Polyamino acid nanoparticles with lung targeting properties and preparation method and application thereof

Abstract

The present invention relates to a polyamino acid nanoparticle with lung targeting, and its preparation method and application. The polyamino acid nanoparticle of the present invention comprises inorganic nanoparticles and a polyamino acid modified on the surface of the inorganic nanoparticle; the polyamino acid comprises a first amino acid residue and a second amino acid residue; the first amino acid residue is a glutamic acid residue; the second amino acid residue comprises one or more of a valine residue, an isoleucine residue, a tryptophan residue, a norvaline residue, and a leucine residue. The present invention clarifies the relationship between structural parameters and lung targeting behavior by regulating the size and type of inorganic nanoparticles, the proportion of polyamino acids, and the feed ratio of reaction raw materials. The polyamino acid nanoparticles with lung targeting properties of the present invention can relieve lung inflammation and treat acute lung injury.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to lung-targeted polyamino acid nanoparticles and a preparation method and application thereof. Background Art

[0002] Nanomedicines offer significant advantages in targeted therapy. Through surface modification or functionalization, they can specifically recognize diseased cells or tissues, enabling precise drug delivery, thereby increasing drug accumulation at the lesion site and minimizing toxic side effects in normal tissues. Furthermore, nanocarriers can enhance drug stability, solubility, and bioavailability, and through responsive material design, achieve controlled drug release. They can even integrate diagnostic and therapeutic functions for integrated diagnosis and treatment. For nanomedicines, the liver is the primary organ for nanodrug accumulation and metabolism during systemic administration, responsible for the degradation and clearance of most nanosized particles. Therefore, the development of nanomedicines with extrahepatic targeting is particularly important. Furthermore, off-target effects are a major bottleneck limiting the development of nanomedicines, due to limitations such as nonspecific systemic distribution and clearance by the mononuclear phagocyte system. On the one hand, drug accumulation in healthy non-target organs can lead to toxic side effects; on the other hand, low drug availability due to off-target effects directly impacts therapeutic efficacy.

[0003] In recent years, the design of extrahepatic targeting systems has mainly focused on two strategies: one is through surface modification of targeting molecules such as antibodies, and the other is to regulate the physicochemical properties of nanomedicines. The former successfully achieves the selective delivery of drug molecules to organs such as the lungs and spleen through affinity interactions between nanomedicines and specific proteins or cells. Although surface modification of targeting molecules can significantly improve the enrichment efficiency of drugs in lesions and reduce nonspecific distribution and toxic side effects to normal tissues, its effect is often limited by factors such as target heterogeneity, biological barriers in the body, and clearance by the immune system. Further optimization is still needed to enhance its clinical application value. In contrast, strategies for regulating the physicochemical properties of nanomedicines show greater advantages. By precisely regulating the size, shape, surface charge and other characteristics of nanomedicines, their interactions with biological molecules and cells in the body can be optimized, thereby achieving more efficient organ-specific targeted delivery.

[0004] While lipid nanoparticle delivery systems have demonstrated significant advantages in the field of drug delivery, they still have several limitations. First, lipid nanoparticles can leak drugs during storage and circulation, compromising their stability and therapeutic efficacy. Certain lipid components can trigger immune responses, leading to inflammation or allergic reactions, limiting their safety for long-term use. Furthermore, the synthesis of lipid nanoparticles is complex, requiring precise proportions and optimization of multiple lipid components. Production conditions (such as temperature, pH, and mixing speed) are also critical, potentially leading to batch-to-batch variability and posing technical challenges for large-scale production. Compared to lipid nanoparticle delivery systems, polymer-based delivery systems offer significant potential due to their flexible chemical structure, ease of functionalization, simple synthesis, and scalable production. For example, polymer carriers can optimize interactions with biomolecules and cells by precisely manipulating parameters such as molecular weight, hydrophobicity, and charge distribution, as well as nanoparticle size and type, to achieve efficient drug delivery. However, research on polymer delivery systems still lacks a systematic understanding, particularly regarding their interaction mechanisms with biomolecules, in vivo metabolism, and organ-targeting efficiency, which require further exploration and optimization. Summary of the Invention

[0005] In order to solve the above problems, the present invention provides a lung-targeted polyamino acid nanoparticle and its preparation method and application. N A polyamino acid nanoparticle library was constructed using a polyamino acid (PAN) polymerization method with carboxylic acid cyclic anhydride (NCA) polymerization. PANs with lung-targeting properties were screened using in vitro animal imaging. Compared to other PANs, PANs incorporating valine, isoleucine, and tryptophan exhibited 7-25-fold increased lung targeting efficiency. Further studies demonstrated that PANs accumulate in the lungs after interaction with erythrocytes. This is primarily due to the steric hindrance of the β-side branching in monomers such as valine (NCA), which results in a lower polymerization rate. Consequently, copolymerization with glutamic acid (NCA) monomers results in a sequence structure similar to a block copolymer. This sequence structure directly influences the spatial distribution of hydrophilic and hydrophobic segments on the nanoparticle surface, resulting in stronger affinity for erythrocyte membranes for the polyvaline hydrophobic segments closer to the nanoparticle surface. Based on this understanding of the mechanism, the present invention designed block copolymer PANs based on norvaline and leucine. Results showed that the PANs, after adjusting the hydrophobic segment distribution through block copolymerization, also exhibited selective lung targeting. Furthermore, by manipulating the size and type of nanoparticles, the molar ratio of the polyamino acid copolymer components, and the ratio of amino groups to NCA (which determines the mass ratio of the inorganic nanocore to polyamino acid in the final nanoparticles), the relationship between structural parameters and lung-targeting behavior was clarified. Ultimately, the lung-targeting polyamino acid nanoparticles alleviated lung inflammation and restored lung function.

[0006] The present invention is achieved through the following technical solutions:

[0007] The first object of the present invention is to provide a lung-targeted polyamino acid nanoparticle, wherein the polyamino acid nanoparticle comprises an inorganic nanoparticle and a polyamino acid modified on the surface of the inorganic nanoparticle; the inorganic nanoparticle and the polyamino acid are connected via an amide bond;

[0008] The polyamino acid includes a first amino acid residue and a second amino acid residue; the first amino acid residue is a glutamic acid residue; the second amino acid residue includes one or more of a valine residue, an isoleucine residue, a tryptophan residue, a norvaline residue and a leucine residue.

[0009] In one embodiment of the present invention, the structure of the lung-targeted polyamino acid nanoparticles is as follows:

[0010] , where m≥1, n≥1;

[0011] .

[0012] In one embodiment of the present invention, the second amino acid residue accounts for 5%-20% of the total molar ratio of the polyamino acid.

[0013] In one embodiment of the present invention, the mass ratio of the inorganic nanoparticles to the polyamino acid is 1:1-1:4.

[0014] In one embodiment of the present invention, the size of the polyamino acid nanoparticles is less than 55 nm.

[0015] In one embodiment of the present invention, the inorganic nanoparticles are selected from one or more of SiO2, QDs, Fe3O4 and CeO2.

[0016] In one embodiment of the present invention, the polyamino acid is obtained by polymerizing a first N-carboxyl intracyclic anhydride monomer and a second N-carboxyl intracyclic anhydride monomer; the first N-carboxyl intracyclic anhydride monomer is selected from γ-tert-butyl- L -glutamic acid NCA; the second N-carboxyl ring anhydride monomer is selected from L -Valine NCA, L -Isoleucine NCA, L -Tryptophan NCA, L -Norvaline NCA and L - one or more of leucine, NCA.

[0017] A second object of the present invention is to provide a method for preparing the lung-targeting polyamino acid nanoparticles, comprising the following steps:

[0018] (1) dissolving the first N-carboxyl intracyclic anhydride monomer and the second N-carboxyl intracyclic anhydride monomer in an organic solvent;

[0019] (2) mixing the solution obtained in step (1) with inorganic nanoparticles modified with amino groups to obtain polyamino acid nanoparticles with lung targeting properties;

[0020] The first N-carboxyl intracyclic anhydride monomer is selected from γ-tert-butyl- L -glutamic acid NCA; the second N-carboxyl ring anhydride monomer is selected from L -Valine NCA, L -Tryptophan NCA and L - one or more of isoleucine NCA;

[0021] or,

[0022] (1) mixing a first N-carboxyl intracyclic anhydride monomer and an inorganic nanoparticle modified with an amino group in an organic solvent for reaction;

[0023] (2) adding a second N-carboxyl ring anhydride monomer to continue the reaction to obtain lung-targeted polyamino acid nanoparticles;

[0024] The first N-carboxyl intracyclic anhydride monomer is selected from γ-tert-butyl- L -glutamic acid NCA; the second N-carboxyl ring anhydride monomer is hydrophobic NCA; the hydrophobic NCA is selected from L -Leucine NCA and / or L -Norvaline NCA.

[0025] In one embodiment of the present invention, the organic solvent is selected from one or more of N,N-dimethylformamide, tetrahydrofuran, N-methylpyrrolidone, dichloromethane and chloroform.

[0026] In one embodiment of the present invention, the inorganic nanoparticles in the amino-surface-modified inorganic nanoparticles are selected from one of SiO2, QDs, Fe3O4 and CeO2.

[0027] In one embodiment of the present invention, the particle size of the inorganic nanoparticles in the amino-surface-modified nanoparticles is less than or equal to 30 nm.

[0028] The third object of the present invention is to provide the use of the lung-targeting polyamino acid nanoparticles in the preparation of drugs for treating acute lung injury.

[0029] The mechanism of action of the present invention:

[0030] The present invention provides a lung-targeting polyamino acid nanoparticle and its preparation method and application ( Figure 1 The present invention utilizes a method for in situ initiation of NCA monomer polymerization on the surface of aminated nanoparticles to efficiently construct and screen a polyamino acid nanoparticle library. On the one hand, the polyamino acid nanoparticles obtained through screening (nanoparticles incorporating valine, isoleucine, and tryptophan) play an important role in selective lung targeting. The polyamino acid nanoparticles of the present invention interact with blood cells, enabling them to reach lung tissue. This is because the polyamino acid nanoparticles have a strong binding effect with red blood cells and bind to the cell membrane surface of red blood cells. After binding to red blood cells, the polyamino acid nanoparticles reach lung tissue and are detached from the red blood cell surface by the shear force of the lungs and enriched in the lung tissue. On the other hand, measurement of the polymerization rate during the polyamino acid copolymerization process further elucidates the mechanism of interaction between nanoparticles and red blood cells and helps design polyamino acid nanoparticles with lung targeting (nanoparticles incorporating norvaline and leucine). The polyamino acid nanoparticles of the present invention can reach lung tissue, alleviate lung inflammation, and restore the lungs to normal levels, demonstrating excellent therapeutic effects for acute lung injury.

[0031] The above technical solution of the present invention has the following advantages over the prior art:

[0032] (1) By utilizing the diversity of NCA side chains and the method of in situ initiating NCA monomer polymerization on the surface of nanoparticles, polyamino acid nanoparticles with different physical and chemical properties can be efficiently constructed.

[0033] (2) The surface of nanoparticles has a negative charge, which prolongs their circulation time in the body.

[0034] (3) Structure-activity relationship studies can improve the understanding of the relationship between structural parameters and organ targeting.

[0035] (4) The overall preparation process is simple and efficient, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0037] Figure 1 Figure a is the preparation route of the polyamino acid nanoparticles in Example 1, b is the preparation route of the polyamino acid nanoparticles in Example 2, and c is a diagram for lung targeting research;

[0038] Figure 2The preparation and characterization of polyamino acid nanoparticles in Example 1 are described; wherein a is the preparation route of different polyamino acid nanoparticles; b is the size and zeta potential characterization of polyamino acid nanoparticles; c is a representative TEM image of SiEV nanoparticles; d is the nuclear magnetic resonance characterization of SiEV nanoparticles. t ratio of Bu-Glu NCA to Val NCA;

[0039] Figure 3 The images show the in vitro organ distribution of different polyamino acid nanoparticles 4 hours after tail vein injection in Example 1, and the lung accumulation of SiEV nanoparticles at different time points after tail vein injection.

[0040] Figure 4 Imaging of the organ distribution of SiEV and SiENV in Test Example 1; wherein a is a schematic diagram of the structure of SiEV and SiENV; b is the in vivo distribution of SiEV and SiENV;

[0041] Figure 5 The distribution of SiEV and SiENV in plasma and blood cells in Test Example 2;

[0042] Figure 6 The results of the study on the lung targeting mechanism of SiEV and SiENV in Test Example 2 are shown in Figure 2; a is the binding of SiEV and SiENV to blood cells observed by confocal laser scanning microscopy; b is the binding of SiEV and SiENV to blood cells observed by scanning electron microscopy; c is the binding of SiEV and SiENV to blood cells detected by flow cytometry; d is the binding of SiEV to blood cells and shedding under shear force detected by flow cytometry;

[0043] Figure 7 is the nuclear magnetic dynamics analysis of the NCA monomer copolymerization process in Test Example 3; a is t NMR dynamics analysis of Bu-Glu NCA and ValNCA; b is t NMR dynamics analysis of Bu-Glu NCA and Nva NCA;

[0044] Figure 8 The effect of block copolymer sequence on lung targeting behavior of test example 4 is studied; wherein a is Si(E- b- b is the distribution of Si(E- b -L) In vivo distribution of nanoparticles;

[0045] Figure 9 The effect of nanoparticle size on lung targeting behavior in Test Example 5; wherein a is the characterization of polyamino acid nanoparticles of different sizes; b is the in vivo distribution of polyamino acid nanoparticles of different sizes;

[0046] Figure 10 The effect of the type of nanoparticles on lung targeting behavior in Test Example 6; where a is the in vivo distribution of QDs and QEV; b is the in vivo distribution of Fe3O4 and FeEV; c is the in vivo distribution of CeO2 and CeEV; * p <0.5,** p <0.01, n = 3;

[0047] Figure 11 To test the effect of the valine ratio on lung targeting behavior in Example 7;

[0048] Figure 12 To test the effect of different mass ratios of SiO2 and NCA on lung targeting behavior in Example 8;

[0049] Figure 13 The therapeutic effect of polyamino acid nanoparticles on acute lung injury in Test Example 9; wherein, a is the observation of lung pathology by hematoxylin-eosin staining; b is the evaluation of the expression of TNF-α, IL-6 and IL-1β by immunohistochemistry; c is the quantitative analysis; wherein, ns = not significant, * p <0.5,** p <0.01,*** p <0.001, n = 8. DETAILED DESCRIPTION

[0050] The present invention will be further described below with reference to 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.

[0051] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are all commercially available unless otherwise specified.

[0052] The CAS numbers of the reagents used in the examples of the present invention are as follows:

[0053]

[0054] Amino-surface-modified QDs were purchased from Beijing Zhongke Keyou Nanotechnology Co., Ltd. (Beijing, China).

[0055] Amino-surface-modified Fe3O4 was purchased from Jiangsu Zhichuan Technology Co., Ltd. (Jiangsu, China).

[0056] Amino-modified CeO2 was purchased from Xi’an Qiyue Biotechnology Co., Ltd. (Xi’an, China).

[0057] BALB / c mice (6–8 weeks) were purchased from Cavens Model Animal Co., Ltd. (Changzhou, China). They were housed in a specific pathogen-free (SPF) room at 25°C. All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH Publication 85-23 Rev. 1985) and approved by the Animal Ethics Committee of Soochow University.

[0058] Example 1:

[0059] This example provides a method for synthesizing polyglutamic acid / valine nanoparticles (SiEV), as follows:

[0060] (1) Silica nanoparticles were synthesized using the Stöber method: ethanol (100 mL), deionized water (2.5 mL), and ammonia (25%, 4.0 mL) were mixed in a 250 mL round-bottom flask and shaken for 15 min. Ethanol (25 mL) and TEOS (tetraethyl orthosilicate, 3 mL) were then mixed and shaken for 25 min. The two solutions were slowly stirred at 70 °C for 2 h. Ammonia (2.0 mL) was then added and stirring continued for 3 h. After the reaction, the silica nanoparticles were purified by centrifugation and washing (deionized water, three times) and redispersed in ethanol to obtain an emulsion suspension; the particle size of the silica nanoparticles was 15 nm.

[0061] (2) In order to introduce amino groups on the surface of nanoparticles, APTES (3-aminopropyltriethoxysilane) was pre-hydrolyzed to obtain silanols, which were then mixed with silica nanoparticles as follows:

[0062] First, an ethanol / water solution (90 mL, 95:5, v / v) was added to a 250 mL round-bottom flask and acidified to pH 5.0 by the addition of glacial acetic acid (60 μL). APTES (4.4 mL, 19 mmol) was added to the acidic solution and stirred at room temperature for 5 minutes. Simultaneously, the resulting silica nanoparticles (1.0 g) were ultrasonically dispersed in ethanol (10 mL) to obtain a milky white suspension. The APTES silanol solution was added to the suspension, and the mixture was stirred at room temperature for 2 hours. The amino-modified silica nanoparticles were purified by centrifugation and washing (three times each with ethanol, acetone, and THF) and dried in an oven at 50°C overnight (yield: 821 mg).

[0063] (3) Using the amino-modified silica obtained above, the polymerization of NCA monomers is directly initiated on the surface to obtain polyamino acid-modified silica nanoparticles:

[0064] Weigh γ-tert-butyl- L -Glutamate NCA ( t Bu-Glu NCA, 19.95 mg, 0.087mmol), L -valine NCA (Val NCA, 1.4 mg, 0.0097 mmol) and amino-functionalized silica nanoparticles (7 mg, containing 9 μmol amino groups), t The molar ratio of Bu-Glu NCA to Val NCA is 9:1. N,N - Dissolve in an equal volume mixture of dimethylformamide (DMF) and dichloromethane (DCM) (250 μL, 1:1, v / v) t Bu-Glu NCA and Val NCA were added to the polymerization flask containing amino-modified silica nanoparticles. The reaction was stirred at room temperature. The reaction kinetics were monitored by infrared spectroscopy. The reaction was completed when the monomer conversion rate was greater than 99% (3 h). The polyamino acid-modified nanoparticles were purified by precipitation with ether / n-hexane (1:1, v / v). Impurities were then removed by washing with ether and tetrahydrofuran (3-5 times). The co-polyamino acid nanoparticles were dissolved in TFA / DCM (500 μL, 1:1, v / v) and stirred in an ice bath at 0°C for 3 h for side chain deprotection to remove the poly(γ-tert-butyl- L The tert-butyl protecting group on the side chain of the (-glutamic acid) residue was removed (the final nanoparticle-to-polyamino acid mass ratio was approximately 1:2). After deprotection, water-soluble nanoparticles were obtained by precipitation with ether / hexane (twice).

[0065] In order to trace polyamino acid nanoparticles in animal experiments, they were fluorescently labeled by copolymerizing about 1% lysine residues. The specific operation was similar to step (3), with the only difference being that lysine was added during the synthesis of polyamino acid. N ε -tert-Butyloxycarbonyl- L -Lysine NCA (0.26 mg, 0.97 μmol). The deprotected polyamino acid nanoparticles were dissolved in a 0.2 M aqueous solution of NaHCO3 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 then dialyzed and lyophilized to obtain fluorescently labeled SiEV nanoparticles.

[0066] Using a method similar to Example 1, other polyamino acid nanoparticles and fluorescently labeled polyamino acid nanoparticles were prepared (preparation process as shown in Figure 2 The only difference is that: L -valine NCA replaced by γ-tert-butyl- L -Glutamate NCA,L -Isoleucine NCA, N ε -tert-Butyloxycarbonyl- L -Lysine NCA, N ε -2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl- L -Arginine, N -trityl- L -Glutamine NCA, O -tert-butyl- L -Serine NCA, L -Threonine NCA, O -tert-butyl- L -Tyrosine NCA, L -Alanine NCA, L -Leucine NCA, L -Tryptophan NCA or L -phenylalanine NCA, the prepared polyamino acid nanoparticles were polyglutamic acid nanoparticles (SiE), polyglutamic acid / isoleucine nanoparticles (SiEI), polyglutamic acid / lysine nanoparticles (SiEK), polyglutamic acid / arginine nanoparticles (SiEK), polyglutamic acid / glutamine nanoparticles (SiEQ), polyglutamic acid / serine nanoparticles (SiES), polyglutamic acid / threonine nanoparticles (SiET), polyglutamic acid / tyrosine nanoparticles (SiEY), polyglutamic acid / alanine nanoparticles (SiEA), polyglutamic acid / leucine nanoparticles (SiEL), polyglutamic acid / tryptophan nanoparticles (SiEW) or polyglutamic acid / phenylalanine nanoparticles (SiEF).

[0067] The characterization diagram of the polyamino acid nanoparticles obtained above is as follows Figure 2 shown by Figure 2 It can be seen that the size of polyamino acid nanoparticles containing different components is relatively consistent, with a size of 35-40 nm; and the surface of the nanoparticles has a negative charge; the nuclear magnetic resonance characterization results show t The ratio of Bu-Glu NCA to Val NCA is 8.9:1.1, which is close to the feed ratio (9:1), demonstrating the controllability of the polymerization.

[0068] The prepared fluorescently labeled polyamino acid nanoparticles (100 μL, 1 mg / mL) were injected into the tail vein of mice (repeated 3 times for each group of nanoparticles). Among them, polyglutamic acid nanoparticles (SiE) served as the control group, and nanoparticles incorporated with different amino acid residues served as the experimental group. After 4 hours, the mice were euthanized, and the main organs such as the heart, liver, spleen, lungs, and kidneys were removed and subjected to in vitro imaging to observe the organ distribution phenomenon. The results are as follows Figure 3 shown by Figure 3 As can be seen, compared to other co-polyamino acid nanoparticles, when valine, isoleucine, and tryptophan were incorporated at a molar percentage of 10%, the nanoparticles exhibited selective lung targeting, with lung targeting efficiency increased by 7-25 times, and peak lung accumulation within 12 hours. Leucine, which has a similar side chain structure to isoleucine, exhibited a completely different in vivo distribution. Therefore, it is hypothesized that the side chain structure of the polyamino acid plays a key role in selective lung targeting.

[0069] Test Example 1:

[0070] SiEV, which has the highest lung-targeting efficiency, was used as the research object. Polyglutamic acid / norvaline nanoparticles (SiENV), prepared from norvaline, which has a similar structure to valine but no branches, were selected as the control group to observe differences in their in vivo distribution and explore their mechanisms. The preparation methods of SiEV and SiENV can be referred to Example 1.

[0071] Fluorescently labeled polyamino acid nanoparticles (100 μL, 1 mg / mL) were injected into mice via the tail vein (repeated 3 times for each group of nanoparticles). The mice were euthanized 12 hours later, and the main organs such as the heart, liver, spleen, lungs and kidneys were removed for in vitro imaging to observe the organ distribution phenomenon. The results are as follows: Figure 4 shown by Figure 4 It can be seen that SiENV has no obvious lung targeting behavior. This result once again shows that the side chain structure of polyamino acids plays a key and decisive role in the lung targeting efficiency of nanoparticles.

[0072] Test Example 2:

[0073] To further explore the lung targeting mechanism of polyamino acid nanoparticles, SiEV and SiENV were used as research objects. First, the two polyamino acid nanoparticles were incubated with mouse blood in vitro (37°C), and blood added with PBS solution was used as a control group. The distribution of polyamino acid nanoparticles in the blood was observed by centrifugation. The results are shown in Figure 2. Figure 5 shown by Figure 5 As can be seen, compared to the control group, the plasma layer above SiENV appeared blue, indicating that SiENV remained dispersed in the plasma; whereas, the blue color of the plasma layer above SiEV became lighter, indicating a stronger interaction between SiEV and blood cells. Subsequently, 100 μL of each of the upper plasma layer and the lower blood cells were taken for quantitative analysis, and the results were consistent with those observed in the images. This result suggests that SiEV may be targeted to lung tissue by hitching a ride on blood cells after binding.

[0074] Fluorescently labeled SiEV and SiENV nanoparticles (100 μL, 1 mg / mL) were injected into the tail vein of mice. One hour later, blood was collected by orbital bleeding and then centrifuged (800 × g , 10 min), and then the blood cells were collected and examined using scanning electron microscopy, confocal laser scanning microscopy, and flow cytometry to observe the binding of blood cells to polyamino acid nanoparticles. The results are as follows Figure 6 shown by Figure 6 As can be seen, SiEVs strongly bind to red blood cells, primarily localizing to the cell membrane. After binding to red blood cells, the nanoparticles hitch a ride to the lungs. Due to the shear forces of the smaller lung capillaries, SiEVs detach from the red blood cell surfaces and accumulate in the lungs. In contrast, SiENVs show no significant red blood cell binding.

[0075] Test Example 3:

[0076] use 1 H NMR monitored the CD2Cl2 and DMF- d 7. The polymerization kinetics in mixed solutions were used to study the copolymerization sequence structure of polyamino acids on the surface of nanoparticles. Specifically, t Bu-Glu NCA (66 mg, 0.288 mmol) and Val NCA (4.58 mg, 0.032 mmol) were dissolved in CD2Cl2:DMF- d 7 (800 μL, 1:1, v / v). The mixture was then mixed evenly and transferred to an NMR tube. The H NMR spectra (with t The end of Bu-Glu NCA reaction was the final monitoring time point). t α-H signal of Bu-Glu NCA ( δ = 4.30 ppm) and the α-H signal of Val NCA ( δ = 4.11 ppm) and compared with t = 0 (i.e., 100% residual NCA) was normalized to calculate the conversion rate of NCA monomer. t The polymerization kinetics of Bu-Glu NCA and Nva NCA were monitored. Figure 7 shown by Figure 7 It can be seen that t There is no significant difference in the reaction rates between Bu-Glu NCA and Nva NCA, indicating that the copolymerized amino acids on the surface of SiENV can be approximately regarded as a random sequence; in comparison, tAfter the Bu-Glu NCA reaction, about 50% of ValNCA monomers remained, indicating that the copolymerized amino acids on the SiEV surface were more similar to a gradient sequence, with a purely hydrophobic polyvaline homopolymer at the N-terminus away from the inorganic nanocore.

[0077] Example 2:

[0078] This embodiment provides a method for synthesizing polyglutamic acid- b -Norvaline nanoparticles (Si(E- b -NV)) method (preparation process as Figure 1 ), specifically as follows:

[0079] (1) Synthesis of silica nanoparticles by Stöber method: Take a 250 mL round-bottom flask, mix ethanol (100 mL), deionized water (2.5 mL) and ammonia (25%, 4.0 mL) and shake for 15 min, then mix ethanol (25 mL) and TEOS (tetraethyl orthosilicate, 3 mL) and shake for 25 min, and the two solutions are stirred at 70 o The mixture was stirred slowly at 40°C for 2 hours. Ammonia (2.0 mL) was then added and stirring continued for 3 hours. After the reaction, the silica nanoparticles were purified by centrifugation and washed (three times with deionized water) and redispersed in ethanol to obtain a milky suspension. The silica nanoparticles had a particle size of 15 nm.

[0080] (2) In order to introduce amino groups on the surface of nanoparticles, APTES (3-aminopropyltriethoxysilane) was pre-hydrolyzed to obtain silanols, which were then mixed with silica nanoparticles as follows:

[0081] First, an ethanol / water solution (90 mL, 95:5, v / v) was added to a 250 mL round-bottom flask and acidified to pH 5.0 by adding glacial acetic acid (60 μL). APTES (4.4 mL, 19 mmol) was added to the acidic solution and stirred at room temperature for 5 min. At the same time, the obtained silica nanoparticles (1.0 g) were ultrasonically dispersed in ethanol (10 mL) to obtain a milky white suspension. APTES silanol solution was added to the suspension, and the mixture was stirred at room temperature for 2 h. The amino-surface-modified silica nanoparticles were purified by centrifugation and washing (ethanol, acetone, and THF three times each) and precipitated at 50 o C and dried in an oven overnight (yield 821 mg).

[0082] (3) Using the amino-modified silica obtained above, the polymerization of NCA monomers is directly initiated on the surface to obtain polyamino acid-modified silica nanoparticles:

[0083] Weigh γ-tert-butyl- L -Glutamate NCA ( t Bu-Glu NCA, 19.95 mg, 0.087mmol), L -Norvaline NCA (Nva NCA, 1.4 mg, 0.0097 mmol) and amino-functionalized silica nanoparticles (7 mg, containing 9 μmol amino groups), t The molar ratio of Bu-Glu NCA to Nva NCA is 9:1. N,N - An equal volume mixture of dimethylformamide (DMF) and dichloromethane (DCM) (218 μL, 1:1, v / v) was first dissolved t Bu-Glu NCA, the solution was added to the polymerization bottle containing amino-surface-modified silica nanoparticles. The reaction was stirred at room temperature. The reaction kinetics were monitored by infrared spectroscopy. When the monomer conversion rate was >99%, the reaction was completed (3 h). Then, Nva NCA was dissolved in an equal volume mixed solvent of anhydrous DMF and DCM (32 μL, 1:1, v / v) and added to the polyglutamic acid nanoparticle solution to continue the reaction. The polyamino acid-modified nanoparticles were purified by precipitation with ether / n-hexane (1:1, v / v). Impurities were then removed by washing with ether and tetrahydrofuran (3-5 times). The co-polyamino acid nanoparticles were dissolved in TFA / DCM (500 μL, 1:1, v / v) and stirred in an ice bath at 0 ° C for 3 h for side chain deprotection to remove the poly(γ-tert-butyl- L The tert-butyl protecting group on the side chain of the (-glutamic acid) residue was removed. After deprotection, water-soluble nanoparticles were obtained by precipitation with ether / n-hexane (twice).

[0084] In order to trace polyamino acid nanoparticles in animal experiments, about 1% lysine residues were copolymerized to fluorescently label them. The specific operation was similar to step (3) of this example, with the only difference being that: when copolymerizing amino acids, 1% lysine residues were added to the nanoparticles. N ε -tert-Butyloxycarbonyl- L -Lysine NCA (0.26 mg, 0.97 μmol). The deprotected polyamino acid nanoparticles were dissolved in a NaHCO3 (0.2 M) aqueous solution and the pH value of the solution was adjusted to 8. Cy5 NHS ester (50 μL, 1.6 μmol) was then added and stirred overnight for reaction. The fluorescently labeled Si(E- b -NV) nanoparticles.

[0085] Using a method similar to that of Example 2, Si(E- b -L) nanoparticles, the only difference is: L -Norvaline NCA is replaced byL -Leucine NCA; Prepared Si(E- b -L) nanoparticles and fluorescently labeled Si(E- b -L) nanoparticles.

[0086] Test Example 4:

[0087] Fluorescently labeled Si(E- b -NV) (100 μL, 1 mg / mL) and Si(E- b -L) (100 μL, 1 mg / mL) were injected into the tail vein of mice (each group of nanoparticles was repeated 3 times). After 12 hours, the mice were euthanized and the main organs such as heart, liver, spleen, lung and kidney were removed. The organ distribution phenomenon was observed by in vitro imaging. The results are as follows Figure 8 shown by Figure 8 It can be seen that Si(E- b -NV) and Si(E- b -L) showed obvious lung targeting behavior, proving that the sequence structure plays a decisive role in lung targeting.

[0088] Example 3:

[0089] This example provides a method for synthesizing SiEV nanoparticles, similar to Example 1, except that the particle size of the silica nanoparticles is changed to 10 nm. The 10 nm silica nanoparticles are prepared by changing the volume ratio of TEOS to ammonia to 2:1 and replacing the ethanol solvent with methanol.

[0090] Example 4:

[0091] This example provides a method for synthesizing SiEV nanoparticles, similar to Example 1, except that the particle size of the silica nanoparticles is changed to 20 nm. The 20 nm silica nanoparticles were prepared by changing the volume ratio of TEOS to ammonia to 2.5:1.

[0092] Example 5:

[0093] This example provides a method for synthesizing SiEV nanoparticles, similar to Example 1, except that the particle size of the silica nanoparticles is changed to 30 nm. The 30 nm silica nanoparticles were prepared by changing the volume ratio of TEOS to ammonia to 2.5:1.7.

[0094] Comparative Example 1:

[0095] This comparative example provides a method for synthesizing SiEV nanoparticles, similar to Example 1, except that the particle size of the silica nanoparticles is changed to 50 nm. The 50 nm silica nanoparticles were prepared by changing the volume ratio of TEOS to ammonia to 2.5:2.

[0096] Comparative Example 2:

[0097] This comparative example provides a method for synthesizing SiEV nanoparticles, which is similar to Example 1, except that the particle size of the silica nanoparticles is replaced with 80 nm. The silica nanoparticles with a particle size of 80 nm are synthesized by changing the volume ratio of TEOS to ammonia water to 1:1 and the reaction temperature to 40 o C was prepared.

[0098] Test Example 5:

[0099] To clarify the structure-activity relationship between nanoparticle size and selective lung targeting, the polyamino acid nanoparticles (100 μL, 1 mg / mL) obtained in Examples 1, 3-5 and Comparative Examples 1 and 2 were injected into mice via the tail vein (repeated 3 times for each group of nanoparticles). Twelve hours later, the mice were euthanized, and the main organs such as the heart, liver, spleen, lungs and kidneys were removed for in vitro imaging to observe organ distribution. The results are shown in Figure 2. Figure 9 shown by Figure 9 It can be seen that when the size of the selected silica nanoparticles is below 30 nm, the corresponding polyamino acid nanoparticles are <55 nm, which has significant selective lung targeting behavior.

[0100] Example 6:

[0101] This example provides a method for synthesizing SiEV nanoparticles, which is similar to Example 1, except that the amino-surface-modified silica nanoparticles are replaced with amino-surface-modified QDs.

[0102] Example 7:

[0103] This embodiment provides a method for synthesizing SiEV nanoparticles, which is similar to that of Example 1, except that the amino-surface-modified silica nanoparticles are replaced with amino-surface-modified Fe3O4.

[0104] Example 8:

[0105] This embodiment provides a method for synthesizing SiEV nanoparticles, which is similar to that of Example 1, except that the amino-surface-modified silica nanoparticles are replaced with amino-surface-modified CeO2.

[0106] Test Example 6:

[0107] To clarify the structure-activity relationship between the type of nanoparticles and selective lung targeting, different types of polyamino acid nanoparticles (100 μL, 1 mg / mL) prepared in Example 1 and Examples 6-8 were injected into mice via the tail vein (repeated 3 times for each group of nanoparticles). After 12 hours, the mice were euthanized, and the main organs such as the heart, liver, spleen, lungs and kidneys were removed and their organ distribution was observed by in vitro imaging. The results are as follows: Figure 10 shown by Figure 10 It can be seen that the type of nanoparticles has no significant effect on the selective lung targeting behavior. Figure 10 The difference in targeting efficiency is mainly due to the difference in surface amino group density of the inorganic nanoparticles modified with different amino groups. Compared with the nanoparticles modified with amino groups, the nanoparticles modified with polyamino acids incorporating 10% molar percentage of valine can target lung tissue.

[0108] Example 9:

[0109] This example provides a method for synthesizing SiEV nanoparticles, which is similar to Example 1, except that the feed ratio (molar ratio) of valine to the total NCA monomers is replaced from 10% to 5%.

[0110] Example 10:

[0111] This example provides a method for synthesizing SiEV nanoparticles, which is similar to Example 1, except that the feed ratio (molar ratio) of valine to the total NCA monomers is replaced from 10% to 15%.

[0112] Example 11:

[0113] This example provides a method for synthesizing SiEV nanoparticles, which is similar to Example 1, except that the feed ratio (molar ratio) of valine to the total NCA monomers is replaced from 10% to 20%.

[0114] Comparative Example 3:

[0115] This comparative example provides a method for synthesizing SiEV nanoparticles, which is similar to Example 1, except that the feed ratio (molar ratio) of valine to the total NCA monomers is replaced from 10% to 1%.

[0116] Comparative Example 4:

[0117] This comparative example provides a method for synthesizing SiEV nanoparticles, similar to Example 1, except that the molar ratio of valine to total NCA monomers is replaced by 25% instead of 10%. However, due to its high hydrophobicity, the resulting polyamino acid nanoparticles were insoluble and therefore unsuitable for subsequent in vivo distribution experiments.

[0118] Test Example 7:

[0119] To clarify the structure-activity relationship between the valine ratio and selective lung targeting, nanoparticles (100 μL, 1 mg / mL) with different polyamino acid ratios obtained in Example 1, Examples 9-11, and Comparative Example 3 were injected into mice via the tail vein (repeated 3 times for each group of nanoparticles). The mice were euthanized 12 hours later, and the main organs such as the heart, liver, spleen, lungs, and kidneys were removed for in vitro imaging to observe the organ distribution phenomenon. The results are as follows: Figure 11 shown by Figure 11 It can be seen that nanoparticles with only 5% molar percentage of valine can be targeted to lung tissue, and as the proportion of valine increases, the lung targeting efficiency gradually increases.

[0120] Example 12:

[0121] This example provides a method for synthesizing SiEV nanoparticles, which is similar to Example 1, except that the feed ratio (mass ratio) of amino-surface-modified silica nanoparticles to NCA is replaced from 1:3 to 2:3 (the mass ratio of nanoparticles to polyamino acids in the final obtained SiEV nanoparticles is 1:1).

[0122] Example 13:

[0123] This example provides a method for synthesizing SiEV rice particles, which is similar to Example 1, except that the feed ratio (mass ratio) of amino-modified silica nanoparticles to NCA is replaced from 1:3 to 1:6 (the mass ratio of nanoparticles to polyamino acid in the final obtained SiEV nanoparticles is 1:4).

[0124] Test Example 8:

[0125] To clarify the structure-activity relationship of different mass ratios of SiO2 to NCA and selective lung targeting, the nanoparticles (100 μL, 1 mg / mL) with different mass ratios obtained in Examples 1, 12, and 13 were injected into mice via the tail vein (repeated 3 times for each group of nanoparticles). After 12 hours, the mice were euthanized, and the main organs such as the heart, liver, spleen, lungs, and kidneys were removed for in vitro imaging to observe the organ distribution phenomenon. The results are shown in the figure below. Figure 12 shown by Figure 12 It can be seen that the lung targeting behavior of polyamino acid nanoparticles is linearly related to the mass ratio, and the increase in the NCA ratio improves the lung targeting efficiency of the nanoparticles.

[0126] Test Example 9:

[0127] The polyamino acid nanoparticles prepared in Example 8 were used to treat acute lung injury. First, lipopolysaccharide was administered through the trachea to establish an acute lung injury model. 2 hours later, nanoparticles were injected into the tail vein (each group of nanoparticles was repeated 3 times), including CeO2, CeO2 doped with 10% valine by molar percentage (CeEV), and CeO2 doped with 10% norvaline by molar percentage (CeENV). Mice injected with PBS solution in the tail vein served as the control group, and mice without an acute lung injury model were served as the sham operation group. The preparation method of CeENV was similar to that in Example 8, except that Val NCA was replaced with Nva NCA. After 22 hours, the mice were euthanized, and the lung tissues were removed. The sections were observed and analyzed using hematoxylin-eosin staining and immunohistochemistry. The results are shown in the figure. Figure 13 shown by Figure 13 As can be seen, compared with the control group, CeO2 and CeENV did not have a good therapeutic effect on acute lung injury because their nanoparticles did not accumulate in the lungs. However, the incorporation of valine endowed CeEV with lung-targeting properties, thereby alleviating lung inflammation and restoring it to a level comparable to the sham operation group, demonstrating a good therapeutic effect for acute lung injury.

[0128] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A lung-targeted polyamino acid nanoparticle, characterized in that: The polyamino acid nanoparticles include inorganic nanoparticles and polyamino acids modified on the surface of the inorganic nanoparticles; The polyamino acid consists of a first amino acid residue and a second amino acid residue; the first amino acid residue is a glutamic acid residue; the second amino acid residue is one of a valine residue, an isoleucine residue and a tryptophan residue; The size of the polyamino acid nanoparticles is less than 55 nm; The inorganic nanoparticles are selected from one or more of SiO2, QDs, Fe3O4 and CeO2; The method for preparing the lung-targeted polyamino acid nanoparticles comprises the following steps: (1) dissolving the first N-carboxyl intracyclic anhydride monomer and the second N-carboxyl intracyclic anhydride monomer in an organic solvent; (2) mixing the solution obtained in step (1) with inorganic nanoparticles modified with amino groups to obtain polyamino acid nanoparticles with lung targeting properties; The first N-carboxyl intracyclic anhydride monomer is selected from γ-tert-butyl- L -glutamic acid NCA; the second N-carboxyl ring anhydride monomer is selected from L -Valine NCA, L -Tryptophan NCA and L -One of the NCAs isoleucine.

2. A lung-targeted polyamino acid nanoparticle, characterized in that: The polyamino acid nanoparticles include inorganic nanoparticles and polyamino acids modified on the surface of the inorganic nanoparticles; The polyamino acid consists of a first amino acid residue and a second amino acid residue; the first amino acid residue is a glutamic acid residue; the second amino acid residue is one of a valine residue, an isoleucine residue, a tryptophan residue, a norvaline residue and a leucine residue; The size of the polyamino acid nanoparticles is less than 55 nm; The inorganic nanoparticles are selected from one or more of SiO2, QDs, Fe3O4 and CeO2; The method for preparing the lung-targeted polyamino acid nanoparticles comprises the following steps: (1) mixing a first N-carboxyl intracyclic anhydride monomer and an inorganic nanoparticle modified with an amino group in an organic solvent for reaction; (2) adding a second N-carboxyl ring anhydride monomer to continue the reaction to obtain lung-targeted polyamino acid nanoparticles; The first N-carboxyl intracyclic anhydride monomer is selected from γ-tert-butyl- L -glutamic acid NCA; the second N-carboxyl ring anhydride monomer is L -Valine NCA, L -Tryptophan NCA, L -Isoleucine NCA, L -Leucine NCA and L -Norvaline, a type of NCA.

3. The polyamino acid nanoparticles according to claim 1 or 2, characterized in that The second amino acid residue accounts for 5%-20% of the total molar ratio of the polyamino acid.

4. The polyamino acid nanoparticles according to claim 1, characterized in that The mass ratio of the inorganic nanoparticles to the polyamino acid is 1:1-1:

4.

5. The polyamino acid nanoparticles according to claim 1, characterized in that The particle size of the inorganic nanoparticles in the amino-surface-modified nanoparticles is less than or equal to 30 nm.

6. Use of the lung-targeted polyamino acid nanoparticles according to any one of claims 1 to 5 in the preparation of a drug for treating acute lung injury, characterized in that: The inorganic nanoparticles in the polyamino acid nanoparticles are CeO2; the first amino acid residue is a glutamic acid residue; and the second amino acid residue is a valine residue.

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