Polypeptide molecular gate boron micelle drug delivery system as well as preparation method and application thereof

Abstract

The invention provides a polypeptide molecular gate boron micelle drug delivery system as well as a preparation method and application thereof. The drug delivery system comprises a carrier skeleton, a polypeptide molecular gate regulation and control layer, a targeting ligand and an entrapped 10B-containing drug, the carrier skeleton is of a core-shell structure formed by co-assembling at least two amphiphilic copolymers; the amphiphilic copolymer comprises a block copolymer; the hydrophobic inner core of the core-shell structure is entrapped with a 10B-containing drug. According to the polypeptide molecule gate boron micelle, PLGA-mPEG is used as a carrier skeleton, a hydrophobic core stably entraps a 10B-containing drug, and the problem of solubility of the 10B-containing drug is solved; the polypeptide molecular gate realizes tumor microenvironment response release, and the targeting ligand enhances tumor enrichment; the material has good biocompatibility, and can improve the concentration of 10B in tumors, enhance the curative effect of BNCT, reduce damage to normal tissues and guarantee medication safety.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of nanobiomedicine, and relates to a poly-polypeptide molecular porodex micelle drug delivery system, a preparation method thereof and application thereof. BACKGROUND

[0002] Boron neutron capture therapy is a new and rapidly developing precise diagnosis and treatment technology in the international oncology field in recent years, and is the "fifth therapy" after surgery, traditional radiotherapy, anticancer drugs and immunotherapy.

[0003] Boron neutron capture therapy is a binary radiotherapy technology that can accurately target tumor sites. The principle is to inject a boron-containing targeted molecule drug specific to the tumor into the human body. This drug can make 10 B specifically enriched in tumor tissue, and then irradiate the tumor with a directional second low-energy epithermal neutron beam outside the body. After the neutron enters the human body, it is captured by 10 B in the drug enriched in the tumor site, and then undergoes nuclear reaction to release 7 Li particles. Due to the short range of the released alpha particle fragments, only about one cell size, and the high energy line density and relative biological effect of the particle and 7 Li particles, the DNA double helix structure of tumor cells is irreversibly broken, resulting in the complete death of tumor cells, thereby achieving cell-scale point killing of cancer cells without damaging normal tissue cells. It is suitable for infiltration, diffusion, metastasis and other cancers, and is considered to be the best means for treating brain glioma, melanoma and the like. Boron neutron capture therapy only needs 1-2 irradiations, greatly reducing the burden on patients and hospital pressure.

[0004] Currently, mercaptoundecahydrododecaborane, also known as mercaptododecaborane disodium salt and L-p-boronylphenylalanine, is the most widely studied drug in boron neutron capture therapy clinical trials. Based on the relative non-selectivity of the two drugs currently used in clinical practice, the development of new and more selective boron drug delivery systems is the biggest demand for the future development of boron neutron capture therapy. The development of boron delivery agents for boron neutron capture therapy began more than 60 years ago, and it is a continuous and arduous task. The most important requirements for a successful boron delivery agent are as follows: (1) low systemic toxicity; (2) low normal tissue uptake and high tumor uptake, with high tumor / normal tissue and tumor / blood boron concentration ratios; (3) rapid clearance from blood and normal tissues during boron neutron capture therapy, and sustained retention in tumors for several hours. However, none of the boron delivery agents currently meets all these criteria, especially the poor tumor tissue distribution of boron drugs, which results in lower clinical benefit. Therefore, the main challenge for future 10 B drugs is to selectively target tumor cells and tumor tissues with effective whole distribution of boron drugs, as well as higher target and non-target ratios.

[0005] Poly-peptide is a kind of protein mimics with excellent biocompatibility and biodegradability, which shows great application potential in biomedical field. Similar to natural polypeptides, poly-peptide can form ordered secondary structures such as α-helix and β-sheet through hydrogen bonding interaction between peptide bonds in the backbone. Thus, they confer conformation-specific assembly behavior and biomedical functions. Among them, α-amino acid-N-carboxylic anhydride ring-opening polymerization is the most convenient method for preparing poly-peptide. α-Amino acid-N-carboxylic anhydride has high polymerization activity, and through protection of amino acid side group functional groups and post-modification of polymerization products, α-amino acid-N-carboxylic anhydride polymerization can synthesize polyamino acids with various functional groups such as thiol, carboxyl, hydroxyl and ester, which are applied in different fields. Through modification of the side chain of poly-peptide, poly-peptide can be used as an effective molecular gate to regulate the release of drugs in nano-drug carriers. For example, through the mutual attraction of positive and negative charges, the polypeptide is curled, which causes the molecular gate to close and encapsulate the hydrophobic drug. Various enzymes present in cancer cells can remove the functional groups modified on the side chain, thereby opening the molecular gate and releasing the hydrophobic drug. The natural polymer formed by the connection of α-amino acids in the form of peptide bond has various important biological functions.

[0006] Multifunctional nano-drugs with good biocompatibility and safety have been widely concerned and shown good potential in tumor radiotherapy applications. Compared with traditional drugs, nano-drugs can passively enrich in tumor tissue sites through the high permeability and retention effect of solid tumors, achieving the purpose of drug targeting tumor. Due to the small size and special surface properties of nano-drugs, they can more easily penetrate the blood vessel wall and interstitial space, achieve more effective tumor penetration, and thus improve the therapeutic effect of boron neutron capture therapy. At the same time, nano-drugs can coat a large number of boron atoms to increase their concentration in tumor cells, thereby enhancing the efficacy of boron neutron capture therapy. Nano-drugs can achieve multifunctionality through surface modification or structural design, such as simultaneous diagnosis and treatment, thereby achieving personalized treatment and monitoring. Therefore, nano-drugs can improve the effectiveness and safety of boron neutron capture therapy, and the development of a new type of nano-boron drug with tumor targeting selectivity is an urgent need for boron neutron capture therapy research. SUMMARY

[0007] In view of the deficiencies of the prior art, the purpose of the present application is to provide a poly-peptide molecular gate boron micelle drug delivery system and a preparation method and application thereof.

[0008] To achieve this purpose, the technical scheme adopted by the present application is as follows: In a first aspect, the present application provides a poly-peptide molecular gate boron micelle drug delivery system, which comprises a carrier, a poly-peptide molecular gate control layer, a targeting ligand and a loaded boron-containing drug.10 B drugs; the carrier is co-assembled by at least two amphiphilic copolymers to form a core-shell structure; the amphiphilic copolymers comprise block copolymers; the hydrophobic inner core of the core-shell structure stably encapsulates the hydrophobic drug 10 B drugs; the first amphiphilic copolymer comprises a block copolymer modified by a targeting ligand through a disulfide bond; The second amphiphilic copolymer comprises a conformation-switchable poly-peptide segment as a molecular gate structure. The poly-peptide is prepared from N-carboxylic anhydride.

[0009] The carrier skeleton of the poly-peptide molecular gate boron gel micelle drug delivery system is co-assembled by at least two amphiphilic copolymers to form a core-shell structure, and the hydrophobic inner core of the core-shell structure can stably encapsulate the hydrophobic drug 10 The B drugs can solve the problem of poor drug solubility and ensure effective drug loading and delivery; wherein the first amphiphilic copolymer is modified by a targeting ligand through a disulfide bond, which can not only precisely recognize tumor cells with the help of the targeting ligand to improve the enrichment of the micelles at the tumor site, but also can break the disulfide bond under the high reduction environment of the tumor microenvironment to assist drug release and reduce drug exposure to normal tissues; at the same time, the core-shell structure can also improve the stability of the micelles in the body fluid to avoid premature drug leakage, and the hydrophobic inner core can efficiently encapsulate the hydrophobic drug 10 The B drugs can improve the intracellular 10 B concentration, and the targeting enrichment characteristics make 10 The B drugs can accumulate at the tumor site, and after neutron irradiation, the high-energy particles released by the nuclear reaction can precisely destroy tumor cells, thereby enhancing the cancer-killing effect of boron neutron capture therapy.

[0010] Preferably, the targeting ligand is selected from any one or a combination of at least two of folate, hyaluronic acid, transferrin, RGD peptide, monoclonal antibody, epidermal growth factor, heparan sulfate, or chondroitin sulfate.

[0011] Further preferably, the targeting ligand is a combination of hyaluronic acid and folate.

[0012] Under this preferred scheme, hyaluronic acid and folate can specifically recognize the corresponding receptors on the surface of tumor cells, respectively, to synergistically improve the targeting binding capacity of the micelles to tumor cells, further enhance the enrichment of the micelles at the tumor site, and reduce the distribution of drugs in normal tissues; at the same time, the stable encapsulation of the hydrophobic drug 10 B drugs by the hydrophobic inner core of the core-shell structure of the carrier skeleton, and the responsive breaking characteristics of the disulfide bond in the tumor microenvironment can more efficiently deliver the drug 10 B to the tumor site and release it, laying the foundation for improving the effect of boron neutron capture therapy.

[0013] Preferably, the block copolymer of the first amphiphilic copolymer and the second amphiphilic copolymer each comprises a hydrophobic segment and a hydrophilic segment; the hydrophobic segment is selected from any one of polylactic-co-glycolic acid copolymer, polycaprolactone, polyglycolide, polylactide, polyacrylate, polystyrene, spermine or short-chain phospholipid; the hydrophilic segment is selected from any one of methoxypolyethylene glycol, polyethylene glycol, polyvinyl alcohol, polyhydroxypropyl methacrylamide, polyethyleneimine, poly-N-isopropyl acrylamide or polyvinylpyrrolidone.

[0014] Preferably, the hydrophobic segment of the first and second amphiphilic copolymer is polylactic-co-glycolic acid copolymer.

[0015] Preferably, the hydrophilic segment is methoxypolyethylene glycol.

[0016] Preferably, the molecular weight of the polylactic-co-glycolic acid copolymer is 1-10 kDa.

[0017] The molecular weight of the PLGA is 1-10 kDa, and the specific point values of 1-10 can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0018] Preferably, the molecular weight of the methoxypolyethylene glycol is 1-5 kDa.

[0019] The molecular weight of the mPEG is 1-5 kDa, and the specific point values of 1-5 can be 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, and the specific point values in the above numerical range can be selected.

[0020] Preferably, polylactic-co-glycolic acid copolymer is used as the hydrophobic segment of the amphiphilic copolymer, which can form a hydrophobic inner core to stably encapsulate hydrophobic drugs. 10 B drugs, while its good biocompatibility and degradable performance reduce the systemic toxicity of micelles, ensuring the effective delivery of drugs and the safety of use.

[0021] Preferably, the N-carboxylic acid anhydride is a natural amino acid-derived N-carboxylic acid anhydride, glycine, and its derivatives; the N-carboxylic acid anhydride is selected from any one or a combination of at least two of the following: alanine N-carboxylic acid anhydride and its derivatives, valine N-carboxylic acid anhydride and its derivatives, leucine N-carboxylic acid anhydride and its derivatives, isoleucine N-carboxylic acid anhydride and its derivatives, methionine N-carboxylic acid anhydride and its derivatives, tryptophan N-carboxylic acid anhydride and its derivatives, serine N-carboxylic acid anhydride and its derivatives, tyrosine N-carboxylic acid anhydride and its derivatives, phenylalanine N-carboxylic acid anhydride and its derivatives, threonine N-carboxylic acid anhydride and its derivatives, aspartic acid N-carboxylic acid anhydride and its derivatives, glutamic acid N-carboxylic acid anhydride and its derivatives, lysine N-carboxylic acid anhydride and its derivatives, arginine N-carboxylic acid anhydride and its derivatives, and histidine N-carboxylic acid anhydride and its derivatives; the side chains of the polypeptide chain are modified by phosphorylation.

[0022] The aforementioned polypeptide segments, acting as the molecular gate control layer of micelles, can maintain a closed "molecular gate" under normal conditions to prevent drug leakage after side chain modification (such as linking polyethyleneimine to the anhydride polymerization end of the γ-benzyl-L-glutamic acid-N-carboxyl ring, or phosphorylation of the anhydride polymerization end of the L-tyrosine-N-carboxyl ring). However, in the tumor microenvironment, they can open the "molecular gate" through conformational switching to release the drug. 10 Drug B enables controlled drug release while ensuring the precision and effectiveness of micelles in tumor treatment.

[0023] Preferably, the containing 10 Drug B is selected from any one or a combination of at least two of 4-dihydroxyborylphenylalanine, mercaptoundecylhydrogendoborane, boric acid, and carborane.

[0024] Secondly, the present invention provides a method for preparing the polypeptide molecular gate boron micelle drug delivery system of the present invention, comprising the following steps: (S1) The first amphiphilic copolymer is obtained by reacting the targeted ligand with the activated block copolymer after being modified by disulfide bonds; (S2) After ring-opening polymerization of N-carboxylic acid anhydride, it is linked with block copolymer and modified by side chain phosphorylation to obtain a second amphiphilic copolymer; (S3) Dissolve the first amphiphilic copolymer and the second amphiphilic copolymer in a solution containing... 10 The delivery system is obtained by mixing drug solution B with ultrasound and then passing it through a membrane.

[0025] The preparation method of this invention synthesizes key components stepwise and assembles them into micelles, ensuring the effective combination of each functional layer of the micelles, namely the targeting layer, the molecular gate regulation layer, and the drug loading layer.

[0026] Preferably, the block copolymer in step S1 is activated by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide solution.

[0027] Preferably, the molar ratio of the block copolymer, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide in step S1 is 1:1:1-20:20:1.

[0028] The specific point value of 1:1:1-20:20:1 can be 1:2:1, 2:3:1, 3:5:1, 4:6:1, 5:8:1, 6:9:1, 7:10:1, 8:12:1, 9:15:1, 10:18:1, 12:15:1, 14:18:1, 16:19:1, 18:20:1, 20:18:1, and the like.

[0029] Further preferably, the molar ratio of the block copolymer, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide in step S1 is 5:5:1.

[0030] The ring-opening polymerization temperature of N-carboxylic anhydride in step S2 is 25-40℃, and the reaction time is 12-36h.

[0031] Preferably, the ultrasonic time in step S3 is 3-8min, and the membrane pore size is 100-400nm.

[0032] Further preferably, the ultrasonic time in step S3 is 5min.

[0033] In a third aspect, the application provides a use of the poly-polypeptide molecular boron glue bundle drug delivery system in the first aspect in the preparation of a boron neutron capture therapy drug.

[0034] The poly-polypeptide molecular boron glue bundle drug delivery system can be used in the preparation of a drug for treating malignant brain tumor, malignant melanoma, head and neck tumor (including oral cancer, tongue cancer, pharynx cancer, larynx cancer, thyroid cancer, parotid cancer, external ear cancer, middle ear cancer), liver cancer, lung cancer, cervical cancer, breast cancer, gastric cancer, esophageal cancer, colorectal cancer, osteosarcoma, prostate cancer, bladder cancer, mesothelioma.

[0035] Compared with the prior art, the application has the following beneficial effects: (1) solve the problem of containing 10 B drug solubility: the carrier skeleton hydrophobic core can load hydrophobic 10B-containing drugs (such as BPA), improve the solubility, and ensure stable drug delivery.

[0036] (2) Realize controllable release of drug: the polypeptide molecular gate control layer is closed in normal environment, and is opened to release drug in tumor microenvironment, thereby reducing damage to normal tissue.

[0037] (3) Enhance tumor targeting: the targeting ligand (such as HA, FA) combines with the nano-carrier EPR effect, thereby improving enrichment amount at tumor site and improving tumor / normal tissue drug concentration ratio.

[0038] (4) Improve BNCT efficacy: improve 10B concentration in tumor cells, release high-energy particles to precisely destroy tumor cell DNA after neutron irradiation, and enhance cancer killing effect.

[0039] (5) Good biocompatibility: the materials used are degradable and low in toxicity, experiments show that the micelles have no obvious toxicity to tumor cells, and are high in safety.

[0040] (6) In the preparation process of the application, the copolymer connection efficiency, micelle particle size distribution and drug loading rate can be precisely controlled through steps such as EDC / NHS activation, ultrafiltration / dialysis purification, ice bath ultrasonic and membrane filtration; especially the process parameter optimization of NCA ring-opening polymerization can ensure uniform polypeptide chain segment structure, thereby providing guarantee for stable realization of "molecular gate" function. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 is a Fourier infrared spectrum of HA-SS; Figure 2 is a transmission electron microscope morphology diagram of the polypeptide molecular gate micelles; Figure 3 is a hydrated particle size diagram of the polypeptide molecular gate micelles; Figure 4 is an electrokinetic potential diagram of the polypeptide molecular gate micelles; Figure 5 is a reduced glutathione and pH response diagram of the polypeptide molecular gate micelles; Figure 6 is a cell uptake diagram of the polypeptide molecular gate micelles; Figure 7 is a cell toxicity detection result diagram of the polypeptide molecular gate micelles. DETAILED DESCRIPTION

[0042] The application will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the application and not to limit the scope of the application, and those skilled in the art can make various modifications or changes to the application after reading the content taught by the application. These equivalent forms also fall within the scope defined by the claims attached to the present application.

[0043] In the following examples, all components without special instructions are commercially available.

[0044] Example 1 This example provides a method for synthesizing hyaluronic acid-disulfide intermediate, hereinafter referred to as HA-SS.

[0045] (1) Activation of hyaluronic acid: 1.3 g of hyaluronic acid, hereinafter referred to as HA, was dissolved in pure water, 2.6 g of EDC and 2.6 g of NHS were added respectively, and argon was passed for 10 min, and stirred at room temperature for 1 hour. The molecular weight of HA is 5 kDa.

[0046] (2) 0.6 g of activated HA was added to an ultrafiltration tube for purification, at a speed of 4000 rpm for 30 min, and ultrafiltration was performed 3 times. After each ultrafiltration, pure water was added and ultrafiltration was performed again. The purified liquid was re-added to the flask, and 1.2 g of cystamine dihydrochloride was added, stirred at room temperature for 24 hours, and then added to the ultrafiltration tube for purification 3 times. After purification, HA-SS was obtained by freeze-drying.

[0047] Example 2 This example provides a method for synthesizing HA-SS-PLGA-mPEG.

[0048] (1) 0.8 g of PLGA-mPEG-COOH was dissolved in 10 mL of DMSO, 1.6 g of EDC and 1.6 g of NHS were added respectively, and argon was passed for 10 min, and stirred at 23±2°C for 1 hour.

[0049] (2) The activated PLGA-mPEG-COOH was added to an ultrafiltration tube for purification, at a speed of 4000 rpm for 30 min, and ultrafiltration was performed 3 times. After each ultrafiltration, DMSO was added and ultrafiltration was performed again. The purified liquid was re-added to the flask.

[0050] (3) HA-SS was dissolved in formamide, and the ultrafiltration-purified PLGA-mPEG-COOH was added dropwise, and stirred at room temperature for 24 hours. The obtained solution was dialyzed in a mixed system of DMSO and deionized water v:v=1:1 for 1 day, and then dialyzed in deionized water for 2 days. After dialysis, the solution was filtered and freeze-dried, and stored at 4°C.

[0051] Example 3 This example provides a method for synthesizing 2D-BPA.

[0052] 1.0 g of BPA was dissolved in basic pure water, 0.5 g of D-fructose and 0.5 g of D-sorbitol were added respectively, and stirred at room temperature for 24 hours. The pH of the solution was titrated to pH=7 using hydrochloric acid, and the obtained solution was filtered to obtain a 2D-BPA solution.

[0053] Example 4 This example provides a method for obtaining a poly-polypeptide molecular door boron glue bundle drug delivery system.

[0054] After mixing 0.2 g of HA-SS-PLGA-mPEG synthesized in Example 2 with 0.2 g of PLGA-mPEG-FA, adding the 2D-BPA drug solution prepared in Example 3, vortexing and mixing, and then placing on ice for ultrasonic treatment for 5 minutes, the polymeric polypeptide molecular gate boro-hydrogel drug delivery system with HA and FA double targeting ligands can be obtained by extruding a polycarbonate membrane with a pore size of 400-100 nm and removing free BPA by dialysis.

[0055] Example 5 This example characterizes the polymeric polypeptide molecular gate boro-hydrogel drug delivery system.

[0056] (1) The polymeric polypeptide molecular gate boro-hydrogel drug delivery system was tested using a Bruker INVENIO series Fourier infrared spectrometer. The test results are shown in Figure 1

[0057] Figure 1 The Fourier infrared spectra of hyaluronic acid (HA), disulfide bond components (SS), and HA-SS conjugates with different modification ratios were compared by comparing the transmittance at different wave numbers.

[0058] As shown in the figure, by comparing the infrared spectra of hyaluronic acid (HA), disulfide bond components (SS), and HA-SS conjugates (HA-SS(2), HA-SS(5), HA-SS(10)) with different modification ratios: In the ~3500 cm -1 hydroxyl characteristic peak region, the changes in peak shape and intensity of the HA-SS series samples compared with pure HA prove that the disulfide bond is combined with HA through chemical reaction, changing the chemical environment of the hydroxyl group in the HA molecule, directly proving the realization of the technical feature in the claim that "the targeting ligand (herein, HA as an example) is modified by a disulfide bond and combined with a block copolymer".

[0059] In the ~1500-1700 cm -1 carbonyl characteristic peak region, the peak shape difference of the HA-SS series samples with different modification ratios quantitatively reflects the controllability of the disulfide bond modification degree, and illustrates the performance advantages of the delivery system in drug loading, targeting, etc.

[0060] (2) The polymeric polypeptide molecular gate boro-hydrogel drug delivery system was characterized using a Thermo Fisher Iliad series transmission electron microscope, as shown in Figure 2 The particle size of the polymeric polypeptide molecular gate boro-hydrogel drug delivery system under the transmission electron microscope is about 180 nm.​

[0061] The hydrated particle size and Zeta potential of the poly-polypeptide molecular gate boron micelle drug delivery system prepared in Example 4 were measured by Malvern Zetasizer Nano ZS90. The measurement results are shown in Figures 3-4 .

[0062] The hydrated particle size of the poly-polypeptide molecular gate boron micelle drug delivery system prepared in the foregoing examples was 185±10 nm, which was consistent with the characterization results under transmission electron microscopy, indicating that the micelle morphology was uniform and the dispersibility was good. The Zeta potential of PLGA-mPEG-HA was - (5±2.4) mV, and the Zeta potential of PLGA-mPEG-HA-FA was - (10±1.5) mV. The Zeta potential was negative and stable in value, indicating that the micelles had strong stability in the solution, which met the application requirements of the nano drug delivery system.

[0063] Experimental Example 1 Poly-polypeptide molecular gate micelle GSH and pH response experiment: The poly-polypeptide molecular gate micelles were placed in different pH and GSH buffer solutions (pH 5.4+0mM GSH, pH 7.4+0mM GSH, pH 5.4+20mM GSH and pH 7.4+20mM GSH) to study their in vitro release behavior. Among them, pH 5.4+20mM GSH simulates the tumor environment, and pH 7.4 simulates the normal body fluid environment. Four groups of 2.0 g micelles were respectively placed in EP tubes, and placed on a shaking bed at 65 rpm. The particle sizes of the four groups were measured at 0, 10, 30 and 60 min, respectively. The results are shown in Figure 4 . When pH 7.4+0mM GSH, the micelle particle size hardly changed and was stable at about 150 nm. pH 5.4+0mM GSH and pH 7.4+20mM GSH showed that the micelles had GSH and pH response, and the response effect of GSH was better than that of pH. The particle size of pH 5.4+20mM GSH decreased the fastest, indicating that the micelles had fast responsiveness under the conditions of the tumor microenvironment.

[0064] Experimental Example 2 Containing 10 Cell uptake experiment of B-targeting micelles: 4T1 cells in the logarithmic growth phase were taken, and 1×10 5 cells per well were uniformly inoculated on sterile six-well cell culture plates and placed in a cell culture incubator with a setting of 37°C and 5% CO2 for 24 hours. After the cells adhered, the old culture solution was removed, and the targeting B-containing micelles of Example 4 were added, respectively. 10B micelles were used to set up a series of gradients (0, 100, 200, 500, 1000 μg / mL). After 6 hours, the six-well plate was removed, the original culture medium was discarded, and each well was washed three times with PBS buffer. 500 nmol of concentrated nitric acid was added to each well to digest the cells, and the concentration of each sample was measured by ICP-MS. 10 The concentration of B, the results are as follows Figure 5 As shown, with increasing BPA concentration in micelles (0, 100, 200, 500, 1000 μg / mL), the cells' response to BPA containing... 10 The uptake of B micelles increases, peaking at 1000 μg / mL.

[0065] Experimental Example 3 Cytotoxicity studies: Mouse breast cancer cell line in logarithmic growth phase, hereinafter referred to as 4T1 cells, was obtained. Cells at the bottom of the culture dish were digested with trypsin, centrifuged, and resuspended to prepare a cell suspension. Cells were counted using a cell counting chamber at 2 × 10⁶ cells per well. 3 A uniform number of cells were seeded into sterile 96-well plates and placed in a cell culture incubator at 37°C and 5% CO2 for 24 hours. The old culture medium was removed, and the targeted cell culture medium from Example 4 was added to each plate. 10 B micelles were used to set up a series of gradients (0, 100, 200, 500, 1000 μg / mL) and cultured for 24 hours. After 24 hours, the 96-well plate was removed, the original culture medium was discarded, and 100 nL of medium containing CCK-8 reagent (CCK-8 reagent to 1640 medium volume ratio 1:9) was added to each well. The plate was then incubated for another 2 hours. Finally, the absorbance of each group at 450 nm was measured using a microplate reader, and cell viability was calculated. The results are as follows: Figure 6 and Figure 7 As shown, compared with the control group (PBS), different concentrations of [unclear text - possibly a specific product or treatment]... 10 4T1 cells cultured in B micelles (0, 100, 200, 500, 1000 μg / mL) proliferated normally, indicating that the micelles had good biocompatibility and no cytotoxicity.

Claims

1. A polymeric polypeptide micellar drug delivery system, comprising, the delivery system comprises a carrier, a polymeric polypeptide molecular gate regulatory layer, a targeting ligand, and an encapsulated cargo comprising 10 B drugs; the carrier is co-assembled from at least two amphiphilic copolymers into a core-shell structure; the amphiphilic copolymers comprise block copolymers; the hydrophobic inner core of the core-shell structure encapsulates a cargo comprising 10 B drugs; the first amphiphilic copolymer comprises a block copolymer modified with a targeting ligand linked by a disulfide bond; The second amphiphilic copolymer comprises a conformation-switchable poly-peptide segment as a molecular gate structure. The poly-peptide is prepared from an N-carboxylic anhydride.

2. The delivery system of claim 1, wherein, The targeting ligand is selected from any one of folate, hyaluronic acid, transferrin, RGD peptide, monoclonal antibody, epidermal growth factor, heparan sulfate or chondroitin sulfate or a combination of at least two thereof.

3. The delivery system of claim 1, wherein, The block copolymer of the first and second amphiphilic copolymers each comprises a hydrophobic segment and a hydrophilic segment; the hydrophobic segment is selected from any one of polylactic-co-glycolic acid, polycaprolactone, polyglycolide, polylactide, polyacrylate, polystyrene, spermine or short-chain phospholipid; and the hydrophilic segment is selected from any one of methoxypolyethylene glycol, polyethylene glycol, polyvinyl alcohol, polyhydroxypropyl methacrylamide, polyethylene imine, poly-N-isopropyl acrylamide or polyvinyl pyrrolidone.

4. The delivery system of claim 3, wherein, The hydrophobic segment of the first and second amphiphilic copolymers is polylactic-co-glycolic acid, and the hydrophilic segment is methoxypolyethylene glycol; the molecular weight of the polylactic-co-glycolic acid is 1-10 kDa; and the molecular weight of the methoxypolyethylene glycol is 1-5 kDa.

5. The delivery system of claim 1, wherein, The N-carboxylic anhydride is N-carboxylic anhydride glycine derived from a natural amino acid and derivatives thereof; the N-carboxylic anhydride is selected from any one of alanine N-carboxylic anhydride and derivatives thereof, valine N-carboxylic anhydride and derivatives thereof, leucine N-carboxylic anhydride and derivatives thereof, isoleucine N-carboxylic anhydride and derivatives thereof, methionine N-carboxylic anhydride and derivatives thereof, tryptophan N-carboxylic anhydride and derivatives thereof, serine N-carboxylic anhydride and derivatives thereof, tyrosine N-carboxylic anhydride and derivatives thereof, phenylalanine N-carboxylic anhydride and derivatives thereof, threonine N-carboxylic anhydride and derivatives thereof, aspartic acid N-carboxylic anhydride and derivatives thereof, glutamic acid N-carboxylic anhydride and derivatives thereof, lysine N-carboxylic anhydride and derivatives thereof, arginine N-carboxylic anhydride and derivatives thereof, and histidine N-carboxylic anhydride and derivatives thereof or a combination of at least two thereof; and the side chain of the poly-peptide segment is modified by phosphorylation.

6. The delivery system of claim 1, wherein, The content 10 B drug is selected from any one of 4-dihydroxyboronyl phenylalanine, mercaptoundecahydrododecaborane, boric acid, carborane or a combination of at least two thereof.

7. A process for the preparation of the polymeric polypeptide micellar drug delivery system as claimed in any one of claims 1 to 6, wherein the process comprises the steps of: a) dissolving the polymeric polypeptide in a suitable solvent; b) adding the drug to the solution; c) evaporating the solvent; and d) lyophilizing the product. 5 The method comprises the following steps: (S1) reacting a targeting ligand modified by a disulfide bond with an activated block copolymer to obtain a first amphiphilic copolymer; (S2) connecting a block copolymer to an N-carboxylic anhydride after ring-opening polymerization of the N-carboxylic anhydride and modifying the side chain by phosphorylation to obtain a second amphiphilic copolymer; (S3) dissolving the first amphiphilic copolymer, the second amphiphilic copolymer in a solvent containing 10 The drug solution is mixed with the ultrasound and the delivery system is obtained by passing through a membrane.

8. The method of claim 7, wherein, In step S1, the disulfide bond modification of the targeting ligand is achieved by coupling the amino group of cystamine dihydrochloride with the carboxyl group of the targeting ligand to form a disulfide bond; In step S1, the block copolymer is activated by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide solution; In step S1, the molar ratio of the block copolymer, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide is 1:1:1-20:20:1; In steps S1 and S2, the purification includes ultrafiltration purification and / or dialysis purification.

9. The method of claim 7, wherein, In step S2, the ring-opening polymerization temperature of the N-carboxylic anhydride is 25-40℃, and the reaction time is 12-36 h; The solvent of the drug solution in step S3 contains 10 The solvent of the drug solution in step S3 contains any one of PBS solution, mannitol solution, glucose solution, and physiological saline. The ultrasonic time in step S3 is 3-8 min, and the membrane pore size is 100-400 nm.

10. Use of the polymeric polypeptide micelle drug delivery system of any one of claims 1-6 in the preparation of a boron neutron capture therapy drug.

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