阅读说明：本技术 双酶响应性哑铃形超两亲分子及其制备方法和用途 (Double-enzyme responsive dumbbell-shaped super-amphiphilic molecule and preparation method and application thereof ) 是由 毕韵梅 危俊吾 林峰 游丹 钱杨杨 王雨佳 于 2019-09-18 设计创作，主要内容包括：双酶响应性哑铃形超两亲分子及其制备方法和用途,属于功能高分子材料领域。产品的分子结构如下：<Image he="654" wi="700" file="DDA0002206071900000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>式<Sup>+</Sup>H<Sub>3</Sub>N-G<Sub>3</Sub>-b-PNVP-b-G<Sub>3</Sub>-NH<Sub>3</Sub><Sup>+</Sup>中n＝40～80。经多个步骤制得的产品用于在水溶液中自组装形成球形胶束,用胶束包载小分子或药物,特别适用于抗癌药物的靶向快速释放,能避免对药物产生污染对和残留物对机体产生危害。(A double-enzyme responsive dumbbell-shaped super-amphiphilic molecule, a preparation method and application thereof, belonging to the field of functional polymer materials. The molecular structure of the product is as follows: formula (II) + H 3 N‑G 3 ‑b‑PNVP‑b‑G 3 ‑NH 3 + Wherein n is 40 to 80. The product prepared through multiple steps is used for self-assembling in an aqueous solution to form spherical micelles, and the micelles are used for encapsulating small molecules or drugs, so that the product is particularly suitable for targeted rapid release of anticancer drugs, and can avoid pollution to the drugs and harm to organisms caused by residues.)

1. The double-enzyme responsive dumbbell-shaped super-amphiphilic molecule is characterized in that: the third generation of dumbbell-shaped (dendritic-linear-dendritic) block copolymer taking phenylalanyl-lysine dipeptide as dendron and poly (N-vinyl pyrrolidone) (PNVP) as linear chain⁺H₃N-G₃-b-PNVP-b-G₃-NH₃ ⁺And adenosine triphosphate are compounded to form the compound,⁺H₃N-G₃-b-PNVP-b-G₃-NH₃ ⁺the molecular structure of (a) is as follows:

formula (II)⁺H₃N-G₃-b-PNVP-b-G₃-NH₃ ⁺Wherein n is 40 to 80.

2. The preparation method of the double-enzyme responsive dumbbell-shaped super-amphiphilic molecule is characterized by comprising the following steps:

(1) the chain transfer agent CTA with alpha, omega-double terminal xanthate group is prepared, and the molecular structure is as follows:

(2) and preparing the dimeric (N-vinyl pyrrolidone) with alpha, omega-double terminal xanthate groups, wherein the molecular structure of the dimeric (N-vinyl pyrrolidone) is as follows:

(3) preparing tert-butyl (2-acrylamidoethyl) carbamate, the molecular structure of which is as follows:

(4) preparing BocNH-PNVP-NHBoc:

adding X-PNVP-X, dichloromethane and n-butylamine into a reaction container, wherein the mass ratio of the X-PNVP-X to the n-butylamine is 1: 80-120, 2-4 g of X-PNVP-X is dissolved in every mL of dichloromethane, tributylphosphine accounting for 0.1-0.5% of the total mass of the X-PNVP-X and the n-butylamine is added according to the mass ratio, vacuumizing and charging nitrogen for 5-20 min, adding tri (2-carboxyethyl) phosphine (TCEP) and tert-butyl (2-acrylamidoethyl) carbamate, wherein the mass ratio of the X-PNVP-X, the tert-butyl (2-acrylamidoethyl) carbamate and the tert-butyl (2-carboxyethyl) phosphine is 1: 1.5-2.5: 2-4, and stirring and reacting for 4-8 h. Preferably, the reaction is carried out in a dry reaction vessel. Coprecipitating the crude product with petroleum ether for 2-5 times, dialyzing in deionized water for 2-4 days, and freeze-drying to obtain the BocNH-PNVP-NHBoc, wherein the molecular structure of the BocNH-PNVP-NHBoc is as follows:

(5) preparation of NH₂-PNVP-NH₂：

Adding BocNH-PNVP-NHBoc, dichloromethane and trifluoroacetic acid into a reaction container, wherein 0.01-0.05 mmol of BocNH-PNVP-NHBoc is dissolved in each mL of dichloromethane, the volume ratio of trifluoroacetic acid to dichloromethane is 1: 1-3, stirring for reacting for 2-6 h, adjusting the pH to be neutral by using 1-2 mol/L of NaOH, filtering, performing rotary evaporation, dialyzing in deionized water for 2-4 days, and performing freeze drying to obtain NH₂-PNVP-NH₂，NH₂-PNVP-NH₂The molecular structure of (a) is as follows:

(6) (FmocNH-Phe) -b-PNVP-b- (Phe-NHFmoc) was prepared,

adding NH into a reaction flask₂-PNVP-NH₂1-hydroxybenzotriazole, N-fluorenylmethoxycarbonyl-L-phenylalanine and N, N-Dimethylformamide (DMF), wherein NH₂-PNVP-NH₂The mass ratio of N-fluorenylmethoxycarbonyl-L-phenylalanine to 1-hydroxybenzotriazole is 1: 1.5-3.5: 4-8, and 0.01-0.03 mmol of NH is dissolved in each mL of DMF₂-PNVP-NH₂Stirring for 20-60 min, and adding N, N-diisopropyl carbodiimide (DIC) and NH₂-PNVP-NH₂And N, N-diisopropylcarbodiimide in a mass ratio of 1: 5-10, stirring for reaction for 20-48 h, adding cold diethyl ether for precipitation, dialyzing in deionized water for 2-4 days, and freeze-drying to obtain (FmocNH-Phe) -b-PNVP-b- (Phe-NHFmoc), (FmocNH-Phe) -b-PNVP-b- (Phe-NHFmoc) with the following molecular structure:

(7) preparation of (H)₂N-Phe)-b-PNVP-b-(Phe-NH₂)：

Adding (FmocNH-Phe) -b-PNVP-b- (Phe-NHFmoc), 4-methylpiperidine and DMF into a reaction bottle, wherein the mass ratio of (FmocNH-Phe) -b-PNVP-b- (Phe-NHFmoc) to 4-methylpiperidine is 1: 6-10, the volume ratio of 4-methylpiperidine to DMF is 1: 3-6, each mL of the mixture of 4-methylpiperidine and DMF contains 0.01-0.04 mmol of (FmocNH-Phe) -b-PNVP-b- (Phe-NHFmoc), stirring for reaction for 20-60 min, adding cold diethyl ether for precipitation, dialyzing in deionized water for 2-4 days, and freeze-drying to obtain (H)₂N-Phe)-b-PNVP-b-(Phe-NH₂)，(H₂N-Phe)-b-PNVP-b-(Phe-NH₂) The molecular structure of (a) is as follows:

(8) preparation of FmocNH-G₁-b-PNVP-b-G₁-NHFmoc：

Adding (H) into a reaction flask₂N-Phe)-b-PNVP-b-(Phe-NH₂) 1-hydroxybenzotriazole (HOBt), N' -bifluoromethoxycarbonyl-L-lysine (Fmoc-Lys (Fmoc) -OH) and N, N-Dimethylformamide (DMF), wherein (H)₂N-Phe)-b-PNVP-b-(Phe-NH₂) The mass ratio of Fmoc-Lys (Fmoc) -OH to HOBt is 1: 5-8: 2-4, and 0.01-0.03 mmol of (H) is dissolved in each mL of DMF₂N-Phe)-b-PNVP-b-(Phe-NH₂) Stirring for 20-60 min, and adding N, N-Diisopropylcarbodiimide (DIC), (H)₂N-Phe)-b-PNVP-b-(Phe-NH₂) The mass ratio of DIC and DIC is 1: 5-10, stirring and reacting for 20-48 h, adding cold ether for precipitation, dialyzing in deionized water for 2-4 days, and freeze drying to obtain G₁-b-PNVP-b-G₁，G₁-b-PNVP-b-G₁The molecular structure of (a) is as follows:

(9) preparation of NH₂-G₁-b-PNVP-b-G₁-NH₂：

Adding (FmocNH-Phe) -b-PNVP-b- (Phe-NHFmoc), 4-methylpiperidine and DMF into a reaction bottle, wherein the mass ratio of (FmocNH-Phe) -b-PNVP-b- (Phe-NHFmoc) to 4-methylpiperidine is 1: 6-10, the volume ratio of 4-methylpiperidine to DMF is 1: 3-6, each mL of the mixture of 4-methylpiperidine and DMF contains 0.01-0.04 mmol of (FmocNH-Phe) -b-PNVP-b- (Phe-NHFmoc), stirring for reaction for 20-60 min, adding cold diethyl ether for precipitation, dialyzing in deionized water for 2-4 days, and freeze-drying to obtain (H)₂N-Phe)-b-PNVP-b-(Phe-NH₂)；

(10) Preparation of FmocNH-G₂-b-PNVP-b-G₂-NHFmoc：

With NH obtained in step (9)₂-G₁-b-PNVP-b-G₁-NH₂Replacing NH in step (6)₂-PNVP-NH₂And (5) repeating the steps (6), (7), (8) and (9) to obtain FmocNH-G₂-b-PNVP-b-G₂-NHFmoc；

(11) Preparation of FmocNH-G₃-b-PNVP-b-G₃-NHFmoc：

Using FmocNH-G obtained in step (10)₂-b-PNVP-b-G₂-NHFmoc instead of FmocNH-G in step (9)₁-b-PNVP-b-G₁-NHFmoc, repeating steps (9) and (10) to obtain FmocNH-G₃-b-PNVP-b-G₃-NHFmoc，

(12) Preparing a double-enzyme responsive super-amphiphilic molecule:

adding FmocNH-G into a reaction bottle₃-b-PNVP-b-G₃-NHFmoc, 4-methylpiperidine and DMF, wherein FmocNH-G₃-b-PNVP-b-G₃The mass ratio of the-NHFmoc to the 4-methylpiperidine is 1: 18-20, the volume ratio of the 4-methylpiperidine to the DMF is 1: 3-6, and each mL of the mixed solution of the 4-methylpiperidine and the DMF contains 0.005-0.008 mmol of FmocNH-G₃-b-PNVP-b-G₃-NHFmoc, stirring for 20-60 min, adding cold diethyl ether for precipitation, dialyzing in deionized water for 2-4 days, and freeze-drying to obtain H₂N-G₃-b-PNVP-b-G₃-NH₂；

Respectively preparing ATP solution and H by using buffer solution with pH of 6.5 as solvent₂N-G₃-b-PNVP-b-G₃-NH₂Solution of H₂N-G₃-b-PNVP-b-G₃-NH₂The mass concentration ratio of the ATP solution to the ATP solution is 1: 15-25, and the prepared ATP solution is dripped into H according to the proportion₂N-G₃-b-PNVP-b-G₃-NH₂Solution of, making⁺H₃N-G₃-b-PNVP-b-G₃-NH₃ ⁺And (3) standing for 4-10 h when the charge ratio of the amphiphilic substance to ATP (adenosine triphosphate) is 1:4 to prepare the double-enzyme-responsive dumbbell-shaped super-amphiphilic molecule.

3. The application of the double-enzyme responsive dumbbell-shaped super-amphiphilic molecule is characterized in that the double-enzyme responsive dumbbell-shaped super-amphiphilic molecule is used for self-assembling in an aqueous solution to form spherical micelles, and the micelles are used for encapsulating small molecules or drugs.

Technical Field

The invention belongs to the field of functional polymer materials, and particularly relates to a dumbbell-shaped (dendritic-linear-dendritic) hybrid block copolymer and adenosine triphosphate composite super-amphiphilic molecule which has response characteristics to two enzymes, takes poly (N-vinyl pyrrolidone) (PNVP) as a linear chain and takes phenylalanyl-lysine dipeptide as a dendron, and a preparation method and application thereof.

Background

A super-amphiphile is an amphiphile formed by combining a hydrophilic moiety with a hydrophobic moiety through non-covalent interactions such as electrostatic attraction, hydrogen bonding, host-guest interactions, and the like. In the super-amphiphilic molecule, the building elements are connected through non-covalent bond interaction, so that other complex chemical synthesis reactions can be effectively avoided, and the utilization rate of the building elements is greatly improved. And in the presence of non-covalent bonds, other functional moieties can be more conveniently introduced. In addition, the non-covalent bond also has good reversibility and controllability. Therefore, the amphiphilic property of the composite material can be regulated and controlled through external stimulus response, and controllable self-assembly and disassembly are obtained. The nano drug transport system with stimulation responsiveness formed by self-assembly of the super amphiphilic molecules has attracted attention in the treatment and research fields of diseases represented by malignant tumors in recent years due to the advantages of effectively improving the bioavailability of drugs, prolonging the circulation and residence time of drugs in blood, increasing the stability of the system and the like. The super-amphiphile can be divided into micromolecule type super-amphiphile and macromolecule type super-amphiphile according to the constitutional structure. At present, most of polymers forming the macromolecular super-amphiphilic molecules are linear macromolecules, and literature search does not show literature reports of the super-amphiphilic molecules based on dumbbell-shaped (dendritic-linear-dendritic) hybrid block copolymers.

Dendritic-linear-dendritic (dumbbell-shaped) triblock copolymers combine the ease of synthesis and processability of linear macromolecules with regular and well-defined dendrimers (motifs) which, because of their exact number of peripheral groups, can be modified to introduce functionality. The amphiphilic dendritic-linear-dendritic (dumbbell-shaped) triblock copolymer is self-assembled in an aqueous solution to form a nano container, and can encapsulate and release drug molecules. In drug delivery systems, the use of amphiphilic dendritic-linear-dendritic (dumbbell-shaped) triblock copolymer self-assembled aggregates as carriers has the advantage that they combine the multifunctional functionality imparted by the dendritic macromolecule with the high loading capacity of the block copolymer, which not only improves the stability of the self-assembled aggregates in aqueous solutions, but also allows optimization of the drug loading of the copolymer drug carrier and modulation of the drug release rate through molecular design, as compared to assemblies formed solely from amphiphilic linear block copolymers or dendrimers.

The enzyme responsive polymer is a promising carrier for a targeted drug delivery system due to excellent biocompatibility and high selectivity, and because some enzymes are often over-expressed in inflamed or tumor tissues, a chemical bond which can be cut off by a specific enzyme is introduced into a drug carrier, so that the targeted release of the drug can be realized, which has important significance in reducing the harm to healthy cells and tissues. And the release rate of the loaded drug can be controlled by adjusting the structure of the enzyme-responsive polymer according to the expression level of the enzyme in a specific region. In addition, the enzyme responsive polymer also has potential application value in the aspects of long-term circulation in vivo and targeted therapy and disease diagnosis. However, although some enzyme-responsive nonlinear polymers have been studied, they all respond to only one enzyme, and the biological environment in humans is complex, and some diseases may be associated with the expression disorder of more than one enzyme, for example, Matrix Metalloproteinases (MMPs), cathepsins (cathepsins B), alkaline phosphatase, etc., which have been reported as enzymes highly expressed in tumor cells. If one could design a non-linear polymer that would respond to two or more enzymes, one would expect to have a drug carrier that is more compatible with the physiological environment in vivo.

Amino acid-based polymers have received considerable attention in pharmaceutical and other medical applications due to their excellent biocompatibility and good biodegradability. L-lysine has been widely used as a polymer having a branched structure for the construction of polymers for biomedical applications such as drug delivery, tissue engineering and gene therapy. However, no amino acid-containing enzyme-responsive dendritic-linear-dendritic (dumbbell-shaped) triblock copolymers have been reported in the literature. On the other hand, some small peptides consisting of several different amino acids can be cleaved by extracellular proteases (such as matrix metalloproteinases) or lysosomal cysteine proteases (such as cathepsin B and trypsin) associated with tumors. Trypsin, previously thought to be a digestive enzyme produced by pancreatic acinar cells, has been shown to be expressed in a variety of cancers and is thought to be involved in tumor proliferation, invasion and metastasis, whereas trypsin only acts enzymatically at the C-terminus of lysine or arginine. Adenosine Triphosphate (ATP), an important energy molecule in the human body, has four negative charges and can be combined with positive ions through electrostatic force. Under the action of alkaline phosphatase, the phosphate bond in ATP can be cleaved, resulting in cleavage of the complex. ATP can form a super-amphiphilic molecule with a polymer with positive charges, the research on the ATP-based super-amphiphilic molecule is mostly concentrated on the aspect of linear polymers at present, and the literature search shows no literature report that ATP is compounded with dendritic-linear-dendritic (dumbbell-shaped) block copolymers to form the super-amphiphilic molecule.

Disclosure of Invention

The invention aims to provide a double-enzyme responsive dumbbell-shaped super-amphiphile, a preparation method and application thereof, in particular to a dumbbell-shaped (dendritic-linear-dendritic) block copolymer and Adenosine Triphosphate (ATP) super-amphiphile which is responsive to trypsin or cathepsin B and alkaline phosphatase (CIAP) and takes phenylalanyl-lysine dipeptide as a dendron, a preparation method and application thereof.

The product of the invention is double-enzyme responsive dumbbell-shaped super-amphiphilicThe block copolymer is characterized in that the third generation of dumbbell-shaped (dendritic-linear-dendritic) block copolymer which takes phenylalanyl-lysine dipeptide as dendron and takes poly (N-vinyl pyrrolidone) (PNVP) as linear chain⁺H₃N-G₃-b-PNVP-b-G₃-NH₃ ⁺And adenosine triphosphate are compounded to form the compound,

⁺H₃N-G₃-b-PNVP-b-G₃-NH₃ ⁺the molecular structure of (a) is as follows:

formula (II)⁺H₃N-G₃-b-PNVP-b-G₃-NH₃ ⁺Wherein n is 40 to 80.

The preparation method of the product of the double-enzyme responsive dumbbell-shaped super-amphiphilic molecule comprises the following steps:

1. preparation of chain transfer agent CTA with alpha, omega-double terminal xanthate groups

According to the prior art (see for example Taton D, Wilczewska A, Destarac M. Macromol. Rapid Commun.2001,22(18): 1497-. The molecular structure of CTA is as follows:

2. preparation of dimeric (N-vinylpyrrolidone) with alpha, omega-bisterminal xanthate groups

Poly (N-vinylpyrrolidone) X-PNVP-X of α, ω -bis-terminal xanthate groups was prepared according to the prior art (see, for example, Wan D, Satoh K, Kamigaito M, Okamoto y. macromolecules,2005,38(25), 10397-. The molecular structure of X-PNVP-X is as follows:

3. preparation of tert-butyl (2-acrylamidoethyl) carbamate

Tert-butyl (2-acrylamidoethyl) carbamate is prepared by the prior art (see, for example, Hobson L J, Feast W J. Polymer,1999,40(5): 1279-1297) by reacting tert-butyl (2-aminoethyl) carbamate with acryloyl chloride using triethylamine as an acid-binding agent. The molecular structure of tert-butyl (2-acrylamidoethyl) carbamate is as follows:

4. preparation of BocNH-PNVP-NHBoc

Adding X-PNVP-X, dichloromethane and n-butylamine into a reaction container, wherein the mass ratio of the X-PNVP-X to the n-butylamine is 1: 80-120, 2-4 g of X-PNVP-X is dissolved in every mL of dichloromethane, tributylphosphine accounting for 0.1-0.5% of the total mass of the X-PNVP-X and the n-butylamine is added according to the mass ratio, vacuumizing and charging nitrogen for 5-20 min, adding tri (2-carboxyethyl) phosphine (TCEP) and tert-butyl (2-acrylamidoethyl) carbamate, wherein the mass ratio of the X-PNVP-X, the tert-butyl (2-acrylamidoethyl) carbamate and the tert-butyl (2-carboxyethyl) phosphine is 1: 1.5-2.5: 2-4, and stirring and reacting for 4-8 h. Preferably, the reaction is carried out in a dry reaction vessel. And coprecipitating the crude product with petroleum ether for 2-5 times, dialyzing in deionized water for 2-4 days, and freeze-drying to obtain the BocNH-PNVP-NHBoc. The molecular structure of BocNH-PNVP-NHBoc is as follows:

5. preparation of NH₂-PNVP-NH₂

Adding BocNH-PNVP-NHBoc, dichloromethane and trifluoroacetic acid into a reaction container, wherein 0.01-0 part of dichloromethane is dissolved in each mL of dichloromethane05mmol of BocNH-PNVP-NHBoc, the volume ratio of trifluoroacetic acid to dichloromethane is 1: 1-3, stirring for reaction for 2-6 h, adjusting the pH to be neutral by using 1-2 mol/L NaOH, filtering, rotary steaming, dialyzing in deionized water for 2-4 days, and freeze-drying to obtain NH₂-PNVP-NH₂。NH₂-PNVP-NH₂The molecular structure of (a) is as follows:

6. preparation (FmocNH-Phe) -b-PNVP-b- (Phe-NHFmoc)

Adding NH into a reaction flask₂-PNVP-NH₂1-hydroxybenzotriazole, N-fluorenylmethoxycarbonyl-L-phenylalanine and N, N-Dimethylformamide (DMF), wherein NH₂-PNVP-NH₂The mass ratio of N-fluorenylmethoxycarbonyl-L-phenylalanine to 1-hydroxybenzotriazole is 1: 1.5-3.5: 4-8, and 0.01-0.03 mmol of NH is dissolved in each mL of DMF₂-PNVP-NH₂Stirring for 20-60 min, and adding N, N-diisopropyl carbodiimide (DIC) and NH₂-PNVP-NH₂And N, N-diisopropylcarbodiimide in a mass ratio of 1: 5-10, stirring for 20-48 h, adding cold diethyl ether for precipitation, dialyzing in deionized water for 2-4 days, and freeze-drying to obtain (FmocNH-Phe) -b-PNVP-b- (Phe-NHFmoc). The molecular structure of (FmocNH-Phe) -b-PNVP-b- (Phe-NHFmoc) is as follows:

7. preparation (H)₂N-Phe)-b-PNVP-b-(Phe-NH₂)

Adding (FmocNH-Phe) -b-PNVP-b- (Phe-NHFmoc), 4-methylpiperidine and DMF into a reaction bottle, wherein the mass ratio of (FmocNH-Phe) -b-PNVP-b- (Phe-NHFmoc) to 4-methylpiperidine is 1: 6-10, the volume ratio of 4-methylpiperidine to DMF is 1: 3-6, each mL of the mixture of 4-methylpiperidine and DMF contains 0.01-0.04 mmol of (FmocNH-Phe) -b-PNVP-b- (Phe-NHFmoc), stirring for reaction for 20-60 min, adding cold diethyl ether for precipitation, dialyzing in deionized water for 2-4 days, and freeze-drying to obtain (H)₂N-Phe)-b-PNVP-b-(Phe-NH₂)。(H₂N-Phe)-b-PNVP-b-(Phe-NH₂) The molecular structure of (a) is as follows:

8. preparation of FmocNH-G₁-b-PNVP-b-G₁-NHFmoc

Adding (H) into a reaction flask₂N-Phe)-b-PNVP-b-(Phe-NH₂) 1-hydroxybenzotriazole (HOBt), N' -bifluoromethoxycarbonyl-L-lysine (Fmoc-Lys (Fmoc) -OH) and N, N-Dimethylformamide (DMF), wherein (H)₂N-Phe)-b-PNVP-b-(Phe-NH₂) The mass ratio of Fmoc-Lys (Fmoc) -OH to HOBt is 1: 5-8: 2-4, and 0.01-0.03 mmol of (H) is dissolved in each mL of DMF₂N-Phe)-b-PNVP-b-(Phe-NH₂) Stirring for 20-60 min, and adding N, N-Diisopropylcarbodiimide (DIC), (H)₂N-Phe)-b-PNVP-b-(Phe-NH₂) The mass ratio of DIC and DIC is 1: 5-10, stirring and reacting for 20-48 h, adding cold ether for precipitation, dialyzing in deionized water for 2-4 days, and freeze drying to obtain G₁-b-PNVP-b-G₁。G₁-b-PNVP-b-G₁The molecular structure of (a) is as follows:

9. preparation of NH₂-G₁-b-PNVP-b-G₁-NH₂

Adding (FmocNH-Phe) -b-PNVP-b- (Phe-NHFmoc), 4-methylpiperidine and DMF into a reaction bottle, wherein the mass ratio of (FmocNH-Phe) -b-PNVP-b- (Phe-NHFmoc) to 4-methylpiperidine is 1: 6-10, the volume ratio of 4-methylpiperidine to DMF is 1: 3-6, each mL of the mixture of 4-methylpiperidine and DMF contains 0.01-0.04 mmol of (FmocNH-Phe) -b-PNVP-b- (Phe-NHFmoc), stirring for reaction for 20-60 min, adding cold diethyl ether for precipitation, dialyzing in deionized water for 2-4 days, and freeze-drying to obtain (H)₂N-Phe)-b-PNVP-b-(Phe-NH₂)。

10. Preparation of FmocNH-G₂-b-PNVP-b-G₂-NHFmoc

With NH obtained in step 9₂-G₁-b-PNVP-b-G₁-NH₂Replacing NH in step 6₂-PNVP-NH₂And repeating the steps 6, 7, 8 and 9 to obtain FmocNH-G₂-b-PNVP-b-G₂-NHFmoc。FmocNH-G₂-b-PNVP-b-G₂The molecular structure of-NHFmoc is as follows:

11. preparation of FmocNH-G₃-b-PNVP-b-G₃-NHFmoc

Using FmocNH-G obtained in step 10₂-b-PNVP-b-G₂-NHFmoc instead of FmocNH-G in step 9₁-b-PNVP-b-G₁-NHFmoc, repeating steps 9 and 10 to obtain FmocNH-G₃-b-PNVP-b-G₃-NHFmoc。FmocNH-G₃-b-PNVP-b-G₃The molecular structure of-NHFmoc is as follows:

12. preparation of double-enzyme responsive super-amphiphilic molecule

Adding FmocNH-G into a reaction bottle₃-b-PNVP-b-G₃-NHFmoc, 4-methylpiperidine and DMF, wherein FmocNH-G₃-b-PNVP-b-G₃The mass ratio of the-NHFmoc to the 4-methylpiperidine is 1: 18-20, the volume ratio of the 4-methylpiperidine to the DMF is 1: 3-6, and each mL of the mixed solution of the 4-methylpiperidine and the DMF contains 0.005-0.008 mmol of FmocNH-G₃-b-PNVP-b-G₃-NHFmoc, stirring for 20-60 min, adding cold diethyl ether for precipitation, dialyzing in deionized water for 2-4 days, and freeze-drying to obtain H₂N-G₃-b-PNVP-b-G₃-NH₂。

Respectively preparing ATP solution and H by using buffer solution with pH of 6.5 as solvent₂N-G₃-b-PNVP-b-G₃-NH₂Solution of H₂N-G₃-b-PNVP-b-G₃-NH₂The mass concentration ratio of the solution to the ATP solution is 1: 15-25. Due to 1moL⁺H₃N-G₃-b-PNVP-b-G₃-NH₃ ⁺(in a buffer solution of pH 6.5, H₂N-G₃-b-PNVP-b-G₃-NH₂Has become in fact⁺H₃N-G₃-b-PNVP-b-G₃-NH₃ ⁺) Carrying 16 positive charges and 1moL ATP carrying 4 negative charges, and dripping the prepared ATP solution into H according to a certain proportion₂N-G₃-b-PNVP-b-G₃-NH₂Solution of, making⁺H₃N-G₃-b-PNVP-b-G₃-NH₃ ⁺The charge ratio to ATP, N, is 1: 4. Standing for 4-10 h to obtain the double-enzyme responsive dumbbell-shaped super-amphiphilic molecules.

The procedures of filtration, dialysis, freeze drying and the like in the steps are the same as the conventional technology.

In the present invention, H₂N-G₃-b-PNVP-b-G₃-NH₂Protonation in buffer solution of pH 6.5⁺H₃N-G₃-b-PNVP-b-G₃-NH₃ ⁺The compound is compounded with ATP with negative charge to form double-enzyme responsive dumbbell-shaped super amphiphilic molecules, and the super amphiphilic molecules can be formed by¹H NMR spectrum, ultraviolet spectrum, fluorescence spectrum, particle size measurement, Zate potential measurement, and Transmission Electron Microscope (TEM).

The application of the double-enzyme responsive dumbbell-shaped super-amphiphilic molecule comprises the following steps: the micelle is used for self-assembling in aqueous solution to form spherical micelle, and the micelle is used for encapsulating small molecules or drugs.

The invention has the beneficial effects that: the third generation dumbbell-shaped (dendritic-linear-dendritic) block copolymer prepared by the invention takes phenylalanyl-lysine dipeptide as dendronized element⁺H₃N-G₃-b-PNVP-b-G₃-NH₃ ⁺The super-amphiphile compounded with Adenosine Triphosphate (ATP) can be self-assembled in an aqueous solution to form a spherical micelle, the micelle can encapsulate small molecules or drugs, and can realize the controllable release of the loaded small molecules or drugs under the action of Trypsin (Trypsin) or cathepsin B (cathepsin B) or bovine small intestine alkaline phosphatase (CIAP); and when two enzymes (Trypsin and CIAP, or Cathepsin B and CIA)P) is simultaneously existed, the release rate of the small molecule or the drug is greatly accelerated, namely the super-amphiphile has double-enzyme responsiveness. In the dumbbell-shaped super-amphiphile structure developed by the invention, poly (N-vinyl pyrrolidone) (PNVP) is a hydrophilic linear chain, amino positive ions at the tail ends of phenylalanyl-lysine dipeptide dendronization elements at two ends are compounded with ATP to form an amphiphilic super-amphiphile, reversible addition-fragmentation chain transfer Radical (RAFT) polymerization in active/controllable polymerization is adopted to prepare poly (N-vinyl pyrrolidone) (PNVP), the length of the PNVP linear chain can be changed, and thus the amphipathy of the super-amphiphile can be adjusted by changing the length of the poly (N-vinyl pyrrolidone) (PNVP) linear chain. Cathepsin B, trypsin and alkaline phosphatase (CIAP) are over-expressed in some tumor tissues, so the super amphiphilic molecule developed by the invention can be used for targeted release of anticancer drugs. In addition, the release of the traditional enzyme-responsive amphiphilic molecules formed by chemical bonds to the loaded drugs is generally finished after 24-48 hours, and the super-amphiphilic molecule assembly can completely release the loaded drugs within a few hours, so that the super-amphiphilic molecule assembly is particularly suitable for targeted rapid release of anticancer drugs. The preparation of an enzyme-responsive amphiphilic molecule drug-carrying system formed by a traditional chemical bond usually requires the induction of an organic solvent and the like, so that not only is the drug polluted, but also residues can cause harm to organisms. The drug-carrying system of the super amphiphilic molecule assembly developed by the invention is formed only by adding⁺H₃N-G₃-b-PNVP-b-G₃-NH₃ ⁺The drug and ATP can form a drug-loaded assembly, and the pollution and harm can be successfully avoided.

Drawings

FIG. 1 is a Transmission Electron Microscope (TEM) image of micelles formed by self-assembly of the example super-amphiphiles in an aqueous solution.

FIG. 2 is a graph showing the release profiles of the example of the fluorescent small molecule micelle carrying the super amphiphile after adding no enzyme (no Trypsin and CIAP), adding Trypsin (With Trypsin), adding alkaline phosphatase (With CIAP), and adding Trypsin and alkaline phosphatase (With Trypsin and CIAP).

Detailed Description

The present invention will be described in further detail with reference to examples, but the present invention is not limited thereto.