A bortezomib-loaded nanogel drug delivery system and its preparation method

Abstract

The present invention belongs to the technical field of drug carriers, and particularly relates to a nano-gel drug delivery system loaded with bortezomib and a preparation method thereof. The present invention uses a gelatin-dopamine (Gel-Dopa) complex and oxidized hyaluronic acid (OHA) as substrates to construct a nano-gel-based drug delivery system loaded with the chemotherapeutic drug BTZ. According to the characteristics of low pH in tumor tissues and overexpression of MMP-2 enzyme, a nano-gel with dual sensitivity to pH and MMP-2 enzyme is designed, and at the same time, the active targeting ability of HA is combined to improve the delivery efficiency of BTZ to tumor tissues, reduce the toxic and side effects of BTZ on normal cell tissues, and achieve controlled release of the drug.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug carriers, and particularly relates to a nano-gel drug delivery system loaded with bortezomib and a preparation method thereof. Background Art

[0002] Disclosing the information of this background art section is only intended to increase the understanding of the overall background of the present invention, and is not necessarily regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Bortezomib (BTZ) is a common chemotherapeutic drug, a 26S proteasome inhibitor, which can cause the death of tumor cells by blocking the degradation of various proteins that regulate apoptosis and signal transduction in cells. Research shows that BTZ has a therapeutic effect on a variety of tumor cells, such as multiple myeloma, lymphoma, lung cancer, prostate cancer, breast cancer, etc. However, BTZ has low water solubility, and the boric acid group in its structure can chelate with certain active functional groups present in plasma proteins, resulting in poor stability and high blood toxicity.

[0004] Delivery of chemotherapeutic drugs based on nano-carriers is currently one of the effective ways to enhance the targeting of chemotherapeutic drugs and reduce their toxic and side effects on normal cells. Nano-drug carriers mainly include nano-gels, polymer micelles, liposomes, metal nanoparticles, dendrimers, etc. Among them, nano-gels are a systemic drug delivery carrier, a hydrogel with a three-dimensional (3D) porous structure, and its three-dimensional structure can encapsulate hydrophobic or hydrophilic drugs in the internal network, thereby protecting these drugs from hydrolysis or enzymatic hydrolysis during storage or in vivo delivery. Through certain design, nano-gels can have the characteristics of responding to environmental stimuli such as pH, temperature, enzymes, redox potential, ultrasound, magnetic field, etc. When the environmental conditions change, the structural conformation of the nano-gel will change and then release the drug. This characteristic can achieve the targeted release of drugs, reduce the release of drugs in non-target tissues, and is beneficial to reducing the side effects of drugs.

[0005] The characteristics of rapid growth, invasion and metastasis of tumor cells make the environment around and inside tumor tissues different from normal cell tissues to a certain extent. For example, the pH value at the tumor site is low, the temperature is high, MMP-2 enzyme is overexpressed, and the glutathione concentration is high. Due to this particularity, the stimulus-responsive characteristics of nano-gels have been widely used in targeted delivery to tumor sites and have shown good therapeutic effects. However, single-stimulus-responsive nano-gels are usually greatly affected by the environment and cannot show obvious stimulus-responsive characteristics in a complex environment, having certain limitations. Summary of the Invention

[0006] To address the deficiencies of the prior art, the objective of the present invention is to provide a bortezomib-loaded nanogel drug delivery system and its preparation method. The present invention constructs a nanogel-based drug delivery system loaded with the chemotherapeutic drug BTZ using a gelatin-dopamine (Gel-Dopa) complex and oxidized hyaluronic acid (OHA) as substrates. Based on the characteristics of low pH in tumor tissues and overexpression of MMP-2 enzyme, a nanogel with dual sensitivity to pH and MMP-2 enzyme is designed. Meanwhile, combined with the active targeting ability of HA, the delivery efficiency of BTZ to tumor tissues is improved, the toxic and side effects of BTZ on normal cell tissues are reduced, and the controlled release of the drug is achieved.

[0007] To achieve the above objective, the present invention is realized through the following technical solutions:

[0008] In the first aspect, the present invention provides a preparation method for a bortezomib-loaded nanogel drug delivery system, comprising the following steps:

[0009] S1. Dissolve span 80 in cyclohexane, stir evenly to obtain an oil phase;

[0010] S2. Dissolve oxidized hyaluronic acid in a borax solution, stir until completely dissolved to obtain an oxidized hyaluronic acid-borax solution. Drop the oxidized hyaluronic acid-borax solution into the oil phase under rapid stirring, stir for coarse emulsification, and perform ultrasonic fine emulsification to obtain emulsion A;

[0011] S3. Dissolve the gelatin-dopamine complex in water to obtain a gelatin-dopamine complex solution, dissolve bortezomib in DMSO to obtain a bortezomib solution. Drop the bortezomib solution into the gelatin-dopamine complex solution under stirring conditions, and stir in the dark to obtain a gelatin-dopamine-bortezomib solution. Drop the gelatin-dopamine-bortezomib solution into the oil phase under rapid stirring, stir for coarse emulsification, and perform ultrasonic fine emulsification to obtain emulsion B;

[0012] S4. Mix emulsion A and emulsion B, perform ultrasonic treatment with an ultrasonic crusher to obtain a nanogel emulsion. Drop the nanogel emulsion into acetone for washing, centrifuge to remove the supernatant, wash the precipitate with acetone and water respectively, and perform freeze-drying to obtain a bortezomib-loaded nanogel drug delivery system.

[0013] In the second aspect, the present invention provides a bortezomib-loaded nanogel drug delivery system, characterized in that it is obtained by the preparation method described in the first aspect.

[0014] The beneficial effects obtained by one or more of the above technical solutions of the present invention are as follows:

[0015] In view of the special characteristics of the low pH value of the tumor microenvironment and the overexpression of MMP-2 enzyme, a nano-gel drug delivery system with dual responsiveness to pH and MMP-2 enzyme was designed. Meanwhile, by combining the property of HA actively targeting the CD44 receptor overexpressed on tumor cells, the combination of passive targeting and active targeting of tumor cells was achieved.

[0016] With the decrease of pH, the cumulative drug release amount of the nano-gel drug delivery system loaded with bortezomib showed an increasing trend, demonstrating obvious pH-responsive characteristics. After co-incubating the nano-gel drug delivery system loaded with bortezomib with MMP-2 enzyme for a period of time, it was found that the particle size distribution of the nano-gel drug delivery system loaded with bortezomib became wider and small-sized particles appeared, confirming its MMP-2 enzyme-responsive characteristics.

[0017] The nano-gel drug delivery system loaded with bortezomib had good blood compatibility (hemolysis rate < 5%); the results of the protein adsorption experiment showed that within 24 hours, the adsorption rate of the nano-gel drug delivery system loaded with bortezomib to BSA was less than 15%, indicating good injection safety. The cytotoxicity experiment, cell uptake experiment, and apoptosis experiment showed that the nano-gel drug delivery system loaded with bortezomib could be taken up by MCF-7 cells and exert a tumor-killing effect similar to that of free BTZ. Description of the Drawings

[0018] The specification drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0019] Figure 1 In which, a is the standard curve of Dopa, and b is the ultraviolet spectral scanning results of Gel, Dopa, and the Gel-Dopa complex in Example 1;

[0020] Figure 2 is the FT-IR spectrum of Gel and the Gel-Dopa complex in Example 1;

[0021] Figure 3 is the concentration-absorbance standard curve of the reaction between glutaraldehyde and Schiff reagent;

[0022] Figure 4 is for HA and OHA in Example 1 1 1H-NMR spectrum;

[0023] Figure 5 is the influence of ultrasonic time on the particle size and PDI of the mixed emulsion in Example 2;

[0024] Figure 6FT-IR spectra (a) and their partial enlarged views (b) of Gel-Dopa, BTZ, and Gel-Dopa-BTZ in Example 3;

[0025] Figure 7 1H-NMR spectra of Gel-Dopa, BTZ, Gel-Dopa-BTZ, and the Gel-Dopa / BTZ mixture in Example 3; 1

[0026] Figure 8 SEM images (a) and particle size distribution diagrams (b) of NG-BTZ in Example 4;

[0027] Figure 9 Standard curve of concentration-absorbance of BTZ;

[0028] Figure 10 Drug cumulative release curves of free BTZ (a) and NG-BTZ (b) at different pH values;

[0029] Figure 11 Particle size distribution results of NG-BTZ before (a) and after (b) incubation with MMP-2 enzyme;

[0030] Figure 12 Hemolysis experiment images (a) and hemolysis rate result diagrams (b) of NG-BTZ solutions with different concentrations;

[0031] Figure 13 In which, a is the standard curve of concentration-absorbance of BSA, and b is the result of the protein adsorption experiment of NG-BTZ;

[0032] Figure 14 Cytotoxicity results of NG-BTZ and free BTZ against MCF-7 cells;

[0033] Figure 15 Live / dead cell staining diagrams of MCF-7 cells co-incubated with free BTZ and NG-BTZ;

[0034] Figure 16 Results of the cell uptake experiment of MCF-7 cells co-incubated with free C6 and NG-C6;

[0035] Figure 17 Intracellular fluorescence intensities of MCF-7 cells co-incubated with the free C6 group, NG-C6 group, and HA-NG-C6 group;

[0036] Figure 18 Results of the apoptotic status of MCF-7 cells co-incubated with the free BTZ group and NG-BTZ group; Detailed implementation manners ​

[0037] In the first typical embodiment of the present invention, a preparation method of a bortezomib-loaded nanogel drug delivery system includes the following steps:

[0038] S1. Dissolve Span 80 in cyclohexane and stir evenly to obtain an oil phase;

[0039] S2. Dissolve oxidized hyaluronic acid in a borax solution, stir until completely dissolved to obtain an oxidized hyaluronic acid-borax solution, and drop the oxidized hyaluronic acid-borax solution into the oil phase under rapid stirring, stir for coarse emulsification, and perform ultrasonic fine emulsification to obtain emulsion A;

[0040] S3. Dissolve the gelatin-dopamine complex in water to obtain a gelatin-dopamine complex solution, dissolve bortezomib in DMSO to obtain a bortezomib solution, drop the bortezomib solution into the gelatin-dopamine complex solution under stirring conditions, and stir in the dark to obtain a gelatin-dopamine-bortezomib solution. The gelatin-dopamine-bortezomib solution is dropped into the oil phase under rapid stirring, stirred for coarse emulsification, and ultrasonically finely emulsified to obtain emulsion B;

[0041] S4. Mix emulsion A and emulsion B, perform ultrasonic treatment with an ultrasonic crusher to obtain a nanogel emulsion, drop the nanogel emulsion into acetone for washing, centrifuge to remove the supernatant, wash the precipitate with acetone and water respectively, and freeze-dry to obtain a bortezomib-loaded nanogel drug delivery system.

[0042] Gel is one of the substrates of matrix metalloproteinase-2 (MMP-2) overexpressed in tumor sites, which endows the system with the property of MMP-2 enzyme response; HA can actively target tumor cells by specifically binding to CD44, a transmembrane glycoprotein receptor overexpressed on the surface of tumor cells. Gelatin-dopamine (Gel-Dopa) complex and oxidized hyaluronic acid (OHA) were prepared by graft modification. OHA can be used as a cross-linking agent, and the Schiff base reaction between the aldehyde group of OHA and the amino group of Gel-Dopa can be used to cross-link and construct nanogels. The grafting of Dopa introduces catechol groups into the system, and these groups can connect with the boric acid groups of BTZ to form pH-responsive borate bonds, which not only realizes the encapsulation of drugs but also endows the system with pH-responsive properties.

[0043] In one or more embodiments of this embodiment, the oxidation degree of the oxidized hyaluronic acid is 15-20%.

[0044] In one or more embodiments of this embodiment, the preparation method of the oxidized hyaluronic acid is:

[0045] Dissolve 2 g of hyaluronic acid in 200 mL of deionized water to obtain a hyaluronic acid solution, and dissolve 1.128 g of sodium periodate in deionized water to obtain a 0.5 mol / L sodium periodate solution; add the sodium periodate solution dropwise to the hyaluronic acid solution, stir for 6 h in the dark, then add ethylene glycol with a molar amount three times that of sodium periodate, and continue to stir the mixed solution for 1 h; place the stirred mixed solution in deionized water for dialysis for three days, and after dialysis, freeze-dry to obtain oxidized hyaluronic acid.

[0046] In one or more embodiments of this embodiment, the content of dopamine in the gelatin-dopamine complex is 99.3 - 109.1 μmol / g.

[0047] In one or more embodiments of this embodiment, the preparation method of the gelatin-dopamine complex is as follows:

[0048] Dissolve 1 g of gelatin in 100 mL of deionized water at 60 °C and stir for 30 min to obtain a gelatin solution; after the gelatin solution is cooled to room temperature, add 0.5 g of EDC and stir for 15 min, then add 0.3 g of NHS and stir for 15 min to obtain a mixed solution; dissolve 1.0 g of dopamine hydrochloride in 2 mL of deionized water and add it dropwise to the mixed solution, stir at room temperature for 24 h in the dark, filter to obtain a filtrate, dialyze it in acidified deionized water with a pH of 3 for 24 hours, then dialyze it in deionized water for 48 hours, and after cooling and drying, obtain the gelatin-dopamine complex.

[0049] In one or more embodiments of this embodiment, in step S1:

[0050] The content of span 80 in the oil phase is 1 - 3%;

[0051] The stirring time is 10 - 20 min.

[0052] In one or more embodiments of this embodiment, in step S2:

[0053] The concentration of the borax solution is 1 - 2 mol / L;

[0054] The concentration of oxidized hyaluronic acid in the oxidized hyaluronic acid-borax solution is 40 - 60 mg / mL;

[0055] The volume ratio of the oxidized hyaluronic acid-borax solution to the oil phase is 1 - 3:40;

[0056] The stirring time is 10 - 20 min;

[0057] The ultrasonic time is 15 - 30 min.

[0058] In one or more embodiments of this embodiment, in step S3:

[0059] The concentration of the gelatin-dopamine complex in the gelatin-dopamine complex solution is 40-60 mg / mL;

[0060] The concentration of bortezomib in the bortezomib solution is 6-10 mg / mL;

[0061] The mass ratio of bortezomib to the gelatin-dopamine complex is 1:5-10;

[0062] The volume ratio of the gelatin-dopamine-bortezomib solution to the oil phase is 1-3:40;

[0063] The stirring time is 10-20 min;

[0064] The ultrasonic time is 15-30 min.

[0065] In one or more embodiments of this embodiment, in step S4:

[0066] The volume ratio of emulsion A to emulsion B is 1:1;

[0067] The ultrasonic time is 6-48 min.

[0068] The second typical embodiment of the present invention, a nano-gel drug delivery system loaded with bortezomib, is obtained by the preparation method described in the first typical embodiment.

[0069] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in combination with specific examples and comparative examples.

[0070] In the present invention: Gel is gelatin; Gel-Dopa is a gelatin-dopamine complex; HA is hyaluronic acid; OHA is oxidized hyaluronic acid; MMP-2 is matrix metalloproteinase-2; EDC is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; NHS is N-hydroxysuccinimide; DMSO is dimethyl sulfoxide; FBS is fetal bovine serum; BTZ is bortezomib; Gel-Dopa-BTZ is a gelatin-dopamine-bortezomib complex; BSA is bovine serum albumin; C6 is coumarin 6; NG-BTZ is a nano-gel drug delivery system loaded with bortezomib.

[0071] Example 1

[0072] Preparation of Gel-Dopa Complex

[0073] At 60 °C, 1 g of porcine skin type A gelatin (Gel) was dissolved in 100 mL of deionized water and stirred for 30 min to obtain an aqueous Gel solution. After the solution was cooled to room temperature in air, 0.5 g of EDC was added and stirred for 15 min, then 0.3 g of NHS was added and stirred for 15 min. Subsequently, 1.0 g of dopamine hydrochloride in 2 mL of deionized water was added dropwise to the mixed solution. After stirring in the dark at 37 °C for 24 h, a small amount of precipitate was filtered off, and the filtrate was dialyzed in acidified deionized water with a pH of 3 for 24 h, and then dialyzed in deionized water for 48 h. The molecular weight cut-off of the dialysis bag was 7000 Da, and freeze-dried at -80 °C for 48 h to obtain the Gel-Dopa complex.

[0074] The Dopa solution was scanned by ultraviolet spectroscopy, and the absorbance of Dopa standard solutions at different concentrations was measured at the maximum absorption peak of Dopa. A standard curve of Dopa was plotted with the concentration of Dopa as the abscissa and the absorbance as the ordinate, as shown in Figure 1 a of. By comparing the absorbance of Gel-Dopa with the Dopa standard curve, the grafting degree of Dopa in Gel-Dopa was obtained. As shown in Figure 1 b of, Gel did not show an obvious ultraviolet characteristic absorption peak at 280 nm, while Dopa showed an obvious ultraviolet characteristic absorption peak at 280 nm. Gel-Dopa showed a strong ultraviolet absorption peak at 280 nm, and this absorption peak belonged to the catechol structure of Dopa. This result indicated that Dopa was successfully grafted onto the Gel molecule. According to the standard curve shown in Figure 1 a of, the content of Dopa in the Gel-Dopa complex was calculated to be 104.2 ± 4.9 μmol / g.

[0075] As shown in Figure 2 , the characteristic peaks of the Gel-Dopa complex included the characteristic peaks of Gel, indicating that the grafting of Dopa did not affect the overall structure of Gel. In addition, in the spectrum of the Gel-Dopa complex, a new characteristic absorption peak belonging to the benzene ring of the Dopa molecule appeared at 1450 cm -1 ; a stretching vibration peak belonging to the C-N bond appeared at 1238 cm -1 . This indicated that the Gel-Dopa complex was successfully prepared by the EDC / NHS coupling reaction.

[0076] Preparation of OHA

[0077] At room temperature, 2.0 g of HA was dissolved in 200 mL of deionized water and stirred until completely dissolved. 1.128 g of sodium periodate (NaIO4) was weighed and dissolved in a certain amount of deionized water to make a 0.5 mol / L solution. Subsequently, the NaIO4 solution was added dropwise to the HA solution, and the mixture was stirred in the dark for 6 h. A certain amount of ethylene glycol (three times the molar amount of sodium periodate) was added to the reaction solution to quench the unreacted NaIO4, and stirring was continued for 1 h to end the reaction. The reaction solution was dialyzed in deionized water for three days, and the fresh deionized water was replaced regularly. The molecular weight cut-off of the dialysis bag was 7000 Da. After dialysis, it was freeze-dried in a freeze dryer at -80 °C for 48 h to obtain the product OHA.

[0078] The oxidation degree of OHA was determined using Schiff reagent. The standard curve of the reaction of glutaraldehyde with Schiff reagent is shown as Figure 3 shown. By referring to this standard curve, the oxidation degree of OHA was calculated to be 16.19%.

[0079] As Figure 4 shown, by comparing the 1 1H-NMR spectra of HA, in the spectrum of OHA, three new signal peaks appeared at around 5.0 ppm. These may be the proton peaks of the stable hemiacetal formed by the combination of the generated aldehyde group and the hydroxyl group on the ring, or may be due to the chemical shift change of the hydrogen atoms adjacent to the aldehyde group caused by the presence of the aldehyde group, proving that HA was oxidized to OHA.

[0080] Example 2

[0081] The preparation of the Gel-Dopa complex and OHA was the same as in Example 1.

[0082] Preparation of blank nanogel (NG)

[0083] Weigh a certain amount of surfactant Span 80 into a beaker, add 10 mL of cyclohexane, and stir for 10 minutes to obtain an oil phase. Weigh a certain amount of OHA and dissolve it in a 1 mol / L borax solution until it is completely dissolved. The OHA concentration in the solution is 50 mg / mL. Take a certain amount of OHA solution as the water phase and add it to the oil phase, stirring it dropwise while stirring rapidly. Stir for 15 minutes for rough emulsification, and then use an ultrasonic crusher to sonicate for 20 minutes to finely emulsify to obtain emulsion A. Weigh a certain amount of Gel-Dopa and dissolve it in deionized water to completely dissolve to obtain a 50 mg / mL Gel-Dopa solution. Similarly, take a certain amount of Gel-Dopa solution as the water phase and add it to the oil phase, stirring it dropwise while stirring rapidly. Stir for 15 minutes for rough emulsification, and then use an ultrasonic crusher to sonicate for 20 minutes to finely emulsify to obtain emulsion B. Mix emulsions A and B, and then use an ultrasonic crusher to ultrasonicate to obtain a nanogel emulsion. The emulsion is added dropwise to 80 mL of acetone for washing, and then centrifuged at 4000 rpm for 10 min, the supernatant is removed, and then acetone is repeated twice, and water is repeated for washing, and finally freeze-dried at -80 ° C for 48 hours to obtain NG powder. Explore the effect of ultrasonic time after mixing emulsions A and B on the particle size and PDI of the mixed emulsion, and investigate the effects of surfactant concentration and water-oil ratio on nanogel particle size and PDI through single factor experiments, and optimize.

[0084] like Figure 5 As shown in the figure, between 0 and 18 minutes, with the increase of ultrasonic time, the particle size and PDI of NG both show a significant decreasing trend. The fusion of Gel-Dopa emulsion droplets and OHA emulsion droplets is rapid. In the early stage of fusion, the particle size of the emulsion droplets is large. Under the cavitation and shear force of ultrasound, the particle size of the mixed emulsion droplets gradually decreases. When the ultrasonic time is between 18 and 30 minutes, the size of the emulsion droplets remains basically stable, and the PDI still shows a downward trend, indicating that after the continuous fusion and fission process induced by high shear, the size of the emulsion has reached a stable state, and the polydispersity of the emulsion gradually decreases, indicating that the uniformity of the emulsion is constantly increasing. However, when the ultrasonic action time is greater than 30 minutes, the particle size and PDI of the emulsion both show a significant upward trend. This may be because excessive ultrasonic treatment accelerates the collision between droplets, causing small droplets to re-aggregate into large droplets. It may also be because more heat is generated during the ultrasonic action process, causing the Gel to denature, thereby increasing the instability of the emulsion and causing droplet aggregation.

[0085] Table 1 shows the experimental results of optimizing the preparation conditions of NG using a single factor test. The ultrasonic time and the volume of the oil phase were fixed, and the effects of surfactant concentration and water phase content on the NG particle size and PDI size were explored. As can be seen from the table, when the surfactant content is consistent, the larger the water phase volume, the larger the NG particle size; when the water phase volume remains consistent, the higher the surfactant content, the smaller the NG particle size. Within a certain range, a sufficient amount of surfactant can stabilize the droplets, while when the surfactant content is too low, the emulsion becomes unstable, resulting in the combination of droplets and an increase in the droplet size.

[0086] Table 1 Optimization results of NG preparation conditions

[0087]

[0088] Example 3

[0089] The preparation of Gel-Dopa complex is the same as that in Example 1.

[0090] Preparation of Gel-Dopa-BTZ

[0091] Accurately weigh 50 mg of freeze-dried Gel-Dopa complex, dissolve in 10 mL of water, and fully dissolve to obtain a 5 mg / mL Gel-Dopa complex solution. Accurately weigh 8 mg of bortezomib (BTZ) powder, dissolve in 1 mL of DMSO, and fully dissolve to obtain a BTZ solution. Slowly add the BTZ solution to the Gel-Dopa complex solution, stirring while adding, and react for 24 hours under light-proof conditions. Then transfer the reaction solution to a dialysis bag (molecular weight cutoff of 7000 Da), dialyze in a pH 8 buffer for 2 days, and change the dialysate regularly to remove DMSO and unreacted BTZ. After the dialysis is completed, freeze dry it at -80 ° C for 48 hours using a freeze dryer to obtain the product Gel-Dopa-BTZ.

[0092] like Figure 6 As shown in the figure, the main characteristic peaks of the FI-IR spectra of Gel-Dopa and Gel-Dopa-BTZ are consistent, indicating that the grafting of BTZ did not destroy the overall structure of Gel-Dopa. Figure 6 As shown in b, 1402cm -1 The stretching vibration peak of the BO bond of free BTZ appeared at 1355 cm-1. -1 This indicates that the B-OH group of free BTZ reacted with the catechol group of Dopa to form a borate ester bond, and Gel-Dopa-BTZ was successfully composited.

[0093] Characterizations of Gel-Dopa, BTZ, Gel-Dopa-BTZ, and the simple mixture of Gel-Dopa / BTZ were performed 1 by 1H-NMR, as Figure 7 shown. By observing and comparing the spectra of Gel-Dopa and Gel-Dopa-BTZ, it was found that the integral of the protons attributed to the catechol ring at 6.62 ppm - 6.41 ppm decreased and shifted to 5.74 ppm - 5.58 ppm. Comparing the spectra of the Gel-Dopa-BTZ complex and the simple mixture of Gel-Dopa / BTZ, obvious differences were found between the two spectra, and the shift of the catechol ring signal peak did not occur in the latter.

[0094] Example 4

[0095] The preparation of the Gel-Dopa complex and OHA was the same as in Example 1.

[0096] Preparation of the bortezomib-loaded nanogel drug delivery system (NG-BTZ)

[0097] Weigh 0.2 g of Span 80 into a beaker, add 10 mL of cyclohexane, and stir for 10 min to obtain the oil phase. Weigh a certain amount of OHA and dissolve it in 1 mol / L borax solution until completely dissolved to form an OHA solution with a concentration of 50 mg / mL. Pipette 500 μL of the OHA solution as the aqueous phase and add it to 10 mL of the oil phase while stirring rapidly. Stir for 15 min for coarse emulsification, and then ultrasonicate with an ultrasonic crusher for 20 min for fine emulsification to obtain Emulsion A. Weigh 50 mg of the Gel-Dopa complex and dissolve it in 10 mL of water to fully dissolve and obtain a 5 mg / mL Gel-Dopa complex solution. Accurately weigh 8 mg of bortezomib (BTZ) powder and dissolve it in 1 mL of DMSO to fully dissolve and obtain a BTZ solution. Slowly add the BTZ solution dropwise to the Gel-Dopa complex solution while stirring, and react for 24 hours under dark conditions to obtain a mixed solution. 500 μL of the mixed solution is used as the aqueous phase and added to 10 mL of the oil phase while stirring rapidly. Stir for 15 min for coarse emulsification. Then ultrasonicate with an ultrasonic crusher for 20 min for fine emulsification to obtain Emulsion B; mix Emulsions A and B, and then ultrasonicate with an ultrasonic crusher to obtain a nanogel emulsion. Drop the emulsion into 80 mL of acetone for washing, then centrifuge at 4000 rpm for 10 min to remove the supernatant, and repeat twice with acetone and wash with water as described above. Finally, freeze-dry at -80 °C for 48 hours to obtain NG-BTZ powder.

[0098] As Figure 8As shown, NG-BTZ presents a spherical shape with a particle size of 254.4 ± 13.2 nm and a uniform particle size distribution. Compared with NG in Example 2, the particle size of NG-BTZ increases, which may be due to the presence of the drug BTZ, resulting in a reduction in the crosslinking between Gel-Dopa and OHA. The standard curve of BTZ is as Figure 9 shown, and the drug loading of NG-BTZ is calculated to be (2.00 ± 0.09)%.

[0099] Environments with pH values of 7.4, 6.5, and 5.0 are used to simulate the normal human tissue and blood environment, the extracellular microenvironment of tumor cells, and lysosomes respectively, and the cumulative drug release of NG-BTZ and free BTZ under different pH conditions is compared. As Figure 10 shown in a of Figure 10 , there is no significant difference in the cumulative release curves of free BTZ at the three pH values, that is, the release of free BTZ does not have pH-responsive characteristics. As

[0100] shown in b of Figure 11 , the release rate and cumulative release amount of NG-BTZ releasing BTZ increase with the decrease of pH. NG-BTZ has pH-responsive characteristics and is more likely to release drugs under acidic conditions, which is beneficial to the targeted delivery of anticancer drugs.

[0101] The blood compatibility results of NG-BTZ are as Figure 12 shown. It can be seen from the figure that compared with the positive control group (water), the supernatants of the negative control group (PBS) and the NG-BTZ experimental group are both clear and transparent, without obvious hemolysis phenomenon. After calculation, with the increase of the concentration of NG-BTZ, the hemolysis rate shows an increasing trend. In the range of 0.1 mg / mL - 2 mg / mL, the hemolysis rate of NG-BTZ is less than 5%, showing a significant difference from the positive control group. NG-BTZ basically has no hemolytic toxicity and has good blood compatibility during intravenous injection.

[0102] The adsorption of NG-BTZ to proteins in BSA solution is used to simulate the adsorption of NG-BTZ to proteins in blood. The standard curve of BSA is as Figure 13 shown in a of Figure 13As shown in b, within 24 hours, the adsorption rate of NG-BTZ to BSA was less than 15%, remaining at a low level. This indicates that NG-BTZ can maintain a certain stability in the BSA solution, which is beneficial for the circulation of NG-BTZ in the blood.

[0103] The results of the cytotoxicity experiments of free BTZ and NG-BTZ on MCF-7 cells are as Figure 14 shown. With the increase in the concentration of BTZ, the toxicity of free BTZ and NG-BTZ to cells showed a significant increasing trend. Compared with free BTZ, the cytotoxicity of NG-BTZ was weaker. This may be because NG-BTZ needs to enter cells through endocytosis mediated by the CD44 receptor, and then, under the action of the slightly acidic intracellular environment, the borate ester bond breaks to release the drug. While the free drug BTZ only needs to enter cells through diffusion to exert its effect. Therefore, within the same time, the killing effect of free BTZ on cells is better. Generally speaking, NG-BTZ can effectively deliver the drug BTZ into tumor cells and play a certain killing role on tumor cells.

[0104] The free BTZ group and the NG-BTZ group with a drug concentration of 25 μg / mL were selected for the live-dead cell staining experiment. As Figure 15 shown, when the drug concentration was 25 μg / mL, the effects of the free BTZ group and the NG-BTZ group were similar, and NG-BTZ could play a good anti-tumor role.

[0105] Since BTZ has no fluorescence, C6 was used instead of BTZ to prepare NG-C6 for cell uptake and active targeting verification. As Figure 16 shown, the appearance of green fluorescence could be observed in both the free C6 group and the NG-C6 group, and in their Merged images, it could be seen that the green fluorescence was evenly dispersed around the blue fluorescence. This indicates that both free C6 and C6 embedded in the nanogel can enter the cells. As Figure 17 shown, the fluorescence intensity of the group saturated with HA (HA-NG-C6) decreased compared with that of the NG-C6 group without HA saturation. This shows that after MCF-7 cells were co-incubated with HA solution, HA bound to the CD44 receptor on the surface of MCF-7 cells, resulting in a slower rate of NG-C6 entering the cells, that is, the cells reduced the uptake of NG-C6, leading to a decrease in the intracellular fluorescence intensity finally. This indicates that NG-BTZ has the characteristic of actively targeting the overexpressed CD44 receptor on the surface of tumor cells, and this characteristic is beneficial for the uptake of NG-BTZ by cancer cells.

[0106] The process of NG-BTZ-induced apoptosis was explored by observing the state of the cell nuclei of the free BTZ and NG-BTZ groups co-incubated with MCF-7 cells at different magnifications after 24 hours, and the results are asFigure 18 As shown, under the 10X objective lens, it can be observed that the number of cells in the free BTZ group is close to that in the NG-BTZ group, and both are significantly less than that in the blank control group. Under the 20X objective lens, it is observed that the nuclear contours in the blank control group are clear, the morphology is complete, the blue fluorescence staining is uniform, and the nuclear pores are clearly visible. However, obvious changes occur in the nuclear morphology in both the free BTZ group and the NG-BTZ group. Some chromatin agglutinates, the nucleus shrinks, and even the nucleus ruptures, and the blue fluorescence staining is uneven. These phenomena are all characteristics of apoptosis. The results of the apoptosis experiment show that, like free BTZ, NG-BTZ can also induce apoptosis of MCF-7 cells and play a killing role on cancer cells.

[0107] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A preparation method of a bortezomib-loaded nanogel drug delivery system, characterized in that, It includes the following steps: S1. Dissolve Span 80 in cyclohexane and stir evenly to obtain an oil phase; S2. Dissolve oxidized hyaluronic acid in a borax solution, stir until completely dissolved to obtain an oxidized hyaluronic acid-borax solution, drop the oxidized hyaluronic acid-borax solution into the oil phase under rapid stirring, stir for coarse emulsification, and perform ultrasonic fine emulsification to obtain emulsion A; S3. Dissolve the gelatin-dopamine complex in water to obtain a gelatin-dopamine complex solution, dissolve bortezomib in DMSO to obtain a bortezomib solution, drop the bortezomib solution into the gelatin-dopamine complex solution under stirring conditions, and stir in the dark to obtain a gelatin-dopamine-bortezomib solution. The gelatin-dopamine-bortezomib solution is dropped into the oil phase under rapid stirring, stirred for coarse emulsification, and ultrasonically finely emulsified to obtain emulsion B; S4. Mix emulsion A and emulsion B, perform ultrasonic treatment with an ultrasonic crusher to obtain a nano-gel emulsion. The nano-gel emulsion is dropped into acetone for washing, centrifuged to remove the supernatant, and the precipitate is washed with acetone and water respectively, and freeze-dried to obtain a nano-gel drug delivery system loaded with bortezomib.

2. The preparation method according to claim 1, wherein The oxidation degree of the oxidized hyaluronic acid is 15-20%.

3. The preparation method according to claim 2, characterized in that, The preparation method of the oxidized hyaluronic acid is as follows: Dissolve 2 g of hyaluronic acid in 200 mL of deionized water to obtain a hyaluronic acid solution, and dissolve 1.128 g of sodium periodate in deionized water to obtain a 0.5 mol / L sodium periodate solution; Drop the sodium periodate solution into the hyaluronic acid solution, stir in the dark for 6 h, then add ethylene glycol with a molar amount three times that of sodium periodate, and continue to stir the mixed solution for 1 h; Place the stirred mixed solution in deionized water for dialysis for three days, and freeze-dry after dialysis to obtain oxidized hyaluronic acid.

4. The preparation method according to claim 1, characterized in that The dopamine content in the gelatin-dopamine complex is 99.3-109.1 μmol / g.

5. The preparation method according to claim 4, characterized in that, The preparation method of the gelatin-dopamine complex is as follows: Dissolve 1 g of gelatin in 100 mL of deionized water at 60 °C and stir for 30 min to obtain a gelatin solution; After the gelatin solution is cooled to room temperature, add 0.5 g of EDC and stir for 15 min, then add 0.3 g of NHS and stir for 15 min to obtain a mixed solution; Dissolve 1.0 g of dopamine hydrochloride in 2 mL of deionized water and drop it into the mixed solution, stir at room temperature in the dark for 24 h, filter to obtain a filtrate, dialyze it in acidified deionized water with a pH of 3 for 24 h, then dialyze it in deionized water for 48 h, and cool and dry to obtain a gelatin-dopamine complex.

6. The preparation method according to claim 1, characterized in that, In step S1: The content of Span 80 in the oil phase is 1-3%; The stirring time is 10-20 min.

7. The preparation method according to claim 1, characterized in that In step S2: The concentration of the borax solution is 1-2 mol / L; The concentration of oxidized hyaluronic acid in the oxidized hyaluronic acid-borax solution is 40-60 mg / mL; The volume ratio of the oxidized hyaluronic acid-borax solution to the oil phase is 1-3:40; The stirring time is 10-20 min; The ultrasonic time is 15-30 min.

8. The preparation method according to claim 1, wherein, In step S3: The concentration of the gelatin-dopamine complex in the gelatin-dopamine complex solution is 40-60 mg / mL; The concentration of bortezomib in the bortezomib solution is 6-10 mg / mL; The mass ratio of bortezomib to the gelatin-dopamine complex is 1:5-10; The volume ratio of the gelatin-dopamine-bortezomib solution to the oil phase is 1-3:40; The stirring time is 10-20 min; The ultrasonic time is 15-30 min.

9. The preparation method according to claim 1, characterized in that, In step S4: The volume ratio of emulsion A to emulsion B is 1:1; The ultrasonic time is 6-48 min.

10. A bortezomib-loaded nanogel drug delivery system, characterized in that, Obtained by the preparation method according to any one of claims 1-9.

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