Angiopep-2 modified fluorescent polyamide-amine dendritic polymer and its preparation method and preparation method of nanoparticles based thereon

Abstract

This invention discloses an Angiopep-2 modified fluorescent polyamide-amine dendritic polymer and its preparation method, as well as a method for preparing nanoparticles based thereon, belonging to the field of nanomaterials technology. The preparation method includes the following steps: mixing the polyamide-amine dendritic polymer with a first phosphate buffer to obtain solution I; mixing maleimide-polyethylene glycol-N-hydroxysuccinimide with the first phosphate buffer to obtain solution II; mixing solution I and solution II and reacting them, followed by ultrafiltration purification to obtain intermediate A; dissolving Angiopep-2 in a second phosphate buffer and reacting it with intermediate A, followed by freeze-drying to obtain intermediate B; dissolving intermediate B in a dimethyl sulfoxide solution to obtain solution III; dissolving fluorescein isothiocyanate in a dimethyl sulfoxide solution to obtain solution IV; mixing solutions III and IV and reacting them under a protective gas atmosphere in the dark; freeze-drying to obtain the Angiopep-2 modified fluorescent polyamide-amine dendritic polymer.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterials technology, specifically relating to an Angiopep-2 modified fluorescent polyamide-amine dendritic polymer and its preparation method and the preparation method of nanoparticles based thereon. Background Technology

[0002] Gliomas are the most common primary intracranial tumors, characterized by high malignancy, high mortality, high disability, and high recurrence rates. Clinical treatment primarily involves surgical resection combined with radiotherapy, chemotherapy, and other treatment methods. However, the inherent heterogeneity and invasiveness of gliomas make incomplete or excessive resection during surgery a common problem, leading to recurrence or damage to functional areas. Furthermore, the blood-brain barrier and chemotherapy resistance significantly hinder drug delivery to the lesion site or greatly reduce the efficacy of chemotherapy drugs. Utilizing nanotechnology to encapsulate drugs in nanoscale drug delivery systems can reduce drug leakage, alleviate systemic toxicity, prolong blood circulation time, and increase the amount of drug reaching the brain through small size and osmotic retention effects, thereby improving therapeutic efficacy.

[0003] Based on this, in order to improve brain targeting efficiency, existing technologies link arginine-glycine-aspartic acid (RGD) or folic acid to nanocarriers, so that the nanocarriers can specifically bind to integrin αvβ3 or folic acid receptors on glioma cells, thus possessing both physical and active targeting capabilities. However, the targeted drug delivery systems used lack selective aggregation in the blood-brain barrier region. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, the present invention aims to provide an Angiopep-2 modified fluorescent polyamide-amine dendritic polymer and its preparation method and nanoparticles based thereon. The nanoscale particle size of the PAMAM dendritic macromolecules in the Angiopep-2 modified fluorescent polyamide-amine dendritic polymer (PAMAM) gives it physical size targeting. PAMAM carries a positive charge and can generate electrostatic interaction with negatively charged brain capillary endothelial cells, thereby enhancing its targeting. In particular, by binding Angiopep-2 to PAMAM branches, an active targeting drug delivery system is constructed. Angiopep-2 specifically binds to low-density lipoprotein receptor-associated protein 1 (LRP1), which is highly expressed on brain capillary endothelial cells and glioma cells, thereby enhancing the brain targeting of the drug delivery system in multiple ways and achieving selective aggregation in the blood-brain barrier region.

[0005] To achieve the above objectives, the present invention employs the following technical solution:

[0006] This invention provides a method for preparing angiopep-2 modified fluorescent polyamide-amine dendritic polymer, comprising the following steps:

[0007] Solution I was prepared by mixing polyamide-amine dendritic polymer with first phosphate buffer, and solution II was prepared by mixing maleimide-polyethylene glycol-N-hydroxysuccinimide with first phosphate buffer. Solution I and solution II were mixed to carry out the first reaction, followed by ultrafiltration purification to obtain intermediate A.

[0008] Angiopep-2 was dissolved in a second phosphate buffer and then mixed with intermediate A to carry out a second reaction, followed by a first freeze-drying to obtain intermediate B;

[0009] Intermediate B was dissolved in dimethyl sulfoxide solution to obtain solution III, and fluorescein isothiocyanate was dissolved in dimethyl sulfoxide solution to obtain solution IV. Solutions III and IV were mixed and subjected to a third reaction under a protective gas atmosphere in the dark. After a second freeze-drying, Angiopep-2 modified fluorescent polyamide-amine dendritic polymer was obtained.

[0010] In the specific implementation process, the average molecular weight of the polyamide-amine dendritic polymer is 13.5-14.5 kDa; the terminal group of the polyamide-amine dendritic polymer is amino; the average molecular weight of the maleimide-polyethylene glycol-N-hydroxysuccinimide is 2 kDa; the pH value of the first phosphate buffer is 8; and the pH value of the second phosphate buffer is 7.

[0011] In the specific implementation process, the mass ratio of the polyamide-amine dendritic polymer to maleimide-polyethylene glycol-N-hydroxysuccinimide is 10:(5.6-8.4);

[0012] The volume ratio of the first phosphate buffer in solution I to the first phosphate buffer in solution II is (3-5):1.

[0013] In the specific implementation process, the mass ratio of the polyamide-amine dendritic polymer in intermediate A to Angiopep-2 is 10:(2-3).

[0014] In the specific implementation process, the mass ratio of intermediate B to fluorescein isothiocyanate is (2-2.5):1;

[0015] The volume ratio of the dimethyl sulfoxide solution in solution III to the dimethyl sulfoxide solution in solution IV is (2-4):1.

[0016] The present invention provides an Angiopep-2 modified fluorescent polyamide-amine dendritic polymer prepared according to any one of the preparation methods of the Angiopep-2 modified fluorescent polyamide-amine dendritic polymer.

[0017] This invention also provides a method for preparing nanoparticles based on Angiopep-2 modified fluorescent polyamide-amine dendritic polymer, comprising the following steps:

[0018] An aqueous solution of Angiopep-2 modified fluorescent polyamide-amine dendritic polymer was prepared by dissolving Angiopep-2 modified fluorescent polyamide-amine dendritic polymer in water.

[0019] An aqueous solution of Angiopep-2 modified fluorescent polyamide-amine dendritic polymer was mixed with the drug solution to be loaded and stirred for a set time. The mixture was then centrifuged at a set speed for a set time and then freeze-dried to obtain nanoparticles with both targeting and fluorescence properties.

[0020] In the specific implementation process, the drug solution to be encapsulated consists of the drug to be encapsulated and the encapsulation solution. The drug to be encapsulated is doxorubicin hydrochloride or paclitaxel, and the encapsulation solution is a methanol solution.

[0021] In the specific implementation process, the mass ratio of the drug to be loaded to the Angiopep-2 modified fluorescent polyamide-amine dendritic polymer is 1:(2-10); the stirring time is set to 12-15h; the set rotation speed is 7000rpm; and the centrifugation time is set to 5-10min.

[0022] In the specific implementation process, the volume ratio of the methanol solution to the water in the aqueous solution of the Angiopep-2 modified fluorescent polyamide-amine dendritic polymer is 1:(8-12).

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] This invention provides a method for preparing Angiopep-2 modified fluorescent polyamide-amine dendritic polymers. By adding maleimide-polyethylene glycol-N-hydroxysuccinimide to react with the amino groups at the ends of the polyamide-amine dendritic polymer, an active maleimide group is introduced. The modification of the polyamide-amine dendritic polymer with Angiopep-2 is achieved through an addition reaction between the maleimide and the terminal thiol groups of Angiopep-2. Furthermore, the Angiopep-2 modified polyamide-amine dendritic polymer material is labeled with fluorescein isothiocyanate. The isothiocyanate groups of fluorescein react with the remaining active amino groups at the ends of the fourth-generation polyamide-amine dendritic polymer, thereby preparing a fluorescently labeled carrier material. This invention utilizes chemical synthesis technology to chemically bond Angiopep-2 and fluorescein isothiocyanate to a polyamide-amine dendritic polymer and modify its structure. This allows the polyamide-amine dendritic polymer, which originally lacks glioma targeting and optical properties, to emit green fluorescence after absorbing light of a specific wavelength and to specifically bind to the cell membrane receptor LRP-1, thus becoming a functional polymer material.

[0025] In the preparation of the above-mentioned Angiopep-2 modified fluorescent polyamide-amine dendritic polymer, the physical targeting ability of polyamide-amine dendritic macromolecules (PAMAM) based on their nanoscale particle size is utilized first. Secondly, the targeting ability is enhanced by the electrostatic interaction between the positive surface charge of PAMAM and the negatively charged brain capillary endothelial cells. In particular, angiopep-2 is bonded to the PAMAM branches to achieve specific binding with low-density lipoprotein receptor-associated protein 1 (LRP1), which is highly expressed on brain capillary endothelial cells and glioma cells, thereby enhancing the brain targeting ability of the drug delivery system in multiple ways.

[0026] Furthermore, to facilitate the evaluation of the brain delivery efficiency of the nanocarrier-delivered drug, the present invention employs fluorescent labeling, labeling the green fluorescent dye fluorescein isothiocyanate onto PAMAM, thereby enabling tracer analysis of the drug delivery system and monitoring of the drug delivery behavior of the nanocarrier system.

[0027] Furthermore, this invention selects a fourth-generation polyamide-amine dendritic polymer, which, compared to other generations of polyamide-amine dendritic polymers, has more active groups at the ends than lower-generation polyamide-amine dendritic polymers and has a smaller particle size than higher-generation polyamide-amine dendritic polymers, making it easier to pass through biological barriers; and selects maleimide-polyethylene glycol-N-hydroxysuccinimide (MAL-PEG-NHS) with a lower molecular weight, which makes the obtained intermediate A have low steric hindrance, making it easier to subsequently bond targeting groups.

[0028] Another aspect of this invention provides a method for preparing nanoparticles based on Angiopep-2-modified fluorescent polyamide-amine dendritic polymers. The method involves preparing an aqueous solution of the Angiopep-2-modified polyamide-amine dendritic polymer labeled with fluorescein isothiocyanate, adding a drug solution to be loaded, mixing, stirring, centrifuging to remove unloaded drug, and then lyophilizing to obtain targeted fluorescent nanoparticles. The fluorescein isothiocyanate-labeled Angiopep-2-modified polyamide-amine dendritic polymer serves as the carrier for the nanoparticles. Upon excitation by light of a specific wavelength, it emits green fluorescence, and the distribution behavior of the nanoparticle carrier can be monitored using a fluorescence spectrophotometer. Furthermore, the release and distribution behavior of the drug loaded onto the nanoparticles can be monitored based on the characteristics of the loaded drug.

[0029] Furthermore, when paclitaxel is loaded, monitoring can be achieved using high-performance liquid chromatography (HPLC); when doxorubicin hydrochloride is loaded, the drug emits red fluorescence after specific fluorescence excitation. By monitoring fluorescence at different wavelengths, not only can the distribution behavior of the nanoparticle carrier be monitored, but also the release and distribution behavior of the drug loaded in the nanoparticles can be monitored, further evaluating the targeting performance of the nanoparticles in vitro and in vivo. The nanoparticles prepared by the above methods have high detection sensitivity and uniform particle size distribution, showing good application prospects. Attached Figure Description

[0030] Figure 1 The fluorescence emission spectrum of the Angiopep-2 modified fluorescent polyamide-amine dendritic polymer prepared in Example 2 of the present invention is shown in Figure (a), where the test substance is fluorescein isothiocyanate and the test substance is the Angiopep-2 modified fluorescent polyamide-amine dendritic polymer.

[0031] Figure 2 This is a fluorescence microscope image of the targeted fluorescent nanoparticles prepared in Example 8 of the present invention;

[0032] Figure 3 This is a transmission electron microscope image of the targeted fluorescent nanoparticles prepared in Example 10 of the present invention. Detailed Implementation

[0033] To enable those skilled in the art to understand the features and effects of the present invention, the terms and expressions used in the specification and claims are explained and defined in general below. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in case of conflict, the definitions in this specification shall prevail.

[0034] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0035] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0036] In this article, unless otherwise specified, “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of,” for example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a.”

[0037] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0038] This invention provides an Angiopep-2 modified fluorescent polyamide-amine dendritic polymer and a method for preparing nanoparticles based thereon.

[0039] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0040] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications conventional in the art. In this specification and the following examples, unless otherwise specified, "%" refers to a percentage by mass, "parts" refers to parts by weight, and "ratio" refers to a mass ratio.

[0041] The first aspect of this invention provides a method for preparing angiopep-2 modified fluorescent polyamide-amine dendritic polymer, using polyamide-amine dendritic polymer (PAMAM), angiopep-2, and fluorescein isothiocyanate as raw materials. The specific principle is as follows:

[0042] Polyamide-amine dendrimers (PAMAMs) are a class of polymers synthesized from ammonia or ethylenediamine as starting monomers, with the addition of methyl acrylate via a Michael addition reaction. PMAMs are widely considered as drug carriers due to their non-immunogenicity, low cytotoxicity, the presence of hydrophobic cavities in their structure that can encapsulate drugs, and the abundance of easily modifiable active groups on their surface. The numerous terminal active groups in the PMAM structure can be used to bond targeting ligands, enabling the construction of active targeted drug delivery systems based on PMAMs.

[0043] This invention uses a fourth-generation polyamide-amine dendritic polymer, and the characteristic proton peaks in the G4 PAMAM structure are assigned as follows: δ 2.31ppm (br, -NCH2CH2CONH-), 2.55ppm (br, -CONHCH2CH2N-), 2.70ppm (br, -NCH2CH2CONH-), 3.08ppm (br, -CONHCH2CH2NH2), 3.17ppm (br, -CONHCH2CH2NH2), 3.27ppm (br, -CONHCH2CH2N-).

[0044] Angiopep-2 (sequence: TFFYGGSRGKRNNFKTEEY) is a highly endocytic targeting peptide derived from the amino acid sequence of aprotinin, which specifically binds to low-density lipoprotein receptor-associated protein 1 (LRP-1). LRP-1 is highly expressed in brain capillary endothelial cells and various glioma cells, such as U87 and C6. Therefore, Angiopep-2 is a promising glioma targeting ligand.

[0045] Fluorescein isothiocyanate is one of the most widely used green fluorescent dyes. Its isothiocyanate group can react with the terminal amino groups of proteins or polyamide-amine dendritic polymers to achieve labeling. It has a maximum absorption wavelength of 490 nm and a maximum emission wavelength of 520 nm, exhibiting bright yellow-green fluorescence. Due to its high absorption rate, excellent fluorescence quantum yield, and good water solubility, fluorescein is widely used in biological and pharmaceutical fields.

[0046] The preparation method of the Angiopep-2 modified fluorescent polyamide-amine dendritic polymer of the present invention includes the following steps:

[0047] Step 1: Polyamide-amine dendritic polymer and maleimide-polyethylene glycol-N-hydroxysuccinimide (MAL-PEG-NHS) are dissolved in first phosphate buffer (pH 8.0) to obtain solution I and solution II. The two solutions I and II are mixed and reacted at room temperature for 2-3 hours. After ultrafiltration purification, intermediate A is obtained.

[0048] The polyamide-amine dendritic polymer used has an average molecular weight of 13.5–14.5 kDa and an amino terminal group; the selected polyamide-amine dendritic polymer is a fourth-generation polyamide-amine dendritic polymer; the average molecular weight of maleimide-polyethylene glycol-N-hydroxysuccinimide is 2 kDa; MAL-PEG-NHS with a molecular weight of 2 kDa was selected to ensure that intermediate A has low steric hindrance, which facilitates subsequent bonding of targeting groups.

[0049] In the specific implementation process, the mass ratio of polyamide-amine dendritic polymer to maleimide-polyethylene glycol-N-hydroxysuccinimide is 10:(5.6~8.4); the volume ratio of the first phosphate buffer in solution I to the first phosphate buffer in solution II is (3~5):1.

[0050] The purpose of step one is to introduce the active group maleimide, which is introduced by reacting maleimide-polyethylene glycol-N-hydroxysuccinimide with the amino group at the end of the polyamide-amine dendritic polymer.

[0051] Step 2: Angiopep-2 was dissolved in second phosphate buffer (pH 7.0) and mixed with intermediate A. The mixture was reacted at room temperature for 20-24 hours. After the reaction was completed, the mixture was freeze-dried at a pressure of 5-10 Pa and a temperature of -80 °C to obtain intermediate B.

[0052] In this intermediate, the mass ratio of the polyamide-amine dendritic polymer in intermediate A to Angiopep-2 is 10:(2-3).

[0053] The purpose of step two is to modify Angiopep-2 on the polyamide-amine dendrimer by means of the addition reaction between maleimide and the terminal thiol group of Angiopep-2.

[0054] Step 3: Take intermediate B and fluorescein isothiocyanate, dissolve them in dimethyl sulfoxide to obtain solution III and solution IV, then mix solution III and solution IV, react at room temperature for 12-15 h under nitrogen protection and light protection, and freeze-dry at a pressure of 5-10 Pa and a temperature of -80 °C to obtain Angiopep-2 modified fluorescent polyamide-amine dendritic polymer.

[0055] The mass ratio of intermediate B to fluorescein isothiocyanate is (2-2.5):1; the volume ratio of dimethyl sulfoxide solution in solution III to dimethyl sulfoxide solution in solution IV is (2-4):1.

[0056] The purpose of step three is to react the isothiocyanate groups of fluorescein isothiocyanate with the remaining active amino groups at the ends of the fourth-generation polyamide-amine dendritic polymer, thereby preparing a fluorescently labeled carrier material.

[0057] The above synthesis method uses simple and readily available raw materials, is inexpensive, and operates under mild reaction conditions. It does not affect the optical, physicochemical, and biological properties of the raw materials. The resulting fluorescent biomaterials exhibit good stability and significant fluorescence, and have promising application prospects.

[0058] The second aspect of the present invention provides an Angiopep-2 modified fluorescent polyamide-amine dendritic polymer obtained by the above preparation method.

[0059] The aforementioned Angiopep-2-modified fluorescent polyamide-amine dendritic polymer (PAMAM) has nanoscale particle size, giving it physical size targeting capabilities. PAMAM carries a positive charge and can generate electrostatic interactions with negatively charged brain capillary endothelial cells, thereby enhancing its targeting. In particular, this invention constructs an active targeting drug delivery system by binding Angiopep-2 to PAMAM, allowing Angiopep-2 to specifically bind to low-density lipoprotein receptor-associated protein 1 (LRP1), which is highly expressed on brain capillary endothelial cells and glioma cells, further enhancing targeting.

[0060] Simultaneously, the green fluorescent dye fluorescein isothiocyanate is labeled on PAMAM to achieve qualitative and quantitative tracking of the drug delivery system, monitor the delivery behavior of the drug delivery system, facilitate the evaluation of the efficiency of drug delivery into the brain by nanocarriers, and provide a convenient monitoring method to ensure the therapeutic dosage of drugs.

[0061] A third aspect of this invention provides a method for preparing nanoparticles based on the above-mentioned Angiopep-2 modified fluorescent polyamide-amine dendritic polymer, the specific steps of which are as follows:

[0062] An aqueous solution of Angiopep-2 modified fluorescent polyamide-amine dendritic polymer was prepared by dissolving Angiopep-2 modified fluorescent polyamide-amine dendritic polymer in water.

[0063] An aqueous solution of Angiopep-2 modified fluorescent polyamide-amine dendritic polymer was mixed with the drug solution to be loaded and stirred for 12-15 h, centrifuged at 7000 rpm for 5-10 min, and then freeze-dried to obtain nanoparticles with both targeting and fluorescence properties.

[0064] In the specific implementation process, an aqueous solution of Angiopep-2 modified fluorescent polyamide-amine dendritic polymer was prepared, and then the drug solution to be loaded was added. After mixing, the mixture was stirred for 12-15 hours, centrifuged at 7000 rpm for 5-10 minutes, and freeze-dried to obtain nanoparticles with both targeting and fluorescence properties. The Angiopep-2 modified polyamide-amine dendritic polymer labeled with fluorescein isothiocyanate was used as the carrier of the nanoparticles.

[0065] The drug-to-encapsulate solution consists of the drug to be encapsulated and the encapsulation solution. The drug to be encapsulated is doxorubicin hydrochloride or paclitaxel. The mass ratio of the drug to be encapsulated to the Angiopep-2 modified fluorescent polyamide-amine dendritic polymer is 1:(2-10). The drug-encapsulating solution is a methanol solution, and the volume ratio of the methanol solution of the drug-encapsulated solution to the water in the aqueous solution of the Angiopep-2 modified fluorescent polyamide-amine dendritic polymer is 1:(8-12).

[0066] Example 1

[0067] Synthesis of FITC-labeled angiopep-2 modified PAMAM material

[0068] 20 mg of polyamide-amine dendritic polymer was completely dissolved in 8 mL of phosphate buffer (pH 8.0), and 12 mg of MAL-PEG-NHS was dissolved in 2 mL of phosphate buffer (pH 8.0). The two solutions were mixed and reacted at room temperature for 2 h. After ultrafiltration purification, intermediate A was obtained.

[0069] 4 mg of Angiopep-2 was dissolved in 8 mL of phosphate buffer (pH 7.0), then mixed with intermediate A, and reacted at room temperature for 24 h. After the reaction was completed, the intermediate B was obtained by freeze drying.

[0070] 4 mg of intermediate B was completely dissolved in 4 mL of dimethyl sulfoxide, and 2 mg of fluorescein isothiocyanate was dissolved in 2 mL of dimethyl sulfoxide. The reaction was carried out under nitrogen protection, light protection and room temperature for 12 h, and then freeze-dried to obtain the Angiopep-2 modified fluorescent polyamide-amine dendritic polymer.

[0071] Example 2

[0072] Synthesis of FITC-labeled angiopep-2 modified PAMAM material

[0073] 50 mg of polyamide-amine dendritic polymer was completely dissolved in 32 mL of phosphate buffer (pH 8.0), and 28 mg of MAL-PEG-NHS was dissolved in 8 mL of phosphate buffer (pH 8.0). The two solutions were mixed and reacted at room temperature for 3 h. After ultrafiltration purification, intermediate A was obtained.

[0074] 10 mg of Angiopep-2 was dissolved in 8 mL of phosphate buffer (pH 7.0), then mixed with intermediate A, and reacted at room temperature for 24 h. After the reaction was completed, the intermediate B was obtained by freeze drying.

[0075] 8 mg of intermediate B was completely dissolved in 12 mL of dimethyl sulfoxide, and 4 mg of fluorescein isothiocyanate was dissolved in 4 mL of dimethyl sulfoxide. The reaction was carried out under nitrogen protection, light protection and room temperature for 12 h, and then freeze-dried to obtain the Angiopep-2 modified fluorescent polyamide-amine dendritic polymer, namely FITC-angiopep-PEG-PAMAM.

[0076] The dried FITC-angiopep-PEG-PAMAM was measured using a nuclear magnetic resonance spectrometer and a Fourier transform infrared spectrometer to perform characteristic peak analysis.

[0077] FITC-angiopep-PEG-PAMAM 1 The 1H NMR spectrum showed characteristic peaks of polyamide-amine dendritic polymers near chemical shift values ​​(δ) 2.3-3.3 ppm, peaks of the MAL-PEG-NHS structure near 3.70 ppm, characteristic peaks of Angiopep-2 near 7.14 ppm, and characteristic proton peaks of fluorescein isothiocyanate structure near (δ) 6.4-7.5 ppm, indicating that the FITC-labeled angiopep-2 modified PAMAM material was successfully synthesized.

[0078] The infrared spectrum of FITC-angiopep-PEG-PAMAM shows that in the 1650-1430 cm⁻¹ range... -1 The stretching vibration peaks of the aromatic ring skeleton and the 3000-3100 cm⁻¹ peaks were observed. -1 The stretching vibration peak of aromatic hydrogens, and the peak not observed at 2039 cm⁻¹ -1 A cumulative double bond peak of FITC (S=C=N) appears nearby, combined with... 1 The H NMR results indicate that FITC was successfully bonded to Angiopep-PEG-PAMAM.

[0079] Example 3

[0080] Synthesis of FITC-labeled angiopep-2 modified PAMAM material

[0081] 50 mg of polyamide-amine dendritic polymer was completely dissolved in 35 mL of phosphate buffer (pH 8.0), and 42 mg of MAL-PEG-NHS was dissolved in 7 mL of phosphate buffer (pH 8.0). The two solutions were mixed and reacted at room temperature for 3 h. After ultrafiltration purification, intermediate A was obtained.

[0082] 10 mg of Angiopep-2 was dissolved in 8 mL of phosphate buffer (pH 7.0), then mixed with intermediate A, and reacted at room temperature for 24 h. After the reaction was completed, the intermediate B was obtained by freeze drying.

[0083] 10 mg of intermediate B was completely dissolved in 16 mL of dimethyl sulfoxide, and 4 mg of fluorescein isothiocyanate was dissolved in 4 mL of dimethyl sulfoxide. The reaction was carried out under nitrogen protection, light protection and room temperature for 15 h, and then freeze-dried to obtain the Angiopep-2 modified fluorescent polyamide-amine dendritic polymer.

[0084] Example 4

[0085] Synthesis of FITC-labeled angiopep-2 modified PAMAM material

[0086] 50 mg of polyamide-amine dendritic polymer was completely dissolved in 32 mL of phosphate buffer (pH 8.0), and 28 mg of MAL-PEG-NHS was dissolved in 7 mL of phosphate buffer (pH 8.0). The two solutions were mixed and reacted at room temperature for 3 h. After ultrafiltration purification, intermediate A was obtained.

[0087] 15 mg of Angiopep-2 was dissolved in 8 mL of phosphate buffer (pH 7.0), then mixed with intermediate A, and reacted at room temperature for 20 h. After the reaction was completed, the intermediate B was obtained by freeze drying.

[0088] 8 mg of intermediate B was completely dissolved in 12 mL of dimethyl sulfoxide, and 4 mg of fluorescein isothiocyanate was dissolved in 4 mL of dimethyl sulfoxide. The reaction was carried out under nitrogen protection, light protection and room temperature for 14 h, and then freeze-dried to obtain the Angiopep-2 modified fluorescent polyamide-amine dendritic polymer.

[0089] Example 5

[0090] Synthesis of FITC-labeled angiopep-2 modified PAMAM material

[0091] 50 mg of polyamide-amine dendritic polymer was completely dissolved in 32 mL of phosphate buffer (pH 8.0), and 28 mg of MAL-PEG-NHS was dissolved in 7 mL of phosphate buffer (pH 8.0). The two solutions were mixed and reacted at room temperature for 3 h. After ultrafiltration purification, intermediate A was obtained.

[0092] 12 mg of Angiopep-2 was dissolved in 8 mL of phosphate buffer (pH 7.0), then mixed with intermediate A, and reacted at room temperature for 22 h. After the reaction was completed, the intermediate B was obtained by freeze drying.

[0093] 8 mg of intermediate B was completely dissolved in 12 mL of dimethyl sulfoxide, and 4 mg of fluorescein isothiocyanate was dissolved in 4 mL of dimethyl sulfoxide. The reaction was carried out under nitrogen protection, light protection and room temperature for 12 h, and then freeze-dried to obtain the Angiopep-2 modified fluorescent polyamide-amine dendritic polymer.

[0094] Example 6

[0095] Fluorescence properties of FITC-labeled angiopep-2-modified PAMAM materials

[0096] The Angiopep-2 modified fluorescent polyamide-amine dendritic polymer prepared in Example 2 was completely dissolved in triple-distilled water to obtain an aqueous solution of Angiopep-2 modified fluorescent polyamide-amine dendritic polymer with a concentration of 0.1 mg / mL. 0.5 mL of the Angiopep-2 modified fluorescent polyamide-amine dendritic polymer aqueous solution was taken and added to triple-distilled water to obtain an aqueous solution of FITC-labeled angiopep-2 modified PAMAM with a concentration of 5 μg / mL. The FITC aqueous solution was prepared in a similar manner. The spectra were detected using a fluorescence spectrophotometer and are shown in the figure below. Figure 1 Figure (a) and Figure (b); by Figure 1 The spectrum shows that the FITC-labeled angiopep-2 modified PAMAM material exhibits good light emission performance after photoexcitation. The emission curve is similar to the FITC light emission curve, and the emission wavelength peak is located at 520nm at the same excitation wavelength of 490nm.

[0097] Example 7

[0098] Preparation of targeted fluorescent nanoparticles

[0099] 12 mg of doxorubicin hydrochloride was dissolved in 2.4 mL of methanol, and 30 μL of triethylamine was added to adjust the pH of the solution to alkaline. 24 mg of the Angiopep-2 modified fluorescent polyamide-amine dendritic polymer prepared in Example 1 was dissolved in 19.2 mL of aqueous solution, and the doxorubicin solution was mixed with it. The mixture was stirred for 12 h, centrifuged at 7000 rpm for 5 min, and then freeze-dried to obtain targeted fluorescent nanoparticles.

[0100] Example 8

[0101] Preparation of targeted fluorescent nanoparticles

[0102] 16 mg of doxorubicin hydrochloride was dissolved in 3.2 mL of methanol, and 30 μL of triethylamine was added to adjust the pH of the solution to alkaline. 160 mg of the Angiopep-2 modified fluorescent polyamide-amine dendritic polymer prepared in Example 1 was dissolved in 32 mL of aqueous solution, and the doxorubicin solution was mixed with it. The mixture was stirred for 14 h, centrifuged at 7000 rpm for 10 min, and then freeze-dried to obtain targeted fluorescent nanoparticles.

[0103] Example 9

[0104] Investigation of fluorescence signal of targeted fluorescent nanoparticles

[0105] The targeted fluorescent nanoparticles prepared in Example 8 were dispersed in a buffer solution and observed under natural light and a fluorescence microscope, respectively. See details [link to example]. Figure 2 .

[0106] like Figure 2 As shown, under blue excitation light in a fluorescence microscope, the nanoparticles exhibited a green fluorescence signal of the Angiopep-2 modified fluorescent polyamide-amine dendritic polymer, indicating that the prepared targeted fluorescent nanoparticles can emit a green fluorescence signal after being excited by light of a specific wavelength.

[0107] Example 10

[0108] Preparation of targeted fluorescent nanoparticles

[0109] 8 mg of paclitaxel was dissolved in 20 mL of methanol. 20 mg of the Angiopep-2 modified fluorescent polyamide-amine dendritic polymer prepared in Example 1 was dissolved in 240 mL of aqueous solution, and the paclitaxel solution was mixed with it. The mixture was stirred for 15 h, centrifuged at 7000 rpm for 5 min, and then freeze-dried to obtain targeted fluorescent nanoparticles.

[0110] The morphology of nanoparticles was observed under a transmission electron microscope, such as... Figure 3 As shown, the nanoparticles are spherical or near-spherical in shape, with a particle size of approximately 40 nm.

[0111] As can be seen from the above embodiments, the targeted fluorescent nanoparticles prepared by the present invention using Angiopep-2 modified fluorescent polyamide-amine dendritic polymer have a smooth and intact surface, uniform particle size distribution, and can emit green fluorescent signals.

[0112] Comparative Example 1

[0113] Synthesis of FITC-labeled angiopep-2 modified PAMAM material

[0114] 10 mg of polyamide-amine dendritic polymer was completely dissolved in 6 mL of phosphate buffer (pH 8.0), and 85 mg of MAL-PEG-NHS was dissolved in 24 mL of phosphate buffer (pH 8.0). The two solutions were mixed and reacted at room temperature for 3 h. After ultrafiltration purification, intermediate A was obtained.

[0115] Intermediate A and 10 mg Angiopep-2 were dissolved in 8 mL of phosphate buffer (pH 7.0) and reacted at room temperature for 20 h. After the reaction was completed, the mixture was freeze-dried to obtain intermediate B.

[0116] 8 mg of intermediate B was completely dissolved in 12 mL of dimethyl sulfoxide, and 4 mg of fluorescein isothiocyanate was dissolved in 4 mL of dimethyl sulfoxide. The mixture was reacted under nitrogen protection and in the dark at room temperature for 12 h. After freeze drying, the Angiopep-2 modified fluorescent polyamide-amine dendritic polymer, namely FITC-angiopep-PEG-PAMAM, was obtained.

[0117] The dried FITC-angiopep-PEG-PAMAM was measured using a nuclear magnetic resonance spectrometer to analyze its characteristic peaks.

[0118] FITC-angiopep-PEG-PAMAM 1 The 1H NMR spectrum showed characteristic peaks of the polyamide-amine dendritic polymer around 2.3-3.3 ppm, peaks of the MAL-PEG-NHS structure around 3.70 ppm, and characteristic peaks of Angiopep-2 around 7.14 ppm. No characteristic proton peaks of the fluorescein isothiocyanate structure were observed around 6.4-7.5 ppm, indicating that the FITC-labeled angiopep-2 modified PAMAM material was not synthesized. This is because the mass ratio of the polyamide-amine dendritic polymer to MAL-PEG-NHS was 10:85, which far exceeds the range of 10:(5.6-8.4) of the present invention, leading to unsuccessful synthesis.

[0119] Comparative Example 2

[0120] Synthesis of FITC-labeled angiopep-2 modified PAMAM material

[0121] 50 mg of polyamide-amine dendritic polymer was completely dissolved in 32 mL of phosphate buffer (pH 8.0), and 28 mg of MAL-PEG-NHS (molecular weight 5 kDa) was dissolved in 8 mL of phosphate buffer (pH 8.0). The two solutions were mixed and reacted at room temperature for 3 h. After ultrafiltration purification, intermediate A was obtained.

[0122] Intermediate A and 10 mg Angiopep-2 were dissolved in 8 mL of phosphate buffer (pH 7.0) and reacted at room temperature for 20 h. After the reaction was completed, the mixture was freeze-dried to obtain intermediate B.

[0123] 8 mg of intermediate B was completely dissolved in 12 mL of dimethyl sulfoxide, and 4 mg of fluorescein isothiocyanate was dissolved in 4 mL of dimethyl sulfoxide. The mixture was reacted under nitrogen protection and in the dark at room temperature for 12 h. After freeze drying, the Angiopep-2 modified fluorescent polyamide-amine dendritic polymer, namely FITC-angiopep-PEG-PAMAM, was obtained.

[0124] The dried FITC-angiopep-PEG-PAMAM was measured using a nuclear magnetic resonance spectrometer to analyze its characteristic peaks.

[0125] FITC-angiopep-PEG-PAMAM 1 The 1H NMR spectrum showed characteristic peaks of polyamide-amine dendritic polymers around chemical shift values ​​(δ) 2.3-3.3 ppm, but no peaks of the MAL-PEG-NHS structure around 3.70 ppm. This indicates that FITC-labeled angiopep-2 modified PAMAM material was not synthesized. The reason for this is that 5 kDa MAL-PEG-NHS was selected instead of 2 kDa MAL-PEG-NHS, resulting in unsuccessful synthesis.

[0126] This invention provides an Angiopep-2 modified fluorescent polyamide-amine dendritic polymer and its preparation method, as well as a method for preparing nanoparticles based thereon. The method utilizes a chemical synthesis reaction to bond Angiopep-2 to the polyamide-amine dendritic polymer, giving it LRP-1 targeting properties. Specifically, Angiopep-2 is covalently linked to the polyamide-amine dendritic polymer via maleimide groups to prepare a biocompatible material with targeting function: the Angiopep-2 modified polyamide-amine dendritic polymer. The targeting material is then labeled with fluorescein isothiocyanate, enabling it to emit fluorescence upon photoexcitation. This material is further used to encapsulate antitumor drugs, thereby preparing nanoparticles with both targeting and fluorescence properties. The preparation method of this invention uses readily available raw materials, operates under mild reaction conditions, and produces nanoparticles with uniform particle size distribution and excellent fluorescence emission performance.

[0127] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A method for preparing an Angiopep-2 modified fluorescent polyamide-amine dendritic polymer, characterized in that, Includes the following steps: Solution I was prepared by mixing a polyamide-amine dendritic polymer with a first phosphate buffer. Solution II was prepared by mixing maleimide-polyethylene glycol-N-hydroxysuccinimide with the first phosphate buffer. Solution I and Solution II were then mixed for a first reaction, followed by ultrafiltration purification to obtain intermediate A. The average molecular weight of the polyamide-amine dendritic polymer was 13.5~14.5 kDa. The terminal groups of the polyamide-amine dendritic polymer were amino groups. The average molecular weight of the maleimide-polyethylene glycol-N-hydroxysuccinimide was 2 kDa. The mass ratio of the polyamide-amine dendritic polymer to the maleimide-polyethylene glycol-N-hydroxysuccinimide was 10:(5.6~8.4). Angiopep-2 was dissolved in a second phosphate buffer and then mixed with intermediate A to carry out a second reaction, followed by a first freeze-drying to obtain intermediate B; Intermediate B was dissolved in dimethyl sulfoxide solution to obtain solution III, and fluorescein isothiocyanate was dissolved in dimethyl sulfoxide solution to obtain solution IV. Solutions III and IV were mixed and subjected to a third reaction under a protective gas atmosphere in the dark. After a second freeze-drying, Angiopep-2 modified fluorescent polyamide-amine dendritic polymer was obtained.

2. The method for preparing the Angiopep-2 modified fluorescent polyamide-amine dendritic polymer according to claim 1, characterized in that, The first phosphate buffer has a pH of 8; the second phosphate buffer has a pH of 7.

3. The method for preparing the Angiopep-2 modified fluorescent polyamide-amine dendritic polymer according to claim 1, characterized in that, The volume ratio of the first phosphate buffer in solution I to the first phosphate buffer in solution II is (3~5):

1.

4. The method for preparing the Angiopep-2 modified fluorescent polyamide-amine dendritic polymer according to claim 1, characterized in that, The mass ratio of the polyamide-amine dendritic polymer in intermediate A to Angiopep-2 is 10:(2~3).

5. The method for preparing the Angiopep-2 modified fluorescent polyamide-amine dendritic polymer according to claim 1, characterized in that, The mass ratio of intermediate B to fluorescein isothiocyanate is (2~2.5):1; The volume ratio of the dimethyl sulfoxide solution in solution III to the dimethyl sulfoxide solution in solution IV is (2~4):

1.

6. An Angiopep-2 modified fluorescent polyamide-amine dendritic polymer prepared by the method according to any one of claims 1 to 5, characterized in that, The Angiopep-2 modified fluorescent polyamide-amine dendritic polymer is a polyamide-amine dendritic polymer modified with Angiopep-2 labeled with fluorescein isothiocyanate. Specifically, the active group maleimide is introduced by reacting maleimide-polyethylene glycol-N-hydroxysuccinimide with the amino group at the end of the polyamide-amine dendritic polymer. The modification of the polyamide-amine dendritic polymer with Angiopep-2 is achieved through an addition reaction between maleimide and the terminal thiol group of Angiopep-2. The isothiocyanate group of fluorescein isothiocyanate reacts with the remaining active amino group at the end of the fourth-generation polyamide-amine dendritic polymer. The Angiopep-2 modified fluorescent polyamide-amine dendritic polymer emits green fluorescence at an emission wavelength of 520 nm when excited at a wavelength of 490 nm.

7. A method for preparing nanoparticles based on Angiopep-2 modified fluorescent polyamide-amine dendritic polymer, characterized in that, Includes the following steps: An aqueous solution of Angiopep-2 modified fluorescent polyamide-amine dendritic polymer as described in claim 6 was prepared by dissolving it in water. An aqueous solution of Angiopep-2 modified fluorescent polyamide-amine dendritic polymer was mixed with the drug solution to be loaded and stirred for a set time, then centrifuged at a set speed for a set time, and then freeze-dried to obtain nanoparticles with both targeting and fluorescence properties. The drug solution to be encapsulated consists of the drug to be encapsulated and the encapsulation solution, wherein the drug to be encapsulated is doxorubicin hydrochloride or paclitaxel. The mass ratio of the drug to be loaded to the Angiopep-2 modified fluorescent polyamide-amine dendritic polymer is 1:(2~10).

8. The method for preparing nanoparticles according to claim 7, characterized in that, The loading solution is a methanol solution; the volume ratio of the methanol solution to water in the aqueous solution of the Angiopep-2 modified fluorescent polyamide-amine dendritic polymer is 1:(8~12).

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