its derivatives are the main effective components of arnebia euchroma (Royle) Johnst, and their antitumor effects have been confirmed in a large number of studies. The action mechanism mainly comprises the ways of inducing cell necrosis and apoptosis, inhibiting topoisomerase, inhibiting protein tyrosine kinase, resisting tumor angiogenesis, influencing tumor cell signal transmission and the like. For example, Chinese patents ZL201310044118, ZL201310044877 and the like disclose a series of purpurin oxime derivatives obtained by oxygen alkylation carbonyl oximation of a naphthazarin parent nucleus, the compounds show very strong antitumor activity in vitro but hardly affect the growth of normal cells of a human body, and DMAKO-20 is a representative compound in the alkannin oxime derivatives, and the chemical structural formula of the DMAKO-20 is shown as follows:
because the compound has poor water solubility, the solubility of the compound influences the application of the compound as an antitumor drug.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide the serum albumin nanoparticle loaded with the anti-tumor active drug and the preparation method thereof.
The purpose of the invention can be realized by the following technical scheme:
one of the technical schemes of the invention is to provide a serum albumin nanoparticle loaded with an anti-tumor active drug, which comprises the following components in parts by weight: 1-3 parts of alkannin oxime derivative, 7-9 parts of serum albumin and 0-1600 parts of freeze-drying protective agent.
Further, the serum albumin is HSA or BSA.
Further, the freeze-drying protective agent is one or more of lactose, glucose, mannitol, trehalose or sucrose.
Further, the alkannin oxime derivative is DMAKO-20.
The second technical scheme of the invention is to provide a preparation method of serum albumin nanoparticles loaded with anti-tumor active drugs, which comprises the following steps:
(1) weighing shikonin oxime derivative DMAKO-201-3 parts, and dissolving in an organic solvent to obtain an organic phase;
(2) weighing 7-9 parts of serum albumin, and dissolving in deionized water to obtain a water phase;
(3) adding the organic phase into the water phase under the stirring action, and stirring to obtain an emulsion;
(4) then homogenizing the emulsion under high pressure to obtain a homogenized product;
(5) and then separating the obtained homogeneous product, adding 0-1600 parts of freeze-drying protective agent (wherein, when the addition amount of the freeze-drying protective agent is 0 part, the freeze-drying protective agent is not added, the supernatant is directly subjected to freeze drying), and continuously performing freeze drying to obtain the target product.
Further, the serum albumin is HSA or BSA.
Further, the freeze-drying protective agent is one or more of lactose, glucose, mannitol, trehalose or sucrose.
Further, the alkannin oxime derivative is DMAKO-20.
Further, the organic solvent is chloroform.
Further, in the step (3), the mixing volume ratio of the organic phase and the water phase is 1-4: 80. Namely, the volume of the organic phase added into 80mL of aqueous phase is designed to be different from 1 to 4mL, and the target nanoparticles can be successfully prepared within the volume change range.
Further, in the step (4), the high-pressure homogenization process specifically comprises: homogenizing under 750bar pressure for 8min, and homogenizing under 1000bar pressure for 3 min.
Further, in the step (5), the separation process is as follows: centrifuging at 2000rpm below 4 deg.C for 30 min.
Further, the freeze drying process specifically comprises: completely freezing at-20 deg.C, and freeze drying at-70 deg.C.
Further, in the step (5), when the addition amount of the lyoprotectant is not 0, the total concentration of the lyoprotectant in the supernatant is up to 100 g/L.
The invention utilizes serum albumin such as HSA, BSA and the like as carriers, and the alkannin oxime derivative is wrapped in HSA nanoparticles in a molecular form, so that the solubility and the stability of the alkannin oxime derivative in an aqueous solution can be effectively improved. In addition, the alkannin oxime derivative can realize passive targeting drug delivery of a tumor part along with an HSA nano carrier because the tumor tissue has high permeability and retention (EPR) effect.
The invention is different from the traditional method of curing serum albumin by using cross-linking agent glutaraldehyde and the like in the compounding preparation process of each component, innovatively cures the serum albumin by using high-pressure homogenization technology, since the HSA structure is composed of 685 amino acid residues, containing 17 disulfide bonds and 1 free thiol group (similarly, Bovine Serum Albumin (BSA) is composed of 581 amino acid residues, containing 17 disulfide bonds and 1 free thiol group), under the high shear force in the high pressure homogenization process, cavitation is generated in the liquid to cause local hyperthermia, superoxide ions which can cross-link polymers are generated, sulfhydryl residues in serum albumin are oxidized or existing disulfide bonds in/among serum albumin molecules are broken, new disulfide bonds are formed by the internal/internal cross-linking of the serum albumin, thereby forming a cross-linked polymer shell around the water-insoluble shikonin oxime derivative. The method is simple, convenient and controllable, reduces the introduction of cross-linking agents, avoids that the traditional cross-linking agents such as glutaraldehyde and the like can release aldehyde residues in organisms and have certain toxic and side effects on the organisms, and improves the safety of the preparation to a certain extent.
Compared with the prior art, the invention has the following advantages:
(1) the preparation process of the serum albumin nanoparticles is improved, the reagent use types are reduced, the preparation safety is improved, and the preparation process is simple and controllable.
(2) Develops a new formulation of the alkannin oxime derivative, effectively improves the solubility and the stability of the alkannin oxime derivative in aqueous solution, and can realize the passive targeted drug delivery of tumor parts by utilizing EPR effect.
Detailed Description
The present invention will be described in detail with reference to specific examples. The present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and a specific operation process are given, but the scope of the present invention is not limited to the following embodiments.
In the following examples, the specific sources of the reagents are as follows:
| reagent | Purity of | Manufacturer of the product |
| BSA(Fraktion V) | Biotechnology Grade | Xibao Biotechnology (Shanghai) Ltd |
| Human serum albumin (component five) | 96-99％ | Shanghai Mielin Biochemical technology Ltd |
| Trichloromethane | Analytical purity | Chemical reagents of national drug group Co Ltd |
| α -lactose monohydrate | Analytical purity | Chemical reagents of national drug group Co Ltd |
| Anhydrous glucose | Analytical purity | Shanghai Lingfeng Chemicals Co., Ltd |
| Mannitol | Analytical purity | Chemical reagents of national drug group Co Ltd |
| D (+) -anhydrous trehalose | 99％ | Chemical reagents of national drug group Co Ltd |
| Sucrose | Analytical purity | Chemical reagents of national drug group Co Ltd |
| Sodium alginate | Analytical purity | Shanghai Aladdin Biotechnology Ltd |
In addition, sources of alkannin oxime derivatives were prepared with reference to the following references: zhang X, Cui J, Zhou W, equivalent. design, synthesis and anticipator activity of shikonin and alkannin derivatives with differential subsistents on the naphthazarin scaffold [ J ]. Chemical Research in Chinese Universities,2015,31(3):394- "400.
And the rest of the raw material reagents or treatment techniques which are not specifically described are conventional commercial raw materials or conventional treatment techniques in the field.
Example 1:
preparation of BSA nanoparticles
1. Measuring 4mL of trichloromethane as an organic phase;
2. weighing 45mg of BSA, and dissolving in 80mL of deionized water to obtain a water phase;
3. rapidly adding the organic phase into the water phase under magnetic stirring, and stirring at 570rpm for 5 min;
4. homogenizing the emulsion with a high pressure homogenizer at 750bar for 8min, homogenizing at 1000bar for 3min, and collecting the product;
5. centrifuging the product at 4 deg.C and 2000rpm for 30min, collecting supernatant, completely freezing at-20 deg.C, and freeze drying at-70 deg.C to obtain white flocculent lyophilized powder.
Example 2:
preparation of alkannin oxime derivative DMAKO-20-loaded BSA nanoparticles
1. Weighing 5mg of shikonin oxime derivative DMAKO-20, dissolving in 4mL of chloroform to obtain an organic phase;
2. weighing 45mg of BSA, and dissolving in 80mL of deionized water to obtain a water phase;
3. rapidly adding the organic phase into the water phase under magnetic stirring, and stirring at 570rpm for 5 min;
4. homogenizing the emulsion with a high pressure homogenizer at 750bar for 8min, homogenizing at 1000bar for 3min, and collecting the product;
5. centrifuging the product at 4 deg.C and 2000rpm for 30min, collecting supernatant, completely freezing at-20 deg.C, and freeze-drying at-70 deg.C to obtain yellowish flocculent lyophilized powder.
Table 1 single factor study of drug loading of BSA nanoparticles loaded with alkannin oxime derivatives
P <0.05, higher than the factor investigation level I
Table 2 orthodox test table L for loading alkannin oxime derivative BSA nanoparticle loading dose9(34)
Tables 1 and 2 show that the factors and the optimal experimental conditions which have the greatest influence on the target nanoparticle drug loading are screened out by fixing other experimental variables and only changing the target investigation factor experiment and the orthogonal experiment based on the target investigation factor experiment.
Single factor review with respect to table 1. The first observation factor shows that in the set mass ratio of the three alkannin oxime derivatives to BSA, the ratio of 10:40 can ensure that the drug loading is optimal, namely, the feeding amount of the alkannin oxime derivative is not too high or too low. The second factor shows that the volume of the organic phase should not be too large, but the drug loading is higher when the volume is smaller, and the drug loading is not different greatly when the volume is smaller. The third observation factor shows that the rotating speed in the stirring and emulsifying process has little influence on the drug loading capacity of the nanoparticles (no significant difference among three groups), but after low-temperature centrifugation for 30min, the rotating speed is 840rpm, and the group has the least precipitation amount, so that the rotating speed of 840rpm is adopted in subsequent orthogonal experiments and finally determined experimental schemes. The fourth factor is considered to indicate that the mode of adding the organic phase into the stirring water phase can obviously influence the drug loading of the nanoparticles, so that the direct addition (i.e. pouring) is optimal. And a fifth observation factor indicates that the stirring and emulsifying time can obviously influence the drug loading of the nanoparticles, and the longer the emulsifying time is, the higher the drug loading is.
The orthogonal experiment of table 2 is used to finally determine experimental conditions by mutual combination between levels after a single factor investigation experiment screens out factors having a significant influence on a target result. Experimental grouping was designed according to the existing four-factor three-level orthogonal table and experimental results were shown in table 2. Wherein each of the following KIThe sum of the results of the experiments for level I intended for this list, i.e. the factors examined, KII、KIIIAnd vice versa. Extreme difference R (R ═ K)max-Kmin) The strength of the influence of each influence factor on the experimental result is reflected by the size from left to rightThe following range differences are respectively 24.91, 9.07, 10.08 and 18.26, which show that the influence of various factors on the drug loading capacity of the nanoparticles is respectively the mass ratio of the alkannin oxime derivative to BSA, the stirring time, the mode of adding the organic phase into the aqueous phase and the volume of the organic phase from large to small. The combination of the maximum K value levels under all the influencing factors is the finally determined optimal method for preparing the nanoparticles.
Example 3
The preparation method of the alkannin oxime derivative-loaded BSA nanoparticle comprises the following steps:
1. weighing 15mg of alkannin oxime derivative, and dissolving in 4mL of trichloromethane to obtain an organic phase;
2. weighing 35mg of BSA, and dissolving in 80mL of deionized water to obtain a water phase;
3. rapidly adding the organic phase into the water phase under magnetic stirring, and stirring at 840rpm for 5 h;
4. homogenizing the emulsion with a high pressure homogenizer at 750bar for 8min, homogenizing at 1000bar for 3min, and collecting the product;
5. centrifuging the product at 4 deg.C and 2000rpm for 30min, collecting supernatant, completely freezing at-20 deg.C, and freeze-drying at-70 deg.C to obtain yellowish flocculent lyophilized powder. The drug loading was determined to be 17.60 ± 1.74% using High Performance Liquid Chromatography (HPLC).
In the above embodiment, HAS may be replaced with an equal amount of BSA.
Example 4:
preparation of HSA nano-particle loaded with alkannin oxime derivative
1. Weighing 15mg of alkannin oxime derivative, and dissolving in 4mL of trichloromethane to obtain an organic phase;
2. weighing 35mg of HSA, and dissolving in 80mL of deionized water to obtain a water phase;
3. rapidly adding the organic phase into the water phase under magnetic stirring, and stirring at 840rpm for 5 h;
4. homogenizing the emulsion with a high pressure homogenizer at 750bar for 8min, homogenizing at 1000bar for 3min, and collecting the product;
5. centrifuging the product at 4 deg.C and 2000rpm for 30min, collecting supernatant, completely freezing at-20 deg.C, and freeze-drying at-70 deg.C to obtain yellowish flocculent lyophilized powder. Drug loading was determined to be 26.56 ± 0.30% using HPLC method. The particle size of the redissolved lyophilized powder is 648.9 + -19.2 nm and the Polydispersity (PDI) is 0.439 + -0.037 measured by Malvern laser particle sizer.
Example 5:
preparation of HSA (human serum albumin) nanoparticle loaded with alkannin oxime derivative and containing freeze-drying protective agent
1. Weighing 15mg of alkannin oxime derivative, and dissolving in 4mL of trichloromethane to obtain an organic phase;
2. weighing 35mg of HSA, and dissolving in 80mL of deionized water to obtain a water phase;
3. rapidly adding the organic phase into the water phase under magnetic stirring, and stirring at 840rpm for 5 h;
4. homogenizing the emulsion with a high pressure homogenizer at 750bar for 8min, homogenizing at 1000bar for 3min, and collecting the product;
5. centrifuging the product at 2000rpm at 4 deg.C for 30min, collecting supernatant, dividing into multiple groups, and adding different kinds of lyophilized protectant respectively, i.e. dissolving sodium alginate, lactose, glucose, mannitol, trehalose, sucrose or trehalose-mannitol 1:1(w/w)) in the supernatant respectively, and except that the concentration of sodium alginate in the supernatant is 0.5g/L, the concentration of the rest sucrose and other kinds of lyophilized protectant in the supernatant is 100 g/L.
Then, after completely freezing at-20 ℃, freeze-drying at-70 ℃ to obtain light yellow freeze-dried powder. The redissolution particle size of the lyophilized powder was 278.7 + -9.9 nm and the Polydispersity (PDI) was 0.430 + -0.054 as measured by a Malvern laser particle sizer.
TABLE 3 influence of lyoprotectant type on particle size and solubility of lyophilized powder
The type of the freeze-drying protective agent influences the dispersibility of the freeze-dried and re-dissolved nanoparticles, and it can be seen that the sugar freeze-drying protective agents except sodium alginate have large particle size difference after freeze-drying under the same mass concentration, wherein the optimal, namely the smallest particle size is trehalose, glucose, sucrose and trehalose-mannitol 1:1 (w/w); even at a very low dosage, sodium alginate still cannot reduce or even aggravate particle aggregation of the lyophilized powder after reconstitution. Another screening index is the re-dissolving capacity of the nanoparticle freeze-dried powder at high concentration, namely observing whether the nanoparticles can be completely re-dissolved (no precipitate is generated) under the condition that the re-dissolving is a certain reduced alkannin oxime derivative concentration. The final results may indicate that sucrose as a lyoprotectant can tolerate reconstitution to higher concentrations in comparison.
Example 6
Preparation of HSA (human serum albumin) nanoparticle loaded with alkannin oxime derivative and containing freeze-drying protective agent
1. Weighing 15mg of alkannin oxime derivative, and dissolving in 4mL of trichloromethane to obtain an organic phase;
2. weighing 35mg of HSA, and dissolving in 80mL of deionized water to obtain a water phase;
3. rapidly adding the organic phase into the water phase under magnetic stirring, and stirring at 840rpm for 5 h;
4. homogenizing the emulsion with a high pressure homogenizer at 750bar for 8min, homogenizing at 1000bar for 3min, and collecting the product;
5. the product was centrifuged at 2000rpm for 30min at 4 ℃ and the supernatant was collected. Sucrose was added and dissolved in the supernatant to give a concentration of 25 g/L. Completely freezing at-20 deg.C, and freeze drying at-70 deg.C to obtain yellowish lyophilized powder. Drug loading was 1.02 ± 0.09% using HPLC method. The particle size of the lyophilized powder is 316.0 + -4.4 nm, and the Polydispersity (PDI) is 0.325 + -0.042.
TABLE 4 influence of lyoprotectant content on lyophilized powder particle size
| Sucrose concentration (g/L) before lyophilization | Particle size (nm) | PDI |
| 100 | 282.7±11.7 | 0.313±0.028 |
| 75 | 369.2±19.6 | 0.395±0.054 |
| 50 | 299.8±3.7 | 0.309±0.039 |
| 25 | 316.0±4.4 | 0.325±0.042 |
| 10 | 366.7±11.4 | 0.357±0.060 |
| 5 | 391.1±19.6 | 0.374±0.036 |
| 1 | 437.6±6.0 | 0.475±0.052 |
| 0 | 531.7±59.2 | 0.528±0.119 |
The result shows that the particle size of the nanoparticles after freeze-drying and redissolving is increased along with the reduction of the sucrose dosage, the effect of maintaining the smaller particle size after freeze-drying and redissolving cannot be well achieved by excessively low dosage, but the serious moisture absorption effect of the freeze-drying powder is caused by excessively high sucrose dosage, and the freeze-drying powder is difficult to store.
Example 7
HSA nanoparticle stability acceleration experiment of shikonin oxime derivative loaded with freeze-drying protective agent
3 batches of HSA nanoparticle freeze-dried powder loaded with alkannin oxime derivatives and without a freeze-drying protective agent and with a freeze-drying protective agent of sucrose (the concentration is 25g/L before freeze-drying) and sodium alginate (the concentration is 0.5g/L before freeze-drying) are respectively taken, the HSA nanoparticle freeze-dried powder is packaged at the temperature of 30 +/-2 ℃ according to the market and stored for 6 months under the condition of relative humidity of 65 +/-5 percent, samples are respectively taken once at 0, 1, 2, 3 and 6 months, and the change of drug loading (by using an HPLC method) and the change of the redissolved particle size of the freeze-dried powder (by using a Markov laser.
TABLE 5 acceleration of the drug loading of lyophilized powders
| Time (moon) | Non-freeze-drying protective agent | Sucrose | Sodium alginate |
| 0 | 26.56±0.30％ | 1.02±0.09％ | 10.56±0.08％ |
| 1 | 26.02±0.14％ | 1.00±0.12％ | 10.38±0.12％ |
| 2 | 25.69±0.37％ | 1.01±0.05％ | 10.11±0.09％ |
| 3 | 25.22±0.20％ | 0.97±0.10％ | 9.99±0.16％ |
| 6 | 24.27±0.58％ | 0.94±0.09％ | 9.72±0.12％ |
TABLE 6 acceleration of particle size of lyophilized powder
Table 5 accelerated experiment results of the drug loading of the lyophilized powder are not much related to the usage of the lyoprotectant, but it can be seen that the drug loading of the nanoparticle lyophilized powder is stable within 6 months and the decrease range is very small no matter whether the lyoprotectant is added or not.
Table 6 accelerated particle size test results of lyophilized powders are much related to the use of lyophilized powders. The sodium alginate group may not be considered. The particle size of the group without the lyoprotectant is larger than that of the group with the sucrose after the lyophilization and the reconstitution, and the particle size of the group without the lyoprotectant is always larger than that of the group with the sucrose in the experimental period of 6 months.
Comparative example 1:
preparing HSA nano-particles of the alkannin oxime derivative loaded with the freeze-drying protective agent by a desolvation crosslinking method:
1. weighing 15mg of alkannin oxime derivative, and dissolving in 60mL of ethanol;
2. weighing 35mg of HSA, and dissolving in 10mL of deionized water;
3. slowly dropwise adding the ethanol solution into the aqueous solution under magnetic stirring, stirring at 840rpm for 30min, adding 3 mu L of 25% glutaraldehyde solution, and curing at room temperature for 12 h;
4. the ethanol was removed by rotary evaporation at 40 ℃ and the product was centrifuged at 2000rpm for 30min at 4 ℃ and the supernatant was collected. Sucrose was added and dissolved in the supernatant to give a concentration of 25 g/L. Completely freezing at-20 deg.C, and freeze drying at-70 deg.C to obtain yellowish lyophilized powder. Drug loading was determined to be 0.68. + -. 0.12% using HPLC. The particle size of the lyophilized powder redissolved by Malvern laser particle size analyzer is 368.0 + -10.2 nm, and PDI is 0.288 + -0.029.
The comparison between the drug loading rate and the freeze-drying reconstitution particle size of example 6 and comparative example 1 shows that the two have no obvious particle size difference, but the drug loading rate of comparative example 1 is obviously reduced compared with that of example 6, and the importance and the necessity of the high-pressure homogenization technology defined by the invention can be proved.
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Claims (9)
1. A serum albumin nanoparticle loaded with an anti-tumor active drug is characterized by comprising the following components in parts by weight: 1-3 parts of alkannin oxime derivative, 7-9 parts of serum albumin and 0-1600 parts of freeze-drying protective agent.
2. The serum albumin nanoparticle loaded with anti-tumor active drugs according to claim 1, wherein the serum albumin is HSA or BSA.
3. The serum albumin nanoparticle loaded with anti-tumor active drugs according to claim 1, wherein the lyoprotectant is one or more of lactose, glucose, mannitol, trehalose, or sucrose.
4. The serum albumin nanoparticle loaded with anti-tumor active drugs according to claim 1, wherein the alkannin oxime derivative is DMAKO-20.
5. A preparation method of serum albumin nanoparticles loaded with anti-tumor active drugs is characterized by comprising the following steps:
(1) weighing 1-3 parts of alkannin oxime derivative, and dissolving in an organic solvent to obtain an organic phase;
(2) weighing 7-9 parts of serum albumin, and dissolving in deionized water to obtain a water phase;
(3) adding the organic phase into the water phase under the stirring action, and stirring to obtain an emulsion;
(4) then homogenizing the emulsion under high pressure to obtain a homogenized product;
(5) and then separating the obtained homogeneous product, adding 0-1600 parts of freeze-drying protective agent into the obtained supernatant, and continuously freezing and drying to obtain the target product.
6. The method of claim 5, wherein the serum albumin nanoparticle is HSA or BSA;
the freeze-drying protective agent is one or more of lactose, glucose, mannitol, trehalose or sucrose;
the alkannin oxime derivative is DMAKO-20;
the organic solvent is trichloromethane.
7. The method for preparing serum albumin nanoparticles loaded with anti-tumor active drugs according to claim 5, wherein in the step (3), the mixing volume ratio of the organic phase to the aqueous phase is 1-4: 80.
8. The method for preparing the serum albumin nanoparticles loaded with the anti-tumor active drug according to claim 5, wherein in the step (4), the high-pressure homogenization process specifically comprises: homogenizing under 750bar pressure for 8min, and homogenizing under 1000bar pressure for 3 min.
9. The method for preparing serum albumin nanoparticles loaded with anti-tumor active drugs according to claim 5, wherein in the step (5), the separation process comprises: centrifuging at 2000rpm below 4 deg.C for 30 min;
the freeze drying process specifically comprises the following steps: completely freezing at-20 deg.C, and freeze drying at-70 deg.C.
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| CN106943379A (en) * | 2017-05-12 | 2017-07-14 | 辽宁大学 | A kind of gambogicacid albumin nano granular and preparation method thereof |
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| CN111449055A (en) * | 2020-05-23 | 2020-07-28 | 河北康腾生物科技有限公司 | Adipose tissue cryopreservation liquid, preparation method and adipose tissue storage method |
| CN111449055B (en) * | 2020-05-23 | 2021-07-23 | 河北康腾生物科技有限公司 | Adipose tissue cryopreservation liquid, preparation method and adipose tissue storage method |
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