Preparation method and application of a new drug carrier

Abstract

The invention discloses a preparation method and application of a novel drug carrier, wherein the preparation method inoculates Lactobacillus rhamnosus in a liquid culture medium provided with a calcium source by crustacean waste for cultivation, centrifuges to obtain a precipitate and a supernatant, treats the precipitate with hydrochloric acid and centrifuges to obtain a supernatant and a precipitate, inoculates Bacillus subtilis in a culture medium containing the acid-treated precipitate and cultivates the precipitate and centrifuges to obtain a precipitate and a supernatant, treats the precipitate with NaOH, centrifuges to obtain a supernatant and a precipitate, mixes the above-mentioned supernatants and inoculates Bacillus velez, collects the precipitate after the cultivation, and washes and dries to obtain a novel drug carrier. The drug carrier has excellent drug loading capacity for positively charged drugs; has good buffering capacity for acidic environments, can slowly release antibiotics as a drug carrier in an environment with a pH value of less than or equal to 6, has excellent sustained-release properties, helps to reduce the toxic and side effects of the human body, and the preparation method is economical and environmentally friendly.

Description

Technical Field

[0001] The invention relates to the preparation of a drug carrier, and in particular to a preparation method and application of spherical calcium carbonate as a novel drug carrier. Background Art

[0002] Drug carriers are an important component of the drug release system in the clinical treatment of diseases and are also the main factor affecting drug efficacy. Traditional treatment methods can only carry drugs to the lesions. Improving the targeting, selectivity, sustained release and controllability of drugs is a key issue in current clinical treatment. Compared with free drug administration treatment methods, drug controlled release systems have a series of advantages. They can effectively control the drug release rate, have little toxicity and side effects on normal cells, can reduce the dosage and frequency of administration, thereby improving the bioavailability of drugs and giving full play to the efficacy of drugs. Therefore, in recent years, the key to drug controlled release carriers is to regulate the release rate of drugs in drug controlled release systems. At present, the materials used for drug controlled release carriers are divided into natural polymer materials, semi-synthetic polymer materials, synthetic polymer materials, etc. according to their sources. However, the above materials have problems such as low drug loading and encapsulation rate, easy hydrolysis of the complex, complex synthesis process and high synthesis cost, which limit their application in the field of drug carriers.

[0003] There are many reports on calcium carbonate synthesized by non-living organisms as drug carriers, but the drug loading capacity is not high and the stability is not strong. With the rapid development of medical level, the invention of a new drug-carrying material with low cost, high drug loading capacity and strong stability has positive significance for the clinical treatment of diseases. At present, there is research on vaterite synthesized by microorganisms. Due to its certain content of organic matter, its adsorption and stability are greatly improved, but at present, it is only explored as a heavy metal ion adsorbent for the treatment of environmental heavy metal pollution, and there is no report on it as a drug carrier. Summary of the invention

[0004] Purpose of the invention: In view of the problems existing in the prior art, the present invention provides a method for preparing a novel drug carrier. The present invention uses crustacean waste as a calcium source to prepare a novel drug sustained-release carrier, which effectively solves the problems of low drug loading, weak stability, and poor sustained-release effect.

[0005] The present invention also provides application of the novel drug carrier.

[0006] Technical solution: In order to achieve the above-mentioned purpose, the preparation method of a novel drug carrier described in the present invention comprises the following steps: inoculating Lactobacillus rhamnosus in a liquid culture medium provided with a calcium source by crustacean waste for culturing, centrifuging to obtain a precipitate A and a supernatant A1, treating the precipitate A with hydrochloric acid, centrifuging after no bubbles are generated to obtain a supernatant B1 and a precipitate B, inoculating Bacillus subtilis in a fermentation culture medium containing the precipitate B for culturing, centrifuging to obtain a precipitate C and a supernatant C1, treating the precipitate C with NaOH, centrifuging after no bubbles are generated to obtain a supernatant D1 and a precipitate D, mixing the supernatants A1, B1, C1 and D1, inoculating Bacillus Velez for culturing, and after the cultivation is completed, centrifuging to collect the precipitate, washing and drying to obtain the novel drug carrier.

[0007] Wherein, the liquid culture medium with a calcium source provided by crustacean waste is an MRS liquid culture medium with a calcium source provided by crustacean waste.

[0008] Wherein, the Lactobacillus rhamnosus, Bacillus subtilis and Bacillus velez are cultured to a bacterial solution concentration of 7-9×10 8 cfu / mL, and inoculated into the culture medium at a volume ratio of 2-5%.

[0009] Wherein, the hydrochloric acid and NaOH are both low-concentration solutions, the hydrochloric acid does not exceed 0.5 mol / L, and the NaOH does not exceed 1 mol / L.

[0010] Among them, after inoculation, Lactobacillus rhamnosus is cultured at 30-37°C for 2-6 days; after inoculation, Bacillus subtilis is cultured at 30-37°C for 2-4 days; after inoculation, Bacillus velezii is cultured at 30-37°C for 4-7 days.

[0011] Among them, Lactobacillus rhamnosus is CCTCC DB 20082412; Bacillus subtilis is CCTCC AB209260, and Bacillus velez is CGMCC No.1.12669.

[0012] A novel drug carrier prepared by the preparation method of the present invention.

[0013] The surface of the drug carrier carries organic functional groups such as hydroxyl, amino and carboxyl groups.

[0014] The application of the novel drug carrier of the present invention in the preparation of a novel drug sustained-release and controlled-release carrier.

[0015] Among them, the loaded drug should have the characteristic of being positively charged; the new drug sustained-release carrier has a sustained-release effect in a weakly acidic environment with a pH value of less than or equal to 6.

[0016] The present invention also provides a drug carrier loaded with antibiotics, comprising the biogenic spherical calcium carbonate prepared using crustacean waste as a calcium source.

[0017] The novel drug carrier of the present invention utilizes Lactobacillus rhamnosus to release calcium ions in crustacean waste, then utilizes Bacillus subtilis to degrade organic matter such as protein therein, and finally utilizes Bacillus velez to induce synthesis of a spherical calcium carbonate organic complex having porous lamination characteristics. The present invention uses biological and chemical combined treatment, and bacteria are used as the leading part of decalcification and deproteinization processes in the synthesis process, thereby effectively reducing the subsequent use amount of acid-base solvents. Meanwhile, the prepared product has better effect than the previous pure chemical treatment, and the drug loading amount is significantly increased. The spherical calcium carbonate has excellent drug loading capacity for positively charged drugs, for example, the maximum adsorption amount of anticancer drug doxorubicin hydrochloride (DOX) is as high as 447.579 mg / g; it has good buffering capacity for acidic environments, and as a drug carrier, it can slowly release antibiotics in an environment with a pH value of less than or equal to 6, has excellent sustained-release characteristics, helps to reduce toxic and side effects of human body, and the preparation method is economical and environmentally friendly.

[0018] The spherical calcium carbonate prepared in the present invention has good biocompatibility, large drug loading, and good sustained release effect. In addition to inducing the synthesis of the spherical calcium carbonate organic complex with porous lamination characteristics by using Bacillus Velezii, the secondary bacterial treatment in the early stage is very important. Compared with the decalcification and deproteinization by pure chemical methods, the spherical calcium carbonate prepared after the specific biological treatment of the present invention significantly increases the drug loading. At the same time, the present invention effectively treats crustacean waste, killing two birds with one stone.

[0019] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0020] 1. The novel drug sustained-release carrier prepared by the present invention is a biogenic spherical calcium carbonate synthesized with crustacean waste as a calcium source, that is, calcium salts and nutrients in the crustacean waste are dissolved by secondary bacterial treatment to prepare an improved liquid culture medium, and finally a spherical calcium carbonate organic complex with porous layered characteristics is synthesized by inducing Bacillus Velezii. The spherical calcium carbonate organic complex contains vaterite calcium carbonate and bacterial metabolites, and is a biomineral material with high-efficiency antibiotic drug-carrying characteristics developed based on the biological mineralization mechanism, which provides a new idea for realizing the deep utilization of waste crustacean resources and reducing environmental pollution.

[0021] 2. The novel drug sustained-release carrier of the present invention has excellent loading capacity for positively charged drugs. For example, the maximum loading capacity of the anticancer drug doxorubicin hydrochloride is as high as 447.579 mg / g, and the encapsulation rate can reach about 94%.

[0022] 3. The novel drug sustained-release carrier of the present invention can slowly release drugs in a weakly acidic environment with a pH value of less than or equal to 6, thereby achieving the effect of reducing the dosage and toxic side effects.

[0023] 4. The preparation method of the novel drug sustained-release carrier synthesized by using crustacean waste as a calcium source is economical, environmentally friendly, easy to operate, low in cost, and has a wide range of applications. The obtained biogenic spherical calcium carbonate has good stability and well-developed mesopores. In the synthesis process, bacteria are the dominant force in the decalcification and deproteinization process, which effectively reduces the subsequent use of acid and alkali solvents, and avoids secondary pollution through the utilization of waste liquid. It has no potential risks to the environment, has good application prospects in drug loading, and realizes the rational utilization of waste resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a structural and morphological characteristic diagram of the biogenic spherical calcium carbonate drug carrier of Example 1;

[0025] Figure 2 The effect of the biogenic spherical calcium carbonate drug carrier on the drug loading of doxorubicin hydrochloride at different time periods in Example 2;

[0026] Figure 3 This is a morphological characteristic diagram of the biogenic spherical calcium carbonate drug carrier loaded with doxorubicin hydrochloride in Example 3;

[0027] Figure 4 This is a structural characteristic diagram of the bio-source spherical calcium carbonate drug carrier loaded with doxorubicin hydrochloride in Example 4;

[0028] Figure 5 This is the in vitro release curve of the mineral complex of doxorubicin hydrochloride loaded with the biogenic spherical calcium carbonate drug carrier of Example 5 at 37°C. DETAILED DESCRIPTION

[0029] The present invention will be further explained below in conjunction with the embodiments and drawings.

[0030] Unless otherwise specified, the materials and reagents used in the examples can be obtained from commercial sources.

[0031] Among them, Lactobacillus rhamnosus, Bacillus subtilis, and Bacillus velez were all purchased from the strain library, Lactobacillus rhamnosus was CCTCC DB 20082412, Bacillus subtilis was CCTCC AB 209260, and Bacillus velez was CGMCCNo.1.12669.

[0032] Example 1

[0033] The preparation method of biogenic spherical calcium carbonate is as follows:

[0034] 1. Seed solution preparation:

[0035] Lactobacillus rhamnosus seed culture medium: MRS medium (DeMan-Rogosa-Sharpe medium) was used for culture, which consists of: peptone 10g / L, beef extract 10g / L, yeast extract 5g / L, glucose 5g / L, sodium acetate 5g / L, diammonium citrate 2g / L, Tween 80 1g / L, dipotassium hydrogen phosphate 2g / L, magnesium sulfate heptahydrate 0.2g / L, manganese sulfate monohydrate 0.05g / L, calcium carbonate 20g / L, pH 6.8, sterilized at 115℃ for 30min. Two rings of Lactobacillus were inoculated into the seed culture medium (100 / mL), and cultured at 37℃ for 10h. The bacterial liquid concentration was about 8×10 8 cfu / mL.

[0036] Bacillus subtilis seed culture medium: Liquid LB medium, inoculate 2 loops of Bacillus subtilis into the seed culture medium (100 mL), and culture in a shaking flask at 37°C and 180 r / min for 12 h. The bacterial liquid concentration is about 8×10 8 cfu / mL.

[0037] Velez Bacillus seed culture medium: Liquid LB medium, inoculate 2 rings of Velez Bacillus into seed culture medium (100 mL), and culture in a flask at 37°C and 180 r / min for 12 h. The bacterial liquid concentration is about 8×10 8 cfu / mL.

[0038] 2. Decalcification, deproteinization and synthesis of spherical calcium carbonate

[0039] The Chinese mitten crab (Eriocheir sinensis) was used as the experimental material. An appropriate amount of the crab shell of the Chinese mitten crab was taken, cleaned, dried, and crushed into 60 meshes.

[0040] Decalcification: Lactobacillus rhamnosus fermentation medium: deionized water is solvent, glucose 10g / 100mL, crab shell powder 5g / 100mL L, 115 ℃ sterilization 30min. Access the lactobacillus fermentation medium (100 / 250mL) after activation with a bacterial inoculum of 4% by volume, place 37 ℃, 100r / min fermentation 2d. Centrifuge the fermented liquid after the reaction (8000rpm, 15 minutes), retain the filtrate (filtrate A1) and filter residue A, add 0.5mol / L HCl solution to filter residue A and leave it standstill for 2h, and the addition amount is that each portion of crab shell powder (5g) handles the residue and adds 5mL HCl solution. This process will remove the calcium carbonate that is not completely taken off, and after the reaction is completed, the same centrifugal collection filter residue B and acidic waste liquid B1 are standby. Filter residue B is used as the next step of deproteinization.

[0041] Deproteinization: All filter residues B after lactobacillus decalcification were added to subtilis fermentation medium (deionized water as solvent, soluble starch 9.37g / L, peptone 10.85g / L, yeast extract 1.67g / L, manganese sulfate 0.07g / L, ferrous sulfate 0.07g / L, magnesium sulfate 0.07g / L, Tween-200.07g / L, sodium dihydrogen phosphate 3.61g / L, dipotassium hydrogen phosphate 0.63g / L) at 5g / 100mL, sterilized at 115°C for 30min. Access subtilis seed solution with a volume ratio of 4%, and culture medium was placed at 37°C, 180r / min for fermentation for 2d. The fermented liquid after the reaction was centrifuged (8000rpm, 15 minutes), and filtrate (filtrate C1) and filter residue C were retained. Taking 5g of initial crab shell powder as an example, add 50mL of 1mol / L NaOH solution to the filter residue C and place it in a 90℃ water bath for 2h. This process will remove the unremoved protein. After the reaction is completed, centrifuge to collect the filter residue D and alkaline waste liquid D1 for later use.

[0042] Synthesis of spherical calcium carbonate: Taking the initial 5g crab shell powder added to the fermentation medium of Lactobacillus rhamnosus as an example, all the above filtrates A1, B1, C1 and D1 are mixed, deionized water is added, the volume is adjusted to 500mL, and 2.5g peptone, 2.5g yeast extract and 5g NaCl are added. Adjust the pH value to about 7.2 and sterilize at 115℃ for 30min. Take the activated Bacillus Velez seed liquid, inoculate it with 2% bacteria by volume, and place it in a shaking table at 37℃ and 180r / min for 5d. After the culture is completed, the fermentation liquid is centrifuged (8000r / min, 15 minutes) to collect the precipitate. After washing with deionized water, the collected precipitate is placed in an oven at 55℃ for drying and grinding to obtain spherical calcium carbonate.

[0043] 4. XRD, FTIR, and SEM-EDS were used to identify the structure and morphology of the precipitate prepared above. The results of XRD and FTIR showed that the precipitate was mainly a complex formed by spherical calcium carbonate and organic matter ( Figure 1 a and b). SEM can observe spherical and a small amount of amorphous calcium carbonate, which can form spherical porous layered aggregates ( Figure 1 c, d and e).

[0044] Example 2

[0045] Loading characteristics of biogenic spherical calcium carbonate for the drug doxorubicin hydrochloride:

[0046] Take the biosource spherical calcium carbonate prepared in Example 1 above for loading positively charged doxorubicin hydrochloride (DOX) as an example: according to the adsorbent dosage of 0.02g / 30mL, the biosource spherical calcium carbonate prepared above is added to the DOX solution with a concentration of (20mg / 30mL), and loaded in a shaker at 25℃ and 200r / min for 36h. After the drug loading is completed, centrifuge (8000r / min, 15min), collect the precipitate and supernatant, and use a UV-visible spectrophotometer to measure the absorbance of DOX in the supernatant. Calculate the encapsulation rate (%) of the drug carrier for antibiotics and the drug loading capacity (mg / g) of the drug carrier as follows: Figure 2 By analyzing the adsorption data using the Freundlich adsorption model, the maximum loading capacity Q of the drug carrier for antibiotics can be obtained. max (mg / g) is 447.579 mg / g, and the encapsulation rate can reach about 94%.

[0047] The Freundlich model parameters of biogenic spherical calcium carbonate for DOX loading are shown in Table 1.

[0048]

[0049]

[0050]

[0051] Where: C is the concentration of DOX in HCl solution, mg / L; C0 is the initial concentration of DOX, mg / L; Ce is the concentration of DOX in the solution when adsorption reaches equilibrium, mg / L; V is the volume of the adsorption system, L; M is the amount of adsorbent vaterite added, g; R is the drug encapsulation rate. e is the amount of DOX adsorbed by unit adsorbent vaterite when adsorption reaches equilibrium, mg / g; C e is the concentration of DOX in the solution when adsorption reaches equilibrium, mg / L; K f is the Freundlich equation constant, mg / L; 1 / n is the Freundlich adsorption intensity constant.

[0052] Table 1 Freundlich model parameters of biogenic spherical calcium carbonate for DOX loading

[0053]

[0054] From Table 1 and Figure 2 It can be seen that the drug carrier prepared by the present invention has a higher drug loading capacity and encapsulation efficiency, indicating that its surface adsorption is mainly multi-layer surface adsorption.

[0055] In addition, under the condition of the same drug concentration, the higher the amount of spherical calcium carbonate prepared by the present invention is added, the lower its encapsulation efficiency and drug loading are. When the adsorbent is added at a dosage of 0.04 g / 30 mL, the loading amount is less than half of the adsorbent dosage of 0.02 g / 30 mL. The loading amount will gradually decrease if the dosage is further increased, which further proves the particularity of the spherical calcium carbonate prepared specifically by the present invention.

[0056] Example 3

[0057] Morphological and structural characteristics of biogenic spherical calcium carbonate loaded with doxorubicin hydrochloride:

[0058] The surface of the mineral complex (i.e., the precipitate collected in Example 2) after loading DOX with spherical calcium carbonate prepared in Example 2 was observed by SEM-EDS. The porous structure of the surface of the biogenic spherical calcium carbonate adsorbed a large amount of DOX ( Figure 3 a), and the spherical calcium carbonate attaches DOX on its surface based on its spherical structure, and the organic matter contained in the spherical calcium carbonate helps the spherical calcium carbonate to adsorb DOX ( Figure 3 bd). The abundant negatively charged organic functional groups such as hydroxyl and carboxyl groups on the surface of spherical calcium carbonate minerals and their mesoporous distribution will adsorb the positively charged DOX on the surface, thus having excellent drug loading capacity for DOX.

[0059] Example 4

[0060] In order to ensure that the DOX drug has been loaded in the spherical calcium carbonate particles, the sample obtained after the spherical calcium carbonate loaded with DOX precipitation and drying prepared in Example 2 was subjected to XRD, FTIR and TG-DTG analysis to observe the structural changes before and after the spherical calcium carbonate adsorbed DOX. The XRD analysis results show that the mineral composite particles maintain the crystal characteristics of vaterite calcium carbonate as the main component ( Figure 4 a), indicating that the loading of DOX did not change the crystal form of spherical calcium carbonate and thus affect its stability. Figure 4 b), the characteristic peaks of calcium carbonate such as vaterite can be clearly observed, and this characteristic peak also appears in the infrared spectrum of the mineral complex after loading DOX. This result can prove that the antibiotic can remain stable during the loading process. Based on the above results, it can be inferred that spherical calcium carbonate has successfully encapsulated DOX effectively.

[0061] Example 5

[0062] Release characteristics of biogenic spherical calcium carbonate as a drug carrier under acidic conditions:

[0063] In order to confirm that the micron or nanometer-sized spherical calcium carbonate particles prepared in the experiment have the potential to deliver drugs to tumor cells (the extracellular pH of tumor cells is about 6), the drug release in a weak acid environment of pH=6 was detected over a period of 8 days using the prepared spherical calcium carbonate-DOX mineral composite particles (precipitated and dried in Example 2 of the present invention). Figure 5 As shown, the drug release in this environment showed an extended slow release behavior, and the initial drug release was low. On the 6th day, i.e., 156 hours, the release began to slowly tend to equilibrium. The main reason was that the prepared spherical calcium carbonate-DOX mineral complex was relatively stable and acid-resistant, which reduced the degradation rate of BV and enabled DOX to be released slowly.

[0064] Example 6

[0065] To explore the drug loading capacity of doxorubicin hydrochloride by biogenic spherical calcium carbonate and inorganic calcium carbonate.

[0066] According to the method in Example 2 above, inorganic calcium carbonate and biological spherical calcium carbonate (prepared in Example 1) are loaded with doxorubicin hydrochloride respectively, and the drug loading of the two drug carriers to the drug is calculated according to the formula. The drug loading of inorganic calcium carbonate to DOX is 116 mg / g, and the drug loading of biological spherical calcium carbonate to DOX is 447.579 mg / g. Among them, inorganic vaterite is a reference cited, and the specific preparation method refers to Dou et al., (2020) (Preparation of non-sphericalvaterite CaCO3 particles by flash nano precipitation technique for targetedand extended drug delivery).

[0067] In addition, the present invention found that the improved liquid culture medium prepared by secondary bacterial treatment and finally induced by Velez Bacillus to synthesize spherical calcium carbonate organic complex has high drug loading properties. If the conventional chemical method is used directly or only one bacterial treatment is used, the loading performance is obviously not as good as the present invention.

[0068] Comparative Example 1

[0069] Removal of calcium carbonate from crab shells: Take 5g of crab shell powder (passed through a 60-mesh sieve) at room temperature and soak the crab shells in 5mL of 1.0mol / L HCl solution until no bubbles are generated (about 24h) to remove calcium carbonate. Filter and retain the filtrate, and use the residue for the next step.

[0070] Removal of protein from crab shells: The filter residue was washed with deionized water and dried. The filter residue was treated with 50 mL of 2.0 mol / L NaOH, and then placed in a water bath, reacted at 90°C for 4 h, filtered to remove the alkali solution, and then the filter residue was washed with deionized water, and the filtrate was retained.

[0071] Synthesis of spherical calcium carbonate: Combine the above two steps to obtain the filtrate, add deionized water to 500 mL, add 2.5 g of peptone, 2.5 g of yeast extract and 5 g of NaCl. According to the spherical calcium carbonate synthesis method in Example 1, a spherical calcium carbonate product is obtained, and then the obtained product is loaded with doxorubicin hydrochloride according to the method of Example 2, and the drug loading capacity of DOX is about 325 mg / g.

[0072] Comparative Example 2

[0073] Lactobacillus rhamnosus fermentation medium: deionized water is solvent, glucose 100g / L, crab shell powder 50g / L, 115 ℃ of sterilization 30min. Access the lactobacillus fermentation medium (100 / 250mL) after activation with the bacterial inoculum amount of 4% by volume, place 37 ℃, 100r / min fermentation 2d. The fermented liquid after reaction is centrifuged (8000rpm, 15 minutes), retain filtrate (filtrate A2) and filter residue A2, add the HCl solution of 0.5mol / L in filter residue A and leave standstill 2h, and addition is that every part of crab shell powder (5g) handles the residue and adds 5mL HCl solution. This process will remove the calcium carbonate that does not take off completely, and after reaction is finished, the same centrifugal collection filter residue B2 and acidic waste liquid B2 are standby.

[0074] Synthesis of calcium carbonate: Taking the initial 5g crab shell powder added to the fermentation medium of Lactobacillus rhamnosus as an example, after the filtrate A2 and the filtrate B2 are mixed, deionized water is added to 500mL, 2.5g peptone, 2.5g yeast extract and 5g NaCl are added, the pH value is adjusted to about 7.2, and sterilized at 115°C for 30min. Take the activated Bacillus Velez seed liquid, inoculate with 2% bacteria by volume, and place it in a shaking table at 37°C and 180r / min for 5d. After the culture is completed, the fermentation liquid is centrifuged (8000r / min, 15 minutes) to collect the precipitate, and the collected precipitate is placed in an oven at 55°C for drying and grinding after washing with deionized water, and no spherical calcium carbonate is formed. The product is loaded with doxorubicin hydrochloride according to the method of Example 2, and its drug loading capacity for DOX is less than 200mg / g.

[0075] Comparative Example 3

[0076] Add 50g / L of crab shell powder to the fermentation medium of Bacillus subtilis (deionized water as solvent, 9.37g / L of soluble starch, 10.85g / L of peptone, 1.67g / L of yeast extract, 0.07g / L of manganese sulfate, 0.07g / L of ferrous sulfate, 0.07g / L of magnesium sulfate, 0.07g / L of Tween-20, 3.61g / L of sodium dihydrogen phosphate, 0.63g / L of potassium dihydrogen phosphate), and sterilize at 115°C for 30min. Inoculate 4% of the volume ratio of Bacillus subtilis seed liquid, place the culture medium at 37°C and 180r / min for fermentation for 2d. Centrifuge the fermentation liquid after the reaction (8000rpm, 15 minutes), and retain the filtrate (filtrate C2) and the residue C2. Add 50mL of 1mol / L NaOH solution to the residue C2, and let it stand in a 90°C water bath for 2h. This process will remove the unremoved proteins. After the reaction is completed, the filter residue D2 and alkaline waste liquid D2 are collected by centrifugation as above for later use.

[0077] Synthesis of calcium carbonate: Taking the initial 5g crab shell powder added to the fermentation medium of Lactobacillus rhamnosus as an example, after the filtrate A2 and the filtrate B2 are mixed, deionized water is added to 500mL, 2.5g peptone, 2.5g yeast extract and 5g NaCl are added, the pH value is adjusted to about 7.2, and sterilized at 115°C for 30min. Take the activated Bacillus Velez seed liquid, inoculate with 2% bacteria by volume, and place it in a shaking table at 37°C and 180r / min for 5d. After the culture is completed, the fermentation liquid is centrifuged (8000r / min, 15 minutes) to collect the precipitate, and the collected precipitate is placed in an oven at 55°C for drying and grinding after washing with deionized water, and all are amorphous calcium carbonate. The product loaded with doxorubicin hydrochloride is obtained according to the method of Example 2, and its drug loading capacity for DOX is less than 150mg / g.

[0078] The above experiments show that the two bacterial treatments in the present invention have a very significant effect on the later synthesis of biological spherical calcium carbonate loaded with doxorubicin hydrochloride, and the effect is significantly better than chemical treatment or single bacterial treatment, and has a very good synergistic effect. In addition, high-concentration chemical reagent treatment is used in Comparative Example 1. If a low-concentration chemical reagent treatment is used (such as Example 1), the loading amount will be further reduced.

[0079] The novel drug carrier prepared by the invention has a large drug loading capacity and high stability, can realize the slow release of drugs, and has broad application prospects as a drug carrier.

Claims

1. A method for preparing a novel drug carrier, characterized in that: The method comprises the following steps: inoculating Lactobacillus rhamnosus in a liquid culture medium provided with a calcium source by crustacean waste for culturing, centrifuging to obtain a precipitate A and a supernatant A1, treating the precipitate A with hydrochloric acid, centrifuging after no bubbles are generated to obtain a supernatant B1 and a precipitate B, inoculating Bacillus subtilis in a fermentation culture medium containing the precipitate B for culturing, centrifuging to obtain a precipitate C and a supernatant C1, treating the precipitate C with NaOH, centrifuging after no bubbles are generated to obtain a supernatant D1 and a precipitate D, mixing the supernatants A1, B1, C1 and D1, inoculating Bacillus Velez for culturing, and after the culturing is completed, centrifuging to collect the precipitate, washing and drying to obtain a novel drug carrier; The hydrochloric acid and NaOH are both low-concentration solutions, with the hydrochloric acid not exceeding 0.5 mol / L and the NaOH not exceeding 1 mol / L; The Lactobacillus rhamnosus, Bacillus subtilis and Bacillus velezii are cultured to a bacterial solution concentration of 7-9×10 8 cfu / mL, inoculated into the culture medium at a volume ratio of 2-5%; The Lactobacillus rhamnosus is CCTCC DB 20082412; the Bacillus subtilis is CCTCC AB 209260, and the Bacillus velezii is CGMCC No. 1.12669.

2. The preparation method according to claim 1, characterized in that: The liquid culture medium with a calcium source provided by crustacean waste is an MRS liquid culture medium with a calcium source provided by crustacean waste treated with Lactobacillus rhamnosus.

3. The preparation method according to claim 1, characterized in that: After inoculation of Lactobacillus rhamnosus, the culture is cultured at 30 ~ 37°C for 2-6 days; after inoculation of Bacillus subtilis, the culture is cultured at 30 ~ 37°C for 2-4 days; after inoculation of Bacillus Velez, the culture is cultured at 30 ~ 37°C for 4-7 days.

4. A novel drug carrier prepared by the preparation method according to claim 1.

5. Use of the novel drug carrier according to claim 4 in preparing a novel drug sustained-release carrier.

6. The use according to claim 5, characterized in that: The loaded drug should have the characteristic of being positively charged; the novel drug sustained-release carrier has a sustained-release effect in a weakly acidic environment with a pH value less than or equal to 6.

Images