Star-shaped polyamino acid nanoparticles with long circulation property and preparation method and application thereof

Abstract

This invention discloses a star-shaped polyamino acid nanoparticle with long-cycle properties, its preparation method, and its applications, belonging to the field of biomedical technology. The nanoparticles of this invention have a star-shaped structure with a polyamide-amine dendritic macromolecule as the core and copolymerized amino acids as arms; the nanoparticles are first initiated by polyamide-amine as an initiator. N -Carboxylic acid ring anhydride and second N The nanoparticles are prepared by random copolymerization of anhydride monomers within a carboxylic acid ring in an organic solvent. The organic solvent is a mixture of a low-polarity haloalkane and a high-polarity amide or cyclic ether solvent in a volume ratio of 9:1. This preparation method is simple and efficient. This invention, by controlling the types of amino acids, the charge of the nanoparticles, and their topological structure, demonstrates that the star-shaped topology and glutamic acid / lysine side chains play a crucial role in achieving long-term cycling. These nanoparticles have broad application prospects in the preparation of therapeutic tumor drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a star-shaped polyamino acid nanoparticle with long-cycle properties, its preparation method, and its application. Background Technology

[0002] Over the past few decades, nanoparticles have been designed to encapsulate drugs, improving their pharmacokinetics and pharmacodynamics, reducing drug metabolism, and prolonging circulating half-life. However, the efficacy of nanomedicines remains low, and the clearance of nanoparticles from the body has become a biological obstacle that must be overcome. Specifically, nanoparticles should be designed to bypass clearance mechanisms, especially the mononuclear phagocytic system (MPS)-mediated clearance pathway, such as monocytes in the blood and Kupffer cells in the liver, ultimately achieving long-term circulation in the bloodstream.

[0003] To prolong the blood circulation time of nanoparticles, researchers have developed various strategies. Surface functionalization is one of the most classic methods, introducing functional coatings on the surface of nanocarriers to shield them from the immune system's recognition. Poly(ethylene glycol) (PEG) modification is a typical example of this strategy: the high flexibility of PEG molecular chains and the steric hindrance effect generated by the hydration layer can effectively enhance the nanocarrier's resistance to antibody binding, enzyme degradation, and macrophage phagocytosis, thereby significantly prolonging its blood circulation time. The mRNA delivery system based on PEGylated lipid nanoparticles (LNPs) has been successfully applied in COVID-19 vaccines, fully validating the effectiveness of this strategy. Furthermore, zwitterionic polymers, due to their ability to form stable hydration layers through ionic solvation in physiological environments, exhibit excellent anti-protein adsorption and anti-cell adhesion properties, and are considered promising antifouling materials. Biomimetic strategies have also been used to enhance the long circulation capacity of nanoparticles, such as coating the surface of nanoparticles with natural cell membranes such as red blood cell membranes, white blood cell membranes, or platelet membranes. By transmitting "self-markers" to macrophages, these membranes can reduce phagocytosis and thus prolong blood circulation time.

[0004] While the aforementioned strategies have achieved some success in prolonging the blood circulation of nanoparticles, significant limitations remain. For example, repeated administration of PEGylated nanoparticles may induce the production of anti-PEG antibodies, leading to accelerated blood clearance (ABC effect), which not only reduces drug bioavailability but may also trigger adverse reactions such as allergies. Similarly, some zwitterionic materials are based on polymer backbones that are difficult to biodegrade (such as polysulfonate betaine and polyphosphocholine), and long-term retention may induce inflammatory responses. Cell membrane encapsulation technology faces challenges such as complex preparation processes, poor batch reproducibility, low yield, and high cost, limiting its clinical translation.

[0005] In recent years, polyamino acids have become one of the most mainstream synthetic polymer materials in the biomedical field (especially for drug delivery) due to their excellent biocompatibility and structural designability. Studies have shown that mixed-charge pseudo-zwitterionic polymers obtained by amino acid polymerization can prevent protein adsorption and have the potential to improve the blood circulation of nanoparticles. Amino acid polymerization (most commonly the ring-opening polymerization of amino acid-N-carboxylic acid intracyclic anhydride (NCA)) is one of the core methods for preparing polyamino acid materials. The preparation steps are simple, and the molecular weight of the obtained polyamino acid materials is controllable, showing broad application prospects. Polyamino acid materials have many outstanding advantages, and the preparation methods are becoming increasingly mature. However, there is still a lack of systematic and in-depth research on the biological properties of polyamino acid materials, especially regarding the core requirements for drug delivery, such as avoiding immune recognition and clearance to significantly prolong blood circulation time. Further in-depth exploration and optimization are still needed. Summary of the Invention

[0006] Purpose of the invention: The first purpose of this invention is to provide a star-shaped polyamino acid nanoparticle with long circulation properties; the second purpose of this invention is to provide a method for preparing the star-shaped polyamino acid nanoparticle with long circulation properties and its application; this invention enhances the steric hindrance effect and surface hydration layer stability through a unique star-shaped topological structure design, effectively avoids MPS-mediated clearance mechanism, significantly prolongs blood circulation time, thereby improving the delivery efficiency and therapeutic effect of antitumor drugs.

[0007] Technical Solution: The present invention discloses a star-shaped polyamino acid nanoparticle with long-cycle properties, comprising polyamide-amine (PAMAM) dendritic macromolecules and copolyamino acids, characterized in that the nanoparticles have a star structure with polyamide-amine (PAMAM) dendritic macromolecules as the core and copolyamino acids as arms; the nanoparticles are initiated by polyamide-amine (PAMAM) as an initiator to initiate the first... N -Carboxylic acid ring anhydride monomer and second N- It is prepared by random copolymerization of anhydride monomers within a carboxylic acid ring in an organic solvent; wherein the organic solvent is a mixture of low-polarity haloalkanes and high-polarity amides or cyclic ethers in a volume ratio of (6-9):(1-4).

[0008] Furthermore, the volume ratio of the low-polarity haloalkane to the high-polarity amide solvent is preferably (8-9):(1-2).

[0009] Further, the low-polarity haloalkane is selected from either dichloromethane or trichloromethane; the high-polarity amide or cyclic ether solvent is selected from... N , N -Dimethylformamide, N , N -Dimethylacetamide, N Either methylpyrrolidone or tetrahydrofuran.

[0010] Preferably, the low-polarity haloalkane is dichloromethane; the high-polarity amide solvent is... N , N -Dimethylformamide; wherein dichloromethane and N , N - The volume ratio of dimethylformamide is 9:1.

[0011] Furthermore, the first N -The anhydride monomer within the carboxylic acid ring is selected from γ-tert-butyl- L -Glutamic acid NCA, γ-Benzyl- L -Any of glutamate NCA; the second N -The anhydride monomer within the carboxylic acid ring is selected from... N ε -tert-Butyloxycarbonyl- L -Lysine NCA, N ε -Benzyloxycarbonyl- L -Lysine NCA, L -Phenylanine NCA, L -Leucine NCA, O-tert-butyl- L -Serine NCA, O-tert-butyl- L -Tyrosine NCA, N δ -2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl- L -Any of arginine NCA.

[0012] Preferably, the first N -The anhydride monomer within the carboxylic acid ring is γ-tert-butyl- L -Glutamic acid NCA; the second N-The anhydride monomer within the carboxylic acid ring is N ε -tert-Butyloxycarbonyl- L -Lysine NCA.

[0013] Furthermore, the polyamide-amine (PAMAM) dendritic macromolecule is G0-G3 with 4-32 arms.

[0014] Preferably, the polyamide-amine (PAMAM) dendritic macromolecule is G0-G2 with 4-16 arms.

[0015] More preferably, the polyamide-amine (PAMAM) dendritic macromolecule is G1 with 8 arms.

[0016] Furthermore, the second N - The anhydride monomer in the carboxylic acid ring accounts for 20%-40% of the total molar ratio of the copolyamino acid; the molar ratio of amino group to copolyamino acid in the polyamide-amine (PAMAM) is 1:50-1:200.

[0017] Preferably, the second N - The anhydride monomer in the carboxylic acid ring accounts for 30% of the total molar ratio of the copolyamino acid; the molar ratio of amino group to copolyamino acid in the polyamide-amine (PAMAM) is 1:100.

[0018] The method for preparing star-shaped polyamino acid nanoparticles with long-cycle properties according to the present invention includes the following steps:

[0019] (1) The first N -Carboxylic acid ring anhydride monomer and second N -The anhydride monomer within the carboxylic acid ring dissolves in organic solvents;

[0020] (2) The solution obtained in step (1) is mixed with polyamide-amine (PAMAM) to react and obtain star-shaped polyamino acid nanoparticles.

[0021] The application of the star-shaped polyamino acid nanoparticles with long-cycle properties described in this invention in the preparation of drugs for treating tumors.

[0022] The present invention relates to the application of the combination of star-shaped polyamino acid nanoparticles with long-cycle properties and a sonosensitive agent in the preparation of drugs for treating tumors.

[0023] Furthermore, the sound-sensitive agent is selected from any one of tetra(4-carboxyphenyl)porphyrin (TCPP), tetra(4-sulfonic phenyl)porphyrin (TPPS), hematoporphyrin, hematoporphyrin monomethyl ether, protoporphyrin IX, and dihydroporphyrin e6.

[0024] Preferably, the sound-sensitive agent is tetra(4-carboxyphenyl)porphyrin (TCPP).

[0025] Furthermore, the sound-sensitizing agent is grafted onto star-shaped polyamino acid nanoparticles with excellent long-cycle capability, and the grafting ratio is expressed as a molar ratio, where the sound-sensitizing agent and the second component of the star-shaped polyamino acid nanoparticles... N - The ratio of amino acid repeating units in the anhydride monomer within the carboxylic acid ring is 5%-15%.

[0026] Preferably, the sound-sensitizing agent is grafted onto star-shaped polyamino acid nanoparticles with excellent long-cycle capability, and the grafting ratio is expressed as a molar ratio, wherein the sound-sensitizing agent and the second component of the star-shaped polyamino acid nanoparticles... N - The ratio of amino acid repeating units in the anhydride monomer within the carboxylic acid ring is 10%.

[0027] Furthermore, the method for verifying the long-cycle capability of the star-shaped polyamino acid nanoparticles described in this invention includes the following steps:

[0028] (1) Fluorescently labeled polyamino acid nanoparticles were injected into mice via the tail vein. Blood was collected from the orbital cavity at time points, and the red blood cells in the blood were lysed and centrifuged (1000 × rpm, 5 min). The supernatant was collected and detected by an enzyme-linked immunosorbent assay (ELISA) reader. The content of polyamino acid nanoparticles in the blood at different time points was calculated, pharmacokinetic curves were plotted, and the distribution half-life and elimination half-life of the nanoparticles in vivo were calculated.

[0029] (2) A tumor model was established subcutaneously in mice, and fluorescently labeled polyamino acid nanoparticles were injected into the mice via the tail vein. After a period of time, the mice were euthanized, and major organs such as the heart, liver, spleen, lungs, and kidneys, as well as tumor tissues, were removed and their distribution was observed through in vitro imaging.

[0030] The star-shaped polyamino acid nanoparticles described in this invention are initiated by polyamide-amine (PAMAM). N The ring-opening polymerization of intracyclic carboxylic acid anhydrides (NCA) is performed using a DCM:DMF solvent system of 9:1. Compared to traditional ring-opening polymerization in DMF, the novel polymerization conditions employed in this invention yield polymers with narrower molecular weight distributions that are closer to the theoretical molecular weight. Furthermore, compared to novel ring-opening polymerization in DCM, this invention has the advantage of being compatible with a wider range of NCA monomers. Based on this, a star-shaped copolyamino acid material library based on polyglutamic acid was constructed and screened.

[0031] This invention discovers that introducing lysine during the polymerization process results in nanoparticles with a longer blood circulation time compared to pure glutamic acid systems. Typically, intravenously administered nanomedicines require prolonged circulation in the blood chamber. Utilizing the abnormal vascular structure and function formed by rapid tumor growth, nanoparticles passively accumulate in solid tumors through enhanced permeability and retention (i.e., the EPR effect). Based on this, this invention verifies the blood circulation capability of nanoparticles by observing their accumulation in tumors. First, the type of the second amino acid residue was controlled, and tumor-targeting polyamino acid nanoparticles were screened using in vitro small animal imaging. Compared to other polyamino acid nanoparticles, star-shaped nanoparticles based on polyglutamic acid-polylysine random copolymers exhibited better tumor accumulation behavior. Since lysine is a positively charged amino acid, further control of the lysine ratio and the type of positively charged amino acid (arginine as an alternative) showed that 30% lysine incorporation into glutamic acid nanoparticles resulted in the best tumor accumulation. Subsequently, altering the chain length of the star-shaped polyamino acid and the type of polyamide-amine (PAMAM) clarified the relationship between structural parameters and tumor-targeting behavior. This invention quantitatively evaluates the blood circulation capacity of nanoparticles through systematic pharmacokinetic studies, discovering that nanoparticles with good tumor enrichment effects all exhibit longer blood circulation times, thus constructing a complete functional validation system of "long circulation-EPR enrichment". The results show that the preferred star-shaped random copolymer amino acids of this invention have longer blood circulation times, and their distribution half-life is superior to that of PEG-modified nanoparticles with similar structures.

[0032] After screening for star-shaped copolymer amino acids with long-cycle properties, we constructed a nanomedicine delivery system loaded with a sonosensitive agent. The sonosensitive agent was covalently grafted onto the long-cycle copolymer amino acid to explore the antitumor effect of the nanosonosensitive agent. Results showed that the nanosonosensitive agent could generate a large amount of reactive oxygen species and significantly kill tumor cells under ultrasound. In a 4T1 subcutaneous tumor-bearing mouse model, the tumor inhibition rate reached over 75%, indicating that this therapeutic system has excellent therapeutic efficacy.

[0033] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0034] 1. This invention successfully prepared star-shaped polyamino acid nanoparticles with well-defined structures and controllable polymerization by initiating random copolymerization of two N-carboxylic acid anhydrides in a mixed solvent system with a specific ratio and using polyamide-amine (PAMAM) dendritic macromolecules as initiators. By changing the types of amino acids incorporated, the charge of the nanoparticles, and the topology, long-circulating nanomedicine nanoparticles suitable for anti-tumor therapy were screened and optimized.

[0035] 2. This invention improves the polyamide-amine (PAMAM) initiation method. NBy determining the conditions for ring-opening polymerization of intracyclic anhydrides (NCA) of α-carboxylic acids, star-shaped polyamino acid nanoparticles with highly controllable polymerization and different physicochemical properties were prepared. The preparation method is simple, efficient, and has broad application prospects.

[0036] 3. This invention constructs a series of polyamino acid nanoparticles with long-cycle properties through structural optimization, elucidates the influence of star-shaped copolymer amino acid structural parameters on their in vivo cycling half-life, and provides a theoretical basis for the design of long-cycle nanocarriers.

[0037] 4. This invention screened and obtained polyamino acid materials with high tumor enrichment. Due to the long cycle time, it can exceed the tumor enrichment capacity of PEG in a short time and exhibits excellent anti-tumor ability after drug loading. Attached Figure Description

[0038] Figure 1 The synthetic route for star-shaped polyamino acid nanoparticles;

[0039] Figure 2 To determine the optimal polymerization conditions for star-shaped polyamino acid nanoparticles, among which, Figure 2 'a' in S8-E 70 K 30 Normalized GPC-light scattering plots of nanoparticles at different DCM:DMF volume ratios; Figure 2 b in the context is S8-E 70 K 30 Normalized GPC-light scattering diagrams of nanoparticles in different mixed solvent systems under a 9:1 ratio;

[0040] Figure 3 S8-E 70 K 30 Nuclear magnetic resonance kinetics of the nanoparticle polymerization process;

[0041] Figure 4 S8-E 70 K 30 1H NMR spectrum of nanoparticles;

[0042] Figure 5 For the cytotoxicity of different types of star-shaped polyamino acid nanoparticles, n = 3;

[0043] Figure 6 S8-E 70 K 30 The degradation of nanoparticles under the action of different enzymes showed that the greater the increase in fluorescence signal, the more obvious the degradation, n = 3;

[0044] Figure 7 S8-E 70 K 30 With S8-E100 Pharmacokinetic curves of nanoparticles, n = 3;

[0045] Figure 8 Star-shaped polyamino acids were incorporated with a second type of amino acid nanoparticle (S8-E) 70 X 30 Distribution of tumor-bearing cells in 4T1 tumor-bearing mice in vivo. p < 0.01, n = 3;

[0046] Figure 9 Star-shaped polyamino acids containing 30% arginine nanoparticles (S8-E) 70 R 30 Enrichment analysis of 4T1 tumors p < 0.01, n = 3;

[0047] Figure 10 For linear polyamino acid materials (LE) 70 K 30 Enrichment analysis of 4T1 tumors p < 0.001, n = 3;

[0048] Figure 11 Star-shaped polyamino acid nanoparticles (S8-E) with different copolymerization ratios a K b Enrichment analysis of 4T1 tumors p < 0.5, n = 3;

[0049] Figure 12 For different arm numbers (Sm-E) 70 K 30 Enrichment analysis of nanoparticles in 4T1 tumors. p < 0.5, n = 3;

[0050] Figure 13 For star-shaped polyamino acid nanoparticles with different chain lengths (S8-(E a K b ) n Enrichment analysis plot of 4T1 tumors (a / b = 7:3), n = 3;

[0051] Figure 14 Pharmacokinetic diagrams for different polyamino acid materials, n = 3;

[0052] Figure 15 The synthetic route for star-shaped nanoparticles (S8-PEG) composed of PEG is shown.

[0053] Figure 16The pharmacokinetic curves are for comparison between nanoparticles and PEG, a commonly used long-cycle material, n = 3;

[0054] Figure 17 For the stability test of different star-shaped nanoparticles in a culture medium containing 10% fetal bovine serum, n = 3;

[0055] Figure 18 To verify S8-E 70 K 30 The universality of nanoparticles and commonly used long-cycle material PEG in different tumors. Figure 18 In the figure, 'a' represents the tumor enrichment analysis of 4T1 breast cancer tumor mice; Figure 18 In the figure, b represents the tumor enrichment analysis of B16F10 melanoma tumor mice. Figure 18 In the figure, c represents the tumor enrichment analysis of CT26 colon cancer mice; p < 0.5, p < 0.01, p < 0.001, n = 3;

[0056] Figure 19 S8-E 70 K 30 -TCPP synthesis roadmap;

[0057] Figure 20 S8-E 70 K 30 -In vitro studies of TCPP Figure 20 In the figure, 'a' represents the ROS generated by solutions loaded with different proportions of acoustic sensor nanoparticles after ultrasound. Figure 20 In this context, 'b' represents the toxicity of nanoparticles loaded with different proportions of acoustic sensitizers to 4T1 tumor cells. p < 0.001, n = 3;

[0058] Figure 21 A confocal image showing the generation of reactive oxygen species in 4T1 tumor cells after incubation with nanoparticles loaded with 10% acoustic sensitizer and ultrasound.

[0059] Figure 22 To determine S8-E 70 K 30 -Optimal ultrasound time for TCPP Figure 22 'a' in S8-E 70 K 30 4T1 tumor enrichment analysis at different time points; Figure 22 b in the context is S8-E 70 K 30 Enrichment analysis of 4T1 tumors after loading with 10% sonosensitive agent for 4 h, n = 3;

[0060] Figure 23 S8-E 70 K 30 -In vivo treatment of TCPP Figure 23 In this context, 'a' represents the drug administration strategy for treatment. Figure 23 In this context, 'b' represents the overall treatment situation. p < 0.001, n = 6;

[0061] Figure 24 These are pathological sections of tumor tissue after treatment. Figure 24 In the image, 'a' represents hematoxylin-eosin staining for observing the pathological condition of tumor tissue. Figure 24 In the figure, b represents the observation of tumor cell apoptosis in tumor tissue using a nick-end labeling method mediated by deoxyribonucleotide terminal transferase. Figure 24 c in the figure represents the observation of reactive oxygen species production in tumor tissue by staining with reactive oxygen probes.

[0062] Figure 25 The image shows S8-E. 70 K 30 A schematic diagram used for tumor treatment. Detailed Implementation

[0063] To further illustrate the preparation and application of the star-shaped polyamino acid nanoparticles with long-cycle properties of the present invention and to achieve the intended purpose of the invention, the present invention will be further described in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0064] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0065] The reagents used in the embodiments of this invention are from the following manufacturers and catalog numbers: Cy5 NHS ester: purchased from Suzhou Ketong Biomedical Technology Co., Ltd., catalog number 40795ES03; polyamide-amine (G1, 8 arms): purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., catalog number 412384; polyamide-amine (G2, 16 arms): purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., catalog number 664138-1KT; polyamide-amine (G3, 32 arms): purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., catalog number 412422; proteinase K: purchased from Shanghai... Aladdin Biochemical Technology Co., Ltd., catalog number P109033; trypsin purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number T105532; SOSG probe purchased from Shanghai Beyotime Biotechnology Co., Ltd., catalog number S0067; DCFH-DA fluorescent probe purchased from Shanghai Beyotime Biotechnology Co., Ltd., catalog number S0033S; isothiocyanate (FITC) purchased from Jiangsu Argon Krypton Xenon Materials Technology Co., Ltd., catalog number E080600; γ-tert-butyl- L - Glutamic acid was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number S135595; N ε -tert-Butyloxycarbonyl- L -Lysine was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number L110998; O -Terbutyl- L - Serine was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., product number O803197; O -Terbutyl- L -Tyrosine was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., product number O803199; L Leucine was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., product number L812333; L -Phenylalanine was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., product number P6248; N δ -2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl- L Arginine was purchased from Jiangsu Argon Krypton Xenon Materials Technology Co., Ltd., item number A070418; trifluoroacetic acid was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., item number T281759; tetrakis(4-carboxyphenyl)porphyrin (TCPP) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., item number P816654; 3-(4,5-dimethyl-2-thiazole)-2,5-diphenyltetrazolium bromide thiazole blue (MTT) was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., item number 40201ES80.

[0066] Female BALB / c mice, 6-8 weeks old and weighing 18-20 g, were purchased from Changzhou Cavens Laboratory Animal Co., Ltd. and housed in a specific pathogen-free (SPF) grade animal laboratory. All animal experiments were conducted in accordance with the guidelines of the Laboratory Animal Management and Ethics Committee of Soochow University.

[0067] The mouse mononuclear macrophage leukemia cells (RAW 264.7) were donated by Professor Zheng Yiran's research group at Soochow University, while the mouse breast cancer cells (4T1) and human umbilical vein endothelial cells (HUVEC) were donated by Professor Yin Lichen's research group at Soochow University. The 4T1 cells were cultured in RPMI-1640 medium containing 10% FBS, and the remaining cells were cultured in DMEM medium containing 10% FBS.

[0068] Example 1: Preparation of polyglutamic acid / lysine star nanoparticles (S8-E) 70 K 30 ) method

[0069] This embodiment provides a method for synthesizing polyglutamic acid / lysine star-shaped nanoparticles (S8-E). 70 K 30 The method and synthesis route are described in [reference needed]. Figure 1 The details are as follows:

[0070] Star-shaped polyamino acid nanoparticles were prepared by ring-opening polymerization (ROP) of NCA monomers using polyamide-amine (G1-PAMAM, 8 arms) as an initiator. Taking the synthesis of poly(glutamic acid / lysine) star-shaped nanoparticles as an example, 19.3 mg of γ-tert-butyl- L -Glutamate NCA ( t Bu-Glu-NCA, 0.084 mmol), 9.8 mg N ε -tert-Butyloxycarbonyl- L -Lysine NCA (30% of the total molar ratio of polyamino acids, BLL-NCA, 0.036 mmol) and 1.1 mg polyamide-amine (PAMAM) initiator (0.15 μmol, molar ratio of amino to NCA in PAMAM is 1:100), anhydrous N , N - Dimethylformamide (DMF) and dichloromethane (DCM). Dissolve in anhydrous DCM:DMF (300 μL, 9:1, v / v). tBu-Glu-NCA and BLL-NCA were both treated with anhydrous DCM:DMF (55 μL, 9:1, v / v) to dissolve the PAMAM initiator. Both solutions were added to a polymerization flask and stirred at room temperature. Reaction kinetics were monitored using infrared spectroscopy, and the reaction was considered complete when monomer conversion > 99% (1.5 h). The nanoparticles were purified by precipitation with diethyl ether:n-hexane (1:1, v / v), and the copolyamino acid nanoparticles were dissolved in DCM (500 μL). Trifluoroacetic acid (500 μL) was then added to the solution at 0°C. o Deprotection was performed by stirring in an ice bath for 3 h. After deprotection, polyamino acid nanoparticles were precipitated with diethyl ether: n-hexane. The precipitated polyamino acid nanoparticles were dissolved in a 0.2 M NaHCO3 aqueous solution, and the pH of the solution was adjusted to 8. Dialysis (dialysis bag molecular weight cutoff of 3500 Da, 6 h) and lyophilization were performed to obtain S8-E. 70 K 30 Nanoparticles (yield 53%).

[0071] To trace polyamino acid nanoparticles in cell and animal experiments, an additional 1 wt% of [a specific ingredient] was added during copolymerization. N ε -tert-Butyloxycarbonyl- L -Lysine NCA (BLL-NCA, 0.33 mg, 1.2 μmol). Deprotected and precipitated polyamino acid nanoparticles were dissolved in NaHCO3 (0.2 M) aqueous solution, and the pH was adjusted to 8. Cy5 NHS ester (50 μL, 1.6 μmol) was then added and the mixture was stirred overnight. The reaction mixture was dialyzed and lyophilized to obtain fluorescently labeled S8-E. 70 K 30 Nanoparticles.

[0072] Other star-shaped polyamino acid nanoparticles and fluorescently labeled star-shaped polyamino acid nanoparticles (such as those in Example 1) were prepared using the same preparation method as in Example 1. Figure 1 As shown), the difference is: N ε -tert-Butyloxycarbonyl- L -Lysine NCA was replaced with γ-tert-butyl-L-glutamic acid NCA, L -Phenylanine NCA, L -Leucine NCA, O-tert-butyl- L -Serine NCA and O-tert-butyl- L -Tyrosine NCA was used to prepare polyamino acid nanoparticles, namely polyglutamic acid nanoparticles (S8-E). 100 ), polyglutamic acid / phenylalanine nanoparticles (S8-E) 70 F 30 ), polyglutamic acid / leucine nanoparticles (S8-E)70 L 30 ), polyglutamic acid / serine nanoparticles (S8-E) 70 S 30 ) or polyglutamic acid / tyrosine nanoparticles (S8-E) 70 Y 30 ) and its fluorescently labeled star-shaped amino acid nanoparticles.

[0073] Example 2: Polyglutamic acid / lysine star nanoparticles (S8-E) in solvent systems with different mixing ratios 70 K 30 Preparation of )

[0074] Example 2 and Example 1: Synthesis of polyglutamic acid / lysine star nanoparticles (S8-E) 70 K 30 The method is the same, except that the solvent dichloromethane (DCM) is used during polymerization. N , N The volume ratio of dimethylformamide (DMF) was changed from 9:1 to 8:2, 7:3, and 6:4 respectively.

[0075] Example 3: Polyglutamic acid / lysine star nanoparticles (S8-E) in different types of solvent systems 70 K 30 Preparation of )

[0076] Example 3 and Example 1: Synthesis of polyglutamic acid / lysine star nanoparticles (S8-E) 70 K 30 The method is the same, except that dichloromethane (DCM) with a volume ratio of 9:1 during polymerization is used. N , N The mixed solvent of dimethylformamide (DMF) was replaced with an equal volume ratio of chloroform (CHCl3) and... N , N Mixed solvents of dimethylformamide (DMF), tetrahydrofuran (THF), and... N , N Mixed solvents of dimethylformamide (DMF) and dichloromethane (DCM) and N-methylpyrrolidone (NMP).

[0077] Comparative Example 1: Poly(glutamic acid / lysine) star-shaped nanoparticles (S8-E) 70 K 30 DMF solvent method for preparation

[0078] Comparative Example 1 and Example 1 synthesized polyglutamic acid / lysine star nanoparticles (S8-E) 70 K 30The method is the same, except that dichloromethane (DCM) in a volume ratio of 9:1 is used with... N , N The mixed solvent of dimethylformamide (DMF) was replaced with anhydrous... N , N - Dimethylformamide (DMF, 300 μL, 0:10) and a mixed solvent of DCM and DMF in a volume ratio of 5:5.

[0079] During polymerization, dichloromethane (DCM) is reacted with... N , N When the mixed solvent system of dimethylformamide (DMF) is changed to dichloromethane (DCM), N ε -tert-Butyloxycarbonyl- L -Lysine NCA has extremely poor solubility in DCM, which prevents the polymerization reaction from proceeding normally.

[0080] Experimental Example 1

[0081] To determine the optimal polymerization conditions, star-shaped polyamino acid nanoparticles (S8-E) prepared in Examples 1, 2, and 3, as well as Comparative Example 1, were used. 70 K 30 For example, the number-average molecular weight and molecular weight distribution of star-shaped copolyamino acids were determined by GPC. Unprotected copolyamino acids were dissolved in chromatographic grade DMF containing LiBr (0.1 M) to prepare a polymer DMF solution (5 mg / mL). The polymer solution was filtered through a polytetrafluoroethylene filter membrane (0.22 μm) and then analyzed by GPC.

[0082] Polyglutamic acid / lysine star nanoparticles (S8-E) prepared in Examples 1 and 2 and Comparative Example 1 70 K 30 The molecular weight and dispersity test results of the sample are shown in Table 1:

[0083] Table 1 S8-E 70 K 30 Molecular weight and dispersity test results at different mixing ratios of DCM:DMF

[0084]

[0085] Example 3 Preparation of polyglutamic acid / lysine star nanoparticles (S8-E) 70 K 30 The molecular weight and dispersity test results of the sample are shown in Table 2:

[0086] Table 2 S8-E 70 K 30Molecular weight and dispersity test results in different types of mixed solvents

[0087]

[0088] Combining Table 1 and Figure 2 In the analysis of α, as the proportion of DCM increases, S8-E 70 K 30 The molecular weight gradually increases. When DCM:DMF = 9:1, S8-E 70 K 30 The molecular weight is 161 kDa, which agrees well with the theoretical value (160 kDa), and the dispersity is below 1.06, exhibiting a narrow molecular weight distribution. In polymerization systems with DCM:DMF ratios of 8:2 and 7:3, S8-E... 70 K 30 The molecular weight is close to the theoretical molecular weight, and the dispersion is also low, but not as excellent as under the 9:1 condition; when the DCM ratio is reduced to 6, the molecular weight differs significantly from the theoretical molecular weight; in the polymerization system with a DCM:DMF ratio of 5:5, not only is the molecular weight much smaller than the theoretical molecular weight, but the molecular weight distribution is also relatively wide, which cannot meet the requirements for subsequent nanoparticle preparation; in the single polymerization system, the product obtained by polymerization in pure DMF solvent has a molecular weight of only 69.1 kDa, which is also much smaller than the theoretical value, and the dispersion is large; based on the data in Table 1, DCM:DMF = 9:1 is the optimal ratio for preparing poly(glutamic acid / lysine) star nanoparticles (S8-E). 70 K 30 The optimal solvent ratio is determined under these conditions: the molecular weight is highest (160.8 kDa), close to the theoretical molecular weight; the dispersion is lowest (1.05), the molecular weight distribution is narrow, and the polymerization controllability is good.

[0089] After determining that 9:1 was the optimal polymerization ratio, the S8-E was replaced. 70 K 30 The polymerization solvent system, combined with Table 2 and Figure 2 Analysis of b in the data shows that dichloromethane (DCM) and... N , N The mixed solvent system of dimethylformamide (DMF) was replaced with chloroform (CHCl3) and... N , N A mixed solvent system of dimethylformamide (DMF) (9:1) and dichloromethane (DCM) with... N In a 9:1 mixed solvent system of NMP (methylpyrrolidone), the molecular weight is close to the theoretical value (160 kDa), and the dispersion is below 1.06, exhibiting a narrow molecular weight distribution. This indicates that both mixed solvent systems are good solvent systems for polymerization, but the polymerization effect is still not as good as that of dichloromethane (DCM) and...N , N - A mixed solvent system of dimethylformamide (DMF); instead, tetrahydrofuran (THF) and N , N The polymerization of dimethylformamide (DMF) in a mixed solvent system (9:1) resulted in a molecular weight much higher than the theoretical value and a high degree of dispersion, indicating that the polymerization process was difficult to control under this mixed solvent system.

[0090] In summary, this invention uses a mixed solvent system of DCM:DMF = 9:1 for subsequent experiments.

[0091] Experimental Example 2

[0092] use 1 H NMR was used to monitor S8-E 70 K 30 In CD2Cl2 and DMF- d 7. Polymerization kinetics of NCA in mixed solutions, with S8-E 70 K 30 For example, t Bu-Glu NCA and BLL NCA were dissolved in CD2Cl2:DMF-d7 (800 μL, 9:1, v / v), mixed thoroughly, and then G1-PAMAM was added and mixed again before being transferred to an NMR tube. The proton NMR spectra were monitored every 90 s after polymerization began, and the results were calculated from each spectrum. t The residual rate of NCA monomers was calculated by integrating the α-H signals of Bu-Glu NCA (δ = 4.40 ppm) and BLL NCA (δ = 4.31 ppm) and normalizing the α-H integrated signal at time t = 0 (i.e., 100% NCA remaining). Figure 3 The results show that the remaining percentages of the two NCAs are similar within the same time period, indicating that the reaction rates of tBu-Glu-NCA and BLL-NCA are similar, which is consistent with the characteristics of random copolymerization. Figure 4 China adopts 1 The polymerization ratio of different star-shaped copolymer amino acids was determined by ¹H NMR, using S8-E NMR as the criterion. 30 K 70 For example, S8-E was dissolved using TFA-d (500 μL). 30 K 70 Polymer (5 mg), performed 1 HNMR testing, analyzed using... 1 The integral area of ​​each characteristic peak in the H NMR spectrum was used to calculate the actual polymerization ratio of different components of the star polymer. The NMR characterization results showed... tThe ratio of Bu-Glu NCA to BLL-NCA was 6.9:3.1, which is close to the feed ratio (7:3). These test results demonstrate the controllability of the polymerization.

[0093] Experimental Example 3

[0094] To further explore the biocompatibility of the nanoparticles, mouse mononuclear macrophage leukemia cells (RAW264.7) and human umbilical vein endothelial cells (HUVECs) were used to assess the cytotoxicity of the nanoparticles. 5 × 10⁻⁶ nanoparticles were used in this study. 3 After seeding cells in 96-well plates and incubating overnight, different types (S8-E prepared in Example 1) were then added. 70 K 30 S8-E 100 S8-E 70 F 30 S8-E 70 L 30 S8-E 70 S 30 S8-E 70 Y 30 Star-shaped polyamino acid nanoparticles (100 μL, 1 mg / mL) were co-incubated with cells for 24 h; simultaneously, cells were co-incubated with the same volume of PBS (100 μL) for 24 h, followed by incubation with MTT (100 μL / well, 1 mg / mL) for 4 h. After carefully aspirating the supernatant, DMSO (100 μL) was added to dissolve formazan crystals, and cytotoxicity was detected by measuring absorbance at 570 nm using a microplate reader. Figure 5 As can be seen, with PBS as the control group, the cells still had high cell activity in the high concentration of materials, which verifies the biosafety of nanoparticles.

[0095] Test Example 4

[0096] The degradation of nanoparticles was characterized by fluorescence aggregation-induced quenching. Specifically, star-shaped polyamino acid nanoparticles S8-E prepared in Example 1 were used. 70 K 30 For the experimental group, 10 mol% (the amount of FITC relative to S8-E) was grafted onto 20 mg of polyamino acid chains. 70 K 30Fluorescein isothiocyanate (FITC) (1.12 mg, 3.0 mmol) containing the molar percentage of lysine repeating units in polyamino acids, normally exhibits fluorescence aggregation-induced quenching, resulting in abundant grafted fluorescence but weak detected fluorescence intensity. Solutions were prepared with concentration ratios of trypsin:nanoparticles = 1:3 and proteinase K:nanoparticles = 1:3. A control group with the same volume of PBS added to the nanoparticle solution was used. The solutions were placed in a 37°C water bath, and the fluorescence intensity was measured at different time points using a microplate reader. Figure 6 The results indicated that, compared to the PBS group, the presence of these two enzymes cleaved the peptide bonds containing lysine residues, preventing luciferin aggregation and increasing fluorescence intensity, indicating that S8-E... 70 K 30 The nanoparticles are degraded by two enzymes, making the material biodegradable.

[0097] Experimental Example 5

[0098] Preliminary exploration of the blood circulation ability of nanoparticles was conducted using fluorescently labeled polyglutamic acid nanoparticles (S8-E) prepared in Example 1. 100 (S8-E) served as the control group, with fluorescently labeled S8-E as the control group. 70 K 30 Nanoparticles were used as the experimental group for pharmacokinetic analysis. First, fluorescently labeled polyamino acid nanoparticles (200 μL, 1 mg / mL) were injected into mice via tail vein (each group of nanoparticles was repeated 3 times). Then, blood was collected from the orbital sinus at specific time points. The blood was treated with erythrocyte lysis buffer (1 mL, 5 min) and centrifuged (1200 × rpm, 5 min). The supernatant was collected, and the fluorescence intensity was detected using an ELISA reader. The nanoparticle content in the blood at different time points was calculated. The blood drug concentration-time curve was fitted using a two-compartment model to calculate the distribution half-life and elimination half-life. The results are shown in Table 3.

[0099] Table 3 Star-shaped nanoparticles (S8-E) 100 S8-E 70 K 30 Pharmacokinetic parameters of )

[0100]

[0101] Combined with Table 3 and Figure 7 Analysis shows that S8-E 70 K 30 Both the distribution half-life and elimination half-life are significantly increased, resulting in a longer blood circulation time. Therefore, the addition of a second amino acid may affect the blood circulation behavior of nanoparticles.

[0102] Experimental Example 6

[0103] Because prolonged blood circulation is a prerequisite and guarantee for high tumor enrichment efficiency, this study explored the enrichment of different star-shaped nanoparticles in tumors, and further investigated the effect of adding a second amino acid on the blood circulation behavior of nanoparticles. (The text then abruptly shifts to a seemingly unrelated topic about 1×10⁻⁶ amino acids.) 6 4T1 cells were dissolved in 50 μL PBS and subcutaneously injected into mice to establish a 4T1 tumor model. The tumor volume was increased to 150 mm². 3 At approximately 10:00 AM, mice were randomly divided into an experimental group and a control group (PBS group). The experimental group received a tail vein injection (1 mg / mL, 200 μL) of fluorescently labeled polyamino acid nanoparticles of different types (fluorescently labeled star-shaped polyamino acid nanoparticles S8-E prepared in Example 1). 70 K 30 S8-E 100 S8-E 70 F 30 S8-E 70 L 30 S8-E 70 S 30 S8-E 70 Y 30 The control group (PBS group) received an equal volume of PBS (200 μL) via tail vein injection, with three replicates per group. Eight hours later, mice were euthanized, and major organs such as the heart, liver, spleen, lungs, and kidneys, as well as tumors, were removed for in vitro imaging to observe their distribution. Figure 8 It can be seen that when the molar percentage of lysine in the nanoparticles is 30% (accounting for 30% of the total molar amount of polyamino acids), compared to other copolymerized amino acid nanoparticles, the S8-E nanoparticles prepared in Example 1... 70 K 30 It exhibits a significant enrichment advantage in tumor tissues, and also has a high distribution in other organs.

[0104] These results confirm that lysine side chains endow nanoparticles with long-cycle properties and promote their efficient accumulation in tumor tissues through the EPR effect. Although nanoparticles are also distributed in other organs, this phenomenon is a normal manifestation of their long-cycle properties.

[0105] Comparative Example 2: Preparation of Polyglutamic Acid / Arginine Nanoparticles

[0106] This embodiment provides a method for synthesizing nanoparticles doped with different positively charged amino acids. The synthesis method is the same as in Example 1, except that... N ε -tert-Butyloxycarbonyl- L -Replace lysine NCA with N δ -2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl-L -Arginine NCA was used to prepare polyglutamic acid / arginine nanoparticles and corresponding fluorescently labeled copolyamino acid star-shaped nanoparticles (S8-E). 70 R 30 ).

[0107] Comparative Example 3: Preparation of linear polyamino acid nanoparticles

[0108] This embodiment provides a method for synthesizing linear polyamino acid nanoparticles (LE). 70 K 30 The synthesis method is the same as in Example 1, except that the polyamide-amine (G1-PAMAM) initiator is replaced with n-hexylamine initiator to prepare the corresponding linear polyamino acid nanoparticles (LE). 70 K 30 ( ) and corresponding fluorescently labeled copolyamino acid star-shaped nanoparticles.

[0109] Experimental Example 7

[0110] To further clarify the effects of amino acid side chains and star-shaped structures of nanoparticles on long-term cycling, a 4T1 tumor model was established according to the procedure in Example 6, and mice were randomly divided into a control group and an experimental group. Fluorescently labeled poly(glutamate / lysine) star-shaped nanoparticles S8-E prepared in Example 1 were used. 70 K 30 As control group 2, the polyglutamic acid / arginine fluorescently labeled copolyamino acid star nanoparticles (S8-E) prepared in comparative example 2... 70 R 30 The fluorescently labeled linear polyamino acid nanoparticles (LE) prepared for experimental group 1 and comparative example 3 are shown. 70 K 30 Group 2 consisted of mice injected intravenously via tail vein with fluorescently labeled polyamino acid nanoparticles of different types (1 mg / mL, 200 μL). Control group 1 (PBS group) was administered an equal volume of PBS via tail vein injection. Each group had three replicates. Eight hours later, the mice were euthanized, and major organs such as the heart, liver, spleen, lungs, and kidneys, as well as tumors, were removed for in vitro imaging to observe their distribution. (See also...) Figure 9 The preparation of Example 1 N ε -tert-Butyloxycarbonyl- L -Lysine NCA replaced with N δ -2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl- L When the amino side chain of lysine is replaced with the guanidinyl side chain of arginine (-arginine NCA), the tumor enrichment effect of nanoparticles is significantly reduced (S8-E). 70 K30 It's S8-E 70 R 30 The tumor enrichment efficiency of nanoparticles was 4.4 times higher; when the star-shaped structure of the nanomaterial prepared in Example 1 was replaced with the linear structure of Comparative Example 3, as... Figure 10 As shown, small animal imaging results indicate S8-E 70 K 30 Nanoparticles are LE 70 K 30 The tumor enrichment efficiency was 12.8 times that of linear nanoparticles, while the tumor enrichment effect of linear nanoparticles was significantly reduced. This indicates that both lysine side chains and star structures are crucial for tumor enrichment, and thus, both are key factors in regulating blood circulation.

[0111] Example 4: Star-shaped nanoparticles (S8-E) with different glutamic acid / lysine ratios a K b Preparation of )

[0112] This embodiment provides a method for synthesizing nanoparticles (S8-E) with different amino acid ratios. a K b The method of synthesis is the same as in Example 1, except that: N ε -tert-Butyloxycarbonyl- L - The molar ratio of lysine NCA to total NCA monomers was replaced from 30% to 10%, 20%, and 40%, respectively. Similarly, γ-tert-butyl- L - By replacing the molar ratio of glutamic acid (NCA) to total NCA monomers from 70% to 90%, 80%, and 60%, fluorescently labeled poly(glutamic acid / lysine) star-shaped nanoparticles S8-E with different copolymerization ratios were prepared. 60 K 40 S8-E 80 K 20 S8-E 90 K 10 .

[0113] Experimental Example 8

[0114] To further clarify the effect of the lysine side chain on long-term cycling, star-shaped polyamino acid nanoparticles with different polymerization ratios were prepared. A 4T1 tumor model was established according to the method in Example 6, and fluorescently labeled poly(glutamate / lysine) star-shaped nanoparticles S8-E prepared in Example 1 were used. 70 K 30 Fluorescently labeled poly(glutamic acid / lysine) star nanoparticles (S8-E) with different ratios prepared in Example 4 60 K 40 S8-E 80 K20 S8-E 90 K 10 The experimental group consisted of mice labeled with fluorescently labeled polyamino acid nanoparticles of different types (1 mg / mL, 200 μL) injected via the tail vein, while the control group (PBS group) consisted of mice injected via the tail vein with an equal volume of PBS (200 μL). Each group had three replicates. Eight hours later, the mice were euthanized, and major organs such as the heart, liver, spleen, lungs, and kidneys, as well as tumors, were removed for in vitro imaging to observe their distribution. Figure 11 It can be seen that, compared with the amino acid star nanoparticles prepared in other proportions in Example 3, when 30% N is incorporated... ε -tert-Butyloxycarbonyl- L -Lysine NCA showed high enrichment in tumor tissue. This suggests that the proportion of lysine side chains in star-type polyamino acid nanoparticles affects their blood circulation capacity.

[0115] Example 5: Star-shaped nanoparticles (Sm-E) with different numbers of polyamino acid arms 70 K 30 Preparation of )

[0116] This embodiment provides a method for synthesizing star-shaped nanoparticles (Sm-E) with different arm numbers. 70 K 30 The synthesis method is the same as in Example 1, except that the polyamide-amine (PAMAM) is replaced by G2 or G3 instead of G1, and the generation of the polyamide-amine (PAMAM) is changed, correspondingly changing the number of arms from 8 to 16 or 32. Fluorescently labeled poly(glutamic acid / lysine) star nanoparticles S8-E with different arm numbers were prepared. 70 K 30 S16-E 70 K 30 S32-E 70 K 30 .

[0117] Example 6: Synthesis of nanoparticles with different chain lengths (S8-(E a K b ) n Preparation of (a / b = 7:3)

[0118] This embodiment provides a method for synthesizing nanoparticles with different chain lengths (S8-(E)). a K b ) nThe synthesis method (a / b = 7:3, n = 50 / 100 / 200) is the same as in Example 1, except that the molar ratio of amino groups to NCA in the polyamide-amine (PAMAM) is replaced from 1:100 to 1:50 or 1:200. Fluorescently labeled poly(glutamic acid / lysine) star-shaped nanoparticles S8-E with different chain lengths were prepared. 35 K 15 S32-E 140 K 60 .

[0119] Experimental Example 9

[0120] To further clarify the influence of nanoparticle structure on long-term cycling, star-shaped polyamino acid nanoparticles with different arm numbers and chain lengths were prepared in Examples 5 and 6. A 4T1 tumor model was established as described in Example 6, and fluorescently labeled poly(glutamate / lysine) star-shaped nanoparticles S8-E prepared in Example 1 were used. 70 K 30 The fluorescently labeled poly(glutamate / lysine) star-shaped nanoparticles prepared in Examples 5 and 6 served as the experimental group. Mice were intravenously injected with 200 μL (1 mg / mL) of the fluorescently labeled polyamino acid nanoparticles via the tail vein, while the control group (PBS group) received an equal volume of PBS (200 μL) via the tail vein. Each group had three replicates. Eight hours later, the mice were euthanized, and major organs such as the heart, liver, spleen, lungs, and kidneys, as well as tumors, were removed for in vitro imaging to observe their distribution. See [link to relevant documentation]. Figure 12 Polyamino acid nanoparticles with different arm numbers exhibit varying enrichment effects in tumor tissues, with nanoparticles having lower arm numbers showing better tumor enrichment. Therefore, S8-E 70 K 30 The tumor enrichment efficiency is the highest; Figure 13 In the study, polyamino acid nanoparticles of different chain lengths showed similar enrichment effects in tumor tissues, but combined with quantitative fluorescence analysis data, it was observed that S8-E... 70 K 30 It still exhibits the highest tumor enrichment efficiency. This suggests that the number of arms of the star-shaped polyamino acid nanoparticles affects the tumor enrichment behavior of the nanoparticles, but the chain length has a smaller impact on the tumor enrichment behavior of the nanoparticles. Furthermore, it suggests that the number of arms of the star-shaped structure affects the blood circulation capacity of the nanoparticles.

[0121] Experimental Example 10

[0122] The above experimental examples have confirmed that the aforementioned factors all affect the tumor enrichment effect; therefore, pharmacokinetic analysis was used to evaluate the blood circulation capacity of typical nanoparticles. Some typical nanoparticles were selected, using the S8-E nanoparticles from Example 1. 70 K 30 Comparative Example 2 S8-E70 R 30 And Comparative Example 3 LE 70 K 30 S8-E in Example 4 60 K 40 S16-E in Example 5 70 K 30 and S8-E in Example 6 140 K 60 (The molar ratio of amino to NCA in G1-PAMAM was 1:200). These fluorescently labeled polyamino acid nanoparticles (200 μL, 1 mg / mL) were injected into mice via tail vein injection (each group of nanoparticles was repeated 3 times). Blood was collected from the orbital sinus at time points. The blood was treated with erythrocyte lysis buffer (1 mL, 5 min) and centrifuged (1000 × rpm, 5 min). The supernatant was collected, and the fluorescence intensity was detected using an ELISA reader. The nanoparticle content in the blood at different time points was calculated. The blood drug concentration-time curve was fitted using a two-compartment model, and the distribution half-life and elimination half-life were calculated. The results are shown in Table 4.

[0123] Table 4 Pharmacokinetic parameters of different copolymer amino acid materials

[0124]

[0125] Distribution half-life reflects the initial retention capacity of nanoparticles in the blood and is a primary condition determining whether they can reach the tumor site via the EPR effect. For passive tumor targeting, sufficient blood circulation time is a prerequisite for tumor accumulation. Based on this, we focus on using this parameter as an evaluation index for analysis. (See Table 4 and...) Figure 14 Analysis shows that the pharmacokinetic results are highly consistent with the tumor enrichment results, compared to the S8-E prepared in Example 1. 70 K 30 Example 2: Nanoparticles (S8-E) incorporating 30% arginine 70 R 30 ) and Comparative Example 3 linear polyamino acid (LE) 70 K 30 All of them have a relatively short distribution half-life. The 16-arm star-shaped polyamino acid nanoparticles prepared in Example 5 also have a shortened distribution half-life, but the S8-E in Example 6... 140 K 60 The distribution half-life showed no significant change. This demonstrates that different structures and amino acid types affect blood circulation time, thus resulting in different tumor enrichment effects, particularly for S8-E. 70 K 30 It has a longer blood circulation time and a better tumor enrichment effect.

[0126] Comparative Example 4: Star-shaped nanoparticles composed of PEG (S8-PEG)

[0127] This comparative example provides a method for synthesizing star-shaped nanoparticles (S8-PEG) composed of PEG. See [link to relevant documentation]. Figure 15 The specific steps are as follows:

[0128] (1) First, weigh a certain amount of triphosgene (1.7 mg, 5.8 μmol) and polyethylene glycol (PEG-OH, 24 mg, 4.8 μmol) and transfer them to a vacuum glove box. Dissolve the triphosgene in anhydrous DCM (103 μL) and then slowly add the polyethylene glycol dissolved in anhydrous DCM (480 μL) to the triphosgene solution. Stir the reaction in the glove box at room temperature. After 2 h, transfer the reaction to the glove box and use a double-row tube to evacuate the vacuum to remove DCM and impurities.

[0129] (2) The dried product was transferred to a glove box and dissolved in anhydrous DCM (985 μL). Then, G1-PAMAM (4.3 mg, 4.8 μmol) initiator was dissolved in anhydrous DCM (215 μL). The two solutions were added to the polymerization flask and stirred at room temperature for 12 h. After the reaction was completed, the nanoparticles were purified by precipitation with diethyl ether: n-hexane (1:1, v / v). The precipitated polyamino acid nanoparticles were dissolved in NaHCO3 (0.2 M) aqueous solution and the pH of the solution was adjusted to 8. The solution was dialyzed and lyophilized to obtain S8-PEG nanoparticles.

[0130] To trace polyamino acid nanoparticles in cell and animal experiments, an additional 1 wt% was added during copolymerization. N ε -tert-Butyloxycarbonyl- L -Lysine NCA was used for fluorescent labeling, and the specific operation was similar to the steps above, except that when performing (2), polyamide-amine (PAMAM) was first reacted with... N ε -tert-Butyloxycarbonyl- L The reaction was initiated with lysine NCA (0.33 mg, 1.2 μmol), and the reaction kinetics were detected using infrared spectroscopy. The reaction was considered complete when the monomer conversion was > 99% (1.5 h). The product was then reacted with the dried product. After the reaction was complete, the nanoparticles were purified by precipitation with diethyl ether:n-hexane (1:1, v / v). The copolyamino acid nanoparticles were then dissolved in DCM (500 μL), and trifluoroacetic acid (20 μL) was added. oDeprotection was performed by stirring in an ice bath for 3 h. After deprotection, water-soluble nanoparticles were obtained by precipitation with diethyl ether:n-hexane. The deprotected nanoparticles were dissolved in NaHCO3 (0.2 M) aqueous solution, and the pH of the solution was adjusted to 8. Cy5 NHS ester (50 μL, 1.6 μmol) was then added and stirred overnight. The reaction was followed by dialysis (dialysis bag molecular weight cutoff of 3500 Da, 8 h) and lyophilization to obtain fluorescently labeled S8-PEG nanoparticles.

[0131] Experimental Example 11

[0132] S8-E prepared in Example 1 is known. 70 K 30 It exhibits a longer blood circulation time and better tumor enrichment effect. The objective effect was observed by comparing it with commonly used PEG materials. The fluorescently labeled poly(glutamate / lysine) star-shaped nanoparticles S8-E prepared in Example 1 were then compared. 70 K 30 Polyglutamic acid nanoparticles (S8-E) 100 The fluorescently labeled S8-PEG nanoparticles (200 μL, 1 mg / mL) prepared in Comparative Example 4 were injected into mice via tail vein injection (each group of nanoparticles was repeated 3 times). Blood was collected from the orbital sinus at time points, and the blood was treated with erythrocyte lysis buffer (1 mL, 5 min) and centrifuged (1000 × rpm, 5 min). The supernatant was collected and analyzed using an ELISA reader to calculate the content of polyamino acid nanoparticles in the blood at different time points. The blood drug concentration-time curve was fitted using a two-compartment model, and the distribution half-life and elimination half-life were calculated, as shown in Table 5.

[0133] Table 5. Pharmacokinetic parameters of different star-shaped nanoparticles

[0134]

[0135] Combined with Table 6 and Figure 16 Analysis of the pharmacokinetic results shows that the S8-PEG nanoparticles prepared in Comparative Example 4 exhibit typical long-cycle characteristics, but the S8-E nanoparticles... 70 K 30 Compared to S8-PEG, the distribution half-life is longer, while the elimination half-life is shorter. However, in the field of tumor nanomedicine delivery, the distribution half-life is more critical than the elimination half-life because the distribution loop half-life ensures that the nanoparticles can fully utilize the EPR effect, repeatedly flowing through the tumor and effectively infiltrating before being eliminated, which is fundamental to achieving efficient delivery. Therefore, the fluorescently labeled poly(glutamate / lysine) star-shaped nanoparticles S8-E prepared in Example 1... 70 K 30The excellent blood circulation and tumor targeting capabilities demonstrate the significant advantages of the poly(glutamate / lysine) star structure as a tumor nanodrug delivery carrier.

[0136] Experimental Example 12

[0137] from Figure 16 It can be known that S8-E 70 K 30 The distribution half-life is relatively long. To explore its mechanism, the stability of different nanoparticles was further investigated, and their protein adsorption behavior was analyzed. Polyglutamic acid nanoparticles (S8-E) prepared in Example 1 were used. 100 As a control group, S8-E prepared in Example 1 was used. 70 K 30 The S8-PEG prepared in Comparative Example 3 was used as the experimental group. Nanoparticles from each group were dispersed in a culture medium containing 10% fetal bovine serum (1 mg / mL) and incubated at 37°C. The particle size changes of the nanoparticles were monitored at different time points, and the results are as follows: Figure 17 As shown. S8-E 100 No significant protein adsorption was observed in the S8-PEG group, consistent with expectations. However, protein adsorption occurred earlier in the S8-PEG group. Adsorbed proteins may be more quickly recognized by the mononuclear phagocytic system and subsequently rapidly cleared, which may explain the shorter distribution half-life of S8-PEG. In contrast, S8-E... 70 K 30 Protein adsorption only occurred in the group after 8 hours, effectively avoiding protein adsorption and immune recognition in the bloodstream, thus maintaining a longer blood circulation time. In Experiment 4, it was shown that S8-E... 70 K 30 It will be effectively degraded through enzymatic hydrolysis, combined with Figure 16 The pharmacokinetic results showed that after circulating in vivo for a certain period, the nanoparticles were effectively degraded and eliminated through enzymatic hydrolysis, which is consistent with their short elimination half-life in vivo. This also indicates that S8-E... 70 K 30 After tumor enrichment, it can be effectively cleared by the body, avoiding the risk of long-term in vivo accumulation that may occur due to the difficulty in degradation of PEG-modified nanoparticles.

[0138] Experimental Example 13

[0139] Whether it is the S8-E prepared in Example 1 70 K 30The S8-PEG nanoparticles prepared in Comparative Example 4 all exhibited longer in vivo blood circulation time. Based on the high permeability and retention effect (EPR effect), nanocarriers with excellent long circulation characteristics are more likely to achieve effective enrichment at tumor sites. To evaluate the tumor enrichment efficiency and delivery universality of the nanoparticles, three representative mouse subcutaneous xenograft models were constructed (established by: 1 × 10⁻⁶ nanoparticles were placed in a 1 × 10⁻⁶ nanoparticle container). 6 A corresponding tumor model was established by subcutaneous injection of 50 μL of PBS into mice, and the tumor volume was increased to 150 mm². 3 When the corresponding models are established (around 10:00-1000), the models are: breast cancer 4T1 model, melanoma B16F10 model, and colon cancer CT26 model. Mice with the same tumor model were randomly divided into experimental group and control group (PBS group). Fluorescently labeled poly(glutamate / lysine) star-shaped nanoparticles S8-E prepared in Example 1 were used. 70 K 30 Polyglutamic acid nanoparticles S8-E100 and fluorescently labeled S8-PEG nanoparticles prepared in Comparative Example 4 were used as experimental groups. The fluorescently labeled polyglutamic acid nanoparticles (1 mg / mL, 200 μL) were injected into mice via the tail vein, while an equal volume of PBS (200 μL) was injected via the tail vein as the control group. Each group had three replicates. Eight hours later, the mice were euthanized, and major organs such as the heart, liver, spleen, lungs, and kidneys, as well as tumors, were removed for in vitro imaging to observe their distribution. See [link to relevant documentation]. Figure 18 Analysis of points a, b, and c shows that, regardless of the tumor model, S8-PEG is superior to S8-E. 100 Both showed high enrichment in tumors, consistent with objective results; however, the S8-E prepared in Example 1... 70 K 30 The higher tumor enrichment indicates that the material prepared in this invention has excellent blood circulation ability and a high enrichment effect in tumors.

[0140] Example 7: S8-E 70 K 30 Grafted acoustic sensitizer TCPP nanomedicine (S8-E) 70 K 30 Synthesis of -TCPP

[0141] This embodiment provides a method for synthesizing S8-E. 70 K 30 Grafted acoustic sensitizer tetra(4-carboxyphenyl)porphyrin (TCPP) nanomedicine (S8-E) 70 K 30- For TCPP methods, see Figure 19 The details are as follows:

[0142] (1) With S8-E70 K 30 Taking a 10% TCPP response as an example (where 10% refers to TCPP in S8-E...), 70 K 30 The percentage of lysine repeating units in the copolymerized amino acid nanoparticles was determined by weighing a certain amount of sound-sensitive agent (TCPP, 11.84 mg, 15.0 μmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 4.3 mg, 22.5 μmol), N,N-diisopropylethylamine (DIEA, 7.74 mg, 60 μmol), and N-hydroxysuccinimide (NHS, 1.72 mg, 15 μmol) in a glove box. TCPP was dissolved in anhydrous dimethyl sulfoxide (DMSO) (237 μL), and then added to the DMSO solution of EDC, NHS, and DIEA. The mixture was reacted in a glove box for 2 h.

[0143] (2) Transfer the reacted solution out of the glove box, and then add dropwise until it contains S8-E. 70 K 30 The solution was reacted in 20 mL of water (100 mg, 15.0 μmol) for 24 h, and then dialyzed and lyophilized to obtain S8-E. 70 K 30 -TCPP nanomedicine.

[0144] Example 8: S8-E 70 K 30 -Optimized preparation of TCPP nanomedicine grafting ratio

[0145] This embodiment provides a method for synthesizing S8-E. 70 K 30 Method for grafting different proportions of the sound-sensitizing agent TCPP nanomedicine: The preparation method is the same as in Example 7, except that the TCPP feed ratio is increased from S8 to E. 70 K 30 The 10% of the total molar amount of amino group can be replaced with 2%, 5% or 20%.

[0146] Test Example 14

[0147] Based on the above experimental results, the S8-E prepared in Example 1 70 K 30 It was determined to be the optimal drug delivery carrier. To further investigate the actual drug loading capacity of this carrier, the series S8-E prepared in Examples 7 and 8 were compared. 70 K 30 The loading of sonosensitive agents in TCPP nanomedicines (with different TCPP grafting ratios) was determined by ultraviolet spectrophotometry. Grafting rate data are shown in Table 6.

[0148] Table 6. S8-E with different TCPP grafting ratios 70 K 30 - Actual grafting rate of TCPP nanomedicines

[0149]

[0150] As can be seen from Table 6, the actual grafting rates are quite close to the theoretical grafting rates, indicating the successful grafting of the sound-sensitive agent TCPP.

[0151] Experimental Example 15

[0152] To test the actual effects of different TCPP grafting rates, various tests were conducted. First, different nanomedicine solutions prepared in Examples 7 and 8 (at the same TCPP concentration (5 μg / mL, 500 μL)) were sonicated together with an SOSG probe (3 μL, 10 μM) (30 kHz, 10 W, 10 min). Since SOSG reacts with reactive oxygen species (ROS) to produce green fluorescence, the fluorescence intensity was detected using a microplate reader after sonication. (See [link to microplate reader]). Figure 20 Analysis a in the study showed that nanomedicines with grafting rates of 5% and 10% exhibited higher fluorescence intensity, indicating that these two grafting rates could generate more reactive oxygen species. Simultaneously, the cell-killing ability of nanomedicines with different grafting rates under ultrasound was examined; see [reference needed]. Figure 20 Analysis b in the study showed that nanomedicines with a 10% grafting rate exhibited higher cell-killing ability under ultrasound irradiation, indicating that a 10% grafting rate is more conducive to the anti-tumor effect of the sonosensitive agent. Further analysis using the DCFH-DA probe (1:1000, 10 mM) to detect reactive oxygen species (ROS) in cells... Figure 21 The results showed that the nanomedicine (S8-E) with a 10% grafting rate 70 K 30 -TCPP can generate a large amount of ROS under the action of ultrasound, which promotes cell death.

[0153] Experimental Example 16

[0154] To ensure the smooth implementation of subsequent in vivo nanomedicine therapy, this study further investigated S8-E. 70 K 30 The tumor enrichment kinetics characteristics. A 4T1 tumor model was established according to the method of Example 6, and the S8-E prepared in Example 7 was used. 70 K 30The experimental group consisted of mice injected via tail vein with TCPP nanomedicine (1 mg / mL, 200 μL), while the control group consisted of mice injected via tail vein with an equal volume of PBS (200 μL). Each group was repeated in triplicate. Mice were euthanized at different time points, and major organs such as the heart, liver, spleen, lungs, and kidneys, as well as tumor tissue, were collected for in vitro fluorescence imaging analysis. See also... Figure 22 Analysis of α in S8-E 70 K 30 The highest tumor enrichment efficiency was observed 4 hours after intravenous injection, indicating that this time point is the optimal treatment window.

[0155] To further verify the enrichment effect of nanoparticle-grafted TCPP in tumors, a 4T1 tumor model was established. (See [link to relevant documentation]). Figure 22 Analysis b in the data shows that during the injection of S8-E 70 K 30 -After TCPP for 4 h, the nanoparticles showed good enrichment in tumor tissue.

[0156] Experimental Example 17

[0157] After confirming the good efficacy of nanomedicines, their therapeutic ability against tumors in mice was tested. First, 1×10⁻⁶ nanoparticles were used... 6 One 4T1 cell was dissolved in 50 μL PBS and subcutaneously injected into mice to establish a 4T1 tumor model. After 7 days, when the tumor volume reached 100 mm, the tumor was allowed to grow. 3 Mice were treated at different times, with tumor-bearing mice (G0) receiving tail vein injection of PBS solution serving as the control group, and divided into S8-E groups. 70 K 30 Materials group (G1), Ultrasound group (G2), S8-E 70 K 30 -TCPP administration group (G3) and S8-E 70 K 30 - Five groups were included: TCPP administration + ultrasound (US) group (G4). See also... Figure 23 Analysis a in the figure specifically illustrates the anti-tumor treatment strategy in mice. Taking group G4 as an example, each mouse was injected with a nanomedicine (200 μL, 10 mg / mL). Four hours later, the tumor site was subjected to ultrasound (30 kHz, 10 W, 10 min). Each mouse was treated three times, with one treatment session every other day. The mice were weighed and the tumor volume was measured every other day. After 15 days, the tumor volume in the control group exceeded 1000 mm. 3 The mice were euthanized, and the tumor tissue was removed for analysis.

[0158] See Figure 23Analysis b in the study, based on tumor growth curves and final tumor weight, showed that compared to the control group, G4 (S8-E) 70 K 30 The -TCPP+US group significantly inhibited tumor growth, with a tumor volume of approximately 250 mm³ and a weight of approximately 90 mg at 15 days; the tumor inhibition rate reached over 75%; while G1 (S8-E) significantly inhibited tumor growth. 70 K 30 The blank vector group (G2 (US)) showed no significant tumor-killing ability, and its tumor growth curve was basically consistent with that of the PBS control group, indicating that the vector itself does not possess anti-tumor activity. G2 (US) and G3 (S8-E) 70 K 30 The -TCPP group showed some tumor-suppressing ability, but the therapeutic effect was limited. In summary, the nanocarrier grafted with the sonosensitive agent had a significant anti-tumor effect under the action of ultrasound.

[0159] Experimental Example 18

[0160] After treatment, the tumor tissue extracted from Case 17 was analyzed. Hematoxylin-eosin staining of the tumor sections (HE sections) was used to observe the pathological condition of the tumor tissue. (See [link to relevant documentation]). Figure 24 In the a-analysis, G0 (PBS control group) tumor cells were tightly packed and morphologically intact, with almost no necrotic areas. Compared with the control group, G4 (S8-E) tumor cells were more densely packed and morphologically intact. 70 K 30 In the -TCPP + US group, large areas of necrosis were found in the tumor tissue, with only a small number of intact tumor cells remaining, indicating that ultrasound-mediated nanomaterials can achieve efficient tumor tissue killing. Tumor cell apoptosis in tumor tissue sections was observed using a nick-end labeling method mediated by deoxyribonucleotide terminal transferase (TUNEL staining), see [link to relevant documentation]. Figure 24 Analysis b in the image is consistent with the HE staining results. In the TUNEL staining results (green represents apoptosis-positive signals, blue represents cell nuclei), the green signal in group G4 was significantly increased, indicating that S8-E... 70 K 30 -TCPP induced a large number of tumor cell apoptosis under ultrasound, indicating that the G4 group can achieve a highly efficient in vivo anti-tumor effect by inducing tumor cell apoptosis and tissue necrosis. (See also: [link to previous text]) Figure 24 The c-analysis observed the production of reactive oxygen species (ROS) in tumor tissue sections (green represents ROS). The results showed that, compared to the control group G0, G3 (S8-E) produced significantly more reactive oxygen species (ROS). 70 K 30 -TCPP) and G2 (US group) showed a small amount of fluorescence signal, possibly due to the production of a small amount of ROS during apoptosis. The blank carrier in group G1 showed no tumor killing effect and the fluorescence signal was very weak. The section results of group G4 indicated that the ultrasound-mediated nano-sound sensor (S8-E) 70 K30 TCPP therapy can generate a large amount of ROS, thereby killing tumor cells and achieving excellent anti-tumor therapeutic effects. The above results demonstrate that through structural optimization and screening, long-term cycling and efficient enrichment of the material in tumors were achieved, and it was verified that using it as a carrier can efficiently deliver the sonosensitive agent TCPP to the tumor site, exerting excellent anti-tumor effects in sonodynamic therapy. Figure 25 ).

Claims

1. A star-shaped polyamino acid nanoparticle with long-cycle properties, comprising a polyamide-amine dendritic macromolecule and a copolymerized amino acid, characterized in that, The nanoparticles have a star-shaped structure with polyamide-amine dendritic macromolecules as the core and copolymerized amino acids as arms; the nanoparticles are initiated by polyamide-amine as an initiator to initiate the first... N -Carboxylic acid ring anhydride monomer and second N - It is prepared by random copolymerization of anhydride monomers within a carboxylic acid ring in an organic solvent; wherein the organic solvent is a mixture of low-polarity haloalkanes and high-polarity amides or cyclic ethers in a volume ratio of (6-9):(1-4).

2. The star-shaped polyamino acid nanoparticles with long-cycle properties according to claim 1, characterized in that, The low-polarity haloalkane is selected from either dichloromethane or trichloromethane; the high-polarity amide or cyclic ether solvent is selected from... N , N -Dimethylformamide, N , N -Dimethylacetamide, N Either methylpyrrolidone or tetrahydrofuran.

3. The star-shaped polyamino acid nanoparticles with long-cycle properties according to claim 1, characterized in that, The first N -The anhydride monomer within the carboxylic acid ring is selected from γ-tert-butyl- L -Glutamic acid NCA, γ-Benzyl- L -Any of glutamate NCA; the second N -The anhydride monomer within the carboxylic acid ring is selected from... N ε -tert-Butyloxycarbonyl- L -Lysine NCA, N ε -Benzyloxycarbonyl- L -Lysine NCA, L -Phenylanine NCA, L -Leucine NCA, O-tert-butyl- L -Serine NCA, O-tert-butyl- L -Tyrosine NCA, N δ -2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl- L -Any of arginine NCA.

4. The star-shaped polyamino acid nanoparticles with long-cycle properties according to claim 1, characterized in that, The polyamide-amine dendritic macromolecule has an algebraic number of G0-G3 and a number of arms of 4-32.

5. The star-shaped polyamino acid nanoparticles with long-cycle properties according to claim 1, characterized in that, The second N - The anhydride monomer in the carboxylic acid ring accounts for 20%-40% of the total molar ratio of the copolymer amino acid; the molar ratio of amino to copolymer amino acid in the polyamide-amine is 1:50-1:

200.

6. A method for preparing star-shaped polyamino acid nanoparticles with long-cycle properties as described in claim 1, characterized in that, Includes the following steps: (1) The first N -Carboxylic acid ring anhydride monomer and second N -The anhydride monomer within the carboxylic acid ring dissolves in organic solvents; (2) The solution obtained in step (1) is mixed with polyamide-amine to react and obtain star-shaped polyamino acid nanoparticles.

7. The use of the star-shaped polyamino acid nanoparticles with long-cycle properties as described in claim 1 in the preparation of drugs for treating tumors.

8. The application according to claim 7, characterized in that, The application of the combination of star-shaped polyamino acid nanoparticles with long-cycle properties and a sonosensitive agent in the preparation of drugs for treating tumors.

9. Use according to claim 8, characterized in that, The sound-sensitive agent is selected from any one of tetra(4-carboxyphenyl)porphyrin, tetra(4-sulfonic phenyl)porphyrin, hematoporphyrin, hematoporphyrin monomethyl ether, protoporphyrin IX, and dihydroporphyrin e6.

10. Use according to claim 8, characterized in that, The sound-sensitizing agent is grafted onto star-shaped polyamino acid nanoparticles with long-cycle properties, and the grafting ratio is expressed as a molar ratio. The sound-sensitizing agent and the second component of the star-shaped polyamino acid nanoparticles... N - The ratio of amino acid repeating units in the anhydride monomer within the carboxylic acid ring is 5%-15%.

Images