Glucose-responsive polyamino acid multifunctional nanovesicles and their preparation and application

Abstract

The present invention relates to the field of smart materials technology, specifically to glucose-responsive polyamino acid multifunctional nanoparticles, their preparation method, and applications. The present invention provides polyamino acid multifunctional nanoparticles comprising a polyamino acid derivative and glucose oxidase; the structure of the polyamino acid derivative is shown in Formula I. The present invention provides sugar-responsive polyamino acid multifunctional nanovesicles. The resulting nanomaterial has excellent sugar sensitivity, capable of undergoing structural and functional changes under high-sugar conditions. It exhibits high specificity and strong controllability, along with long-lasting circulation, effectively mitigating the risk of hypoglycemia. Its intrinsic fluorescence properties provide it with excellent tracing capabilities. Furthermore, its preparation method is simple and its biocompatibility is excellent. This, in turn, offers promising application potential in areas such as self-assembly, biosensing, drug delivery, disease diagnosis, and treatment. #imgabs0#

Description

Technical Field

[0001] The present invention relates to the technical field of smart materials, and in particular to a glucose-responsive polyamino acid multifunctional nanoparticle, a preparation method and an application thereof. Background Art

[0002] Diabetes is primarily treated with oral medications and insulin injections. Oral medications have limited applicability and unsatisfactory efficacy; the majority of diabetic patients require insulin injections. Currently, over 10 million diabetic patients in my country require long-term, repeated insulin injections, with an average annual cost per person exceeding 10,000 yuan, totaling 100 billion yuan. In addition to the significant expense, direct insulin injections present numerous challenges, including low controllability, the susceptibility to side effects such as hypoglycemia, and poor patient compliance due to their short duration of action and high injection frequency. Therefore, safer, more effective, and more economical diabetes treatments are urgently needed.

[0003] The rapid development of nanotechnology has had a profound impact on diabetes treatment, significantly improving the efficacy of diabetes treatment regimens. Nanoparticles offer excellent biocompatibility and degradability, customizable size and surface properties, high drug loading and delivery efficiency, and excellent circulatory stability. These properties not only enhance drug bioavailability but also reduce the side effects of systemic administration. Consequently, nanoparticles are widely used in drug delivery systems. Polymers can self-assemble into diverse morphologies, with vesicles, a nanoassembly with a hydrophobic bilayer enclosing a hydrophilic cavity, closely resembling the structure of cell membranes. These vesicles are more complex than micelles with a simple shell-core structure. Due to the hydrophobic interactions and chain entanglement of polymer chains within their bilayers, polymer vesicles exhibit enhanced stability in diverse physical and chemical environments. Since their development, the potential applications of polymer vesicles have broadened to include disease diagnosis and treatment, nanoreactors, artificial organs, energy storage, antimicrobial therapy, and wastewater treatment.

[0004] Although nanovesicles have a stable structure, their membrane permeability is often poor. Some common small molecules, ions, and even water molecules in some systems have difficulty passing through the double-layer membrane structure of the vesicles. Large molecular drugs such as insulin are even more difficult to penetrate the membrane and be released. This greatly limits the application of polymer vesicles in the field of disease treatment. Although scientists have designed a series of stimulus-responsive vesicles, most of them are disintegrating vesicles, which can lead to explosive release of drugs and bring about a series of side effects. The design of non-disintegrating vesicles also has many drawbacks, such as difficulty in regulating drug release rate and specificity, complex processes, and high costs. Therefore, it is of great significance to design new sugar-responsive nanoparticles to intelligently control drug release. Summary of the Invention

[0005] To address these deficiencies, the present invention provides sugar-responsive polyamino acid multifunctional nanovesicles. The resulting nanomaterial exhibits excellent sugar sensitivity, enabling structural and functional changes under high-glucose conditions. It possesses high specificity and strong controllability, along with long-lasting circulation, effectively mitigating the risk of hypoglycemia. Its bulk fluorescence imparts excellent tracing properties, while its preparation method is simple and its biocompatibility is excellent. This leads to promising applications in areas such as self-assembly, biosensing, drug delivery, and disease diagnosis and treatment.

[0006] The technical solution of the present invention:

[0007] The first technical problem to be solved by the present invention is to provide a polyamino acid multifunctional nanoparticle, wherein the polyamino acid multifunctional nanoparticle comprises a polyamino acid derivative and glucose oxidase; the structure of the polyamino acid derivative is shown in Formula I:

[0008]

[0009] Wherein, R1 and R2 are substituents containing M groups, and the M groups are: R'-phenyl, C2-C 20 Straight chain alkyl, C3-C 60 At least one of a branched alkyl group, a biphenyl group, an R"-biphenyl group, a dehydrogenation group of a hydrophobic fluorescent dye molecule and its derivatives, a dehydrogenation group of a hydrophobic bioactive molecule and its derivatives, and a straight or branched alkyl group containing multiple cyclic structures, wherein R' is C2-C 20 Straight chain alkyl or C3-C 60 Branched alkyl, R "is C1-C 20 Straight chain alkyl or C3-C 60 branched alkyl, n (polymerization degree) ≥ 5, m = 1, 2 or 4;

[0010] The Y is a group containing Z, wherein Z is a group obtained by deamination of polyethylene glycol, polyvinyl alcohol, polyacrylic acid, dextran or polymannose derivatives; the A is an amide bond ester bond ether bond or thioether bond In the present invention, Refers to random copolymers.

[0011] Furthermore, 10≤n≤100, preferably 15≤n≤50.

[0012] Further, the Y is a methoxy polyethylene glycol amine deamination group 20≤x≤150, desaccharin amino deamination group 15≤y≤100; more preferably a methoxypolyethylene glycol amine deamino group.

[0013] Furthermore, in R1 and R2, the dehydrogenation groups of the hydrophobic fluorescent dye molecules and their derivatives are selected from: Nile red and its derivative dehydrogenation groups, fluorescein isothiocyanate and its derivative dehydrogenation groups, anthocyanin fluorescent dyes and their dehydrogenation groups, pyrene and its derivative dehydrogenation groups, dehydrogenation groups of molecules with aggregation-induced emission effect, perylene and its derivative dehydrogenation groups or lipophilic carbocyanine dyes and their derivative dehydrogenation groups; the dehydrogenation groups of the hydrophobic bioactive molecules and their derivatives are selected from: doxorubicin and its derivatives The dehydrogenation group of the derivative, the dehydrogenation group of paclitaxel and its derivatives, the dehydrogenation group of camptothecin and its derivatives, the dehydrogenation group of ciprofloxacin and its derivatives or the dehydrogenation group of plant amino alcohol derivatives and their dehydrogenation groups; the straight chain or branched alkyl group containing multiple ring structures is selected from: azophenyl, naphthyl, R "-naphthyl, phenanthrenyl, R "-phenanthrenyl, anthracenyl, R "-anthracenyl, fluoranthrenyl, R "-fluoranthrenyl, chrysene, R "-chrysene, fluorenyl, R "-fluorenyl, acenaphthenyl, R "-acenaphthenyl or hexadecyl; R " is C1-C 20 Straight chain alkyl or C3-C 60 branched alkyl.

[0014] Preferably, the M group in R1 and R2 is selected from one of the following structural formulas:

[0015]

[0016]

[0017] Furthermore, the structure and function of the polyamino acid derivative represented by Formula I can be transformed under acidic conditions.

[0018] The second technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned polyamino acid multifunctional nanoparticles, wherein the preparation method comprises: preparing the polyamino acid multifunctional nanoparticles by self-assembly of the polyamino acid derivative represented by formula I and glucose oxidase.

[0019] Furthermore, in the polyamino acid multifunctional nanoparticles, the mass ratio of the glucose oxidase loading to the polyamino acid derivative is 1:20-100, that is, the mass ratio of the actual mass of the glucose oxidase finally self-assembled in the polyamino acid derivative to the mass of the polyamino acid derivative.

[0020] Furthermore, the polyamino acid derivative represented by formula I is selected from: polyethylene glycol-polylysine benzene-polyglutamic acid biphenyl, polyethylene glycol-polylysine anthracene-polyglutamic acid anthracene, polyethylene glycol-polylysine dodecyl-polyglutamic acid fluorene, polyethylene glycol-polylysine benzene-polyglutamic acid pyrene, polyethylene glycol-polylysine anthracene-polyglutamic acid tetraphenylethylene, polyethylene glycol-polylysine benzene-polyglutamic acid perylene or polyethylene glycol-polylysine anthracene-polyglutamic acid sterane.

[0021] Furthermore, the polyamino acid derivative represented by formula I is prepared by any of the following methods:

[0022] Method 1: The polyamino acid derivative of formula I is prepared by ring-opening polymerization of the lysine derivative intracyclic carboxylic anhydride of formula II, the amino acid derivative intracyclic carboxylic anhydride of formula III, and the substance of formula IV;

[0023]

[0024] Wherein, A is an amide bond ester bond ether bond or thioether bond m = 1, 2, or 4;

[0025] or:

[0026] Method 2: A polyamino acid is obtained by ring-opening polymerization of a lysine ring carboxylic anhydride protected by benzyloxycarbonyl, tert-butyloxycarbonyl or trifluoroacetyl, an amino acid derivative ring carboxylic anhydride represented by formula III, and a substance represented by formula IV; then, the polyamino acid with exposed lysine side chain amino groups is obtained by deprotection; finally, the substance represented by formula V is reacted with the polyamino acid with exposed lysine side chain amino groups to obtain the polyamino acid derivative represented by formula I; wherein the structure of the substance represented by formula V is The R1 is as defined above.

[0027] Furthermore, the lysine intracyclic carboxylic anhydride protected by benzyloxycarbonyl, tert-butyloxycarbonyl or trifluoroacetyl is selected from: N6-benzyloxycarbonyl-L-lysine intracyclic anhydride, N-α-tert-butyloxycarbonyl-L-lysine intracyclic anhydride or Nε-trifluoroacetyl-L-lysine intracyclic anhydride.

[0028] Preferably, the compound of formula IV is methoxypolyethylene glycol amine.

[0029] Furthermore, in the above-mentioned method 1 and method 2, in the ring-opening polymerization, the reaction temperature is 25 to 50° C. (preferably 35° C.), and the reaction time is 48 to 96 hours, preferably 72 hours.

[0030] Furthermore, the polyamino acid multifunctional nanoparticles are the following nanomaterials:

[0031] The nanomaterial formed by self-assembly of polyethylene glycol-polylysine benzene-polyglutamic acid biphenyl and glucose oxidase; or:

[0032] The nanomaterial formed by self-assembly of polyethylene glycol-polylysine anthracene-polyglutamic acid anthracene and glucose oxidase; or:

[0033] The nano material is formed by self-assembly of polyethylene glycol-polylysine dodecyl-polyglutamate fluorene and glucose oxidase.

[0034] Furthermore, the self-assembly method is: co-assembly or dropwise addition method.

[0035] The third technical problem to be solved by the present invention is to point out the use of the above-mentioned polyamino acid nanoparticles in biosensing or drug delivery.

[0036] Beneficial effects of the present invention:

[0037] Compared with the existing technology, the sugar-responsive polyamino acid multifunctional nanoparticles provided by the present invention have excellent sugar sensitivity and can quickly respond to high sugar environments; under the action of high sugar environments, they can efficiently and quickly achieve rapid transformation of the structure and function of the nanomaterial; and without the need for additional cross-linking and other means, they can ensure the integrity of the nanovesicles and achieve efficient and long-lasting release and long-term circulation of drugs; effectively avoid the risk of drug burst release and improve the bioavailability of drugs; in addition, the sugar-responsive polyamino acid multifunctional nanoparticles have good fluorescence properties without the need for additional introduction of other fluorescent molecules and can be traced in vivo. Unlike existing nanovesicle permeability control technologies, the sugar-responsive polyamino acid multifunctional nanoparticles provided by the present invention have stronger controllability and structural stability, excellent sugar sensitivity and bulk fluorescence tracing performance, and the preparation method is relatively simple. Therefore, the functional polyamino acid derivatives provided by the present invention have huge application potential in self-assembly, biosensing, drug delivery, disease diagnosis and treatment, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the fluorescence spectrum of the polyethylene glycol-polylysine benzene-polyglutamic acid biphenyl / glucose oxidase nanoparticles encapsulating R6G obtained in Example 2.

[0039] Figure 2 This is a transmission electron microscopy image of the polyethylene glycol-polylysine benzene-polyglutamic acid biphenyl / glucose oxidase nanoparticles obtained in Example 2.

[0040] Figure 3 This is the fluorescence spectrum of the polyethylene glycol-polylysine benzene-polyglutamic acid biphenyl / glucose oxidase nanoparticles obtained in Example 2.

[0041] Figure 4 The polyethylene glycol-polylysine benzene-polyglutamic acid biphenyl / glucose oxidase nanoparticles encapsulated R6G in a high concentration glucose environment (400 mg dL -1 ) under fluorescence spectrum.

[0042] Figure 5The polyethylene glycol-polylysine benzene-polyglutamic acid biphenyl / glucose oxidase nanoparticles obtained in Example 3 were subjected to high concentration glucose (400 mg dL -1 ) under transmission electron microscopy.

[0043] Figure 6 These are the fluorescence spectra of the polyethylene glycol-polylysine anthracene-polyglutamic acid anthracene / glucose oxidase nanoparticles encapsulating R6G obtained in Example 5 under different environments.

[0044] Figure 7 These are transmission electron micrographs of the polyethylene glycol-polylysine anthracene-polyglutamic acid anthracene / glucose oxidase nanoparticles obtained in Example 5 under different environments.

[0045] Figure 8 This is the fluorescence spectrum of the polyethylene glycol-polylysine benzene / glucose oxidase nanoparticles obtained in Example 7.

[0046] Figure 9 This is a transmission electron microscopy image of the polyethylene glycol-polylysine benzene / glucose oxidase nanoparticles obtained in Example 7.

[0047] Figure 10 This is a CD transmission electron microscopy image of the polyethylene glycol-polylysine benzene / glucose oxidase nanomaterial obtained in Example 7 in a high sugar environment.

[0048] Figure 11 This is the release curve of R6G in Experimental Example 1 from polyethylene glycol-polylysine benzene-polyglutamic acid biphenyl / glucose oxidase in a high sugar environment.

[0049] Figure 12 This is the insulin release curve of polyethylene glycol-polylysine benzene-polyglutamic acid biphenyl / glucose oxidase in Experimental Example 1 under different sugar concentration environments.

[0050] Figure 13 The polyethylene glycol-polylysine anthracene-polyglutamic acid anthracene / glucose oxidase in the high sugar environment (400 mg dL -1 ) release curve of R6G in .

[0051] Figure 14 This is the fluorescence imaging of the polyethylene glycol-polylysine benzene-polyglutamic acid biphenyl / glucose oxidase nanoparticles loaded with FITC-labeled insulin in Experimental Example 2 24 hours after injection into diabetic mice.

[0052] Figure 15 The changes in insulin content in the blood of diabetic mice after the nanoparticles prepared in Example 5 and Example 7 loaded with insulin in Experimental Example 2 were injected into the body.

[0053] Figure 16The changes in insulin content in diabetic mice were observed after the insulin-loaded nanoparticles prepared in Example 2, Example 5 and Example 6 in Experimental Example 3 were injected into the body.

[0054] Figure 17 The changes in blood sugar levels after the nanoparticles loaded with insulin prepared in Example 2 in Experimental Example 3 were injected into normal mice ( Figure 17 a) and low glycemic index ( Figure 17 b). DETAILED DESCRIPTION

[0055] The present invention provides sugar-responsive polyamino acid multifunctional nanoparticles, which are self-assembled from a polyamino acid derivative represented by Formula I and glucose oxidase. Nanoparticles prepared by encapsulating insulin in this sugar-responsive polyamino acid multifunctional nanomaterial undergo structural transformation in a high-sugar environment, effectively improving vesicle membrane permeability, rapidly and stably releasing the encapsulated insulin, and controlling blood glucose levels within a normal range for an extended period of time. Furthermore, the speed of structural transformation and functional regulation is closely related to the concentration of glucose oxidase and blood glucose concentration, resulting in excellent controllability and no side effects such as hypoglycemia. Furthermore, the polyamino acid derivative represented by Formula I exhibits inherent fluorescent properties without the introduction of other fluorescent molecules, enabling in vivo tracing.

[0056] In the polyamino acid derivatives represented by Formula I of the present invention, the benzylimide bond in the side chain of the lysine derivative breaks under weakly acidic conditions, causing the hydrophobic R1 group to fall off and expose the hydrophilic amino group. However, the side chain of the amino acid derivative containing the R2 group does not contain an acid-sensitive bond. The hydrophobic interaction and π-π stacking interaction between the rigid hydrophobic groups in the side chain can maintain the integrity of the nanoparticles. Therefore, under weakly acidic conditions, the nanoparticles formed by the polyamino acid derivatives form microchannels composed of hydrophilic amino groups while maintaining their integrity, thereby releasing the entrapped drug.

[0057] The following examples are given to specifically describe the present invention. However, it is worth noting that the following examples are only used to further illustrate the present invention and are not to be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made to the present invention by ordinary professionals in this field based on the contents of the above invention still fall within the scope of protection of the present invention.

[0058] Example 1 Preparation of polyethylene glycol-polylysine benzene-polyglutamic acid biphenyl

[0059] 1) Preparation of lysine-benzene

[0060] Under argon protection, lysine (4.0 g) and 4A molecular sieves were dissolved in N,N-dimethylformamide (DMF) (50 mL), stirred at 40°C, and triethylamine was added to adjust the base. Then, 4-phenylbenzaldehyde was added. After two days of reaction, the product was filtered out, precipitated with diethyl ether three times, and dried in vacuo to obtain lysine-benzene monomer (yield 70%).

[0061] 2) Preparation of glutamic acid-biphenyl

[0062] Benzyl glutamic acid (4.0 g) was dissolved in N,N-dimethylformamide (DMF) (50 mL), and 4-phenylaniline (2.0 g) was added. The mixture was reacted at 45°C for 48 h. After the reaction was stopped, the mixture was concentrated under reduced pressure, and the product was extracted with dichloromethane. The resulting oil phase was washed twice with a saturated aqueous sodium bicarbonate solution and a saturated sodium chloride solution. After the aqueous phase was reversely washed, the organic phases were combined and dried over anhydrous sodium sulfate overnight. The mixture was filtered and concentrated, and the resulting solid was dried and purified by column chromatography to obtain glutamic acid-biphenyl monomer (yield 60%).

[0063] 3) Preparation of lysine-benzene ring carboxylic acid anhydride

[0064] The lysine-benzene monomer (3.2 g) prepared above was dissolved in 30 mL of anhydrous tetrahydrofuran, and a tetrahydrofuran solution (20 mL) of triphosgene (0.56 g) was slowly added. The reaction was incubated at 50°C for 4 h. The tetrahydrofuran was then concentrated, and the resulting product was recrystallized three times from a tetrahydrofuran / n-hexane mixed solvent and then dried for storage to obtain lysine-benzene ring carboxylic anhydride with a yield of 65%.

[0065] 4) Preparation of glutamic acid-biphenyl ring carboxylic anhydride

[0066] The glutamic acid-biphenyl monomer prepared above (3 g) was dissolved in 30 mL of anhydrous tetrahydrofuran, and a tetrahydrofuran solution (20 mL) of triphosgene (0.56 g) was slowly added. The reaction was incubated at 50° C. for 4 h. The tetrahydrofuran was then concentrated, and the resulting product was recrystallized three times from a tetrahydrofuran / n-hexane mixed solvent and then dried to obtain glutamic acid-biphenyl ring carboxylic anhydride with a yield of 60%.

[0067] 5) Preparation of functional polyethylene glycol-polylysine benzene-polyglutamic acid biphenyl

[0068] Under argon, the lysine-benzene ring carboxylic anhydride (2.41 g) and glutamic acid-biphenyl ring carboxylic anhydride (1.8 g) obtained above were placed in a Schlenk flask and dissolved in 30 mL of DMF. Methoxypolyethylene glycolamine (MPEG-NH2, Mw 5000) (0.96 g) was azeotropically dehydrated with toluene and dissolved in 10 mL of anhydrous DMF. This was then added to the reaction system, stirred at 35°C with argon bubbling, and allowed to react for 3 days. After completion of the reaction, the mixture was precipitated three times in icy ether and vacuum dried for 48 hours to obtain polyethylene glycol-polylysine benzene-polyglutamic acid biphenyl.

[0069] Example 2 Preparation of Sugar-Responsive Polyethylene Glycol-Polylysine Phenyl-Polyglutamic Acid Biphenyl / Glucose Oxidase Nanoparticles

[0070] 10 mg of the polyethylene glycol-polylysine benzene-polyglutamic acid biphenyl obtained in Example 1 was dissolved in 1 mL of DMF and slowly added dropwise at a rate of 30 s / d to 8 mL of rapidly stirred deionized water containing 2 mg of glucose oxidase. After the addition was complete, stirring was continued for half an hour. The liquid was then transferred to a dialysis bag with a MWCO of 3500 and dialyzed in deionized water for 3 days, with the water changed every 3 hours. The liquid was then centrifuged (3500 r / min), filtered (0.45 μm), and the volume was adjusted to 10 mL to obtain polyethylene glycol-polylysine benzene-polyglutamic acid biphenyl / glucose oxidase nanoparticles.

[0071] The encapsulation amount of glucose oxidase in the polyethylene glycol-polylysine benzene-polyglutamic acid biphenyl / glucose oxidase nanoparticles (encapsulation amount, i.e., the actual loading amount of glucose oxidase) was 0.035 mg / mL by protein detection agent. The particle size of the prepared nanomaterial was determined by dynamic light scattering (DLS), and the result was 136 nm. The morphology of the nanomaterial was characterized by transmission electron microscopy (TEM) and fluorescence encapsulation method. The results are as follows: Figure 1 and 2 The fluorescence properties of polyethylene glycol-polylysine benzene-polyglutamic acid biphenyl / glucose oxidase nanoparticles were tested by fluorescence spectrometer. Figure 3 As shown, the obtained nanomaterials exhibit certain fluorescence properties.

[0072] Example 3 Polyethylene glycol-polylysine benzene-polyglutamic acid biphenyl / glucose oxidase responsiveness test

[0073] The polyethylene glycol-polylysine benzene-polyglutamic acid biphenyl / glucose oxidase nanoparticles (3 mL) prepared in Example 2 were placed in different sugar concentration environments (normal physiological environment, high sugar environment 400 mg dL -1 Dynamic light scattering (DLS) was used to determine the concentration of 1% β-glucose in a high sugar environment (400 mg dL-1 The particle size of the nanoparticles was 152 nm. The morphology of the nanoparticles was characterized by transmission electron microscopy (TEM) and fluorescence encapsulation method. The results are as follows Figure 4 and 5 As shown in the figure, the prepared nanomaterial still has a complete vesicle structure.

[0074] Example 4 Preparation of polyethylene glycol-polylysine anthracene-polyglutamic acid anthracene

[0075] 1) Preparation of lysine-anthracene

[0076] Under argon protection, lysine (4.0 g) and 4A molecular sieves were dissolved in N,N-dimethylformamide (DMF) (50 mL), stirred at 40°C, and triethylamine was added to adjust the base. 3-(anthracen-10-yl)benzaldehyde was added. After two days of reaction, the product was filtered out, precipitated with ether three times, and dried in vacuo to obtain lysine-anthracene monomer (yield 68%).

[0077] 2) Preparation of glutamic acid-anthracene

[0078] Benzyl glutamic acid (4.0 g) was dissolved in N,N-dimethylformamide (DMF) (50 mL), and anthracene-9-ylmethylamine (2.1 g) was added. The mixture was reacted at 45° C. for 48 h. After the reaction was stopped, the mixture was concentrated under reduced pressure, and the product was extracted with dichloromethane. The resulting oil phase was washed twice with a saturated aqueous sodium bicarbonate solution and a saturated sodium chloride solution. After the aqueous phase was reversely washed, the organic phases were combined and dried over anhydrous sodium sulfate overnight. The mixture was filtered and concentrated, and the resulting solid was dried and purified by column chromatography to obtain a glutamic acid-anthracene monomer (yield 62%).

[0079] 3) Preparation of lysine-anthracycline carboxylic anhydride

[0080] The lysine-anthracene monomer (3.8 g) prepared above was dissolved in 30 mL of anhydrous tetrahydrofuran, and a tetrahydrofuran solution (20 mL) of triphosgene (1.26 g) was slowly added. The reaction was incubated at 50°C for 4 h. The tetrahydrofuran was then concentrated, and the resulting product was recrystallized three times from a tetrahydrofuran / n-hexane mixed solvent and then dried to obtain lysine-anthracene ring carboxylic anhydride with a yield of 67%.

[0081] 4) Preparation of glutamic acid-anthracenyl carboxylic anhydride

[0082] The glutamic acid-anthracene monomer prepared above (3.4 g) was dissolved in 30 mL of anhydrous tetrahydrofuran, and a tetrahydrofuran solution (20 mL) of triphosgene (1.26 g) was slowly added. The reaction was incubated at 50°C for 4 h. The tetrahydrofuran was then concentrated, and the resulting product was recrystallized three times from a tetrahydrofuran / n-hexane mixed solvent and then dried to obtain glutamic acid-anthracene ring carboxylic anhydride with a yield of 62%.

[0083] 5) Preparation of functional polyethylene glycol-polylysine anthracene-polyglutamic acid anthracene

[0084] Under argon, the lysine-anthracycline carboxylic anhydride (1.6 g) and glutamic acid-anthracycline carboxylic anhydride (3.3 g) obtained above were placed in a Schlenk flask and dissolved in 30 mL of DMF. Methoxypolyethylene glycolamine (MPEG-NH2, Mw 5000) (0.96 g) was azeotropically dehydrated with toluene and then dissolved in 10 mL of anhydrous DMF. The mixture was stirred at 35°C with argon bubbling and allowed to react for 3 days. After completion of the reaction, the mixture was precipitated three times in icy ether and dried under vacuum for 48 hours to obtain polyethylene glycol-polylysine anthracene-polyglutamic acid anthracene.

[0085] Example 5 Preparation and responsiveness test of sugar-responsive polyethylene glycol-polylysine anthracene-polyglutamic acid anthracene / glucose oxidase nanomaterials

[0086] 10 mg of the polyethylene glycol-polylysine anthracene-polyglutamic acid anthracene obtained in Example 4 was dissolved in 1 mL of dichloromethane and slowly added dropwise to rapidly stirred deionized water (9 mL) at a rate of 30 s / d. After completion of the addition, stirring was continued for half an hour, and then the liquid was transferred to a dialysis bag with a MWCO of 3500 and dialyzed in deionized water for 3 days, with the water changed every 3 hours. The liquid was then centrifuged (3500 r / min), filtered (0.45 μm), and constant volume was obtained to obtain a polyethylene glycol-polylysine anthracene-polyglutamic acid anthracene nanomaterial. 3 mg of glucose oxidase was dissolved in 0.5 mL of DMSO and then slowly added dropwise to the rapidly stirred polylysine-polycysteine anthracene-polyglutamic acid anthracene nanomaterial solution at a rate of 30 s / d. After the addition was complete, stirring was continued for half an hour, and then the liquid was transferred to a dialysis bag with a MWCO of 3500 and dialyzed in deionized water for 3 days, with the water being changed every 3 hours. The liquid was then centrifuged (3500 r / min), filtered (0.45 μm), and the volume was adjusted to 10 mL to obtain polyethylene glycol-polylysine anthracene-polyglutamic acid anthracene / glucose oxidase nanomaterial.

[0087] The polyethylene glycol-polylysine anthracene-polyglutamic acid anthracene / glucose oxidase nanoparticles (3 mL) were placed in different sugar concentration environments. The loading amount of glucose oxidase was 0.041 mg / mL as determined by protein detection reagent. Dynamic light scattering (DLS) was used to determine the loading amount of glucose oxidase in normal environment and high sugar environment (400 mg dL). -1 The particle size of the nanoparticles is 125nm under normal conditions and 132nm under high sugar conditions. The morphology of the nanoparticles in normal and high sugar environments was characterized by transmission electron microscopy (TEM) and fluorescence encapsulation method. The results are as follows Figure 6 and 7As shown in Figure 2, the prepared nanoparticles are vesicle structures under normal and high sugar environments. The fluorescence properties of polyethylene glycol-polylysine benzene-polyglutamic acid biphenyl / glucose oxidase nanoparticles were tested by fluorescence spectrometer. The results are shown in Figure 2. Figure 8 As shown, the obtained nanomaterials exhibit certain fluorescence properties.

[0088] Example 6 Preparation of polyethylene glycol-polylysine dodecyl-polyglutamic acid fluorene / glucose oxidase nanomaterials

[0089] 1) Preparation of lysine-dodecyl

[0090] Under argon protection, lysine (4.0 g) and 4A molecular sieves were dissolved in N,N-dimethylformamide (DMF) (50 mL), stirred at 40°C, and triethylamine was added to adjust the base. Then, dodecylbenzaldehyde was added. After two days of reaction, the product was filtered out, precipitated with ether three times, and dried in vacuo to obtain lysine-dodecyl monomer (yield 68%).

[0091] 2) Preparation of glutamic acid-fluorene

[0092] Benzyl glutamic acid (4.0 g) was dissolved in N,N-dimethylformamide (DMF) (50 mL), and 2-aminofluorene (1 g) was added. The mixture was reacted at 45°C for 48 h. After the reaction was stopped, the mixture was concentrated under reduced pressure, and the product was extracted with dichloromethane. The resulting oil phase was washed twice with a saturated aqueous sodium bicarbonate solution and a saturated sodium chloride solution. After the aqueous phase was reversely washed, the organic phases were combined and dried over anhydrous sodium sulfate overnight. The mixture was filtered and concentrated, and the resulting solid was dried and purified by column chromatography to obtain a glutamic acid-fluorene monomer (yield 62%).

[0093] 3) Preparation of lysine-dodecyl ring carboxylic anhydride

[0094] The lysine-dodecyl monomer prepared above (3.6 g) was dissolved in 30 mL of anhydrous tetrahydrofuran, and a tetrahydrofuran solution (20 mL) of triphosgene (0.15 g) was slowly added. The reaction was incubated at 50° C. for 4 h. The tetrahydrofuran was then concentrated, and the resulting product was recrystallized three times from a tetrahydrofuran / n-hexane mixed solvent and then dried for storage to obtain lysine-dodecyl intracyclic carboxylic anhydride with a yield of 71%.

[0095] 4) Preparation of glutamic acid-fluorene ring carboxylic anhydride

[0096] The glutamic acid-fluorene monomer prepared above (3.5 g) was dissolved in 30 mL of anhydrous tetrahydrofuran, and a tetrahydrofuran solution (20 mL) of triphosgene (1.2 g) was slowly added. The mixture was reacted at 50°C for 4 h. The tetrahydrofuran was then concentrated, and the resulting product was recrystallized three times from a tetrahydrofuran / n-hexane mixed solvent and then dried for storage to obtain glutamic acid-fluorene ring carboxylic anhydride; the yield was 64%.

[0097] 5) Preparation of functional polyethylene glycol-polylysine dodecyl-polyglutamic acid fluorene

[0098] Under argon, the above-obtained lysine-dodecyl carboxylic anhydride (1.6 g) and glutamic acid-fluorene carboxylic anhydride (2.6 g) were placed in a Schlenk flask and dissolved in 30 mL of DMF. Methoxypolyethylene glycolamine (MPEG-NH2, Mw 5000) (0.96 g) was azeotropically dehydrated with toluene and then dissolved in 10 mL of anhydrous DMF. The mixture was stirred at 35°C with argon bubbling and allowed to react for 3 days. After completion of the reaction, the mixture was precipitated three times in icy ether and vacuum dried for 48 hours to obtain polyethylene glycol-polylysine-dodecyl-polyglutamic acid-fluorene.

[0099] 6) Preparation of nanomaterials

[0100] 10 mg of the polyethylene glycol-polylysine dodecyl-polyglutamic acid fluorene obtained above was dissolved in 1 mL of DMF and slowly added dropwise at a rate of 30 s / d to 8 mL of rapidly stirring deionized water containing 1.4 mg of glucose oxidase. Stirring was continued for half an hour after the addition was complete. The solution was then transferred to a dialysis bag with a MWCO of 3500 and dialyzed against deionized water for three days, with the water changed every three hours. The solution was then centrifuged (3500 rpm), filtered (0.45 μm), and the volume was adjusted to 10 mL to obtain polyethylene glycol-polylysine dodecyl-polyglutamic acid fluorene / glucose oxidase nanoparticles. A protein detection assay showed a glucose oxidase loading of 0.022 mg / mL.

[0101] Example 7 Preparation of Sugar-Responsive Polyethylene Glycol-Polylysine Benzene / Glucose Oxidase Nanoparticles (Comparative Example)

[0102] Under argon, 3.1 g of the lysine-benzene ring carboxylic anhydride obtained in Example 1 was placed in a Schlenk flask and dissolved in 30 mL of DMF. Methoxypolyethylene glycolamine (MPEG-NH2, Mw 5000) (0.96 g) was azeotropically dehydrated with toluene and then dissolved in 10 mL of anhydrous DMF. The mixture was stirred at 35°C with argon bubbling and allowed to react for 3 days. After completion of the reaction, the mixture was precipitated three times in icy ether and vacuum dried for 48 hours to obtain polyethylene glycol-polylysine benzene.

[0103] 10 mg of the polyethylene glycol-polylysine benzene obtained above was dissolved in 1 mL of DMF and slowly added dropwise at a rate of 30 s / d to 8 mL of rapidly stirred deionized water containing 2 mg of glucose oxidase. After the addition was complete, stirring was continued for half an hour. The liquid was then transferred to a dialysis bag with a MWCO of 3500 and dialyzed in deionized water for 3 days, with the water changed every 3 hours. The liquid was then centrifuged (3500 r / min), filtered (0.45 μm), and the volume was adjusted to 10 mL to obtain polyethylene glycol-polylysine benzene / glucose oxidase nanoparticles.

[0104] The loading amount of glucose oxidase was detected by protein detection agent and was 0.029 mg / mL. The particle size of the prepared nanomaterial was determined by dynamic light scattering (DLS) and the result was 127 nm. The morphology of the nanomaterial under different environments was characterized by transmission electron microscopy (TEM). The results are as follows: Figure 9 and 10 (High sugar environment) shows that the prepared nanomaterial has a vesicle structure under normal conditions, but the vesicle structure ruptures under high sugar conditions, indicating that it cannot maintain a stable vesicle structure under high sugar conditions.

[0105] Experimental Example 1 Study on the drug release behavior of sugar-responsive polyamino acid multifunctional nanomaterials

[0106] 200 μL of R6G (rhodamine 6G) and FITC (fluorescein isothiocyanate) labeled insulin aqueous solution (0.2 mg mL -1 ) was added dropwise to 2 mL of the nanomaterials obtained in Example 2 and Example 5 (0.2 mg mL -1 ) solution, stirred for 30 minutes, sonicated for 2 hours, and then dialyzed for 12 hours using a dialysis bag with MWCO 3500, with the water changed every 4 hours. The drug release behavior of the polymer nanoparticles loaded with fluorescent dye under different treatment conditions was detected by fluorescence spectrophotometry. The results of the material obtained in Example 2 are shown in FIG. Figure 11 and Figure 12 As shown in the figure: the polyethylene glycol-polylysine benzene-polyglutamic acid biphenyl nanoparticles obtained in Example 2 have excellent drug release behavior in a high sugar environment, the vesicle membrane permeability is improved, and the release is zero-order release without burst release. In addition, there is basically no significant release in a normal sugar environment, and the release behavior is sugar concentration dependent; the polyethylene glycol-polylysine anthracene-polyglutamic acid anthracene / glucose oxidase nanoparticles obtained in Example 5 have excellent drug release behavior in a high sugar environment, the vesicle membrane permeability is improved ( Figure 13This is mainly due to the fact that in a high-sugar environment, glucose reacts with glucose oxidase to produce a large amount of gluconic acid, which in turn breaks the benzyl imine bond of the lysine side chain, causing the hydrophobic group of the lysine side chain to fall off, generating hydrophilic amino groups. This causes a large number of hydrophilic microbends to appear in the hydrophobic membrane of the vesicles, forming a large number of drug-release microchannels.

[0107] Experimental Example 2: Distribution of Sugar-responsive Polyamino Acid Multifunctional Nanoparticles in Vivo

[0108] The nanomaterial prepared in Example 2 was selected to study the long-term circulation performance of sugar-responsive polyamino acid nanoparticles in vivo. Sugar-responsive polyamino acid nanoparticles loaded with glucose oxidase and FITC-labeled insulin were injected into mice with type I diabetes and healthy mice. Free FITC-labeled insulin (referred to as the free insulin group) was injected into diabetic mice as a control. Figure 14 As shown, in mice injected with free insulin, fluorescence decreased significantly 4 hours after injection, reaching virtually no fluorescence at 24 hours. However, in diabetic mice injected with sugar-responsive polyamino acid nanoparticles, fluorescence was still detected 24 hours after injection, indicating that insulin encapsulated in sugar-responsive vesicles has a longer circulation time than free insulin. Furthermore, when sugar-responsive nanoparticles were injected into healthy mice, they still exhibited significant fluorescence 24 hours after injection, demonstrating that the particles are highly glucose-responsive and release insulin only in a high-glucose environment.

[0109] Furthermore, the nanomaterials prepared in Example 5 and Example 7 were selected to study the circulation time of the insulin they encapsulated in the body. Nanoparticles encapsulating glucose oxidase and insulin were injected into mice with type I diabetes, with free insulin as a control. The insulin content in the mice injected with free insulin and the mice injected with insulin-loaded nanoparticles was monitored using a human insulin enzyme-linked immunosorbent assay. Figure 15 As shown in the figure, the insulin content in mice injected with insulin-loaded polyethylene glycol-polylysine anthracene-polyglutamic acid anthracene / glucose oxidase nanoparticles was significantly higher than that in mice injected with free insulin within 12 hours. This result is basically consistent with the in vivo imaging result of the nanomaterial prepared in Example 2. The insulin content in mice injected with insulin-encapsulated polyethylene glycol-polylysine benzene / glucose oxidase nanoparticles was also significantly lower than that encapsulated by the nanomaterial prepared in Example 7. The insulin content was also significantly lower than that in mice injected with insulin-loaded polyethylene glycol-polylysine anthracene-polyglutamic acid anthracene / glucose oxidase nanoparticles. This is mainly because in a high-sugar environment, the polyethylene glycol-polylysine benzene nanoparticles disintegrated, resulting in a burst release of insulin, which was quickly metabolized, resulting in its low bioavailability.

[0110] Experimental Example 3: In vivo study of diabetes treatment using sugar-responsive polyamino acid multifunctional nanoparticles

[0111] Nanomaterials prepared in Example 2, Example 5 and Example 6 were selected to study the in vivo diabetes treatment performance of sugar-responsive polyamino acid nanoparticles. Insulin and sugar-responsive polyamino acid nanoparticles loaded with glucose oxidase and insulin were added at a concentration of 10 mg kg -1 The blood glucose of mice injected with insulin and the nanomaterials prepared in Examples 2, 5 and 6 loaded with insulin dropped to 95 mg dL within 1 hour. -1 However, the blood sugar level of the free insulin group quickly lost control and returned to the initial level in a short time. However, the blood sugar level of the mice injected with the self-assembly of the nanomaterials prepared in Example 2, Example 5 and Example 6 loaded with insulin maintained a normal range (<200 mg dL -1 ) for 10 h, and then gradually increased to the initial level ( Figure 16 This is mainly due to its glucose-responsive insulin-releasing properties, which enable it to maintain blood sugar stability over a longer period of time.

[0112] Insulin injection often produces symptoms of hypoglycemia. To assess the risk of hypoglycemia caused by sugar-responsive polyamino acid nanoparticles, we injected free insulin and the insulin-loaded nanomaterials prepared in Example 2 into healthy mice. The results showed that the blood sugar of mice injected with free insulin decreased significantly, while the blood sugar of mice injected with the insulin-loaded nanomaterials prepared in Example 2 remained essentially unchanged. Figure 17 a). In addition, we also studied the hypoglycemic index, and the results showed that the mice injected with the nanomaterial prepared in Example 2 loaded with insulin had a lower hypoglycemic index ( Figure 17 b) These results indicate that in healthy mice, the glucose-responsive polyamino acid vesicles did not increase membrane permeability, and essentially no insulin was released, resulting in no hypoglycemic symptoms.

Claims

1. A polyamino acid multifunctional nanoparticle, characterized in that: The polyamino acid multifunctional nanoparticles include a polyamino acid derivative and glucose oxidase; wherein the structure of the polyamino acid derivative is as shown in Formula I: Formula I; Where R1 is C2-C 20 A straight-chain alkyl or anthracenyl group, R2 is a biphenyl group, anthracenyl group or a fluorenyl group, n≥5, m=1, 2 or 4; The Y is a group obtained by deaminating a polyethylene glycol derivative; and the A is an amide bond, an ester bond, an ether bond, or a thioether bond.

2. The polyamino acid multifunctional nanoparticle according to claim 1, characterized in that: 10≤n≤100。 3. The polyamino acid multifunctional nanoparticle according to claim 2, characterized in that: 15≤n≤50。 4. The polyamino acid multifunctional nanoparticle according to any one of claims 1 to 3, characterized in that: The Y is ; Among them, 20≤x≤150.

5. The polyamino acid multifunctional nanoparticles according to claim 1, characterized in that: The polyamino acid derivative is selected from polyethylene glycol-polylysine anthracene-polyglutamic acid anthracene and polyethylene glycol-polylysine dodecyl-polyglutamic acid fluorene.

6. The method for preparing the polyamino acid multifunctional nanoparticles according to any one of claims 1 to 5, characterized in that: The preparation method comprises the following steps: preparing polyamino acid multifunctional nanoparticles by self-assembly of the polyamino acid derivative and glucose oxidase.

7. The method for preparing polyamino acid multifunctional nanoparticles according to claim 6, characterized in that: The mass ratio of the loading amount of glucose oxidase to the polyamino acid derivative is 1:20-100.

8. The method for preparing polyamino acid multifunctional nanoparticles according to claim 6, characterized in that: The polyamino acid derivative is prepared by any of the following methods: Method 1: The polyamino acid derivative is prepared by ring-opening polymerization of the lysine derivative intracyclic carboxylic anhydride represented by formula II, the amino acid derivative intracyclic carboxylic anhydride represented by formula III, and the substance represented by formula IV; Formula II, Formula III, Formula IV; Method 2: A polyamino acid is obtained by ring-opening polymerization of a lysine ring carboxylic anhydride protected by benzyloxycarbonyl, tert-butyloxycarbonyl or trifluoroacetyl, an amino acid derivative ring carboxylic anhydride represented by formula III and a substance represented by formula IV; then, the polyamino acid with exposed lysine side chain amino groups is obtained by deprotection; finally, the substance represented by formula V is reacted with the polyamino acid with exposed lysine side chain amino groups to obtain the polyamino acid derivative; wherein formula III is , Formula IV is , the structural formula of the substance shown in formula V is .

9. The method for preparing polyamino acid multifunctional nanoparticles according to claim 8, characterized in that: The lysine intracyclic carboxylic anhydride protected by benzyloxycarbonyl, tert-butyloxycarbonyl or trifluoroacetyl is selected from: N6-benzyloxycarbonyl-L-lysine intracyclic anhydride, N-α-tert-butyloxycarbonyl-L-lysine intracyclic anhydride or Nε-trifluoroacetyl-L-lysine intracyclic anhydride.

10. The method for preparing polyamino acid multifunctional nanoparticles according to claim 8, characterized in that: In the method 1 and the method 2, in the ring-opening polymerization, the reaction temperature is 25 to 50° C., and the reaction time is 48 to 96 h.

11. Use of polyamino acid multifunctional nanoparticles in preparing biosensing or drug delivery materials, wherein the polyamino acid multifunctional nanoparticles are the nanoparticles according to any one of claims 1 to 5, or are nanoparticles prepared by the method according to any one of claims 6 to 10.

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