Multi-pH / ROS responsive controllable sustained-release drug polymer prodrug nanomicelles, prodrug drug-loaded micelles, preparation method and application

Abstract

The present application relates to a kind of multiple pH / ROS response type controllable sustained-release drug polymer prodrug nanomicelles, drug-loaded micelles and preparation method and application, prodrug micelles are with CS as raw material, synthesis OCS as basic skeleton by oxidation aldehyde group reaction, graft APBA and CUR modified hydrophobic end, modification is carried out to CS, by Schiff base bond and phenylboronic acid ester bond bonding amphiphilic polymer prodrug in water medium Self-assembly is formed, prodrug micelles are loaded with drug CUR to form nanometer polymer prodrug drug-loaded micelles, drug-loaded nanomicelles have suitable drug loading and encapsulation efficiency, preparation process is simple, mild, good stability;With the characteristics of prolonging blood circulation time, improving drug bioavailability and easy to purify;Multiple pH and ROS sensitivity are introduced to give the nanosystem pH / ROS trigger type drug release behavior, ensure rapid, sufficient, accurate targeted drug release;It can reduce the dosage of administration, can improve the anti-inflammatory or antitumor therapeutic effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical nanotechnology, specifically relating to an inflammation and tumor-targeting drug-loaded nanopolymer micelle, its preparation method, and its application. In particular, it relates to a novel amphiphilic nanopolymer prodrug that is linked to a small molecule compound and a basic polysaccharide via Schiff base bonds and phenylboronic acid ester bonds, which self-assembles in an aqueous medium to form an inflammation and tumor-targeting, multi-pH and reactive oxygen species (ROS) responsive, controllable sustained-release polymer prodrug nanocarrier micelle, its preparation method, and its application. Background Technology

[0002] According to WHO reports, inflammation-related diseases and cancer are among the greatest threats to human health, and are two of the leading causes of death worldwide. To better deliver drugs to inflamed and tumor tissues, and considering the low pH and high reactive oxygen species in the inflammatory or tumor microenvironment, increasing research is being conducted on drug delivery systems. These systems demonstrate good targeting and therapeutic efficacy against inflammation and tumors, showing great application potential. Currently, drug therapy remains an important treatment method in clinical practice, but its application is widely limited due to poor drug solubility, instability, low bioavailability, and cytotoxicity. Therefore, effective targeted delivery to inflamed or tumor cells is crucial for improving drug efficacy and reducing toxic side effects.

[0003] Chitosan (CS) is a major natural positively charged polysaccharide with advantages such as biodegradability, biocompatibility, antibacterial properties, and anti-angiogenic and anti-inflammatory effects through immunomodulation, and has been applied in drug delivery systems. However, its use is limited by drawbacks such as poor solubility in water and organic solvents, inability to effectively encapsulate lipophilic drugs, and lack of target specificity.

[0004] Currently, modifying CS (Chemical Substrate) with functional compounds can increase drug cycle time, stability, solubility, biocompatibility, and bioavailability. Existing technologies disclose hydrophobic or hydrophilic modifications of CS, particularly amphiphilic derivatives, which can be used to prepare hydrophobic drug carriers. Methods such as carboxymethylation, O-glycosylation, trimethylation, and quaternization have also been disclosed to modify the CS structure, improving its poor solubility and drug loading capacity. However, these methods are cumbersome, requiring the addition of various organic or toxic reagents to aid in the synthesis of nanomaterials. The synthesis of copolymers often requires complex synthetic routes, specific experimental conditions, and complex procedures. Furthermore, excessive drug retention in the micelle core leads to insufficient drug release into tissues when micelles accumulate in a slightly acidic environment.

[0005] In summary, existing delivery systems for the treatment of inflammatory diseases and anti-tumor drugs have shortcomings such as poor tissue selectivity, weak drug loading capacity, numerous experimental steps required for preparation, the need to add various related organic or toxic reagents to assist in the synthesis of nanomaterials, relatively complex synthetic routes, special experimental conditions, and complicated experimental operations. These shortcomings greatly limit the clinical application of targeted drug therapy. Summary of the Invention

[0006] The purpose of this invention is to provide amphiphilic, multi-pH / ROS-responsive, controllable sustained-release polymer prodrug nanomicelles and prodrug-loaded micelles, and to provide a simple method and application for synthesizing and self-assembling these amphiphilic, multi-pH / ROS-responsive, controllable sustained-release polymer prodrug nanomicelles and drug-loaded micelles. This addresses the problem of micelle instability caused by pH / ROS-sensitive intelligent responsiveness, thus improving the selectivity and efficiency of targeted drug delivery to tissue cells. This invention introduces a novel, highly active aminophenylboronic acid (APBA) and curcumin into a natural polymeric basic polysaccharide. The method for synthesizing a multi-pH / ROS-sensitive prodrug micelle delivery system can be conveniently implemented under conventional experimental conditions. The self-assembly of amphiphilic polymer prodrugs bonded by Schiff base bonds and phenylboronic ester bonds to construct targeted drug-loaded polymer prodrug nanomicelles as a potential drug delivery system represents a significant breakthrough compared to previous methods for constructing functionalized polymer micelles.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A multi-pH / ROS-responsive controllable sustained-release polymer prodrug nanomicelles are described. The nanopolymer prodrug nanomicelles are synthesized from CS as a raw material via an oxidative aldehyde reaction to form OCS as the basic framework. The hydrophobic ends are modified by grafting 3-aminophenylboronic acid (APBA) and curcumin (CUR), and the CS is amphiphilic modified. The amphiphilic polymer is synthesized by bonding through Schiff base bonds and phenylboronic acid ester bonds, and then self-assembled in an aqueous medium to form a structure with pH-sensitive / ROS-responsive properties that can target the acidic microenvironment of inflammatory and tumor tissue cells.

[0009] A method for preparing multi-pH / ROS-responsive controllable sustained-release polymer prodrug nanomicelles includes the following steps:

[0010] A. Preparation of OCS-APBA-CUR:

[0011] A1. Add sodium periodate solution dropwise to chitosan solution and react at 40°C in the dark for 12 hours; add 4 mL of ethylene glycol and react in the dark for 1 hour; adjust the pH to 7-8, add 300 mL of anhydrous ethanol, react at 4°C for 6 hours, then filter and purify, and OCS can be obtained at room temperature or freeze-dried; wherein, the molecular weight of CS is 20 kD and the degree of deacetylation is 95% or higher.

[0012] A2. Dissolve OCS in pure water, add 3-APBA solution, adjust the pH to 4, react at 40℃ in the dark for 6 hours, adjust the pH to 7-8, add 300 mL of anhydrous ethanol, react at 4℃ for 6 hours, then filter and purify, and obtain OCS-APBA at room temperature or freeze-dry.

[0013] A3. Dissolve OCS-APBA in pure water, adjust the pH to 8, add curcumin methanol solution (w / w = 10:1), react at room temperature in the dark for 30 min, and then rotary evaporate at 40℃ until the liquid is completely evaporated. Then take out the solid, add anhydrous ethanol and wash repeatedly. The obtained filter cake can be obtained by drying at room temperature or freeze-drying.

[0014] B. Dissolve the OCS-APBA-CUR prepared in step A3 in purified water, adjust the pH to 7.4, centrifuge for 5 min, add purified water, and prepare and obtain OCS-APBA-CUR, a prodrug nanopolymer with amphiphilic multi-pH and reactive oxygen species (ROS) responsive controllable sustained-release properties, with an average particle size of 103 nm-127 nm.

[0015] Further, the method is characterized in that: in step A1, the chitosan solution is prepared by dissolving chitosan in 1% acetic acid solution, the sodium periodate solution is prepared by dissolving sodium periodate in purified water, and the molar ratio of chitosan to potassium periodate solution is 1:1; in step A2, the optimal molar ratio of 3-APBA to OCS is 5:1; and in step A3, the optimal mass ratio of CUR to OCS-APBA is 1:10-2:10.

[0016] Further, in step B, the centrifuge speed is 12000 rpm / min.

[0017] Application of a multi-pH / ROS-responsive controllable sustained-release polymer prodrug nanomicelle in inflammation or tumor-targeted drugs.

[0018] A multi-pH / ROS-responsive controllable sustained-release polymer prodrug-loaded micelle is obtained by encapsulating the therapeutic drug CUR in the amphiphilic multi-pH / ROS-responsive controllable sustained-release polymer prodrug nanomicelles, with an optimal drug loading and encapsulation efficiency of 12.2% and 92.2%, respectively.

[0019] A method for preparing multi-pH / ROS-responsive controllable sustained-release drug polymer prodrug loading micelles includes the following steps:

[0020] C. Dissolve OCS-APBA-CUR in purified water. Add the CUR drug with anti-inflammatory, antioxidant, or anti-tumor functions to the ethanol solution and dissolve it. Add the curcumin ethanol solution dropwise to the OCS-APBA-CUR solution. Then adjust the pH to 7.4, centrifuge for 5 min, and add purified water to obtain amphiphilic multi-pH and ROS-responsive controllable sustained-release drug nanopolymer prodrug-loaded micelles CUR / OCS-APBA-CUR with an average particle size of 132.7 nm-226.5 nm and a critical micelle concentration of 28.1 μg / mL.

[0021] Further, in step C, the centrifuge speed is 12000 rpm / min.

[0022] Application of a multi-pH / ROS-responsive controllable sustained-release polymer prodrug micelle in the therapeutic probe for inflammation or tumor targeting.

[0023] Compared with the prior art, the beneficial effects of the present invention are:

[0024] This invention provides a controllable sustained-release prodrug nanomicelle for targeting inflammation or tumors, its preparation method, and its application. The prepared OCS-APBA-CUR polymer, through oxidative modification of CS, uses OCS as the basic framework, enabling the nanomicelles to exhibit pH / ROS-sensitive targeting effects on inflammation or tumors. CS is a biodegradable functional polymeric organic compound with good biocompatibility, non-toxicity, and excellent film-forming properties. By introducing pH-sensitive imine bonds (Schiff bases) and pH / ROS-sensitive phenylboronic acid ester bonds into the carrier, the nanosystem is endowed with pH and ROS-triggered drug release behavior, ensuring rapid, sufficient, and precise drug release in the cellular microenvironment of inflamed or tumor tissues. CS modification can increase the in vivo retention time of the nanodrug delivery system, improve the plasma half-life, and increase accumulation at inflamed or tumor sites.

[0025] The CUR encapsulated in the OCS-APBA-CUR polymer prodrug nanocarrier system prepared in this invention can exert anti-inflammatory, antioxidant, or anti-tumor effects in the acidic or reactive oxygen species microenvironment of inflamed or tumor tissue cells. At the same time, the amino protonation, imine bond (Schiff base), and phenylboronic ester bond in the OCS-APBA-CUR polymer can undergo charge reversal and specific cleavage of imine and phenylboronic ester bonds in response to pH-sensitive stimuli. In addition, the phenylboronic ester bond has reactive oxygen species responsive properties, thereby reaching the site of inflammation or tumor to release therapeutic drugs in a targeted manner, thereby improving the anti-inflammatory, antioxidant, or anti-tumor effects.

[0026] The OCS-APBA-CUR prodrug nanocarrier system prepared in this invention, with OCS as the basic framework, has good biocompatibility and degradability, overcomes the defect of poor water solubility of poorly soluble drugs, greatly improves the solubility of poorly soluble anti-inflammatory, antioxidant or anti-tumor drugs, and is low in cost and easy to obtain. The preparation process is simple and the preparation conditions are mild. It is an excellent nanocarrier for targeted delivery of anti-inflammatory, antioxidant or anti-cancer drugs.

[0027] The prodrug-loaded nanomicelles prepared by this invention have small particle size, complete morphology and uniform spherical shape, with suitable drug loading and encapsulation efficiency, and good stability;

[0028] The nanoprodrug micelles prepared by this invention simultaneously encapsulate hydrophobic CUR within the nanomicelles, reducing the dosage and improving the anti-inflammatory or anti-tumor therapeutic effect. Attached Figure Description

[0029] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1A Synthetic route for OCS-APBA-CUR, a multi-pH / ROS-sensitive amphiphilic polymer prodrug;

[0031] Figure 1B For CS, OCS, 3-APBA and OCS-APBA-CUR 1 H NMR structural spectrum;

[0032] Figure 1C Infrared spectra of CS, OCS, 3-APBA, and OCS-APBA-CUR;

[0033] Figure 1D The ultraviolet spectra of CS, OCS, 3-APBA, and OCS-APBA-CUR are shown.

[0034] Figure 1E Ultraviolet illumination images of OCS(a), 3-APBA(b), and OCS-APBA(c);

[0035] Figure 2A The effect of the drug-to-carrier molar ratio of OCS-APBA-CUR on encapsulation efficiency and drug loading;

[0036] Figure 2BThe effect of the molar ratio of OCS-APBA-CUR drug to carrier on particle size and PDI;

[0037] Figure 2C The effect of the molar ratio of OCS-APBA-CUR drug to carrier on the zeta potential;

[0038] Figure 3A The distribution of OCS zeta potentials at different oxidation degrees;

[0039] Figure 3B The solubility properties of CS and OCS with different oxidation degrees;

[0040] Figure 4 shows the determination of critical micelle concentration, where Figure 4A The fluorescence emission spectrum of pyrene; Figure 4B The OCS-APBA-CUR fluorescence intensity ratio of pyrene (I 372 / I 382 ) vs. logarithmic concentration graph;

[0041] Figures 5A-5B The effects of nanomicelles on particle size, PDI, and Zeta potential under different pH conditions;

[0042] Figures 5C-5D Transmission electron microscopy (TEM) images of nanomicelles (pH=7.4, pH=5.5); Scale bar: 100 nm;

[0043] Figures 5E-5F The effects of particle size, PDI, and Zeta potential on nanomicelles under reactive oxygen species conditions;

[0044] Figure 6 Line graph showing the cumulative release percentage of curcumin-encapsulated CUR / OCS-APBA-CUR nanomicelles in vitro;

[0045] Figure 7 The effect of the nanocarrier drug delivery system on LPS-induced macrophage proliferation activity (***p<0.001).

[0046] Figure 8A The toxic effects of OCS-APBA-CUR nanomicelles on macrophages were evaluated (**p<0.01, ***p<0.001).

[0047] Figure 8B The toxic effects of CUR / OCS-APBA-CUR nanomicelles on macrophages were evaluated (**p<0.01, ***p<0.001).

[0048] Figure 9A The effect of nanomicelles on hemolysis (***p<0.001);

[0049] Figure 9B The effect of nanomicelles on platelet adhesion (***p<0.001);

[0050] Figure 10 To illustrate the effect of nano-drug delivery systems on DPPH radical scavenging, the half-inhibitory concentration (IC50) for DPPH radical scavenging was determined. 50 The free curcumin content was 4.74, the prodrug nanomicelles content was 3.99, and the drug-loaded nanomicelles content was 2.86.

[0051] Figure 11A The effect of OCS-APBA-CUR drug-loaded nanoparticles on stability under storage conditions of 4℃;

[0052] Figure 11B The effect of OCS-APBA-CUR drug-loaded nanoparticles on the stability of serum;

[0053] Figure 12A Colorimetric diagram of the OCS-APBA-CUR solution in response to H2O2;

[0054] Figure 12B The UV-Vis spectrum of the OCS-APBA-CUR response to H2O2. Detailed Implementation

[0055] The present invention will be further described below with reference to embodiments:

[0056] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it. Furthermore, it should be noted that, for ease of description, the accompanying drawings show only the parts relevant to the present invention, and not all of the structures.

[0057] It should be noted that similar reference numerals and letters in the following figures indicate similar items; therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures. Furthermore, in the description of this invention, terms such as "first," "second," etc., are used only to distinguish descriptions and should not be construed as indicating or implying relative importance.

[0058] Chitosan (CS) is a widely used biodegradable natural polymeric polysaccharide drug carrier material in recent years. To construct intelligent, stimulus-responsive drug carriers, nano-controlled-release drug carrier systems constructed from natural polymeric polysaccharides using CS as an excipient have attracted considerable attention. The protonation of chitosan amino groups can lead to specific cleavage in response to pH-sensitive stimuli, thereby achieving targeted release of therapeutic drugs at the inflammation or tumor target sites, effectively improving drug utilization. Furthermore, CS is a natural polymeric material used for encapsulating, modifying, or covalently modifying poorly soluble drugs with low efficacy and significant side effects. CS possesses advantages such as biocompatibility, non-toxicity, low immunogenicity, antibacterial, antitumor, lipid-lowering, and immune-enhancing properties, making it one of the most promising natural polymeric materials currently available. CS modification can increase drug retention time in vivo, improve plasma half-life, and prolong absorption time. CS modification can reduce the frequency of drug administration, improve efficacy, enhance tolerability, and reduce the severity of toxic side effects and the incidence of adverse events. Therefore, CS is often used as a drug delivery technology, which can directly encapsulate drugs, conjugate with drugs, or attach to the drug surface and encapsulate them together in nanocarrier materials.

[0059] Therefore, the nanopolymer prodrug micelles of the present invention, with OCS as the basic framework, have the following advantages:

[0060] First, CS is a biodegradable natural functional high-molecular-weight organic compound with biocompatibility, non-toxicity, and good film-forming properties. Oxidative aldehyde modification of CS increases its solubility. Second, grafting oxidized chitosan (OCS) with 3-APBA introduces pH-sensitive imine bonds (Schiff bases) into the carrier, endowing the nanosystem with dual pH-triggered drug release behavior. Subsequently, grafting OCS-APBA with CUR introduces pH and ROS-sensitive phenylboronic acid ester bonds into the carrier, endowing the nanosystem with multiple pH / ROS-stimulated drug release behavior, ensuring rapid, sufficient, and precise intracellular drug release from inflammatory cells. Third, CS modification can... This invention increases the in vivo retention time of the nanomedicine delivery system, enhances plasma half-life, and increases accumulation at inflammatory sites. Furthermore, the use of multiple pH and ROS responses enables the nanomicelles to target inflammation. Finally, the introduction of conjugated structures of APBA, OCS, and APBA into the nanomicelles demonstrates their fluorescence, making them fluorescent molecules. Simultaneously, the inclusion of curcumin (CUR), a classic therapeutic drug, within the nanomicelles, also results in fluorescence within a specific wavelength range, allowing for both therapeutic effects and detection. Additionally, OCS responds to pH-sensitive stimuli with specific cleavage, achieving targeted release of the therapeutic drug at the inflammatory site, synergistically enhancing anti-inflammatory, antioxidant, or anti-tumor effects. Moreover, the nanomicelles prepared in this invention have their hydrophobic ends modified by phenylboronic acid and curcumin during self-assembly. The introduction of pH-sensitive imine and phenylboronic acid ester bonds endows the carrier material with multiple pH / ROS-sensitive targeting properties, demonstrating a reasonable design concept and simple, easy-to-implement operation.

[0061] This invention utilizes pH and ROS-sensitive smart responsiveness to induce micellar instability, thereby improving the selectivity and efficiency of drug delivery to target cells and significantly enhancing the performance of CS. Modification of CS can improve its solubility, and the introduction of different chemical groups can expand its application range.

[0062] The present invention relates to a multi-pH and ROS-responsive controllable sustained-release drug polymer prodrug nanomicelle, which uses oxidized chitosan (OCS) as the basic framework and grafts 3-aminophenylboronic acid (APBA) and curcumin (CUR) to modify the hydrophobic ends, thereby performing amphiphilic modification on CS.

[0063] The present invention discloses a method for preparing multi-pH and ROS-responsive controllable sustained-release polymer prodrug nanomicelles, comprising the following steps:

[0064] 1. Preparation of OCS-APBA-CUR:

[0065] Chitosan was dissolved in 1% acetic acid solution to prepare a chitosan solution; sodium periodate was dissolved in purified water to prepare a sodium periodate solution, which was added dropwise to the chitosan solution and reacted at 40°C in the dark for 12 hours; the molar ratio of chitosan to potassium periodate was 1:1; 4 mL of ethylene glycol was added dropwise and reacted in the dark for 1 hour; the pH was adjusted to 7-8, 300 mL of anhydrous ethanol was added, and the reaction was carried out at 4°C for 6 hours, followed by filtration and purification. OCS was obtained by room temperature or freeze-drying; OCS was dissolved in purified water, and 3-APBA solution was added, with a molar ratio of OCS to 3-APBA solution of 1:5. The pH was adjusted to 4, and the reaction was carried out at 40°C in the dark. After 6 hours, adjust the pH to 7-8, add 300 mL of anhydrous ethanol, and react at 4°C for 6 hours. Then, filter and purify, and obtain OCS-APBA by room temperature or freeze-drying. Dissolve OCS-APBA in pure water, with a CUR to OCS-APBA mass ratio of 1:10-2:10, adjust the pH to 8, add curcumin methanol solution (w / w = 10:1), react at room temperature in the dark for 30 minutes, and then rotary evaporate at 40°C until the liquid is completely evaporated. Then, take out the solid, wash repeatedly with anhydrous ethanol, and obtain OCS-APBA-CUR by room temperature or freeze-drying. The molecular weight of CS is 20 kDa, and the degree of deacetylation is 95% or higher.

[0066] 2. Preparation of amphiphilic, multi-pH and ROS-responsive controllable sustained-release polymer prodrug nanomicelles OCS-APBA-CUR: with an average particle size of 103nm-127nm.

[0067] Dissolve the OCS-APBA-CUR prepared in step 1 in purified water, adjust the pH to 7.4, centrifuge at 12000 rpm / min for 5 min, add purified water, and prepare and obtain amphiphilic multi-pH / ROS responsive controllable sustained-release drug polymer prodrug nanomicelles (OCS-APBA-CUR prodrug nanomicelles).

[0068] This invention relates to the application of amphiphilic, multi-pH and ROS-responsive, controllable sustained-release polymer prodrug nanomicelles in the preparation of targeted drugs for inflammation or tumors.

[0069] The present invention discloses a method for preparing multi-pH and ROS-responsive controllable sustained-release polymer prodrug-loaded micelles CUR / OCS-APBA-CUR (nano-drug delivery system), comprising the following steps:

[0070] 3. Dissolve the OCS-APBA-CUR prepared in step 1 in purified water. Add the CUR containing anti-inflammatory, antioxidant, or antitumor functions to an ethanol solution and dissolve it. Add the curcumin ethanol solution dropwise to the OCS-APBA-CUR solution. Then adjust the pH to 7.4, centrifuge at 12000 rpm / min for 5 min, and add purified water to obtain amphiphilic, multi-pH, and ROS-responsive controllable sustained-release drug nanopolymer prodrug-loaded micelles CUR / OCS-APBA-CUR. These nanomicelles encapsulate anti-inflammatory, antioxidant, or antitumor drugs and have inflammation or tumor-targeting functions, making them suitable for inflammation or tumor targeting. The optimal drug loading and encapsulation efficiency are 12.2% and 92.2%, respectively.

[0071] This invention also provides an application of multi-pH and ROS-responsive controllable sustained-release polymer prodrug-loaded micelles, specifically, for use as a therapeutic probe for targeting inflammation or tumors. The therapeutic probe includes the amphiphilic multi-pH and ROS-responsive controllable sustained-release polymer nanoparticle prodrug-loaded micelles (CUR / OCS-APBA-CUR nanomicelles) prepared by the above preparation method.

[0072] Example 1

[0073] A multi-pH / ROS-responsive controllable sustained-release drug polymer prodrug nanomicelle is based on oxidized chitosan (OCS) as the basic framework, with APBA and CUR grafted onto the hydrophobic ends to perform amphiphilic modification of CS.

[0074] Example 2

[0075] The preparation of OCS-APBA-CUR includes the following steps:

[0076] OCS Synthesis: OCS is synthesized from CS via an aldehyde reaction. The synthetic route is as follows: Figure 1A As shown, sodium periodate aqueous solution was added dropwise to a chitosan solution prepared by dissolving chitosan in 1% acetic acid solution, with a molar ratio of chitosan to potassium periodate of 1:1. The reaction was carried out at 40°C in the dark for 12 hours, followed by the dropwise addition of 4 mL of ethylene glycol and a further 1 hour of reaction in the dark. The pH of the solution was adjusted to 7-8, and 300 mL of anhydrous ethanol was added. The reaction was carried out at 4°C for 6 hours, followed by purification by filtration. OCS with an average oxidation degree of 30-48% was obtained by room temperature or freeze-drying.

[0077] Synthesis of OCS-APBA-CUR: OCS was dissolved in purified water, and then 3-APBA solution was added at a molar ratio of 1:5. The pH was adjusted to 4, and the reaction was carried out at 40°C in the dark for 6 hours. The pH was then adjusted to 7-8, and 300 mL of anhydrous ethanol was added. The reaction was carried out at 4°C for 6 hours, followed by filtration and purification. OCS-APBA was obtained by room temperature or freeze-drying. The average maximum grafting rate was 7.9%. Alternatively, OCS-APBA was dissolved in purified water at a CUR to OCS-APBA mass ratio of 1:10-2:10. The pH was adjusted to 8, and curcumin methanol solution (w / w = 10:1) was added. The reaction was carried out at room temperature in the dark for 30 minutes. The liquid was then completely evaporated by rotary evaporation at 40°C. The solid was then removed, and the mixture was washed repeatedly with anhydrous ethanol. The resulting filter cake was obtained by room temperature or freeze-drying to obtain OCS-APBA-CUR. The average maximum grafting rate was 3.5%. See Table 1.

[0078] Table 1

[0079]

[0080] CS,OCS,3-APBA,,CUR,OCS-APBA-CUR 1 The H NMR structure spectrum is shown in [reference]. Figure 1B This confirms that the oxidation of CS successfully synthesized OCS, and also confirms that APBA and CUR were successfully grafted onto OCS.

[0081] Infrared spectra of CS, OCS, 3-APBA, CUR, and OCS-APBA-CUR are shown below. Figure 1C This confirms the successful synthesis of OCS and OCS-APBA-CUR.

[0082] The UV spectra of CS, OCS, 3-APBA, CUR, and OCS-APBA-CUR are shown below. Figure 1D This confirms the successful synthesis of OCS-APBA-CUR.

[0083] The fluorescence spectrum of OCS-APBA is shown below. Figure 1E The yellow-green fluorescence of OCS-APBA confirmed the successful synthesis of OCS-APBA.

[0084] Example 3

[0085] A method for preparing multi-pH / ROS-responsive controllable sustained-release polymer prodrug nanomicelles, wherein the prepared polymer prodrug nanomicelles can target the pH-stimulated response and the reactive oxygen species-stimulated response of the acidic microenvironment of inflammation or tumors. The preparation method includes the following steps:

[0086] OCS-APBA-CUR was dissolved in purified water, the pH was adjusted to 7.4, centrifuged at 12000 rpm / min for 5 min, and purified water was added to prepare and obtain OCS-APBA-CUR nanopolymer prodrug micelles with amphiphilic multi-pH / ROS responsive controllable sustained release.

[0087] Example 4

[0088] Synthesis of a multi-pH / ROS-responsive controlled-release polymer prodrug-loaded micelles (prodrug nanomicelles encapsulating the inflammation- or tumor-targeting drug CUR):

[0089] OCS-APBA-CUR was dissolved in purified water, and CUR was dissolved in ethanol solution. Curcumin ethanol solution was added dropwise to the OCS-APBA-CUR solution. The pH was then adjusted to 7.4, and the mixture was centrifuged at 12000 rpm / min for 5 min. Purified water was then added to prepare and obtain amphiphilic, multi-pH / ROS-responsive, controllable sustained-release drug CUR / OCS-APBA-CUR nanopolymer prodrug micelles encapsulated with the therapeutic drug CUR. The results, measured by UV spectrophotometry, showed that the optimal drug loading and encapsulation efficiency were 12.2% and 92.2%, respectively (see Figure 2).

[0090] In this process, the CS was oxidized and modified to synthesize OCS, with the aim of modifying APBA and CUR onto CS to synthesize amphiphilic polymer prodrug nanomicelles.

[0091] Example 5

[0092] In the OCS solubility experiment, OCS was soluble in acidic, alkaline solutions and distilled water, indicating that OCS improved the solubility of CS (see Figure 3).

[0093] Example 6

[0094] Determination of critical micelle concentration in nanomicelles.

[0095] Critical micelle concentration (CAC) was determined using a pyrene fluorescent probe method. 4 mL of sample solutions of different concentrations were added to 200 μL of a 6x10⁻⁶ ppm solution. -4 The sample was placed in a pyrene solution at 37℃ and shaken for 24 hours. The fluorescence intensity of each sample at 350–450 nm was scanned using a fluorescence spectrophotometer at an excitation wavelength of 334 nm. The ratio of fluorescence intensity at 372 nm and 382 nm was measured, and a curve of I372 / I382 versus Log C was plotted. The calculated CAC was 28.1 μg / mL (see Figure 4).

[0096] The prodrug nanomicelles with inflammation or tumor targeting effects obtained above were observed using a particle size analyzer and a transmission electron microscope. As shown in Figure 5, the particle size analyzer image and the transmission electron microscope image show that the nanoparticles are uniformly spherical with a particle size of 132.7 nm-226.7 nm. They have suitable size, good uniformity, good pH / ROS sensitivity, and good biocompatibility.

[0097] Example 7

[0098] In vitro release assay of CUR / OCS-APBA-CUR nanomicelles loaded with CUR.

[0099] In this embodiment, the release rate of CUR was determined by dialysis: 0.75 μL of freshly prepared CUR (2 mg / mL) anhydrous ethanol solution was transferred to a dialysis bag (8-12 kDa), and then release medium (PBS containing 1% Tween 80) was added at 37°C to a final volume of 3 mL. The solution was then placed in the dialysis bag and shaken steadily at 50 rpm / min. 5 mL of 1 mg / mL CUR / OCS-APBA-CUR solution (PBS containing 1% Tween 80) was transferred to a dialysis bag (8-12 kDa) and shaken steadily at 50 rpm / min at 37°C. At set time intervals, 4 mL of the released solution was collected, and an equal volume of fresh release medium was added. The CUR concentration was determined using ultraviolet spectrophotometry, and the cumulative release percentage was calculated. Figure 6 As shown.

[0100] Example 8

[0101] In vitro inflammatory targeting experiments of CUR / OCS-APBA-CUR nanomicelles encapsulating the therapeutic drug CUR.

[0102] In this embodiment, the CCK-8 assay was used to investigate the inflammation-targeting ability of primary macrophages (mouse-derived) nanomicelles using an LPS-induced macrophage inflammatory response model. CUR / OCS-APBA-CUR nanomicelles were prepared using the method described in Example 4. Cells in logarithmic growth phase were seeded at a density of 5 × 10⁴ cells / well in 96-well plates and incubated at 37°C and 0.5% CO₂ for 24 h. After incubation for 1 h in fresh DMEM medium containing different concentrations of Free CUR, OCS-APBA-CUR, and CUR / OCS-APBA-CUR, 10 μL of LPS (10 μg / mL) was added to the wells, and incubation continued for 24 h. After washing three times with PBS, 90 μL of fresh culture medium and 10 μL of CCK-8 were added to the wells, and incubation continued for 6 h. The absorbance of CUR was measured at 450 nm using a microplate reader, and cell viability was calculated using the following formula: Figure 7 As shown.

[0103]

[0104] Wherein, ODcontrol is the absorbance of the control group, ODsample is the absorbance of the sample group, and ODblank is the absorbance of the culture medium containing DMEM. The results indicate that, compared to free drug, the drug encapsulated in these micelles can target inflammatory cells more effectively.

[0105] Example 9

[0106] The cytotoxicity of OCS-APBA-CUR and OCS-APBA-CUR nanomicelles loaded with the therapeutic drug CUR to macrophages was investigated using the CCK-8 assay. Cells in logarithmic growth phase were seeded at a density of 5 × 10⁴ cells / well in 96-well plates. After overnight culture, 100 μL of free drug and fresh culture medium containing different micelle solutions were added and incubated for 4 h. After 24 h of incubation, the cells were washed with PBS, and 10 μL of CCK-8 was added. The cells were incubated at 37 °C for 24 h. The culture medium was discarded, the cells were washed three times with PBS, and 10 μL of CCK-8 was added. The cells were incubated at 37 °C for 4 h, and the absorbance (OD) was measured at 450 nm using a microplate reader. Cell viability was calculated as follows:

[0107] Cell viability (%) = ((OD sample - OD blank) / (OD control - OD blank)) × 100%

[0108] As shown in Figure 8, none of the nanomicelles exhibited significant cytotoxic effects. However, the cytotoxicity of the nanomicelles loaded with curcumin was significantly lower than that of the nanomicelles, indicating that nanomicelles are sufficient to more efficiently encapsulate drugs and better exert anti-inflammatory effects.

[0109] Example 10

[0110] Blood compatibility experiment of OCS-APBA-CUR nanogel bundles.

[0111] In this embodiment, the biocompatibility of OCS and OCS-APBA-CUR nanomicelles was investigated using a erythrocyte hemolysis test. OCS and OCS-APBA-CUR nanomicelles were prepared using the method described above. Subsequently, 1 mL of mouse blood was mixed with 3.8% sodium citrate (v / v = 1:9) and added to 2 mL of PBS. The mixture was centrifuged at 3000 rpm for 5 min to separate erythrocytes. After washing with PBS, the erythrocytes were resuspended in 10 mL of PBS. 0.5 mL of erythrocytes were added to 0.5 mL of 1% polyoctylphenyl ether PBS solution containing different concentrations of OCS and nanomicelles, respectively. The mixture was incubated at 37°C for 3 h, and then centrifuged at 3000 rpm for 5 min. The supernatant was collected, and the absorbance (OD) was measured at 540 nm using a UV spectrophotometer. The formula for calculating the hemolysis rate is shown below:

[0112] Hemolysis rate (%) = ((A) sample (sample OD)-A blank (PBS OD)) / (A control (1% Polyethylene Glycol Octylphenyl Ether OD)-A blank (PBS OD)))×100%.

[0113] The results are as follows Figure 9A As shown, none of the nanomicelles exhibited significant hemolytic activity, indicating that the aforementioned nanoparticles all possess good biocompatibility.

[0114] Example 11

[0115] Platelet adhesion assay of OCS-APBA-CUR prodrug nanomicelles.

[0116] In this embodiment, the biocompatibility of OCS and OCS-APBA-CUR prodrug nanomicelles was investigated using a platelet adhesion assay. OCS and OCS-APBA-CUR prodrug nanomicelles were prepared using the method described above. Subsequently, a 1 mg / mL nanoparticle solution was coated onto a glass tube and dried in a drying oven for 12 h to coat the film. 1 mL of mouse blood was mixed with 3.8% sodium citrate (v / v = 1:9), centrifuged at 1000 rpm / min for 5 min to obtain plasma. Platelets were diluted with PBS, and 0.5 mL of the plasma sample containing 5 × 10⁵ platelets / mL was added to a glass tube containing the CS and OCS-APBA-CUR film. The mixture was stirred steadily at 75 rpm / min at 37°C and incubated for 1 h before counting the platelets. The platelet adhesion rate was calculated as follows:

[0117] Platelet adhesion rate (%) = ((N) control (Platelet count in plasma before incubation) - N sample(Platelet count in plasma after incubation) / N control (Platelet count in plasma before incubation) × 100%

[0118] The results are as follows Figure 9B As shown, none of the nanomicelles exhibited significant platelet adhesion. This indicates that the introduction of phenylboronic acid into the nanoparticles endows the nanomicelles with good biocompatibility and can significantly improve the biocompatibility of CS.

[0119] Example 12

[0120] DPPH radical scavenging experiments of OCS-APBA-CUR and CUR / OCS-APBA-CUR nanomicelles

[0121] In this embodiment, the antioxidant effect of OCS-APBA-CUR and CUR / OCS-APBA-CUR nanomicelles was investigated using a DPPH free radical scavenging experiment. OCS-APBA-CUR nanomicelles were prepared using the method in Example 3, and CUR / OCS-APBA-CUR nanomicelles were prepared using the method in Example 4. Subsequently, 2 mL of CUR ethanol solution, OCS-APBA-CUR, and CUR / OCS-APBA-CUR nanomicelle solutions containing different concentrations were added to 2 mL of DPPH solution (40 μg / mL), respectively, and incubated at 37°C for 1 h in the dark. The absorbance (OD) was then measured at 517 nm using a UV spectrophotometer. The formula for calculating the DPPH free radical scavenging rate is shown below:

[0122] DPPH radical scavenging rate (%) = 1 - (Asample (sample OD) / Ablank (blank OD)) × 100%, see Figure 10 .

[0123] Example 13

[0124] Storage stability of OCS-APBA-CUR and CUR / OCS-APBA-CUR nanomicelles

[0125] In this embodiment, the stability of OCS-APBA-CUR and CUR / OCS-APBA-CUR nanomicelles was investigated using a storage stability experiment. (See...) Figure 11A OCS-APBA-CUR nanomicelles were prepared using the method described in Example 3, and CUR / OCS-APBA-CUR nanomicelles were prepared using the method described in Example 4. Subsequently, 1 mg / mL drug-loaded nanoparticle solutions of OCS-APBA-CUR and CUR / OCS-APBA-CUR were stored at 4°C in the dark, and the particle size and PDI of the drug-loaded nanoparticles were measured using a nano ZS Malvern laser particle size analyzer at different time points.

[0126] Example 14

[0127] Serum stability of OCS-APBA-CUR and CUR / OCS-APBA-CUR nanomicelles

[0128] In this embodiment, the stability of OCS-APBA-CUR and CUR / OCS-APBA-CUR nanomicelles was investigated using a serum stability experiment. (See attached image) Figure 11B OCS-APBA-CUR nanomicelles were prepared using the method described in Example 3, and CUR / OCS-APBA-CUR nanomicelles were prepared using the method described in Example 4. Subsequently, 1 mL of the above drug-loaded nanoparticle solution (1 mg / mL) was added to 4 mL of serum, and samples were taken after incubation at 37°C for different times. The particle size and PDI of the drug-loaded nanoparticles were determined using a nano ZS Malvern laser particle size analyzer.

[0129] Example 15

[0130] Reactive oxygen species (ROS) were detected using ultraviolet spectrophotometry and colorimetry to investigate their scavenging ability. (See Figure 12). 0.1 mL of 3% H₂O₂ solution and purified water were added to 1 mL of 1 mg / mL OCS-APBA-CUR solution. The spectra from 300 nm to 600 nm were scanned at 0.5 h, 1 h, 1.5 h, 2 h, 3 h, and 4 h, and observed colorimetrically.

[0131] This invention provides a nanomedicine delivery system based on OCS (Optical Characteristic of Sodium Chloride). By using APBA and CUR as hydrophobic end modifications, the nanomicelles exhibit multi-pH / ROS targeting effects on inflammation or tumors. The resulting nanomicelles are small in size and possess good biocompatibility, biodegradability, drug loading capacity, and encapsulation efficiency. Furthermore, the use of APBA and CUR as hydrophobic end modifications enables the nanomicelles to target inflammation or tumors. CS is a degradable functional high-molecular-weight organic compound with good biocompatibility, non-toxicity, and excellent film-forming properties. By introducing pH-sensitive imine bonds (Schiff bases) and pH / ROS-sensitive phenylboronic acid ester bonds into the carrier, the nanosystem is endowed with pH / ROS-triggered drug release behavior. Under specific pH acidic environment stimulation, the OCS amino groups in the nanomicelles undergo protonation, causing potential reversal, and the imine bonds (Schiff bases) of the nanomicelles specifically break in response to acidic environment stimulation. Meanwhile, the phenylboronic acid ester bonds simultaneously respond to both acidic environment stimulation and reactive oxygen species stimulation, thus ensuring rapid, sufficient, and precise drug release within the cellular environment of inflamed or tumor tissues, effectively improving the utilization rate of anti-inflammatory, antioxidant, or antitumor drugs. CS modification can increase the in vivo retention time of drugs, improve plasma half-life, prolong absorption time, and increase accumulation at inflamed or tumor sites. CS-modified nanomedicines can reduce the frequency of administration, improve efficacy, improve tolerability, and reduce the severity and incidence of adverse events. The intelligent, controlled-release prodrug nanomicelles for targeting inflammation or tumors provided by this invention have promising application prospects as carriers for anti-inflammatory, antioxidant, or antitumor drugs.

[0132] Simultaneously, this invention utilizes CUR to encapsulate it within a nanocarrier. Furthermore, the hydrophobic ends of the nanoparticles are modified with APBA and CUR to mediate the pH / ROS-responsive targeted uptake of the nanomicelles by inflammatory or tumor cells. Moreover, the conjugated structure of CUR and the APBA nanopolymer molecule possesses fluorescent properties, thus integrating fluorescence imaging and therapy. This allows for real-time fluorescence imaging of inflammation or tumors while also serving as a navigation tool for drug treatment—a so-called therapeutic probe.

[0133] Note that the above description is merely a preferred embodiment of the present invention and the technical principles employed. Those skilled in the art will understand that the present invention is not limited to the specific embodiments described herein, and various obvious changes, readjustments, and substitutions can be made without departing from the scope of protection of the present invention. Therefore, although the present invention has been described in detail through the above embodiments, the present invention is not limited to the above embodiments, and may include many other equivalent embodiments without departing from the concept of the present invention, the scope of which is determined by the scope of the appended claims.

Claims

1. A method for preparing multi-pH / ROS-responsive controllable sustained-release polymer prodrug nanomicelles, characterized in that, The polymer prodrug nanomicelles, which are pH / ROS responsive and controllable sustained-release polymer prodrugs, are synthesized from chitosan (CS) via oxidative aldehyde reaction. OCS serves as the basic framework, and the hydrophobic ends are modified with 3-aminophenylboronic acid (3-APBA) and curcumin (CUR). The CS is then amphiphilically modified, and the amphiphilic polymer prodrug is synthesized through Schiff base and phenylboronic ester bonds. This polymer prodrug then self-assembles in an aqueous medium, exhibiting pH and ROS-sensitive response characteristics that can target the acidic microenvironment of inflammation and tumors. The preparation method specifically includes the following steps: A. Preparation of OCS-APBA-CUR: A1. Add sodium periodate solution dropwise to chitosan solution and react at 40°C in the dark for 12 h; add 4 mL ethylene glycol dropwise and react in the dark for 1 h; adjust the pH to 7-8, add 300 mL anhydrous ethanol, react at 4°C for 6 h, then filter and purify, and obtain OCS at room temperature or freeze-dry; wherein, the molecular weight of CS is 20 kD and the degree of deacetylation is 95% or higher; A2. Dissolve OCS in pure water, add 3-APBA solution, adjust the pH to 4, react at 40℃ in the dark for 6 hours, adjust the pH to 7-8, add 300 mL of anhydrous ethanol, react at 4℃ for 6 hours, then filter and purify, and obtain OCS-APBA at room temperature or freeze-dry. A3. Dissolve OCS-APBA in pure water, adjust the pH to 8, add curcumin methanol solution, react at room temperature in the dark for 30 min, and then evaporate at 40℃ until the liquid is completely evaporated. Then take out the solid, add anhydrous ethanol and wash repeatedly. The obtained filter cake can be obtained by drying at room temperature or freeze-drying. B. Dissolve the OCS-APBA-CUR prepared in step A3 in pure water, adjust the pH to 7.4, centrifuge for 5 min, add pure water, and prepare and obtain multi-pH / ROS responsive controllable sustained-release drug polymer prodrug nanomicelles with an average particle size of 103 nm-127 nm. Step A1, the chitosan solution is prepared by dissolving chitosan in 1% acetic acid solution, and the sodium periodate solution is prepared by dissolving sodium periodate in purified water; Step B: The centrifuge speed is 12000 rpm / min.

2. A multi-pH / ROS-responsive controllable sustained-release polymer prodrug loading micelle, characterized in that: The polymer prodrug-loaded micelles were prepared by the method described in claim 1, and the therapeutic drug CUR was encapsulated in multiple pH / ROS-responsive controllable sustained-release polymer prodrug nanomicelles. The suitable drug loading and encapsulation efficiency were 12.2% and 92.2%, respectively. A method for preparing multi-pH / ROS-responsive controllable sustained-release drug polymer prodrug loading micelles includes the following steps: C. Dissolve OCS-APBA-CUR in purified water. Add the CUR drug with anti-inflammatory, antioxidant, or anti-tumor functions to the ethanol solution and dissolve it. Add the curcumin ethanol solution dropwise to the OCS-APBA-CUR solution. Then adjust the pH to 7.4, centrifuge for 5 minutes at a speed of 12,000 rpm, and add purified water to obtain multi-pH / ROS-responsive controllable sustained-release polymer prodrug-loaded micelles CUR / OCS-APBA-CUR.

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