Lung selective biomacromolecule delivery system and construction method and application thereof

Abstract

The invention relates to a lung selective biomacromolecule delivery system as well as a construction method and application thereof, and belongs to the field of biomedical materials. The biomacromolecule delivery system disclosed by the invention comprises a biomacromolecule compound, wherein the biomacromolecule compound is a polymer; the biomacromolecular compound is obtained by self-assembly of a metal-organic cage, polyethylene glycol, polyethyleneimine and a biomacromolecular drug. The delivery system disclosed by the invention has efficient lung selectivity on an active matter level, and the bioavailability of a biomacromolecular drug in lung tissues is remarkably improved, so that the delivery system has good safety and wide application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedical materials, and in particular to a lung-selective biomacromolecule delivery system and a construction method and application thereof. Background Art

[0002] Due to the lack of effective treatments and lung drug delivery vehicles, respiratory diseases, especially lung infections, have become the world's leading cause of death, posing serious health risks to humans. Traditional small molecule drugs have shortcomings such as drug resistance, poor lung accumulation, and severe side effects.

[0003] Compared with traditional small molecule drugs, protein drugs have the advantages of good efficacy, strong specificity and few side effects, so they are of great value in clinical applications. However, due to their large molecular weight, strong hydrophilicity and complex three-dimensional structure, most protein drugs cannot spontaneously penetrate the cell membrane and enter the cell, which greatly limits the current treatment methods for intracellular protein components. In order to make protein drugs enter cells more efficiently, a variety of protein drug carriers have been developed and applied, such as liposomes, viruses, polymers, etc. At present, the main challenge of protein drug delivery is how to achieve tissue-specific delivery. To achieve lung-specific protein delivery at the level of living animals, most existing carriers use atomization administration, which has the disadvantages of cumbersome operation, easy inactivation of proteins during sample preparation, and biomacromolecule complexes must be atomized to 1-10 microns to achieve effective lung deposition. Therefore, the development of new protein drug delivery carriers to achieve lung-selective protein delivery through intravenous administration has a broader application prospect. Summary of the invention

[0004] The purpose of the present invention is to provide a lung-selective biomacromolecule delivery system and its construction method and application. The biomacromolecule delivery system of the present invention includes a metal coordination-guided supramolecular carrier, which has high lung selectivity at the level of living animals, can achieve more efficient retention of biomacromolecule drugs in lung tissue, and improve the bioavailability of biomacromolecule drugs in lung tissue.

[0005] The solution provided by the present invention is as follows:

[0006] A biomacromolecule delivery system comprises a biomacromolecule complex.

[0007] According to an embodiment of the present invention, the biomacromolecule complex is obtained by self-assembly of metal-organic cage, polyethylene glycol, polyethyleneimine and biomacromolecule drugs.

[0008] According to an embodiment of the present invention, the biomacromolecule drug is selected from biomacromolecule drugs known in the art, for example, selected from nucleic acid macromolecule drugs, protein macromolecule drugs, for example, selected from but not limited to at least one of albumin, alcohol dehydrogenase, β-galactosidase, catalase, and DUF5 protein.

[0009] According to an embodiment of the present invention, in the biomacromolecule complex, the metal-organic cage serves as the main skeleton and is assembled into the biomacromolecule complex with the biomacromolecule drug under the action of polyethylene glycol and polyethyleneimine.

[0010] According to an embodiment of the present invention, the assembly refers to the assembly of biomacromolecule drugs through, for example, host-guest interaction, click chemistry modification or cross-linking.

[0011] According to an embodiment of the present invention, the mass ratio of the polyethyleneimine, the metal-organic cage and the polyethylene glycol is 100:(5-20):(80-120), for example, 100:12.5:102.

[0012] According to an embodiment of the present invention, the mass ratio of the polyethyleneimine to the biomacromolecule drug is 100:(10-230), for example, 100:100.

[0013] According to an embodiment of the present invention, the metal-organic cage is selected from ligands and metal ions coordinated and assembled.

[0014] According to an embodiment of the present invention, the ligand is selected from the compound shown in formula I.

[0015]

[0016] Wherein, R1 is selected from C 3-12 Heterocyclic group, C5-C 14 Aryl;

[0017] -L- represents a linking group, which can be selected from unsubstituted C 1-50 Alkylene, or optionally containing one or more heteroatoms C 1-50 The heteroatom may be selected from O and S.

[0018] R2 is selected from a group having a host-guest effect and / or a cross-linking effect, and can be selected from an adamantane group, an alkynyl group, and an azide group.

[0019] According to an exemplary embodiment of the present invention, the ligand is selected from the compounds shown in the following formulas Ⅰ-1 to Ⅰ-4:

[0020]

[0021] According to an embodiment of the present invention, the metal ion is an ion with coordination function known in the art, and can be selected from palladium ions and platinum ions.

[0022] According to an embodiment of the present invention, the assembly of the ligand and the metal is a coordination self-assembly process that can be understood in the art.

[0023] According to an embodiment of the present invention, the metal-organic cage is selected from a compound having a structure as shown in Formula A, wherein: refers to the connection point, and m is selected from 1-100, for example, 1, 3, 5, 10, 20, 30, 40.

[0024] Exemplarily, when m=3, the compound of the structure shown in Formula A is named TPE-MOC.

[0025]

[0026] According to an embodiment of the present invention, the polyethylene glycol is selected from polyethylene glycol containing a first modifying group, and its structure is shown in Formula B:

[0027]

[0028] Wherein, R3 is the first modifying group selected from adamantane, azide, and alkynyl; n is selected from 1-300, for example, 10, 20, 30, and 40.

[0029] According to an embodiment of the present invention, the molecular weight of the polyethylene glycol is 100-10000, for example, 1900.

[0030] According to an embodiment of the present invention, the polyethylene glycol is selected from compounds having at least one structure of the following formulas B-1, B-2 and B-3:

[0031]

[0032] Wherein, in formulas B-1, B-2 and B-3, n is selected from 1-300, for example, 10, 20, 30, 40, 50.

[0033] According to an exemplary embodiment of the present invention, the polyethylene glycol is selected from a compound having a structure of formula B-1, that is, polyethylene glycol containing an adamantyl group; for example, when n is 44, the molecular weight of the polyethylene glycol containing an adamantyl group is 1900, and is named Ada-PEG1900.

[0034] According to an embodiment of the present invention, the polyethyleneimine is selected from polyethyleneimine containing a second modifying group, which contains a repeating unit as shown in formula C;

[0035]

[0036] Wherein, R4 is a second modifying group selected from, for example, a cyclodextrin group, an azide group, an alkyne group, and the like.

[0037] According to an embodiment of the present invention, the molecular weight of the polyethyleneimine is 100-50000, for example, 600-50000, preferably 600, 1800, 5000, 10000, 25000.

[0038] According to a preferred embodiment of the present invention, the polyethyleneimine is selected from polyethyleneimine PEI-βCD containing a cyclodextrin group. Preferably, the polyethyleneimine PEI-βCD has a structure as shown in Formula C-1, wherein βCD is a cyclodextrin group.

[0039]

[0040] According to an embodiment of the present invention, the structure of βCD in Formula C-1 is as shown in Formula D.

[0041]

[0042] According to an embodiment of the present invention, the mass ratio of the polyethyleneimine, the metal-organic cage and the polyethylene glycol is 100:(5-20):(80-120), for example, 100:12.5:102.

[0043] According to an embodiment of the present invention, the mass ratio of the polyethyleneimine to the biomacromolecule drug is 100:(10-230), for example, 100:100.

[0044] According to an exemplary embodiment of the present invention, the biomacromolecule complex is obtained by self-assembly of a metal-organic cage TPE-MOC containing an adamantyl group, polyethylene glycol Ada-PEG1900 containing an adamantyl group, polyethyleneimine PEI-βCD containing a cyclodextrin group and a biomacromolecule drug.

[0045] According to an embodiment of the present invention, the administration method of the biomacromolecule delivery system is intravenous administration.

[0046] The present invention also provides a method for constructing the above-mentioned biomacromolecule delivery system, which comprises: self-assembling the metal-organic cage, polyethylene glycol, polyethyleneimine and biomacromolecule drugs to obtain a biomacromolecule complex, namely the biomacromolecule delivery system.

[0047] According to an embodiment of the present invention, the construction method specifically comprises the following steps:

[0048] (1) preparing a storage solution of a biomacromolecule drug, a storage solution of a metal-organic cage, a storage solution of polyethyleneimine, and a storage solution of polyethylene glycol respectively;

[0049] (2) The storage solution of polyethyleneimine, the storage solution of biomacromolecule drugs, the storage solution of polyethylene glycol and the storage solution of metal-organic cages in step (1) are mixed and stirred to obtain a mixed solution, and a biomacromolecule complex is obtained by self-assembly of metal-organic cages, polyethylene glycol, polyethyleneimine and biomacromolecule drugs. After centrifugal purification, the biomacromolecule complex is dispersed in a buffer solution to obtain the biomacromolecule delivery system.

[0050] According to an embodiment of the present invention, in step (1), the storage solution of the metal-organic cage refers to dissolving the metal-organic cage in an organic solvent, and the metal-organic cage has the meaning as described above. Preferably, the organic solvent can be selected from organic solvents known in the art, for example, dimethyl sulfoxide (abbreviated as DMSO).

[0051] According to an embodiment of the present invention, in step (1), the storage solution of polyethyleneimine refers to dissolving the polyethyleneimine in a solvent, and the polyethyleneimine has the meaning as described above. Preferably, the solvent can be selected from solvents known in the art, such as pure water.

[0052] According to an embodiment of the present invention, in step (1), the storage solution of polyethylene glycol refers to dissolving the polyethylene glycol in a solvent, and the polyethylene glycol has the meaning as described above. Preferably, the solvent can be selected from solvents known in the art, such as pure water.

[0053] According to an embodiment of the present invention, in step (2), in the mixed solution, the mass ratio of the polyethyleneimine, the metal-organic cage and the polyethylene glycol is 100:(5-20):(80-120), for example, 100:12.5:102.

[0054] According to an embodiment of the present invention, in step (2), in the mixed solution, the mass ratio of the polyethyleneimine to the biomacromolecule drug is 100:(10-230), for example, 100:100.

[0055] According to an embodiment of the present invention, in step (2), the concentration of the biomacromolecule drug in the mixed solution is 0.1-1 mg / mL, for example, 1 mg / mL.

[0056] According to an embodiment of the present invention, in step (2), the concentration of polyethyleneimine in the mixed solution is 0.5-4 mg / mL, for example, 1 mg / mL.

[0057] According to an embodiment of the present invention, in step (2), the concentration of the metal-organic cage in the mixed solution is 0.025-0.8 mg / mL, for example, 0.1 mg / mL.

[0058] According to an embodiment of the present invention, in step (2), the concentration of polyethylene glycol in the mixed solution is 0.4-5 mg / mL, for example, 1.02 mg / mL.

[0059] According to an embodiment of the present invention, in step (2), the mixing and stirring conditions are: stirring at room temperature for 0.25-2h.

[0060] According to an embodiment of the present invention, room temperature refers to 20-26°C.

[0061] According to an embodiment of the present invention, in step (2), during centrifugal purification, the centrifugal speed is 10000-15000 r / min.

[0062] According to an embodiment of the present invention, in step (2), the buffer solution may be a buffer solution known in the art, such as PBS buffer solution.

[0063] According to an exemplary scheme of the present invention, the construction method specifically includes the following steps: self-assembling the metal-organic cage TPE-MOC containing adamantyl, polyethylene glycol Ada-PEG1900 containing adamantyl, polyethyleneimine PEI-βCD containing cyclodextrin and biomacromolecule drugs to obtain the biomacromolecule delivery system.

[0064] The present invention also provides the application of the above biomacromolecule delivery system in selective drug delivery to lung tissue and enzyme catalysis, preferably in selective drug delivery to lung tissue.

[0065] Beneficial effects:

[0066] The present invention uses metal-organic cage as the main skeleton, adds polyethyleneimine and polyethylene glycol modified with cyclodextrin groups to assemble biomacromolecule drugs, constructs a protein nano delivery system, and then uses the protein nano delivery system to conduct live animal experiments. The present invention constructs a biomacromolecule delivery system that can be used for lung selectivity, and the process of the constructed biomacromolecule delivery system is simple, and the raw materials are cheap and easy to obtain. It can be seen by projection electron microscopy that the formed biomacromolecule complex has a uniform particle size and a stable complex. The experimental results show that the biomacromolecule delivery system of the present invention can efficiently deliver biomacromolecule drugs (such as proteins) selectively to lung tissue without losing the biological activity of the protein, and can be applied to the treatment of lung-related diseases and the field of gene editing.

[0067] The biomacromolecule delivery system constructed by the present invention prolongs the circulation time of the biomacromolecule complex in the animal body. The experimental results of the present invention show that compared with the two-component carriers of polyethyleneimine and metal-organic cage reported in previous literature that can only deliver proteins to the liver, the biomacromolecule delivery system of the present invention can efficiently deliver proteins to lung tissue, expanding the application scope of biomacromolecule drugs in life systems, and can be used in the treatment of lung diseases with biomacromolecule drugs.

[0068] The biomacromolecule delivery system constructed by the present invention can efficiently deliver β-galactosidase to lung tissue, and then catalyze 5-bromo-4-chloro-3-indole-β-D-galactoside (X-Gal) to produce a color reaction, which significantly improves the targeting of biomacromolecule drugs and reduces the effects of biomacromolecule drugs in other tissues or organs.

[0069] The delivery system of the present invention has high lung selectivity at the level of living animals, significantly improving the bioavailability of biomacromolecule drugs in lung tissue, indicating that it has good safety and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] In order to more clearly illustrate the embodiments of the present invention, the drawings of the experiments required in the embodiments are briefly introduced below. The drawings in the following description are only some embodiments of the present invention and are not to be considered as limiting the present invention.

[0071] Figure 1 Schematic diagram of selective delivery of biomacromolecule drugs to the lungs;

[0072] Figure 2 Transmission electron microscopy (TEM) images of PEI-βCD / TPE-MOC / Ada-PEG1900 / BSA-Cy5;

[0073] Figure 3 Fluorescence photos of major organs of mice injected with BSA-Cy5 loaded by tail vein;

[0074] Figure 4 The content of metal Pt in major organs after tail vein injection of PEI-βCD / TPE-MOC / Ada-PEG1900 / BSA-Cy5 complex in mice;

[0075] Figure 5 Fluorescence photos of the main organs of mice injected with CAT-Cy5-encapsulated cells through the tail vein;

[0076] Figure 6 Fluorescence photos of the main organs of mice injected with DUF-Cy5-encapsulated cells through the tail vein;

[0077] Figure 7Fluorescence photos of the main organs of mice injected with ADH-Cy5 loaded by tail vein;

[0078] Figure 8 Fluorescence photos of the main organs of mice injected with β-Gal-Cy5-encapsulated carriers through the tail vein;

[0079] Fig. 9 The absorbance of the mouse main organs after injection of β-Gal-Cy5 into the tail vein was measured after grinding and incubation with the substrate X-Gal. DETAILED DESCRIPTION

[0080] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0081] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0082] Preparation Example 1

[0083] The synthesis of BSA-Cy5 is as follows: BSA (10 mg, 0.15 μmol) and Cy5-SE (1 mg, 1.3 μmol) are dissolved in 1 mL of sodium carbonate-sodium bicarbonate buffer (pH 8.5, 0.1 M). The mixture is reacted at room temperature in the dark for 1 h. After the reaction is completed, the mixture is centrifuged at 13000 g, the supernatant is purified by ultrafiltration (50 KDa), replaced with PBS buffer, and the concentration of protein BSA-Cy5 is quantified by BCA, and stored at 4°C.

[0084] Preparation Example 2

[0085] The synthesis of ADH-Cy5 is as follows: alcohol dehydrogenase (ADH, 10 mg, 0.07 μmol) and Cy5-SE (1 mg, 1.3 μmol) are dissolved in 1 mL of sodium carbonate-sodium bicarbonate buffer (pH 8.5, 0.1 M). The reaction is carried out at room temperature in the dark for 1 h. After the reaction is completed, centrifuge at 13000 g, and the supernatant is purified by ultrafiltration (100 KDa), replaced with PBS buffer, and the concentration of protein ADH-Cy5 is quantified by BCA, and stored at 4°C.

[0086] Preparation Example 3

[0087] The synthesis of CAT-Cy5 is as follows: catalase (CAT, 10 mg, 0.04 μmol) and Cy5-SE (1 mg, 1.3 μmol) are dissolved in 1 mL of sodium carbonate-sodium bicarbonate buffer (pH 8.5, 0.1 M). The reaction is carried out at room temperature in the dark for 1 h. After the reaction is completed, centrifuge at 13000 g, and the supernatant is purified by ultrafiltration (100 KDa), replaced with PBS buffer, and the concentration of protein CAT-Cy5 is quantified by BCA, and stored at 4°C.

[0088] Preparation Example 4

[0089] The synthesis of β-Gal-Cy5 is as follows: β-galactosidase (β-Gal, 10 mg, 0.02 μmol) and Cy5-SE (1 mg, 1.3 μmol) are dissolved in 1 mL of sodium carbonate-sodium bicarbonate buffer (pH 8.5, 0.1 M). The reaction is carried out at room temperature in the dark for 1 hour. After the reaction is completed, centrifuge at 13000g, and the supernatant is purified by ultrafiltration (100 KDa), replaced with PBS buffer, and the concentration of protein β-Gal-Cy5 is quantified by BCA, and stored at 4°C.

[0090] Preparation Example 5

[0091] The synthesis of DUF5-Cy5 is as follows: DUF5 (10 mg, 0.02 μmol) and Cy5-SE (1 mg, 1.3 μmol) were dissolved in 1 mL of sodium carbonate-sodium bicarbonate buffer (pH 8.5, 0.1 M). The reaction was allowed to proceed for 1 h at room temperature in the dark. After the reaction was completed, the supernatant was centrifuged at 13000 g, purified by ultrafiltration (30 KDa), replaced with PBS buffer, and the concentration of protein DUF5-Cy5 was quantified by BCA, and stored at 4°C.

[0092] Example 1

[0093] The construction method of the biomacromolecule delivery system PEI-βCD / TPE-MOC / Ada-PEG1900 / BSA-Cy5 is as follows:

[0094] (1) Synthesis of PEI-βCD and TPE-MOC: The synthesis of PEI-βCD and TPE-MOC can be carried out according to the synthesis methods of PEI-βCD and TPE-MOC in the literature (specifically, Liu J., Sheng JH, Shao L.H., et al. Tetraphenylethylene-featured fluorescent supramolecular nanoparticles for intracellular trafficking of protein delivery and neuroprotection [J]. Angew. Chem. Int. Ed. 2021, 60, 26740-26746), respectively. Figure 1 The structures shown in (a) and (b).

[0095] (2) Synthesis of Ada-PEG1900: The synthesis of mPEG-AD can be carried out by referring to the synthesis method of mPEG-AD in the literature (specifically Dong RJ, Chen HY, Wang D.L., et al. Supramolecular fluorescent nanoparticles for targeted cancer imaging [J]. ACS Macro Lett. 2012, 1, 1208-1211) to obtain adamantane-modified PEG with a molecular weight of 1900, denoted as Ada-PEG1900, and the structure is as follows: Figure 1 As shown in (c).

[0096] (3) Preparation of BSA-Cy5 (Preparation Example 1), PEI-βCD, TPE-MOC, and Ada-PEG1900 storage solutions: Prepare protein storage solution at a concentration of 10 mg / mL in PBS buffer (0.01 M, pH 7.2); prepare PEI-βCD and Ada-PEG1900 storage solutions at concentrations of 10 mg / mL and 10 mg / mL in pure water; prepare TPE-MOC storage solution at a concentration of 10 mg / mL in DMSO.

[0097] (4) In a clean 2 mL centrifuge tube, PEI-βCD, BSA-Cy5, Ada-PEG1900 and TPE-MOC storage solutions were added dropwise in sequence, so that the final PEI-βCD concentration was 1 mg / mL, the BSA-Cy5 concentration was 0.125 mg / mL, the Ada-PEG1900 concentration was 1.02 mg / mL, and the TPE-MOC concentration was 0.1 mg / mL. The resulting mixture was stirred at room temperature for 1 hour, then centrifuged at 15000 r / min for 15 minutes, and the nanoparticles were resuspended in PBS buffer so that the final concentration of PEI-βCD was 1 mg / mL to obtain a biomacromolecule delivery system.

[0098] Comparative Example 1

[0099] The construction method of the biomacromolecule delivery system PEI-βCD / TPE-MOC / BSA-Cy5 in this comparative example is basically the same as that in Example 1, except that:

[0100] Ada-PEG1900 was not added in steps (3) and (4).

[0101] Test Example 1

[0102] The TEM test of the biomacromolecule delivery system is as follows:

[0103] The protein nano-delivery system constructed in Example 1 was diluted 10 times with pure water, and 5 μL was dropped on a copper mesh. After standing overnight at room temperature to dry, a TEM test was performed to obtain the following results: Figure 2 The TEM images shown are from Figure 2 It can be seen that the biomacromolecule complex obtained by the protein nano-delivery system has a spherical morphology, a uniform particle size and a nanoscale dimension, and the particle size is basically between 100nm and 150nm.

[0104] Test Example 2

[0105] (A) BALB / c mice (4-6 weeks old) were injected with 200 μL of the biomacromolecule delivery system prepared in Example 1 via the tail vein. Six hours later, the mice were anesthetized and killed. The main organs (heart, liver, spleen, lung and kidney) were imaged with an excitation wavelength of 640 nm and a filter of 680 nm. The test results are shown in the figure. Figure 3 middle

[0106] PEI-βCD / TPE-MOC / Ada-PEG1900. Three control groups were selected: Group 1 was intravenously injected with 200 μL PBS buffer, denoted as PBS; Group 2 was injected with 200 μL PBS solution of BSA-Cy5 with the same concentration as the biomacromolecule delivery system, denoted as BSA-Cy5 alone; Group 3 was injected with 200 μL of the biomacromolecule delivery system prepared in Comparative Example 1, denoted as PEI-βCD / TPE-MOC. Figure 3 It can be seen that the PEI-βCD / TPE-MOC / BSA-Cy5 of comparative example 1 delivers the protein BSA-Cy5 to the liver, but almost no fluorescence signal is seen in the lungs. The biomacromolecule delivery system PEI-βCD / TPE-MOC / Ada-PEG1900 / BSA-Cy5 constructed by the present invention can selectively deliver proteins to lung tissues, see Figure 3 When PEI-βCD / TPE-MOC / Ada-PEG1900 / BSA-Cy5 was used, the lungs of mice showed obvious fluorescence.

[0107] (B) After intravenous injection of the biomacromolecule delivery system, the Pt content in the main organs of mice was determined as follows:

[0108] Weigh appropriate amounts of the above-mentioned heart, liver, spleen, lung and kidney organ samples into a polytetrafluoroethylene digestion tank, and add 5 mL of nitric acid. After standing until the reaction is completed, cover and seal, and place in a microwave digestion instrument. The digestion program is 100°C, 3 min; 140°C, 3 min; 160°C, 3 min; 180°C, 3 min; 190°C, 15 min. After the temperature cools to below 50°C, take out the digestion tank and place it in a fume hood. Open the digestion tank, rinse it with ultrapure water 3-4 times, transfer it to a 25 mL volumetric flask, make up to volume with ultrapure water, and perform ICP-MS test. The test results are as follows: Figure 4 As shown. Figure 4 It can be seen that compared with other tissues, Pt is mainly in the lung tissue, indicating that the biomacromolecule delivery system constructed by the present invention is mainly retained in the lung.

[0109] Example 2

[0110] The construction method of the biomacromolecule delivery system PEI-βCD / TPE-MOC / Ada-PEG1900 / ADH-Cy5 is basically the same as that of Example 1, except that:

[0111] In step (3), ADH-Cy5 prepared in Preparation Example 2 is used instead of BSA-Cy5 to prepare ADH-Cy5, PEI-βCD, TPE-MOC, and Ada-PEG1900 storage solutions: a protein storage solution is prepared at a concentration of 10 mg / mL in PBS buffer (0.01 M, pH 7.2); PEI-βCD and Ada-PEG1900 storage solutions are prepared at concentrations of 10 mg / mL and 10 mg / mL in pure water; and a TPE-MOC storage solution is prepared at a concentration of 10 mg / mL in DMSO.

[0112] In step (4), PEI-βCD, ADH-Cy5, Ada-PEG1900 and TPE-MOC storage solutions are sequentially added dropwise in a clean 2 mL centrifuge tube, so that the final PEI-βCD concentration is 1 mg / mL, the BSA-Cy5 concentration is 0.125 mg / mL, the Ada-PEG1900 concentration is 1.02 mg / mL, and the TPE-MOC concentration is 0.1 mg / mL. The resulting mixture is stirred at room temperature for 1 hour, then centrifuged at 15000 r / min for 15 minutes, and the nanoparticles are resuspended in PBS buffer so that the final concentration of PEI-βCD is 1 mg / mL to obtain a biomacromolecule delivery system.

[0113] Example 3

[0114] The construction method of the biomacromolecule delivery system PEI-βCD / TPE-MOC / Ada-PEG1900 / CAT-Cy5 is basically the same as that of Example 1, except that:

[0115] In step (3), CAT-Cy5 of Preparation Example 3 is used instead of BSA-Cy5 to prepare CAT-Cy5, PEI-βCD, TPE-MOC, and Ada-PEG1900 storage solutions: a protein storage solution is prepared at a concentration of 10 mg / mL in PBS buffer (0.01 M, pH 7.2); PEI-βCD and Ada-PEG1900 storage solutions are prepared at concentrations of 10 mg / mL and 10 mg / mL in pure water; a TPE-MOC storage solution is prepared at a concentration of 10 mg / mL in DMSO.

[0116] In step (4), PEI-βCD, CAT-Cy5, Ada-PEG1900 and TPE-MOC storage solutions are sequentially added dropwise to a clean 2 mL centrifuge tube, so that the final PEI-βCD concentration is 1 mg / mL, the BSA-Cy5 concentration is 0.125 mg / mL, the Ada-PEG1900 concentration is 1.02 mg / mL, and the TPE-MOC concentration is 0.1 mg / mL. The resulting mixture is stirred at room temperature for 1 hour, then centrifuged at 15000 r / min for 15 minutes, and the nanoparticles are resuspended in PBS buffer so that the final concentration of PEI-βCD is 1 mg / mL to obtain a biomacromolecule delivery system.

[0117] Example 4

[0118] The construction method of the biomacromolecule delivery system PEI-βCD / TPE-MOC / Ada-PEG1900 / β-Gal-Cy5 is basically the same as that of Example 1, except that:

[0119] In step (3), β-Gal-Cy5 of Preparation Example 4 is used instead of BSA-Cy5 to prepare β-Gal-Cy5, PEI-βCD, TPE-MOC, and Ada-PEG1900 storage solutions: a protein storage solution is prepared at a concentration of 10 mg / mL in PBS buffer (0.01 M, pH 7.2); PEI-βCD and Ada-PEG1900 storage solutions are prepared at concentrations of 10 mg / mL and 10 mg / mL in pure water; a TPE-MOC storage solution is prepared at a concentration of 10 mg / mL in DMSO.

[0120] In step (4), PEI-βCD, β-Gal-Cy5, Ada-PEG1900 and TPE-MOC storage solutions are sequentially added dropwise in a clean 2 mL centrifuge tube, so that the final PEI-βCD concentration is 1 mg / mL, the BSA-Cy5 concentration is 0.125 mg / mL, the Ada-PEG1900 concentration is 1.02 mg / mL, and the TPE-MOC concentration is 0.1 mg / mL. The resulting mixture is stirred at room temperature for 1 hour, then centrifuged at 15000 r / min for 15 minutes, and the nanoparticles are resuspended in PBS buffer so that the final concentration of PEI-βCD is 1 mg / mL to obtain a biomacromolecule delivery system.

[0121] Example 5

[0122] The construction method of the biomacromolecule delivery system PEI-βCD / TPE-MOC / Ada-PEG1900 / DUF5-Cy5 is basically the same as that of Example 1, except that:

[0123] In step (3), DUF5-Cy5 of Preparation Example 5 is used instead of BSA-Cy5 to prepare DUF5-Cy5, PEI-βCD, TPE-MOC, and Ada-PEG1900 storage solutions: a protein storage solution is prepared at a concentration of 10 mg / mL in PBS buffer (0.01 M, pH 7.2); PEI-βCD and Ada-PEG1900 storage solutions are prepared at concentrations of 10 mg / mL and 10 mg / mL in pure water; a TPE-MOC storage solution is prepared at a concentration of 10 mg / mL in DMSO.

[0124] In step (4), PEI-βCD, DUF5-Cy5, Ada-PEG1900 and TPE-MOC storage solutions are sequentially added dropwise in a clean 2 mL centrifuge tube, so that the final PEI-βCD concentration is 1 mg / mL, the BSA-Cy5 concentration is 0.125 mg / mL, the Ada-PEG1900 concentration is 1.02 mg / mL, and the TPE-MOC concentration is 0.1 mg / mL. The resulting mixture is stirred at room temperature for 1 hour, then centrifuged at 15000 r / min for 15 minutes, and the nanoparticles are resuspended in PBS buffer so that the final concentration of PEI-βCD is 1 mg / mL to obtain a biomacromolecule delivery system.

[0125] Test Example 3

[0126] (1) BALB / c mice (4-6 weeks old) were selected and injected with 200 μL of the biomacromolecule delivery system prepared in Example 3 via the tail vein. Six hours later, the mice were anesthetized and killed, and the main organs (heart, liver, spleen, lung and kidney) were imaged. The excitation wavelength was 640 nm and the filter was 680 nm. The test results were as follows: Figure 5 PEI-βCD / TPE-MOC / Ada-PEG1900 was shown in the figure. Two control groups were selected: Group 1 was intravenously injected with 200 μL PBS buffer, denoted as PBS; Group 2 was injected with 200 μL CAT-Cy5 (same as Preparation Example 3), denoted as CAT-Cy5 alone. Figure 5 It can be seen that the biomacromolecule delivery system constructed by the present invention can selectively deliver CAT protein to lung tissue, see Figure 5 When the biomacromolecule delivery system of Example 3 was used, the lungs of the mice showed obvious fluorescence; while when other delivery systems were used, the lungs of the mice had no obvious fluorescence.

[0127] (2) BALB / c mice (4-6 weeks old) were selected and injected with 200 μL of the biomacromolecule delivery system prepared in Example 5 via the tail vein. Six hours later, the mice were anesthetized and killed, and the main organs (heart, liver, spleen, lung and kidney) were imaged. The excitation wavelength was 640 nm and the filter was 680 nm. The test results were as follows: Figure 6 PEI-βCD / TPE-MOC / Ada-PEG1900 was shown in Figure 2. Two control groups were selected: Group 1 was intravenously injected with 200 μL PBS buffer, denoted as PBS; Group 2 was injected with 200 μL DUF5-Cy5 (same as Preparation Example 5), denoted as DUF5-Cy5 alone. Figure 6 It can be seen that the biomacromolecule delivery system constructed by the present invention can selectively deliver DUF5 protein to lung tissue, see Figure 6 When the biomacromolecule delivery system of Example 5 was used, the lungs of the mice showed obvious fluorescence; while when other delivery systems were used, the lungs of the mice showed no obvious fluorescence.

[0128] (3) BALB / c mice (4-6 weeks old) were selected and injected with 200 μL of the biomacromolecule delivery system prepared in Example 2 via the tail vein. Six hours later, the mice were anesthetized and killed, and the main organs (heart, liver, spleen, lung and kidney) were imaged. The excitation wavelength was 640 nm and the filter was 680 nm. The test results were as follows: Figure 7 PEI-βCD / TPE-MOC / Ada-PEG1900 was shown in the figure. Two control groups were selected: Group 1 was intravenously injected with 200 μL PBS buffer, denoted as PBS; Group 2 was injected with 200 μL ADH-Cy5 (same as Preparation Example 2), denoted as ADH-Cy5 alone. Figure 7 It can be seen that the biomacromolecule delivery system constructed by the present invention can selectively deliver ADH protein to lung tissue, see Figure 7 When the biomacromolecule delivery system of Example 2 was used, the lungs of the mice showed obvious fluorescence; while when other delivery systems were used, the lungs of the mice had no obvious fluorescence.

[0129] (4) BALB / c mice (4-6 weeks old) were selected and injected with 200 μL of the biomacromolecule delivery system prepared in Example 4 via the tail vein. Six hours later, the mice were anesthetized and killed, and the main organs (heart, liver, spleen, lung and kidney) were imaged. The excitation wavelength was 640 nm and the filter was 680 nm. The test results were as follows: Figure 8 PEI-βCD / TPE-MOC / Ada-PEG1900 was shown in the figure. Two control groups were selected: Group 1 was intravenously injected with 200 μL PBS buffer, denoted as PBS; Group 2 was injected with 200 μL β-Gal-Cy5 (same as Preparation Example 4), denoted as β-Gal-Cy5 alone. Figure 8 It can be seen that the biomacromolecule delivery system constructed by the present invention can selectively deliver β-Gal protein to lung tissue, see Figure 8When the biomacromolecule delivery system of Example 4 was used, the lungs of the mice showed obvious fluorescence; while when other delivery systems were used, the lungs of the mice had no obvious fluorescence.

[0130] Test Example 4

[0131] The β-Gal-Cy5 enzyme activity test in the main organs of mice is as follows:

[0132] Mice were injected with the biomacromolecule delivery system of Example 4 via the tail vein. Six hours later, the main organs (heart, liver, spleen, lung and kidney) were taken out, minced, added with 1 mL of PBS and ground, centrifuged at 8000 r / min, and the supernatant was taken for BCA quantification of protein concentration. 100 μg of total protein was taken and incubated with X-Gal at a final concentration of 1 mg / mL at 37°C for 1-4 h, and the absorbance at 633 nm was measured. The results are as follows: Fig. 9 As shown. Fig. 9 It can be seen that compared with other tissues, lung tissue has the highest β-Gal enzyme activity, indicating that the biomacromolecule delivery system constructed by the present invention can selectively deliver β-Gal to lung tissue, and this process does not damage the catalytic activity of β-Gal enzyme.

[0133] In the present invention, when polyacetamide, polyethylene glycol or metal organic cage has other groups with host-guest effect or click chemistry modification, cross-linking effect, it has similar cross-linking function and can obtain the same result. For example, when polyacetamide, polyethylene glycol or metal organic cage has azide and terminal alkyne, click reaction will occur, and biological macromolecular drugs such as nucleic acid and protein can also be loaded on the metal organic cage, which will not be described one by one here. The selected groups include but are not limited to the azide and terminal alkyne listed above, so the above cross-linking effect can be achieved through click chemistry and other effects.

[0134] The above is a description of the exemplary embodiments of the present invention. However, the protection scope of the present application is not limited to the above embodiments. Any modification, equivalent substitution, improvement, etc. made by those skilled in the art within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A biomacromolecule delivery system, characterized in that: The biomacromolecule delivery system includes a biomacromolecule complex; The biomacromolecule complex is obtained by self-assembly of metal-organic cage, polyethylene glycol, polyethyleneimine and biomacromolecule drugs.

2. The biomacromolecule delivery system according to claim 1, characterized in that: In the biomacromolecule complex, the metal-organic cage is used as the main skeleton and is assembled into the biomacromolecule complex with the biomacromolecule drug under the action of polyethylene glycol and polyethyleneimine. Preferably, the biomacromolecule drug is selected from nucleic acid macromolecule drugs and protein macromolecule drugs, for example, selected from but not limited to at least one of albumin, alcohol dehydrogenase, β-galactosidase, catalase, and DUF5 protein. Preferably, the mass ratio of the polyethyleneimine, the metal-organic cage and the polyethylene glycol is 100:(5-20):(80-120). Preferably, the mass ratio of the polyethyleneimine to the biomacromolecule drug is 100:(10-230).

3. The biomacromolecule delivery system according to claim 1 or 2, characterized in that: The metal-organic cage is selected from ligands and metal ions and assembled by coordination. Preferably, the ligand is selected from the compounds shown in formula I. Wherein, R1 is selected from C 3-12 Heterocyclic group, C5-C 14 Aryl; -L- represents a linking group, which can be selected from unsubstituted C 1-50 Alkylene, or optionally containing one or more heteroatoms C 1-50 The heteroatom may be selected from O and S. R2 is selected from a group having a host-guest effect and / or a cross-linking effect, and can be selected from an adamantane group, an alkynyl group, and an azide group. Preferably, the metal-organic cage is selected from a compound having a structure as shown in Formula A, wherein: It refers to the connection point, and m is selected from 1 to 100.

4. The biomacromolecule delivery system according to any one of claims 1 to 3, characterized in that: The polyethylene glycol is selected from polyethylene glycol containing a first modification group, and its structure is shown in Formula B: Wherein, R3 is the first modifying group selected from adamantane, azide, and alkynyl; and n is selected from 1-300. Preferably, the molecular weight of the polyethylene glycol is 100-10000. Preferably, the polyethylene glycol is selected from compounds having at least one structure of the following formulas B-1, B-2 and B-3: Wherein, in formulas B-1, B-2 and B-3, n is selected from 1-300.

5. The biomacromolecule delivery system according to any one of claims 1 to 4, characterized in that: The polyethyleneimine is selected from polyethyleneimine containing a second modifying group, which contains a repeating unit as shown in formula C; Wherein, R4 is a second modifying group selected from a cyclodextrin group, an azide group, and an alkyne group. Preferably, the molecular weight of the polyethyleneimine is 100-50000. Preferably, the polyethyleneimine is selected from polyethyleneimine PEI-βCD containing a cyclodextrin group. Preferably, the polyethyleneimine PEI-βCD has a structure as shown in Formula C-1, wherein βCD is a cyclodextrin group. Preferably, the structure of βCD in formula C-1 is as shown in formula D.

6. The biomacromolecule delivery system according to any one of claims 1 to 5, characterized in that: The mass ratio of the polyethyleneimine, the metal-organic cage and the polyethylene glycol is 100:(5-20):(80-120). Preferably, the mass ratio of the polyethyleneimine to the biomacromolecule drug is 100:(10-230). Preferably, the administration method of the biomacromolecule delivery system is intravenous administration.

7. The method for constructing a biomacromolecule delivery system according to any one of claims 1 to 6, characterized in that: The construction method comprises: self-assembling the metal-organic cage, polyethylene glycol, polyethyleneimine and biomacromolecule drugs to obtain a biomacromolecule complex, which is the biomacromolecule delivery system.

8. The construction method according to claim 7, characterized in that: The construction method specifically comprises the following steps: (1) preparing a storage solution of a biomacromolecule drug, a storage solution of a metal-organic cage, a storage solution of polyethyleneimine, and a storage solution of polyethylene glycol respectively; (2) The storage solution of polyethyleneimine, the storage solution of biomacromolecule drugs, the storage solution of polyethylene glycol and the storage solution of metal-organic cages in step (1) are mixed and stirred to obtain a mixed solution, and a biomacromolecule complex is obtained by self-assembly of metal-organic cages, polyethylene glycol, polyethyleneimine and biomacromolecule drugs. After centrifugal purification, the biomacromolecule complex is dispersed in a buffer solution to obtain the biomacromolecule delivery system.

9. The construction method according to claim 7 or 8, characterized in that: In step (2), in the mixed solution, the mass ratio of polyethyleneimine, metal-organic cage and polyethylene glycol is 100:(5-20):(80-120). Preferably, in step (2), in the mixed solution, the mass ratio of the polyethyleneimine to the biomacromolecule drug is 100:(10-230). Preferably, in step (2), the concentration of the biomacromolecule drug in the mixed solution is 0.1-1 mg / mL. Preferably, in step (2), the concentration of polyethyleneimine in the mixed solution is 0.5-4 mg / mL. Preferably, in step (2), the concentration of the metal-organic cage in the mixed solution is 0.025-0.8 mg / mL. Preferably, in step (2), the concentration of polyethylene glycol in the mixed solution is 0.4-5 mg / mL. Preferably, in step (2), the mixing and stirring conditions are: stirring at room temperature for 0.25-2h.

10. Use of the biomacromolecule delivery system according to any one of claims 1 to 6 in lung tissue selective drug delivery and enzyme catalysis.

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