Preparation method of reversible covalent bond controlled slow-release bio-based nano-drug
By oxidizing and lateral linking branches on cellulose nanocrystals, a bio-based nanodrug carrier that controls sustained release is formed with reversible covalent bonds, which solves the problems of unstable drug release and poor carrier functional modification in the prior art, and achieves efficient drug targeting and sustained release therapeutic effects.
Patent Information
- Application Number
- CN202510020542.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-07
- Publication Date
- 2025-04-04
AI Technical Summary
In the prior art, it is difficult for nanodrug carriers to achieve sustainable and stable drug release in the body, resulting in poor therapeutic effects, complex synthesis process, high cost and poor functional modification.
Dialdehyde cellulose nanoparticles were obtained by oxidizing cellulose nanoparticles, and the methoxy polyethylene glycol side chain and amino-containing anti-cancer drugs were grafted through Schiff base reaction to form a bio-based nanodrug carrier that controls sustained release by reversible covalent bonds.
The drug is sustained release with high drug loading, high salt tolerance and low cytotoxicity. The drug can be responsively released under acidic conditions, which is suitable for targeted treatment of tumor sites.
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Abstract
Description
Technical Field
[0001] The invention relates to a method for preparing a reversible covalent bond controlled sustained-release bio-based nano drug, belonging to the technical field of anticancer drug preparation. Background Art
[0002] At present, cancer has become a major disease that endangers human health. After cancer is diagnosed, chemotherapy is an indispensable and important link. The best chemotherapy regimen requires the quantitative release of the corresponding therapeutic drug molecules to the designated location, but because the therapeutic drug molecules in the body are too small to be quickly cleared by metabolic organs, they cannot produce a sustained therapeutic effect at the tumor site. However, nanoscale polymers can be selectively distributed in tumor cells through the EPR effect, release drugs under the special internal environment of tumor cells, and use the hydrophilic and hydrophobic groups of nanocarrier materials and the intermolecular forces to simultaneously encapsulate or adsorb drug molecules to construct a stable drug delivery system, solve the problem of free small molecules being easily metabolized and cleared in the body, improve drug stability and targeting, and reduce drug systemic toxicity, which is an important strategy for precision treatment.
[0003] Common nano drug carriers include inorganic nanoparticles, liposomes, polymer nanoparticles, etc., but most of them still face problems such as complex synthesis process, high cost, and poor functional modification. In recent years, polysaccharide polymers, such as cellulose, starch, chitosan, etc., have the advantages of low cost, biocompatibility and modifiability, and have been widely studied and applied in the field of nano drug carriers. As the smallest physical structural unit of natural cellulose, cellulose nanocrystals (CNCs) are one-dimensional nanorods with a width of 5-15 nm and a length of 100-300 nm. They also have unique properties such as high crystallinity, high specific surface area, high hydrophilicity and ultrafine structure, and have gradually become one of the research hotspots in the field of nanobiomedicine. Wu et al. grafted thermosensitive poly N, N-dimethylacrylamide (PNIPAM) polymer brushes on the surface of CNCs and used their thermosensitive conformational changes to achieve drug loading and sustained release. However, the physical loading method still has problems such as low drug loading and instability (BioResources, 2016, 11(3), 7026-7035). In another study, Lee et al. used multifunctional polymer DNA produced by rolling circle amplification (RCA) to form polymer DNA-modified CNCs (pDCs) through electrostatic interaction with surface cationized CNCs, and used them for cancer-targeted intracellular delivery and pH-responsive drug release. Due to its aptamer and i-motif functions, pDCs showed anticancer properties in vitro without side effects. As a nanodrug carrier platform, it has good application prospects in targeted cancer therapy (Carbohydrate Polymers, 2024, 340, 122270). At present, there are few studies on the use of CNCs' unique nanostructure for sustained drug release, which is still in its infancy. Summary of the invention
[0004] Purpose of the invention: In order to overcome the deficiencies in the prior art, the present invention provides a method for preparing a bio-based nano drug with reversible covalent bond-controlled sustained release.
[0005] Technical solution: To achieve the above purpose, the technical solution adopted by the present invention is: A method for preparing a bio-based nano drug carrier with reversible covalent bond controlled sustained release comprises the following steps: Step 1: oxidizing cellulose nanocrystals to obtain dialdehyde cellulose nanoparticles.
[0006] Step 2: reacting dialdehyde cellulose nanoparticles, methoxy polyethylene glycol amine, and sodium cyanoborohydride to obtain cellulose nanoparticles grafted with methoxy polyethylene glycol side chains.
[0007] Step 3, adding the amino-containing anticancer drug to the dispersion of cellulose nanoparticles grafted with methoxypolyethylene glycol side chains, so that the amino groups in the amino-containing anticancer drug and the aldehyde groups on the surface of the cellulose nanoparticles are covalently bonded through a Schiff base reaction, thereby obtaining a reversible covalent bond (imine bond) controlled sustained release bio-based nano drug carrier.
[0008] Preferably: the method of oxidizing cellulose nanocrystals to obtain dialdehyde cellulose nanoparticles in step 1: cellulose nanocrystals, sodium periodate, zinc chloride and water are added to a round-bottom flask. Then, the oxidation reaction is carried out under stirring at room temperature in a light-shielded environment, and ethylene glycol is added to quench the residual periodate to terminate the reaction. Thereafter, purified dialdehyde cellulose nanoparticles are obtained after dialyzing in water. Finally, the supernatant is collected and freeze-dried to obtain dry dialdehyde cellulose nanoparticles.
[0009] Preferably, the weight components of cellulose nanocrystals, sodium periodate, zinc chloride and water are: 100 parts of water, 0.5-2 parts of cellulose nanocrystals, 3-12 parts of sodium periodate and 0.5-2 parts of zinc chloride.
[0010] Preferably, the oxidation reaction is carried out by stirring at a speed of 200-300 rpm for 60-84 hours at room temperature in a light-shielded environment.
[0011] Preferably, the preparation method of cellulose nanoparticles grafted with methoxy polyethylene glycol side chains in step 2 is: take dry dialdehyde cellulose nanoparticles and evenly disperse them in N, N-dimethylformyl solvent, stir and heat and keep at 30°C, then add methoxy polyethylene glycol amine PEG-NH2 to react for a period of time, finally add sodium cyanoborohydride and continue to react at a constant temperature for a period of time, and obtain cellulose nanoparticles grafted with methoxy polyethylene glycol side chains after dialysis treatment.
[0012] Preferably, the concentration of the dialdehyde cellulose nanoparticle dispersion is 2-3 mg / mL.
[0013] Preferably: heating to 25-35° C. under stirring, fully dissolving and mixing, adding 25-35 parts of methoxy polyethylene glycol amine to the dialdehyde cellulose nanoparticle dispersion maintained at 25-35° C. under stirring, and reacting for 12 hours. Subsequently, adding 0.5-1.5 parts of sodium cyanoborohydride and continuing the constant temperature reaction for 12 hours. Then, dialysis is performed for three days to remove unreacted reagents and impurities, and finally obtaining cellulose nanoparticles grafted with methoxy polyethylene glycol side chains.
[0014] Preferably, 1-3 parts of an amino-containing anticancer drug are added to 0.5-1.5 parts of a dispersion of cellulose nanoparticles grafted with methoxy polyethylene glycol side chains, and the resulting mixture is shaken at room temperature in the dark for 12-36 hours. After completion, the sample is transferred to an ultrafiltration tube, and centrifuged at a relative centrifugal force of 3000-5000 rcf for 20-30 minutes to remove the non-covalently bound anticancer drug molecules, and the centrifugal washing is repeated several times until the clear liquid under the ultrafiltration tube is clear, and finally a reversible covalent bond controlled sustained release bio-based nano drug carrier is obtained.
[0015] Preferably, the amino-containing anticancer drug is one of doxorubicin hydrochloride, amine quinazoline, eribulin and gemcitabine.
[0016] Compared with the prior art, the present invention has the following beneficial effects: First, dialdehyde cellulose nanoparticles (DAC) were prepared by oxidizing cellulose nanocrystals with periodate, and amino anticancer drugs were grafted onto dialdehyde cellulose nanoparticles through Schiff base reaction to obtain reversible covalent bond-controlled sustained-release bio-based nanodrugs with high drug loading, high salt tolerance and low cytotoxicity. Due to the reversibility of the Schiff base bond, the drug was responsively released under acidic conditions, which matches the pH decreasing trend from normal tissue to tumor tissue, and can be used as a nanodrug carrier for targeted and sustained-release treatment of cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 Schematic diagram of the synthesis route of bio-based nano drug carriers with reversible covalent bond-controlled sustained release.
[0018] Figure 2 are FTIR spectrum, C spectrum, and N spectrum, among which, Figure 2 a is the FTIR spectra of DAC, DAC-PEG, and DOX-DAC-PEG, Figure 2 b is the XPS high-resolution C spectrum of DOX-DAC-PEG. Figure 2 c is the N spectrum of DOX-DAC-PEG.
[0019] Figure 3 For TEM photos, Figure 3 a is the TEM photo of DAC, Figure 3 b is the TEM image of PEG-DAC, Figure 3 c is the TEM image of DOX-DAC-PEG, Figure 3 d is the Zeta potential diagram, Figure 3 e is the particle size distribution diagram.
[0020] Figure 4 are the spectra of DAC, PEG-DAC, and DOX-DAC-PEG, where Figure 4a is the absorption spectrum, Figure 4 b is the fluorescence excitation spectrum, Figure 4 c is the fluorescence emission spectrum.
[0021] Figure 5 The drug release curves of DOX-DAC-PEG nanodrugs at different pH values. DETAILED DESCRIPTION
[0022] The present invention is further explained below in conjunction with the accompanying drawings and specific embodiments. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. After reading the present invention, various equivalent forms of modifications to the present invention by those skilled in the art all fall within the scope defined by the claims attached to this application.
[0023] Example 1 Doxorubicin (DOX) is a broad-spectrum anticancer chemotherapy drug in clinical applications, but DOX is often accompanied by severe cardiotoxicity and systemic toxicity in clinical applications. In order to reduce the toxic side effects of DOX on healthy tissues and maintain and improve its chemotherapy effect, a variety of DOX-loaded nanodrugs (such as DOX acid-sensitive prodrugs and DOX liposomes, etc.) have been developed. This embodiment provides a method for preparing a bio-based nano drug carrier with reversible covalent bond controlled sustained release: cellulose nanocrystals (CNC) prepared by sulfuric acid hydrolysis are used as carriers. Since C6 is usually occupied by sulfonic acid groups, it has good water dispersion stability through strong electrostatic repulsion, but it also has the problem that the reactivity of the hydroxyl groups at C2 and C3 is not high enough. In order to further improve the reactivity of CNC, periodate (NaIO 4 ) Selectively oxidize the vicinal diols at the C2 and C3 positions to obtain a high aldehyde content. It is well known that the water dispersion stability of CNC is crucial for its subsequent modification and application. Based on this, a portion of the aldehyde groups on its surface are used to graft polyethylene glycol (PEG) side chains to improve the salt resistance stability of CNC. On this basis, the remaining aldehyde groups are further used to chemically bond DOX through the Schiff base reaction to obtain a high drug loading capacity bound by covalent bonds. Due to the reversible hydrolysis characteristics of the Schiff base structure, it can achieve controlled sustained release of DOX. Figure 1 As shown, the specific steps include: Step 1, Preparation of dialdehyde cellulose nanoparticles (DAC) CNC (1 g), NaIO 4(8g), zinc chloride (1g) and 100mL water. Then, the oxidation reaction was carried out by stirring at a speed of 250rpm for 72h at room temperature in a light-shielded environment. Ethylene glycol was added to quench the residual periodate to terminate the reaction. Then, purified dialdehyde cellulose nanoparticles (DAC) were obtained after dialysis in water. The DAC suspension was heated at 80°C in an oil bath for 4h, and then the sample was centrifuged at a speed of 14,000rpm for 30min to remove residual solids. Finally, the supernatant was collected and dried and stored in a desiccator. The concentration of the obtained dialdehyde cellulose nanoparticle dispersion was 2-3mg / mL.
[0024] Step 2, Preparation of Cellulose Nanoparticles Grafted with Methoxy Polyethylene Glycol Side Chains (DAC-PEG) A certain amount of DAC was dispersed in DMF solvent to prepare a DAC dispersion with a concentration of 2.5 mg / mL. The dispersion was heated to 30°C under stirring. After being fully dissolved and mixed, methoxy polyethylene glycol amine (PEG-NH2, 30 mg) was added to the DMF solution maintained at 30°C. At the same time, sodium cyanoborohydride (NaBH 3 CN, 1 mg) was reacted at a constant temperature for 24 h to obtain the desired product solution. The pH of the product solution was adjusted to 7.2 by HCl, and then it was dialyzed for three days to finally obtain the desired cellulose nanoparticles grafted with methoxy polyethylene glycol side chains (DAC-PEG).
[0025] Step 3, Preparation of reversible covalent bond controlled sustained release bio-based nano drug carrier (DOX-DAC-PEG) There are two types of groups that can be used to covalently couple doxorubicin (DOX) to carriers, namely amino and keto groups. In this example, DOX was covalently grafted via a Schiff base bond formed between the amino group and the aldehyde group (–CHO) of DAC.
[0026] First, DOX (2.06 mg) was added to 1 mL of dialyzed DAC-PEG solution, and the resulting mixture was shaken at room temperature in the dark for 24 h. Afterwards, the sample was transferred to an ultrafiltration tube and centrifuged at a relative centrifugal force of 4000 rcf for 25 min to remove the ungrafted DOX. This was repeated several times until the clear liquid under the ultrafiltration tube was clear, and the final volume was 1 mg / mL, which was a reversible covalent bond controlled sustained release bio-based nano drug carrier (DOX-DAC-PEG) dispersion.
[0027] Example 2 This embodiment provides a method for preparing a bio-based nano drug carrier with reversible covalent bond controlled sustained release, comprising the following steps: Step 1, Preparation of DAC CNC (0.5 g), NaIO4 (3 g), zinc chloride (0.5 g) and 100 mL of water were added to a round-bottom flask. Then, the oxidation reaction was carried out by stirring at 200 rpm for 84 h at room temperature in a light-shielded environment. Ethylene glycol was added to quench the residual periodate to terminate the reaction. Then, purified DAC was obtained after dialysis in water. The DAC suspension was heated at 75°C in an oil bath for 5 h, and then the sample was centrifuged at 13,000 rpm for 50 min to remove residual solids. Finally, the supernatant was collected and dried and stored in a desiccator.
[0028] Step 3, Preparation of DAC-PEG A certain amount of DAC was dispersed in DMF solvent to prepare a DAC dispersion with a concentration of 2.5 mg / mL. The dispersion was heated to 25 °C under stirring and fully dissolved and mixed. 2 (25 mg) was added to the DMF solution maintained at 25 °C, and NaBH 3 CN (0.5 mg) is continuously reacted at a constant temperature to obtain the desired product solution. The pH of the product solution is adjusted to 7.2 by HCl, and then the solution is dialyzed for three days to finally obtain the desired DAC-PEG.
[0029] Step 4, Preparation of DOX-DAC-PEG First, DOX (1 mg) was added to 0.5 mL of dialyzed DAC-PEG solution, and the resulting mixture was shaken at room temperature in the dark for 12 h. After completion, the sample was transferred to an ultrafiltration tube and centrifuged at a relative centrifugal force of 5000 rcf for 20 min to remove the ungrafted DOX. This was repeated several times until the supernatant under the ultrafiltration tube was clear. The DOX-DAC-PEG dispersion was finally fixed to 1 mg / mL, which was a reversible covalent bond controlled sustained release bio-based nano drug carrier.
[0030] Example 3 This embodiment provides a method for preparing a bio-based nano drug carrier with reversible covalent bond controlled sustained release, comprising the following steps: Step 1, Preparation of DAC CNC (2 g), NaIO 4 (12 g), zinc chloride (2 g) and 100 mL of water. Then, the oxidation reaction was carried out by stirring at 300 rpm for 60 h at room temperature in a light-shielded environment. Ethylene glycol was added to quench the residual periodate to terminate the reaction. Then, purified DAC was obtained after dialysis in water. The DAC suspension was heated at 85°C for 3 h in an oil bath, and then the sample was centrifuged at 15,000 rpm for 10 min to remove residual solids. Finally, the supernatant was collected and dried and stored in a desiccator.
[0031] Step 2, Preparation of DAC-PEG A certain amount of DAC was dispersed in DMF solvent to prepare a DAC dispersion with a concentration of 2.5 mg / mL. The dispersion was heated to 35 °C under stirring and fully dissolved and mixed. 2 (35 mg) was added to a DMF solution maintained at 35°C, and NaBH3CN (1.5 mg) was added to continue the constant temperature reaction to obtain the desired product solution. The pH of the product solution was adjusted to 7.2 by HCl, and then it was dialyzed for three days to finally obtain DAC-PEG.
[0032] Step 3, Preparation of DOX-DAC-PEG First, DOX (3 mg) was added to 1.5 mL of dialyzed DAC-PEG solution, and the resulting mixture was shaken at room temperature in the dark for 36 h. After completion, the sample was transferred to an ultrafiltration tube and centrifuged at a relative centrifugal force of 3000 rcf for 30 min to remove the ungrafted DOX. This was repeated several times until the clear liquid under the ultrafiltration tube was clear, and the final volume was 1 mg / mL of PEG-DAC-DOX solution, which was a reversible covalent bond controlled sustained release bio-based nano drug carrier.
[0033] In another embodiment, the amino-containing anticancer drug is one of amine quinazoline, eribulin, and gemcitabine.
[0034] Example 4 This example tests the reversible covalent bond controlled sustained-release bio-based nano drug carrier obtained in Example 1.
[0035] like Figure 2 The CNC, DAC, DAC-PEG, and DOX-DAC-PEG shown in a were confirmed by FTIR analysis: the 1730 and 880 cm -1 The absorption band at represents the characteristic band of aldehyde group ( Figure 2 a) The weak absorption peak of the stretching of aldehyde groups on DAC may be due to the formation of a hemiacetal structure between the aldehyde group and the nearby hydroxyl group. In addition, the absorption peaks at 1730 cm-1 in DAC-PEG and DOX-DAC-PEG -1 The characteristic peak at 1000 nm disappeared, indicating that the Schiff base coupling converted the dialdehyde group into an imine group. The prepared PEG-DAC-DOX showed an N1s peak in the range of 397-403 eV ( Figure 2 c), indicating that the product contains nitrogen, further confirming the covalent binding of DAC-PEG and DOX.
[0036] Research generally believes that nanoparticles with a particle size of less than 500 nm can achieve passive targeted enrichment in tumor tissues by virtue of the EPR effect, but have no such effect in normal tissues. Figure 3 As shown in c, DOX-DAC-PEG is spherical with an average diameter of 40 to 80 nm and good stability in aqueous solution. This is conducive to the passive targeting of tumor tissues by the EPR effect, achieving targeted aggregation at the tumor, and improving the delivery efficiency and therapeutic effect. In addition, the dynamic light scattering (DLS) test results show that in a 10 mmol / L PBS solution, the Zeta potential value of the round DOX-DAC-PEG (0.01wt%) is -4eV ( Figure 3 d). Negatively charged DOX-DAC-PEG can avoid adsorption and aggregation with proteins and can exist stably in the blood.
[0037] like Figure 4 As shown, the UV-visible absorption and fluorescence spectra of DOX-DAC-PEG both showed the characteristic absorption peaks and fluorescence emission peaks of DOX, confirming that DOX was successfully grafted onto DAC through covalent bonds.
[0038] The nano drug carrier can be stably present in an alkaline environment or at a neutral pH value, but can quickly break the Schiff base structure at an acidic pH value, thereby releasing the drug. Figure 5 As shown in the figure, the drug release behavior of DOX-DAC-PEG nanoparticles showed obvious pH-sensitive characteristics, and DOX was released faster in acidic media (pH 5.5 and 3.0) than in pH 7.4. At pH 7.4, the cumulative release efficiency of DOX-DAC-PEG nanoparticles was 16% after 10 h and 27% after 48 h; at pH 5.5, the Schiff base bonds were broken, and DOX was released by 25% after 10 h and 50% after 48 h; at pH 3.0, the Schiff base bonds were broken in large quantities, resulting in faster DOX release, 58% after 10 h and 88% after 48 h. The pH-responsive drug release behavior of DOX-DAC-PEG nanocarriers will be beneficial to the targeted release of DOX at tumor sites.
[0039] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. A method for preparing a bio-based nano drug carrier with reversible covalent bond controlled sustained release, characterized in that: The following steps are involved: Step 1, oxidizing cellulose nanocrystals to obtain dialdehyde cellulose nanoparticles; Step 2, reacting dialdehyde cellulose nanoparticles, methoxy polyethylene glycol amine, and sodium cyanoborohydride to obtain cellulose nanoparticles grafted with methoxy polyethylene glycol side chains; Step 3, adding the amino-containing anticancer drug to the dispersion of cellulose nanoparticles grafted with methoxypolyethylene glycol side chains, so that the amino groups in the amino-containing anticancer drug and the aldehyde groups on the surface of the cellulose nanoparticles are covalently bonded through a Schiff base reaction, thereby obtaining a reversible covalent bond-controlled sustained-release bio-based nano-drug carrier.
2. The method for preparing a bio-based nano drug carrier with reversible covalent bond controlled sustained release according to claim 1, characterized in that: The method of oxidizing cellulose nanocrystals to obtain dialdehyde cellulose nanoparticles in step 1 is as follows: cellulose nanocrystals, sodium periodate, zinc chloride and water are added to a round-bottom flask; then, the oxidation reaction is carried out under stirring at room temperature in a light-shielded environment, and ethylene glycol is added to quench the residual periodate to terminate the reaction; thereafter, purified dialdehyde cellulose nanoparticles are obtained after dialyzing in water; finally, the supernatant is collected and freeze-dried to obtain dry dialdehyde cellulose nanoparticles.
3. The method for preparing a bio-based nano drug carrier with reversible covalent bond controlled sustained release according to claim 2, characterized in that: The weight components of cellulose nanocrystal, sodium periodate, zinc chloride and water are respectively: 100 parts of water, 0.5-2 parts of cellulose nanocrystal, 3-12 parts of sodium periodate and 0.5-2 parts of zinc chloride.
4. The method for preparing a bio-based nano drug carrier with reversible covalent bond controlled sustained release according to claim 3, characterized in that: The oxidation reaction is carried out by stirring at a speed of 200-300 rpm for 60-84 hours at room temperature in a light-shielded environment.
5. The method for preparing a bio-based nano drug carrier with reversible covalent bond controlled sustained release according to claim 4, characterized in that: The preparation method of cellulose nanoparticles grafted with methoxy polyethylene glycol side chains in step 2 is: take dry dialdehyde cellulose nanoparticles and evenly disperse them in N, N-dimethylformyl solvent, stir and heat and maintain, then add methoxy polyethylene glycol amine PEG-NH2 to react for a period of time, and finally add sodium cyanoborohydride to continue constant temperature reaction for a period of time, and obtain cellulose nanoparticles grafted with methoxy polyethylene glycol side chains after dialysis treatment.
6. The method for preparing a bio-based nano drug carrier with reversible covalent bond controlled sustained release according to claim 5, characterized in that: The concentration of the dialdehyde cellulose nanoparticle dispersion is 2-3 mg / mL.
7. The method for preparing a bio-based nano drug carrier with reversible covalent bond controlled sustained release according to claim 6, characterized in that: The mixture was heated to 25-35° C. with stirring, and after being fully dissolved and mixed, 25-35 parts of methoxypolyethylene glycol amine was added to the dialdehyde cellulose nanoparticle dispersion maintained at 25-35° C. with stirring, and the reaction was continued for 12 hours; then 0.5-1.5 parts of sodium cyanoborohydride were added and the reaction was continued at a constant temperature for 12 hours; and then the unreacted reagents and impurities were removed by dialysis for three days to finally obtain cellulose nanoparticles grafted with methoxypolyethylene glycol side chains.
8. The method for preparing a bio-based nano drug carrier with reversible covalent bond controlled sustained release according to claim 7, characterized in that: 1-3 parts of amino-containing anticancer drugs are added to 0.5-1.5 parts of cellulose nanoparticle dispersion grafted with methoxypolyethylene glycol side chains, and the resulting mixture is oscillated in the dark at room temperature for 12-36 hours; after completion, the sample is transferred into an ultrafiltration tube, and centrifuged at a relative centrifugal force of 3000-5000rcf for 20-30 minutes to remove the non-covalently bound anticancer drug molecules, and the centrifugal washing is repeated several times until the clear liquid under the ultrafiltration tube is clear, and finally a bio-based nano drug carrier with reversible covalent bond-controlled sustained release is obtained.
9. The method for preparing a bio-based nano drug carrier with reversible covalent bond controlled sustained release according to claim 8, characterized in that: The amino-containing anticancer drug is one of doxorubicin hydrochloride, amine quinazoline, eribulin and gemcitabine.