L-4-dihydroxyboronphenylalanine-n-carboxyanhydride monomers and polyamino acids, and methods of making and using the same

Abstract

The application discloses an L-4-dihydroxyboron phenylalanine-N-carboxylic acid inner anhydride monomer and polyamino acid, a preparation method and application thereof, constructs a poly-polypeptide nanomaterial with biological responsiveness and good biocompatibility, and particularly relates to synthesis of L-4-dihydroxyboron phenylalanine-N-carboxylic acid inner anhydride, and a series of polymers and applications in drug delivery prepared by ring-opening polymerization thereof. N The polymers disclosed by the application have excellent biocompatibility, can be used for preparing (tumor-targeting) polymer micelles, and can be applied to efficient loading and delivery of hydrophilic or hydrophobic chemical drugs, cis-1,2 or 1,3-diol drugs, polypeptide drugs, protein drugs and nucleic acid drugs.

Description

TECHNICAL FIELD

[0001] The present application constructs a poly-peptide nanomaterial with biological responsiveness and good biocompatibility, and particularly relates to an L-4-dihydroxyboron phenylalanine N synthesis of carboxylic endo-anhydride, and a series of polymers prepared by ring-opening polymerization thereof and applications in drug delivery. BACKGROUND

[0002] Poly-peptides are widely used in drug delivery, tissue engineering and other fields due to their unique secondary structure and biocompatibility. However, existing poly-peptide materials usually have insufficient functionality, complex and cumbersome introduction of functional groups, harsh preparation conditions and other problems. In addition, although poly-peptide-based nanodrugs can improve drug solubility and pharmacokinetics, reduce toxicity to normal cells, and expand the therapeutic window, they still have limitations such as poor stability and uncontrollable drug release (i.e. premature release of drugs during circulation and slow release of drugs at tumor sites). Therefore, it has become a research hotspot to construct poly-peptide nanodrug carriers with good biocompatibility and biological responsiveness (reduction responsiveness, pH responsiveness, ROS responsiveness or enzyme responsiveness, etc.) release. SUMMARY

[0003] The present application designs and synthesizes an L-4-dihydroxyboron phenylalanine N carboxylic endo-anhydride monomer, and initiates L-4-dihydroxyboron phenylalanine N carboxylic endo-anhydride monomer or / and other types N carboxylic endo-anhydride monomer ring-opening polymerization to prepare a series of block copolymers, which can self-assemble into polymer micelles for drug delivery.

[0004] To achieve the above purpose, the technical scheme adopted by the present application is: an L-4-dihydroxyboron phenylalanine N carboxylic endo-anhydride with the structure of formula I:

[0005]

[0006] A linear block copolymer with the structure of formula II:

[0007]

[0008] wherein R1 is a terminal functional group from the initiator polyethylene glycol, and is preferably , , , or and the like; R2 is from other types of amino acids, and preferably different types of amino acids are L-tyrosine, Nε -Boc-L-lysine, β -benzyl-L-aspartic acid; m is 70-210, x is 5-30, y is 0-15, n is 5-45.

[0009] In the linear block copolymer of the present application with the structure of formula II, when y is 0, it is linear polyethylene glycol initiating L-4-dihydroxyphenylalanine N In the linear block copolymer of the present application with the structure of formula II, when y is 0, it is linear polyethylene glycol initiating L-4-dihydroxyphenylalanine N In the linear block copolymer of the present application with the structure of formula II, when y is 0, it is linear polyethylene glycol initiating L-4-dihydroxyphenylalanine N In the linear block copolymer of the present application with the structure of formula II, when y is 0, it is linear polyethylene glycol initiating L-4-dihydroxyphenylalanine N ε -Boc-L-lysine, β -benzyl-L-aspartic acid, etc.

[0010] In the present application, when y is 0, the polyethylene glycol-poly(L-4-dihydroxyphenylalanine) copolymer is as formula III:

[0011]

[0012] wherein R3 is from the initiator polyethylene glycol, which is the terminal functional group thereof, and is preferably , , , or etc.; m is 70-210, n is 5-70; preferably m is 90-150, n is 10-30.

[0013] A branched block copolymer with the structure of formula IV:

[0014]

[0015] wherein m is 20-150, n is 2-20, x is 2-8; preferably m is 50-120, n is 4-8, x is 4 or 8; R4 is the branching center of the initiator branched polyethylene glycol, and the present application preferably uses four-arm or eight-arm polyethylene glycol amine as the initiator.

[0016] The present application discloses the preparation method of the above-mentioned L-4-dihydroxyphenylalanine N -carboxylic anhydride, using L-4-dihydroxyphenylalanine, αpinene, triphosgene as reactants, reacting in anhydrous tetrahydrofuran, to prepare the L-4-dihydroxyphenylalanine N carboxylic acid anhydride. The specific reaction process is as follows: under the protection of nitrogen, L-4-dihydroxyphenylalanine and α pinene is added to an anhydrous tetrahydrofuran solution, and then a tetrahydrofuran solution of triphosgene is added dropwise, and the reaction is carried out at 50-60℃ for 3-7 hours. After the reaction is completed, the reactants that do not participate in the reaction are filtered off, and then the reaction liquid is concentrated by rotary evaporation, and the crude product is precipitated in ice petroleum ether. The crude product is redissolved in tetrahydrofuran, and then precipitated again, and the operation is repeated for 2-3 times, to obtain the final white powder solid, which is L-4-dihydroxyphenylalanine N carboxylic acid anhydride (BPA-NCA).

[0017] In the above technical solution, L-4-dihydroxyphenylalanine, α The molar ratio of pinene and triphosgene is 2:4-6:1-2, and preferably 2:5:1.

[0018] The present application uses linear polyethylene glycol as an initiator, L-4-dihydroxyphenylalanine N carboxylic acid anhydride as a monomer, or L-4-dihydroxyphenylalanine N carboxylic acid anhydride and other types N carboxylic acid anhydride as a monomer, and ring-opening polymerization is carried out to obtain a linear block copolymer. Branched polyethylene glycol is used as an initiator, L-4-dihydroxyphenylalanine N carboxylic acid anhydride as a monomer, and ring-opening polymerization is carried out to obtain a branched block copolymer.

[0019] The preparation of the above linear or branched block copolymer is carried out in an organic solvent such as N , N dimethylformamide (DMF), dichloromethane (DCM), chloroform, tetrahydrofuran (THF), and the present application preferably uses N , N dimethylformamide (DMF) as a solvent; the temperature of ring-opening polymerization is 30-90℃, and the present application preferably uses a polymerization temperature of 80℃; and the polymerization reaction time is 1-5 days, and the present application preferably uses a reaction time of 3 days.

[0020] The present application discloses a kind of targeted block copolymer, which is obtained by coupling targeting molecule from linear block copolymer with formula II structure, or from branched block copolymer with formula IV structure. In the block polymer of the present application, PEG end or amino acid end can be chemically coupled specific targeting molecule, including short peptide (ApoE, Angiopep-2, cRGD, cNGQ, etc.), small molecule targeting molecule (glucose, folic acid, etc.), antibody and antibody fragment, etc.

[0021] This invention further discloses the application of the above-mentioned block copolymers, especially the amphiphilic polymer polyethylene glycol-poly(L-4-dihydroxyboronylphenylalanine), in the self-assembly of nanomicelles for drug delivery; the drugs are hydrophilic / hydrophobic chemical drugs, drugs containing cis-1,2 or 1,3-diols, peptide drugs, protein drugs, or nucleic acid drugs, preferably curcumin (Cur) and / or sorafenib tosylate (Sor). Specifically, the above-mentioned block copolymers, especially the amphiphilic polymer polyethylene glycol-poly(L-4-dihydroxyboronylphenylalanine), are used in the preparation of drug carriers. Because the polymers of this invention have a suitable hydrophilic / hydrophobic ratio, a series of polymer micelles with controllable sizes can be prepared by solvent displacement method. Specifically, the polymer micelles are prepared by solvent displacement method, that is, the block copolymer is dissolved in dimethyl sulfoxide (DMSO) or... N , N A polymer solution is obtained by adding dimethylformamide (DMF), and then the polymer solution is added dropwise to a buffer solution such as HEPES or PB. Finally, the organic solvent is removed by dialyzing using a dialysis bag with a molecular weight cutoff (MWCO) of 7000, resulting in polymer micelles with a particle size of 18–200 nm. This invention can prepare specifically targeted polymer micelles by adjusting the ratio of targeted block copolymers to non-targeted block copolymers. Furthermore, targeting molecules can be introduced onto the surface of the prepared non-targeted polymer micelles through post-modification, such as introducing short peptides (ApoE, Angiopep-2, cRGD, cNGQ, etc.), small molecule targeting molecules (glucose, folic acid, etc.), antibodies, and antibody fragments at the PEG end of the micelles.

[0022] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0023] The present invention prepared α -amino acid- N -Carboxylated anhydride is a novel anhydride monomer that can be used to obtain polypeptides with controllable performance through ring-opening polymerization. Compared with other polyamino acids, this type of polyamino acid has better bioresponsiveness and wider applicability.

[0024] This invention utilizes polyethylene glycol amine as an initiator to obtain polymers with controllable molecular weight and narrow molecular weight distribution through ring-opening polymerization, which greatly expands the types of amphiphilic polypeptides.

[0025] The polymer disclosed in this invention has excellent biocompatibility and can be used to prepare (tumor-targeted) polymer micelles, which are suitable for the efficient loading and delivery of hydrophilic and hydrophobic drugs, drugs containing cis-1,2 or 1,3-diol, peptide drugs, protein drugs, and nucleic acid drugs.

[0026] L-4-dihydroxyboronphenylalanine rich in boron-10 is an important boron carrying agent for boron neutron capture therapy, the synthetic process disclosed in the present application is also applicable to the synthesis of poly-peptide rich in boron-10, and the obtained poly-peptide is also applicable to the preparation process of the micelles, which will widen the application range of L-4-dihydroxyboronphenylalanine, and is expected to be used as a new boron carrying agent for boron neutron capture therapy.

[0027] The preparation method is simple, and the raw materials are widely sourced, thereby having good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 is a reaction schematic diagram of example two, example three and example four.

[0029] Figure 2 is the nuclear magnetic hydrogen spectrum (A), the nuclear magnetic carbon spectrum (B) of BPA-NCA in example one, and the nuclear magnetic hydrogen spectrum (C) of PEG 5k -PBPA 4k , the infrared monitoring chart (C) and the nuclear magnetic hydrogen spectrum (D) of PEG 5k -PBPA 4k in example two.

[0030] Figure 3 is the nuclear magnetic hydrogen spectrum (A) of PEG 5k -P(Tyr 1k -BPA 4k ) in example three, the nuclear magnetic hydrogen spectrum (B) of PEG 5k -P(BLA 1k -BPA 4k ), the nuclear magnetic hydrogen spectrum (C) of PEG 5k -P(Lys 1k -BPA 4k ) and the nuclear magnetic hydrogen spectrum (D) of 4-arm-PEG 20k -PBPA 3k .

[0031] Figure 4 is the nuclear magnetic hydrogen spectrum (A) of ApoE-PEG 5k -PBPA 4k in example four, the nuclear magnetic hydrogen spectrum (B) of 4-arm-PEG 20k -PBPA 1.5k -Glucose.

[0032] Figure 5 is the particle size distribution (embedded TEM diagram) of micelles formed by self-assembly of polymer PEG-PBPA in example five (A), dilution, serum and long-term storage stability (B) and in-vitro responsiveness (C) and in-vitro release (D).

[0033] Figure 6 Figure is the cytotoxicity results graph of the hollow polymer micelles in Example 6 on L929 mouse fibroblasts, U87 MG-luc human glioma cells and B16F10 mouse melanoma cells and cell toxicity graph (A), free Cur and Sor and single drug-loaded micelles PBN-Cur and PBN-Sor on U87 MG-luc cells (B), polymer micelles loaded with curcumin and sorafenib tosylate on U87 MG-luc mouse melanoma cells (C) and cell long-term inhibition effect graph (D).

[0034] Figure 7 Figure is the hemolysis experiment (A) and hemolysis rate (B) of red blood cells treated with Cur, Sor, PBN-Cur, PBN-Sor, PBN-Cur / Sor (low) and PBN-Cur / Sor (high).

[0035] Figure 8 Figure is the apoptosis of U87 MG-luc cells treated with Cur, Sor, PBN-Cur, PBN-Sor and PBN-Cur / Sor (Cur: 1.5 μg / mL, Sor: 1.5 μg / mL) (A) and cell cycle arrest of U87 MG-luc cells treated with Cur and PBN-Cur (Cur: 1.5 μg / mL) (B).

[0036] Figure 9 Figure is the boron-10 enriched 10 BPA-NCA (A) and mPEG-P 10 BPA nuclear magnetic spectrum (B). DETAILED DESCRIPTION

[0037] The present application discloses L-4-dihydroxyboron phenylalanine N -carboxyl end anhydride, which is complexed with cis 1,2 or 1,3-dihydroxy compounds and monosaccharides, polysaccharides, nucleic acids, etc. to form reversible five or six-membered ring lactone or boron-nitrogen coordination binding, thereby constructing nanomedicine or hydrogel carrier, which can realize stable and efficient loading of various drugs, and the formed borate ester bond and boron-nitrogen coordination binding is a dynamic action, which can dissociate and release the loaded drugs under certain conditions (such as ROS, low pH), and can also act with sialic acid highly expressed on the surface of tumor cells. Therefore, the present application is used in many fields such as biosensing and drug delivery.

[0038] The preparation scheme of the above L-4-dihydroxyboron phenylalanine N -carboxyl end anhydride (BPA-NCA) can be represented as follows:

[0039]

[0040] This invention also discloses the initiation of the above-mentioned L-4-dihydroxyboronphenylalanine using linear or branched polyethylene glycol as an initiator. N A block copolymer (polyethylene glycol-poly(L-4-dihydroxyboronphenylalanine) copolymer) prepared by ring-opening polymerization of a carboxylic acid anhydride monomer, wherein the molecular weight of polyethylene glycol is 2000-40000 and the molecular weight of poly(L-4-dihydroxyboronphenylalanine) is 1000-50000.

[0041] The linear block copolymer is initiated by linear polyethylene glycol to produce the above-mentioned L-4-dihydroxyboronphenylalanine- N The chemical structure of the -carboxylic acid anhydride monomer prepared by ring-opening polymerization is shown below:

[0042]

[0043] Wherein, m is 70–210, and n is 5–70; preferably, m is 90–150, and n is 10–30; R3 is the terminal functional group of the polyethylene glycol initiator, and the present invention preferably uses a polyethylene glycol amino initiator with the following molecular formula:

[0044]

[0045] Among them, R3 is preferably , , , or wait.

[0046] Branched polymers are produced by initiating the above-mentioned L-4-dihydroxyboronphenylalanine- through branched polyethylene glycol. N The chemical structure of the -carboxylic acid anhydride monomer, prepared by ring-opening polymerization, is shown below:

[0047]

[0048] Wherein, m is 20-150, n is 2-20, and x is 2-8; preferably, m is 50-120, n is 4-8, and x is 4 or 8; R2 is the branching center of the branched polyethylene glycol initiator, and the present invention preferably uses existing four-armed or eight-armed polyethylene glycol amine as the initiator.

[0049] Four-arm polyethylene glycol amine:

[0050]

[0051] Eight-arm polyethylene glycol amine:

[0052]

[0053] This invention also discloses the use of linear polyethylene glycol as an initiator to initiate the above-mentioned L-4-dihydroxyboronphenylalanine- N -Carboxylic anhydride monomers and other types N Block copolymers prepared by random copolymerization of -carboxylic acid anhydride monomers, wherein the molecular weight of polyethylene glycol is 2000-20000, and the molecular weight of poly(amino acid-) co The molecular weight of 1,000–50,000 for β-L-4-dihydroxyboronylphenylalanine (amino acid) refers to other types of amino acids. N The product obtained by polymerization of L-4-dihydroxyboronylalanine monomers. The polymerization is initiated by linear polyethylene glycol as an initiator. N -Carboxylic anhydride monomers and other types N Block copolymers were prepared by random copolymerization of carboxyl anhydride monomers, and the chemical structural formula is shown below:

[0054]

[0055] Wherein, m is 70–210, x is 5–30, y is 0–15, and n is 5–45; R1 is the terminal functional group of the polyethylene glycol initiator, preferably... α -Methoxy- ω -Amino-polyethylene glycol and α -maleimide- ω -Amino-polyethylene glycol is used as the initiator; R2 is a different type of amino acid, preferably L-tyrosine in this invention. N ε -Boc-L-lysine, β -Benzyl-L-aspartic acid.

[0056] The preparation process of the above polymers uses organic solvents such as... N , N The process is carried out in dimethylformamide (DMF), dichloromethane (DCM), chloroform, and tetrahydrofuran (THF), and is preferably carried out in this invention. N , N - Dimethylformamide (DMF) is used as a solvent; the ring-opening polymerization temperature is 30-90°C, and the preferred polymerization temperature is 80°C; the polymerization reaction time is 1-5 days, and the preferred reaction time is 3 days.

[0057] The reaction of this invention is described in [reference needed]. Figure 1 The raw materials used are all commercially available products, and the specific preparation and testing methods are all conventional techniques. The invention will be further described below with reference to the accompanying drawings and embodiments:

[0058] Example 1: L-4-Dihydroxyboronylalanine N Synthesis of β-carboxylic acid anhydride (BPA-NCA)

[0059] L-4-dihydroxyphenylalanine (500 mg, 2.39 mmol) and α - Pinene (948 μL, 5.97 mmol) was added to a dry three-necked round bottom flask, followed by the addition of anhydrous tetrahydrofuran (THF, 150 mL), and then a THF solution of triphosgene was added dropwise to the suspension. The reaction was stirred in an oil bath at 55 °C for 5 h, and then allowed to cool to room temperature. The reaction was filtered through a fritted funnel, and the clear filtrate was concentrated to 10 mL by rotary evaporation. The crude product was precipitated in pre-cooled petroleum ether to obtain a white powder solid, which was then re-dissolved in THF and precipitated again. This process was repeated three times to obtain L-4-dihydroxyphenylalanine N - BPA-NCA (yield: 60%). The NMR characterization of BPA-NCA is shown in the attached Figure 2 , 1 H NMR (400 MHz, DMSO- d 6 , δ ): 9.09 (s, 1H, -CON H -), 8.03 (s, 2H, -B(O H )2), 7.71 and 7.14 (d, J = 7.6 Hz, 4H, -C6 H 4 -), 4.79(t, J = 5.2 Hz, 1H, -COC H NH-), 3.03 (d, J = 5.2 Hz, 2H, -C6H4C H 2 -); 13 C NMR (100MHz, DMSO- d 6, δ ): 171.27, 152.09, 137.05, 134.67, 133.31, 129.19, 58.62,36.81. BPA-NCA's elemental analysis: C, 53.19; H, 4.85; N, 5.53 (theoretical: C, 51.11; H, 4.29; N, 5.96); Mass: MS (m / z): 235.1 (theoretical: 235.1).

[0060] Example 2 Preparation of block copolymer PEG-PBPA

[0061] The present application uses polyethylene glycol with amino group at the end as initiator to initiate L-4-dihydroxyboron phenylalanine-NCA ring-opening polymerization by adjusting the molar ratio of polyethylene glycol and L-4-dihydroxyboron phenylalanine-NCA-carboxyl internal anhydride to prepare polymers PEG-PBPA with different chain lengths (Table 1). N N The molar ratio of polyethylene glycol and L-4-dihydroxyboron phenylalanine-NCA-carboxyl internal anhydride can be adjusted to prepare polymers PEG-PBPA with different chain lengths (Table 1). M n = 5.0-4.0 kg / mol)as an example: under nitrogen atmosphere, PEG-NH2(0.25 g, 0.05 mmol)in DMF was added to a sealed reactor, and BPA-NCA(0.25 g, 1.06 mmol)in DMF was added to the sealed reactor under stirring, and the reaction was carried out in a constant temperature oil bath at 80℃ for 3 days (the corresponding carbonyl stretching peak of BPA-NCA monomer in infrared spectrum 1770 cm -1 and 1845 cm -1 completely disappeared, indicating that the polymerization was complete). After the reaction was completed, the polymer solution was precipitated with ice anhydrous ether, centrifuged, then redissolved with methanol, and repeated precipitation 3 times, and the solid precipitate was collected and vacuum dried for 48 hours to obtain white solid product, yield: 81%. The NMR characterization of PEG-PBPA block copolymer is shown in the attached Figure 2 . 1 H NMR (DMSO- d 6 / CD3OD (v / v = 2 / 1), 400 MHz, δ ): 7.68 and7.22 (- C 6 H 4 B(OH)2), 4.49 (-CO CH NH-), 3.51 (-O CH 2 CH 2 O-), 2.96-2.78 (-C6H4 CH 2 -)。

[0062] ​Similarly, BPA-based branched polymers were synthesized using 4 / 8-arm PEG-NH2 as an initiator. For example, four-arm PEG-NH2 (1.32 g, 0.066 mmol) dissolved in anhydrous DMF was added to a BPA-NCA (0.250 g, 1.06 mmol) solution under a nitrogen atmosphere, and the reaction was carried out at 80 °C for 24 hours. After the reaction, the polymer solution was precipitated with ice-cold anhydrous diethyl ether, centrifuged, and then reconstituted with methanol. The precipitation was repeated three times, and the solid precipitate was collected and vacuum dried for 48 hours to obtain 4-arm-PEG-PBPA ( M n = 20.0-3.0 kg / mol). 1 H NMR (400 MHz, DMSO- d 6 / CD3OD(v / v = 2 / 1), δ ):7.68 and 7.22 (-C6 H 4 B(OH)2), 4.50 (-COC H NH-), 3.51 (-OC H 2C H 2O-), 2.98-2.74 (-C6H4 CH 2 -); Similarly, using eight-arm PEG-NH2, 8-arm-PEG-PBPA was obtained ( M n = 20.0-12.0 kg / mol), 1 HNMR (400 MHz, DMSO- d 6 / CD3OD (v / v = 2 / 1), δ ):7.68 and 7.22 (-C6 H 4 B(OH)2), 4.50 (-COC H NH-), 3.51 (-OC H 2C H 2O-), 2.98-2.74 (-C6H4 CH 2 -).

[0063]

[0064]

[0065] Example 3 Block copolymer PEG-P(Tyr-co -BPA), PEG-P(Lys- co -BPA) and PEG-P(BLA- co Synthesis of PEG-P(BPA) and other copolymers

[0066] The present application uses linear polyethylene glycol as an initiator to initiate the above L-4-dihydroxyboron phenylalanine- N -Carboxyl internal anhydride monomers and other types N -Carboxyl internal anhydride monomers (such as Tyr-NCA, Lys(Boc)-NCA and BLA-NCA, etc.) are randomly copolymerized to obtain phenylboronic acid-based poly-peptide materials with different molecular structures, different hydrophilic and hydrophobic properties, different charges and different functional groups (Table 3). For example, PEG-P(Tyr- co -BPA) (PEG-P(Tyr- M n = 5.0-1.0-4.0 kg / mol): methoxyl polyethylene glycol amine was used as an initiator, and a DMF solution of PEG-NH2 (0.25 g, 1.06 mmol) was added to a DMF solution of Tyr-NCA (0.121 g, 0.396 mmol) and BPA-NCA (0.25 g, 1.06 mmol) under nitrogen environment, and then reacted at 80°C for 3 days. After the reaction was completed, the polymer solution was precipitated with ice anhydrous ether, centrifuged, then redissolved with methanol, and repeated precipitation for 3 times, and the solid precipitate was collected and vacuum dried for 48 hours to obtain a gray-white product, yield: 78%. PEG-P(Tyr- co The NMR characterization of PEG-P(Tyr- Figure 3 . 1 H NMR (DMSO- d 6 / CD3OD (v / v = 2 / 1), 400 MHz, δ ): 7.68 and 7.22 (-C6 H 4 B(OH)2), 6.95 and 6.61 (- C 6 H 4 OH), 4.47 and 4.37 (-CO CH NH-), 3.52 (-O CH 2 CH 2 O-), 2.82-2.63 (-C6H4 CH 2 -)。

[0067] PEG-P(Lys(Boc)-co -BPA), PEG-P(BLA- co -BPA) and 4-arm-PEG-PBPA were synthesized following the same procedure as described above. PEG-P(Lys(Boc)- co -BPA) after deprotection in trifluoroacetic acid to give PEG-P(Lys- co -BPA).

[0068]

[0069] Example Four Synthesis of ApoE-modified PEG-PBPA polymer (ApoE-PEG-PBPA)

[0070] Referring to the above method, the present application uses maleimide-modified polyethylene glycol as an initiator to initiate the above L-4-dihydroxyphenylalanine- N -carboxyl endo anhydride monomer and / or other types N -carboxyl endo anhydride monomer to obtain (Mal-PEG-PBA or Mal-PEG-P(BPA- co -Tyr) and other block polymers, and then the PEG end thereof can be chemically coupled to specific targeting molecules such as ApoE, Angiopep-2, cRGD, etc. Take the synthesis of ApoE-PEG-PBPA as an example: under a nitrogen atmosphere, dissolve Mal-PEG-PBPA (100 mg, 0.011 mmol) and ApoE peptide (30.7 mg, 0.013 mmol) in deoxygenated DMSO / CH3OH (v / v = 9 / 1, 0.1 mL) and react at 37°C for 24 hours to obtain a polypeptide solution. After the reaction is completed, dialyze the polypeptide solution in DMSO / CH3OH for 24 hours, then dialyze in double-distilled water to replace the organic solvent, and finally lyophilize to obtain a white solid product ApoE-PEG-PBPA, yield: 78%. The NMR characterization of ApoE-PEG-PBPA block copolymer is shown in the attached Figure 4 A. 1 H NMR (400MHz, DMSO- d 6 / CD3OD (v / v = 2 / 1), δ ): 7.68 and 7.22 (-C6 H 4 B(OH)2), 4.49 (-CO CH NH-),3.51 (-O CH 2 CH 2 O-), 2.98-2.74 (-CH 2 NH-), 0.78-2.41 (ApoE). The grafting efficiency of ApoE was 92% as determined by the 9,10-phenanthrenequinone method. Similarly, glucose molecules can be modified at the polymer ends, and attached... Figure 4 B is the NMR spectrum of the glucose-modified polymer, yield: 82%. NMR analysis indicates that approximately 3.6 glucose molecules were modified onto the polymer 4-arm-PEG-PBPA.

[0071] Example 5: Loading curcumin and sorafenib tosylate into PEG-PBPA polymer micelles (PBN) and their in vitro release.

[0072] Polymer PEG-PBPA ( M n = 5.0-4.0 kg / mol), curcumin (Cur), and sorafenib tosylate (Sor) were dissolved in DMSO solution, and mixed in certain amounts before being added dropwise to HEPES, PB buffer solution or ultrapure water under stirring. Then, the organic solvent and free drug were removed by dialysis using a dialysis bag with a molecular weight cutoff of 7000. The dialysis process was carried out in HEPES, PB buffer solution or pure water at pH = 7.4 (Table 4). Taking the co-encapsulation of Cur and Sor (theoretical drug loading of 10 wt% for both) as an example: 10 μL of dissolved PEG-PBPA polymer solution (100 mg / mL), 5.55 μL of Cur solution and 5.55 μL of Sor solution were thoroughly mixed and added dropwise to 979 μL of HEPES buffer solution. Dialysis was then performed in HEPES buffer solution to remove organic solvent and unencapsulated drug. Finally, the size of the polymer micelles was determined to be 95 nm using dynamic light scattering particle size analyzer (DLS), with a narrow particle size distribution (<0.2). The actual drug loadings of Cur and Sor were determined to be 7.8 wt% and 9.8 wt%, respectively, by HPLC. Furthermore, the polymer micelles exhibited good stability under high-dilution (50-fold), 10% fetal bovine serum (FBS), and long-term storage at 4°C.

[0073]

[0074] In vitro release experiments of Cur and Sor were conducted in a constant-temperature shaker at 37°C and 200 rpm. Specifically, 1.0 mL of PBN-Cur / Sor (Cur and Sor drug loading of 10 wt%) micelle solution was transferred to a release bag (MWCO = 12-14 KD) and immersed in 25.0 mL of release medium under different conditions (pH 7.4, 100 μM H2O2). At predetermined time points, 5.0 mL of release medium was aspirated and an equal volume of fresh release medium was added. After all samples were freeze-dried, they were reconstituted with 0.3 mL of acetonitrile, and the contents of Cur and Sor were determined by HPLC. (See attached image) Figure 5 D represents the cumulative release of Cur and Sor over time. The figure shows that drug-loaded micelles can release drugs through diffusion.

[0075] Example 6: MTT assay for cytotoxicity of empty polymer micelles and drug-loaded micelles

[0076] The cytotoxicity of empty polymeric micelles (PBN) to tumor cells (B16F10 cells, U87 MG-luc cells) and normal mouse fibroblasts (L929 cells) was evaluated using the MTT assay. Specifically, cells were seeded into 96-well plates (80 μL, 5 × 10⁻⁶ cells per well). 3 Cells were incubated in wells at 37°C for 24 hours. Then, 20 μL of PBN micelle solution at different concentration gradients was added to each well, resulting in final concentrations of 0.05, 0.1, 0.2, 0.5, 0.75, and 1.0 mg / mL. After a total incubation of 48 hours, 10 μL of 5.0 mg / mL MTT solution was added to each well for an additional 4 hours of incubation. The culture medium was then removed, and 150 μL of DMSO was added to dissolve the purple formazan crystals produced by the interaction of MTT with live cells. Finally, the absorbance at 570 nm was measured in each well using a multi-mode microplate reader. Cell viability was determined by the ratio of the absorbance of each sample group to that of the blank control group (which contained an equal volume of PBS). Each group had five parallel wells. Figure 6 A is the polymer PEG-PBPA ( M n The cytotoxicity results of the self-assembled micelles (5.0-4.0 kg / mol) against B16F10, U87 MG-luc, and L929 cells showed that when the concentration of the polymer micelles increased from 0.1 to 1.0 mg / mL, the survival rate of cells in all groups was still higher than 80%, indicating that the polymer micelles have good biocompatibility.

[0077] Toxicity of the drug-loaded micelles (PBN-Cur, PBN-Sor and PBN-Cur / Sor) of Example 5 on U87 MG-luc cells was studied, and the cell culture operation was consistent with the above. After different concentrations of PBN-Cur, PBN-Sor (Cur or Sor final concentration of 0.01, 0.1, 0.5, 1, 2, 4, 6, 8 and 10 μg / mL) and different drug ratios of PBN-Cur / Sor micelle solution were added to the cells and incubated for 48 hours, MTT was added, and the treatment and determination of absorbance were consistent with the above. The experimental results are shown in the following table. Figure 6 B&C, the results show that Cur and Sor both exhibit a certain killing effect, and the killing effect of PBN-Cur and PBN-Sor is improved compared with free drugs. For the combined action of the two drugs on U87 MG-luc, when the mass ratio of the two drugs is 1 / 1, the synergistic effect (CI = C CA / C A +C CB / C B ; C CA : IC 50 concentration of A, C CB : IC 50 concentration of B, C A and C B are the IC 50 concentrations of A and B, respectively) of the combined drugs A and B is the most significant, and the half lethal concentration (IC 50 ) is 1.47 μg / mL. It shows that the micelles of the application have good delivery ability of Cur and Sor, and can achieve effective release to ultimately kill tumor cells.

[0078] In addition, cell colony formation experiments were used to verify the cytotoxicity of PBN-Cur, PBN-Sor and PBN-Cur / Sor. The specific steps are as follows: U87 MG-luc cells (2×10 5 cells / well) were inoculated in a 6-well plate, and after 24 hours of culture, Cur, Sor, PBN-Cur, PBN-Sor and PBN-Cur / Sor (Cur: 1.5 μg / mL, Sor: 1.5 μg / mL, Cur / Sor: 1.5 / 1.5 μg / mL) were added respectively for 48 hours of co-incubation. Then the cells were trypsinized, and the cells in each sample group (300 cells / well) were re-inoculated in a 6-well plate and placed in a cell culture incubator for 9 days, during which the culture medium was replaced every three days. Finally, the culture medium was aspirated, washed twice with PBS, and the cells were fixed with 4% paraformaldehyde solution for 15 minutes, then stained with crystal violet staining solution, and finally the crystal violet was slowly washed off and air dried. From Figure 6It can be seen that PBN-Cur (Cur: 1.5 μg / mL) and PBN-Sor (Sor: 1.5 μg / mL) both have a certain long-term inhibitory effect on tumor cell proliferation, and the inhibitory effect of the PBN-Sor group is slightly better than that of the PBN-Cur group, and the PBN-Cur / Sor group has the most excellent inhibitory effect, which is consistent with the MTT experiment results.

[0079] Example Seven Hemolysis Results of Drug-loaded Micelles

[0080] In this experiment, deionized water was used as a positive control and 0.9% NaCl as a negative control to study the hemolysis of Cur, Sor, PBN-Cur, PBN-Sor, PBN-Cur / Sor (low) and PBN-Cur / Sor (high). Figure 7 A). The drug concentration was set to 50 μg / mL, except for the PBN-Cur / Sor (high; Cur: 110 μg / mL, Sor: 110 μg / mL) group. Figure 7 The results of B show that free Sor has obvious hemotoxicity, inducing about 45% hemolysis at a concentration of 50 μg / mL, while the nano-drug PBN-Sor only shows slight hemolysis (HR of 4.3%) at the same concentration. In addition, PBN-Cur / Sor at low concentration (Cur: 50 μg / mL; Sor: 50 μg / mL) and high concentration also showed superior blood compatibility, with an HR of about 2.3%, which can provide an effective strategy for simultaneous intravenous injection of Cur and Sor.

[0081] Example Eight Apoptosis and Cell Cloning Experiments of Drug-loaded Micelles

[0082] The present application uses Annexin V-FITC / Propidium iodine (PI) double staining technology to study the apoptosis effect of Cur, Sor, PBN-Cur, PBN-Sor and PBN-Cur / Sor on U87 MG-luc cells using a flow cytometer. Specifically, log phase U87 MG-luc cells (2×10 5U87 MG-luc cells (2 x 10 6 The percentage of cells in each stage of apoptosis was detected by flow cytometry. The double-stained samples of the control group were treated in the same way as the sample group. In addition, early apoptosis samples were prepared by placing the cells in a 50°C water bath for 5 minutes, and late apoptosis samples were prepared by adding 4% paraformaldehyde for 5 minutes. All data were analyzed using Flowjo software. Figure 8 As can be seen from Figure B, most of the cells in the PBS group were in the G1 phase (67.28%), the S phase cells accounted for only 23.98%, and the G2 / M phase content was only 8.74%. The cycle arrest of U87 MG-luc cells by Cur and PBN-Cur was better than that of the PBS group, and PBN-Cur had a slight improvement compared with Cur.

[0083] In addition, it is reported in the literature that Cur has the ability to inhibit the cell cycle, so the cell cycle arrest ability of PBN-Cur on U87 MG-luc cells was also tested accordingly. Specifically, U87 MG-luc cells (2 x 10 5 The cells were collected by trypsinization and washed with 4°C pre-cooled PBS three times, and finally dispersed in 1.0 mL of PBS. The cell suspension was added dropwise to 4.0 mL of 95% ethanol oscillating at a constant speed, and fixed at 4°C for 24 hours. Then the cells were stained with a cell cycle kit for 30 minutes, and the cell cycle was detected by flow cytometry. From Figure 8 As can be seen from Figure B, most of the cells in the PBS group were in the G1 phase (67.28%), the S phase cells accounted for only 23.98%, and the G2 / M phase content was only 8.74%. The cycle arrest of U87 MG-luc cells by Cur and PBN-Cur was better than that of the PBS group, and PBN-Cur had a slight improvement compared with Cur.

[0084] Example Eight Boron-10 Enriched 10 Synthesis of BPA-NCA and mPEG-P 10 Synthesis of BPA

[0085] 10 Synthesis of BPA-NCA and mPEG-P 10 The synthesis of BPA followed Example One and Example Two, respectively. The only difference was that during the monomer synthesis, boron-10 enriched BPA was used, Figure 9 A&B are its characterization. 10 Synthesis of BPA-NCA 1 H NMR (400 MHz, DMSO- d 6, δ ): 9.09 (s, 1H), 8.03 (s, 2H), 7.71 (d, J = 7.6 Hz, 2H),7.14 (d, J = 7.6 Hz, 2H), 4.79 (t, J = 5.2 Hz, 1H), 3.03 (d, J = 5.2 Hz, 2H). mPEG-P 10 Synthesis of BPA 1 H NMR (DMSO- d 6 / CD3OD (v / v = 2 / 1), 400 MHz, δ ): 7.68 ( C 6 H 2 B(OH)2), 7.22(- C 6 H 2 CH2-), 4.49 (-CO CH NH-), 3.51 (-O CH 2 CH 2 O-), 3.26-2.85 (-C6H4 CH 2 -).

Claims

1. A linear block copolymer having the structure of formula II: ###0001### ； wherein R1is from an initiator polyethylene glycol selected from , , , or ; R2is from another type of amino acid, another type of amino acid means an amino acid other than poly(L-4-dihydroxyphenylalanine), selected from L-tyrosine, N ε -Boc-L-lysine, β -benzyl-L-aspartic acid; m is 70 to 210, x is 5 to 30, y is 0 to 15, n is 5 to 45; The linear block copolymer is prepared by using linear polyethylene glycol as an initiator, L-4-dihydroxyboron phenylalanine as a monomer or L-4-dihydroxyboron phenylalanine and other types of monomers N - carboxylic acid anhydride as a monomer, ring-opening polymerization to obtain a linear block copolymer; y is 0, is linear polyethylene glycol-initiated L-4-dihydroxyphenylalanine N - carboxylic internal anhydride ring opening polymerization to form polyethylene glycol-poly(L-4-dihydroxyphenylalanine) copolymer; y when n is not 0, linear polyethylene glycol initiates L-4-dihydroxyphenylalanine N - carboxylic internal anhydrides and other types N - ring opening polymerization of carboxylic internal anhydrides to form polyethylene glycol-poly(L-4-dihydroxyphenylalanine)-poly other type amino acid copolymers; Other types of amino acids are amino acids other than poly(L-4- dihydroxyphenylalanine) and are selected from the group consisting of L-tyrosine, N ε -Boc-L-lysine, β -benzyl-L-aspartic acid; Initiator linear polyethylene glycol with end functional groups of , , , or .

2. A branched block copolymer having the structure of formula IV: ###0002### ； wherein m is 20-150, n is 2-20, x is 2-8; R4 is the branching center of the initiator branched polyethylene glycol; The branched block copolymer is prepared by ring-opening polymerization using branched polyethylene glycol as an initiator, L-4-dihydroxyboron phenylalanine as a monomer, to obtain a branched block copolymer; the initiator is tetra-armed or octa-armed polyethylene glycol amine; The structure is: ###0003### Tetra-armed polyethylene glycol amine: ###0004### ； Octa-armed polyethylene glycol amine: ###0005### 。 3. A targeted block copolymer characterized in that, obtained by coupling a targeting molecule to the linear block copolymer of claim 1, or obtained by coupling a targeting molecule to the branched block copolymer of claim 2.

4. A drug delivery system, characterized by obtained by loading a drug into the linear block copolymer of claim 1 or the branched block copolymer of claim 2; or obtained by coupling a targeting molecule after loading a drug into the linear block copolymer of claim 1 or the branched block copolymer of claim 2; or obtained by co-loading a drug into the linear block copolymer of claim 1 and the targeting block copolymer of claim 3; or obtained by co-loading a drug into the branched block copolymer of claim 2 and the targeting block copolymer of claim 3.

5. Use of the linear block copolymer of claim 1, the branched block copolymer of claim 2, the targeting block copolymer of claim 3, or the drug delivery system of claim 4 in the preparation of a medicament.

6. Use of the linear block copolymer of claim 1, the branched block copolymer of claim 2, or the targeting block copolymer of claim 3 in the preparation of a drug carrier.

Images