Lycium barbarum polysaccharide-based nano-micelle drug delivery system as well as preparation method and application thereof

Abstract

The invention relates to a lycium barbarum polysaccharide-based nano-micelle capable of responsively delivering hydrophobic chemotherapeutic drugs in a tumor high glutathione (GSH) microenvironment, and belongs to the technical field of medicines. The delivery system is composed of amphiphilic diblock copolymer self-assembled nano-micelles. The amphipathic diblock copolymer is synthesized from lycium barbarum polysaccharide and a lipophilic compound containing disulfide bonds and carboxyl through esterification reaction; the nano-micelle is of a structure with a hydrophobic core formed by self-assembly of a synthesized amphiphilic diblock copolymer in water, and can effectively entrap hydrophobic chemotherapeutic drugs. In a tumor high-GSH microenvironment, GSH and disulfide bonds in the nano-micelles are subjected to an oxidation-reduction reaction to trigger responsive release of a drug, so that precise drug delivery at a tumor part is realized. Research proves that lycium barbarum polysaccharide has an anti-tumor synergistic effect and an immunoregulation effect, not only can be used as a carrier material in the nano-micelle, but also has chemotherapy synergistic and immunoregulation potentials, and has an attenuated synergistic potential of combining medicines with auxiliary medicines.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a Lycium barbarum polysaccharide-based nano-micelle drug delivery system, a preparation method thereof, and an application thereof. Background Art

[0002] Hydrophobic chemotherapeutic drugs are an important part of clinical anti-tumor treatment. However, their poor water solubility, low bioavailability, and severe toxic and side effects caused by non-specific distribution greatly limit their clinical application. To solve these problems, nano-delivery systems have received extensive attention as a new drug delivery strategy. However, traditional nano-carriers have defects such as uncontrollable drug release and single function. The concentration of glutathione (GSH) in the tumor tissue microenvironment is significantly higher than that in normal tissues (about 4-10 times that of normal tissues), and this characteristic provides a theoretical basis for the development of GSH-responsive nano-delivery systems. Nano-carriers containing disulfide bonds can achieve targeted drug release through redox reactions in a high-GSH environment.

[0003] Lycium barbarum polysaccharide (LBP), as a natural active polysaccharide, has been proven to have significant anti-tumor and immunomodulatory activities. Using LBP to construct a nano-drug delivery system has the potential of "combining drug and adjuvant" to "reduce toxicity and increase efficacy". In the prior art, Patent CN 108078939 A discloses an anti-cancer drug carrier preparation, and the raw materials of the drug carrier are succinic anhydride, Lycium barbarum polysaccharide, and α-linolenic acid, which are natural and non-toxic, and have good biocompatibility and biodegradability; Patent CN 115120560 B discloses an anti-tumor targeted drug delivery system, which consists of Lycium barbarum polysaccharide, tumor antigen CD155 plasmid, and liposome. The Lycium barbarum polysaccharide and the tumor antigen CD155 plasmid are encapsulated in the liposome, reducing the toxic and side effects of the anti-tumor targeted drug.

[0004] Although LBP has great development potential as both a carrier material and an active auxiliary component in the field of nano-delivery systems, its potential as a carrier material for nano-drug delivery systems has not been fully developed. At present, combining Lycium barbarum polysaccharide with disulfide bond-containing compounds to construct an intelligent responsive delivery system, enabling it to "combine drug and adjuvant" in the nano-carrier, and thus possessing the potential of "reducing toxicity and increasing efficacy", is still blank in the field of anti-tumor nano-drug research, and has important research value and application prospects. Summary of the Invention

[0005] The purpose of the present invention is to provide a Lycium barbarum polysaccharide-based nano-micelle drug delivery system, a preparation method thereof, and an application thereof to solve the above problems. The nano-micelle drug delivery system provided by the present invention undergoes a redox reaction between GSH and the disulfide bond in the nano-micelle in the tumor high-GSH microenvironment, triggering the responsive release of the drug and achieving precise drug delivery to the tumor site.

[0006] The object of the present invention is achieved by the following technical solutions:

[0007] A Lycium barbarum polysaccharide-based nano-micelle drug delivery system, which is composed of amphiphilic diblock copolymer self-assembled nano-micelles with a hydrophobic environment inside for encapsulating hydrophobic chemotherapeutic drugs; wherein, the amphiphilic diblock copolymer is synthesized by the esterification reaction of Lycium barbarum polysaccharide and a lipophilic compound containing a disulfide bond and a carboxyl group.

[0008] The nano-micelle drug delivery system of the present invention is self-assembled from a synthesized amphiphilic diblock copolymer in water, with a hydrophobic environment inside for encapsulating hydrophobic chemotherapeutic drugs. The tumor microenvironment responsiveness means that in the high-GSH microenvironment of tumors, GSH undergoes a redox reaction with the disulfide bond in the nano-micelles, triggering the responsive release of drugs, thereby achieving precise drug delivery to the tumor site.

[0009] As a preferred technical solution of the present invention, the lipophilic compound includes lipoic acid (LA), dithioctic acid (DSA), and preferably lipoic acid (LA).

[0010] As a preferred technical solution of the present invention, in the amphiphilic diblock copolymer, the mass ratio of the Lycium barbarum polysaccharide to the lipophilic compound is 1:1 - 1:5, preferably 1:1 - 1:3.

[0011] As a preferred technical solution of the present invention, the hydrophobic chemotherapeutic drug is selected from at least one of irinotecan, 7-ethyl-10-hydroxycamptothecin (SN38), paclitaxel, docetaxel, doxorubicin, and is further preferably SN38.

[0012] As a preferred technical solution of the present invention, the mass ratio of the amphiphilic diblock copolymer to the hydrophobic chemotherapeutic drug is 10:1 - 10:5, preferably 10:1 - 10:3.

[0013] As a preferred technical solution of the present invention, the drug loading DL (%) of the hydrophobic chemotherapeutic drug in the delivery system nano-micelles is 2% - 6%, preferably 5.94 ± 0.05%, and the encapsulation efficiency EE (%) is 10% - 66%, preferably 65.33 ± 0.58%.

[0014] As a preferred technical solution of the present invention, the particle size of the amphiphilic diblock copolymer microparticles is 150 - 400 nm, preferably 200 - 250 nm, and further preferably 220 - 240 nm, and the PDI is 0.01 - 0.5, preferably the PDI is 0.01 - 0.3.

[0015] A preparation method of the above-mentioned delivery system, comprising the following steps:

[0016] (1) The polysaccharide of Lycium barbarum and lipophilic compounds are synthesized into amphiphilic diblock copolymers through esterification reaction;

[0017] (2) Hydrophobic chemotherapeutic drugs and the prepared amphiphilic diblock copolymers are used to prepare nanomicelles by nanoparticle co-precipitation method.

[0018] As a preferred technical solution of the present invention, the specific method of step (1) is: dissolving lipophilic compounds and coupling reagents in a solvent and stirring evenly; adding a dimethyl sulfoxide solution containing polysaccharide of Lycium barbarum and a catalyst; stirring and reacting the mixed solution at room temperature, and after the reaction is completed, dialyzing with a dialysis bag and freeze-drying to obtain amphiphilic diblock copolymers;

[0019] And / or, the coupling reagent includes N,N'-dicyclohexylcarbodiimide (DCC);

[0020] And / or, the solvent includes dichloromethane;

[0021] And / or, the catalyst includes 4-dimethylaminopyridine (DMAP);

[0022] And / or, lipophilic compounds containing disulfide bonds and carboxyl groups and coupling reagents are dissolved in a solvent and stirred at room temperature for 20 - 28 h. After stirring is completed, the solution is filtered through a filter membrane to remove impurities formed during the reaction process;

[0023] And / or, the molar ratio of lipophilic compounds containing disulfide bonds and carboxyl groups, coupling reagents, and catalysts is 2:2:1;

[0024] And / or, the mixed solution is stirred and reacted at room temperature for 36 - 72 h, then the solution is dialyzed with a dialysis bag for 18 - 36 h, and stored at -4°C after freeze-drying.

[0025] As a preferred technical solution of the present invention, the specific method of step (2) is: dissolving amphiphilic diblock copolymers and hydrophobic chemotherapeutic drugs in a solvent, continuously stirring at a speed of 200 - 400 rpm at room temperature for 0.5 - 1.5 h, adding water dropwise, and continuing to stir for 2 - 6 h. After completion, the sample is placed in a dialysis bag, and after dialysis for 18 - 32 h, drug-loaded nanoparticles are obtained. The drug-loaded nanoparticle solution is ultrasonically treated and centrifuged to obtain nanomicelles;

[0026] And / or, the solvent includes dimethyl sulfoxide;

[0027] And / or, the drug-loaded nanoparticle solution is ultrasonically treated at 100 - 150 W for 1 - 10 min and centrifuged at 2000 - 3000 rpm for 10 - 20 min.

[0028] The application of the described delivery system in the preparation of anti-tumor drugs, especially in the treatment of colorectal cancer.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. The "drug-excipient integration" design strategy of Lycium barbarum polysaccharide

[0031] The present invention innovatively uses Lycium barbarum polysaccharide as the hydrophilic segment in amphiphilic diblock copolymers, breaking through the limitation that the carrier material in traditional delivery systems only serves as a drug-loading excipient. Lycium barbarum polysaccharide has been proven to have significant anti-tumor synergistic effects and immunomodulatory functions. In this system, it not only provides good biocompatibility and stability but also can produce a synergistic anti-tumor effect with chemotherapeutic drugs. This "drug-excipient integration" design concept expands the function of the carrier material from simple physical drug loading to participating in biological activities, endowing the delivery system with therapeutic activity itself. Compared with traditional inert carrier materials, this design may enhance the chemotherapeutic effect through immunomodulation and direct anti-tumor effects, while reducing the requirement for the dose of chemotherapeutic drugs, thereby reducing toxic and side effects, representing a new paradigm for the design of nano-delivery systems.

[0032] 2. GSH-responsive disulfide bond drug release mechanism

[0033] The present invention ingeniously utilizes the characteristic that the GSH concentration in the tumor microenvironment is higher than that in normal tissues. By introducing a disulfide bond structure into the amphiphilic copolymer, precise drug release at the tumor site is achieved. When the nano-micelles reach the tumor microenvironment, the high concentration of GSH triggers the redox reaction of the disulfide bond, leading to the disassembly of the nano-micelle structure and realizing the site-specific release of the drug. This design overcomes the limitation of traditional nano-delivery systems lacking a specific release mechanism, significantly increasing the drug concentration at the tumor site while reducing the exposure to normal tissues. Compared with ordinary pH-sensitive or enzyme-sensitive delivery systems, the GSH-responsive mechanism provides higher tumor specificity, especially suitable for the treatment of solid tumors. This precise drug release strategy can not only improve the chemotherapeutic effect but also significantly reduce systemic toxicity, providing a new idea for the targeted delivery of hydrophobic anti-cancer drugs. Description of the drawings

[0034] Figure 1 is the Fourier transform infrared spectroscopy (FT-IR) of LBP, LA, and LL polymers;

[0035] Figure 2 is the nuclear magnetic resonance hydrogen spectrum of LBP, LA, and LL polymers;

[0036] Figure 3 is the X-ray diffraction pattern of LBP, LA, and LL polymers;

[0037] Figure 4 is the differential scanning calorimetry detection chart (DSC chart) of LBP, LA, and LL polymers;

[0038] Figure 5 is the critical micelle concentration (CMC) curve of LL;

[0039] Figure 6 is the transmission electron microscopy image of LLNPs;

[0040] Figure 7 is the particle size distribution diagram of LLNPs;

[0041] Figure 8 is the cumulative release amount of SN38 from SN38-loaded LLNPs in simulated gastric fluid (SGF), simulated intestinal fluid (SIF), simulated colon fluid (SCF), and SCF+GSH environments (n = 3) (GSH-mediated responsive release);

[0042] Figures 9-10 is the cytotoxicity of SN38-loaded LLNPs against colon cancer cell line CT-26 after 24 h and 48 h of treatment (detected by CCK8 method);

[0043] Figure 11 is the cytotoxicity of SN38-loaded LLNPs against colon cancer cell line CT-26 after 24 h of treatment with / without GSH (enhanced cytotoxicity caused by GSH-mediated responsive release). Detailed implementation manners

[0044] The present invention will be described in detail below in conjunction with specific embodiments.

[0045] The definitions of the abbreviations in the embodiments are as follows:

[0046] LBP / LBPs: Lycium barbarum polysaccharide;

[0047] LA: Lipoic acid;

[0048] SN38: 7-Ethyl-10-hydroxycamptothecin;

[0049] LBP-LA, LL: Lycium barbarum polysaccharide-lipoic acid amphiphilic diblock copolymer;

[0050] LLNPs: Lycium barbarum polysaccharide-lipoic acid amphiphilic diblock copolymer self-assembled nanomicelles;

[0051] SN38 / LLNPs: Amphiphilic diblock copolymer self-assembled nanomicelles loaded with SN38;

[0052]

Preparation and characterization of SN38 / LLNPs

[0053] (1) Preparation of LL amphiphilic diblock copolymer

[0054] The LL amphiphilic diblock copolymer was synthesized by an esterification reaction. The specific preparation method is as follows: Weigh 0.537 g of LA and a certain amount of N,N'-dicyclohexylcarbodiimide (DCC) and dissolve them in 5 ml of dichloromethane. Use a thermostatic magnetic stirring water bath to stir at a speed of 300 rpm / min at room temperature for 24 h. After stirring, filter the solution through a 0.45 μm filter membrane to remove impurities of N-N dicyclohexylurea formed during the reaction. Then add LBP dissolved in 5 ml of dimethyl sulfoxide (DMSO) with a mass ratio of 1:1 to 1:5. Finally, add 4-dimethylaminopyridine (DMAP), where the molar ratio of LA:DCC:DMAP is 2:2:1, and further stir the resulting solution at the same speed at room temperature for 48 h. Then dialyze the solution with a pre-treated dialysis bag (cut-off molecular weight: 3500) for 24 h, freeze-dry, and store at -4 °C.

[0055] (2) Determination of the critical micelle concentration of the LL amphiphilic diblock copolymer

[0056] The critical micelle concentration (CMC) of the LL amphiphilic diblock copolymer was determined by pyrene fluorescence spectroscopy: Preparation of the pyrene / methanol solution: Under dark conditions, weigh 12.1 mg of pyrene into a 10 ml brown volumetric flask. After dissolving and making up the volume with methanol, transfer 0.2 ml of this pyrene solution to a 100 ml brown volumetric flask and make up the volume to obtain a 1.2×10 -5 mmol / ml pyrene / methanol solution. Seal it with a sealing film and store it in a 4 °C refrigerator for later use.

[0057] Transfer 0.5 ml of the pyrene / methanol solution with a concentration of 1.2×10 -5 mmol / ml into 15 10 ml brown volumetric flasks. Let the methanol naturally volatilize under dark conditions. Then transfer 10 μl, 15 μl, 25 μl, 35 μl, 50 μl, 100 μl, 150 μl, 250 μl, 350 μl, 500 μl, 750 μl, 1 ml, 1.5 ml, 2 ml, 2.5 ml of the LL amphiphilic diblock copolymer solution into the above-mentioned pyrene-containing brown volumetric flasks respectively, and add ultrapure water to make up the volume to prepare a series of concentration gradients: 0.0005, 0.0015, 0.0025, 0.0035, 0.005, 0.01, 0.015, 0.025, 0.035, 0.05, 0.075, 0.1, 0.15, 0.2, 0.25 mg / ml pyrene-containing LL amphiphilic diblock copolymer solutions, where the final concentration of pyrene is 6×10 -7mol / L. Under dark conditions, after shaking well, place it in a constant temperature water bath oscillator at 37 °C and 100 r / min for 12 h, let it stand for 24 h, and then ultrasonicate for 5 min at 120 W to allow the pyrene probe to fully enter the hydrophobic core of the LL amphiphilic diblock copolymer. Use a fluorescence spectrophotometer for wavelength scanning, and the measurement conditions are as follows: the excitation wavelength is set at 335 nm, the excitation slit is set at 20 nm, the emission slit is set at 5.0 nm, the test temperature is set at 25 °C, and scan the fluorescence emission spectra of the LL amphiphilic diblock copolymer solutions containing pyrene at a series of concentrations at 300 - 500 nm. Record the first peak (I1 = 374 nm) and the third peak (I3 = 382 nm) of the fluorescence emission spectra, and calculate the ratio I1 / I 3， Take the logarithm of the LL concentration LgC as the abscissa and the value of I1 / I3 as the ordinate, plot a graph, and use Oringin8.0 to process the experimental data.

[0058] (3) Characterization of LL

[0059] Characterize the structure of the prepared LL amphiphilic diblock copolymer by nuclear magnetic resonance hydrogen spectrum, infrared spectrum, differential scanning calorimetry, and X-ray diffraction.

[0060] (4) Synthesis of LLNPs

[0061] Prepare LL blank nanomicelles by the nanoparticle co-precipitation method: Weigh 10 mg of the LL amphiphilic diblock copolymer and dissolve it in 2 ml of dimethyl sulfoxide (DMSO). Then, continuously stir at a speed of 300 rpm / min at room temperature for 1 h, and slowly add 3 ml of ultrapure water dropwise. After stirring for 4 h, dialyze the mixture for 1 day using a dialysis bag (cut-off molecular weight: 3500) to remove DMSO, and the final concentration is 1 mg / ml. Then, ultrasonicate the solution for 5 min at 120 W and centrifuge at 2500 rpm for 15 min. Take the supernatant to measure the particle size and observe the morphology of the nanoparticles using a transmission electron microscope. Finally, lyophilize the obtained solution and store it at -4 °C for further use.

[0062] (5) Synthesis of SN38 / LLNPs

[0063] Prepare SN38 / LLNPs by the nanoparticle co-precipitation method: Weigh 10 mg of the LL amphiphilic diblock copolymer and 1 mg of SN38 and dissolve them in 2 ml of DMSO. Continuously stir at a speed of 300 rpm / min at room temperature for 1 h, and then slowly add 3 ml of ultrapure water dropwise. After stirring for 4 h, place the sample in a dialysis bag (cut-off molecular weight: 3500). After dialysis for 24 h, drug-loaded nanoparticles are obtained. Ultrasonicate the drug-loaded nanoparticle solution for 5 min at 120 W and centrifuge at 2500 rpm for 15 min. Take the supernatant to measure its drug loading, encapsulation efficiency, and particle size.

[0064] (6) Establishment of SN38 detection method

[0065] The content of SN38 was detected by an ultraviolet-visible spectrophotometer at a detection wavelength of 390 nm. First, an SN38 standard curve was established: 10 mg of SN38 reference standard was weighed and placed in a 100-ml volumetric flask, and diluted to a constant volume with a mixed solvent (DMSO:H2O = 9:1) to obtain an SN38 stock solution with a concentration of 0.1 mg / ml. Then, it was diluted into a series of standard solutions with concentration gradients of 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0 μg / mL. The ultraviolet-visible spectrophotometer was used for detection, and each absorbance value was recorded. A standard curve graph was made with the SN38 concentration as the abscissa (X-axis) and the absorbance value as the ordinate (Y-axis).

[0066] (7) Determination of SN38 encapsulation efficiency and drug loading

[0067] When preparing SN38 / LLNPs, the supernatant obtained by centrifugation was collected. The absorbance value of SN38 in the supernatant was measured by an ultraviolet-visible spectrophotometer, and the drug content of SN38 in the supernatant was calculated by substituting it into the standard curve established above.

[0068] The drug encapsulation efficiency (EE) and drug loading (DL) of SN38 / LLNPs were calculated according to the following formulas:

[0069] Encapsulation efficiency (EE)% = weight of SN38 in nanoparticles / total drug dosage × 100%

[0070] Drug loading (DL)% = weight of SN38 in nanoparticles / weight of drug-loaded nanoparticles × 100%

[0071] (8) Prescription screening of SN38 / LLNPs

[0072] Weigh 10 mg of LL amphiphilic diblock copolymer and 10:1 - 10:5 mg of SN38 in a mass ratio and dissolve them in 2 ml of DMSO. Stir at 300 rpm / min at room temperature for 1 h, slowly add 3 ml of ultrapure water dropwise, stir for 4 h, and then place the sample in a dialysis bag for dialysis for 24 h to obtain the product. Ultrasonic the solution for 5 min under the condition of 120 W, centrifuge at 2500 rpm for 15 min, take the supernatant to measure its drug loading, encapsulation efficiency and particle size, and optimize the drug-loading ratio.

[0073] (9) In vitro responsive release of SN38 in SN38 / LLNPs mediated by GSH

[0074] The dialysis bag diffusion method was used to evaluate the drug release behavior of the prepared nanoparticles. First, 1 mL of free SN38 (containing 0.5 mg of SN38), SN38 / LL NPs, and SN38 / LL NPs + GSH (10 mM) were placed in a dialysis bag with a molecular weight cut-off of 8000 - 14000 Da. Subsequently, the dialysis bag was immersed in 35 mL of PBS buffer containing simulated gastric fluid (pH 1.2, 0 - 2 h), small intestinal fluid (pH 6.8, 2 - 6 h), and colonic fluid (pH 7.4, 6 - 48 h). Then, 0.5% w / v Tween 80 was added to the release medium to promote the dissolution of SN38. The mixture was shaken on a rotary shaker at 37 °C and 100 rpm. At predetermined time intervals: 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 16 h, 24 h, 48 h, 72 h, 96 h, 3 mL of the supernatant was collected and an equal volume of fresh buffer was added. The absorbance of SN38 at 380 nm was obtained using a UV-visible spectrophotometer. Based on the amount of SN38 released, the cumulative release curve of SN38 was plotted.

[0075]

Performance Verification of SN38 / LLNPs at the Cellular Level

[0076] (1) Evaluation of the in vitro cytotoxicity of SN38 / LLNPs

[0077] The CCK-8 method was used to evaluate the cytotoxicity of free SN38 and SN38 / LLNPs. Specifically, CT-26 cells in the logarithmic growth phase were seeded in a 96-well plate at a density of 1×10 4 cells per well in 100 μL of RPMI-1640 medium containing 10% fetal bovine serum. After culturing the cells overnight at 37 °C in a 5% carbon dioxide atmosphere to allow cell adhesion, the medium was discarded and 100 μL of fresh medium containing free SN38 and SN38 / LLNPs at concentrations of 0.1, 1.25, 2.5, 5, 10 μM was added. The cells were incubated for 24 and 48 hours. After incubation, the medium was discarded and the cells were washed twice with PBS. Subsequently, 100 μl of basal medium and 10 μl of CCK-8 were added to each well and incubated for another 2 h. The absorbance of each well at a wavelength of 450 nm was measured using a microplate reader.

[0078] (2) Effect of GSH-mediated responsive release on in vitro cytotoxicity

[0079] Studies have shown that GSH-OEt and BSO can increase and decrease the intracellular glutathione level, respectively. Therefore, GSH-OEt and BSO were used to study the GSH-responsive release of nanodrugs in cells. The specific steps are as follows: CT-26 cells were seeded in 96-well plates containing 100 μl of complete medium at a density of 1×104 cells per well. After the cells adhered overnight, the medium was discarded, and CT-26 cells were pretreated with 1 mM BSO or 20 mM GSH-OEt for 2 h, and then fresh medium containing SN38 / LLNPs was added. The drug-loaded formulations were incubated at concentrations of 0.1, 1.25, 2.5, 5, and 10 μM for 24 h. After incubation, the medium was discarded, and the cells were washed twice with PBS (pH 7.4, 0.01 M). Subsequently, 100 μl of basal medium and 10 μL of CCK-8 were added to each well and incubated for another 2 h. Untreated cells were used as a control. The absorbance of each well at a wavelength of 450 nm was measured using a microplate reader.

[0080] Experimental results

[0081]

Characterization results of LL

[0082] (1) Fourier transform infrared spectroscopy (FT-IR)

[0083] The preparation of the LL polymer was completed according to the esterification reaction. First, the LL polymer was prepared by DCC-mediated condensation reaction, and its chemical structure was characterized by FT-IR. As Figure 1 shown, in the FT-IR spectrum of LA, the stretching vibration of C=O (carboxylic acid) at 1706 cm-1 was attributed to, and the main characteristic peaks of LBP were the O-H stretching vibration peak at 3404 cm-1, the symmetric contraction vibration peak of methylene at 2941 cm-1, and the asymmetric stretching vibration peak of C=O at 1645 cm-1. When LBP was conjugated with LA, the carboxyl peak of LA shifted from 1706 cm-1 (C=O asymmetric stretching vibration peak) to a higher frequency direction, and the peak position was 1736 cm-1, confirming the synthesis of the LL polymer by esterification reaction.

[0084] (2) Proton nuclear magnetic resonance (1H NMR)

[0085] As Figure 2 shown, in the 1H NMR spectrum of LA, the peaks at 2.55 - 2.95 ppm were the proton peaks of -S-CH- and -S-S-CH2, and the proton peaks of LBP were at 2.50 - 6.22 ppm. When LBP was grafted, a new peak appeared at 1.02 - 1.44, proving the synthesis of the LL polymer.

[0086] (3) X-ray diffraction (XRD)

[0087] To further verify whether the LL polymer was successfully synthesized, XRD was used to characterize the structure. XRD is used to collect qualitative information and determine the crystalline state differences in the physicochemical changes of compounds during synthesis and preparation. The results are as Figure 3 shown. The XRD diffraction pattern of LBP showed crystalline peaks at 2θ = 12.68°, 2θ = 14.57°, and 2θ = 20.64°. When LA was grafted, the diffraction peaks of the LL polymer became weaker at these positions. This indicates that the introduction of LA disrupted the original crystalline state of LBP. These results show that the esterification reaction between LA and LBP changed the molecular structure of LBP.

[0088] (4) Differential scanning calorimetry (DSC)

[0089] In the DSC thermogram ( Figure 4 ), LBP had an endothermic melting peak at 86.3 °C and a broad peak at 131 - 137 °C. However, in the LL polymer, these peaks became a broad peak at 83 - 113 °C and the peak intensity became weaker, indicating that the crystalline state of LBP was completely different from that of the LL polymer. The above results prove that the LL polymer was successfully synthesized.

[0090] (5) Determination of the CMC of the LL polymer

[0091] The CMC (critical micelle concentration) was used to verify the performance of the polymer self-assembly to form nanomicelles. Using pyrene as a hydrophobic fluorescent probe, the CMC of the LL polymer was detected by pyrene fluorescence method. As Figure 5 shown, the CMC of the LL polymer was 0.019 mg / ml. The lower CMC indicates that the nanoparticles have good stability even at high dilution and maintain their structure in vivo without dissociation, thus preventing the rapid release of drugs from the nanoparticles before reaching the target.

[0092]

Prescription screening and characterization results of SN38 / LLNPs

[0093] (1) Screening the optimal reaction ratio of LBP and LA by the single factor method

[0094] The particle size and PDI index of LLNPs were detected using a Malvern nano particle size and zeta potential analyzer. Taking the particle size, PDI index, SN38 drug loading DL(%), and encapsulation efficiency EE(%) as comprehensive indicators, the reaction ratio of LA and LBP was optimized. As shown in Table 1, when the mass ratio of LA:LBP was 3:1, the average particle size of the obtained LLNPs was 227.5±3.44 nm, the polydispersity index (PDI) was 0.248±0.016, the particle size was appropriate, and the distribution range was relatively narrow. At the same time, a relatively high drug loading (5.94±0.05%) and encapsulation efficiency (65.33±0.58%) could be obtained. Therefore, the LL amphiphilic diblock copolymer with a mass ratio of LA:LBP of 3:1 was selected for subsequent research.

[0095] Table 1 Screening of the optimal ratio of LBP and LA by the single factor method (n = 3)

[0096]

[0097] (2) Screening of the optimal drug dosage ratio by the single factor method

[0098] To further optimize the formulation, based on the LL amphiphilic diblock copolymer with a mass ratio of LA:LBP of 3:1, the feeding ratio of the LL amphiphilic diblock copolymer and SN38 was optimized. As shown in Table 2, when the mass ratio of the LL polymer to SN38 was 10:1, the average particle size of the obtained SN38 / LLNPs was 236.7±5.22 nm, the PDI was 0.139±0.04, the DL(%) was 5.94±0.05%, and the EE(%) was 65.33±0.58%. Compared with other drug dosage ratios, the particle size was appropriate, the distribution range was relatively narrow, and it had a relatively high drug loading and encapsulation efficiency. Therefore, the feeding ratio of the LL amphiphilic diblock copolymer and SN38 of 10:1 was selected.

[0099] Table 2 Screening of the optimal drug loading ratio by the single factor method (n = 3)

[0100]

[0101] (3) Particle size and morphology characterization of LL NPs

[0102] LLNPs prepared from the LL amphiphilic diblock copolymer with a mass ratio of LA:LBP of 3:1 were spherical and evenly distributed under transmission electron microscopy (see Figure 6 ), and the particle size distribution diagram is shown in Figure 7 .

[0103] (4) GSH-mediated in vitro responsive release of SN38 / LLNPs

[0104] As shown in Figure 8As shown, in the normal colonic environment (pH = 7.4), the release of SN38 from SN38 / LL NPs at 96 h was only 60.52%. The SN38 release rate was significantly accelerated in the GSH environment, and the release amount reached 80.64%, which was significantly higher than that in the normal colonic environment, revealing that SN38 / LL NPs have the ability of GSH-responsive release.

[0105]

Performance Evaluation Results of SN38 / LL NPs at the Cellular Level

[0106] (1) In vitro cytotoxicity of SN38 / LL NPs

[0107] The in vitro cytotoxicity of SN38 / LL NPs against CT-26 cells at 24 h and 48 h was evaluated by the CCK-8 method. As Figures 9-10 shown, the inhibitory effect of SN38 / LL NPs on CT-26 cells was significantly stronger than that of the free drug SN38, and was concentration- and time-dependent.

[0108] (2) GSH-mediated responsive release enhances the cytotoxicity of SN38 / LL NPs

[0109] Studies have shown that buthionine sulfoximine (BSO) downregulates the intracellular glutathione level by inhibiting γ-glutamylcysteine synthetase; glutathione ethyl ester (GSH-oet) is a derivative of GSH and can be used to partially replenish GSH intracellularly in cells experiencing cysteine and / or GSH depletion, and was used to detect the redox-responsive release of SN38 intracellularly. Therefore, using CT-26 cells as a model, the intracellular GSH response of SN38 / LL NPs was studied. As Figure 11 shown, the survival rate of cells without any pretreatment was significantly lower than that of cells treated with BSO, while the survival rate of cells treated with GSH-oet was significantly lower than that of cells pretreated with BSO and untreated cells. After treatment with GSH-oet, the survival rate of cells treated with SN38 / LL NPs was significantly reduced, confirming that GSH-mediated responsive release significantly enhanced the cytotoxicity of SN38 / LL NPs.

[0110] The above description of the embodiments is for the convenience of those of ordinary skill in the art to understand and use the invention. Those familiar with the technology can obviously make various modifications to these embodiments easily and apply the general principles described herein to other embodiments without creative labor. Therefore, the present invention is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art without departing from the scope of the present invention according to the disclosure of the present invention should be within the protection scope of the present invention.

Claims

1. A Lycium barbarum polysaccharide-based nano-micelle drug delivery system, characterized in that: The delivery system is composed of amphiphilic diblock copolymer self-assembled nanomicelles, which have a hydrophobic environment inside and encapsulate the hydrophobic chemotherapeutic drugs; The amphiphilic diblock copolymer is synthesized by esterification reaction of wolfberry polysaccharide and a lipophilic compound containing disulfide bonds and carboxyl groups.

2. The delivery system according to claim 1, characterized in that The lipophilic compound includes lipoic acid (LA) and dithioic acid (DSA), preferably lipoic acid (LA).

3. The delivery system according to claim 1, characterized in that In the amphiphilic diblock copolymer, the mass ratio of the wolfberry polysaccharide to the lipophilic compound is 1:1-1:5, preferably 1:1-1:

3.

4. The delivery system according to claim 1, characterized in that The hydrophobic chemotherapy drug is selected from at least one of irinotecan, 7-ethyl-10-hydroxycamptothecin (SN38), paclitaxel, docetaxel, and doxorubicin.

5. The delivery system according to claim 1, characterized in that The mass ratio of the amphiphilic diblock copolymer to the hydrophobic chemotherapeutic drug is 10:1-10:5, preferably 10:1-10:

3.

6. The delivery system according to claim 1, characterized in that The drug loading amount DL (%) of the hydrophobic chemotherapy drug in the delivery system nano micelle is 2%-6%, and the encapsulation efficiency EE (%) is 10%-66%.

7. A method for preparing the delivery system according to claim 1, characterized in that: The following steps are involved: (1) synthesizing an amphiphilic diblock copolymer by esterification of wolfberry polysaccharide and a lipophilic compound; (2) Hydrophobic chemotherapy drugs and amphiphilic diblock copolymers were prepared into nanomicelles by nanoparticle co-precipitation method.

8. The method for preparing the delivery system according to claim 7, characterized in that: The specific method of step (1) is as follows: dissolving the lipophilic compound and the coupling reagent in a solvent and stirring and mixing; adding a dimethyl sulfoxide solution containing wolfberry polysaccharide and a catalyst; stirring the mixed solution at room temperature for reaction, dialyzing with a dialysis bag after the reaction is completed, and freeze-drying to obtain an amphiphilic diblock copolymer; and / or, the coupling reagent comprises N,N'-dicyclohexylcarbocyanine (DCC); and / or, the solvent comprises dichloromethane; and / or, the catalyst comprises 4-dimethylaminopyridine (DMAP); and / or, a lipophilic compound containing a disulfide bond and a carboxyl group and a coupling reagent are dissolved in a solvent and stirred at room temperature for 20-28 hours, and after the stirring is completed, the solution is filtered with a filter membrane to remove impurities formed during the reaction process; and / or, the molar ratio of the lipophilic compound containing a disulfide bond and a carboxyl group to the coupling reagent and the catalyst is 2:2:1; And / or, the mixed solution is stirred for reaction at room temperature for 36-72 hours, then the solution is dialyzed with a dialysis bag for 18-36 hours, and then freeze-dried and stored at -4°C.

9. The method for preparing the delivery system according to claim 7, characterized in that: The specific method of step (2) is as follows: dissolving the amphiphilic diblock copolymer and the hydrophobic chemotherapy drug in a solvent, stirring continuously at a speed of 200-400 rpm for 0.5-1.5 hours at room temperature, adding water dropwise, and continuing stirring for 2-6 hours. After the end, placing the sample in a dialysis bag, dialyzing for 18-32 hours to obtain drug-loaded nanoparticles, and subjecting the drug-loaded nanoparticle solution to ultrasonic and centrifugal treatment to obtain nanomicelles; and / or, the solvent comprises dimethyl sulfoxide; And / or, the drug-loaded nanoparticle solution is sonicated at 100-150 W for 1-10 min, and centrifuged at 2000-3000 rpm for 10-20 min.

10. Use of the delivery system according to claim 1 in the preparation of anti-tumor drugs.

Images