Polyamino acid nano-particles with lung targeting property as well as preparation method and application of polyamino acid nano-particles

Abstract

The invention relates to a lung-targeting polyamino acid nanoparticle as well as a preparation method and application thereof. The polyamino acid nano-particles comprise inorganic nano-particles and polyamino acid modified on the surfaces of the inorganic nano-particles; 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, an n-valine residue and a leucine residue. According to the invention, the relationship between the structural parameters and the lung targeting behavior is defined by regulating and controlling the size and the type of the inorganic nanoparticles, the proportion of the polyamino acid and the feed ratio of the reaction raw materials. The polyamino acid nano-particles with the lung targeting property can relieve lung inflammation and treat acute lung injury.

Description

Technical Field

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

[0002] Nanomedicines have shown significant advantages in targeted therapy. Through surface modification or functionalization, they can specifically identify diseased cells or tissues and achieve precise drug delivery, thereby increasing drug enrichment in the lesion site and reducing toxic side effects on normal tissues. At the same time, nanocarriers can enhance the stability, solubility and bioavailability of drugs, and achieve controlled release of drugs through responsive material design, and even integrate diagnostic and therapeutic functions to achieve integrated diagnosis and treatment. For nanomedicines, the liver is the main accumulation and metabolism organ of nanomedicines during systemic administration, responsible for degrading and clearing most nanosized particles. Therefore, it is particularly important to develop extrahepatic targeted nanomedicines. In addition, due to the nonspecific distribution of nanomedicines throughout the body and clearance by the mononuclear phagocyte system, off-target effects are one of the main bottlenecks restricting the development of nanomedicines. On the one hand, the accumulation of drugs in healthy non-target organs will bring toxic side effects; on the other hand, the low drug utilization caused by off-target effects will also directly affect the therapeutic efficiency of drugs.

[0003] In recent years, the design of extrahepatic targeting systems has mainly focused on two strategies: one is to modify targeting molecules such as antibodies on the surface, 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 the affinity interaction between nanomedicines and specific proteins or cells. Although surface-modified targeting molecules can significantly improve the enrichment efficiency of drugs in lesions and reduce nonspecific distribution and toxic side effects to normal tissues, their effects are often limited by factors such as target heterogeneity, biological barriers in the body, and clearance by the immune system, and further optimization is still needed to enhance the value of clinical applications. 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] Although lipid nanoparticle delivery systems have shown significant advantages in the field of drug delivery, they still have some limitations. First, lipid nanoparticles may leak drugs during storage and circulation in the body, affecting their stability and therapeutic effects. Certain lipid components may trigger immune responses, leading to inflammation or allergic reactions, limiting their safety for long-term use. In addition, the synthesis process of lipid nanoparticles is complex, involving the precise ratio and optimization of multiple lipid components, and has high requirements for production conditions (such as temperature, pH value, mixing speed, etc.), which may lead to batch differences, and large-scale production faces technical difficulties. Compared with lipid nanoparticle delivery systems, polymer-based delivery systems have the advantages of flexible chemical structure, easy functionalization, simple synthesis and large-scale preparation, showing great application potential. For example, polymer carriers can optimize their interactions with biomolecules and cells by precisely controlling parameters such as molecular weight, hydrophobicity, charge distribution, and adjusting parameters such as the size and type of nanoparticles, thereby achieving efficient drug delivery. However, the current research on polymer delivery systems still lacks a systematic understanding, especially in terms of their interaction mechanism with biomolecules, in vivo metabolism, and organ targeting efficiency, which still need further in-depth exploration and optimization. Summary of the invention

[0005] In view of the above problems, the present invention provides a lung-targeted polyamino acid nanoparticle and its preparation method and application. The present invention proposes to use the amino groups on the surface of the nanoparticle to initiate in situ N -Carboxylic acid cyclic anhydride (NCA) polymerization method was used to construct a polyamino acid nanoparticle library, and polyamino acid nanoparticles with lung targeting properties were screened out by in vitro animal imaging. Compared with other polyamino acid nanoparticles, the lung targeting efficiency of nanoparticles incorporating valine, isoleucine and tryptophan can be increased by 7-25 times. Further studies have shown that it is enriched in the lungs after interacting with red blood cells. This is mainly due to the presence of steric hindrance of the β-side branch of the monomer side chain such as valine NCA, which has a lower polymerization rate. Therefore, its copolymerization with glutamic acid NCA monomers obtains a sequence structure similar to a block copolymer. This sequence structure directly affects the spatial distribution of hydrophilic and hydrophobic segments on the surface of the nanoparticles, so that the polyvaline hydrophobic segments closer to the surface of the nanoparticles show stronger affinity for the red blood cell membrane. Based on the understanding of the above mechanism, the present invention designs block copolymerized amino acid nanoparticles based on norvaline and leucine, etc. The results show that the nanoparticles after adjusting the distribution of hydrophobic segments by block copolymerization can also selectively target the lungs. In addition, the relationship between structural parameters and lung targeting behavior was clarified by regulating the size and type of nanoparticles, the molar ratio of the copolymer components of polyamino acids, and the feed ratio of amino groups and NCA (which determines the mass ratio of inorganic nanocores to polyamino acids in the final nanoparticles). Finally, polyamino acid nanoparticles with lung targeting properties alleviated lung inflammation and restored lung function.

[0006] The present invention is achieved through the following technical solutions: The first object of the present invention is to provide a polyamino acid nanoparticle with lung targeting, 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; 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.

[0007] In one embodiment of the present invention, the structure of the lung-targeted polyamino acid nanoparticles is as follows: , where m≥1, n≥1; .

[0008] 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.

[0009] In one embodiment of the present invention, the mass ratio of the inorganic nanoparticles to the polyamino acid is 1:1-1:4.

[0010] In one embodiment of the present invention, the size of the polyamino acid nanoparticles is less than 55 nm.

[0011] In one embodiment of the present invention, the inorganic nanoparticles are selected from SiO 2 、QDs、Fe 3 O 4 and CeO 2 One or more of .

[0012] 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.

[0013] The second object of the present invention is to provide a method for preparing the polyamino acid nanoparticles with lung targeting, comprising the following steps: (1) dissolving a first N-carboxyl intracyclic acid anhydride monomer and a second N-carboxyl intracyclic acid 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 or more of isoleucine, NCA; or, (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 intracyclic anhydride monomer to continue the reaction to obtain polyamino acid nanoparticles with lung targeting; 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.

[0014] 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.

[0015] In one embodiment of the present invention, the inorganic nanoparticles in the amino-surface-modified inorganic nanoparticles are selected from SiO 2 、QDs、Fe 3 O 4 and CeO 2 One of them.

[0016] 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.

[0017] The third object of the present invention is to provide the use of the polyamino acid nanoparticles with lung targeting in the preparation of drugs for treating acute lung injury.

[0018] The mechanism of action of the present invention: The present invention provides a lung-targeted polyamino acid nanoparticle and a preparation method and application thereof ( Figure 1). The present invention utilizes the method of in situ initiating the polymerization of NCA monomers on the surface of aminated nanoparticles to efficiently construct and screen a polyamino acid nanoparticle library. On the one hand, the polyamino acid nanoparticles (nanoparticles incorporating valine, isoleucine and tryptophan) obtained by screening play an important role in selective lung targeting behavior. The polyamino acid nanoparticles of the present invention interact with blood cells to enable them to reach the lung tissue. This is because the polyamino acid nanoparticles have a strong binding effect with red blood cells and are bound to the cell membrane surface of red blood cells. After the polyamino acid nanoparticles bind to the red blood cells, they reach the lung tissue, and through the action of the lung shear force, they fall off the surface of the red blood cells and are enriched in the lung tissue. On the other hand, the determination of the polymerization rate during the polyamino acid copolymerization process further explains the mechanism of the interaction between nanoparticles and red blood cells, and helps design polyamino acid nanoparticles (nanoparticles incorporating norvaline and leucine) with lung targeting behavior. The polyamino acid nanoparticles of the present invention can reach the lung tissue, relieve lung inflammation, restore the lungs to normal levels, and have a good therapeutic effect on acute lung injury.

[0019] The above technical solution of the present invention has the following advantages compared with the prior art: (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.

[0020] (2) The surface of nanoparticles has a negative charge, which prolongs their circulation time in the body.

[0021] (3) Structure-activity relationship studies can improve our understanding of the relationship between structural parameters and organ targeting.

[0022] (4) The overall preparation process is simple and efficient, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] 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: Figure 1 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 lung targeting research diagram; Figure 2 The 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 The ratio of Bu-Glu NCA to Val NCA; Figure 3 The images are the in vitro organ distribution imaging of different polyamino acid nanoparticles 4 hours after tail vein injection in Example 1 and the lung enrichment of SiEV nanoparticles at different time points after tail vein injection; Figure 4 This is an organ distribution imaging 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; Figure 5 The distribution of SiEV and SiENV in plasma and blood cells in Test Example 2; Figure 6 These are the results of the study on the lung targeting mechanism of SiEV and SiENV in Test Example 2; wherein, a is the observation of the binding of SiEV and SiENV to blood cells by confocal laser scanning microscopy; b is the observation of the binding of SiEV and SiENV to blood cells by scanning electron microscopy; c is the detection of the binding of SiEV and SiENV to blood cells by flow cytometry; d is the detection of the binding of SiEV to blood cells and the shedding under the action of shear force by flow cytometry; Figure 7 is the nuclear magnetic dynamics analysis of the copolymerization process of NCA monomer 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; 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 in vivo distribution of Si(E- b -L) In vivo distribution of nanoparticles; Fig. 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; Fig.10 The effect of the types of nanoparticles on lung targeting behavior in Test Example 6; a is the in vivo distribution of QDs and QEV; b is Fe 3 O 4 and FeEV distribution in vivo; c is CeO 2 and the distribution of CeEV in vivo;* p <0.5,** p <0.01, n = 3; Fig.11To test the effect of valine ratio on lung targeting behavior in Example 7; Fig.12 For the SiO in Test Example 8 2 The effect of different mass ratios with NCA on lung targeting behavior; Fig.13 The therapeutic effect of polyamino acid nanoparticles on acute lung injury in Test Example 9; wherein, a is hematoxylin-eosin staining to observe lung pathology; b is immunohistochemistry to evaluate the expression of TNF-α, IL-6 and IL-1β; c is quantitative analysis; wherein, ns = not significant, * p <0.5,** p <0.01,*** p <0.001, n = 8. DETAILED DESCRIPTION

[0024] The present invention is further described below 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, but the embodiments are not intended to limit the present invention.

[0025] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0026] The CAS numbers of the reagents used in the embodiments of the present invention are shown in the following table:

[0027] Amino-surface-modified QDs were purchased from Beijing Zhongke Keyou Nanotechnology Co., Ltd. (Beijing, China).

[0028] Amino-modified Fe 3 O 4 Purchased from Jiangsu Zhichuan Technology Co., Ltd. (Jiangsu, China).

[0029] Amino-modified CeO 2 It was purchased from Xi'an Qiyue Biotechnology Co., Ltd. (Xi'an, China).

[0030] BALB / c mice (6–8 weeks) were purchased from Cavens Model Animal Co., Ltd. (Changzhou, China) and 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.

[0031] Embodiment 1:

[0032] This embodiment provides a method for synthesizing polyglutamic acid / valine nanoparticles (SiEV), which is as follows: (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 slowly stir the two solutions at 70 °C for 2 h. Then add ammonia (2.0 mL) and continue stirring for 3 h. After the reaction, the silica nanoparticles are 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 is 15 nm.

[0033] (2) In order to introduce amino groups on the surface of nanoparticles, APTES (3-aminopropyltriethoxysilane) was pre-hydrolyzed to obtain silanol, which was then mixed with silica nanoparticles as follows: 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 dried in an oven at 50 °C overnight (yield 821 mg).

[0034] (3) Using the amino-modified silica obtained above, directly surface-initiate the polymerization of NCA monomers to obtain polyamino acid-modified silica nanoparticles: 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), where 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 bottle containing amino-modified silica nanoparticles. The reaction was stirred at room temperature and the reaction kinetics were monitored by infrared. The reaction was completed when the monomer conversion rate was >99% (3 h). The polyamino acid modified nanoparticles were purified by precipitation with ether / n-hexane (1:1, v / v), and then washed with ether and tetrahydrofuran (3-5 times) to remove impurities. The copolymerized amino acid nanoparticles were dissolved with 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 poly(γ-tert-butyl- L -Glu) residue side chain tert-butyl protecting group (the final mass ratio of nanoparticles to polyamino acids is about 1:2). After deprotection, water-soluble nanoparticles were obtained by precipitation with ether / n-hexane (twice).

[0035] 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), except that the polyamino acid was added during synthesis. N ε -tert-Butyloxycarbonyl- L -Lysine NCA (0.26 mg, 0.97 μmol). The deprotected polyamino acid nanoparticles were dissolved in NaHCO 3 The solution was diluted with 5% paraformaldehyde (0.2 M) aqueous solution and the pH value was adjusted to 8. Cy5 NHS ester (50 μL, 1.6 μmol) was then added and stirred overnight for reaction. The fluorescently labeled SiEV nanoparticles were obtained by dialyzing and freeze-drying.

[0036] Using a method similar to Example 1, other polyamino acid nanoparticles and fluorescently labeled polyamino acid nanoparticles were prepared (the preparation process is as follows 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 -Triphenylmethyl- 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 are polyglutamate nanoparticles (SiE), polyglutamate / isoleucine nanoparticles (SiEI), polyglutamate / lysine nanoparticles (SiEK), polyglutamate / arginine nanoparticles (SiEK), polyglutamate / glutamine nanoparticles (SiEQ), polyglutamate / serine nanoparticles (SiES), polyglutamate / threonine nanoparticles (SiET), polyglutamate / tyrosine nanoparticles (SiEY), polyglutamate / alanine nanoparticles (SiEA), polyglutamate / leucine nanoparticles (SiEL), polyglutamate / tryptophan nanoparticles (SiEW) or polyglutamate / phenylalanine nanoparticles (SiEF).

[0037] 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; NMR 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), proving the controllability of the polymerization.

[0038] The prepared fluorescently labeled polyamino acid nanoparticles (100 μL, 1 mg / mL) were injected into the tail vein of mice (each group of nanoparticles was repeated 3 times). Among them, polyglutamic acid nanoparticles (SiE) were used as the control group, and nanoparticles with different amino acid residues were used as the experimental group. The mice were euthanized 4 hours later, and the main organs such as heart, liver, spleen, lung and kidney were removed for in vitro imaging to observe the organ distribution phenomenon. The results are as follows Figure 3 shown by Figure 3 It can be seen that compared with other copolymeric amino acid nanoparticles, when valine, isoleucine and tryptophan are incorporated at a molar percentage of 10%, the nanoparticles have selective lung targeting behavior and the lung targeting efficiency is increased by 7-25 times, and the highest enrichment in the lungs is within 12 hours. Leucine, which has a similar side chain structure to isoleucine, has a completely different distribution in the body. Therefore, it is speculated that the side chain structure of polyamino acids plays an important role in selective lung targeting behavior.

[0039] Test Example 1:

[0040] SiEV with the highest lung targeting efficiency was used as the research object, and polyglutamic acid / norvaline nanoparticles (SiENV) prepared from norvaline, which has a similar structure to valine but no branched structure, were selected as the control group to observe the differences in their in vivo distribution and explore the mechanism. The preparation methods of SiEV and SiENV can refer to Example 1.

[0041] Fluorescently labeled polyamino acid nanoparticles (100 μL, 1 mg / mL) were injected into mice via the tail vein (each group of nanoparticles was repeated 3 times). 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.

[0042] Test Example 2:

[0043] In order to further explore the mechanism of lung targeting of polyamino acid nanoparticles, SiEV and SiENV were still used as research objects. First, the two polyamino acid nanoparticles were incubated with mouse blood in vitro (37 °C), and the blood added with PBS solution was used as the control group. The distribution of polyamino acid nanoparticles in the blood was observed by centrifugation. The results are as follows: Figure 5 shown by Figure 5 It can be seen that compared with the control group, the upper plasma of SiENV is blue, indicating that SiENV is still dispersed in the plasma; while the blue color of the upper plasma of SiEV becomes lighter, indicating that SiEV has a strong interaction with blood cells. Subsequently, 100 μL of the upper plasma and the bottom blood cells were taken for quantitative analysis, which was consistent with the results observed in the picture. This result suggests that SiEV may be targeted to the lung tissue by hitchhiking after binding to blood cells.

[0044] Fluorescently labeled SiEV and SiENV nanoparticles (100 μL, 1 mg / mL) were injected into mice via the tail vein. Blood was collected by orbital bleeding 1 h later and then centrifuged (800 × g , 10 min), and then 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 6It can be seen that SiEV has a strong binding effect with red blood cells, mainly located on the cell membrane surface of red blood cells. After the nanoparticles bind to red blood cells, they hitchhike to the lung tissue. Due to the shear force of the thinner capillaries in the lungs, SiEV falls off the surface of red blood cells and accumulates in the lung tissue. In contrast, SiENV does not show obvious binding to red blood cells.

[0045] Test Example 3:

[0046] use 1 H NMR monitored the CD 2 Cl 2 and DMF- d 7 The polymerization kinetics in mixed solution was 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 CD 2 Cl 2 :DMF- d 7 (800 µL, 1:1, v / v). The mixture was then mixed well and transferred to an NMR tube, and 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) 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, t After the Bu-Glu NCA reaction, about 50% of the ValNCA monomers remained, indicating that the copolymerized amino acids on the surface of SiEV were more similar to a gradient sequence, with a section of pure hydrophobic polyvaline homopolymer at the N-terminus far away from the inorganic nanocore.

[0047] Embodiment 2:

[0048] This embodiment provides a method for synthesizing polyglutamic acid-b -Norvaline nanoparticles (Si(E- b -NV)) method (preparation process as Figure 1 ), as follows: (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 water (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. The two solutions are stirred at 70 °C for 1 h. o C and stirred slowly for 2 h. Then, ammonia water (2.0 mL) was added and stirred 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.

[0049] (2) In order to introduce amino groups on the surface of nanoparticles, APTES (3-aminopropyltriethoxysilane) was pre-hydrolyzed to obtain silanol, which was then mixed with silica nanoparticles as follows: 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 heated at 50 o C in an oven overnight (yield: 821 mg).

[0050] (3) Using the amino-modified silica obtained above, directly surface-initiate the polymerization of NCA monomers to obtain polyamino acid-modified silica nanoparticles: 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), where tThe 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-modified silica nanoparticles, the reaction was stirred at room temperature, and the reaction kinetics were detected by infrared. When the monomer conversion rate was >99%, the reaction was completed (3 h), and 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), and then washed with ether and tetrahydrofuran (3-5 times) to remove impurities, and the copolymerized amino acid nanoparticles were dissolved with 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 poly (γ-tert-butyl- L After deprotection, water-soluble nanoparticles were obtained by precipitation with ether / n-hexane (twice).

[0051] In order to trace the polyamino acid nanoparticles in animal experiments, about 1% of lysine residues were copolymerized to fluorescently label them. The specific operation was similar to step (3) of this embodiment, except that: when copolymerizing amino acids, a N ε -tert-Butyloxycarbonyl- L -Lysine NCA (0.26 mg, 0.97 μmol). The deprotected polyamino acid nanoparticles were dissolved in NaHCO 3 (0.2 M) aqueous solution, the solution pH was adjusted to 8, and then Cy5 NHS ester (50 μL, 1.6 μmol) was added and stirred overnight for reaction, and then dialyzed and freeze-dried to obtain fluorescently labeled Si(E- b -NV) nanoparticles.

[0052] Using a method similar to that of Example 2, Si(E- b -L) nanoparticles, the only difference is: L - Norvaline NCA is replaced by L -Leucine NCA; Prepare Si(E- b -L) nanoparticles and fluorescently labeled Si(E- b -L) nanoparticles.

[0053] Test Example 4:

[0054] 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). The mice were euthanized 12 hours later, 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) has obvious lung targeting behavior, proving that the sequence structure plays a decisive role in lung targeting.

[0055] Embodiment 3:

[0056] This embodiment provides a method for synthesizing SiEV nanoparticles, which is similar to Embodiment 1, except that the particle size of the silicon dioxide nanoparticles is replaced with 10 nm. The silicon dioxide nanoparticles with a particle size of 10 nm are prepared by changing the volume ratio of TEOS to ammonia water to 2:1 and replacing the ethanol solvent with methanol.

[0057] Embodiment 4:

[0058] This embodiment provides a method for synthesizing SiEV nanoparticles, which is similar to Embodiment 1, except that the particle size of the silicon dioxide nanoparticles is replaced with 20 nm. The silicon dioxide nanoparticles with a particle size of 20 nm are prepared by changing the volume ratio of TEOS to ammonia water to 2.5:1.

[0059] Embodiment 5:

[0060] This embodiment provides a method for synthesizing SiEV nanoparticles, which is similar to Embodiment 1, except that the particle size of the silicon dioxide nanoparticles is replaced with 30 nm. The silicon dioxide nanoparticles with a particle size of 30 nm are prepared by changing the volume ratio of TEOS to ammonia water to 2.5:1.7.

[0061] Comparative Example 1:

[0062] This comparative example provides a method for synthesizing SiEV nanoparticles, which is similar to Example 1, except that the particle size of the silicon dioxide nanoparticles is replaced with 50 nm. The silicon dioxide nanoparticles with a particle size of 50 nm are prepared by changing the volume ratio of TEOS to ammonia water to 2.5:2.

[0063] Comparative Example 2:

[0064] This comparative example provides a method for synthesizing SiEV nanoparticles, which is similar to Example 1, except that the particle size of the silicon dioxide nanoparticles is replaced with 80 nm. The silicon dioxide 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.

[0065] Test Example 5:

[0066] In order to clarify the structure-activity relationship between the size of nanoparticles 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 tail vein (each group of nanoparticles was repeated 3 times). After 12 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: Fig. 9 shown by Fig. 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.

[0067] Embodiment 6:

[0068] This embodiment provides a method for synthesizing SiEV nanoparticles, which is similar to that of Embodiment 1, except that the amino-surface-modified silica nanoparticles are replaced with amino-surface-modified QDs.

[0069] Embodiment 7:

[0070] This embodiment provides a method for synthesizing SiEV nanoparticles, which is similar to that of embodiment 1, except that the amino-surface-modified silica nanoparticles are replaced by amino-surface-modified Fe 3 O 4 .

[0071] Embodiment 8:

[0072] This embodiment provides a method for synthesizing SiEV nanoparticles, which is similar to that of embodiment 1, except that the amino-surface-modified silica nanoparticles are replaced with amino-surface-modified CeO 2 .

[0073] Test Example 6:

[0074] In order to clarify the structure-activity relationship between the types of nanoparticles and selective lung targeting, the polyamino acid nanoparticles (100 μL, 1 mg / mL) prepared in Example 1 and Example 6-8 were injected into mice through the tail vein (each group of nanoparticles was repeated 3 times). After 12 hours, the mice were euthanized, and the main organs such as the heart, liver, spleen, lung and kidney were removed and their organ distribution was observed by in vitro imaging. The results are as follows: Fig.10 shown by Fig.10 It can be seen that the type of nanoparticles has no significant effect on the selective lung targeting behavior. Fig.10 The difference in targeting efficiency mainly comes from the difference in surface amino density of inorganic nanoparticles modified with different amino groups. Compared with the nanoparticles modified with amino groups, the nanoparticles modified with polyamino acid with 10% valine can target lung tissue.

[0075] Embodiment 9:

[0076] This embodiment provides a method for synthesizing SiEV nanoparticles, which is similar to that of Embodiment 1, except that the feed ratio (molar ratio) of valine to total NCA monomers is replaced from 10% to 5%.

[0077] Embodiment 10:

[0078] This embodiment provides a method for synthesizing SiEV nanoparticles, which is similar to that of Embodiment 1, except that the feed ratio (molar ratio) of valine to total NCA monomers is replaced from 10% to 15%.

[0079] Embodiment 11:

[0080] This embodiment provides a method for synthesizing SiEV nanoparticles, which is similar to that of Embodiment 1, except that the feed ratio (molar ratio) of valine to total NCA monomers is replaced from 10% to 20%.

[0081] Comparative Example 3:

[0082] 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%.

[0083] Comparative Example 4:

[0084] 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 monomer is replaced from 10% to 25%. However, due to its high hydrophobic ratio, the prepared polyamino acid nanoparticles cannot be dissolved and are therefore not suitable for subsequent in vivo distribution experiments.

[0085] Test Example 7:

[0086] In order to clarify the structure-activity relationship between the valine ratio and selective lung targeting, the 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 through the tail vein (each group of nanoparticles was repeated 3 times). After 12 hours, the mice were euthanized, and the main organs such as the heart, liver, spleen, lung and kidney were removed, and the organ distribution phenomenon was observed by in vitro imaging. The results are as follows: Fig.11 shown by Fig.11 It can be seen that nanoparticles doped 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.

[0087] Embodiment 12:

[0088] This embodiment 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 SiEV nanoparticles is 1:1).

[0089] Embodiment 13:

[0090] This embodiment 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 acids in the final SiEV nanoparticles is 1:4).

[0091] Test Example 8:

[0092] To clarify SiO 2 The structure-activity relationship of different mass ratios and selective lung targeting with NCA was studied. The nanoparticles (100 μL, 1 mg / mL) with different mass ratios obtained in Example 1, Example 12 and Example 13 were injected into mice through the tail vein (each group of nanoparticles was repeated 3 times). The mice were euthanized 12 hours later, and the main organs such as the heart, liver, spleen, lung and kidney were removed. The organ distribution phenomenon was observed by in vitro imaging. The results are as follows Fig.12 shown by Fig.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.

[0093] Test Example 9:

[0094] 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. After 2 hours, nanoparticles were injected into the tail vein (each group of nanoparticles was repeated 3 times), including CeO 2 , CeO doped with 10% valine by mole 2 (CeEV), and CeO doped with 10 mol% norvaline 2 (CeENV). Mice injected with PBS solution in the tail vein were used as the control group, and mice without acute lung injury model were used as the sham operation group. The preparation method of CeENV was referred to Example 8, except that Val NCA was replaced by Nva NCA. After 22 hours, the mice were euthanized, and the lung tissues were removed. The sections were observed and analyzed by hematoxylin-eosin staining and immunohistochemistry. The results are as follows Fig.13 shown by Fig.13 It can be seen that compared with the control group, CeO 2 CeENV and CeEV do not have a good therapeutic effect on acute lung injury because their nanoparticles are not enriched in the lungs. However, the incorporation of valine gives CeEV the property of lung targeting, thus alleviating lung inflammation and restoring it to a level comparable to that of the sham operation group, showing a good therapeutic effect on acute lung injury.

[0095] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the 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 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.

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

3. 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.

4. The polyamino acid nanoparticles according to claim 1, characterized in that The size of the polyamino acid nanoparticles is less than 55 nm.

5. The polyamino acid nanoparticles according to claim 1, characterized in that: The inorganic nanoparticles are selected from one or more of SiO2, QDs, Fe3O4 and CeO2.

6. The polyamino acid nanoparticles according to claim 1, characterized in that The polyamino acid is obtained by polymerizing a first N-carboxyl intracyclic acid anhydride monomer and a second N-carboxyl intracyclic acid anhydride monomer; the first N-carboxyl intracyclic acid 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.

7. The method for preparing the lung-targeted polyamino acid nanoparticles according to any one of claims 1 to 6, characterized in that: The following steps are involved: (1) dissolving a first N-carboxyl intracyclic acid anhydride monomer and a second N-carboxyl intracyclic acid 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 or more of isoleucine, NCA; or, (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 intracyclic anhydride monomer to continue the reaction to obtain polyamino acid nanoparticles with lung targeting; The first N-carboxyl intracyclic anhydride monomer is selected from γ-tert-butyl- L -glutamic acid NCA; the second N-carboxyl intracyclic anhydride monomer is hydrophobic NCA.

8. The preparation method according to claim 7, characterized in that: The inorganic nanoparticles in the amino-surface-modified inorganic nanoparticles are selected from one of SiO2, QDs, Fe3O4 and CeO2.

9. The preparation method according to claim 7, characterized in that: The particle size of the inorganic nanoparticles in the amino-surface-modified nanoparticles is less than or equal to 30 nm.

10. Use of the lung-targeted polyamino acid nanoparticles according to any one of claims 1 to 6 in the preparation of a drug for treating acute lung injury.

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