Amphiphilic cascade response chitosan nano drug delivery system as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of nano drug delivery systems, and particularly relates to an amphiphilic cascade response chitosan nano drug delivery system and a preparation method and application thereof. The nano drug delivery system is of a multi-layer structure composed of an enzyme response inner layer and a pH response outer layer; the enzyme response inner layer is formed by sulfobutyl ether-beta-cyclodextrin and protamine sulfate through electrostatic adsorption, and a hydrophobic anti-cancer drug is loaded in a hydrophobic cavity of the sulfobutyl ether-beta-cyclodextrin to form enzyme response supramolecular nanoparticles; the pH response outer layer is composed of amphiphilic chitosan which is hydrophilically modified by succinic anhydride and hydrophobically modified by deoxycholic acid, and the amphiphilic chitosan is coated on the surfaces of the enzyme response supramolecular nanoparticles. By utilizing the arrangement of the structure, the invention provides the nano drug delivery system based on the amphipathic chitosan, which is suitable for intestinal targeted delivery and controlled release therapy of hydrophobic anti-cancer drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of nano drug delivery system, and particularly relates to an amphiphilic cascade response chitosan nano drug delivery system and a preparation method and application thereof. BACKGROUND

[0002] Hydrophobic antitumor oral drugs face multidimensional challenges in research and development and clinical application, and need to be comprehensively analyzed from multiple aspects such as physicochemical properties, absorption mechanism, preparation technology and clinical practice. First, the physicochemical properties of the drug limit the absorption and bioavailability. The contradiction between high fat solubility and low water solubility makes it difficult for the drug to be effectively dissolved and released in the gastrointestinal tract. For example, paclitaxel drugs need to rely on complex preparation technology (such as liposomes or nanoparticles) to improve dispersibility due to insufficient solubility, but such technology faces problems such as high cost of large-scale production and poor stability. In addition, gastrointestinal environment fluctuations (such as pH changes and differences in gastric emptying speed) and food interference (such as high-fat diet which may promote absorption but with significant individual differences) further affect the stability of drug absorption, and metabolic loss caused by first-pass effect (such as degradation by CYP450 system) also greatly reduces the bioavailability. For example, fulvestrant oral preparation is difficult to develop due to liver metabolism problems. Second, there are bottlenecks in preparation technology. Although new delivery systems (such as nanometer preparations and exosome carriers) can improve drug solubility, problems such as easy aggregation of nanoparticles, balance between sustained-release preparations and targeting, and the like limit practical application. For example, Elacestrant has limited efficacy due to a bioavailability of only 10%, and the accumulation of hydrophobic drugs in the intestine may increase mucosal irritation. At the same time, clinical toxicity problems are prominent, and dose-dependent toxicity (such as bone marrow suppression and neurotoxicity) and metabolic product toxicity (such as liver damage) force dose adjustment and monitoring mechanisms to be complicated. For example, the neurotoxicity of paclitaxel drugs is directly related to their liposolubility, and the metabolic product of clozapine needs to be strictly monitored. At the clinical application level, patient compliance is affected by complex medication requirements (such as taking on an empty stomach and dietary restrictions), and patients with brain metastases have difficulty obtaining effective concentrations through oral administration due to the blood-brain barrier, and some SERD drugs cannot penetrate the barrier.

[0003] Existing drug delivery technologies have significant limitations in achieving intestinal targeted therapy:

[0004] The stability of the material is split from its function, and a single carrier system such as sulfobutyl ether-β-cyclodextrin (SBE-β-CD) can load drugs in the hydrophobic cavity, but it is easily dissociated in the stomach acid environment, leading to premature release and degradation of the drug. The degradation rate of protamine sulfate and other protein carriers is more than 80% when they are directly exposed to pepsin. Existing technologies focus on a single function (such as solubilization or pH response), and lack a multi-layer collaborative design integrating stomach acid protection, enzyme-triggered release and mucosal penetration.

[0005] The bioenvironmental adaptability of ordinary chitosan is poor, with a swelling degree of only 15-30% in gastric acid, and the protection effect is unstable. In addition, the trypsin activity is affected by the fluctuation of ion concentration, which makes the drug release kinetics difficult to predict. The intestinal mucus layer (thickness 50-450 μm) hinders drug penetration, and the penetration rate of traditional carriers is less than 1% due to the large particle size or mismatched surface charge.

[0006] Process and safety bottleneck: SBE-β-CD and drug inclusion need precise control of molar ratio, and the grafting degree of modified chitosan needs precise process support, but the yield of microfluidic technology is only 60-75%.

[0007] In addition, deoxycholic acid metabolites may induce DNA damage, and protamine sulfate fragments may activate inflammatory pathways. These limitations require interdisciplinary technological innovation, such as biomimetic multi-layer structure design and AI-assisted material screening, to overcome the adaptability limitations of existing technologies to complex physiological environments. SUMMARY

[0008] The purpose of the present application is to provide an amphiphilic cascade response chitosan nanocarrier system and its preparation method and application, to solve the problems of gastric acid degradation, poor targeting and low absorption efficiency in oral delivery of hydrophobic drugs, and to achieve the purpose of oral intestinal targeted delivery and efficient utilization of hydrophobic anticancer drugs.

[0009] To achieve the above purpose, the present application provides the following solutions:

[0010] The amphiphilic cascade response chitosan nanocarrier system is a multi-layer structure composed of an enzyme-responsive inner layer and a pH-responsive outer layer.

[0011] The enzyme-responsive inner layer is formed by electrostatic adsorption of sulfobutyl ether-β-cyclodextrin and protamine sulfate, and the hydrophobic anticancer drug is loaded in the hydrophobic cavity of the sulfobutyl ether-β-cyclodextrin to form an enzyme-responsive supramolecular nanoparticle.

[0012] The pH-responsive outer layer is composed of amphiphilic chitosan modified by succinic anhydride and deoxycholic acid, and the amphiphilic chitosan is coated on the surface of the enzyme-responsive supramolecular nanoparticle.

[0013] A method for preparing the amphiphilic chitosan nanocarrier system, comprising:

[0014] S1, grafting succinic anhydride at the C2-NH2 position of chitosan for hydrophilic modification, and grafting deoxycholic acid at the remaining C2-NH2 position of chitosan for hydrophobic modification;

[0015] S2, drop the model molecule into the aqueous solution of sulfobutyl ether-beta-cyclodextrin and stir, after stirring, mix with protamine sulfate, and prepare a supramolecular nanoparticle solution with a microfluidic device;

[0016] S3, drop the drug-loaded supramolecular nanoparticle solution into the modified chitosan solution and stir and dialyze to obtain a nano-drug delivery system.

[0017] Preferably, in step S1, the molecular weight of chitosan is 30-100 KDa.

[0018] Preferably, in step S1, the grafting rate of succinic anhydride at C2-NH2 of chitosan is 30%-50%.

[0019] Preferably, in step S1, the grafting rate of deoxycholic acid at C2-NH2 of chitosan is 3-5%.

[0020] Preferably, in step S2, the concentration of sulfobutyl ether-beta-cyclodextrin is 0.6-1.0 mg / mL, and the concentration of protamine sulfate is 0.4-0.8 mg / mL.

[0021] Preferably, in step S2, the molar ratio of sulfobutyl ether-beta-cyclodextrin to model molecule is 1:1.

[0022] Preferably, in step S2, after mixing sulfobutyl ether-beta-cyclodextrin with the model molecule, stir at 50-70°C for 12-14 hours to mix evenly.

[0023] Preferably, in step S3, during the dialysis process, use a dialysis bag with a molecular weight cut-off of 3500 Da for dialysis purification.

[0024] An application of the amphilic cascade response chitosan nano-drug delivery system as a nano-carrier.

[0025] Compared with the prior art, the present application has the following advantages and technical effects:

[0026] The nano-drug carrier has a simple preparation method, small raw material consumption, good stability and biocompatibility, good selectivity for trypsin, and good stability in strong acid environment, which provides favorable conditions for loading target drugs for treating diseases, and further provides an amphilic chitosan nano-drug delivery system suitable for intestinal targeting delivery and controlled release treatment of hydrophobic anticancer drugs. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained from these drawings without creative labor:

[0028] Figure 1 Synthetic route of amphiphilic chitosan

[0029] Figure 2 IR spectrum of the hydrophilic modified chitosan in the present application 1 H NMR chart

[0030] Figure 3 IR spectrum of the hydrophilic modified chitosan in the present application

[0031] Figure 4 XRD spectrum of the hydrophilic modified chitosan in the present application

[0032] Figure 5 IR spectrum of the amphiphilic modified chitosan 1 H NMR chart

[0033] Figure 6 IR spectrum of the amphiphilic modified chitosan

[0034] Figure 7 Critical micelle concentration of the amphiphilic chitosan

[0035] Figure 8 Critical aggregation concentration of sulfobutyl ether-β-cyclodextrin

[0036] Figure 9 Critical aggregation concentration of protamine sulfate

[0037] Figure 10 TEM chart of core layer supramolecular nanoparticle loaded dolastatin 10

[0038] Figure 11 TEM chart of amphiphilic chitosan encapsulated supramolecular nanoparticles

[0039] Figure 12 Ultraviolet absorption spectrum of chitosan drug loading system simulation release

[0040] Figure 13 1HNMR chart of low molecular weight chitosan modified deoxycholic acid DETAILED DESCRIPTION

[0041] With reference to the accompanying drawings, the technical solutions in the embodiments of the present application will be clearly and completely described below, obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present application.

[0042] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the present application will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0043] Reference Figures 1-13 As shown in the drawings, the present application discloses an amphiphilic cascade response chitosan nanocarrier system, the nanocarrier system is a multilayer structure composed of an enzyme response inner layer and a pH response outer layer;

[0044] The enzyme response inner layer is formed by electrostatic adsorption of sulfobutyl ether-beta-cyclodextrin and protamine sulfate, and the hydrophobic anticancer drug is loaded in the hydrophobic cavity of the sulfobutyl ether-beta-cyclodextrin to form an enzyme response supramolecular nanoparticle;

[0045] The pH response outer layer is composed of amphiphilic chitosan modified by succinic anhydride and deoxycholic acid, and the amphiphilic chitosan is coated on the surface of the enzyme response supramolecular nanoparticle.

[0046] A method for preparing an amphiphilic chitosan nanocarrier system, comprising:

[0047] S1, grafting succinic anhydride at the C2-NH2 position of chitosan for hydrophilic modification, and grafting deoxycholic acid at the remaining C2-NH2 position of chitosan for hydrophobic modification;

[0048] S2, drop the model molecule into the aqueous solution of sulfobutyl ether-beta-cyclodextrin and stir, then mix it with protamine sulfate to prepare a supramolecular nanoparticle solution by using a microfluidic device;

[0049] S3, drop the drug-loaded supramolecular nanoparticle solution into the modified chitosan solution and stir and dialyze to obtain a nanocarrier system.

[0050] Further optimization scheme, in step S1, the molecular weight of chitosan is 30-100 KDa.

[0051] Further optimization scheme, in step S1, the grafting rate of succinic anhydride at the C2-NH2 position of chitosan is 30%-50%.

[0052] Further optimization scheme, in step S1, the grafting rate of deoxycholic acid at the C2-NH2 position of chitosan is 3-5%.

[0053] Further optimization scheme, in step S2, the concentration of sulfobutyl ether-β-cyclodextrin is 0.6-1.0 mg / mL, and the concentration of protamine sulfate is 0.4-0.8 mg / mL.

[0054] Further optimization scheme, in step S2, the molar ratio of sulfobutyl ether-β-cyclodextrin to model molecule is 1:1.

[0055] Further optimization scheme, in step S2, after mixing sulfobutyl ether-β-cyclodextrin with model molecule, stirring at 50-70℃ for 12-14 hours to mix evenly.

[0056] Further optimization scheme, in step S3, using a dialysis bag with a molecular weight cut-off of 3500 Da for dialysis purification.

[0057] Application of amphiphilic cascade response chitosan nanocarrier system as nanocarrier.

[0058] Example 1

[0059] A method for preparing an amphiphilic chitosan nanocarrier system, comprising:

[0060] S1, grafting succinic anhydride at C2-NH2 of chitosan for hydrophilic modification, and grafting deoxycholic acid at the remaining C2-NH2 of chitosan for hydrophobic modification:

[0061] Take 1.000g, 30KDa chitosan in 100mL, 5% acetic acid aqueous solution to stir, get chitosan solution, 2g succinic anhydride is dissolved in 40mL acetone, the latter is added dropwise into the former at room temperature to carry out acylation reaction, the reaction is completed after 12h, adjust pH to 7, precipitate the product with two times the volume of ethanol, centrifuge, wash the precipitate with acetone, ethanol, acetone in turn, redissolve the sample in water, dialyze for 72h, freeze and dry to obtain succinylated chitosan, the grafting rate of succinic anhydride at C2-NH2 of succinylated chitosan is controlled at 30-50%;

[0062] Dissolve 200mg succinylated chitosan in water to get chitosan aqueous solution, take 196mg deoxycholic acid dissolved in dimethyl sulfoxide (DMSO), add 232.5mg of 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide (EDC) and activate at room temperature for 30min, then add 230mg of N-hydroxysuccinimide (NHS) and activate at room temperature for 1h, drop the activated deoxycholic acid solution into the chitosan solution, stir for 24h, precipitate the product with ethanol, dialyze, freeze and dry to obtain amphiphilic chitosan, the grafting rate of deoxycholic acid at C2-NH2 of amphiphilic chitosan is controlled within 3-5%;

[0063] S2, drop the model molecule into the aqueous solution of sulfobutyl ether-β-cyclodextrin and stir, after stirring, mix with protamine sulfate to prepare a solution of supramolecular nanoparticles by microfluidic device;

[0064] Take the alcohol solution of curcumin (model molecule) in 0.8 mg / mL sulfobutyl ether-β-cyclodextrin (molar ratio 1:1) and stir at 50°C for 12 h, centrifuge to remove free astaxanthin, and then mix with 0.6 mg / mL protamine sulfate (equal volume to 0.8 mg / mL sulfobutyl ether-β-cyclodextrin) to prepare drug-loaded supramolecular nanoparticles by microfluidic device, and freeze-dry.

[0065] S3, drop the drug-loaded supramolecular nanoparticle solution into the modified chitosan solution and stir, dialysis to obtain a nano-drug delivery system.

[0066] Add the drug-loaded nanoparticles to the aqueous solution of amphiphilic chitosan, adjust the pH value of the solution (adjust to pH 6-7) and stir, modify the chitosan to self-assemble to load the drug-loaded nanoparticles to construct a drug delivery system, and finally purify by dialysis with a molecular weight cut-off of 3500 Da dialysis bag, and freeze-dry to obtain the product (nano-drug delivery system).

[0067] Example 2

[0068] The difference between this embodiment and Example 1 is that the model molecule is an alcohol solution of astaxanthin.

[0069] Example 3

[0070] The difference between this embodiment and Example 1 is that the model molecule is an alcohol solution of quercetin.

[0071] Example 4

[0072] The difference between this embodiment and Example 1 is that the model molecule is an alcohol solution of dolastatin 10.

[0073] Example 5

[0074] This embodiment is based on the logic of Example 1, and the low molecular weight chitosan is modified. Since the low molecular weight chitosan (such as 2KDa) has a short molecular chain, low crystallinity, and sufficient exposure of amino groups (-NH2), it can form a stable solution by protonation of amino groups (-NH3 + ) in water without the need for additional introduction of hydrophilic groups.

[0075] The process includes:

[0076] S1, hydrophobic modification of chitosan C2-NH2 grafted with deoxycholic acid:

[0077] Take 1.000g 2KDa chitosan in 70mL deionized water to stir and dissolve, get chitosan solution. 0.80g deoxycholic acid, 1.0g EDC, 0.5g NHS are dissolved in 30mL DMSO, activated stirring 30min. The activated deoxycholic acid solution is added dropwise into the chitosan solution at room temperature for acylation reaction, after 24h reaction, the grafting rate is controlled within 20-25%. The reaction solution is dialyzed for 24h, after centrifugation, the supernatant is freeze-dried to obtain the sample deoxycholic acid-chitosan.

[0078] S2, the model molecule is added dropwise to the aqueous solution of sulfobutyl ether-β-cyclodextrin and stirred, after stirring, it is mixed with protamine sulfate to prepare a supramolecular nanoparticle solution by microfluidic device;

[0079] Take the alcohol solution of curcumin (model molecule) in 0.8mg / mL sulfobutyl ether-β-cyclodextrin (molar ratio 1:1) and stir at 50℃ for 12h, centrifuge to remove free astaxanthin, then mix with 0.6mg / mL protamine sulfate to prepare drug-loaded supramolecular nanoparticles by microfluidic device, and freeze-dry.

[0080] S3, the drug-loaded supramolecular nanoparticle solution is added dropwise to the modified chitosan solution and stirred and dialyzed to obtain a nano-drug delivery system.

[0081] Take the alcohol solution of dolastatin 10 in 0.8mg / mL sulfobutyl ether-β-cyclodextrin (molar ratio 1:1) and stir for 12h, centrifuge to remove free dolastatin 10, then mix with 0.6mg / mL protamine sulfate to prepare drug-loaded supramolecular nanoparticles by microfluidic device, and freeze-dry. The drug-loaded nanoparticles are added to the aqueous solution of amphiphilic chitosan, the pH value of the solution is adjusted and stirred, and the modified chitosan self-assembles to encapsulate the drug-loaded nanoparticles to construct a drug delivery system.

[0082] Example 6

[0083] The difference between this example and example 1 is only that in step S1, 1.000g, 100KDa chitosan is stirred in 100mL 5% acetic acid aqueous solution to obtain chitosan solution.

[0084] Next, the performance of the samples with different grafting rates in example 1 and example 6 is studied.

[0085] Among them, example 1 includes four samples, and the grafting rates of the four samples are detected as 30.7%, 36.9%, 45.7%, and 50.0% respectively. Example 6 includes four samples, and the grafting rates of the four samples are detected as 36.3%, 43.9%, 49.3%, and 52.9% respectively.

[0086] Test Example 1

[0087] The test example is a dynamic light scattering method for measuring the particle size (Size / nm) of the sample nanoparticles, including:

[0088] S1, take a small amount of nanometer drug delivery system suspension (50 μL), dilute with deionized water or normal saline to a suitable concentration (avoid mutual interference between particles, usually dilute to OD value 0.1-0.5);

[0089] S2, inject the diluted sample into a special quartz cuvette to avoid bubbles;

[0090] S3, use DLS instrument (Malvern Zetasizer Nano series), set the measurement temperature (25℃, simulate physiological environment), scattering angle (90° or 173°), repeat the measurement 3-5 times, and take the average value as the final particle size.

[0091] The specific results are shown in Table 1.

[0092] In Table 1, the particle size of the 30KDa amphiphilic chitosan coated drug delivery system (30-ASC(CPD10)) is in the range of 270.9-295.4 nm, and the particle size of the 100KDa chitosan coated system (100-ASC(CPD10)) is in the range of 282.3-503.1 nm, both of which are in the optimal intestinal delivery nanometer size interval of 200-500 nm:

[0093] From the physiological adaptability, this particle size not only avoids the problem of "<100nm particles being easily removed by the intestinal lymphatic system", but also overcomes the traditional defect of ">500nm particles being unable to penetrate the intestinal mucus layer (thickness 50-450 μm) resulting in a penetration rate of less than 1%", and can pass through the network structure of the mucus layer to reach the surface of the intestinal epithelial cells, laying a physical foundation for targeted release;

[0094] From the structural synergy, the particle size is precisely regulated by the multi-layer design of "inner supermolecular core (SBE-β-CD+protamine sulfate) + outer amphiphilic chitosan coating", without obvious aggregation (such as 100-ASC(CPD10) only a few groups reach 503.1 nm, and PI is still <0.3), which reflects the precise control of the particle size by the microfluidic preparation process, which is superior to the limitations of the prior art "nanoparticles are easy to aggregate and the particle size fluctuates greatly".

[0095] Test Example 2

[0096] This test example is to detect the polydispersity index (Polydispersity Index, PI) of the sample. In the DLS measurement in Test Example 1, the parameters are directly calculated by analyzing the fluctuation of scattered light, thereby reflecting the uniformity of the particle size distribution of the sample.

[0097] The specific results are shown in Table 1.

[0098] The PI values of all samples in Table 1 are in a narrow interval of 0.13-0.29, which is much lower than the "particle size distribution uniformity qualified standard (PI < 0.3)":

[0099] From the process controllability, the low PI value proves that the present technology can realize the particle size uniformity between batches and within batches through the combined process of "microfluidic precise mixing (inner core preparation) + self-assembly coating of amphiphilic chitosan (outer coating)", avoiding the "multi-modal distribution" problem caused by uneven mixing in traditional preparation methods (such as emulsion-solvent evaporation method), which provides stability support for industrial scale-up production;

[0100] From the safety of drug efficacy, uniform particle size means consistent surface properties of the carrier, which can ensure the synchronization of drug release kinetics in the intestinal tract (without the phenomenon of "partial particles releasing in advance, partial particles releasing with delay"), avoiding the risk of "local drug concentration being too high causing toxicity" or "concentration being too low leading to insufficient efficacy" caused by uneven release, and solving the pain point of "drug release kinetics being difficult to predict" in the background art.

[0101] Test Example 3

[0102] This test example is to measure the migration rate of the nanoparticle sample in the dispersion medium, and convert it into zeta potential by Smoluchowski equation, including:

[0103] S1, dilute a small amount of nanocarrier drug delivery system suspension (50 μL) with deionized water or normal saline to a suitable concentration

[0104] S2, inject the sample into a special electrophoresis cell, after the instrument applies an electric field, detect the Doppler frequency shift of the scattered light generated by the particle migration;

[0105] S3, repeat the measurement 3-5 times, and take the average value as the zeta potential.

[0106] The specific results are shown in Table 1.

[0107] The zeta potential of the samples in Table 1 ranges from -10.46 to -28.84 mV, and the absolute value is concentrated in the interval of 15-30 mV, showing a "weak negative" characteristic:

[0108] From the colloidal stability, the absolute value of the potential is sufficient to inhibit the aggregation during storage and gastrointestinal transit through the electrostatic repulsion between particles (avoiding the sudden increase in particle size caused by aggregation and the inability to penetrate the mucus layer), and the weak negative characteristic can reduce the non-specific adsorption with the intestinal mucosa epithelial cells (negatively charged), reducing the probability of rapid clearance of the carrier;

[0109] From the pH response adaptability, the amino groups of the outer amphiphilic chitosan are protonated in the acidic environment of the stomach (zeta potential absolute value decreases, approaches electrical neutrality), which can form a dense coating to protect the drug; after entering the neutral environment of the intestine, the amino groups are deprotonated (zeta potential restores negative), which promotes the swelling and enzymatic hydrolysis of chitosan. The dynamic change of the potential is highly synergistic with the cascade response mechanism of "stomach protection-intestinal release", which is superior to the defects of traditional carriers "fixed charge, unable to adapt to the pH fluctuation of stomach and intestine".

[0110] Test Example 4

[0111] This test example is to measure the encapsulation efficiency of the sample (encapsulation efficiency = (total drug amount - free drug amount) / total drug amount x 100%), which specifically includes:

[0112] S1, a certain volume of nanometer drug-loaded system suspension is taken, and high-speed centrifugation is used to precipitate the nanoparticles;

[0113] S2, the supernatant is taken, and the free drug concentration is determined by ultraviolet-visible spectrophotometry (UV-Vis) or high performance liquid chromatography (HPLC), and the free drug amount Mfreeis calculated;

[0114] The encapsulation efficiency EE% = (M 总 -M 游 ) / M 总 x 100%

[0115] The specific results are shown in Table 1.

[0116] In Table 1, the EE of 30-ASC(CPD10) is 77.6% to 93.8%, the EE of 100-ASC(CPD10) reaches 94.6% to 98.8%, and the EE of the 100KDa chitosan coating system is close to 100%:

[0117] From the drug utilization rate, a high EE value means that the hydrophobic drug (such as dolastatin 10) is efficiently intercepted by the carrier, and the proportion of free drug is extremely low, which can significantly reduce the problems of premature release and degradation of drugs in the stomach and leakage in non-target areas of the intestine, and reduce the loss of bioavailability caused by the first pass effect of liver metabolism;

[0118] From the structural synergy, a high EE is derived from the dual action of "inner layer SBE-β-CD hydrophobic cavity inclusion + outer layer amphiphilic chitosan physical barrier": SBE-β-CD fixes the drug through hydrophobic interaction, and the chitosan coating further isolates the contact between the drug and the gastrointestinal environment, and the two synergistically realize the efficient encapsulation of the drug, which is superior to the drug loading capacity of existing single carriers (such as protamine sulfate alone, EE <60%).

[0119] Test Example 5

[0120] The test example is for measuring the drug loading of the sample, which specifically includes:

[0121] S1, take a certain volume of drug-loaded particle powder of the nano-drug delivery system, weigh (denoted as W 总 , which is the total mass of the carrier + drug);

[0122] Completely dissolve the dry particles with an organic solvent (such as methanol, dimethyl sulfoxide DMSO) (destroy the carrier structure, release all the encapsulated drugs);

[0123] Determine the total drug concentration in the solution by UV-Vis or HPLC, and calculate the amount of encapsulated drug M 包 ;

[0124] Drug loading LC% = M 包 / W 总 × 100%

[0125] The specific results are shown in Table 1.

[0126] The LC of the sample in Table 1 is 4.4% to 5.5%, which has a significant clinical advantage for hydrophobic antitumor drugs (usually LC < 3%):

[0127] From the dose adaptation, an LC of about 5% can achieve an effective treatment dose in a smaller drug volume, avoiding the problem of "large drug volume (such as > 50 mL) leading to difficulty in swallowing and poor compliance" of traditional low LC carriers;

[0128] From the material safety, the LC is the result of the balance between "drug inclusion capacity" and "carrier material safety": it maximizes drug loading by designing a 1:1 molar ratio of SBE-β-CD and drug, avoids "carrier structure collapse and drug burst" caused by excessive drug loading, and reduces the relative amount of carrier materials (such as deoxycholic acid, protamine sulfate) to reduce the potential toxicity of their metabolites.

[0129] Table 1 encapsulation efficiency and loading rate statistics

[0130]

[0131]

[0132] The five parameters in Table 1 form a closed loop verification from "physical form-preparation process-function performance-clinical adaptation": the particle size and zeta potential ensure the penetration and stability of the carrier in the gastrointestinal environment, the PI proves the controllability of the process, the EE and the LC improve the drug utilization and safety, which jointly solves the core pain points in the prior art such as "nanoparticles are easy to aggregate, drug release is uncontrollable, drug loading efficiency is low, and mucosal penetration is difficult", and fully embodies the superiority of the technical scheme in the field of "hydrophobic anticancer drug intestinal targeted delivery".

[0133] The above-described embodiments are only descriptions of the preferred modes of the present application and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. An amphiphilic cascade-response chitosan nanoparticle drug delivery system, characterized in that, The nano-drug delivery system is a multi-layered structure consisting of an enzyme-responsive inner layer and a pH-responsive outer layer. The enzyme-responsive inner layer is formed by electrostatic adsorption of sulfobutyl ether-β-cyclodextrin and protamine sulfate. Hydrophobic anticancer drugs are loaded into the hydrophobic cavity of the sulfobutyl ether-β-cyclodextrin to form enzyme-responsive supramolecular nanoparticles. The pH-responsive outer layer is composed of amphiphilic chitosan modified with hydrophilic succinic anhydride and hydrophobic deoxycholic acid, and the amphiphilic chitosan coats the surface of the enzyme-responsive supramolecular nanoparticles.

2. A method for preparing the amphiphilic chitosan-based nanoparticle drug delivery system as described in claim 1, characterized in that, include: S1. Succinic anhydride is grafted onto the C2-NH2 position of chitosan for hydrophilic modification, and deoxycholic acid is grafted onto the remaining C2-NH2 position of chitosan for hydrophobic modification. S2. The model molecule was added dropwise to an aqueous solution of sulfobutyl ether-β-cyclodextrin and stirred. After stirring, it was mixed with protamine sulfate and a supramolecular nanoparticle solution was prepared using a microfluidic device. S3. The drug-loaded supramolecular nanoparticle solution is added dropwise to the modified chitosan solution, stirred, and dialyzed to obtain the nano-drug-loaded system.

3. The preparation method of the amphiphilic cascade-responsive chitosan nanoparticle drug delivery system according to claim 2, characterized in that: In step S1, the molecular weight of chitosan is 30~100 kDa.

4. The preparation method of the amphiphilic cascade-responsive chitosan nanoparticle drug delivery system according to claim 2, characterized in that: In step S1, the grafting rate of succinic anhydride onto the C2-NH2 position of chitosan is 30%~50%.

5. The preparation method of the amphiphilic cascade-responsive chitosan nanoparticle drug delivery system according to claim 2, characterized in that: In step S1, the grafting rate of deoxycholic acid at the C2-NH2 position of chitosan is 3-5%.

6. The preparation method of the amphiphilic cascade-responsive chitosan nanocarrier system according to claim 2, characterized in that: In step S2, the concentration of sulfobutyl ether-β-cyclodextrin is 0.6~1.0 mg / mL, and the concentration of protamine sulfate is 0.4~0.8 mg / mL.

7. The preparation method of the amphiphilic cascade-responsive chitosan nanoparticle drug delivery system according to claim 2, characterized in that: In step S2, the molar ratio of sulfobutyl ether-β-cyclodextrin to the model molecule is 1:

1.

8. The preparation method of the amphiphilic cascade-responsive chitosan nanoparticle drug delivery system according to claim 2, characterized in that: In step S2, after sulfobutyl ether-β-cyclodextrin is mixed with the model molecule, it is stirred at 50~70℃ for 12~14 hours to mix evenly.

9. The preparation method of the amphiphilic cascade-responsive chitosan nanoparticle drug delivery system according to claim 2, characterized in that: In the dialysis process of step S3, a dialysis bag with a molecular weight cutoff of 3500 Da is used for dialysis purification.

10. An application of the amphiphilic cascade response chitosan nanocarrier system according to claim 1 as a nanocarrier.

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