Preparation method of polymer vesicle capable of crossing blood brain barrier

Abstract

The invention discloses a preparation method of a polymer vesicle capable of crossing a blood brain barrier, and belongs to the field of high polymer materials and medical engineering. The preparation method of the polymer vesicle capable of crossing the blood brain barrier comprises the following steps: synthesizing an amphiphilic block copolymer by utilizing ring-opening polymerization reaction of polycaprolactone and anhydride in an amino acid ring, then assembling the polymer into the polymer vesicle, and modifying a monoclonal antibody of a transferrin receptor on a polypeptide chain. The polymer vesicle prepared by the invention is simple and convenient in preparation process, short in period, good in stability, good in biocompatibility and good in biodegradability, can be used as an excellent carrier of chemotherapeutic drugs, and can effectively realize drug delivery of targeting brain tumor cells across a blood brain barrier. Compared with a commercial drug delivery carrier, the polymer vesicle has the advantage that the permeability of the drug in a bionic blood brain barrier and the targeted uptake rate of brain tumor cells are remarkably improved.

Description

Technical Field

[0001] The invention relates to a method for preparing polymer vesicles capable of crossing the blood-brain barrier, and belongs to the fields of polymer materials and medical engineering. Background Art

[0002] Natural extracellular vesicles (EVs) are tiny vesicles secreted by cells and possess a phospholipid bilayer. They are non-immunogenic, bypassing the immune system and avoiding clearance by the liver and kidneys. EVs are also non-cytotoxic, have good biocompatibility, long circulation time, and tumor homing ability, making them an excellent platform for delivering drugs that can cross the blood-brain barrier.

[0003] Based on this, in the field of polymers, through the self-assembly technology of amphiphilic block polymers, polymer vesicles with a double-layer membrane hollow structure can be obtained. Their structure is more stable and convenient for various functional modifications, and their application range is wider.

[0004] Stimuli-responsive polymersomes are polymersomes that can respond to external stimuli, including temperature, pH, light, ultrasound, magnetic fields, and redox reactions. In response to changes in these stimuli, the composition, morphology, or size of these polymersomes change accordingly. This stimuli-responsive behavior has led to their widespread application in drug delivery, micro- and nanoreactors, and diagnostic contrast agents.

[0005] The blood-brain barrier (BBB) ​​is a protective barrier system between blood and brain tissue. Its primary structure is the endothelium, which is tightly connected and overlaps. The BBB maintains a relatively stable internal environment within the nervous system by strictly controlling the flow of substances in and out, protecting neurons from damage. However, this also presents significant challenges in the treatment of central nervous system diseases. Clinical data show that 98% of small molecule drugs and nearly all large molecule drugs are unable to effectively cross the BBB and reach brain lesions, significantly compromising therapeutic efficacy. For example, the current standard clinical treatment for glioblastoma is surgical resection, radiotherapy, and chemotherapy. While these treatments can prolong patient survival to some extent, the prognosis is poor and recurrence is common. Unlike other tumors, the specific location of brain tumors and the restrictive nature of the BBB make effective drug delivery and accumulation difficult. Therefore, how to deliver drugs across the BBB to brain lesions remains a research hotspot and a challenge in the treatment of central nervous system diseases, and holds significant clinical significance.

[0006] Currently, commonly used nanoparticles for brain tumor drug delivery primarily include liposomes, polymer nanoparticles, and solid lipid nanoparticles. Despite the diverse array of nanoparticles for brain-targeted drug delivery, only a few nanoparticle-based therapies and technologies have been approved, and none have yet been clinically applied to treat central nervous system diseases. Therefore, the development of multifunctional nanoparticles for drug delivery targeting the blood-brain barrier remains an urgent challenge in this field. Summary of the Invention

[0007] In response to the defects and shortcomings of the existing technology, the main purpose of the present invention is to provide a method for preparing polymer vesicles that can cross the blood-brain barrier, which can effectively improve the encapsulation efficiency and stability of chemotherapy drugs, and effectively deliver them to brain tumor cells across the blood-brain barrier, thereby killing brain tumor cells.

[0008] The blood-brain barrier-crossing polymer vesicles prepared by this invention are hollow nanospheres self-assembled from amphiphilic block polymers modified with a monoclonal antibody against the transferrin receptor. They possess a biodegradable polypeptide backbone, excellent ultrasound responsiveness, blood-brain barrier penetration, and brain tumor targeting capabilities. With excellent biocompatibility and biodegradability, they can serve as excellent carriers for chemotherapy drugs, effectively achieving drug delivery across the blood-brain barrier. Compared to commercial drug delivery vehicles, these polymer vesicles significantly improve the blood-brain barrier penetration rate of chemotherapy drugs and the targeted uptake rate of brain tumor cells.

[0009] The first object of the present invention is to provide a method for preparing polymer vesicles that can cross the blood-brain barrier, the method comprising the following steps:

[0010] (1) Synthesis of amphiphilic polymers

[0011] Dissolving polycaprolactone and amino acid cyclic anhydride in a first type solvent, reacting under vacuum at 20-40° C. for 30-50 hours to obtain a polycaprolactone-block-polyamino acid copolymer; then dissolving the obtained polycaprolactone-block-polyamino acid copolymer in trifluoroacetic acid, adding hydrobromic acid and acetic acid solution, reacting at 20-40° C. for 2-5 hours, and precipitating in anhydrous ether to obtain a polycaprolactone-block-polyamino acid copolymer from which tert-butyloxycarbonyl protection has been removed;

[0012] (2) Preparation of polymer vesicles

[0013] The polycaprolactone-block-polyamino acid copolymer obtained from step (1) is dissolved in a second type of solvent, and the initial concentration of the target polymer is controlled to be 0.5-5.0 mg / mL. 1-5 times the volume of deionized water is added dropwise at a rate of 15-45 d / min under stirring. After the addition is completed, the copolymer is first dialyzed in a 1-5 wt% sodium bicarbonate aqueous solution for 10-100 h, and then dialyzed in deionized water for 10-50 h, with the water being changed every 1-10 h. The dialyzed solution is the polymer vesicle solution;

[0014] (3) Targeted modification of polymer vesicles

[0015] The polymer vesicle solution obtained in step (2) is diluted 1 to 10 times, the pH is adjusted to 4 to 6, N-hydroxysuccinimide is added and stirred for 0.5 to 2 hours; 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is then added and stirred for 2 to 5 hours; then, the pH is adjusted to 7.0 to 8.0, streptavidin is added to the above solution, and the solution is dialyzed to remove by-products after stirring for 5 to 15 hours; finally, biotin-labeled transferrin receptor monoclonal antibody is added to the dialyzed solution and stirred for 5 to 15 hours to finally obtain transferrin receptor monoclonal antibody-modified targeted polymer vesicles.

[0016] In one embodiment, the first type of solvent in step (1) is one or more of n-hexane, N,N-dimethylformamide, acetone and dichloromethane.

[0017] In one embodiment, the amino acid anhydride in step (1) is an amino acid anhydride having a reactive group, including any one of L-glutamic acid-γ-benzyl ester anhydride and L-aspartic acid-β-benzyl ester anhydride.

[0018] In one embodiment, the polycaprolactone in step (1) is amino-terminated polycaprolactone.

[0019] In one embodiment, the molar ratio of the polycaprolactone to the amino acid cyclic anhydride in step (1) is 1:(1-30), more preferably 1:13.

[0020] In one embodiment, the volume mass ratio of the trifluoroacetic acid to the polycaprolactone-block-polyamino acid copolymer in step (1) is 10-15:1, mL / g.

[0021] In one embodiment, the volume mass ratio of the hydrobromic acid acetic acid solution to the polycaprolactone-block-polyamino acid copolymer in step (1) is 10-15:1-2, mL / g; wherein the mass fraction of the hydrobromic acid acetic acid solution is 33%.

[0022] In one embodiment, the second type of solvent in step (2) is one or more of N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran or acetone; more preferably N,N-dimethylformamide.

[0023] In one embodiment, in step (2), the initial concentration of the target polymer is controlled at 1 mg / mL.

[0024] In one embodiment, the dialysis conditions in step (2) are: first dialyzing in a 1-5 wt% sodium bicarbonate aqueous solution for 10-100 h, then dialyzing in deionized water for 10-50 h, and changing the water every 1-10 h.

[0025] In one embodiment, the molar ratio of the polymer vesicles to N-hydroxysuccinimide in step (3) is 1:(10-100), more preferably 1:(80-100); the molar ratio of the polymer vesicles to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:(10-100), more preferably 1:(80-100); the molar ratio of the polymer vesicles to streptavidin is 1:(0.01-1); the molar ratio of the polymer vesicles to the biotin-labeled monoclonal antibody to transferrin receptor is 1:(0.0001-1).

[0026] The second object of the present invention is to provide a polymer vesicle that can cross the blood-brain barrier and is prepared by the above method.

[0027] In one embodiment, the polymer vesicles are hollow nanospheres with a unique "inner crown-cavity-membrane layer-outer crown" structure with a diameter of 100 to 500 nm, wherein polycaprolactone constitutes the hydrophobic membrane layer of the nanospheres, and a polypolypeptide modified with a monoclonal antibody to the transferrin receptor constitutes the hydrophilic corona layer of the nanospheres; under the action of pulsed ultrasound, the size change rate of the nanospheres is 5% to 30%.

[0028] The third object of the present invention is to provide a use of the above-mentioned polymer vesicles that can cross the blood-brain barrier as a delivery carrier for chemotherapy drugs for brain glioma cells.

[0029] Beneficial effects of the present invention:

[0030] (1) Traditional small molecule chemotherapy drugs are usually not targeted and cannot cross the blood-brain barrier to reach brain lesions to exert their efficacy after intravenous injection, and they have large toxic and side effects. The polymer vesicles prepared by the present invention have a high affinity for the transferrin receptor monoclonal antibody modified with the hydrophilic corona and the transferrin receptor on the surface of the blood-brain barrier endothelial cells and glioblastoma cells, and can effectively cross the blood-brain barrier to deliver chemotherapy drugs; as a drug delivery carrier targeting the blood-brain barrier, it can efficiently encapsulate chemotherapy drugs and achieve ultrasound-controlled drug encapsulation and release. Therefore, the polymer vesicles of the present invention that can cross the blood-brain barrier can effectively achieve the reduction of toxicity and enhancement of the efficacy of chemotherapy drugs, and can be used as a chemotherapy drug carrier.

[0031] (2) Free chemotherapy drugs are easily cleared by the liver, lack targeting, and are cytotoxic, making it difficult to achieve effective brain tumor treatment. The targeted polymer vesicles prepared by the present invention can achieve efficient encapsulation of doxorubicin hydrochloride under ultrasound, and can cross the biomimetic blood-brain barrier to achieve its significant killing effect on brain tumor cells. Therefore, the targeted polymer vesicles prepared by the present invention can effectively achieve the encapsulation of chemotherapy drugs and the killing of tumor cells, and serve as a drug delivery carrier across the blood-brain barrier.

[0032] (3) The polymer vesicles prepared by the present invention that can cross the blood-brain barrier have good water solubility and dispersibility, and have good dilution stability in both deionized water and PBS. They are evenly dispersed and are not easy to agglomerate when stored for a long time, ensuring the safety of in vivo application; and the polymer vesicles use a polypeptide with reactive groups as the skeleton, the post-modification step is simple and efficient, the design is highly expandable, and they have good biodegradability. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a transmission electron microscopy (TEM) image of the polymer vesicles capable of crossing the blood-brain barrier prepared in Example 1 of the present invention;

[0034] Figure 2 Figure 1 is a graph showing the particle size and distribution (PDI) of the polymer vesicles capable of crossing the blood-brain barrier prepared in Example 1 of the present invention before and after ultrasound treatment;

[0035] Figure 3 This is a graph showing the ultrasound-responsive release curve of the chemotherapy drug doxorubicin hydrochloride encapsulated in the polymer vesicles capable of crossing the blood-brain barrier prepared in Example 1 of the present invention;

[0036] Figure 4 This is a diagram showing the targeted uptake of the chemotherapy drug doxorubicin hydrochloride encapsulated by the blood-brain barrier-crossing polymer vesicles prepared in Example 1 of the present invention in a biomimetic blood-brain barrier transwell model;

[0037] Figure 5This is a diagram showing the permeability of the polymer vesicles capable of crossing the blood-brain barrier prepared in Example 1 of the present invention after encapsulating the chemotherapy drug doxorubicin hydrochloride in a bionic blood-brain barrier 3D multicellular spheroid. DETAILED DESCRIPTION

[0038] The following describes preferred embodiments of the present invention. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0039] Sources of raw materials used in the present invention:

[0040] Streptavidin: Solarbio brand, product number S9171; biotin-labeled transferrin receptor monoclonal antibody: abcam brand, product number ab28116.

[0041] Example 1

[0042] A method for preparing polymer vesicles capable of crossing the blood-brain barrier comprises the following steps:

[0043] (1) Preparation of amphiphilic polymers

[0044] First, amino-terminated polycaprolactone and L-glutamic acid-γ-benzyl ester cyclic anhydride are synthesized. The specific synthesis method is described as follows: 10.0g ε-caprolactone and 150mL toluene solution are added to a round-bottom flask, the mixture is placed in an oil bath at 140°C to azeotropically remove moisture from the system. Under nitrogen protection, 0.019g stannous octoate and 0.353g N-(tert-butoxycarbonyl)ethanolamine are added, and the reaction is carried out at 110°C for 48h, and then purified by ice methanol precipitation to obtain tert-butoxycarbonyl-protected polycaprolactone. Take 5.0g of butoxycarbonyl-protected polycaprolactone and dissolve it in 10mL dichloromethane, add 6mL trifluoroacetic acid, and stir at 25°C for 4h. After the reaction is completed, dialyze and purify, and freeze-dry to obtain amino-terminated polycaprolactone;

[0045] 10.0 g of L-glutamic acid-γ-benzyl ester cyclic anhydride and 29.3 g of α-pinene were reacted in 250 mL of tetrahydrofuran as the reaction solvent, under the catalysis of 9.48 g of triphosgene, at 55°C for 4 h, and then purified with n-hexane to obtain L-glutamic acid-γ-benzyl ester cyclic anhydride.

[0046] 0.500 g of the amino-terminated polycaprolactone synthesized above and 0.500 g of L-glutamic acid-γ-benzyl ester cyclic anhydride (the molar ratio of amino-terminated polycaprolactone to L-glutamic acid-γ-benzyl ester cyclic anhydride is 1:13) were dissolved in 6.0 mL of anhydrous dimethylformamide, and the mixture was vacuum-dried at room temperature for 48 hours. After the reaction was completed, the mixture was dialyzed in deionized water for 48 hours, with the water being changed every 3 hours. The mixture was dried at -45°C and a vacuum degree of 0.07 mBar for 2 days to obtain a polycaprolactone-block-polyglutamic acid copolymer;

[0047] Then, 1.00 g of the obtained polycaprolactone-block-polyglutamic acid copolymer was dissolved in 15 mL of trifluoroacetic acid, and 10.0 mL of a 33.wt% hydrobromic acid acetic acid solution was added. The mixture was reacted at room temperature for 5 h, and precipitated in anhydrous ether three times to obtain a polycaprolactone-block-polyglutamic acid copolymer from which the tert-butyloxycarbonyl protection was removed.

[0048] (2) Preparation of polymer vesicles that can cross the blood-brain barrier

[0049] The target copolymer was dissolved in N,N-dimethylformamide with an initial copolymer concentration of 1.0 mg / mL. Deionized water (2 times the volume of N,N-dimethylformamide) was added dropwise to the polymer solution at a rate of 20 d / min under rapid stirring. After the addition was complete, the solution was dialyzed against a 1-5 wt% sodium bicarbonate aqueous solution for 48 h and then against deionized water for 24 h. The dialyzate was replaced with fresh dialyzate every 3-4 h. The dialyzed solution was a polymer vesicle aqueous solution with a concentration of 0.20 mg / mL.

[0050] The polymersome solution was diluted 10-fold and the pH adjusted to 4-6. 0.408 mg of N-hydroxysuccinimide was added and stirred for 0.5 h. 0.679 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was then added and stirred for 2.5 h. Next, the pH was adjusted to neutral, and 2.27 mg of streptavidin was added to the solution. The mixture was stirred for 12 h and then dialyzed to remove byproducts. Finally, 33 μL of biotinylated transferrin monoclonal antibody was added (the molar ratio of polymersomes: N-hydroxysuccinimide: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride: streptavidin: biotinylated transferrin receptor monoclonal antibody was 1:100:100:1:0.012). The transferrin receptor monoclonal antibody was stirred for 12 h, resulting in transferrin receptor monoclonal antibody-modified polymersomes that could cross the blood-brain barrier.

[0051] Example 2

[0052] The difference from Example 1 is that the molar ratio of amino-terminated polycaprolactone and L-glutamic acid-γ-benzyl ester cyclic anhydride in step (1) is adjusted as shown in Table 1 (1:7), and the other parameters and conditions are the same as those in Example 1.

[0053] Table 1. Feeding of each reactant in step (1)

[0054]

[0055] Example 3

[0056] The difference from Example 1 is that the molar ratio of amino-terminated polycaprolactone to L-glutamic acid-γ-benzyl ester cyclic anhydride in step (1) is adjusted as shown in Table 2 (1:15), and the other parameters and conditions are the same as those in Example 1.

[0057] Table 2. Feeding of each reactant in step (1)

[0058]

[0059] Example 4

[0060] The difference from Example 1 is that in step (2), the target copolymer is dissolved in N,N-dimethylformamide, the initial concentration of the copolymer is adjusted to 0.5 mg / mL, and the other parameters and conditions are the same as in Example 1.

[0061] Example 5

[0062] The difference from Example 1 is that in step (2), the target copolymer is dissolved in N,N-dimethylformamide, the initial concentration of the copolymer is adjusted to 1.5 mg / mL, and the other parameters and conditions are the same as in Example 1.

[0063] Comparative Example 1

[0064] The difference from Example 1 is that in step (2), the target copolymer is dissolved in N,N-dimethylformamide instead of being dissolved in dimethyl sulfoxide, the initial concentration of the copolymer is 1.0 mg / mL, and the other parameters and conditions are the same as in Example 1.

[0065] Comparative Example 2

[0066] The difference from Example 1 is that in step (2), the target copolymer is dissolved in N,N-dimethylformamide instead of being dissolved in dimethyl sulfoxide, and the initial concentration of the copolymer is 1.5 mg / mL. Other parameters and conditions are the same as in Example 1.

[0067] Comparative Example 3

[0068] The difference from Example 1 is that in step (2), the target copolymer is dissolved in N,N-dimethylformamide instead of being dissolved in dimethyl sulfoxide, the initial concentration of the copolymer is 2 mg / mL, and the other parameters and conditions are the same as in Example 1.

[0069] Results Performance Characterization

[0070] 1. Performance measurement of the polymer vesicles capable of crossing the blood-brain barrier prepared in Example 1

[0071] The polymer vesicles prepared in Example 1 that can cross the blood-brain barrier were subjected to TEM measurement. The results are as follows: Figure 1 Shown: By Figure 1 It can be seen that the polymer vesicle has a clear structure, a hollow structure, and is uniformly dispersed; the polymer vesicle that can cross the blood-brain barrier is a hollow nanovesicle self-assembled from a polycaprolactone-polyglutamic acid copolymer modified with a monoclonal antibody to the transferrin receptor; the polycaprolactone constitutes the hydrophobic membrane layer of the nanovesicle, and the polyglutamic acid skeleton modified with a monoclonal antibody to the transferrin receptor constitutes the hydrophilic corona layer of the nanovesicle.

[0072] The concentration of the blood-brain barrier-crossing polymer vesicles prepared in Example 1 was diluted to 0.1 mg / mL, and the particle size was measured before and after ultrasound. The results were as follows: Figure 2 As shown in the figure: under the conditions of ultrasonic frequency of 1.0 MHz, ultrasonic power of 5 W and ultrasonic time of 5 min, the particle sizes of the polymer vesicles before and after ultrasound are 263 nm and 237 nm respectively, the size change rate is 10% and the PDI value is small, indicating that the particle size distribution of the targeted polymer vesicles is relatively uniform.

[0073] 2. The polymer vesicles prepared in Example 1 that can cross the blood-brain barrier were used as a delivery vehicle for the chemotherapy drug doxorubicin hydrochloride to investigate its drug release behavior.

[0074] The experimental procedure involved dissolving 5.0 mg of the target copolymer in 5 mL of N,N-dimethylformamide to prepare a 1.0 mg / mL initial solution. Then, 10 mL of a 0.25 mg / mL aqueous doxorubicin hydrochloride solution was added dropwise with rapid stirring. After the addition, the polymer vesicles were dialyzed against Tris buffer (pH 7.4, 0.01 M) to remove unentrapped doxorubicin hydrochloride, yielding a blood-brain barrier-crossing polymer vesicle solution containing doxorubicin hydrochloride. This solution was then placed in a dialysis bag with a molecular weight cutoff of 8,000 to 14,000. An equal volume and concentration of free doxorubicin hydrochloride in aqueous solution was used as a control, with the doxorubicin hydrochloride concentration in both cases being 80 μg / mL.

[0075] The above solutions were treated as follows:

[0076] (1) Neutral environment ultrasound group of polymer vesicles loaded with doxorubicin hydrochloride: 3 mL of targeted polymer vesicle solution loaded with doxorubicin hydrochloride was ultrasonicated at a frequency of 1.0 MHz, an ultrasonic power of 5 W, and a sonication time of 5 min, and then placed in 80 mL of tris buffer (pH = 7.4, 0.01 M);

[0077] (2) Neutral environment group of polymer vesicles loaded with doxorubicin hydrochloride: 3 mL of targeted polymer vesicle solution loaded with doxorubicin hydrochloride was placed in 80 mL of tris buffer (pH = 7.4, 0.01 M);

[0078] (3) Acidic environment ultrasound group of polymer vesicles loaded with doxorubicin hydrochloride: 3 mL of targeted polymer vesicle solution loaded with doxorubicin hydrochloride was ultrasonicated at a frequency of 1.0 MHz, an ultrasonic power of 5 W, and a sonication time of 5 min, and then placed in 80 mL of tris buffer (pH = 5.0, 0.01 M);

[0079] (4) Acidic environment group of polymer vesicles loaded with doxorubicin hydrochloride: 3 mL of targeted polymer vesicle solution loaded with doxorubicin hydrochloride was placed in 80 mL of tris buffer (pH = 5.0, 0.01 M);

[0080] (5) Free doxorubicin hydrochloride neutral environment group: 3 mL of free doxorubicin hydrochloride aqueous solution was placed in 80 mL of tris buffer (pH = 7.4, 0.01 M);

[0081] (6) Free doxorubicin hydrochloride acidic environment group: 3 mL of free doxorubicin hydrochloride aqueous solution was placed in 80 mL of tris buffer (pH = 5.0, 0.01 M).

[0082] All drug-release experiments were conducted at 37°C and 200 rpm, with three replicates per group. The entire process was conducted in the dark. Fluorescence values ​​of doxorubicin hydrochloride in the beaker were measured at 0, 1, 2, 3, 4, 6, 8, 12, and 24 hours. The released drug content was calculated using a standard curve for doxorubicin hydrochloride, and a drug release curve was plotted.

[0083] Depend on Figure 3The results show that polymersome encapsulation has a sustained drug release effect. Free drug reaches maximum release within 3 hours, while vesicle encapsulation allows for sustained drug release for over 12 hours. Comparing drug release at pH 7.4 and pH 5.0 reveals that a weakly acidic environment can accelerate drug release. After drug injection into the body, release is slow at normal physiological pH, minimizing drug loss and cytotoxicity before reaching the target site. Once the drug reaches the target site, the weakly acidic tumor tissue microenvironment accelerates drug release, leading to better therapeutic efficacy. Comparing drug release with and without ultrasound reveals that ultrasound, while increasing polymersome deformation, also accelerates drug release to a certain extent.

[0084] 3. The polymer vesicles capable of crossing the blood-brain barrier prepared in Example 1 were used as drug delivery vehicles, and their cell-targeted uptake efficiency in the blood-brain barrier transwell model was quantitatively determined by flow cytometry.

[0085] The groups involved in the experiment are described as follows:

[0086] (1) Free drug group: Doxorubicin hydrochloride was dissolved in deionized water and then diluted with sterile PBS to a 10 μg / mL solution;

[0087] (2) Drug-loaded polymersomes: 5.0 mg of the target copolymer was dissolved in 5 mL of N,N-dimethylformamide to prepare a 1.0 mg / mL initial solution. 10 mL of a 0.25 mg / mL aqueous solution of doxorubicin hydrochloride was added dropwise under rapid stirring. After the addition was complete, the solution was dialyzed against Tris buffer (pH = 7.4, 0.01 M) to remove the unentrapped doxorubicin hydrochloride. This resulted in the drug-loaded polymersomes.

[0088] (3) Targeted drug-loaded polymersome group: Take 0.02 mg / mL polymersome solution, adjust the pH to 4-6, add 0.408 mg N-hydroxysuccinimide and stir for 0.5 h. Then add 0.679 mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and stir for 2.5 h. Then, adjust the pH to 7.0, add 2.27 mg streptavidin to the above solution, stir for 12 h and dialyze to remove byproducts. Finally, add 33 μL biotin-labeled transferrin receptor monoclonal antibody (the molar ratio of polymersome: N-hydroxysuccinimide: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride: streptavidin: biotin-labeled transferrin receptor monoclonal antibody is 1:100:100:1:0.012) and stir for 12 h. The solution was then stirred and mixed with a 0.25 mg / mL aqueous solution of doxorubicin hydrochloride, and then dialyzed and purified in a Tris buffer solution (pH=7.4, 0.01 M) to obtain a targeted drug-loaded polymer vesicle group.

[0089] (4) Targeted drug-loaded polymer vesicles plus ultrasound group: The materials used were the same as those used in the preparation of targeted drug-loaded polymer vesicles in (3), except that ultrasound stimulation (1 MHz, 1.0 W / cm 2 ,1min).

[0090] The specific experimental process is as follows: hCMEC / D3 cells were inoculated in the upper chamber of the transwell chamber, and the transendothelial cell resistance was measured every day. When the transendothelial cell resistance value was greater than 200Ω·cm 2 When the concentration of doxorubicin hydrochloride in each group of materials was determined and calculated using ultraviolet and fluorescence spectrophotometers, the concentration was then diluted to 10 μg / mL. After 24 hours of cell culture, the old culture medium was removed, and fresh culture medium containing 10% PBS (negative control) and each group of materials (with the same concentration of doxorubicin hydrochloride) was added to the upper chamber. Culture medium was added to the lower chamber to level the inner and outer liquid levels. After incubation at 37°C for 4 hours, the upper chamber was removed, and the cells in the lower chamber were digested and collected in a centrifuge tube. Subsequently, the cells were centrifuged (3000 rpm, 4 min) and gently washed with PBS, which was repeated 3 times. Finally, the cells were suspended in 500 μL PBS, placed on ice, and analyzed by flow cytometry.

[0091] The results are as follows Figure 4 As shown, the positive cell ratios in the PBS group, drug-loaded polymersome group, free drug group, targeted drug-loaded polymersome group, and targeted drug-loaded polymersome plus ultrasound group were 0.0%, 10.9%, 17.7%, 25.6%, and 33.2%, respectively. The positive cell ratios of the parallel experiments in each group were averaged and analyzed for significant differences. The results showed that there were extremely significant differences between the targeted drug-loaded polymer vesicle group and the drug-loaded polymer vesicle group and the free drug group, p<0.001, indicating that the modified antibody can improve the ability of drug-loaded vesicles to target hCMEC / D3 cells and U-87MG cells; there were also extremely significant differences between the targeted drug-loaded polymer vesicle plus ultrasound group and the drug-loaded polymer vesicle group, the free drug group, and the targeted drug-loaded polymer vesicle group, p<0.001, indicating that the use of ultrasound can significantly improve the permeability of the in vitro blood-brain barrier model, assist the monoclonal antibody modified with transferrin receptor to penetrate the dense hCMEC / D3 cell layer, and increase the uptake rate of U-87MG cells.

[0092] 4. The blood-brain barrier-crossing polymer vesicles prepared in Example 1 were used as drug delivery vehicles, and the blood-brain barrier penetration ability of the blood-brain barrier-crossing polymer vesicles as drug delivery vehicles was evaluated by measuring the mean fluorescence intensity at different depths of the 3D multicellular spheroids.

[0093] The experimental procedure was as follows: 96-well U-shaped plates were coated with 3D cell culture plate coating solution, and equal numbers of hCMEC / D3 cells and U-87MG cells were added to each well. Cell status was observed daily until compact spheroids of appropriate size were formed. The doxorubicin hydrochloride concentration of each material was measured and calculated using UV and fluorescence spectrophotometers, and then diluted to 10 μg / mL. After successful 3D spheroid formation, the old culture medium was aspirated, and fresh culture medium containing 10% PBS (negative control) and the same concentration of doxorubicin hydrochloride for each material group was added to each culture medium. After incubation at 37°C for 4 hours, the old culture medium was removed, and the cells were washed three times with PBS. The spheroids were then transferred to a confocal microplate and scanned in the z-axis under a laser confocal microscope. The fluorescence intensity of doxorubicin hydrochloride in the spheroids was observed at different scanning depths. Quantitative analysis was performed using Image J software to assess the permeability of each material group in the 3D spheroid model of the blood-brain barrier.

[0094] The results are as follows Figure 5 As shown, the fluorescence intensity of the free drug and drug-loaded polymersomes groups reached its maximum at a scanning depth of 45 μm, while that of the targeted drug-loaded polymersomes group reached its maximum at a scanning depth of 75 μm. The fluorescence intensity of the drug-loaded polymersomes plus ultrasound and targeted drug-loaded polymersomes plus ultrasound groups continued to increase with increasing scanning depth. The fluorescence intensity of each group, from lowest to highest, was as follows: drug-loaded polymersomes group, free drug group, targeted drug-loaded polymersomes group, drug-loaded polymersomes plus ultrasound group, and targeted drug-loaded polymersomes plus ultrasound group. This indicates that receptor-mediated transcytosis and ultrasound-enhanced permeability contribute to the enhanced ability of drug-loaded polymersomes to penetrate the biomimetic blood-brain barrier. In summary, these targeted polymersomes can be used as drug delivery vehicles to effectively encapsulate chemotherapeutic drugs and, combined with ultrasound, enhance their permeability in the biomimetic blood-brain barrier 3D multicellular spheroid model.

[0095] 5. Particle size and PDI determination of the polymer vesicles prepared in Example 2 that can cross the blood-brain barrier

[0096] The particle size of the blood-brain barrier-crossing polymer vesicles prepared in Example 2 was 300 nm, and the PDI was 0.342. Compared with the blood-brain barrier-crossing polymer vesicles prepared in Example 1, the polymer vesicles prepared in this example were larger in size and had a wider distribution.

[0097] 6. Particle size and PDI determination of the polymer vesicles prepared in Example 3 that can cross the blood-brain barrier

[0098] The particle size of the blood-brain barrier-crossing polymer vesicles prepared in Example 3 was 375 nm, and the PDI was 0.124. Compared with the blood-brain barrier-crossing polymer vesicles prepared in Example 1, the polymer vesicles prepared in this example were larger in size and had a wider distribution.

[0099] 7. Particle size and PDI determination of the polymer vesicles capable of crossing the blood-brain barrier prepared in Example 4

[0100] The particle size of the blood-brain barrier-crossing polymer vesicles prepared in Example 4 was 274 nm, and the PDI was 0.128. Compared with the blood-brain barrier-crossing polymer vesicles prepared in Example 1, the polymer vesicles prepared in this example had a wider distribution.

[0101] 8. Particle size and PDI determination of the polymer vesicles capable of crossing the blood-brain barrier prepared in Example 5

[0102] The particle size of the blood-brain barrier-crossing polymer vesicles prepared in Example 5 was 348 nm, and the PDI was 0.036. Compared with the blood-brain barrier-crossing polymer vesicles prepared in Example 1, the polymer vesicles prepared in this example were larger in size.

[0103] 9. Particle size and PDI of the polymer vesicles prepared in Comparative Example 1 that can cross the blood-brain barrier

[0104] The particle size of the blood-brain barrier-crossing polymer vesicles prepared in Comparative Example 1 was 546 nm, and the PDI was 0.289. Compared with the blood-brain barrier-crossing polymer vesicles prepared in Example 1, the polymer vesicles prepared in this example were larger in size and had a wider distribution.

[0105] 10. Particle size and PDI of the polymer vesicles prepared in Comparative Example 2 that can cross the blood-brain barrier

[0106] The particle size of the blood-brain barrier-crossing polymer vesicles prepared in Comparative Example 2 was 633 nm, and the PDI was 0.05. Compared with the blood-brain barrier-crossing polymer vesicles prepared in Example 1, the polymer vesicles prepared in this example were larger in size and had a wider distribution.

[0107] 11. Particle size and PDI of the polymer vesicles prepared in Comparative Example 3 that can cross the blood-brain barrier

[0108] The particle size of the blood-brain barrier-crossing polymer vesicles prepared in Comparative Example 3 was 469 nm, and the PDI was 0.372. Compared with the blood-brain barrier-crossing polymer vesicles prepared in Example 1, the polymer vesicles prepared in this example were larger in size and had a wider distribution.

[0109] In summary, the preparation method of the present invention is to utilize amino-terminated polycaprolactone and amino acid cyclic anhydride with reactive groups to carry out ring-opening reaction at room temperature, and the resulting copolymer self-assembles to form polymer vesicles of suitable size and uniform distribution, and then encapsulates the chemotherapy drug doxorubicin hydrochloride and modifies the monoclonal antibody of transferrin receptor. The preparation method is simple and efficient. The monoclonal antibody of transferrin receptor modified by the hydrophilic corona of the polymer vesicle has a high affinity for the transferrin receptor overexpressed on the surface of brain endothelial cells and glioblastoma cells, and can deliver chemotherapy drugs to brain tumor cells across the blood-brain barrier. This polymer vesicle that can cross the blood-brain barrier has good stability and degradability, can effectively achieve drug delivery targeting intracranial lesions, and has broad application prospects in the field of treatment of brain diseases.

[0110] The embodiments provided above are not intended to limit the scope of the present invention, nor are the steps described to limit their execution order. Any obvious improvements to the present invention made by those skilled in the art in combination with existing common knowledge shall fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing polymer vesicles capable of crossing the blood-brain barrier, characterized in that: The steps include: (1) Synthesis of amphiphilic polymers Using polycaprolactone and amino acid cyclic anhydride as raw materials, a polycaprolactone-block-polyamino acid copolymer is prepared, which is dissolved in trifluoroacetic acid, and a hydrobromic acid-acetic acid solution is added to react, and the mixture is precipitated in anhydrous ether to obtain a polycaprolactone-block-polyamino acid copolymer with the tert-butyloxycarbonyl protection removed. (2) Preparation of polymer vesicles The polycaprolactone-block-polyamino acid copolymer obtained in step (1) without tert-butyloxycarbonyl protection is dissolved in a second type of solvent, and the initial concentration of the target polymer is controlled to be 0.5 to 5.0 mg / mL. 1 to 5 times the volume of deionized water is added dropwise. After the addition is completed, dialysis is performed. The dialyzed solution is the polymer vesicle solution; (3) Targeted modification of polymersomes The polymer vesicle solution obtained in step (2) is diluted 1 to 10 times, the pH is adjusted to 4 to 6, N-hydroxysuccinimide is added and stirred, and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is added and stirred. Then, the pH is adjusted to neutral, streptavidin is added to the above solution, and the by-products are dialyzed. Finally, a biotin-labeled monoclonal antibody for transferrin receptor is added to the dialyzed solution and stirred to finally obtain a targeted polymer vesicle modified with a monoclonal antibody for transferrin receptor.

2. The preparation method according to claim 1, characterized in that In step (1), the amino acid cyclic anhydride is an amino acid cyclic anhydride having a reactive group, which is one of L-glutamic acid-γ-benzyl ester cyclic anhydride and L-aspartic acid-β-benzyl ester cyclic anhydride; and the polycaprolactone is amino-terminated polycaprolactone.

3. The preparation method according to claim 1, characterized in that In step (1), the molar ratio of the polycaprolactone to the amino acid cyclic anhydride is 1:

13.

4. The preparation method according to claim 1, characterized in that In step (1), the volume mass ratio of the hydrobromic acid acetic acid solution to the polycaprolactone-block-polyamino acid copolymer is 10-15:1-2, mL / g.

5. The preparation method according to claim 1, characterized in that In step (1), the volume mass ratio of trifluoroacetic acid to polycaprolactone-block-polyamino acid copolymer is 10-15:1, mL / g.

6. The preparation method according to claim 1, characterized in that In step (2), the second type of solvent is N,N-dimethylformamide.

7. The preparation method according to claim 1, characterized in that In step (2), the initial concentration of the target polymer is controlled at 1 mg / mL.

8. The preparation method according to claim 1, characterized in that In step (3), the molar ratios of the polymer vesicle to N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, streptavidin, and biotin-labeled monoclonal antibody to transferrin receptor are 1:(10-100), 1:(10-100), 1:(0.01-1), and 1:(0.0001-1), respectively.

9. Polymer vesicles capable of crossing the blood-brain barrier obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the polymer vesicle capable of crossing the blood-brain barrier according to claim 9 in the preparation of chemotherapy drugs and nucleic acid drug delivery carriers.

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