PD-L1 siRNA-loaded nano boron medicine as well as preparation method and application thereof

Abstract

The invention discloses a triblock polymer containing 10B. The triblock polymer and PD-L1siRNA can be self-assembled to form a nano boron medicine. The invention further discloses a preparation method of the triblock polymer containing 10B. The invention also provides a nano boron medicine loaded with PD-L1siRNA, wherein the nano boron medicine is prepared from the triblock polymer which is used for modifying the cRGD peptide and contains 10B, and the PD-L1siRNA. Cell experiments prove that the nano boron medicine has better tumor cell uptake and intracellular retention performance than a clinical boron medicine fructose BPA; in-vivo anti-tumor treatment experiments prove that the nano boron medicine can co-deliver PD-L1siRNA and 10B atom targeting tumor cells, and the combined anti-tumor effect of boron neutron capture therapy and immunotherapy is achieved. According to the invention, the anti-tumor effect of boron neutron capture therapy is effectively improved, and the application prospect of combination of boron neutron capture therapy and immunotherapy in tumor resistance is broadened.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceutical preparations, and specifically relates to a nano-boron drug loaded with PD-L1 siRNA and a preparation method and application thereof. Background Art

[0002] Boron Neutron Capture Therapy (BNCT) is considered one of the most advanced tumor treatment methods in the world today. 10 B atoms are delivered into tumor cells, and then neutron irradiation is used to induce 10 The nuclear reaction of B releases high-energy rays. The radiation range of the rays is 5-10μm, which is exactly the size of a cell and can accurately kill tumor cells. Therefore, whether BNCT can accurately kill tumor cells depends entirely on the carrier. 10 Can boron drugs with B atoms be enriched in tumor cells for a long time with high concentration and high selectivity? At present, only two small molecule boron drugs, 4-boronic acid-L-phenylalanine (BPA) and disodium thiododecaborane (BSH), have been clinically approved. Among them, BPA is the most widely used and was approved for marketing in Japan in 2020. BPA and BSH have been used in clinical trials for more than 70 years, but only more than a thousand cases have been conducted worldwide, and progress has been very slow. Small molecule boron drugs are limited by defects at the molecular property level, with rapid metabolism and insufficient specificity, which is one of the important reasons limiting the clinical development of BNCT. Compared with small molecule boron drugs, nano boron drugs can show many advantages: long half-life in vivo, high tumor accumulation, solubility and pharmacokinetics can be improved as needed, different tumor cells can be targeted by connecting different targeting molecules, and multi-drug delivery can be achieved through reasonable design.

[0003] Patent CN116271031A discloses a nano-boron delivery agent, which is a nanoparticle formed by a lipid-soluble 3-boric acid phenyl-1-carbamic acid cholesterol ester or a 4-boronic acid-L-phenylalanine cholesterol modification and albumin, wherein the boron in the modification contains 10B. The nano-boron delivery agent can effectively increase the water-soluble concentration of boron-containing drugs, enhance their targeting and killing effects on tumors, and use it as a boron-containing targeted drug to significantly enhance BNCT. Patent CN111281975A discloses a method for preparing a boron-containing nano-targeted drug, which discloses that the boron-containing nano-targeted drug is obtained by loading the boron drug with an amphiphilic polymer. Compared with free BPA, the tumor-blood ratio of the nano-targeted drug is significantly improved, which can significantly improve the therapeutic effect of tumors. It has very good application prospects when used alone as an anti-tumor drug and in combination with anti-tumor drugs such as paclitaxel. PD-L1 (programmed death ligand 1) is a protein expressed on the surface of many cells. It can bind to the programmed death receptor 1 (PD-1) on the surface of T cells, thereby inhibiting the activity of T cells and enabling tumor cells to escape the attack of the body's immune system. PD-L1 siRNA is a small interfering RNA that can specifically target the PD-L1 gene. It can form an RNA-induced silencing complex (RISC) with nucleases in the cytoplasm through the RNA interference (RNAi) mechanism, recognize and bind to the complementary PD-L1 mRNA sequence, and then degrade the mRNA under the action of nucleases, thereby inhibiting the expression of PD-L1 protein and enhancing the body's immune system's ability to recognize and kill tumor cells. However, there is currently no nanoboron drug that can encapsulate PD-L1 siRNA that can achieve the effects of both BNCT and immunotherapy. Summary of the invention

[0004] The purpose of the present invention is to overcome the above-mentioned defects and shortcomings in the prior art and provide a 10 The triblock polymer of B can form a nano boron drug by self-assembly with PD-L1 siRNA.

[0005] The second object of the present invention is to provide the above-mentioned 10 B is a method for preparing a triblock polymer.

[0006] The third object of the present invention is to provide a nanoboron drug loaded with PD-L1 siRNA.

[0007] The fourth object of the present invention is to provide the above-mentioned 10 Application of the triblock polymer of B or the above-mentioned nano-boron drug in the preparation of drugs for treating tumors.

[0008] A fifth object of the present invention is to provide a pharmaceutical composition.

[0009] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0010] The present invention provides a 10B, wherein the triblock polymer is polyethylene glycol-b-polyacrylamide (2-mercaptoethyl)-b-polyacrylamide (benzoborazolyl) (PEG-PME-PBOB), and its structural formula is shown in the following formula (I):

[0011]

[0012] Wherein n= is 42-48, m is 8-12, and p is 55-65.

[0013] The triblock polymer of the present invention can be used to crosslink PD-L1 siRNA and form a nanoboron drug loaded with PD-L1 siRNA through self-assembly. Specifically, the side group of the PBOB block of the polymer is a benzoborazole group, containing 10 B atom. The benzoborazole group can react with the vicinal diol of the ribose group at both ends of the PD-L1 siRNA under neutral or alkaline pH conditions to form a boron ester bond. Therefore, PD-L1 siRNA can cross-link the PBOB block of the polymer PEG-PME-PBOB. Coupled with the intermolecular π-π stacking effect of the benzoborazole group, PD-L1 siRNA and the polymer PEG-PME-PBOB can self-assemble to form nanoparticles in a PBS solution at pH 7.4. Subsequently, the thiol group of the middle layer is oxidized by oxygen to form a disulfide-crosslinked middle layer structure, thereby further stabilizing the nanoparticles and obtaining a disulfide-crosslinked nanoboron drug.

[0014] Furthermore, the 10 The end of the triblock polymer of B is also modified with a cRGD peptide, which is a polymer of cRGD peptide-polyethylene glycol-b-polyacrylamide (2-mercaptoethyl)-b-polyacrylamide (benzoborazole), referred to as cRGD-PEG-PME-PBOB, and its structural formula is shown in the following formula (II):

[0015]

[0016] Wherein n= is 42-48, m is 8-12, and p is 55-65.

[0017] The triblock polymer cRGD-PEG-PME-PBOB is a polymer of formula (I) PEG-PME-PBOB with a PEG-terminal modified cRGD peptide to form a polymer cRGD peptide-polyethylene glycol-b-polyacrylamide (2-mercaptoethyl)-b-polyacrylamide (benzoborazole). The cRGD peptide has tumor cell targeting properties, thereby 10B atom and PD-L1 siRNA are delivered into tumor cells. Wherein, the number of repeating units of polyethylene glycol, polyacrylamide (2-mercaptoethyl) and polyacrylamide (benzoborazole) can be 42 to 48, m is 8 to 12, and p is 55 to 65, respectively.

[0018] Furthermore, n=45, m=10, and p=60.

[0019] The present invention also provides the above-mentioned 10 The preparation method of the triblock polymer of B is to synthesize the triblock by Raft polymerization, which specifically comprises the following steps:

[0020] S1. Allyl-polyethylene glycol-2-(dodecyl trithiocarbonate)-2-methylpropionate, S-trityl-2-mercaptoethylacrylamide and a rich 10 B 5-acrylamide benzoborazole reaction, purification, to obtain a polymer allyl-polyethylene glycol-b-polyacrylamide (S-trityl-2-mercaptoethyl)-b-polyacrylamide (benzoborazole)-2-(dodecyl trithiocarbonate)-2-methylpropionate;

[0021] S2. The polymer allyl-polyethylene glycol-b-polyacrylamide (S-trityl-2-mercaptoethyl)-b-polyacrylamide (benzoborazolyl)-2-(dodecyltrithiocarbonate)-2-methylpropionate is reacted with the cRGD polypeptide and purified to obtain the polymer cRGD peptide-polyethylene glycol-b-polyacrylamide (S-trityl-2-mercaptoethyl)-b-polyacrylamide (benzoborazolyl)-2-(dodecyltrithiocarbonate)-2-methylpropionate.

[0022] S3. After removing the trityl protecting group and DDAT group from the polymer cRGD peptide-polyethylene glycol-b-polyacrylamide (S-trityl-2-mercaptoethyl)-b-polyacrylamide (benzoborazolyl)-2-(dodecyltrithiocarbonate)-2-methylpropionate, purify it to obtain the polymer represented by formula (II): cRGD peptide-polyethylene glycol-b-polyacrylamide (2-mercaptoethyl)-b-polyacrylamide (benzoborazolyl); or after removing the trityl protecting group and DDAT group from the polymer allyl-polyethylene glycol-b-polyacrylamide (S-trityl-2-mercaptoethyl)-b-polyacrylamide (benzoborazolyl)-2-(dodecyltrithiocarbonate)-2-methylpropionate in step S1, purify it to obtain the polymer represented by formula (I): polyethylene glycol-b-polyacrylamide (2-mercaptoethyl)-b-polyacrylamide (benzoborazolyl).

[0023] Furthermore, in step S1, allyl-polyethylene glycol-2-(dodecyl trithiocarbonate)-2-methylpropionate, S-trityl-2-mercaptoethyl acrylamide and 10 The molar ratio of 5-acrylamide benzoborazole in B is 0.1-0.11:1-1.4:6-8.

[0024] Preferably, in step S1, allyl-polyethylene glycol-2-(dodecyl trithiocarbonate)-2-methylpropionate, S-trityl-2-mercaptoethylacrylamide and 10 The molar ratio of 5-acrylamidobenzoborazole in B is 0.106:1.2:7.

[0025] Furthermore, the preparation method of allyl-polyethylene glycol-2-(dodecyl trithiocarbonate)-2-methylpropionate comprises the following steps: after dissolving allyl-polyethylene glycol (CAS: 27274-31-3), adding 2-(dodecyl trithiocarbonate)-2-methylpropionic acid (CAS: 461642-78-4), 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (CAS: 25952-53-8) and 4-dimethylaminopyridine (CAS: 1122-58-3), and after reaction and purification, allyl-polyethylene glycol-2-(dodecyl trithiocarbonate)-2-methylpropionate is obtained.

[0026] Furthermore, the preparation method of S-trityl-2-mercaptoethylacrylamide comprises the following steps: dissolving 2-(tritylthio)ethylamine (CAS: 1095-85-8), adding N,N-diisopropylethylamine and acryloyl chloride, and reacting and concentrating to obtain monomer S-trityl-2-mercaptoethylacrylamide.

[0027] Furthermore, the rich 10 The preparation method of 5-acrylamide benzoborazole of B comprises the following steps:

[0028] S1. (2-formylphenyl) boron 10 B acid (CAS: 269727-10-8) and NaBH 4 Mixed reaction, purified to obtain 10 Benzoborazole of B;

[0029] S2. will be rich in 10 B Benzoborazole and HNO 3 and H 2 SO 4 Mix the reaction and purify to obtain 10 5-Nitrobenzoborazole of B;

[0030] S3.10 B 5-Nitrobenzoborazole is dissolved with NaHCO 3 Mix the reaction and purify to obtain 10 5-aminobenzoborazole of B;

[0031] S4. 10 B 5-aminobenzoborazole with NaHCO 3 , acryloyl chloride, and purified to obtain 10 5-Acrylamidobenzoborazole of B.

[0032] Furthermore, in step S2, the molar ratio of allyl-polyethylene glycol-b-polyacrylamide (S-trityl-2-mercaptoethyl)-b-polyacrylamide (benzoborazolyl)-2-(dodecyl trithiocarbonate)-2-methylpropionate to cRGD polypeptide is 1:9-11.

[0033] Preferably, in step S2, the molar ratio of allyl-polyethylene glycol-b-polyacrylamide (S-trityl-2-mercaptoethyl)-b-polyacrylamide (benzoborazolyl)-2-(dodecyl trithiocarbonate)-2-methylpropionate to cRGD polypeptide is 1:10.

[0034] Furthermore, in the step S3, the trityl protecting group of the side group of the polymer cRGD peptide-polyethylene glycol-b-polyacrylamide (S-trityl-2-mercaptoethyl)-b-polyacrylamide (benzoborazolyl)-2-(dodecyl trithiocarbonate)-2-methylpropionate or the polymer allyl-polyethylene glycol-b-polyacrylamide (S-trityl-2-mercaptoethyl)-b-polyacrylamide (benzoborazolyl)-2-(dodecyl trithiocarbonate)-2-methylpropionate is first removed to generate a thiol group, and then the DDAT group is removed.

[0035] The present invention also provides a nano-boron drug loaded with PD-L1 siRNA 10 B / siPD-L1 NPs, the nano boron drug contains 10 B is composed of a triblock polymer and loaded PD-L1 siRNA.

[0036] Nano boron medicine of the present invention 10 B / siPD-L1 NPs contain 10 The triblock polymer of B is the carrier, as described above, 10 The side groups of the PBOB blocks of the triblock polymer cRGD-PEG-PME-PBOB contain 10The benzoborazole group of the B atom can react with the vicinal diol of the ribose group at both ends of the PD-L1 siRNA under neutral or alkaline conditions to form a boron ester bond. Therefore, PD-L1 siRNA can cross-link the PBOB block of the polymer cRGD-PEG-PME-PBOB. Coupled with the intermolecular π-π stacking effect of the benzoborazole group, PD-L1 siRNA and the polymer cRGD-PEG-PME-PBOB can self-assemble to form nanoparticles in a PBS solution at pH = 7.4. Subsequently, the thiol group of the intermediate layer PME block is oxidized by oxygen to form a disulfide-crosslinked intermediate layer structure, thereby further stabilizing the nanoparticles to obtain disulfide-crosslinked nanoboron drugs. 10 B / siPD-L1 NPs. 10 B / siPD-L1 NPs have cRGD peptide on their surface, which can target tumor cells for entry, thereby 10 B atoms and PD-L1 siRNA delivery into tumor cells. Under neutron irradiation 10 B atoms can undergo nuclear reactions to generate high-energy radiation to kill tumor cells and perform boron neutron capture therapy (BNCT). In tumor cells, high concentrations of glutathione can break down 10 B / siPD-L1 NPs have disulfide cross-linked layers; the high concentration of ATP in tumor cells also contains vicinal diol structures, which can replace and release PD-L1 siRNA to play an immunotherapy role. 10 B / siPD-L1 NPs nanoboron drugs can be loaded and delivered 10 B atoms and PD-L1 siRNA are targeted and taken up by tumor cells, realizing the combination of BNCT and immunotherapy.

[0037] The present invention also provides a method for preparing the nano-boron drug loaded with PD-L1 siRNA, comprising the following steps: dissolving the polymer cRGD-PEG-PME-PBOB, incubating, purifying, and then adding PD-L1 siRNA to mix to obtain the nano-boron drug. 10 B / siPD-L1 NPs.

[0038] Furthermore, the nucleotide sequence of the PD-L1 siRNA is shown in SEQ ID No. 1-2.

[0039] Furthermore, the 10 The mass ratio of B triblock polymer to PD-L1 siRNA is 15-25:1.

[0040] Furthermore, the 10 The mass ratio of B triblock polymer to PD-L1 siRNA is 18-22:1.

[0041] Preferably, the mass ratio of the triblock polymer to the PD-L1 siRNA is 20: 1. When the mass ratio of the polymer to the PD-L1 siRNA is greater than 20: 1, the PD-L1 siRNA is almost completely loaded, so the optimal choice is to use a mass ratio of the polymer to the PD-L1 siRNA of 20: 1 to prepare the nanoboron drug.

[0042] Furthermore, the hydrated particle size of the nano-boron medicine is 90 to 110 nm, and the surface potential is -10 to -20 mV.

[0043] Preferably, the hydrated particle size of the nano-boron drug is 97nm, and the surface potential is -17mV. When the mass ratio of the polymer to PD-L1siRNA is 20:1, the hydrated particle size of the prepared nano-boron drug is 97nm, the surface potential is -17mV, and the morphology is a spherical structure.

[0044] The present invention has proved the successful preparation of nano-boron drugs through characterization such as nuclear magnetic resonance hydrogen spectrum, gel electrophoresis, transmission electron microscopy and particle size potential. Cell experiments have shown that the nano-boron drug has better tumor cell uptake and intracellular retention than the clinical boron drug fructose BPA, and is expected to improve the anti-tumor effect of boron neutron capture therapy. In addition, in vivo anti-tumor treatment experiments have shown that the nano-boron drug can co-deliver PD-L1 siRNA and 10 Targeting tumor cells with B atoms can achieve combined anti-tumor effects of BNC therapy and immunotherapy, which is more effective than BNC therapy alone and broadens the application of BNC therapy.

[0045] Therefore, the present invention provides the above-mentioned 10 Application of triblock polymers of B or nano-boron drugs in the preparation of drugs for treating tumors.

[0046] Furthermore, the tumor includes but is not limited to breast cancer, etc., and is applicable to all tumors that can be treated by BNCT and immunotherapy.

[0047] The present invention also provides a pharmaceutical composition, which comprises the above-mentioned 10 B triblock polymer or nanoboron drug.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] The present invention provides a 10 The present invention also provides a nano-boron drug loaded with PD-L1 siRNA, wherein the nano-boron drug is composed of the above-mentioned modified cRGD peptide 10The nanoboron drug is composed of a triblock polymer of B and loaded PD-L1 siRNA. Cell experiments have shown that the nanoboron drug has better tumor cell uptake and intracellular retention than the clinical boron drug fructose BPA; in vivo anti-tumor treatment experiments have shown that the nanoboron drug can co-deliver PD-L1 siRNA and 10 The targeting of B atoms to tumor cells can achieve the combined anti-tumor effect of BNC therapy and immunotherapy. The present invention effectively improves the anti-tumor effect of BNC therapy and broadens the application prospect of the combined use of BNC therapy and immunotherapy in anti-tumor. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is the H NMR spectrum of the polymer APEG-P(ME-Trt)-PBOB-DDAT.

[0051] Figure 2 This is the H NMR spectrum of the polymer cRGD-PEG-P(ME-Trt)-PBOB-DDAT.

[0052] Figure 3 This is the H NMR spectrum of the polymer cRGD-PEG-PME-PBOB.

[0053] Figure 4 Gel electrophoresis analysis of nanoboron drugs prepared with different polymer and siRNA mass ratios.

[0054] Figure 5 Nanoboron drug 10 Characterization of B / siPD-L1 NPs by transmission electron microscopy, hydrated particle size, and surface potential.

[0055] Figure 6 Nanoboron drug 10 B / siPD-L1 NPs or fructose BPA and 4T1 cells incubated for different time periods 10 B concentration (n=3, mean±SD).

[0056] Figure 7 Nanoboron drug 10 Retention rates of B / siPD-L1 NPs and fructose BPA in 4T1 cells at different times (n=3, mean±SD).

[0057] Figure 8 Nanoboron drug 10 In vivo anti-tumor therapeutic effect of B / siPD-L1 NPs. Figure 8 A is the tumor volume growth curve of different treatment groups (n=5, mean±SD); B is the tumor tissue CD8 + T cells account for CD45+ The proportion of T cells (n=3, mean±SD). P values ​​were determined by one-way analysis of variance (ANOVA) combined with Tukey's multiple comparison test, **P<0.01, ***P<0.001, ****P<0.0001. DETAILED DESCRIPTION

[0058] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0059] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0060] (1) Allyl-polyethylene glycol-2-(dodecyl trithiocarbonate)-2-methylpropionate:

[0061] Allyl-polyethylene glycol (10 g, 5 mmol, CAS: 27274-31-3) with a molecular weight of 2 kDa was added to a Schlenk flask and dried under vacuum at 70°C for 2 hours. After cooling to room temperature, 30 mL of dichloromethane was added to dissolve the allyl-polyethylene glycol. Subsequently, 2-(dodecyl trithiocarbonate)-2-methylpropionic acid (3.6 g, 10 mmol, CAS: 461642-78-4), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.9 g, 10 mmol, CAS: 25952-53-8) and 4-dimethylaminopyridine (1.2 g, 10 mmol, CAS: 1122-58-3) were added under ice bath and nitrogen protection, and the reaction was carried out for 12 hours. After the reaction was completed, the mixture was placed in a dialysis bag (molecular weight cutoff: 500 Da) and dialyzed with methanol for 1 day. After dialysis, the dialysate was concentrated and precipitated with diethyl ether to give allyl-polyethylene glycol-2-(dodecyltrithiocarbonate)-2-methylpropionate (8.5 g, 3.6 mmol).

[0062] (2) S-trityl-2-mercaptoethyl acrylamide:

[0063] 2-(Tritylthio)ethylamine (2 g, 6.51 mmol, CAS: 1095-85-8) was dissolved in dichloromethane, and N,N-diisopropylethylamine (1.32 mL, 13.03 mmol, CAS: 7087-68-5) and acryloyl chloride (0.59 mL, 7.17 mmol) were added. After stirring in the dark for 4 hours, the reaction mixture was concentrated and the crude product was dissolved in ethyl acetate. Subsequently, water, 1 M KHSO4 solution, 5% NaHCO 3Solution and saturated brine, wash the organic phase in turn, and wash with anhydrous Na 2 SO 4 The residue was dried and finally concentrated to obtain the monomer S-trityl-2-mercaptoethylacrylamide (1.82 g, 5.04 mmol).

[0064] (3) Rich in 10 5-Acrylamidobenzoborazole of B:

[0065] First, (2-formylphenyl)borane- 10 B acid (5 g, 33.56 mmol, CAS: 269727-10-8) was dissolved in 50 mL of methanol and NaBH 4 (sodium borohydride, 6.3 g, 166.53 mmol), stirred at 0 ° C for 2 hours. Then, the reaction was terminated by adding 2M HCl to adjust the pH to 1. After removing methanol by rotary evaporation, the mixture was extracted with ethyl acetate (3×50 mL), and the organic layer was washed with saturated brine. The organic phase was dried, filtered and concentrated, and purified by silica gel column chromatography (ethyl acetate / n-hexane=1:1) to obtain a rich 10 B of benzoborazole.

[0066] Subsequently, the rich 10 Benzoborazole (3 g, 22.56 mmol) of B was added to precooled concentrated HNO 3 and H 2 SO 4 The reaction was started at -45°C in the mixed solution (v / v=3:2) and then continued at -20°C for 1 hour. After the reaction was completed, the mixture was poured onto crushed ice and stirred for 15 minutes. The organic layer was extracted with ethyl acetate (3×100 mL), washed with brine, dried, filtered and concentrated, and then precipitated with n-hexane to obtain a rich 10 5-Nitrobenzoborazole of B.

[0067] Next, the rich 10 5-Nitrobenzoborazole (2 g, 11.24 mmol) of B was dissolved in a 1:1 mixture of methanol (40 mL) and 1 M HCl (40 mL), and zinc powder (7.28 g, 112 mmol) was added and stirred at room temperature for 2 hours. After the reaction, the precipitate was filtered and the methanol was evaporated. The saturated NaHCO 3 The reaction mixture was alkalized with ethyl acetate (3×150 mL), and the organic layer was then washed with saturated brine, dried, filtered, concentrated, and precipitated with n-hexane to obtain a rich 10 5-aminobenzoborazole of B.

[0068] Finally, the rich 105-Aminobenzoborazole (1.5 g, 10.14 mmol) of B was dissolved in 15 mL THF and 15 mL NaHCO 3 solution (3.41 g, 40.6 mmol). Subsequently, acryloyl chloride (0.98 mL, 12 mmol) was added dropwise at 0°C, and then stirred at room temperature for 2 hours. After evaporating THF, the suspension was extracted with ethyl acetate, washed with saturated NaCl solution, dried, concentrated and precipitated to finally obtain a rich 10 5-Acrylamidobenzoborazole (1.75 g, 8.67 mmol) of B.

[0069] Example 1 Preparation and characterization of PD-L1 siRNA loaded nanoboron drug

[0070] 1. Experimental Methods

[0071] Contains 10 The synthetic route of polymer B cRGD-PEG-PME-PBOB is shown below:

[0072]

[0073] (1) Using the raft reagent allyl-polyethylene glycol-2-(dodecyl trithiocarbonate)-2-methylpropionate (APEG 2k -DDAT, 0.25 g, 0.106 mmol), S-trityl-2-mercaptoethylacrylamide (AAME-Trt, 0.43 g, 1.2 mmol) and azobisisobutyronitrile (AIBN, 4.9 mg, 0.03 mmol) were dissolved in 10 mL DMF and placed in a Schlenk reaction bottle. After two freeze-thaw cycles to remove oxygen, the reaction was heated to 80 °C for 12 hours. Subsequently, a solution rich in 10 B's 5-acrylamidobenzoborazole (AABOB, 1.41 g, 7 mmol) was added and then freeze-thawed twice for deoxygenation. The reaction was continued at 80°C for 24 hours. After the reaction was completed, the reaction solution was placed in a dialysis bag (molecular weight cutoff: 1 kDa) and dialyzed in methanol. Finally, the solvent in the dialysis bag was removed by rotary evaporation to obtain a polymer allyl-polyethylene glycol-b-polyacrylamide (S-trityl-2-mercaptoethyl)-b-polyacrylamide (benzoborazole)-2-(dodecyl trithiocarbonate)-2-methylpropionate, referred to as APEG-P(ME-Trt)-PBOB-DDAT.

[0074] (2) The polymer APEG-P(ME-Trt)-PBOB-DDAT (1 g, 0.055 mmol), cRGD peptide (c(RGDfC), 318 mg, 0.55 mmol) and AIBN (17 mg, 0.1 mmoll) were dissolved in 5 mL DMF and placed in a Schlenk reaction bottle. After two freeze-thaw cycles to deoxygenate, the reaction mixture was heated to 80°C for 24 hours. After the reaction, the reaction solution was transferred to a dialysis bag (molecular weight cutoff: 3.5 kDa) and dialyzed in methanol. Finally, the solvent in the dialysis bag was removed by rotary evaporation to obtain the polymer cRGD peptide-polyethylene glycol-b-polyacrylamide (S-trityl-2-mercaptoethyl)-b-polyacrylamide (benzoborazolyl)-2-(dodecyl trithiocarbonate)-2-methylpropionate, referred to as cRGD-PEG-P(ME-Trt)-PBOB-DDAT.

[0075] (3) The polymer cRGD-PEG-P(ME-Trt)-PBOB-DDAT (0.5 g, 0.027 mmol) was dissolved in 5 mL of trifluoroacetic acid (TFA) and placed in a Schlenk reaction bottle. The reaction was allowed to react at room temperature for 4 hours to remove the trityl protecting group of the side group and generate a thiol group. After the reaction was completed, the solvent was removed by rotary evaporation. The resulting solid was dissolved in DMF, and n-butylamine (0.2 g, 2.7 mmol) was added and reacted for 1 hour to remove the DDAT group. Subsequently, the reaction solution was placed in a dialysis bag (molecular weight cutoff: 1 kDa) and dialyzed in methanol. Finally, the solvent in the dialysis bag was removed by rotary evaporation to obtain the final polymer cRGD peptide-polyethylene glycol-b-polyacrylamide (2-mercaptoethyl)-b-polyacrylamide (benzoborazolyl), referred to as cRGD-PEG-PME-PBOB.

[0076] (4) 5 mg of polymer cRGD-PEG-PME-PBOB was dissolved in 0.5 mL PBS solution (pH 8.5) containing 10 mM tri(2-carboxyethyl)phosphine and incubated at room temperature for 2 hours to break the disulfide bonds. Subsequently, the polymer solution was purified using a HiTrap Desalting column (Sephadex G-25) with PBS (pH 8.5) as the eluent. Next, PD-L1 siRNA (sequence: sense: 5'-GGC GUU UAC UGC UGC AUA AUU-3', antisense: 5'-UUAUGC AGC AGUAAACGC CUU-3') was dissolved in DEPC water to prepare a solution with a concentration of 1 μg / μL. The polymer solution was mixed with the PD-L1 siRNA solution at different mass ratios and incubated at room temperature for 2 hours. Subsequently, the solution was aerated with oxygen for 30 minutes and further allowed to stand at room temperature for 2 hours to obtain a disulfide-crosslinked nanoboron drug. 10 B / siPD-L1 NPs.

[0077] 2. Experimental Results

[0078] like Figure 1 As shown, the hydrogen atoms on the structure of the polymer APEG-P(ME-Trt)-PBOB-DDAT have corresponding assignments in the H NMR spectrum, proving the successful synthesis of APEG-P(ME-Trt)-PBOB-DDAT.

[0079] like Figure 2 As shown, the hydrogen on the structure of the polymer cRGD-PEG-P(ME-Trt)-PBOB-DDAT has corresponding assignments in the nuclear magnetic resonance hydrogen spectrum, proving the successful synthesis of cRGD-PEG-P(ME-Trt)-PBOB-DDAT.

[0080] like Figure 3 As shown, the hydrogens on the structure of the polymer cRGD-PEG-PME-PBOB have corresponding assignments in the H NMR spectrum, proving the successful synthesis of cRGD-PEG-PME-PBOB. The peak area integration of the H NMR spectrum showed that the number of repeating units was approximately 45, 10, and 60. Based on this number of units, the number average molecular weight of the polymer cRGD-PEG-PME-PBOB is approximately 16 kDa.

[0081] like Figure 4 As shown in the figure, when the mass ratio of polymer to PD-L1 siRNA is greater than 20:1, PD-L1 siRNA is almost completely loaded, so the subsequent experimental conditions use a mass ratio of polymer to PD-L1 siRNA of 20:1 to prepare nanoboron drugs. Figure 5As shown, when the mass ratio of polymer to PD-L1 siRNA is 20:1, the hydrated particle size of the prepared nanoboron drug is 97nm, the surface potential is -17mV, and the morphology is a spherical structure.

[0082] Example 2 Nanoboron Drug 10 Cellular uptake and retention of B / siPD-L1 NPs

[0083] 1. Experimental Methods

[0084] (1) Nanoboron drug 10 B / siPD-L1 NPs and fructose BPA were added to the culture medium and incubated with 4T1 tumor cells. 10 B / siPD-L1 NPs and fructose BPA in culture medium 10 The concentration of B was 35 μg / mL. After incubation for different time periods, the cells were washed three times with PBS and then digested with trypsin. The digested cells were counted and digested with 500 μL of nitric acid at 90 °C. The digested solution was diluted to 20 mL with deionized water and filtered with a 0.22 μm filter membrane. The intracellular 10 B atomic concentration.

[0085] (2) Nanoboron drug 10 After incubating 4T1 tumor cells with B / siPD-L1 NPs and fructose BPA for four hours, fresh medium without boron was replaced and cultured. ICP-MS was used to quantitatively detect the intracellular 10 B concentration, the specific steps are the same as above.

[0086] 2. Experimental Results

[0087] The results are as follows Figure 6 As shown, compared with fructose BPA, nano boron medicine 10 In 4T1 tumor cells after incubation with B / siPD-L1 NPs, 10 The concentration of B atoms was higher. After four hours of incubation, the nanoboron drug 10 B / siPD-L1 NPs and fructose BPA-treated 4T1 cells 10 The concentrations of B atoms were 0.93 and 0.22 μg / 10 6 cells, indicating that nanoboron drugs 10 The tumor cell uptake efficiency of B / siPD-L1 NPs was 4.22 times that of fructose BPA.

[0088] The results are as follows Figure 7 As shown, compared with fructose BPA, nano boron medicine 10B / siPD-L1 NPs had a higher retention rate in 4T1 tumor cells. 10 The intratumor cell retention rate of B / siPD-L1 NPs is still 49.9%, while the intratumor cell retention rate of fructose BPA is only 3.4%.

[0089] In summary, the nano boron drug of the present invention 10 B / siPD-L1 NPs have better tumor cell uptake and intracellular retention properties than the clinically used small molecule boron drug fructose BPA.

[0090] Example 3 Nano boron medicine 10 In vivo antitumor therapeutic effect of B / siPD-L1 NPs

[0091] First, a BALB / c mouse 4T1 tumor model was established. When the tumor volume grew to 80-100 mm 3 The treatment experiment was started at 14:00. 10 B / siNC NPs (the sequence of NC-siRNA is as shown in SEQ ID No. 3-4: sense: 5'-UUCUCC GAACGU GUC ACG UUU-3', antisense: 5'-ACG UGACAC GUU CGG AGAAUU-3'), 10 B / siPD-L1 NPs were delivered into tumor-bearing mice. 10 The dose of B was 10 mg / kg. After 24 hours, neutron irradiation was applied to the tumor area with a radiation dose of 2 Gy. A total of two treatments were performed with an interval of 7 days.

[0092] The experimental results are as follows Figure 8 As shown, 10 B / siNC NPs plus neutron irradiation group ( 10 B / siNC+N) and 10 B / si PD-L1NPs plus neutron irradiation group ( 10 B / siPD-L1+N) can inhibit tumor growth, but 10 B / siPD-L1+N ratio 10 B / siNC+N more effectively inhibited tumor growth ( Figure 8 A), indicating that BNCT combined with PD-L1 siRNA promoted the anti-tumor therapeutic effect. In addition, the tumor CD8 + The proportion of T cells found 10 B / siPD-L1+N ratio 10 B / siNC+N more effectively promoted tumor CD8+ Infiltration of T cells ( Figure 8 B), has a stronger anti-tumor immune effect.

Claims

1. A 10 A triblock polymer of B, characterized in that The triblock polymer is polyethylene glycol-b-polyacrylamide (2-mercaptoethyl)-b-polyacrylamide (benzoborazole), and its structural formula is shown in the following formula (I): Wherein n= is 42-48, m is 8-12, and p is 55-65.

2. According to claim 1 10 A triblock polymer of B, characterized in that The said 10 The end of the triblock polymer B is also modified with a cRGD peptide, and its structural formula is shown in the following formula (II): Wherein n= is 42-48, m is 8-12, and p is 55-65.

3. according to any one of claims 1 or 2 containing 10 A triblock polymer of B, characterized in that The n= is 45, m is 10, and p is 60.

4. The method according to any one of claims 1 or 2 10 The method for preparing the triblock polymer of B is characterized in that: The following steps are involved: S1. Allyl-polyethylene glycol-2-(dodecyl trithiocarbonate)-2-methylpropionate, S-trityl-2-mercaptoethylacrylamide and a rich 10 B 5-acrylamide benzoborazole reaction, purification, to obtain a polymer allyl-polyethylene glycol-b-polyacrylamide (S-trityl-2-mercaptoethyl)-b-polyacrylamide (benzoborazole)-2-(dodecyl trithiocarbonate)-2-methylpropionate; S2. reacting the polymer allyl-polyethylene glycol-b-polyacrylamide (S-trityl-2-mercaptoethyl)-b-polyacrylamide (benzoborazolyl)-2-(dodecyl trithiocarbonate)-2-methylpropionate with the cRGD polypeptide, purifying the polymer to obtain the polymer cRGD peptide-polyethylene glycol-b-polyacrylamide (S-trityl-2-mercaptoethyl)-b-polyacrylamide (benzoborazolyl)-2-(dodecyl trithiocarbonate)-2-methylpropionate; S3. After removing the trityl protecting group and DDAT group from the polymer cRGD peptide-polyethylene glycol-b-polyacrylamide (S-trityl-2-mercaptoethyl)-b-polyacrylamide (benzoborazolyl)-2-(dodecyltrithiocarbonate)-2-methylpropionate, purify it to obtain the polymer represented by formula (II): cRGD peptide-polyethylene glycol-b-polyacrylamide (2-mercaptoethyl)-b-polyacrylamide (benzoborazolyl); or after removing the trityl protecting group and DDAT group from the polymer allyl-polyethylene glycol-b-polyacrylamide (S-trityl-2-mercaptoethyl)-b-polyacrylamide (benzoborazolyl)-2-(dodecyltrithiocarbonate)-2-methylpropionate in step S1, purify it to obtain the polymer represented by formula (I): polyethylene glycol-b-polyacrylamide (2-mercaptoethyl)-b-polyacrylamide (benzoborazolyl).

5. A nanoboron drug loaded with PD-L1 siRNA, characterized in that: The nano boron medicine comprises the nano boron medicine according to any one of claims 1 to 3 10 B is composed of a triblock polymer and loaded PD-L1 siRNA.

6. The nano-boron medicine according to claim 5, characterized in that: The nucleotide sequence of the PD-L1 siRNA is shown in SEQ ID No. 1-2.

7. The nano-boron medicine according to claim 5, characterized in that: The said 10 The mass ratio of B triblock polymer to PD-L1 siRNA is 15-25:

1.

8. The nanoboron drug according to any one of claims 5 to 7, characterized in that: The hydrated particle size of the nano-boron medicine is 90 to 110 nm, and the surface potential is -10 to -20 mV.

9. The compound according to any one of claims 1 to 2 10 Use of the triblock polymer of B or the nano boron drug according to any one of claims 5 to 7 in the preparation of drugs for treating tumors.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises any one of claims 1 to 2 10 The triblock polymer of B or the nano boron drug according to any one of claims 5 to 7.

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