Preparation method and application of closed-loop feedback-regulation nano drug delivery system

Abstract

The invention belongs to the technical field of drug carriers, and particularly discloses a preparation method and application of a closed-loop feedback-regulation nano drug delivery system.The preparation method comprises the following steps that a starting material containing a reversible molecular library is provided, and the reversible molecular library is prepared in a buffer salt system; under thermodynamic control, adding anti-cancer drug molecules and guest molecules into the starting material; through a reversible covalent reaction process, molecules in an initial material spontaneously form a self-assembled nano-carrier containing drug molecules under the combined action of the drug molecules and guest molecules. According to the preparation method and application of the closed-loop feedback-regulation nano drug delivery system, reversible covalent reaction, closed-loop feedback-regulation and self-assembly technologies are combined, spontaneous construction of a nano carrier is achieved, and the nano carrier is endowed with excellent drug loading capacity, stability and environmental responsiveness.

Description

Technical Field

[0001] The present invention relates to the technical field of drug carriers, and in particular to a preparation method and application of a closed-loop feedback-regulating nano drug delivery system. Background Art

[0002] Chemotherapy plays a vital role in cancer, a global health challenge. As one of the main means of cancer treatment, chemotherapy directly acts on tumor cells through drugs to inhibit their proliferation and spread. However, although chemotherapy drugs have shown strong anti-cancer activity in laboratories and clinical trials, their efficacy is often limited by the delivery efficiency and targeting of drugs in actual applications. Before reaching the tumor site, traditional chemotherapy drugs often reduce their efficacy due to degradation, nonspecific distribution and rapid clearance in the blood circulation, and may cause serious systemic toxic side effects. In recent years, with the rapid development of nanotechnology and materials science, drug delivery systems based on nanocarriers have gradually become a research hotspot. These systems can minimize damage to normal tissues while ensuring efficacy by precisely controlling the release and distribution of drugs.

[0003] As a powerful nanotechnology, molecular self-assembly technology has been widely used to construct drug delivery carriers. Nanocarriers formed by self-assembly, such as liposomes, linear / multi-arm polymers and dendrimers, can encapsulate drug molecules, improve drug stability and bioavailability, and achieve selective delivery to tumor sites through enhanced permeability and retention (EPR) effect. However, these traditional self-assembled nanocarriers generally have problems such as low drug loading (usually less than 10%), poor stability and lack of intelligent responsiveness, which seriously limit their effectiveness in clinical applications.

[0004] To overcome these challenges, researchers began to explore self-assembly prodrug strategies, directly connecting drug molecules to carrier molecules through chemical bonds to increase drug loading. However, the prodrug strategy also faces problems such as poor solubility, high toxicity, and poor bioavailability and pharmacokinetic properties. The root of these problems lies in the limitations of current material design strategies, that is, most self-assembling molecules and prodrug candidates are obtained through time-consuming and labor-intensive screening processes, lacking spontaneous synthesis and optimization mechanisms at the system level.

[0005] In contrast, biological molecules in nature evolve spontaneously by intelligently responding to environmental changes, forming complex systems with high adaptability and functionality. Inspired by this, researchers began to explore closed-loop feedback-regulation strategies that simulate the natural selection process to achieve self-screening and optimization of functional molecules at the system level. In closed-loop feedback-regulation systems, molecules continuously exchange modules through reversible chemical reactions and reach a thermodynamically controlled equilibrium state. By introducing guest molecules or stimuli, the quantitative amplification and self-assembly of specific molecules can be guided to generate nanomaterials with specific properties and functions.

[0006] In this context, the present invention proposes a method for preparing a nano drug delivery system with a closed-loop feedback-regulation strategy to achieve a drug delivery system with high drug loading, high stability and stimulus responsiveness. The method realizes the spontaneous and quantitative synthesis of modules into octamer and trimer disulfide macrocyclic molecules (8mer, 3mer) by reversible covalent reaction under the joint guidance of guest molecules and anticancer drug paclitaxel (PTX), and further co-assembles with guest molecules into nanorod structures. The nanorod not only has high drug loading and encapsulation efficiency, but also can release drug molecules in an intelligent and responsive manner under reducing and tumor micro-acidic environmental conditions, showing significant anticancer activity and high biosafety, providing a novel and effective solution for cancer chemotherapy. Summary of the invention

[0007] The purpose of the present invention is to provide a preparation method and application of a closed-loop feedback-regulation nano-drug delivery system, which combines closed-loop feedback-regulation and self-assembly technology to achieve the spontaneous construction of nano-carriers and endow them with excellent drug loading capacity, stability and environmental responsiveness.

[0008] To achieve the above object, the present invention provides a method for preparing a closed-loop feedback-regulating nano drug delivery system, comprising the following steps:

[0009] Step 1: providing a starting material comprising a reversible molecule library, wherein the starting material forms a reversible molecule library in a buffer salt system at pH 7.4;

[0010] Step 2: Under thermodynamic control, anticancer drug molecules and guest molecules are added to the starting material, ultrasonicated in a water bath for 30 minutes, vigorously stirred, and completely oxidized in air after 24 hours. Through a closed-loop feedback-regulation chemical process, the starting material molecules spontaneously form self-assembled nanocarriers containing drug molecules under the joint action of drug molecules and guest molecules.

[0011] Preferably, the reversible molecule library comprises a plurality of molecules capable of exchanging reactive groups with guest molecules.

[0012] Preferably, the reversible molecule library comprises a plurality of disulfide bond molecules that can be interconverted by reversible chemical reactions.

[0013] Preferably, the disulfide bond molecule is a dithiol module spontaneously formed by a reversible covalent reaction of a dithiol molecule, which contains a carboxylic acid group and can be deprotonated at physiological pH, so that the oxidized macrocycle has a negative charge. The chemical structure of the dithiol module is as follows:

[0014]

[0015] Preferably, the anticancer drug is a broad-spectrum antitumor drug such as paclitaxel (PTX), mitoxantrone (MTZ), and doxorubicin (DOX).

[0016] Preferably, the guest molecule is a cationic molecule with a long aliphatic tail chain, which can form an amphiphilic complex with the dithiol module molecule and then self-assemble into nanorods; the chemical structure of the cationic molecule is as follows:

[0017]

[0018] Where n = 5, 6, 7, 8;

[0019] Where n = 5, 6;

[0020] Among them, n=5,6,7,8.

[0021] A closed-loop feedback-regulating nano drug delivery system is prepared by the above-mentioned preparation method; it contains anti-tumor drug molecules, the drug loading amount is 40.1%-44.1%, and the encapsulation efficiency is as high as 99.2%.

[0022] Preferably, the nano drug delivery system has a nanorod shape, and the diameter of the nanorod is 20-160 nm.

[0023] The present invention also provides an application of a closed-loop feedback-regulating nano drug delivery system for preparing a new dosage form drug for treating tumors.

[0024] The advantages and beneficial effects of the preparation method and application of the closed-loop feedback-regulating nano drug delivery system of the present invention are:

[0025] 1. High drug loading: The present invention successfully realizes the spontaneous construction and self-assembly process of nanocarriers by adopting a closed-loop feedback-regulation chemical strategy. This innovative method significantly improves the drug loading capacity of nanocarriers. Under the guidance of the closed-loop feedback-regulation strategy, the reversible molecular library of the present invention can quantitatively synthesize macrocyclic molecules that are highly matched with drug molecules and further self-assemble with drug molecules and guest molecules into nanocarriers, thereby ensuring that the maximum drug loading reaches 44.1%. This high drug loading not only improves the efficiency of drug delivery, but also effectively reduces the loss of drugs during the delivery process, providing a more sufficient drug reserve for subsequent cancer treatment.

[0026] 2. High stability: Under physiological conditions, the nano drug delivery system of the present invention exhibits excellent stability. Through carefully designed intermolecular interactions, the nano drug delivery system of the present invention can maintain a stable morphology and structure before reaching the tumor site, effectively preventing the premature release of the drug in the blood circulation. This high stability not only prolongs the circulation time of the nanocarrier, but also ensures that the drug can be accurately delivered to the target tumor tissue, thereby improving the therapeutic effect.

[0027] 3. Dual intelligent responsiveness: The nano drug delivery system is designed as an intelligent drug delivery system with dual responsiveness to acidity and reduction. The tumor microenvironment is usually characterized by acidity and high reduction, and these characteristics are cleverly used to trigger the release of drugs in nanorods. Under acidic conditions, the carboxylic acid groups in the nano drug delivery system will be protonated, causing the nanostructure to become fragile and then release drug molecules; at the same time, the high concentration of glutathione (GSH) in tumor cells can reduce the disulfide bonds in the nanorods, further promoting the release of drugs. This intelligent responsiveness ensures that the drug can be accurately released in the tumor microenvironment, maximizing the concentration of the drug in the lesion site while reducing the toxic side effects to normal tissues.

[0028] 4. Enhanced anticancer activity: Compared with free drugs, the nano drug delivery system can be more effectively taken up by tumor cells and accumulate to a higher concentration in the cells. The nano drug delivery system significantly increases the intracellular concentration of the drug by overcoming the effect of the drug efflux pump, thereby effectively inhibiting the proliferation of tumor cells. In animal experiments, the tumor growth of the nano drug delivery system treatment group was significantly inhibited, and even in some cases, complete tumor regression was achieved. These results show that the nano drug delivery system of the present invention not only significantly enhances the anticancer activity, but also provides new ideas and methods for solving the problem of tumor resistance.

[0029] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1TEM images of the nano drug delivery system loaded with various drugs in the embodiments of the present invention, wherein A is a combined nanorod loaded with PTX (PTX nano), B is a combined nanorod loaded with MTZ (MTZ nano), and C is a combined nanorod loaded with DOX (DOX nano);

[0031] Figure 2 HPLC analysis of the combination of dithiol module BB, guest molecule T and drug molecule in the embodiment of the present invention, wherein (a) is a reversible molecular library formed by dithiol module BB, (b) is the combination of dithiol module BB and guest molecule T, (c) is the combination of dithiol module BB, guest molecule T and drug molecule MTZ, and (d) is the combination of dithiol module BB, guest molecule T and drug molecule PTX;

[0032] Figure 3 It is a trend diagram of the change of PTX released from the PTX-loaded composite nanorods (PTXnano) over time at 37° C. under different pH and GSH conditions in the embodiment of the present invention;

[0033] Figure 4 1 is a comparative diagram of the antiproliferative activity of different types of drugs in the embodiments of the present invention on drug-resistant MCF-7R cells and sensitive MCF-7S cells, wherein (a) is drug-resistant MCF-7R cells, and (b) is sensitive MCF-7S cells;

[0034] Figure 5 This is an image of the cell scratch experiment in an embodiment of the present invention to explore the effect on cell migration ability;

[0035] Figure 6 This is an image of the effect on cell invasion ability explored in the Transwell cell invasion assay in an embodiment of the present invention;

[0036] Figure 7 This is a comparison diagram of the in vivo imaging positioning of PTX nano in an embodiment of the present invention;

[0037] Figure 8 : is a comparative diagram of in vivo drug efficacy in the embodiments of the present invention, wherein (a) is a visualized tumor size, and (b) is a tumor growth curve;

[0038] Fig. 9 It is a preparation flow chart of the closed-loop feedback-regulating nano drug delivery system of the present invention. DETAILED DESCRIPTION

[0039] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0040] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0041] Unless otherwise defined, the reagents and equipment used in the present invention are commonly available from the market.

[0042] Example 1

[0043] Preparation method of closed-loop feedback-regulated nano drug delivery system, such as Fig. 9 As shown, the following steps are included:

[0044] Step 1, providing a starting material containing a reversible molecule library, and preparing a reversible molecule library in a phosphate buffered saline (PBS) (pH 7.4) solution. The reversible molecule library contains multiple molecules that can exchange reactive groups with drug molecules and guest molecules T. The reversible molecule library contains multiple disulfide bond molecules that can be mutually converted through reversible chemical reactions. The disulfide bond molecule is a dithiol module, which contains a carboxylic acid group and can be deprotonated at physiological pH, so that the oxidized macrocycle has a negative charge. The chemical structure of the dithiol module is as follows:

[0045]

[0046] A reversible molecular library is constructed, which is based on dithiols with unique reversible disulfide bond reactivity as the starting building block. In a pH environment close to physiological conditions (pH 7.4), these dithiols units autonomously generate a series of structurally diverse disulfide molecules through a natural oxidation process, covering closed polycyclic macrocycles of different sizes. Under thermodynamic regulation, these molecules maintain a dynamic equilibrium state with the help of reversible disulfide bond exchange reactions, ensuring the continuous adjustment and optimization of the molecular composition in the library.

[0047] Step 2, under thermodynamic control, add drug molecules and guest molecules to the starting material. The concentration of the guest molecule T is 1.88mM, the target concentration of the drug molecule is 1.74mM, and the water bath is ultrasonicated for 30min. The library is stirred vigorously and completely oxidized in air after 24h. The anticancer drugs are paclitaxel (PTX), mitoxantrone (MTZ), and doxorubicin (DOX) with broad-spectrum therapeutic effects. The guest molecule T is a cationic molecule with a long aliphatic tail chain, which can form an amphiphilic complex with a dithiol module molecule and then self-assemble into a nanorod; the chemical structure of the cationic molecule is as follows:

[0048]

[0049] Where n = 5, 6, 7, 8;

[0050] Where n = 5, 6;

[0051] Among them, n=5,6,7,8.

[0052] Cationic compounds with long fatty chains are selected as guest molecules, paclitaxel (PTX), mitoxantrone (MTZ), and doxorubicin (DOX) are selected as drug molecules, and both guest molecules and drug molecules are introduced into the reversible molecule library at the same time. The guest molecules can form stable complexes with specific macrocyclic molecules in the reversible molecule library through specific interactions (such as electrostatic attraction and hydrophobic effects), thereby guiding the spontaneous synthesis of nanoassemblies. The drug molecules serve as one of the driving forces to promote the formation of nanomedicines of specific sizes and structures, and achieve efficient drug loading in the process.

[0053] Step 3, through a closed-loop feedback-regulation process, the molecules in the starting material are spontaneously constructed to form a self-assembled nanorod-like structure containing the drug molecules under the joint action of the drug molecules and the guest molecules.

[0054] The drug-added reversible library remained as a clear, slightly opalescent solution for more than 2 months and produced new macrocyclic multimers quantitatively. This remarkable expansion could be reconstituted with a partial reduction and reoxidation process, indicating that these multimeric macrocycles are thermodynamic products.

[0055] In the closed-loop feedback-regulation process, the introduction of guest molecules T and drug molecules breaks the original dynamic balance of the molecular library. Driven by thermodynamics, disulfide molecules of a specific size are amplified and self-assembled into stable nanocarriers together with guest molecules and drug molecules. These nanocarriers not only have a drug loading capacity of up to 40.1%-44.1%, but also remain stable under physiological conditions to prevent drug leakage during delivery.

[0056] The closed-loop feedback-regulated nano drug delivery system is prepared by the above method; it contains anti-tumor drug molecules with high drug loading, the drug loading is 40.1%-44.1%, and the encapsulation efficiency is 99%. The designed nano drug carrier has excellent pH and redox responsiveness. Under the acidic conditions of the tumor microenvironment (pH <7) and the action of high concentrations of glutathione (GSH), the disulfide bonds in the nanocarrier are broken, resulting in the disintegration of the carrier structure and the release of drug molecules. This intelligent release mechanism ensures efficient accumulation and precise treatment of drugs at the tumor site.

[0057] The nanocarrier has a nanorod shape, and the diameter of the nanorod is 20-160 nm. The combined nanorods loaded with PTX, MTZ and DOX are denoted as PTX nano, MTZ nano and DOX nano, respectively, and their average diameters are 99, 153 and 20 nm, respectively.

[0058] Based on the EPR effect of tumors, nanodrug carriers are used to enhance the accumulation of drugs in tumor tissues, especially to overcome the drug resistance of anti-tumor drugs.

[0059] Example 2

[0060] Construction of a reversible molecular library

[0061] First, accurately weigh an appropriate amount of dithiol module (BB), which contains a carboxylic acid group, to ensure that it can undergo oxidation reaction under physiological pH conditions to form a series of disulfide-linked cyclic molecules of different sizes. Dissolve the weighed module in phosphate buffer (PBS, pH 7.4) and ensure that the module is fully dispersed in the reaction system.

[0062] Subsequently, the dissolved module solution is stirred to evenly distribute the modules in the solution. Next, the solution is left open in the air for natural oxidation. In this process, the modules are oxidized by oxygen in the air to generate a series of cyclic molecules of different sizes, and the concentration distribution of these molecules in the reversible molecule library will gradually reach thermodynamic equilibrium.

[0063] Example 3

[0064] Introduction of guest molecules and drug molecules

[0065] After the reversible molecular library is constructed, a pre-designed and accurately weighed guest molecule T is added thereto. The guest molecule is a cationic compound with a long fatty chain, and its cationic group can specifically bind to the hydrophobic cavity of the cyclic molecule in the reversible molecular library, thereby reducing the energy state of the cyclic molecule and making it easier to form a stable nanostructure. At the same time, hydrophobic drug molecules are added to the reversible molecular library. Through the hydrophobic interaction between the drug molecules and the fatty chains of T, they can enter its hydrophobic interval well and can be effectively encapsulated into the nanocarrier through the self-assembly process.

[0066] Under the condition of continuous stirring, the guest molecules, drug molecules and cyclic molecules in the reversible molecule library interact with each other and form a stable nanocarrier structure through non-covalent bonding. In this process, the guest molecules not only serve as the driving force for the formation of the nanostructure, but also improve the stability of the nanocarrier by binding with the cyclic molecules. By adjusting the concentration ratio of the guest molecules to the module molecules, the drug loading and stability of the nanocarrier can be further optimized to achieve the best drug delivery effect.

[0067] like Figure 1As shown, transmission electron microscopy (TEM) observations revealed that the average diameters of the self-synthesized nanorods were approximately 20, 153, and 99 nm, respectively. This high aspect ratio nanostructure can prevent drugs from being excreted by the kidneys and avoid being sequestered in the liver and spleen.

[0068] like Figure 2 As shown, through high performance liquid chromatography (HPLC) testing, it was found that the reversible molecule library alone was mainly composed of trimer and tetramer disulfide macrocyclic molecules (3mer, 4mer). After the addition of guest molecule T, the reversible molecule library became mainly composed of trimer disulfide macrocyclic molecules (3mer). In the mixed system of reversible molecule library, guest molecule T, and MTZ, the system was mainly composed of trimer disulfide macrocyclic molecules (3mer). In the mixed system of reversible molecule library, guest molecule T, and PTX, the system was mainly composed of octamer and trimer disulfide macrocyclic molecules (8mer, 3mer). In the mixed system of reversible molecule library, template molecule T, and DOX, the system was mainly composed of octamer disulfide macrocyclic molecules (8mer).

[0069] Regarding the pH response characteristics of PTX nanorods, the drug release behavior was studied at pH 5.8 and 7.4, and the release curves were recorded, such as Figure 3 As shown. The results showed that under acidic conditions (pH 5.8), the release of PTX showed a gradual release trend, and the cumulative release in the first 30 hours was 45% at pH 5.8. However, under physiological conditions (pH 7.4), the cumulative release of PTX was less than 5% within 30 hours. Since the microenvironment of tumor tissue is acidic due to the presence of metabolites such as lactic acid and carbon dioxide, the release of PTX from PTX nanorods may be inhibited in normal cells, but can be triggered in tumor tissue. In addition, it was found from the release curve that under acidic conditions, this pH-responsive PTX release behavior is a slow process, which can prolong the administration time and reduce the toxic effects.

[0070] Regarding GSH, the GSH concentration in tumor cells (about 10 mM) is higher than that in the extracellular matrix (about 5 μM). Figure 3As shown, when the GSH concentration increased from 5μM to 5.0mM, PTX release gradually increased from 3% in the first hour to 100%. In the presence of 5μM GSH, about 10% of the cumulative PTX release was observed after 30 hours of incubation, indicating minimal drug leakage during blood circulation. The results show that at a GSH concentration of 5mM, the release of PTX from PTX nano is more efficient and rapid than at a GSH concentration of 5μM. Overall, this pH-promoted and reduction-responsive drug release property allows PTX-loaded nanorods to exhibit a preferential drug release profile at tumor sites, while less release is seen in normal tissues under physiological conditions.

[0071] Example 4

[0072] In vitro cell experiments on self-synthesized nanorods

[0073] The nanorods were subjected to in vitro anti-tumor experiments to evaluate their inhibitory effects on tumor cells, migration and invasion.

[0074] 1. Cytotoxicity assay

[0075] The CCK-8 method was used to detect the effects of each experimental group on the survival rate of MCF-7S sensitive human breast cancer cells and MCF-7R resistant human breast cancer cells. Cells in the logarithmic growth phase were taken, the old culture medium was discarded, and washed twice with PBS. After the PBS was sucked out with a pipette, 1ml of trypsin was added for digestion for 2-3min, 1ml of complete culture medium was added to terminate the digestion, centrifuged (1000r / 5min), the supernatant was discarded, 1ml of complete culture medium was added, and the mixture was mixed with a pipette to form a cell suspension. 10ul of cell suspension was taken, counted with a hemocytometer, and the cells were diluted with complete culture medium and added to a 96-well plate. 100ul of cell suspension (5000 cells / well) was added to each well, and the plate was placed in a cell culture incubator (37°C, 5% CO2) and cultured until the cell density reached 70-80%. When the cell density of each well reached 70-80%, the original culture medium was sucked out, and 100ul of drug-containing culture medium was added to the cells of the drug group. After the 96-well plate was returned to the cell culture incubator for 24 hours, 10 ul of CCK-8 solution was added to each well, shaken well, protected from light, and placed in the cell culture incubator for 2 hours. The absorbance of each group was measured at 450 nm using an ELISA reader, and the cell viability was calculated.

[0076] like Figure 4 As shown, Figure 4This is a comparison of the antiproliferative activities of different drug types on drug-resistant MCF-7R cells and sensitive MCF-7S cells, where (a) is drug-resistant MCF-7R cells, (b) is sensitive MCF-7S cells, blank nano is blank nano, free paclitaxel is free PTX and nanorods loaded with paclitaxel are PTX nano; the IC50 value of free PTX for MCF-7S is about 11.5uM, while the IC50 value of PTX nano is about 7.1uM, indicating that the IC50 value of free PTX for MCF-7S cells is 1.6 times higher than that of PTX nano; in comparison, in MCF-7R cells, the IC50 value of PTX nanorods is about 0.9uM, while the IC50 value of free PTX is about 50uM, and the IC50 value of free PTX for MCF-7R cells is 55 times higher than that of PTX nano. The above toxicity experiments show that PTX nanorods can kill MCF-7S and MCF-7R cells more effectively, showing significant anti-tumor drug resistance effects.

[0077] 2. Cell scratch experiment was used to determine the effect of nanorods on cell migration.

[0078] The cell scratch test is a common method to measure cell migration and repair ability. A 6-well plate is required for the cell scratch test. Before inoculating cells, draw 3 horizontal lines on the back of each well of the 6-well plate, and the spacing between the horizontal lines is the same. The 6-well plate is marked and divided into a control group and a drug-treated group, where the drug-treated groups are free PTX group, blank nano group, and PTX nano. Take MCF-7R cells in the logarithmic growth period, discard the old culture medium, wash twice with PBS, remove the remaining PBS, add an appropriate amount of trypsin digestion solution, put it in a cell culture incubator for digestion for 2-3 minutes, add complete culture medium to terminate the digestion, centrifuge (1000r / 5min), discard the supernatant, add fresh culture medium, count with a hemocytometer, and inoculate in a 6-well plate, 1×10 per well. 6 Cells were placed in a 37°C, 5% CO2 cell culture incubator and cultured to a density of 90-95%. Using a ruler, use a 20ul sterile pipette tip to make cell scratches perpendicular to the well plate and line so that the scratches intersect with the marked line. Aspirate the old culture medium, then wash the cells 3 times with sterile PBS to wash away the scratched cells so that the gaps left are clearly visible to the naked eye. Add 2mL of drug-containing culture medium to the drug-treated group (the concentration of each drug-treated group is 0.04umol / L), observe the migration of cells in each group under an inverted microscope at 0h and 24h of drug administration, take pictures, and calculate the image area using imagej software. Calculate the cell migration rate using the following formula:

[0079] Cell migration rate = [(0 h scratch area - 24 h scratch area) / 0 h scratch area] × 100%;

[0080] Migration rate compared with the control group = [cell migration rate of each group / cell migration rate of the normal group] × 100%.

[0081] like Figure 5 As shown in the figure, after 24 hours of drug treatment, both the free PTX group and the PTX nano group had an inhibitory effect on the migration of MCF-7R cells compared with the control group. Figure 5 It can be seen that the cell scratch made at 0h is the widest. Without drug intervention, the scratch gradually decreases within 24h. Compared with the free PTX drug, the PTX nano preparation group has a stronger effect in inhibiting the migration of MCF-7R cells, indicating that PTX nano is significantly better than free PTX in inhibiting the migration of MCF-7R cells.

[0082] 3. Transwell experiment was used to determine the effect of the preparation on the invasion of human drug-resistant breast cancer cell line (MCF-7R cells).

[0083] Take the MCF-7R cells in the logarithmic growth phase, digest them with trypsin, add complete medium, centrifuge and discard the supernatant. Then add 1mL complete medium. Count with a hemocytometer and divide the 6-well plate into a control group and a drug group. The drug groups are freePTX group and PTX nano group. The cells are inoculated in a 6-well plate, 2×10 4The cells were placed in a 37°C, 5% CO2 incubator for 24 hours, and then the old culture medium was discarded. 2mL (0.04umol / L) of drug-containing culture medium was added to the free PTX group and the PTX nano group, and fresh complete culture medium was added to the control group. The 6-well plate was placed in a cell culture incubator for 24 hours. Each group had 3 parallel controls. The Transwell chamber was placed in a 24-well plate, and the matrix gel was diluted with pre-cooled blank culture medium at a ratio of 8:1, and then evenly added to the upper chamber. 200uL was added to each upper chamber and placed in a cell culture incubator for 3 hours at 37°C. After 3 hours, the unbound matrix gel was carefully aspirated, and 100uL of serum-free culture medium was added. The 24-well plate was placed in a 37°C incubator for 30 minutes for hydration, the liquid in the chamber was removed, and a small amount of blank culture medium was added to check for leaks. If there was no leak, it could be used to inoculate cells. After 24 hours of administration, the drug-containing culture medium was aspirated and discarded, pH7.4 PBS buffer was slowly added to gently wash, PBS was discarded, and an appropriate amount of trypsin was added to digest the cells. After digestion, complete medium was added to terminate digestion. The cells of each group were gently blown down and placed in a sterile centrifuge tube. Centrifuged at 1000 rpm for 5 min, the supernatant was discarded, pH 7.4 PBS was added to wash the cells, and centrifuged again. Finally, the cells were resuspended in blank medium and prepared into 1×10 6 / mL cell suspension, add 600uL complete culture medium to the lower chamber of the 24-well plate, and add 200uL cell suspension to each Transwell upper chamber covered with matrix gel. Place the 24-well plate in a 37°C, 5% CO2 incubator for 24h. After 24h, remove the Transwell chamber, remove the culture medium, gently wash the Transwell chamber with pH 7.4PBS, then add 600uL4% paraformaldehyde fixative to the clean wells of the 24-well plate, put the chamber in for fixation for 30min, discard the fixative, and wash the inside and outside of the chamber with PBS once. Add 600uL crystal violet staining solution to the clean wells of the 24-well plate, put the chamber in for staining for 20min. Take out the chamber and wash the inside and outside of the chamber with PBS 3 times. After proper air drying, select 5 fields of view under the microscope, take pictures, count them with Image J, and calculate the cell invasion rate relative to the control group.

[0084] Invasion rate compared with the control group = [number of invaded cells in the drug group / number of invaded cells in the control group] × 100%.

[0085] Depend on Figure 6As shown in the figure, after 24 hours of incubation with cells, more cells in the control group passed through the chamber, indicating that MCF-7R cells have a certain degree of metastasis. After 24 hours of administration, compared with the control group, the number of cells passing through the chamber in the free PTX group and the PTX nano group was significantly reduced, indicating that the drug group has a certain anti-metastasis ability for MCF-7R cells. The number of crystal violet-stained cells was counted using Image J, and it was found that compared with the control group, the invasion rates of the free PTX group and the PTXnano group decreased by 47.34% and 78.04%, respectively, and the difference was extremely statistically significant. The above results show that PTX nano can significantly inhibit the invasion ability of MCF-7R cells.

[0086] Example 5

[0087] In vivo animal imaging experiment using self-synthesized nanorods

[0088] 1.5 mg DiR was dissolved in 0.5 mL of a mixed solvent (chloroform: methanol = 3:2, vol / vol), and then the DiR solution was injected into a 5 ml PTX nano system, sonicated in a water bath for half an hour, and the unencapsulated DiR was separated by filtration through a 0.45 μm polycarbonate membrane (Millipore Co.), and then dialyzed for 9 hours using a membrane with a molecular weight cutoff of 2,000 Da (water was changed every hour). To determine the in vivo biodistribution, 100 μL of free DiR and PTX / DiRnano nanomicelles were injected into the tail vein of female C57BL / 6 mice bearing 4T1 mouse breast cancer tumors, with a dose equivalent to 270 μg / mL DiR (n = 3 per group). An in vivo imaging system (Photon Imager; Biospace Lab) was used to record the real-time distribution and tumor accumulation of free DiR and PTX / DiRnano nanorods at 2, 8, 24, and 48 hours after injection. Figure 7 The PTX / DiR nano nanosystem has strong tumor targeting. After 24 hours of administration, effective accumulation of nano drugs in the tumor can be observed, and strong DiR fluorescence intensity can still be observed in the tumor after 48 hours, while free DiR can hardly be observed to have obvious intratumoral drug accumulation and significant tumor targeting. The results show that PTX / DiR nano can show rapid tumor aggregation in vivo and good tumor targeting in a short time.

[0089] Example 6

[0090] Animal efficacy experiments of self-synthesized nanorods

[0091] 1.0×10 74T1 cells were suspended in 100 μL of PBS buffer / Matrigel (BD Pharmingen) mixed solution (1:1, vol / vol) and inoculated subcutaneously into the flank region of 5-week-old female C57BL / 6 mice. 3 At 14:1, female C57BL / 6 mice were injected with free PTX and PTX nano as well as free MTZ and MTZnano via the tail vein. The doses of PTX and MTZ were 8 mg / kg and 5.0 mg / kg, respectively, and administered once every other day for a total of 7 doses. The PBS group was used as a control (5 mice per group). The body weight of mice was recorded to evaluate toxicity. Tumor volume was measured with a caliper three times a week, and the calculation formula was: volume = 1 / 2 × LW 2 (L is the longest diameter of the tumor, W is the shortest diameter of the tumor). Finally, all organs were removed and paraffin sections were made. Paraffin sections of all tumors were stained with anti-Ki-67 monoclonal antibody to evaluate tumor proliferation; paraffin sections of all organs were stained with HES for histological analysis. Figure 8 As shown, free drugs can only slowly delay tumor growth, however, after treatment with PTXnano and MTZ nano micelles, mouse tumors were significantly inhibited, and self-synthesized nanorods significantly inhibited tumor growth, with an average tumor volume of only 0.3 times that of the model group, and the tumor of one mouse completely disappeared. In contrast, free drug treatment did not significantly inhibit tumor growth. This was further supported by immunohistochemistry (IHC) analysis to assess tumor cell proliferation.

[0092] In summary, this nano-drug delivery system can not only accumulate more effectively at tumor lesions through the EPR effect, but also penetrate deeper into the tumor, resulting in higher local concentrations of anticancer drugs at the tumor site, ultimately showing better antitumor activity compared with free drugs. Figure 8 As shown, the nanorods exhibited higher anti-tumor activity and lower toxic side effects than free drugs in both in vitro cell experiments and in vivo animal efficacy experiments.

[0093] Therefore, the present invention adopts the preparation method and application of the above-mentioned closed-loop feedback-regulation nano-drug delivery system, which combines the closed-loop feedback-regulation chemical strategy and self-assembly technology to achieve the spontaneous construction of nano-carriers and endow them with excellent drug loading capacity, stability and environmental responsiveness.

[0094] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for preparing a closed-loop feedback-regulated nano drug delivery system, characterized in that: The following steps are involved: Step 1: providing a starting material containing a reversible molecule library, wherein the starting material is used to synthesize the reversible molecule library in a buffer salt system at pH 7.4; Step 2: Under thermodynamic control, anticancer drug molecules and guest molecules are added to the starting material, ultrasonicated in a water bath for 30 minutes, vigorously stirred, and completely oxidized in air after 24 hours. Through a closed-loop feedback-regulation chemical process, the starting material molecules spontaneously form self-assembled nanocarriers containing drug molecules under the joint action of drug molecules and guest molecules.

2. The method for preparing a closed-loop feedback-regulating nano drug delivery system according to claim 1, characterized in that: The reversible molecular library contains a plurality of molecules that are capable of exchanging reactive groups with guest molecules.

3. The method for preparing a closed-loop feedback-regulating nano drug delivery system according to claim 1, characterized in that: The reversible molecule library contains multiple disulfide bond molecules that can be converted into each other through reversible chemical reactions.

4. The method for preparing a closed-loop feedback-regulating nano drug delivery system according to claim 3, characterized in that: The disulfide bond molecule is a dithiol module spontaneously formed by a reversible covalent reaction of dithiol molecules, which contains a carboxylic acid group that can be deprotonated at physiological pH, making the oxidized macrocycle negatively charged. The chemical structure of the dithiol module is as follows:

5. The method for preparing a closed-loop feedback-regulating nano drug delivery system according to claim 1, characterized in that: The anticancer drugs are broad-spectrum antitumor drugs such as paclitaxel, mitoxantrone, and doxorubicin.

6. The method for preparing a closed-loop feedback-regulating nano drug delivery system according to claim 1, characterized in that: The guest molecule is a cationic molecule with a long aliphatic tail chain. The chemical structure of the cationic molecule is as follows: Where n = 5, 6, 7, 8; Where n = 5, 6; Among them, n=5,6,7,8.

7. A closed-loop feedback-regulated nano-drug delivery system, characterized in that: The invention is prepared by the preparation method according to any one of claims 1 to 6; it contains anti-tumor drug molecules, the drug loading amount is 40.1% to 44.1%, and the encapsulation efficiency is as high as 99.2%.

8. A closed-loop feedback-regulating nano-drug delivery system according to claim 7, characterized in that: The nano drug delivery system has the shape of nanorods with a diameter of 20-160 nm.

9. Use of the nano drug delivery system as described in any one of claims 7-8 in preparing a new dosage form of drug for treating tumors.

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