Hyaluronic acid-dopamine-bortezomib polymer, preparation method and application thereof

Abstract

The present invention belongs to the technical field of nanoformulation, and discloses a hyaluronic acid-dopamine-bortezomib polymer, a preparation method and its application, wherein the hyaluronic acid-dopamine-bortezomib polymer is a pH-sensitive drug-carrying amphiphilic polymer HA-DA-BTZ, which is composed of a polymer carrier material hyaluronic acid HA, a small molecule connecting material dopamine DA and a therapeutic drug bortezomib BTZ. The pH-sensitive co-drug-carrying nanoparticles prepared by the present invention have an average particle size between 100-200nm, a simple preparation method, and good encapsulation efficiency and drug loading. The HA-DA-BTZ amphiphilic polymer material synthesized by the present invention can also be used as a drug-carrying platform to encapsulate hydrophobic drugs while realizing the connection to the BTZ drug; HA-DA-BTZ is connected by a boric acid ester bond, has good pH sensitivity, can ensure the responsive release of drugs at the tumor site, and exerts a better therapeutic effect.

Description

Technical Field

[0001] The invention belongs to the technical field of nano preparations, and in particular relates to a hyaluronic acid-dopamine-bortezomib polymer, a preparation method and an application thereof. Background Art

[0002] Tumors exist in a complex tumor microenvironment (TME), including tumor cells, fibroblasts, immune cells, and tumor extracellular matrix. The interactions between different components lead to tumor proliferation, drug resistance, and metastasis. Traditional chemotherapy drugs often fail to treat due to their single mechanism of action.

[0003] Bortezomib (BTZ) is a small molecule chemical drug with strong cytotoxicity to cancer cells. It can exert anti-tumor therapeutic effects by inhibiting proteasome expression. BTZ has been proven to have multiple pharmacological effects. In addition to its excellent direct killing effect on tumor cells, it can also inhibit the activity of CAFs through the pro-apoptotic protein Caspase-3 pathway; in addition, it can also inhibit the expression of MDR1 gene and membrane transport protein P-glycoprotein (P-gp) through the NF-κB pathway to reduce the treatment resistance caused by chemotherapy drugs. BTZ was approved for marketing by the US Food and Drug Administration (FDA) in 2003 for the first-line treatment of hematological cancers such as myeloma and mantle cell lymphoma.

[0004] Although in vitro experiments have confirmed that BTZ also has a good killing effect on a variety of solid tumors, due to the poor specificity of BTZ and its easy binding to plasma proteins in the blood, it is difficult for the drug to aggregate and exert its effect in solid tumors; in addition, BTZ has strong toxic side effects (neurotoxicity, thrombocytopenia), which limits its clinical application in solid tumors. Therefore, improving the drugability of BTZ, reducing its toxic side effects, and applying it to solid tumors has important clinical value.

[0005] In recent years, nano-delivery systems have been widely used in tumor treatment and have become an inevitable trend in the future development of drug preparations. Among them, polymer materials are a common type of drug carrier for the preparation of nano-delivery systems. Generally, the hydrophilic and hydrophobic ends contained in their structures are used to form nanoparticles through self-aggregation. On the one hand, the nanoparticles can improve the apparent solubility of hydrophobic drugs and improve the drugability of drugs. On the other hand, they can also improve the efficiency of drug delivery to tumors, reduce the toxic side effects of drugs, and achieve efficient release of drugs of different properties at the tumor site.

[0006] Publication No. CN 118105509 A provides a "pH-responsive pure drug composite nanoformulation based on boronate structure and its preparation method", which connects bortezomib (drug A) and drug B together by connecting a linker, and can also achieve pH-sensitive release of drugs. However, since both drugs are connected by chemical reaction, there are specific requirements for the structure of drug B, which is limited to drugs containing active amino or hydroxyl groups.

[0007] Hyaluronic acid (HA) is a common drug delivery carrier. As a high molecular weight aminopolysaccharide, it has good biocompatibility and biodegradability. Due to its CD44-mediated tumor targeting and non-toxicity, it is often selected as a carrier for drug delivery. In addition, a large number of studies have shown that low molecular weight HA can also promote the repolarization of M2 macrophages to M1, thereby improving the anti-tumor immune response. Therefore, HA is an ideal drug delivery carrier in the anti-tumor field. Since HA is a hydrophilic polymer material, it is usually connected to hydrophobic materials or drugs through chemical bonds to form amphiphilic polymers, which provides the possibility for further preparation of nanoparticles. However, the chemical connection between the carrier material and the drug may lead to insufficient drug release performance due to stable chemical bond binding; on the other hand, the different positions of chemical bond breakage may also lead to subtle changes in the drug structure after release, thereby changing its original pharmacological effect.

[0008] Through the above analysis, the problems and defects of the prior art are as follows:

[0009] (1) Traditional chemotherapy drugs often lead to treatment failure due to their single mechanism of action.

[0010] (2) Bortezomib is difficult to accumulate and exert its effect in solid tumors; at the same time, bortezomib has strong toxic side effects (neurotoxicity, thrombocytopenia), which limits its clinical application in solid tumors.

[0011] (3) The chemical connection between drug carriers and drugs may lead to changes in the release properties or pharmacological effects of drugs. Summary of the invention

[0012] In order to overcome the problems existing in the related art, the embodiments disclosed in the present invention provide a hyaluronic acid-dopamine-bortezomib polymer, a preparation method and application thereof, and the technical scheme is as follows:

[0013] The present invention is achieved by providing a hyaluronic acid-dopamine-bortezomib polymer, which is a pH-sensitive drug-carrying amphiphilic polymer HA-DA-BTZ, wherein the HA-DA-BTZ is composed of a polymer carrier material hyaluronic acid HA, a small molecule connecting material dopamine DA and a therapeutic drug bortezomib BTZ.

[0014] The HA used in the present invention is a high molecular polymer, which serves as a main chain to achieve drug loading. HA contains multiple block carboxyl structures, and each monomer carboxyl group can be connected with the amino group in DA through reaction.

[0015] Dopamine DA is a small molecule compound that uses its structural characteristics of amino and phthalic acid dihydroxy as an intermediate connecting material. Among them, the amino group can bind to the carboxyl group in HA, and the phthalic acid dihydroxy can specifically bind to the boric acid group in BTZ to form a pH-sensitive boronate ester bond, thereby realizing the chemical connection between HA and the drug BTZ. The boronate ester bond can be broken under the weakly acidic conditions of the tumor microenvironment, thereby achieving the responsive release of the BTZ drug, which is beneficial to improving the therapeutic effect. After the boronate ester bond is broken, BTZ returns to its own chemical structure without any structural changes, which can ensure that the drug exerts its pharmacological effect.

[0016] In the HA-DA-BTZ polymer, since HA has good hydrophilicity and BTZ is a hydrophobic drug, the high molecular weight HA has multiple monomer blocks, and each monomer can be connected to BTZ through DA to form an amphiphilic polymer with HA as the main chain and multiple BTZ molecules attached, which provides the possibility for the subsequent further preparation of nanoparticles.

[0017] Another object of the present invention is to provide a method for preparing a hyaluronic acid-dopamine-bortezomib polymer HA-DA-BTZ, which is used to prepare the hyaluronic acid-dopamine-bortezomib polymer, and the method comprises the following steps:

[0018] S1. After activating the carboxyl group of HA with an acylating agent, add DA aqueous solution, adjust the pH value to weak acidity, react under nitrogen protection, dialyze for 12-24 hours after the reaction, and lyophilize to obtain HA-DA;

[0019] S2. Weigh BTZ and dissolve it in DMSO solution, and dissolve HA-DA in distilled water respectively. After mixing, adjust the pH value to weak alkalinity with NaOH, stir for 4-8 hours to complete the reaction, and dialyze and freeze-dry the product.

[0020] Furthermore, the molecular weight of the HA is in the range of 5000-50000 Da, the molar ratio of the HA to DA is 1:1-1:1000, and the weakly acidic pH value is in the range of 4.5-6.0.

[0021] Furthermore, the mass ratio of BTZ to HA-DA is between 1:5 and 1:100, and the pH value is adjusted by NaOH to a weakly alkaline range of between 8.0 and 10.0.

[0022] Another object of the present invention is to provide a pH-sensitive co-drug-loaded nanoparticle, which comprises the hyaluronic acid-dopamine-bortezomib polymer, and the nanoparticle is loaded with a hydrophobic drug on the basis of HA-DA-BTZ to form a co-drug-loaded nanoparticle.

[0023] Furthermore, the hydrophobic drug is an anti-tumor drug, including paclitaxel, docetaxel, camptothecin, gemcitabine, lenalidomide, sorafenib, lenvatinib, regorafenib, donafenib, imatinib, gefitinib or erlotinib.

[0024] Another object of the present invention is to provide a method for preparing pH-sensitive co-drug-loaded nanoparticles, which is used to prepare the pH-sensitive co-drug-loaded nanoparticles, and the method comprises:

[0025] The HA-DA-BTZ polymer is dissolved in an aqueous solution as the aqueous phase, and the hydrophobic drug is dissolved in an organic solvent as the organic phase;

[0026] The organic phase is added dropwise into the aqueous phase at a certain temperature, stirred and subjected to probe ultrasound; the mixed solution is then placed in a dialysis bag and dialyzed with distilled water;

[0027] After dialysis, the solution was filtered through 0.45 μm and 0.22 μm filter membranes respectively to obtain the co-drug-loaded nanoformulation.

[0028] Furthermore, the molecular weight of the dialysis bag ranges from 1000 to 5000.

[0029] Furthermore, the mass ratio of the hydrophobic drug to the HA-DA-BTZ polymer is 1:5-1:100, and the organic solvent is any one of ethanol, methanol, acetone, tetrahydrofuran or dimethyl sulfoxide.

[0030] Furthermore, when the organic phase is added dropwise into the aqueous phase, the temperature is controlled at 20-65° C., the volume ratio of the organic phase to the aqueous phase is 1:1-1:10, and the stirring time is 2-12 h.

[0031] HA-DA-BTZ is an amphiphilic polymer that can self-polymerize into nanoparticles through hydrophilic-hydrophobic interactions. In this process, hydrophobic drugs can be further encapsulated.

[0032] HA-DA-BTZ amphiphilic polymer can be considered as a drug delivery platform, which can further encapsulate other hydrophobic drugs and overcome the disadvantages of low solubility and poor drugability of hydrophobic drugs. In the encapsulation process, the size of the nanoparticles can be adjusted by adjusting the ratio of hydrophilic and hydrophobic components. At the same time, this process also realizes the combination of different drugs to exert a synergistic anti-tumor effect.

[0033] In theory, all hydrophobic drugs can be encapsulated. However, in practical applications, the significance and necessity of the combination of drugs and BTZ need to be considered. In combination with the clinical application of BTZ, the hydrophobic drugs further encapsulated in the present invention are limited to anti-tumor drugs, which can be used in combination with BTZ to exert potential synergistic therapeutic effects.

[0034] The co-drug-loaded nanoparticles chemically link BTZ and physically encapsulate hydrophobic drugs. After reaching the tumor site, the weakly acidic pH value in the tumor microenvironment and the further decrease in the pH value in the cell lysosome can responsively break the boronate bond on HA-DA-BTZ, thereby disintegrating the nanoparticles and releasing the two therapeutic drugs.

[0035] Another object of the present invention is to provide a method for preparing pH-sensitive co-drug-loaded nanoparticles, which is used to prepare the pH-sensitive co-drug-loaded nanoparticles, and the method comprises:

[0036] (1) dissolving the HA-DA-BTZ polymer in an aqueous solution as the aqueous phase, and dissolving the hydrophobic drug in an organic solvent as the organic phase;

[0037] (2) adding the organic phase dropwise into the aqueous phase at a certain temperature, stirring and performing probe ultrasound; then placing the mixed solution in a dialysis bag and dialyzing it with distilled water;

[0038] (3) After dialysis, the solution was filtered through 0.45 μm and 0.22 μm filter membranes to obtain the co-drug-loaded nanoformulation.

[0039] In step (1), the mass ratio of the hydrophobic drug to the HA-DA-BTZ polymer is 1:5-1:100.

[0040] In step (1), the organic solvent must be miscible with water and may be ethanol, methanol, acetone, tetrahydrofuran or dimethyl sulfoxide.

[0041] In step (2), the temperature of adding the organic phase dropwise to the aqueous phase is in the range of 20-65° C., the volume ratio of the organic phase to the aqueous phase is between 1:1-1:10, and the stirring time is controlled within 2-12 hours.

[0042] In the preparation process of pH-sensitive co-drug-loaded nanoparticles of the present invention, moderate heating, sufficient stirring and probe ultrasound are all conducive to the formation of nanoparticle morphology; dialysis is to remove organic solvents and unencapsulated free drugs; and the filter membrane can control the particle size of the nanoparticles within a suitable range.

[0043] In combination with all the above technical solutions, the beneficial effects of the present invention are as follows:

[0044] First, the pH-sensitive co-drug-loaded nanoparticles prepared by the present invention have an average particle size between 100-200 nm, a simple preparation method, and good encapsulation efficiency and drug loading. The synthesized HA-DA-BTZ amphiphilic polymer material can be used as a drug loading platform to further encapsulate hydrophobic drugs while realizing the connection of BTZ drugs. Moreover, HA-DA-BTZ is connected by borate bonds, has good pH sensitivity, can ensure responsive release of drugs at the tumor site, and exert better therapeutic effects.

[0045] Second, the present invention provides a pH-sensitive hyaluronic acid-dopamine-bortezomib polymer (HA-DA-BTZ) to overcome the defects of BTZ in solid tumors; on the other hand, the polymer can also be used as a drug encapsulation platform to further achieve the combination with other therapeutic drugs to exert a synergistic anti-tumor effect. The co-drug-loaded nanoparticles prepared by the present invention have a particle size between 100-200nm and have a high encapsulation rate and drug loading.

[0046] Third, BTZ is a first-line drug for hematological tumors, with annual global sales of up to 3 billion US dollars and huge market value. If the technical solution of the present invention is transformed, it is expected to expand the indications to the field of solid tumor treatment, further broaden the market scope, and thus have extremely high commercial value. The technical solution of the present invention can effectively overcome the disadvantages of the low cumulative distribution and large toxic and side effects of the drug in solid tumors, and is expected to be applied in solid tumors, further expand the clinical indications of the drug, and fill the technical gap that the drug cannot be applied in solid tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The accompanying drawings herein are incorporated in and constitute a part of the specification, illustrate embodiments consistent with the present disclosure, and together with the description, serve to explain the principles of the present disclosure;

[0048] Figure 1 is a flow chart of the preparation method of the HA-DA-BTZ polymer provided in Example 1 of the present invention;

[0049] Figure 2 The particle size, potential and TEM image of the co-drug-loaded nanoparticles provided in Example 2 of the present invention; (A) particle size distribution diagram; (B) transmission TEM image; (C) BTZ determination standard curve; (D) zeta potential diagram.

[0050] Figure 3 This is a graph showing the particle size change of the co-drug-loaded nanoparticles provided in Example 2 of the present invention within 14 days;

[0051] Figure 4The pH-sensitive responsive disintegration particle size change and TEM photos of the co-drug-loaded nanoparticles provided in Example 2 of the present invention; (A) particle size change trend diagram under different pH values; (B) transmission TEM morphology diagram under different pH values.

[0052] Figure 5 This is a pH-sensitive response drug release diagram of the co-drug-loaded nanoparticles provided in Example 2 of the present invention;

[0053] Figure 6 Schematic diagram of investigating (A) in vitro cytotoxicity and (B) hemolysis rate of the nanocarrier HA-DA provided in an embodiment of the present invention;

[0054] Figure 7 This is a diagram showing the significant enhancement of the anti-tumor effect of HDB / PTX on in situ breast cancer provided in Example 2 of the present invention; (A) the trend of changes in mouse body weight during treatment; (B) the trend of changes in mouse tumor volume during treatment; (C) the tumor inhibition rate calculated based on the tumor volume. DETAILED DESCRIPTION

[0055] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific implementation disclosed below.

[0056] The innovation of the present invention is:

[0057] (1) The structure of the polymer is innovative. The boronate bond formed by the connection between the carrier and the drug has good pH sensitivity, which can ensure the responsive release of the drug at the tumor site, and after breaking, the chemical structure of the drug remains unchanged.

[0058] (2) The polymer can be used in the treatment of solid tumors, which can overcome the disadvantage of bortezomib's own toxic side effects. At the same time, it can also improve its distribution in solid tumors, thereby improving the treatment effect and reducing toxic side effects.

[0059] (3) The polymer can also be used as a drug delivery platform to further encapsulate hydrophobic drugs. On the one hand, it can overcome the disadvantage of low solubility of hydrophobic drugs, and on the other hand, it can also achieve the combined treatment of bortezomib and the drug, further enhancing the tumor treatment effect.

[0060] Example 1. The embodiment of the present invention provides a novel pH-sensitive drug-carrying amphiphilic polymer HA-DA-BTZ, wherein the amphiphilic polymer HA-DA-BTZ is composed of a polymer carrier material hyaluronic acid HA, a small molecule connecting material dopamine DA, and a therapeutic drug bortezomib BTZ.

[0061] HA is a high molecular weight polymer that is used as the main chain to load drugs. HA contains multiple blocks of carboxyl structures, and each monomer carboxyl group can react with the amino group in DA to achieve connection.

[0062] Dopamine DA is a small molecule compound that uses its amino and phthalic acid dihydroxy structures as an intermediate connecting material. The amino group can bind to the carboxyl group in HA, while the phthalic acid dihydroxy group can specifically bind to the boric acid group in BTZ to form a pH-sensitive boronate bond, thereby achieving a chemical connection between HA and the drug BTZ. The boronate bond can be broken under weakly acidic conditions in the tumor microenvironment, thereby achieving responsive release of the BTZ drug, which is beneficial to improving the therapeutic effect.

[0063] In the HA-DA-BTZ polymer, since HA has good hydrophilicity and BTZ is a hydrophobic drug, the high molecular weight HA has multiple monomer blocks, and each monomer can be connected to BTZ through DA to form an amphiphilic polymer with HA as the main chain and multiple BTZ molecules attached, which provides the possibility for the subsequent further preparation of nanoparticles.

[0064] like Figure 1 As shown, the embodiment of the present invention also provides a method for preparing a hyaluronic acid-dopamine-bortezomib polymer HA-DA-BTZ, and the preparation method comprises the following steps:

[0065] S1, after activating the carboxyl group of HA with an acylating agent, add an appropriate amount of DA aqueous solution, adjust the pH value to weak acidity, react under nitrogen protection, dialyze for a certain period of time after the reaction, and lyophilize to obtain HA-DA;

[0066] S2, BTZ was weighed and dissolved in DMSO solution, and HA-DA was dissolved in distilled water, and the pH value was adjusted to weak alkalinity with NaOH after mixing. After stirring and reacting for a certain period of time, the product was dialyzed and freeze-dried to obtain HA-DA-BTZ.

[0067] In step S1, the molecular weight of HA provided in the embodiment of the present invention is in the range of 5000-50000 Da, the molar ratio of HA to DA is 1:1-1:1000, and the weakly acidic pH value is in the range of 4.5-6.0.

[0068] In step S2, the mass ratio of BTZ to HA-DA provided in the embodiment of the present invention is between 1:5-1:100, and NaOH is used to adjust the pH value to a weak alkaline range of between 8.0-10.0.

[0069] In step S2, the molecular weight range of the dialysis bag provided in the embodiment of the present invention is between 1000-5000.

[0070] The synthetic route of HA-DA-BTZ is as follows:

[0071]

[0072] The embodiment of the present invention also provides a pH-sensitive co-drug-loaded nanoparticle, which encapsulates a hydrophobic drug on the basis of HA-DA-BTZ to form a co-drug-loaded nanoparticle. HA-DA-BTZ is an amphiphilic polymer that can self-polymerize into nanoparticles through hydrophilic-hydrophobic interactions. In this process, the encapsulation of hydrophobic drugs can be further achieved.

[0073] HA-DA-BTZ amphiphilic polymer can be considered as a drug-carrying platform to further encapsulate other hydrophobic drugs. In this process, the size of the nanoparticles can be adjusted by adjusting the ratio of hydrophilic and hydrophobic components. On the other hand, this process also enables the combination of different drugs to exert a synergistic anti-tumor effect.

[0074] In theory, all hydrophobic drugs can be encapsulated. However, in practical applications, the significance and necessity of the combination of drugs and BTZ need to be considered. In combination with the clinical application of BTZ, the hydrophobic drugs further encapsulated in the present invention are limited to anti-tumor drugs, which can be used in combination with BTZ to exert potential synergistic therapeutic effects.

[0075] Preferably, the hydrophobic drug can be: paclitaxel, docetaxel, camptothecin, gemcitabine, lenalidomide, sorafenib, lenvatinib, regorafenib, donafenib, imatinib, gefitinib, erlotinib, etc.

[0076] The co-drug-loaded nanoparticles chemically link BTZ and physically encapsulate hydrophobic drugs. After reaching the tumor site, the weakly acidic pH value in the tumor microenvironment and the further decrease in the pH value in the cell lysosome can responsively break the boronate bond on HA-DA-BTZ, thereby disintegrating the nanoparticles and releasing the two therapeutic drugs.

[0077] The embodiment of the present invention also provides a method for preparing pH-sensitive co-drug-loaded nanoparticles, which is used to prepare pH-sensitive co-drug-loaded nanoparticles. The preparation method comprises the following steps:

[0078] (1) dissolving the HA-DA-BTZ polymer in an aqueous solution as the aqueous phase, and dissolving the hydrophobic drug in an organic solvent as the organic phase;

[0079] (2) adding the organic phase dropwise into the aqueous phase at a certain temperature, stirring and performing probe ultrasound; then placing the mixed solution in a dialysis bag and dialyzing it with distilled water;

[0080] (3) After dialysis, the solution was filtered through 0.45 μm and 0.22 μm filter membranes to obtain the co-drug-loaded nanoformulation.

[0081] In step (1), the mass ratio of the hydrophobic drug to the HA-DA-BTZ polymer is 1:5-1:10, and the organic solvent may be ethanol, methanol, acetone, tetrahydrofuran or dimethyl sulfoxide.

[0082] In step (2), the temperature of adding the organic phase dropwise to the aqueous phase is in the range of 20-65° C., the volume ratio of the organic phase to the aqueous phase is between 1:1-1:10, and the stirring time is within 2-12 hours.

[0083] In the preparation process of pH-sensitive co-drug-loaded nanoparticles of the present invention, moderate heating, sufficient stirring and probe ultrasound are all conducive to the formation of nanoparticle morphology; dialysis is to remove organic solvents and unencapsulated free drugs; and the filter membrane can control the particle size of the nanoparticles within a suitable range.

[0084] Example 2: Bortezomib and paclitaxel co-loaded nanoparticles (abbreviated as HDB / PTX) provided in an embodiment of the present invention are composed of HA-DA-BTZ polymer (abbreviated as HDB) and paclitaxel (PTX).

[0085] The preparation method of the bortezomib and paclitaxel co-loaded nanoparticles comprises the following steps:

[0086] Take 100 mg of hyaluronic acid (MW 5000Da) and dissolve it in 10 mL of distilled water. Add 360 μmoL of EDC and NHS and stir at room temperature for 0.5 h to activate the carboxyl group. Take another 50 mg of DA and dissolve it in 1 mL of distilled water. Add the DA aqueous solution drop by drop into the HA solution. Use 1 M HCl to adjust the solution to pH = 5.5. Stir and react for 24 h under nitrogen protection. After dialyzing for 48 h, the reaction solution is freeze-dried to obtain HA-DA.

[0087] Take 100 mg of HA-DA and dissolve it in 10 mL of distilled water, and 5 mg of BTZ and dissolve it in an appropriate amount of DMSO and add it dropwise to the HA-DA solution. Then adjust the pH of the solution to 8.5 with 1 M NaOH solution and stir at room temperature for 24 hours. After the reaction is completed, dialyze and freeze-dry the product to obtain HA-DA-BTZ.

[0088] Weigh 100 mg of HA-DA-BTZ polymer and dissolve it in 5 mL of distilled water. Dissolve 10 mg of paclitaxel in methanol solution and add it dropwise into the HA-DA-BTZ solution and stir vigorously for 4 hours. After thorough mixing, use a probe to perform ice bath ultrasound for 3 times. Then pour the solution into a dialysis bag and dialyze it with distilled water for 24 hours. Finally, pass it through 0.45 μm and 0.22 μm filter membranes to remove free PTX to obtain bortezomib and paclitaxel co-loaded nanoparticles (HDB / PTX).

[0089] By analyzing the characteristic peaks on the bortezomib pyrazine ring group at 8.5-9.5 ppm in the H-NMR spectrum, the grafting rate of BTZ was measured to be 42.3%, and the drug content of BTZ in every 100 mg of HA-DA-BTZ polymer was calculated to be 2.0 mg.

[0090] The prepared HDB / PTX has an average particle size of 120 nm and a zeta potential of -30 mV. Transmission electron microscopy (TEM) images show that the co-loaded nanoparticles are regular spherical ( Figure 2 10% Triton was used to break the emulsion and the PTX drug content was determined by HPLC. The encapsulation efficiency and drug loading were 96.8% and 8.8%, respectively.

[0091] According to the drug content of HDB / PTX, it can be calculated that the BTZ drug concentration in the final preparation is 0.4 mg / mL and the PTX drug concentration is 1.9 mg / mL.

[0092] The nanoparticles were diluted 10 times with culture medium (RPMI-1640) and PBS (pH = 7.4), respectively, and then sterilized by membrane and dispensed into 7 centrifuge tubes. The particle size changes of the nanoparticles in RPMI-1640 and PBS within 14 days were negligible, and the initial stability was good ( Figure 3 ).

[0093] The changes in HDB / PTX under different pH values ​​were investigated by particle size changes and TEM images. Under acidic conditions, the particle size of the nanoparticles gradually increased, indicating that the stability of the nanoparticles was destroyed; and the TEM images showed that under acidic conditions, the particle size of the spherical nanoparticles continued to increase and dissociated ( Figure 4 ). This indicates that the nanoformulation is acid-instable and can achieve responsive rupture and drug release in the tumor tissue microenvironment.

[0094] The drug release of HDB / PTX was investigated by dynamic dialysis. Under acidic conditions, both BTZ and PTX can be rapidly released within 6 hours. The final cumulative release of BTZ at pH 6.5 and 5.0 can reach 68.1% and 81.6%, respectively, while the cumulative release of the drug at pH 7.4 is about 60%. The drug release of PTX is similar to that of BTZ, and acidic conditions can achieve more drug release ( Figure 5 ). This indicates that the boronate bond in HA-DA-BTZ has good pH sensitivity and can achieve efficient drug release under acidic conditions.

[0095] The cell hemolysis performance of the nano-micelle carrier was investigated. The hemolysis rate of the nano-carrier at different concentrations was less than 5%, indicating that the carrier would not cause red blood cell rupture and had good biosafety ( Figure 6 ).

[0096] The therapeutic effect of HDB / PTX was investigated in an orthotopic breast cancer model in mice. Compared with the BTZ and PTX free drug combination group, the tumor volume and weight of mice treated with the nanoformulation were significantly inhibited, and the tumor inhibition rate increased from 37.4% to 82.2%, indicating that the preparation can exert a synergistic therapeutic effect of the two drugs ( Figure 7 ).

[0097] Example 3: Bortezomib and paclitaxel co-loaded nanoparticles (abbreviated as HDB / PTX) provided in an embodiment of the present invention are composed of HA-DA-BTZ polymer (abbreviated as HDB) and paclitaxel (PTX).

[0098] The preparation method of the above-mentioned co-drug-loaded nanoparticles is basically the same as that of Example 2, except that when HA-DA and BTZ are reacted to synthesize HA-DA-BTZ, the ratio of the two is adjusted to 5:1, that is, 100 mg of HA-DA is weighed and dissolved in 10 mL of distilled water, and 20 mg of BTZ is dissolved in 2 mL of DMSO.

[0099] The HDB / PTX prepared by the present invention has a BTZ grafting rate of 68.5%, and the drug content of BTZ in every 100 mg of HA-DA-BTZ polymer is calculated to be 12.0 mg. The PTX drug content is determined by HPLC, and its encapsulation rate and drug loading are 76.4% and 7.1%, respectively.

[0100] The HDB / PTX prepared by the present invention has an average particle size of 178 nm, which is slightly larger than that of Example 1, and a zeta potential of -18 mV. The BTZ drug concentration is measured to be 2.4 mg / mL, and the PTX drug concentration is 1.4 mg / mL.

[0101] Example 4: Bortezomib and sorafenib co-loaded nanoparticles (abbreviated as HDB / SF) provided in an embodiment of the present invention are composed of HA-DA-BTZ polymer (abbreviated as HDB) and sorafenib (SF).

[0102] The preparation method of the bortezomib and sorafenib co-loaded nanoparticles is basically the same as that of Example 2, except that SF is used to replace PTX, and the solvent for dissolving SF is changed to dimethyl sulfoxide.

[0103] The HDB / SF prepared by the present invention has an average particle size of 105 nm and a zeta potential of -25 mV. The measured BTZ drug concentration is 0.4 mg / mL and the SF drug concentration is 1.9 mg / mL.

[0104] Example 5: Bortezomib and docetaxel co-loaded nanoparticles (abbreviated as HDB / DTX) provided in an embodiment of the present invention are composed of HA-DA-BTZ polymer (abbreviated as HDB) and docetaxel (DTX).

[0105] The preparation method of the above co-drug-loaded nanoparticles is basically the same as that of Example 2, except that DTX is used instead of PTX, and the dosage of DTX is adjusted to 20 mg (the ratio of DTX to HA-DA-BTZ is 1:5).

[0106] The HDB / DTX prepared by the present invention has an average particle size of 132 nm and a zeta potential of -28 mV. The measured BTZ drug concentration is 0.4 mg / mL and the DTX drug concentration is 3.1 mg / mL.

[0107] Comparative Example 1, compared with Example 2, during the synthesis of HA-DA, after hyaluronic acid and DA were added dropwise and mixed, 1M NaOH was used to adjust the solution to a weakly alkaline pH of 8.0, but not to a weakly acidic pH range of 4.5-6.0. At this time, the solution color became darker, DA self-aggregated to form polydopamine, and HA-DA could not be successfully synthesized.

[0108] This indicates that the weakly acidic pH range of 4.5-6.0 is a necessary step for the synthesis of HA-DA.

[0109] Comparative Example 2, compared with Example 2, during the synthesis of HA-DA-BTZ, after HA-DA and BTZ were added dropwise and mixed, 1M NaOH was used to adjust the solution to a neutral pH of 7.0, but not to a weakly alkaline pH range of 8.0-10.0. After the reactants were dialyzed to remove DMSO, a precipitate was precipitated, which was determined to be a BTZ drug precipitate, indicating that HA-DA-BTZ was not successfully synthesized.

[0110] This indicates that the weakly alkaline pH range of 8.0-10.0 is a necessary step for the synthesis of HA-DA-BTZ.

[0111] Comparative Example 3, compared with Example 2, the ratio of HA-DA-BTZ polymer to hydrophobic drug PTX was adjusted to 2:1. That is, 100 mg of HA-DA-BTZ polymer was weighed and dissolved in 5 mL of distilled water, 50 mg of paclitaxel was dissolved in methanol solution and added dropwise to the HA-DA-BTZ solution and stirred vigorously for 4 hours. After sufficient mixing, obvious precipitation was precipitated, and the subsequent membrane could not be passed, indicating that HDB / PTX was not successfully prepared.

[0112] This shows that the HA-DA-BTZ polymer encapsulates hydrophobic drugs with saturation, and when the drug ratio is high, it exceeds the maximum loading capacity of the polymer. Therefore, the mass ratio of hydrophobic drugs to HA-DA-BTZ polymer should be in the range of 1:5-1:100.

[0113] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principles of the present invention should be covered within the protection scope of the present invention.

Claims

1. A pH-sensitive co-drug-loaded nanoparticle, characterized in that: The nanoparticles contain hyaluronic acid-dopamine-bortezomib polymer HA-DA-BTZ, and the nanoparticles are loaded with hydrophobic drugs on the basis of HA-DA-BTZ to form co-drug-loaded nanoparticles; The polymer is a pH-sensitive drug-carrying amphiphilic polymer HA-DA-BTZ, and the HA-DA-BTZ is composed of a polymer carrier material hyaluronic acid HA, a small molecule connecting material dopamine DA, and a therapeutic drug bortezomib BTZ; The preparation method of the hyaluronic acid-dopamine-bortezomib polymer HA-DA-BTZ comprises: S1. After activating the carboxyl group of HA with an acylating agent, add DA aqueous solution, adjust the pH value to weak acidity, react under nitrogen protection, dialyze for 12-24 hours after the reaction, and lyophilize to obtain HA-DA; S2. Weigh BTZ and dissolve them in DMSO solution and HA-DA in distilled water respectively, adjust the pH value to weak alkalinity with NaOH after mixing, stir for 4-8h to complete the reaction, and dialyze and freeze-dry the product; In step S1, the molecular weight of HA is in the range of 5000-50000Da, the molar ratio of HA to DA is 1:1-1:1000, and the weakly acidic pH value is in the range of 4.5-6.0; The hydrophobic drug is an anti-tumor drug, including paclitaxel, docetaxel or sorafenib; The mass ratio of the hydrophobic drug to the HA-DA-BTZ polymer is 1:5-1:

100.

2. The pH-sensitive co-drug-loaded nanoparticles according to claim 1, characterized in that: The mass ratio of BTZ to HA-DA is between 1:5 and 1:100, and the pH value is adjusted to a weakly alkaline range of 8.0-10.0 by using NaOH.

3. A method for preparing pH-sensitive co-drug-loaded nanoparticles, characterized in that: The method is used to prepare the pH-sensitive co-drug-loaded nanoparticles according to any one of claims 1 to 2, and the method comprises: The HA-DA-BTZ polymer is dissolved in an aqueous solution as the aqueous phase, and the hydrophobic drug is dissolved in an organic solvent as the organic phase; The organic phase was added dropwise into the aqueous phase, stirred and subjected to probe ultrasound; the mixed solution was then placed in a dialysis bag and dialyzed with distilled water; After dialysis, the solution was filtered through 0.45 μm and 0.22 μm filter membranes respectively to obtain the co-drug-loaded nanoformulation.

4. The method for preparing pH-sensitive co-drug-loaded nanoparticles according to claim 3, characterized in that: The molecular weight of the dialysis bag ranges from 1000 to 5000.

5. The method for preparing pH-sensitive co-drug-loaded nanoparticles according to claim 3, characterized in that: The mass ratio of the hydrophobic drug to the HA-DA-BTZ polymer is 1:5-1:100, and the organic solvent is any one of ethanol, methanol, acetone, tetrahydrofuran or dimethyl sulfoxide.

6. The method for preparing pH-sensitive co-drug-loaded nanoparticles according to claim 3, characterized in that: When the organic phase is added dropwise to the aqueous phase, the temperature is controlled at 20-65° C., the volume ratio of the organic phase to the aqueous phase is 1:1-1:10, and the stirring time is 2-12 h.

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