A polyamino acid imparting lung targeting function to nanoparticles, and a preparation method and application thereof

Abstract

The application discloses a kind of polyamino acids for giving nanoparticle lung targeting function and its preparation method and application, the polyamino acid is tri-block polyamino acid, which is composed of middle hydrophilic block and two end hydrophobic blocks, the hydrophilic block is negative charge alpha-helix conformation, its rigid structure can provide structural support for the full exposure of hydrophobic block;One end hydrophobic block modifies various lipid-based nano delivery systems through hydrophobic interaction, the other end hydrophobic block is exposed to the surface of delivery system and makes it have in situ red blood cell hitchhiking ability and lung targeting ability, and can be used for lung disease targeted therapy;The application also compares the effects of different polyamino acid conformation, hydrophilic block polymerization degree, hydrophobic component and polyamino acid post-insertion ratio, and verifies the feasibility of the polyamino acid for giving nanoparticle lung targeting function through in vitro and in vivo tests, to provide a new technical solution for realizing safe, efficient and universal lung targeted drug delivery.

Description

Technical Field

[0001] This invention relates to a polyamino acid, its preparation method and application, and more particularly to a polyamino acid that endows nanoparticles with lung-targeting function, its preparation method and application. Background Technology

[0002] The lungs are vital functional organs of the respiratory system, playing a crucial role in maintaining oxygenation and acid-base balance. However, due to their continuous and direct contact with the external environment, they are highly vulnerable to pathogen invasion and damage from air pollutants. In recent years, researchers have developed various technological approaches to achieve selective drug delivery to lung tissue, including regulating the formulation of lipid nanoparticles (LNPs), introducing targeted antibody ligands, altering the delivery route, and cell hitching. These strategies have improved drug distribution in vivo to some extent, increased drug concentrations in target organs, and reduced the risk of exposure to non-target tissues.

[0003] Among various lung-targeted drug delivery strategies, the modification of targeting molecules such as antibodies is the most classic and commonly used method, offering advantages such as excellent selectivity and broad applicability. Depending on the application scenario, antibodies targeting specific receptors such as those on lung endothelial cells and alveolar macrophages can be flexibly selected to deliver nanomedicines to lung tissue. For example, antibodies targeting endothelial adhesion molecules such as platelet endothelial cell adhesion molecule-1 (PECAM-1) or intercellular adhesion molecule-1 (ICAM-1) can enhance the accumulation of nanomedicines in lung tissue. Previous studies have reported that PECAM-1 antibody-modified LNPs can be used for lung-targeted mRNA delivery; and ICAM-1 antibody-modified drug-loaded nanoparticles can also be used to improve lung inflammation in acute lung injury models.

[0004] While antibody-modified targeting strategies can selectively increase the concentration of nanomedicines in lung tissue, the activity of antibodies depends on their sophisticated high-order structure, thus requiring stringent storage and usage conditions. The resulting cost and stability issues are major factors limiting the further development of this technology. Furthermore, the application of antibodies is also limited by a deeper understanding and research into the corresponding receptor targets in lung tissue.

[0005] Unlike antibody modification strategies, erythrocyte (RBC) hitchhiking, as an emerging lung-targeted drug delivery strategy, does not rely on specific receptor-targeting molecule interactions. Instead, it has attracted widespread attention due to its unique "surface adsorption-lung shear force-induced desorption" cascade mechanism. Nanomedicines first adsorb onto the RBC surface during in vitro incubation via non-covalent interactions, forming a nanomedicine-RBC complex. When this complex is injected intravenously, it is compressed as it passes through the finer pulmonary capillaries. The resulting shear force and RBC deformation lead to the desorption of the nanomedicine, resulting in its accumulation in lung tissue. Compared to traditional drug delivery methods, the RBC hitchhiking strategy can increase the drug concentration in the lungs by several times or even tens of times. However, while traditional RBC hitchhiking can achieve highly efficient lung targeting, the procedures for separating ex vivo RBCs and subsequent nanomedicine conjugation are cumbersome, and there is a risk of infection during in vitro operations.

[0006] To address this issue, ZL202510520188.0 developed a class of polyamino acid nanoparticles with a hydrophobic outer layer. These nanoparticles do not require in vitro incubation with RBCs; instead, they directly adsorb onto the RBC surface after intravenous injection, utilizing this "hitchhiking" mechanism to achieve efficient enrichment in lung tissue, representing a significant improvement in ease of operation. However, this technology still has significant limitations: the nanoparticle core used in its embodiments is made of inorganic materials such as solid silica. While such inorganic cores provide good structural stability, they are not conducive to the efficient loading and delivery of nucleic acid drugs, limiting their widespread application in emerging therapeutic areas such as gene therapy and mRNA vaccines.

[0007] In summary, lung-targeted drug delivery strategies suffer from the dual drawbacks of high cost and poor stability of traditional antibody modification, as well as the difficulty in loading nucleic acid drugs onto inorganic cores in existing RBC hitchhiking technologies. Summary of the Invention

[0008] Purpose of the invention: The purpose of this invention is to provide a triblock polyamino acid that can be modified onto the surface of lipid nanoparticles via a post-insertion strategy, thereby endowing the nanoparticles with the ability to hitch a ride on red blood cells in situ and highly efficient lung targeting capabilities.

[0009] Technical solution: This invention provides a polyamino acid that endows nanoparticles with lung-targeting function, having the following structure:

[0010]

[0011] Where x = 30-50, y = 5-15, and R² is...

[0012] z = 1-4, R is

[0013] The x segment is a negatively charged α-helical conformation.

[0014] The polyamino acid is a triblock polyamino acid, with a hydrophilic block in the middle and hydrophobic blocks at both ends. The middle hydrophilic block is composed of a negatively charged glutamic acid derivative at the end of the side chain (denoted as E), which is used to improve the water solubility of the polyamino acid and form a rigid α-helical chain segment; the hydrophobic blocks at both ends are composed of hydrophobic polyamino acids or their derivatives, such as polyvaline (V), polyleucine (NL), or polyisoleucine (I), etc. One end of the hydrophobic block can mediate the anchoring of the polyamino acid on the surface of the lipid-based nanodelivery system through hydrophobic interactions, and the other end promotes the binding of the delivery system to RBCs, thereby achieving in situ RBC hitchhiking and lung targeting.

[0015] Furthermore, the conformational state of polyamino acids determines the exposure of their hydrophobic domains. When the middle hydrophilic block is in an α-helix conformation, its rigid structure provides structural support for the full exposure of the hydrophobic block, enabling the polyamino acid-modified delivery system to exhibit higher RBC binding affinity and lung targeting capability. However, when the middle hydrophilic block is in a random coil conformation, the polyamino acid lacks a stable rigid support structure, and the hydrophobic blocks at both ends tend to curl towards the hydrophilic segment, thereby reducing RBC binding affinity and lung targeting capability.

[0016] Preferably, x = 35-40. As the degree of hydrophilic block polymerization increases, the helicity of the polyamino acid also increases accordingly. When the degree of hydrophilic block polymerization is 70-80 (i.e., x = 35-40), the RBC binding and lung targeting effects of the nanoparticles are better, and the optimal degree of hydrophilic block polymerization is 80 (i.e., x = 40).

[0017] Preferably, R is Among the different hydrophobic components, I, NL, and V exhibited good lung-targeting behavior, with valine-based nanoparticles showing the best performance.

[0018] The present invention also provides a method for preparing the polyamino acid, comprising the following steps:

[0019]

[0020] Where x = 30-50, y = 5-15, and R is

[0021] ;

[0022] When reactant POB NCA is added in step 1, R1 is: Step 2: Add hydrophobic NCA; Step 3: Using click chemistry, add PMDETA, CuBr, and azide carboxylic acid, R2 is... z = 1-4;

[0023] When reactant CP NCA is added in step 1, R1 is: Step 2: Add hydrophobic NCA; Step 3: Use click chemistry, first add NaN3, then add PMDETA, CuBr and acetylic acid, R2 is... z = 1-4;

[0024] When reactant AOB NCA is added in step 1, R1 is: Step 2: Add hydrophobic NCA; Step 3: Using click chemistry, add I-2959 and mercapto acid, R2 is... z = 1-4.

[0025] The preparation method belongs to the sequential ring-opening polymerization (ROP) method. 1,6-Diaminohexane is preferably used as the initiator. In the preparation process, the raw materials constituting the hydrophilic block are first mixed with diphenyl phosphate (DPP), and an initiator is added for polymerization; then, the raw materials constituting the hydrophobic block are added for chain extension to obtain a triblock polyamino acid; finally, the side chains of the hydrophilic block are changed through click chemistry to obtain a water-soluble triblock polyamino acid with an α-helical conformation.

[0026] Preferably, the POB NCA, CP NCA, or AOB NCA is an L-configuration.

[0027] The present invention also provides a lipid-based nanodelivery system for surface-modified polyamino acids.

[0028] The polyamino acids are physically embedded into the surface of the lipid-based nanodelivery system using hydrophobic interactions between their hydrophobic segments and the lipid components of the delivery system as a driving force. This method is mild and can maximize the preservation of the original structure of the delivery system and the activity of the loaded material without relying on covalent cross-linking or complex ligand activation steps.

[0029] Preferably, the lipid-based nanodelivery system is an LNP or liposome. The modification process of the polyamino acid relies solely on hydrophobic-hydrophobic physical intercalation and is not limited by the chemical composition of the nanoparticle core material. Therefore, it has excellent versatility and platform adaptability, and can be widely applied to a variety of common lipid-based nanodelivery systems, providing reliable technical support for the development of safe, efficient, and universal lung-targeting nanoplatforms.

[0030] Preferably, the molar ratio of polyamino acids to lipids is 0.1-5%. The lung targeting efficiency of the lipid-based nanodelivery system is closely related to the proportion of polyamino acids, with the optimal lung targeting performance observed when the insertion ratio is 1 mol%.

[0031] The present invention also provides the application of the lipid-based nanodelivery system in the preparation of lung-targeted drugs.

[0032] Preferably, the drug is a drug for treating pulmonary fibrosis.

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

[0034] 1. The polyamino acids can be used to modify lipid nanodelivery systems, giving them excellent lung targeting capabilities: (1) LNPs modified with α-helical conformation polyamino acids have higher RBC binding affinity and lung targeting capabilities, with lung-liver ratios that are 5.6 times and 2.9 times that of unmodified LNPs and LNPs modified with random coil conformation polyamino acids, respectively; (2) When the degree of hydrophilic block polymerization is 80, the modified LNPs exhibit the best RBC binding and lung targeting capabilities; (3) When the hydrophobic component is valine, the modified LNPs exhibit the best lung targeting performance; (4) When the proportion of polyamino acids inserted into the surface of LNPs is 1 mol%, the LNPs exhibit the best lung targeting performance.

[0035] 2. The polyamino acid-modified nanoparticles exhibit an mRNA encapsulation rate exceeding 80% and possess excellent size and dispersion, making them suitable for targeted therapy of lung diseases and beneficial for the delivery of nucleic acid drugs. In vitro experiments show that loading mFoxf1 onto Foxf1-deficient lung endothelial cells with the polyamino acid-modified LNPs can achieve therapeutic recovery of endothelial function. In vivo experiments show that treating mice with idiopathic pulmonary fibrosis with the polyamino acid-modified LNPs resulted in a survival rate of approximately 75% on day 40 after treatment, significantly higher than all other groups. Furthermore, pulmonary edema and inflammation were reduced, and collagen deposition was effectively inhibited, alleviating pulmonary fibrosis.

[0036] 3. The polyamino acid is low in cost, easy to store, and has a simple and efficient preparation process. Attached Figure Description

[0037] Figure 1 The diagram shows the synthetic route and nanoparticle modification of triblock polyamino acid VEV; (a) shows the synthetic route of triblock polyamino acid VEV; and (b) shows the schematic diagram of triblock polyamino acid modified nanoparticles.

[0038] Figure 2 For triblock polyamino acids VEV and VE DL Synthetic routes for V; (a) the synthetic route for the triblock polyamino acid VEV exhibiting an α-helical conformation; and (b) the synthetic route for the triblock polyamino acid VE exhibiting a random coil conformation. DL Synthetic route of V;

[0039] Figure 3 Characterization of different polyamino acids; where (a) is GPC characterization; (b) is CD characterization;

[0040] Figure 4Size characterization of triblock polyamino acid-modified LNPs; where (a) is an unmodified LNP; (b) is a VE. DL (c) is an LNP modified by V; (c) is an LNP modified by VEV;

[0041] Figure 5 Characterization of RBC binding and lung targeting of triblock polyamino acid modified LNPs with different configurations; (a) flow cytometry detection of the binding of three LNPs to RBCs; (b) organ distribution imaging of the three LNPs; (c) lung-liver ratio of the three LNPs.

[0042] Figure 6 Characterization of triblock polyamino acids with different hydrophilic block polymerization degrees; where (a) is GPC characterization; (b) is CD characterization;

[0043] Figure 7 The study aimed to characterize the size, RBC binding, and lung targeting of triblock polyamino acid-modified LNPs with different hydrophilic block polymerization degrees. (a) shows the DLS size characterization of the five LNPs; (b) shows the binding of the five LNPs to RBCs detected by flow cytometry; and (c) shows the lung-liver ratio of the five LNPs.

[0044] Figure 8 The structures and characterizations of triblock polyamino acids with different hydrophobic components are shown; (a) is the structural formula of triblock polyamino acids with different hydrophobic components; (b) is the GPC characterization; (c) is the CD characterization.

[0045] Figure 9 Screening of LNPs modified with different hydrophobic components and their insertion ratios; (a) for size characterization; (b) for PDI characterization; (c) for mRNA encapsulation efficiency characterization;

[0046] Figure 10 Characterization of RBC binding and lung targeting of triblock polyamino acid modified LNPs with different hydrophobic components; (a) flow cytometry detection of the binding of 18 LNPs to RBCs; (b) organ distribution imaging of 18 LNPs; (c) lung-liver ratio of 18 LNPs.

[0047] Figure 11 The study aimed to assess the in vitro therapeutic effects of triblock polyamino acid-modified LNPs delivering mFoxf1. (a) Western blot analysis revealed the expression of CD31, Foxf1, and α-SMA after in vitro treatment; (bc) showed the recovery of endothelial function after in vitro treatment; (de) revealed the expression of inflammatory factors and fibrosis-related factors in endothelial cells after in vitro treatment; and (fg) a transwell assay was performed to detect the effect of treated endothelial cells on the migration of fibroblasts and RAW 264.7 cells after in vitro treatment.

[0048] Figure 12 The study aimed to assess the in vivo therapeutic effect of mFoxf1 delivered by triblock polyamino acid-modified LNP on idiopathic pulmonary fibrosis. (a) shows the construction and treatment of the idiopathic pulmonary fibrosis model; (b) shows the survival of mice after treatment in different groups; (c) shows the lung / body weight ratio of mice after treatment in different groups; (d) shows the dry-wet weight ratio of the lungs of mice after treatment in different groups; (e) shows the changes in hydroxyproline content in the lungs of mice after treatment in different groups; and (f) shows the expression of TGF-β protein in the lungs of mice after treatment in different groups, detected by ELISA. Detailed Implementation

[0049] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0050] Example 1

[0051] This embodiment provides a triblock polyamino acid VEV that endows nanoparticles with lung-targeting function. E is a hydrophilic block and V is a hydrophobic block. The hydrophilic block is composed of glutamic acid derivatives POB NCA, CP NCA, or AOB NCA (L configuration), and the hydrophobic block is composed of Val NCA. These raw materials are prepared via a DPP-mediated ROP method.

[0052] The synthetic route of triblock polyamino acids is as follows: Figure 2 As shown in (a) above, taking POB NCA as an example of a hydrophilic block copolymer raw material:

[0053] Step 1: Mix DPP (0.20 mg, 0.0008 mmol) with a DCM solution of POB NCA (20 mg, 0.063 mmol), and add a DCM solution of initiator 1,6-diaminohexane (C6-diNH2, 0.05 M, 15.8 μL, 0.0008 mmol, [M]0 = 0.2 M, [M]0 / [I]0 = 80) to polymerize, obtaining polyamino acid E. POB ;

[0054] Step 2: After FTIR characterization showed that monomer consumption was >99%, 20 μL of a DCM solution of Val NCA (2.3 mg, 0.016 mmol, [M]0 / [I]0 = 20) was added to the polymerization mixture to extend the chain and obtain the triblock polyamino acid VE. POB V;

[0055] Step 3: Subsequently, the product was purified by hexane precipitation, vacuum dried, and obtained by click chemistry to yield water-soluble α-helical triblock polyamino acid VEV. Specifically, in a glove box, the copolymer (22.3 mg, 0.063 mmol side chain alkynyl groups) was dissolved in DMF (1.0 mL), and then a premixed DMF solution of 3-azidopropionic acid (10.8 mg, 0.095 mmol), PMDETA (5.5 mg, 0.032 mmol), and CuBr (0.9 mg, 0.006 mmol) was added. The reaction mixture was stirred in the glove box at room temperature for 24 h, then removed from the glove box and subjected to air to oxidize cuprous ions to copper ions. The crude polyamino acid was precipitated by diethyl ether / n-hexane (1:1, v / v), dialyzed (MWCO = 3.5 kDa), and lyophilized to obtain water-soluble α-helical triblock polyamino acid VEV (yield: 77%).

[0056] In some embodiments, the raw materials that make up the hydrophilic blocks in the above-mentioned triblock polyamino acid can be replaced with CP NCA, and the reaction conditions in step 3 can be adjusted to: first add NaN3, then add PMDETA, CuBr and acetylic acid.

[0057] In some embodiments, the raw material constituting the hydrophilic block in the above-mentioned triblock polyamino acid can also be replaced with AOBNCA. In this case, the reaction conditions of step 3 are adjusted to: UV irradiation and the addition of I-2959 (CAS: 106797-53-9) and various thiol acids.

[0058] The hydrophilic blocks of the triblock polyamino acid VEVs prepared by the above methods all have negatively charged α-helical conformations, providing a basis for subsequent lung targeting.

[0059] Example 2

[0060] This embodiment provides a triblock polyamino acid VE that imparts lung-targeting function to nanoparticles. DL V. The synthesis steps are as follows: Figure 2 As shown in (b), similar to the VEV of Example 1, the difference lies in the configuration of the hydrophilic block material, where the hydrophilic block material is changed from... L -NCA replaced with DL -NCA (containing both L- and D-forms) to obtain polyamino acid VE in a random coil conformation. DL V.

[0061] Test Example 1

[0062] The intermediate and final products of Examples 1 and 2 were characterized, including:

[0063] Example 1, Step 1 product – Polyamino acid E POB ;

[0064] The product of step 2 in Example 1—triblock polyamino acid VE POB V;

[0065] The product of step 3 in Example 1 is a water-soluble triblock polyamino acid VEV.

[0066] Example 2, Step 1 product – Polyamino acid E POB, DL ;

[0067] The product of step 2 in Example 2—triblock polyamino acid VE POB, DL V;

[0068] The product of step 3 in Example 2—water-soluble triblock polyamino acid VE DL V.

[0069] The intermediate products were characterized by GPC and the final products were characterized by CD.

[0070] See results Figure 3 See Table 1. GPC characterization of the triblock polyamino acids yielded a single peak with low dispersibility (dispersibility = weight-average molecular weight / number-average molecular weight < 1.10), indicating good polymerization control. Compared to the product of step 1, the GPC peak of the second-stage Val NCA monomer showed a significant shift, indicating higher chain-end fidelity. Circular dichroism (CD) analysis revealed that VE with a racemic mesoblock... DL V exhibits a random coil conformation, while VEV exhibits a distinct α-helical conformation (with two characteristic negative peaks near 208 nm and 222 nm), the latter ensuring the exposure of hydrophobic structural domains.

[0071] Table 1. Triblock Polyamino Acid VE POB V and VE POB, DL Characterization of molecular weight and dispersion of V

[0072]

[0073] Example 3

[0074] This embodiment provides a lipid-based nanodelivery system for a surface-modified triblock polyamino acid VEV as described in Example 1, wherein the lipid-based nanodelivery system is selected as LNP.

[0075] The synthetic route of the lipid-based nanodelivery system VEV-LNP is as follows: Figure 1 As shown: Ionizable lipids (SM-102), cofactor lipids (DSPC), and cholesterol (Chol) with PEG-lipids (DMG-PEG) 2000The lipids were bound in ethanol at a molar ratio of 50:10:38.5:1.5. The mRNA solution was diluted to 80.12 ng / μL with sodium citrate buffer (pH = 4.0). Using a laboratory microfluidic LNP production system, the lipid and mRNA solutions were mixed at a volume ratio of 1:3 (lipid:mRNA), N / P = 8, and a total flow rate of 12 mL / min to generate LNPs. The resulting mixture was then incubated at 4... o Excess ethanol was removed by dialysis with PBS at C for 2 h. The purified LNP was mixed with a PBS solution of triblock polyamino acid VEV (polyamino acid:lipid = 0.5%, mol:mol) to obtain lung-targeting activity. 37 o Stir at C for 1 hour, then at 4 o Ultrafiltration (MWCO = 100 kDa, 3000 × g, 15 min), followed by washing three times with PBS to obtain VEV-LNP.

[0076] Example 4

[0077] This embodiment provides a surface modification example 2: triblock polyamino acid VE. DL V lipid-based nanodelivery system VE DL V-LNP, the synthesis route is as described in Example 3.

[0078] Test Example 2

[0079] The lipid-based nanodelivery system VEV-LNP of Example 3 and the lipid-based nanodelivery system VE of Example 4 were compared. DL V-LNP is used for dimensional characterization.

[0080] See results Figure 4 DLS results indicate that the size ratio of the nanoparticles after polyamino acid modification increased compared to the unmodified nanoparticles, and that VEV-LNP was superior to VE. DL The increase in V-LNP size is more pronounced, which may be due to the α-helical conformation of VEV driving the polyamino acid chains to exhibit a more extended rod-like morphology on the LNP surface.

[0081] Test Example 3

[0082] The lipid-based nanodelivery system VEV-LNP of Example 3 and the lipid-based nanodelivery system VE of Example 4 were compared. DL The affinity of V-LNP for binding to RBC was tested.

[0083] Test method: Blood was collected from healthy mice using anticoagulated blood vessels, followed by centrifugation to separate the plasma (900 × g, 10 min). RBCs were then compared with VEV-LNP or VE. DL V-LNP at 37 oIncubate at C for 1 h, then collect the complex by centrifugation (900 × g, 10 min). Quantitatively evaluate VEV-LNP or VE by flow cytometry. DL The combination of V-LNP and RBC.

[0084] The results are as follows Figure 5 As shown in (a) of the diagram. VE DL V-LNP-treated RBCs showed only 5.9% Cy5-positive cells, while VEV-LNP-treated RBCs showed 24.2% Cy5-positive cells, confirming that VEV-LNP has a superior affinity for RBCs compared to VE. DL V-LNP.

[0085] Test Example 4

[0086] After confirming that VEV-LNP exhibits significantly higher RBC binding affinity, further investigation was conducted to determine whether this enhanced binding translates into improved lung targeting in vivo. VEV-LNP and VE were then compared. DL V-LNP was intravenously injected into mice, and its accumulation in various major organs was observed.

[0087] Test method: Healthy C57BL / 6J mice were randomly divided into groups (n = 3) and injected via tail vein with DiD-labeled VEV-LNP or VE. DL V-LNP (10 mg / kg lipids) was administered to animals 6 hours later, and their major organs (heart, liver, spleen, lung, and kidney) were harvested. An IVIS optical imaging system (λ) was used. ex = 620 nm, λ em DiD fluorescence signals within organs were analyzed at a wavelength of 670 nm. Furthermore, lung and liver tissues were homogenized, filtered, and RBCs were lysed. The DiD fluorescence signals in the supernatants were measured and normalized to the intensity per gram of tissue to calculate the lung-to-liver ratio.

[0088] The results are as follows Figure 5 As shown in (b) and (c) in the figure. Unmodified LNP and VE DL V-LNPs primarily accumulate in the liver, likely due to their relatively large size, while VEV-LNPs accumulate significantly more in the lungs, with the lung-to-liver ratio being that of unmodified LNPs and VEs, respectively. DL 5.6 times and 2.9 times that of V-LNP.

[0089] In summary, the α-helical conformation of VEV enhances the binding of LNP to RBC and achieves effective lung targeting.

[0090] Example 5

[0091] This embodiment investigates the effect of the degree of polymerization of hydrophilic blocks in triblock polyamino acids.

[0092] By changing the feed ratio of POB NCA to Val NCA in Example 1, triblock polyamino acids with different degrees of hydrophilic block polymerization (60, 70, 80, 90, 100) were prepared, and a lipid-based nanodelivery system for surface-modified polyamino acids was further prepared.

[0093] Test Example 5

[0094] The triblock polyamino acids with different degrees of hydrophilicity were characterized in Example 5.

[0095] like Figure 6 As shown in Table 2, GPC characterization of the triblock polyamino acids all exhibited a single peak with low dispersibility (dispersibility = weight average molecular weight / number average molecular weight < 1.06), indicating good control of the polymerization process. Furthermore, the GPC peak shifted significantly to the left with increasing degree of polymerization. In addition, CD analysis showed that the helicity of the polyamino acids increased with increasing degree of polymerization of the hydrophilic blocks. The sample with a degree of polymerization of 80% corresponds to the VE described in Example 1. POB V and VEV, their molecular weight and dispersity data are shown in Table 1.

[0096] Table 2. Characterization of molecular weight and dispersity of triblock polyamino acids with different degrees of hydrophilicity.

[0097]

[0098] like Figure 7 As shown, DLS results indicate that the size of the nanoparticles gradually increases as the degree of hydrophilic block polymerization increases from 70 to 100. This is because the helix length increases with increasing degree of polymerization, resulting in longer rod-shaped segments in the polymer chain, thus causing the physical size of the nanoparticles to monotonically increase with increasing molecular chain length. Notably, at a degree of hydrophilic block polymerization of 60, the nanoparticle size increases abnormally, which may be due to the connection of two LNPs by the polyamino acid chain. Furthermore, flow cytometry was used to examine the RBC binding and lung targeting of LNPs modified with different degrees of hydrophilic block polymerization. The results show that the RBC binding and lung targeting of the nanoparticles are optimal when the degree of hydrophilic block polymerization is 80.

[0099] Example 6

[0100] This embodiment explores the effects of different hydrophobic components in polyamino acids.

[0101] By replacing the Val NCA in Example 1 with other hydrophobic NCAs (see details) Figure 8 In (a) of the preparation, triblock polyamino acid XEX with different hydrophobic components (X represents hydrophobic block) was prepared.

[0102] Test Example 6

[0103] The polyamino acids with different hydrophobic components in Example 6 were characterized.

[0104] like Figure 8 As shown in (b) and (c) and Table 3, the triblock polyamino acids with different hydrophobic components obtained by GPC characterization all exhibited a single peak with low dispersibility (dispersibility = weight average molecular weight / number average molecular weight < 1.10), indicating that the polymerization process was well controlled. Furthermore, CD analysis showed that all triblock polyamino acids exhibited a distinct α-helical characteristic peak.

[0105] Table 3. Characterization of molecular weight and dispersity of triblock polyamino acids with different hydrophobic components.

[0106]

[0107] Example 7

[0108] In this embodiment, LNPs were modified with triblock polyamino acids of different hydrophobic components as described in Example 6, and 50 LNP libraries were constructed by changing the molar ratio of polyamino acids to lipids (polyamino acids: lipids = 0.1%, 0.5%, 1%, 2%, 5%).

[0109] Test Example 7

[0110] The 50 LNPs of Example 7 were characterized.

[0111] The results are as follows Figure 9 As shown in the figure, nanoparticles were screened based on size, dispersion, and mRNA encapsulation efficiency. Nanoparticles with a size > 100 nm, dispersion > 0.2, and encapsulation efficiency < 80% were excluded, resulting in 18 high-performance nanoparticles for subsequent RBC binding and lung-targeting studies.

[0112] Test Example 8

[0113] The lung targeting of the 18 nanoparticles selected in Test Case 7 was evaluated using flow cytometry and lung-liver ratio.

[0114] The results are as follows Figure 10 As shown, compared to other nanoparticles, those with hydrophobic components I, NL, and V exhibited good lung-targeting behavior, with valine-based nanoparticles showing the best performance. Furthermore, the lung-targeting efficiency was closely related to the proportion of polyamino acids inserted into the LNP surface, with an insertion ratio of 1 mol% exhibiting the optimal lung-targeting performance.

[0115] Example 8

[0116] In vitro experiments were conducted to evaluate the therapeutic efficacy of polyamino acid-modified LNPs for delivering mFoxf1.

[0117] It has been reported that the deletion of the Foxf1 gene in endothelial cells leads to endothelial-mesenchymal transition. Therefore, after identifying candidate LNP agents with lung-targeting properties, we further evaluated their therapeutic efficacy by delivering mFoxf1 to lung endothelial cells.

[0118] Endothelial cells were seeded into 6-well plates, 2 × 10⁶ cells per well. 5 37 cells o Cells were cultured at C for 24 h. They were then stimulated with tumor necrosis factor-α (TNF-α, 100 ng / mL) and subsequently treated with PBS, mFoxf1, mFoxf1@LNP, and mFoxf1@VE. DL Cells were treated with V-LNP or mFoxf1@VEV-LNP (1 μg mRNA per well) for 24 h. Cells were washed and lysed with RIPA lysis buffer. Western blot was used to detect the expression levels of Foxf1, CD31, and α-SMA proteins.

[0119] To assess whether mFoxf1 delivery could alleviate endothelial dysfunction, transendothelial resistance (TEER) and relative endothelial permeability were measured. Endothelial cells were loaded at 5 × 10⁻⁶ ppm. 4 Cells were added to the transwell chambers at a density of 100 cells / well. The culture medium was changed every two days until a complete monolayer of cells was formed. Cells were stimulated with TNF-α (100 ng / mL), followed by PBS, mFoxf1, mFoxf1@LNP, and mFoxf1@VE. DL V-LNP or mFoxf1@VEV-LNP (1 μg mRNA per well) were used to treat the monolayer membranes for 24 h, with untreated cells serving as a negative control. After treatment, the TEER value of the monolayer membranes was measured using a Millicell ERS-2 volt-ohmmeter.

[0120] Endothelial cells were packed at 5 × 10⁻⁶ 4 Cells were added to the transwell chambers at a density of 100 cells / well. The culture medium was changed every two days until a complete monolayer of cells was formed. Cells were stimulated with TNF-α (100 ng / mL), followed by PBS, mFoxf1, mFoxf1@LNP, and mFoxf1@VE. DL V-LNP or mFoxf1@VEV-LNP (1 μg mRNA per well) were used for 24 h of treatment. After stimulation, the upper chamber medium was replaced with fresh medium containing FITC-glucan (10 μg / mL), and the mixture was incubated at 37°C. o Incubate at C for 1 h. Then, collect an aliquot (100 μL) from the lower chamber and use a microplate reader (λ). ex = 485 nm, λ emFluorescence intensity was measured at 520 nm. Relative endothelial permeability was calculated with the fluorescence intensity of the PBS group (cells not treated with LNP) as 100%.

[0121] Previous studies have reported that the absence of Foxf1 in endothelial cells can promote the secretion of various cytokines, thereby inducing the migration of fibroblasts and macrophages. To evaluate the anti-inflammatory and anti-fibrotic efficacy of mFoxf1@VEV-LNP, the levels of cytokines in the culture supernatant were detected using ELISA. Specifically, endothelial cells were cultured at a concentration of 2 × 10⁶ cells / mL. 5 Cells were seeded at a density of 37 cells / well in 6-well plates. o Cells were incubated at C for 24 h. Cells were stimulated with TNF-α (100 ng / mL), then treated with PBS, mFoxf1, mFoxf1@LNP, and mFoxf1@VE. DL V-LNP or mFoxf1@VEV-LNP (1 μg mRNA per well) were used to treat cells for 24 h. After treatment, cell culture supernatants were collected, and TNF-α, interleukin-6 (IL-6), CXC motif ligand 1 (CXCL1), and chemokine ligand 2 (CCL2) were quantified using a commercial ELISA kit.

[0122] The migration ability of fibroblasts was investigated using a transwell assay. Endothelial cells were added to 24-well plates and stimulated with TNF-α (100 ng / mL), followed by treatment with PBS, mFoxf1, mFoxf1@LNP, and mFoxf1@VE. DL V-LNP or mFoxf1@VEV-LNP (1 μg mRNA per well) were treated for 24 h. Subsequently, fibroblasts were cultured at 5 × 10⁻⁶ mRNA levels. 4 Cells were seeded at a density of 37 cells / well in transwell chambers. o Cultured at C for 24 h. Fixed with 4% paraformaldehyde (PFA) for 40 min, stained with crystal violet for 10 min, washed three times with PBS, and observed under an inverted microscope. The effect of TNF-α-stimulated endothelial cells on the migration of RAW 264.7 cells was investigated using the same method, except that the fibroblasts in the transwell chambers were replaced with RAW 264.7 cells.

[0123] The results are as follows Figure 11As shown in the figure, Western blot results revealed that mFoxf1 delivery upregulated the expression of Foxf1 and the endothelial marker CD31, while downregulating α-SMA (an endothelial-mesenchymal transition marker). Transendothelial resistance (TEER) and relative endothelial permeability results showed that mFoxf1@VEV-LNP treatment restored both to healthy control levels, indicating a recovery of endothelial barrier function. Furthermore, mFoxf1-loaded LNPs, upon delivery to cells, reduced the secretion of pro-inflammatory and pro-fibrotic factors by endothelial cells. In addition, transwell migration assays confirmed that mFoxf1-loaded LNPs significantly reduced the migration ability of fibroblasts and macrophages, further supporting its ability to improve endothelial function and attenuate pro-inflammatory and pro-fibrotic paracrine effects.

[0124] Example 9

[0125] After validating the therapeutic effect in vitro, the in vivo performance of polyamino acid-modified LNPs was further evaluated in a bleomycin-induced idiopathic pulmonary fibrosis (IPF) mouse model.

[0126] An IPF animal model was established by intratracheal infusion of bleomycin hydrochloride (3 mg / kg). At 10, 13, 16, and 19 days after IPF establishment, various LNPs loaded with mFoxf1 (0.5 mg / kg mRNA) were injected via tail vein. Healthy mice received an equal volume of sterile saline as a sham-operated group. Nintedanib (60 mg / kg) was administered orally daily as a positive control group. Mice were euthanized after 21 days, and lung tissue was collected for various analyses. A separate animal group was established for survival analysis and continued observation until day 40 after modeling.

[0127] Pulmonary edema was quantified by calculating the lung-to-body weight ratio and the dry-to-wet weight ratio of lung tissue. After injection of various LNPs loaded with mFoxf1, mice were weighed, and the right lung of IPF mice was removed, washed with PBS, stained, and weighed to obtain the "wet" weight. The lung-to-body weight ratio was then calculated. Subsequently, the lung tissue was subjected to 50... o Dry at C for 24 h, weigh to obtain the "dry" weight, and calculate the wet / dry weight ratio. The calculation method for the wet / dry weight ratio of lung tissue in healthy mice is similar to that in the control group.

[0128] In addition, the lung tissue homogenate was diluted with 12 N HCl (1:1, v / v) at 95°C. o Hydrolyzed at C for 6 h, centrifuged (16000 rpm, 20 min), the supernatant was adjusted to pH 6.0-8.0 with NaOH (10 mol / L) and diluted to 4 mL with distilled water. The hydroxyproline content was determined using a microplate reader (λ = 560 nm).

[0129] Finally, to assess the content of transforming growth factor-β (TGF-β) in lung tissue, lung tissue was collected and homogenized. o After centrifugation at 3000 × g for 20 min, the supernatant was collected, and the level of TGF-β in the supernatant was detected using an ELISA kit.

[0130] The results are as follows Figure 12 As shown, mFoxf1@VEV-LNP, with its superior lung-targeting ability, exhibited the most significant therapeutic effect, with a survival rate of approximately 70% on day 40 post-treatment, significantly higher than all other groups. Furthermore, the lung-to-body weight ratio and dry-to-wet weight ratio of mice in the mFoxf1@VEV-LNP treatment group recovered to levels comparable to the sham-operated group, indicating a reduction in pulmonary edema and inflammation. Consistent with this, the levels of hydroxyproline and TGF-β in lung tissue recovered to levels comparable to the sham-operated group, demonstrating its effective inhibition of collagen deposition and alleviation of pulmonary fibrosis.

Claims

1. A polyamino acid that endows nanoparticles with lung-targeting function, characterized in that, It has a structure: Where x = 30-50, y = 5-15, and R² is... z = 1-4, R is The x segment is a negatively charged α-helical conformation.

2. The polyamino acid according to claim 1, characterized in that, x = 35-40。 3. The polyamino acid according to claim 1, characterized in that, R is .

4. A method for preparing the polyamino acid according to any one of claims 1-3, characterized in that, Includes the following steps: Where x = 30-50, y = 5-15, and R is ； When reactant POB NCA is added in step 1, R1 is: Step 2: Add hydrophobic NCA; Step 3: Using click chemistry, add PMDETA, CuBr, and azide carboxylic acid, R2 is... z = 1-4; When reactant CP NCA is added in step 1, R1 is: Step 2: Add hydrophobic NCA; Step 3: Use click chemistry, first add NaN3, then add PMDETA, CuBr and acetylic acid, R2 is... z = 1-4; When reactant AOB NCA is added in step 1, R1 is: Step 2: Add hydrophobic NCA; Step 3: Using click chemistry, add I-2959 and mercapto acid, R2 is... z = 1-4.

5. The preparation method according to claim 4, characterized in that, The POB NCA, CP NCA, or AOB NCA is of L configuration.

6. A surface-modified lipid nanodelivery system for polyamino acids according to any one of claims 1-3.

7. The lipid-based nanodelivery system according to claim 6, characterized in that, The lipid-based nanodelivery system is a lipid nanoparticle or a liposome.

8. The lipid-based nanodelivery system according to claim 6, characterized in that, The molar ratio of polyamino acids to lipids is 0.1-1%.

9. The use of the lipid-based nanodelivery system according to any one of claims 6-8 in the preparation of lung-targeted drugs.

10. The application according to claim 9, characterized in that, The drug is used to treat pulmonary fibrosis.

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