Liposome nano-particles of polypeptide modified targeted respiratory system and application of liposome nano-particles

Abstract

The invention discloses a polypeptide modified targeted respiratory system liposome nanoparticle and application thereof, the polypeptide modified targeted respiratory system liposome nanoparticle comprises a polypeptide and a liposome, the mass ratio of the polypeptide to the liposome is (0.01-30): 100, and the polypeptide is a polypeptide 1 or a polypeptide 2. The polypeptide modified liposome nano-particles of the targeting respiratory system can target the respiratory system, especially the nasal mucosa, the respiratory tract and the lung, so that the uptake rate of the targeting medicine in the nasal mucosa, respiratory tract cells and lung mucosa cells is improved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a liposome nanoparticle, its preparation method, and its application, and more particularly to a polypeptide-modified liposome nanoparticle targeting the respiratory system and its application. Background Technology

[0002] Liposome nanoparticles (LNPs), as highly efficient non-viral carriers, have been widely used for the delivery of mRNA vaccines and nucleic acid drugs, with key advantages including high encapsulation efficiency, tunable physicochemical properties, and good biocompatibility. However, traditional LNP systems face significant challenges when targeting specific tissues or mucosa. For example, clinically commonly used LNPs often accumulate in the liver, leading to inefficient delivery to extrahepatic organs after systemic administration, and mucosal barriers (such as the mucus layer and ciliary clearance mechanisms of the nasal mucosa) further limit their local accumulation and cellular uptake. Furthermore, in mucosal delivery methods such as nebulization or inhalation, LNPs are prone to aggregation or disintegration due to mechanical shear forces or changes in environmental ionic strength, resulting in decreased delivery efficiency. Therefore, developing novel LNP modification strategies to enhance their mucosal targeting and stability has become a current research hotspot.

[0003] Peptide modification, due to its high specificity, low immunogenicity, and ease of functionalization, is widely used to improve the targeting of nanocarriers. For example, LNPs conjugated with the Angiopep-2 peptide can cross the blood-brain barrier and significantly increase the accumulation of siRNA at brain tumor sites; while charge-assisted stabilization (CAS) technology, by introducing negatively charged peptide-lipid conjugates, enhances the stability of LNPs during nebulization and promotes their targeted delivery to lung dendritic cells. These studies demonstrate that peptides can achieve precise delivery by regulating the surface charge of LNPs, enhancing their binding to specific cell receptors, or improving colloidal stability.

[0004] However, existing research has focused on targeting the liver, spleen, or brain, and there is still a lack of systematic exploration in the design of delivery systems for mucosal cells of the respiratory system, including the nasal cavity and the entire respiratory tract. In particular, how to balance the effects of peptide modification on LNP stability, ability to penetrate the mucus barrier, and immune activation effects still needs in-depth research.

[0005] In summary, there is an urgent need to develop a novel LNP system based on peptide modification. By introducing mucosal penetrating peptides or adhesion peptides, the targeting, stability, and immune activation of LNPs in the nasal mucosa can be synergistically enhanced, providing innovative solutions for drugs such as vaccines and local anti-infective therapies for treating nasal mucosal or respiratory tract infections. Summary of the Invention

[0006] To achieve the above objectives, the present invention aims to provide a polypeptide-modified liposome nanoparticle targeting the respiratory system and its application, which can target mucosal cells of the respiratory system, especially the nasal cavity, respiratory tract and lungs, thereby improving the uptake rate of targeted drugs in nasal mucosal cells, respiratory tract mucosal cells and lung mucosal cells.

[0007] One aspect of the present invention provides a polypeptide, wherein the polypeptide is polypeptide 1, and the amino acid sequence of the polypeptide 1 has at least 80%, 85%, 95%, 96%, 97%, 98%, 99% or more homology with SEQ ID NO:1, and the amino acid sequence of the SEQ ID NO:1 is shown as RGDLDVGFOGER.

[0008] Another aspect of the present invention provides a polypeptide-modified liposome nanoparticle, comprising a polypeptide and a liposome, wherein the polypeptide is the aforementioned polypeptide 1 or polypeptide 2, the mass ratio of the polypeptide to the liposome is (0.01~30):100, and the amino acid sequence of polypeptide 2 has at least 80%, 85%, 95%, 96%, 97%, 98%, 99% or more homology with cyclo(RGDfK).

[0009] In a preferred embodiment of the present invention, when the polypeptide is polypeptide 1, the mass ratio of the polypeptide to the liposome is (0.05~20):100.

[0010] In a preferred embodiment of the present invention, when the polypeptide is polypeptide 2, the mass ratio of the polypeptide to the liposome is (0.05~30):100.

[0011] In a preferred embodiment of the present invention, the polypeptide-modified liposome nanoparticles are liposome nanoparticles targeting the respiratory system, wherein the respiratory system preferably includes the nasal mucosa, respiratory tract and / or lungs.

[0012] In a preferred embodiment of the present invention, when the polypeptide is polypeptide 1, the polypeptide-modified liposome nanoparticles are liposome nanoparticles targeting the nasal mucosa; or

[0013] When the polypeptide is polypeptide 2, the polypeptide-modified liposome nanoparticles are liposome nanoparticles that target the nasal mucosa, respiratory tract and / or lungs.

[0014] In a preferred embodiment of the present invention, when the mass ratio of the polypeptide to the liposome is 30:100, the polypeptide-modified liposome nanoparticles are liposome nanoparticles targeting the lungs.

[0015] In a preferred embodiment of the present invention, the liposome comprises the following components by weight percentage: 49-80% cationic lipids, 8-10% phospholipids, 1-5% polyethylene glycol, and 5-38.5% cholesterol.

[0016] In a preferred embodiment of the present invention, the liposome comprises the following components in weight percentages: 50% cationic lipids, 10% phospholipids, 1.5% polyethylene glycol, and 38.5% cholesterol.

[0017] In a preferred embodiment of the present invention, the cationic lipid is SM-102 or DOTAP, the phospholipid is DSPC or DSPE, the polyethylene glycol is PEG2000, and the cholesterol is CHO-HP.

[0018] In a preferred embodiment of the present invention, the polypeptide-modified liposome nanoparticles are sprayed or dropped.

[0019] In a preferred embodiment of the present invention, the polypeptide-modified liposome nanoparticles are administered via nasal instillation or spraying to induce immunization.

[0020] Another aspect of the present invention provides a method for preparing the above-mentioned polypeptide-modified liposome nanoparticles by means of the following steps: weighing appropriate amounts of each component and mixing them, then adding an equal volume of anhydrous ethanol, mixing evenly, and incubating in a water bath at 37°C in the dark for 1 to 5 hours, preferably 1 hour.

[0021] Another aspect of the present invention provides a pharmaceutical composition comprising the above-mentioned polypeptide-modified liposome nanoparticles and a drug molecule encapsulated in the liposome nanoparticles, wherein the mass ratio of the liposome nanoparticles to the drug molecule is 1:1 to 1:10, preferably 1:3.

[0022] Preferably, the drug molecule is an mRNA, DNA, protein, polysaccharide, or polypeptide drug.

[0023] Another aspect of the present invention provides the use of polypeptide-modified liposome nanoparticles or the above-described pharmaceutical compositions in the preparation of medicaments for the treatment and / or prevention of nasal and respiratory tract-related diseases.

[0024] In a preferred embodiment of the present invention, the respiratory system-related diseases include nasal cavity-related diseases and respiratory tract infection-related diseases.

[0025] In a preferred embodiment of the present invention, the drug for treating and / or preventing respiratory system-related diseases is a vaccine or antibody preparation.

[0026] Another aspect of the present invention provides a method for treating and / or preventing respiratory-related diseases, comprising administering the above-described polypeptide-modified liposome nanoparticles or the above-described pharmaceutical composition to a subject.

[0027] Preferably, the respiratory system-related diseases are administered to the subject by spraying or dripping into the nasal cavity, and the respiratory system-related diseases preferably include nasal-related diseases and respiratory tract infection-related diseases.

[0028] The beneficial effects of this invention are that by designing a polypeptide and using it to modify liposome nanoparticles, polypeptide-modified liposome nanoparticles are obtained, which can target the respiratory system, especially the nasal mucosa, respiratory tract, and lungs, thereby improving the uptake rate of drugs targeting the nasal mucosa, respiratory tract, and lungs in mucosal cells. Experiments show that when drug compositions containing polypeptide-modified liposome nanoparticles are administered to animals, the titer is significantly improved compared to the non-targeted group, indicating that targeted delivery can significantly stimulate antibody production. This overcomes the problems in the prior art where LNPs are difficult to deliver to extrahepatic organs after systemic administration due to the liver enrichment effect, and the mucosal barriers (such as the mucus layer and ciliary clearance mechanism of the nasal mucosa) further limit their local accumulation and cellular uptake.

[0029] The liposome nanoparticles of this invention enable targeted delivery of mRNA. When mixed with mRNA, the liposome nanoparticles significantly enhance targeted delivery to the nasal mucosa, respiratory tract, and lungs, improving the efficacy and reliability of mRNA drugs. Furthermore, these liposome nanoparticles can be used for the delivery of various mRNAs, suitable for mRNAs of different proteins. This characteristic makes them widely applicable in addressing the treatment needs of various nasal mucosa and respiratory tract infection-related diseases. They also demonstrate good efficacy and have broad application prospects in the development of mRNA vaccines, antibodies, and drugs.

[0030] Furthermore, the liposome nanoparticles of the present invention are safe and non-toxic, improving their applicability to existing mRNA vaccines. These liposome nanoparticles can be prepared on a large scale based on in vitro reactions, and are significantly superior to traditional delivery formulations in terms of production cost, production speed, safety, and protection range. This provides a feasible approach for large-scale mRNA vaccine production and also facilitates drug storage and use. Attached Figure Description

[0031] The accompanying drawings, which form part of this specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.

[0032] The invention will be more clearly understood with reference to the accompanying drawings and the following detailed description, wherein:

[0033] Figure 1 The images show in vivo imaging results after LNP immunization with different amounts of polypeptide 1 in Example 5 of the present invention. From left to right, the images show the imaging results of mice immunized with LNP containing polypeptide 1 at a polypeptide to liposome mass ratio of 0:100, 0.05:100, 1:100 and 20:100.

[0034] Figure 2The images show in vivo imaging results after LNP immunization with different amounts of peptide 2 in Example 5 of the present invention. From left to right, the images show the imaging results of mice immunized with LNP containing peptide 2 with a peptide-to-liposome mass ratio of 0:100, 0.05:100, 1:100 and 30:100.

[0035] Figure 3 The results of serum antibody titer detection in mice after nasal immunization with OVA in Example 6 of the present invention;

[0036] Figure 4 The above are the results of spleen lymphocyte detection in mice after intranasal immunization with OVA in Example 6 of the present invention. In this figure, A is a flow cytometry scatter plot, and B is CD4+. + T cell content statistical bar chart, C represents CD8. + T cell content statistical bar chart. Detailed Implementation

[0037] Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0038] The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the invention or its application or use.

[0039] Techniques, methods, and equipment known to those skilled in the art may not be discussed in detail, but where appropriate, such techniques and methods should be considered part of the specification.

[0040] Unless otherwise specified, the “6-8 week old BALB / c female mice” mentioned in this article were all purchased from Vital River Biotechnology Co., Ltd.

[0041] Unless otherwise specified, “ovalbumin mRNA-LNP” in this article is also referred to as “a pharmaceutical composition containing liposome nanoparticles of ovalbumin mRNA”.

[0042] Unless otherwise specified, "LNP" in this article refers to "liposome nanoparticles".

[0043] Example 1. Preparation of peptide-modified liposome nanoparticles

[0044] The sequences of the modified peptides were designed, and peptides 1 and 2 were obtained through screening. Both peptides can promote the retention of LNPs in the nasal mucosa, respiratory tract, or lungs through receptor-ligand binding. The obtained peptides 1-2 were then used to prepare peptide-modified liposome nanoparticles according to the following method.

[0045] Prepare a lipid-ethanol solution containing the following components by weight percentage: 50% SM-102 (dissolved in anhydrous ethanol), 1.5% PEG2000 (dissolved in anhydrous ethanol), 38.5% CHO-HP (dissolved in anhydrous ethanol), and 10% DSCP (dissolved in anhydrous ethanol). Simultaneously, add peptide 1 at a peptide-to-liposome mass ratio of 1:100 (peptide mass to total liposome mass, w / w, peptide dissolved in PBS). After mixing all components, add an equal volume of anhydrous ethanol, mix thoroughly, and incubate in a light-protected 37°C water bath for 1 hour.

[0046] Liposome nanoparticles containing polypeptide 2 were prepared using the same method described above. The unused components were:

[0047] Replace the polypeptide with polypeptide 2, and add polypeptide 2 at a polypeptide to liposome mass ratio of 5:100 (polypeptide mass to total liposome mass, w / w).

[0048] Peptide sequence 1: RGDLDVGFOGER (SEQ ID NO:1)

[0049] Polypeptide sequence 2: cyclo(RGDfK).

[0050] Example 2: mRNA preparation

[0051] This embodiment uses in vitro transcription to synthesize Luciferase mRNA, but is not limited to this method. The gene encoding the epitope protein sequence is inserted into the pUC57 vector, but is not limited to this vector, and then transformed into competent E. coli expression cells for bacterial amplification. The bacteria are harvested when they reach the plateau phase. Subsequently, the mRNA is obtained through plasmid extraction, restriction enzyme digestion and linearization, in vitro transcription, and chromatography purification.

[0052] The specific process for developing the antigen expression vector for mRNA vaccines is as follows: Shanghai Sangon Biotech Co., Ltd. synthesizes the following gene sequences: from the 5' to 3' ends, they are the T7 promoter (SEQ ID NO:2), 5' UTR (SEQ ID NO:3), kozak sequence (GCCACC), tPA signal peptide sequence (SEQ ID NO:4), Luciferase nucleotide sequence (SEQ ID NO:7), 3' UTR (SEQ ID NO:11), and polyA (SEQ ID NO:12). Then, using genetic engineering techniques, the target gene sequence is constructed into a cloning vector, which is then transformed into competent E. coli cells for large-scale bacterial culture and plasmid extraction.

[0053] The sequences designed and synthesized above, as well as the sequences of the antigen proteins, are as follows:

[0054] The DNA sequence of the T7 promoter is shown in SEQ ID NO:2:

[0055] TAATACGACTCACTATAGG

[0056] The DNA sequence of the 5' untranslated region (5'UTR) is shown in SEQ ID NO:3:

[0057] GAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCGCTAGCCTCGAG

[0058] The tPA signal peptide sequence is shown in SEQ ID NO:4:

[0059] ATGGACGCCATGAAGAGGGGGCTGTGCTGCGTGCTGCTGCTGTGCGGAGCCGTGTTCGTGAGCGCCTCC

[0060] The DNA sequence of the 3' untranslated region (3'UTR) is shown in SEQ ID NO:11:

[0061] GATATCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTG

[0062] Polyadenylate (poly A) has the sequence shown in SEQ ID NO:12 and contains 104 bases A:

[0063] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0064] The amino acid sequence of Luciferase is shown in SEQ ID NO:5:

[0065] MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKIAV

[0066] The luciferase mRNA sequence is as shown in SEQ ID NO: 6:

[0067]

[0068] The DNA sequence of Luciferase is shown in SEQ ID NO:7:

[0069]

[0070] The amino acid sequence of ovalbumin is shown in SEQ ID NO:8:

[0071] MGSIGAASMEFCFDVFKELKVHHANENIFYCPIAIMSALAMVYLGAKDSTRTQINKVVRFDKLPGFGDSIEAQCGTSVNVHSSLRDILNQITKPNDVYSFSLASRLYAEERYPILPEYLQCVKELYRGGLEPINFQTAADQARELINSWVESQTNGIIRNVLQPSSVDSQTAMVLVNAIVFKGLWEKAFKDED TQAMPFRVTEQESKPVQMMYQIGLFRVASMASEKMKILELPFASGTMSMLVLLPDEVSGLEQLESIINFEKLTEWTSSNVMEERKIKVYLPRMKMEEKYNLTSVLMAMGITDVFSSSANLSGISSAESLKISQAVHAAHAEINEAGREVVGSAEAGVDAASVSEEFRADHPFLFCIKHIATNAVLFFGRCVSP

[0072] The ovalbumin mRNA sequence is shown in SEQ ID NO:9:

[0073]

[0074] The ovalbumin DNA sequence is shown in SEQ ID NO:10:

[0075]

[0076] Example 3: Preparation of the pharmaceutical composition

[0077] This embodiment uses a droplet microfluidic system to prepare the immune composition. Specifically, the drug composition is prepared as follows: the different peptide-modified liposome nanoparticles obtained in Example 1 are mixed with the mRNA prepared in Example 2 at a mass ratio of 1:3 to obtain liposome nanoparticles containing Luciferase mRNA, liposome nanoparticles containing peptides 1-2 and Luciferase mRNA (for Example 5), and liposome nanoparticles containing peptide 2 and ovalbumin mRNA (for Example 6).

[0078] Example 4: Characterization of the liposome nanoparticle and mRNA composition

[0079] The encapsulation efficiency of the drug composition obtained in Example 3 was detected using the Quant-iTTM RiboGreen assay kit. The results showed that when the mass ratio of peptide to liposome nanoparticles was (0.05~30):100, the encapsulation efficiency was above 80%. Particle size analysis was performed using a particle size analyzer, and the results are shown in Tables 1-2 below.

[0080] Table 1. Statistical results of particle size and encapsulation efficiency after encapsulating LNP with different contents of peptide 1.

[0081] Mass ratio of peptides to liposomes 0:100 0.05:100 0.1:100 1:100 5:100 10:100 15:100 20:100 25:100 30:100 Particle size 103nm 104nm 102nm 98nm 99nm 105nm 101nm 105nm 116nm 141nm Package rate 95.2% 92.3% 94.6% 93.5% 91.8% 89.7% 91.3% 90.8% 88.1% 83.1%

[0082] Table 2. Statistical results of particle size and encapsulation efficiency after encapsulating LNP with different contents of peptide 2.

[0083] Mass ratio of peptides to liposomes 0:100 0.05:100 0.1:100 1:100 5:100 10:100 15:100 20:100 25:100 30:100 Particle size 101nm 102nm 98nm 99nm 102nm 102nm 103nm 105nm 102nm 109nm Package rate 93.1% 91.2% 92.3% 91.3% 92.9% 90.7% 90.6% 91.5% 92.7% 89.5%

[0084] The particle size analysis showed that when the mass ratio of peptide 1 to liposomes was (0.05~20):100, the particle size of the drug composition was mainly distributed around 100 nm. When the mass ratio of peptide 1 to liposomes was greater than or equal to 25:100, the LNP particle size increased significantly and the encapsulation efficiency decreased significantly. Based on these results, the mass ratio of peptide 1 to liposomes was selected as (0.05~20):100.

[0085] The concentration of peptide 2 differs from that of peptide 1. When the mass ratio of peptide 2 to liposomes is close to 30:100, the LNP particle size tends to increase, but still does not exceed 110 nm. The content range of peptide 2 is (0.05~30):100.

[0086] Example 5: Expression of liposome nanoparticles and mRNA complex in mice

[0087] Drug compositions with peptide 1 and liposome mass ratios of 0:100, 0.05, 100, 1:100 and 20:100, and drug compositions with peptide 2 and liposome mass ratios of 0:100, 0.05:100, 1:100 and 30:100, respectively, were prepared according to the method in Example 3. The different drug compositions were then administered intranasally to 6-8 week old BALB / c female mice, with the drug composition without peptides serving as the control group (non-targeted group) at a dose of 50 μL. Two to four hours after immunization, Luciferase substrate was injected intraperitoneally, and the mice were sacrificed 10 minutes later for in vivo imaging.

[0088] The mice were observed, and the results were as follows: Figure 1-2 As shown, from Figure 1 It can be seen that the LNP with a mass ratio of peptide 1 to liposomes of 0.05:100 has the best effect. Significant protein expression was observed in the nasal cavity of mice, but no significant protein expression was observed in the lungs of mice. No protein expression was observed in the nasal cavity of mice in the group without peptide, indicating that the LNP modified with peptide 1 is mainly concentrated in the nasal mucosa and can achieve targeted delivery to the nasal mucosa.

[0089] from Figure 2 It can be seen that protein expression in the nasal mucosa and lungs was observed in mice immunized with peptide 2, and LNP with a peptide 1 to liposome mass ratio of 0.05:100 showed the highest expression in the nasal cavity and lungs. No significant protein expression was observed in the nasal cavity and lungs of mice in the group without peptide.

[0090] Example 6: Validation of the effects of different polypeptide liposome nanoparticles and mRNA complexes on mice.

[0091] The luciferase gene was replaced with the ovalbumin gene sequence, and mRNA was synthesized according to the method in Example 2. During the synthesis process, peptide 1 or peptide 2 was added at a peptide to liposome mass ratio of 0.05:100 to obtain a pharmaceutical composition containing peptide 1 and a pharmaceutical composition containing peptide 2 (referred to as targeted delivery peptide 1 and targeted delivery peptide 2, respectively).

[0092] Six- to eight-week-old female Balb / c mice were nasally instilled with 100 μL of an LNP-mRNA complex. The blank control group received PBS, the non-targeted group (a drug composition containing Luciferase mRNA liposome nanoparticles) received non-targeted ovalbumin mRNA-LNP, and the targeted delivery peptide 1 and targeted delivery peptide 2 groups received their corresponding ovalbumin mRNA-LNP. Two weeks after the initial immunization, mice were immunized again, and booster immunizations were performed two weeks later. Antibody titers in mouse serum were detected using an ELISA kit. Results are shown below. Figure 3 As shown.

[0093] from Figure 3 It can be seen that there was no significant difference between the non-targeted group and the blank control group, while the titers of the two targeted groups were significantly higher than those of the blank group. This indicates that targeted delivery can significantly stimulate antibody production in mice, and the liposome nanoparticle drug combination containing peptide 2 has a better effect on stimulating antibody production. This may be because the lungs contain more tissue-resident lymphocytes, which can stimulate strong humoral immunity. The nasal mucosa contains fewer lymphocytes, resulting in relatively weaker humoral immunity. This does not mean that the peptide 1 targeted delivery system is less effective than peptide 2; in some drug delivery processes for treating nasal allergies, the peptide 1 targeted delivery system may have more advantages.

[0094] Subsequently, the mouse spleen was isolated, and CD4 counts were detected using flow cytometry. + T cells and CD8 + Changes in T cell content, results as follows Figure 4 As shown. From Figure 4 It can be seen that there was no significant difference between the non-targeted group and the blank control group, while the CD4 levels in the two targeted delivery groups were significantly different. + The proportion of T cells increased, while CD8 cells increased. + The decrease in T cell proportion indicates that nasal mucosal immunization with the vaccine can induce cellular immunity in mice, and the reduction was more pronounced in the targeted peptide 2 group than in the targeted peptide 1 group. Since OVA expressed on mRNA is itself an sensitizer, its increased levels in vivo will lead to CD8... + Decreased T cell ratio and CD4 + The increase in the proportion of T cells is consistent with the expected results of this experiment, indicating that more protein is expressed by the target peptide 2 and that it can induce a stronger cellular immune response, which is consistent with the results of humoral immunity.

[0095] In summary, this invention demonstrates through experiments that the targeted delivery system of peptide 1 mainly expresses proteins in the nasal mucosa, which can induce a weaker immune response and is more suitable for the treatment of diseases related to the nasal mucosa. The targeted delivery system of peptide 2 mainly expresses proteins in the lungs and nasal mucosa, which can induce very strong cellular and humoral immunity and is more suitable for the delivery of antiviral drugs and vaccines for the lungs.

[0096] In summary, this invention mainly involves designing a polypeptide and using it to modify liposome nanoparticles to obtain polypeptide-modified liposome nanoparticles that target the nasal mucosa, respiratory tract, and lungs. This solves the problem in the prior art where LNPs are difficult to deliver to extrahepatic organs after systemic administration due to the liver enrichment effect, and the mucosal barrier (such as the mucus layer and ciliary clearance mechanism of the nasal mucosa) further limits its local accumulation and cellular uptake.

[0097] This technology was used to modify liposome nanoparticles, resulting in peptide-modified liposome nanoparticles capable of targeting the nasal mucosa, respiratory tract, and lungs. This improved the uptake rate of drugs targeting these areas in mucosal cells. Experiments showed that drug compositions containing peptide-modified liposome nanoparticles, when administered to animals, exhibited significantly higher potency compared to the non-targeted group, indicating that targeted delivery significantly stimulates antibody production. This invention overcomes the limitations of mRNA-targeted delivery in traditional liposome nanoparticles: after mixing with mRNA, the liposome nanoparticles significantly enhance targeted delivery to the nasal mucosa, respiratory tract, and lungs, improving the efficacy and reliability of mRNA drugs. Furthermore, these liposome nanoparticles can be used for the delivery of various mRNAs, suitable for different protein mRNAs, making them widely applicable to addressing various nasal mucosal diseases. With good results, they show broad application prospects in mRNA vaccine, antibody, and drug development.

[0098] Furthermore, the liposome nanoparticles of the present invention are safe and non-toxic, improving their applicability to existing mRNA vaccines. These liposome nanoparticles can be prepared on a large scale based on in vitro reactions, and are significantly superior to traditional delivery formulations in terms of production cost, production speed, safety, and protection range. This provides a feasible approach for large-scale mRNA vaccine production and also facilitates drug storage and use.

[0099] The description of this invention is given for illustrative and descriptive purposes only and is not intended to be exhaustive or to limit the invention to the forms disclosed. Many modifications and variations will be apparent to those skilled in the art. The embodiments were chosen and described in order to better illustrate the principles and practical application of the invention and to enable those skilled in the art to understand the invention and to design various embodiments with various modifications suitable for a particular purpose.

Claims

1. A polypeptide, wherein the polypeptide is polypeptide 1, and the amino acid sequence of the polypeptide 1 has at least 80%, 85%, 95%, 96%, 97%, 98%, 99% or more homology with SEQ ID NO:1, wherein the amino acid sequence of the SEQ ID NO:1 is shown as RGDLDVGFOGER.

2. A polypeptide-modified liposome nanoparticle, comprising a polypeptide and liposomes, wherein the polypeptide is polypeptide 1 or polypeptide 2 as described in claim 1, the mass ratio of the polypeptide to the liposomes is (0.01~30):100, and the amino acid sequence of polypeptide 2 has at least 80%, 85%, 95%, 96%, 97%, 98%, 99% or more homology with cyclo(RGDfK).

3. The polypeptide-modified liposome nanoparticles according to claim 2, characterized in that, When the polypeptide is polypeptide 1, the mass ratio of the polypeptide to the liposome is (0.05~20):

100.

4. The polypeptide-modified liposome nanoparticles according to claim 2, characterized in that, When the polypeptide is polypeptide 2, the mass ratio of the polypeptide to the liposome is (0.05~30):

100.

5. The polypeptide-modified liposome nanoparticles according to any one of claims 2 to 4, characterized in that, The peptide-modified liposome nanoparticles are liposome nanoparticles targeting the respiratory system, which preferably includes the nasal cavity, respiratory tract and / or lungs. Preferably, when the polypeptide is polypeptide 1, the polypeptide-modified liposome nanoparticles are liposome nanoparticles targeting the nasal mucosa; or When the polypeptide is polypeptide 2, the polypeptide-modified liposome nanoparticles are liposome nanoparticles that target the nasal cavity or lungs. More preferably, when the mass ratio of the polypeptide to the liposome is 30:100, the polypeptide-modified liposome nanoparticles are liposome nanoparticles targeting the lungs.

6. The polypeptide-modified liposome nanoparticles according to any one of claims 2 to 5, characterized in that, The liposomes comprise the following components by weight percentage: cationic lipids 49-80%, phospholipids 8-10%, polyethylene glycol 1-5%, and cholesterol 5-38.5%. Preferably, the liposomes comprise the following components in weight percentages: 50% cationic lipids, 10% phospholipids, 1.5% polyethylene glycol, and 38.5% cholesterol.

7. The polypeptide-modified liposome nanoparticles according to any one of claims 2 to 6, characterized in that, The cationic lipid is SM-102 or DOTAP, the phospholipid is DSPC or DSPE, the polyethylene glycol is PEG2000, and the cholesterol is CHO-HP.

8. The polypeptide-modified liposome nanoparticles according to any one of claims 2 to 7, characterized in that, The polypeptide-modified liposome nanoparticles are in the form of a spray or droplet; Preferably, the polypeptide-modified liposome nanoparticles are administered via nasal dripping or spraying for immunization.

9. The polypeptide-modified liposome nanoparticles according to any one of claims 2 to 8 are prepared by a method comprising the following steps: weighing appropriate amounts of each component and mixing them, then adding an equal volume of anhydrous ethanol, mixing evenly, and incubating in a water bath at 37°C in the dark for 1 to 5 hours, preferably for 1 hour.

10. A pharmaceutical composition comprising peptide-modified liposome nanoparticles according to any one of claims 2 to 8 and a drug molecule, wherein the drug molecule is encapsulated in the peptide-modified liposome nanoparticles, and the mass ratio of the peptide-modified liposome nanoparticles to the drug molecule is 1:1 to 1:10, preferably 1:

3. Preferably, the drug molecule is an mRNA, DNA, protein, polysaccharide, or polypeptide drug.

11. The use of the polypeptide-modified liposome nanoparticles according to any one of claims 2 to 8 or the pharmaceutical composition according to claim 10 in the preparation of a medicament for treating and / or preventing respiratory-related diseases; Preferably, the respiratory system-related diseases include nasal cavity-related diseases and respiratory tract infection-related diseases; More preferably, the drug is a vaccine or antibody preparation.

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