Targeted substance mediated nucleic acid nano preparation and preparation method thereof

Abstract

The invention belongs to but not limited to the technical field of biological medicine, and discloses a target-mediated nucleic acid nano preparation and a preparation method thereof, the target-mediated nucleic acid nano preparation comprises a cationic lipid: C6A1 lipid, a polymer: PLGA, a modified lipid: DSPEPEG2000, and a target: a specific antibody or peptide fragment; a composite structure of cationic lipid and polymer nanoparticles is adopted, a target is modified on the surface of a nano preparation to realize targeted delivery, the target comprises an antibody, a peptide or a small molecule ligand, PEG is modified on the surfaces of the liposome and the nanoparticles, the stability is improved, and the circulation time is prolonged. The targeted nucleic acid nano preparation prepared by the invention can realize efficient and safe nucleic acid and drug delivery.

Description

Technical Field

[0001] The present invention belongs to but is not limited to the field of biomedicine technology, and in particular relates to a target-mediated nucleic acid nanoformulation and a preparation method thereof. Background Art

[0002] Prior Art 1: Liposome Gene Delivery System (US20060013897A1), which discloses a method for preparing cationic liposomes and their application in gene delivery. Cationic liposomes are prepared by mixing phosphatidylcholine, cholesterol, and a cationic lipid (such as distearoylphosphatidylethanolamine, DSPE).

[0003] Existing technical problems:

[0004] 1. Non-targeting: Liposomes lack specific targeting, resulting in low gene delivery efficiency.

[0005] 2. Immune response: Cationic liposomes trigger immune response, affecting the therapeutic effect.

[0006] 3. Poor stability: Liposomes are easily degraded in the in vivo environment, affecting the durability of gene delivery.

[0007] Prior Art 2: Polymer Nanoparticle Drug Delivery System (CN101482685A) discloses a polymer nanoparticle-based drug delivery system for drug delivery. The nanoparticles are prepared by mixing poly(lactic-co-glycolic acid) (PLGA) and polyethylene glycol (PEG)-modified lipids to improve drug bioavailability and stability.

[0008] Existing technical problems:

[0009] 1. Low drug encapsulation efficiency: The drug encapsulation efficiency of nanoparticles is not high, which affects the efficacy of the drug.

[0010] 2. Short circulation time: Nanoparticles have a short circulation time in the blood and are difficult to effectively reach the target tissue.

[0011] 3. Toxicity issues: Some polymer nanoparticles have certain toxicity, which limits their clinical application.

[0012] In view of the above analysis, the technical problems that urgently need to be solved in the existing technology are: a nucleic acid nanoformulation with high targeted delivery efficiency, stability and strong biosafety is needed. Summary of the Invention

[0013] In response to the problems existing in the prior art, the present invention provides a target-mediated nucleic acid nanoformulation and a preparation method thereof.

[0014] The present invention is achieved by providing a target-mediated nucleic acid nanoparticle preparation, the specific components of which include cationic lipid: C6A1 lipid, polymer: PLGA, modified lipid: DSPEPEG2000, and target: specific antibody or peptide segment;

[0015] A composite structure of cationic lipids and polymer nanoparticles is used to modify the surface of the nanoformulation with a target (such as an antibody, peptide or small molecule ligand), and PEG is modified on the surface of the liposome and nanoparticles.

[0016] Cationic lipid: C6A1 lipid, accounting for 20% to 30% of the total weight;

[0017] Polymer: poly(lactic-co-glycolic acid) (PLGA), accounting for 40% to 50% of the total weight;

[0018] Modified lipids: 1,2-distearoyl sn glycerol 3-phosphoethanolamine N [polyethylene glycol 2000] (DSPEPEG2000), accounting for 5% to 10% of the total weight;

[0019] Targeting agent: specific antibody or peptide, accounting for 5% to 10% of the total weight.

[0020] The above-mentioned nanoformulation adopts a composite structure of cationic lipids and polymer nanoparticles, and modifies specific antibodies, peptides or small molecule ligands on the surface of the nanoformulation to ensure that the PEG modification of the surface of liposomes and nanoparticles effectively improves stability and targeting.

[0021] Another object of the present invention is to provide a method for preparing a target-mediated nucleic acid nanoparticle preparation, comprising the following steps:

[0022] Step 1: Prepare the raw polymer PLGA and the modified lipid DSPEPEG2000, synthesize the cationic lipid C6A1 containing a six-carbon chain, and prepare specific antibodies or peptides through chemical synthesis or biotechnology methods;

[0023] Step 2: dissolving PLGA in an organic solvent, adding it dropwise to the aqueous phase for emulsification, and evaporating the organic solvent to form nanoparticles;

[0024] Step 3: mixing the nanoliposomes and polymer nanoparticles and combining them through electrostatic interaction; modifying the target on the surface of the composite nanoparticles by chemical covalent bonding or adsorption;

[0025] Step 4: centrifuge the prepared composite nanoformulation in an ultracentrifuge to remove unbound components and obtain a purified targeted nucleic acid nanoformulation.

[0026] Further, the preparation of liposomes includes:

[0027] (1) C6A1, phosphatidylcholine (PC), Chol, and cholesterol (DSPEPEG2000) were mixed in proportion and dissolved in chloroform to form a lipid solution;

[0028] (2) placing the lipid solution in a rotary evaporator and evaporating the chloroform under reduced pressure to form a uniform lipid film;

[0029] (3) Hydrate the lipid film in an appropriate amount of PBS buffer (pH 7.4) and let it stand for 30 minutes to allow the lipid film to be fully hydrated; use an ultrasonic processor to disperse the hydrated lipid film into nano-sized liposome particles.

[0030] Furthermore, the mass ratio is: C6A1:PC:Chol:DSPEPEG2000=20:50:20:10.

[0031] Furthermore, in step (2), the rotation speed is 80 rpm, the water bath temperature is 45° C.; in step (3), the ultrasonic frequency is 22 kHz, the ultrasonic power is 250 W, and the treatment time is 10 minutes (intermittent, each ultrasonication is 30 seconds, with an interval of 30 seconds).

[0032] Furthermore, the preparation of polymer nanoparticles in step 2 includes:

[0033] 1) Preparing PLGA solution: dissolving PLGA in an appropriate amount of organic solvent (such as ethyl acetate or dichloromethane) to form a uniform PLGA solution;

[0034] 2) Emulsification and evaporation: The PLGA solution is added dropwise to an aqueous phase containing a stabilizer (such as PVA) for emulsification; a high-speed homogenizer is used for emulsification until a nano-emulsion is formed; after emulsification, the organic solvent is evaporated from the emulsion in a rotary evaporator to form PLGA nanoparticles.

[0035] Furthermore, in step 2), the rotation speed is 80 rpm and the water bath temperature is 40°C.

[0036] Furthermore, in step three, the prepared nanoliposomes and PLGA nanoparticles are mixed at a certain volume ratio, that is, the volume ratio of nanoliposomes:PLGA nanoparticles=1:1.

[0037] Furthermore, in step three, the target is connected to the surface of the composite nanoparticle by chemical covalent bonding or adsorption; the linker: such as EDC / NHS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / hydroxysuccinimide); the linking reaction: the reaction is stirred in an ice bath for 2 hours, and then the unbound target is removed by dialysis or ultrafiltration.

[0038] Furthermore, the composite nanoformulation suspension in step 4 is centrifuged in an ultracentrifuge (centrifugal speed: 15000 rpm, centrifugation time: 30 minutes); after centrifugation, the supernatant is collected to obtain a purified targeted nucleic acid nanoformulation, and unbound components and small molecule impurities are further removed by ultrafiltration or dialysis.

[0039] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0040] First, the preparation method of the present invention successfully prepared targeted nucleic acid nanoformulations, achieving efficient and safe nucleic acid and drug delivery, solving the problems of poor targeting, low stability and toxicity in the existing technology. It has the following technical effects:

[0041] 1. Improve targeting: Through target modification, specific targeted delivery can be achieved and delivery efficiency can be improved.

[0042] 2. Enhanced stability: PEG modification improves the stability and circulation time of nanoformulations in the body.

[0043] 3. Improve encapsulation efficiency: The composite structure improves the encapsulation efficiency of nucleic acids and increases the nucleic acid delivery effect.

[0044] 4. Reduce toxicity: Use materials with good biocompatibility to reduce toxicity and improve biosafety.

[0045] Second, the present invention has made innovative improvements to several key issues existing in existing nucleic acid delivery technologies and achieved significant technological progress.

[0046] First, existing nucleic acid delivery vectors often suffer from insufficient stability, making nucleic acids susceptible to degradation or loss of activity in vivo. The present invention utilizes the cationic lipid C6A1 to bind to nucleic acids, forming a stable lipid-nucleic acid complex that effectively protects the nucleic acids from degradation and improves their stability in vivo.

[0047] Secondly, traditional delivery vectors have high nonspecific uptake in the body, resulting in low delivery efficiency. The present invention modifies the nanoparticle surface with DSPEPEG2000, which reduces nonspecific uptake through the long chain effect of PEG, prolongs the circulation time of the nanoformulation in the body, and improves delivery efficiency.

[0048] Furthermore, existing technologies often lack targeting to specific cells, resulting in a lack of precision in nucleic acid delivery. The present invention, by modifying the nanoparticle surface with specific antibodies or peptides, achieves specific recognition and binding to target cells, improving the precision and effectiveness of nucleic acid delivery.

[0049] In summary, the present invention achieves significant technological advancement by addressing the stability, nonspecific uptake, and targeting issues inherent in the prior art. These innovative improvements enable the present invention's target-mediated nucleic acid nanoformulations to have broader application prospects and greater practical value in areas such as gene therapy and drug delivery. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 The figure is a flow chart of the preparation method of the target-mediated nucleic acid nanoformulation provided by the embodiment of the present invention. DETAILED DESCRIPTION

[0051] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0052] An embodiment of the present invention provides a target-mediated nucleic acid nanoformulation, comprising:

[0053] Cationic lipid: C6A1 lipid, provides nucleic acid binding ability;

[0054] Polymer: PLGA, providing structural stability and degradability;

[0055] Modified lipid: DSPEPEG2000, provides stability and extends circulation time;

[0056] Targeting agent: specific antibodies or peptides to achieve targeted delivery.

[0057] The composite structure of cationic lipids and polymer nanoparticles combines the nucleic acid binding ability of cationic lipids with the structural stability and degradability of polymer nanoparticles. The cationic lipids effectively bind to negatively charged nucleic acids to form a stable complex, while the polymer nanoparticles provide additional structural support and protection, ensuring the integrity of the nucleic acid during delivery.

[0058] Modifying the surface of nanoparticles with targeting agents (such as antibodies, peptides, or small molecule ligands) is a key step in achieving targeted delivery. These targeting agents can specifically recognize and bind to receptors on target cells or tissues, thereby increasing the accumulation of nanoparticles in the target area, reducing their distribution in non-target areas, and improving delivery efficiency.

[0059] Modifying the surface of liposomes and nanoparticles with PEG (polyethylene glycol) is a common method for improving stability and extending circulation time. PEG molecules can form a hydration film on the surface of nanoparticles, reducing their interaction with plasma proteins, enzymes, and other components, and reducing the risk of recognition and clearance by the reticuloendothelial system (RES), thereby extending the circulation time of nanoparticles in the blood.

[0060] The following are the specific components and implementation schemes of the target-mediated nucleic acid nanoformulation provided in the embodiments of the present invention.

[0061] Cationic lipids: C6A1 lipids

[0062] Function: Provide nucleic acid binding ability, tightly bind to negatively charged nucleic acids through electrostatic interaction to form a stable complex.

[0063] Implementation: Select a suitable C6A1 lipid with good cationic properties and nucleic acid binding ability. During the preparation process, the C6A1 lipid is mixed with other auxiliary lipids (such as DSPC, cholesterol, etc.) in a certain proportion and dissolved in an organic solvent to form a lipid solution.

[0064] Polymer: PLGA

[0065] Function: Provide structural stability and degradability, protect nucleic acids from damage by the external environment, and gradually degrade and release nucleic acids after reaching the target site.

[0066] Implementation: Select the appropriate PLGA material and adjust its molecular weight and degradation rate as needed. During the preparation process, PLGA can be combined with a lipid solution and formed into PLGA-coated lipid nanoparticles through specific processes (such as emulsification solvent evaporation and nanoprecipitation).

[0067] Modified lipid: DSPEPEG2000

[0068] Function: Provide stability and prolong circulation time, and reduce the interaction between nanoparticles and plasma components through the hydration membrane effect of PEG molecules.

[0069] Implementation: DSPEPEG2000 is added as a modified lipid to a lipid solution, forming a mixed lipid solution with C6A1 lipid and other auxiliary lipids. During nanoparticle formation, DSPEPEG2000 naturally embeds into the nanoparticle surface, exerting its stability and long-circulation efficacy.

[0070] Target: specific antibody or peptide

[0071] Function: Achieve targeted delivery by specifically recognizing and binding to receptors on target cells or tissues, thereby increasing the accumulation of nanoparticles at the target site.

[0072] Implementation: Select an appropriate specific antibody or peptide as a target and modify it onto the nanoparticle surface through methods such as chemical conjugation. During the modification process, it is necessary to ensure that the activity and specificity of the target are not affected, while controlling the modification density to optimize the targeting effect.

[0073] The preparation of nucleic acid nanoformulations involves multiple components and complex processes. By rationally selecting and combining components such as cationic lipids, polymers, modified lipids, and targeting agents, nucleic acid nanoformulations with efficient, targeted delivery capabilities can be prepared. During the preparation process, strict control of process parameters and quality standards at each step is required to ensure the performance and safety of the nanoformulation.

[0074] like Figure 1 As shown, an embodiment of the present invention provides a method for preparing a target-mediated nucleic acid nanoformulation.

[0075] (1) Material preparation:

[0076] Cationic lipid (C6A1): Cationic lipid C6A1 containing a six-carbon chain was synthesized.

[0077] Polymer (PLGA): purchased from a commercial supplier.

[0078] Modified lipid (DSPEPEG2000): purchased from a commercial supplier.

[0079] Targeting agents (antibodies or peptides): Specific antibodies or peptides are prepared by chemical synthesis or biotechnology methods.

[0080] (2) Liposome preparation:

[0081] C6A1, PC, Chol and DSPEPEG2000 were mixed in proportion and dissolved in chloroform to form a lipid solution.

[0082] The lipid film was formed by rotary evaporation, hydrated in PBS buffer, and sonicated to obtain nanoliposomes.

[0083] (3) Preparation of polymer nanoparticles:

[0084] PLGA is dissolved in an organic solvent, added dropwise to the aqueous phase for emulsification, and the organic solvent is evaporated to form nanoparticles.

[0085] (4) Assembly of composite nanoformulations:

[0086] Nanoliposomes and polymer nanoparticles are mixed and bound via electrostatic interaction.

[0087] Surface modification targets (antibodies or peptides) are modified by chemical covalent bonding or adsorption.

[0088] (5) Centrifugal purification:

[0089] The prepared composite nanoformulation is centrifuged in an ultracentrifuge to remove unbound components and obtain a purified targeted nucleic acid nanoformulation.

[0090] The specific preparation steps include:

[0091] 1. Liposome Preparation

[0092] (1) Preparation of lipid solution:

[0093] C6A1, phosphatidylcholine (PC), cholesterol (Chol) and DSPEPEG2000 were mixed according to the proportion (mass ratio: C6A1:PC:Chol:DSPEPEG2000=20:50:20:10).

[0094] The above mixture was dissolved in an appropriate amount of chloroform to form a homogeneous lipid solution.

[0095] (2) Rotary evaporation:

[0096] The lipid solution was placed in a rotary evaporator and chloroform was evaporated under reduced pressure to form a uniform lipid film.

[0097] Rotation speed: 80rpm

[0098] Water bath temperature: 45°C

[0099] (3) Hydration and ultrasonic dispersion:

[0100] The lipid film was hydrated in an appropriate amount of PBS buffer (pH 7.4) and allowed to stand for 30 minutes to allow the lipid film to be fully hydrated.

[0101] The hydrated lipid film was dispersed into nano-sized liposome particles using an ultrasonic processor.

[0102] Ultrasonic frequency: 22kHz

[0103] Ultrasonic power: 250W

[0104] Treatment time: 10 minutes (intermittent, 30 seconds each ultrasound, 30 seconds interval)

[0105] 2. Preparation of Polymer Nanoparticles

[0106] (1) Preparation of PLGA solution:

[0107] PLGA is dissolved in an appropriate amount of an organic solvent (such as ethyl acetate or dichloromethane) to form a uniform PLGA solution.

[0108] (2) Emulsification and evaporation:

[0109] The PLGA solution is added dropwise to the aqueous phase containing a stabilizer (such as PVA) for emulsification.

[0110] Emulsify using a high-speed homogenizer until a nanoemulsion is formed.

[0111] After emulsification, the emulsion was evaporated in a rotary evaporator to evaporate the organic solvent and form PLGA nanoparticles.

[0112] Rotation speed: 80rpm

[0113] Water bath temperature: 40°C

[0114] 3. Assembly of Composite Nanoparticle Formulations

[0115] (1) Mixing and combining:

[0116] The prepared nanoliposomes and PLGA nanoparticles are mixed in a certain proportion and allowed to stand to allow the two to combine through electrostatic interaction.

[0117] Ratio: nanoliposomes: PLGA nanoparticles = 1:1 (volume ratio)

[0118] (2) Surface modified targets:

[0119] Targeting agents (antibodies or peptides) are modified on the surface of composite nanoparticles.

[0120] The targeting agent is connected to the surface of the composite nanoparticles by chemical covalent bonding or adsorption.

[0121] Linker: such as EDC / NHS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / hydroxysuccinimide)

[0122] Ligation reaction: Stir the reaction in an ice bath for 2 hours, and then remove unbound targets by dialysis or ultrafiltration.

[0123] 4. Centrifugation Purification

[0124] (1) Centrifugal separation:

[0125] The prepared composite nanoparticle suspension is centrifuged in an ultracentrifuge.

[0126] Centrifugal speed: 15000rpm

[0127] Centrifugation time: 30 minutes

[0128] (2) Collection and purification:

[0129] The supernatant was collected to obtain the purified targeted nucleic acid nanoparticle preparation.

[0130] Unbound components and small molecule impurities are further removed by ultrafiltration or dialysis.

[0131] Example 1: Targeted gene delivery

[0132] 1. Targeted agents: anti-tumor antibodies (such as Herceptin).

[0133] 2. Nucleic acid: siRNA, targeted silencing of tumor cell genes.

[0134] 3. Preparation method: Prepare targeted nucleic acid nanoparticles according to the above steps.

[0135] 4. Experimental results: In tumor cell models, nucleic acid nanoformulations showed highly effective gene silencing effects with low toxicity.

[0136] Example 2: Targeted drug delivery

[0137] 1. Targeting agent: tumor targeting peptide (such as RGD).

[0138] 2. Drugs: anticancer drugs (such as doxorubicin).

[0139] 3. Preparation method: Prepare targeted drug nanoformulation according to the above steps.

[0140] 4. Experimental results: In the mouse tumor model, the accumulation of drug nanoformulations at the tumor site increased significantly, the tumor volume was significantly reduced, and the therapeutic effect was obvious.

[0141] Working principle of target-mediated nucleic acid nanoformulation.

[0142] 1. Composition and structure

[0143] The target-mediated nucleic acid nanoformulation of the present invention is primarily composed of the cationic lipid C6A1, the polymer PLGA, the modified lipid DSPEPEG2000, and the target (specific antibody or peptide). Its structural characteristics lie in the composite structure of cationic lipid and polymer nanoparticles, and the modification of the target and PEG on the surface of the nanoformulation to optimize its stability and targeting.

[0144] 1) Cationic lipid C6A1: A cationic lipid with a six-carbon chain. Its cationic head group can bind to negatively charged nucleic acids through electrostatic interaction to form a stable complex, protecting nucleic acids from degradation and promoting their cellular uptake.

[0145] 2) Polymer PLGA: A biodegradable polymer with excellent biocompatibility and controlled drug release properties. In this nanoformulation, PLGA forms the nanoparticle backbone, providing a stable carrier for nucleic acid delivery.

[0146] 3) Modified lipid DSPEPEG2000: After DSPE (distearylphosphatidylethanolamine) is combined with PEG (polyethylene glycol), it not only improves the hydrophilicity and stability of the nanoparticles, but also reduces their nonspecific uptake in the body and prolongs their circulation time.

[0147] 4) Targeting agents (specific antibodies or peptides): modified on the surface of nanoparticles through chemical covalent bonding or adsorption, they can specifically recognize and bind to target cells, thereby improving the targeting of nanoformulations.

[0148] 2. Working Principle

[0149] 1) Loading and protection of nucleic acids:

[0150] The cationic lipid C6A1 binds to negatively charged nucleic acids through electrostatic interactions, forming stable lipid-nucleic acid complexes that not only protect nucleic acids from degradation but also facilitate their transmembrane transport.

[0151] 2) Formation and stabilization of nanoparticles:

[0152] PLGA is dissolved in an organic solvent and added dropwise to the aqueous phase via an emulsification method. As the organic solvent evaporates, the PLGA self-assembles into nanoparticles. These nanoparticles provide a stable carrier for nucleic acid delivery.

[0153] DSPEPEG2000 is modified on the surface of nanoparticles, which improves the hydrophilicity and stability of nanoparticles through the long chain effect of PEG and reduces their nonspecific uptake in the body.

[0154] 3) Targeted recognition and cellular uptake:

[0155] Specific antibodies or peptides are modified on the surface of nanoparticles, which specifically recognize target cells through mechanisms such as antigen-antibody reaction or receptor-ligand binding.

[0156] Once the nanoparticles bind to the target cells, they enter the cells through endocytosis and then release nucleic acids in the intracellular environment to achieve their therapeutic or diagnostic functions.

[0157] 4) Ultracentrifugation purification:

[0158] The prepared composite nanoformulation is centrifuged in an ultracentrifuge to remove unbound components (such as lipids, polymers, and targeting agents that are not bound to the nanoparticles) to obtain a purified targeted nucleic acid nanoformulation. This step ensures the uniformity and stability of the nanoformulation.

[0159] In summary, the target-mediated nucleic acid nanoformulation of the present invention, through its unique composition and structural design, achieves efficient nucleic acid loading and protection, stable nanoparticle formation and modification, and targeted recognition and cellular uptake. The synergistic effect of these functions makes this nanoformulation have broad application prospects in gene therapy, drug delivery and other fields.

[0160] Targeted nucleic acid nanoparticles are nanoscale drug carriers used to deliver nucleic acid drugs. They are modified with specific targeting agents (such as antibodies, peptides, or small molecule ligands) to improve their delivery efficiency in targeted cells or tissues. Two specific examples are listed below:

[0161] Example 1: Antibody-modified nucleic acid nanoformulations

[0162] Components:

[0163] Cationic lipids: C6A1 lipids

[0164] Polymer: PLGA (poly(lactic-co-glycolic acid))

[0165] Modified lipid: DSPEPEG2000 (1,2-distearoyl sn-glycerol 3-phosphoethanolamine PEG2000)

[0166] Target: HER2-specific antibody

[0167] Preparation steps:

[0168] 1) Preparation of PLGA nanoparticles: PLGA is dissolved in an organic solvent (such as acetone or chloroform), and then added dropwise to an aqueous solution containing C6A1 lipid and DSPEPEG2000 to obtain PLGA nanoparticles by solvent evaporation.

[0169] 2) Encapsulation of nucleic acid drugs: When preparing PLGA nanoparticles, nucleic acid drugs (such as siRNA or miRNA) are added, and the nucleic acid drugs are encapsulated in the nanoparticles through the self-assembly process of the nanoparticles.

[0170] 3) Antibody modification: A HER2-specific antibody was chemically coupled to DSPEPEG2000 to form an antibody-PEG lipid complex. This complex was then inserted onto the surface of PLGA nanoparticles to produce an antibody-modified nucleic acid nanoparticle.

[0171] This nanoformulation can target cancer cells expressing HER2 receptors, such as breast cancer cells, to achieve targeted delivery and therapeutic effects of nucleic acid drugs.

[0172] In order to demonstrate the research of this nanoformulation and verify its targeting effect and therapeutic effect, the following experimental steps and methods can be designed and implemented:

[0173] 1. Surface modification verification of nanoformulations

[0174] Objective: To confirm whether HER2-specific antibodies are successfully modified on the surface of PLGA nanoparticles.

[0175] method:

[0176] Transmission electron microscopy (TEM): used to observe the morphology and size of nanoparticles.

[0177] Dynamic light scattering (DLS): used to measure the particle size and surface charge of nanoparticles.

[0178] Fourier transform infrared spectroscopy (FTIR): used to detect the characteristic absorption peaks of DSPEPEG2000 and antibodies on the surface of nanoparticles.

[0179] Western blot or ELISA: used to detect HER2 antibodies on the surface of nanoparticles.

[0180] 2. Targeting effect verification

[0181] Objective: To verify the targeting ability of antibody-modified nanoformulations on HER2-positive cancer cells in vitro.

[0182] method:

[0183] Cell culture: Culture HER2-positive and HER2-negative breast cancer cells (such as SKBR3 cells and MDAMB231 cells).

[0184] Cell binding assay:

[0185] The antibody-modified nanoformulations were incubated with both cell lines.

[0186] The binding of nanoparticles to cells was detected by flow cytometry (FACS).

[0187] The distribution of nanoparticles on cells was observed using fluorescence microscopy.

[0188] Competitive binding experiment: After pre-incubation of cells with free HER2 antibody, antibody-modified nanoformulations are added and the changes in binding rate are observed to verify specificity.

[0189] 3. Verification of treatment effect

[0190] Objective: To verify the therapeutic effect of antibody-modified nucleic acid nanoformulations on HER2-positive cancer cells in vitro and in vivo.

[0191] method:

[0192] In vitro experiments:

[0193] Cell proliferation inhibition assay:

[0194] The effects of nanoformulations on the proliferation of HER2-positive and HER2-negative cells were detected using CCK8 or MTT assays.

[0195] Apoptosis detection:

[0196] The cell apoptosis rate was detected by flow cytometry using the Annexin V / PI double staining method.

[0197] Nucleic acid drug release experiment:

[0198] The release of nucleic acid drugs from nanoparticles into cells was measured, and changes in target gene expression of nucleic acid drugs were detected by qPCR or Western blot.

[0199] In vivo experiments:

[0200] Animal model establishment: A HER2-positive breast cancer xenograft tumor model was established in nude mice.

[0201] Nanoformulation injection: Antibody-modified nucleic acid nanoformulations were administered via tail vein injection.

[0202] Tumor growth inhibition effect:

[0203] The tumor volume was measured regularly and the tumor growth inhibition rate was calculated.

[0204] The cell apoptosis in tumor tissues was detected by TUNEL method.

[0205] Biodistribution assay:

[0206] The distribution of nanoformulations in the body, especially their enrichment in tumor sites, is detected by fluorescence imaging or radiotracer technology.

[0207] Data analysis and results:

[0208] 1. Surface modification verification:

[0209] Successful antibody modification should show expected characteristic changes in TEM, DLS, and FTIR tests, and Western blot or ELISA results should show the presence of HER2 antibodies on the surface of the nanoparticles.

[0210] 2. Targeting effect verification:

[0211] HER2-positive cells should exhibit significantly higher nanoparticle binding than HER2-negative cells, and competition binding experiments should show that free antibody can significantly reduce nanoparticle binding.

[0212] 3. Verification of treatment effect:

[0213] In vitro experiments should show that the antibody-modified nanoformulation has significant proliferation inhibition and apoptosis induction effects on HER2-positive cells.

[0214] In vivo experiments should show that tumor growth is significantly inhibited, the nanoformulation is enriched at the tumor site, and exhibits good biodistribution and low toxicity.

[0215] Through the above experimental steps and methods, the targeting effect and therapeutic effect of antibody-modified nucleic acid nanoformulations in targeting cancer cells expressing HER2 receptors can be fully verified, providing strong support for its potential in clinical applications.

[0216] Example 2: Peptide-modified nucleic acid nanoparticles

[0217] Components:

[0218] Cationic lipids: C6A1 lipids

[0219] Polymer: PLGA

[0220] Modified lipid: DSPEPEG2000

[0221] Target: RGD peptide (targeting integrin receptors)

[0222] Preparation steps:

[0223] 1) Preparation of PLGA nanoparticles: PLGA is dissolved in an organic solvent (such as acetone or chloroform), and then added dropwise to an aqueous solution containing C6A1 lipid and DSPEPEG2000 to obtain PLGA nanoparticles by solvent evaporation.

[0224] 2) Encapsulation of nucleic acid drugs: When preparing PLGA nanoparticles, nucleic acid drugs (such as mRNA or antisense oligonucleotides) are added, and the nucleic acid drugs are encapsulated in the nanoparticles through the self-assembly process of the nanoparticles.

[0225] 3) Peptide modification: RGD peptide was chemically coupled to DSPEPEG2000 to form an RGD peptide-PEG lipid complex. This complex was then inserted onto the surface of PLGA nanoparticles to produce a peptide-modified nucleic acid nanoparticle.

[0226] This nanoformulation can target cells expressing integrin receptors, such as tumor vascular endothelial cells, to achieve targeted delivery of nucleic acid drugs and therapeutic effects against tumor angiogenesis.

[0227] To demonstrate the research of this nanoformulation and verify its targeting effect and therapeutic effect, the following is a detailed experimental verification method:

[0228] 1. Surface modification verification of nanoformulations

[0229] Objective: To confirm whether RGD peptide is successfully modified on the surface of PLGA nanoparticles.

[0230] method:

[0231] Transmission electron microscopy (TEM): observe the morphology and size of nanoparticles.

[0232] Dynamic Light Scattering (DLS): measures the particle size and surface charge of nanoparticles.

[0233] Fourier transform infrared spectroscopy (FTIR): Detect the characteristic absorption peaks of DSPEPEG2000 and RGD peptide on the surface of nanoparticles.

[0234] Western blot or ELISA: Detect RGD peptide on the surface of nanoparticles.

[0235] 2. Targeting effect verification

[0236] Objective: To verify the targeting ability of peptide-modified nanoformulations on cells expressing integrin receptors in vitro.

[0237] method:

[0238] Cell culture: Tumor vascular endothelial cells (such as HUVEC cells) expressing integrin receptors and control cells are cultured.

[0239] Cell binding assay:

[0240] The peptide-modified nanoformulation was incubated with the two cell lines.

[0241] The binding of nanoparticles to cells was detected by flow cytometry (FACS).

[0242] The distribution of nanoparticles on cells was observed using fluorescence microscopy.

[0243] Competitive binding experiment: After pre-incubating cells with free RGD peptide, the peptide-modified nanoformulation is added and the changes in binding rate are observed to verify specificity.

[0244] 3. Verification of treatment effect

[0245] Objective: To verify the anti-angiogenic effect of peptide-modified nucleic acid nanoformulation on tumor vascular endothelial cells in vitro and in vivo.

[0246] method:

[0247] In vitro experiments:

[0248] Cell proliferation inhibition assay:

[0249] The CCK8 or MTT assay was used to detect the effect of nanoformulations on the proliferation of tumor vascular endothelial cells.

[0250] Apoptosis detection:

[0251] The cell apoptosis rate was detected by flow cytometry using the Annexin V / PI double staining method.

[0252] Nucleic acid drug release experiment:

[0253] The release of nucleic acid drugs from nanoparticles into cells was measured, and changes in target gene expression of nucleic acid drugs were detected by qPCR or Western blot.

[0254] In vivo experiments:

[0255] Animal model establishment: Tumor xenograft models were established in nude mice.

[0256] Nanoformulation injection: The peptide-modified nucleic acid nanoformulation is administered via tail vein injection.

[0257] Tumor growth inhibition effect:

[0258] The tumor volume was measured regularly and the tumor growth inhibition rate was calculated.

[0259] The cell apoptosis in tumor tissues was detected by TUNEL method.

[0260] Anti-angiogenic effects:

[0261] CD31 immunohistochemical staining was used to detect tumor angiogenesis.

[0262] Biodistribution assay:

[0263] The distribution of nanoformulations in the body, especially their enrichment in tumor sites, is detected by fluorescence imaging or radiotracer technology.

[0264] Data analysis and results:

[0265] 1. Surface modification verification:

[0266] Successful peptide modification should show expected characteristic changes in TEM, DLS, and FTIR tests, and Western blot or ELISA results should show the presence of RGD peptide on the surface of nanoparticles.

[0267] 2. Targeting effect verification:

[0268] Cells expressing integrin receptors should exhibit significantly higher nanoparticle binding than control cells, and competition binding experiments should show that free peptide can significantly reduce nanoparticle binding.

[0269] 3. Verification of treatment effect:

[0270] In vitro experiments should show that peptide-modified nanoformulations have significant proliferation inhibition and apoptosis-inducing effects on tumor vascular endothelial cells.

[0271] In vivo experiments should show that tumor growth is significantly inhibited, the nanoformulation is enriched at the tumor site, and exhibits good anti-angiogenic effects and low toxicity.

[0272] Through the above experimental steps and methods, the targeting effect and anti-tumor angiogenesis therapeutic effect of peptide-modified nucleic acid nanoformulations in cells expressing integrin receptors can be fully verified, providing strong support for its potential in clinical applications.

[0273] These two examples demonstrate the potential of target-mediated nucleic acid nanoformulation in different applications by modifying different target molecules.

[0274] The above description is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with this technical field within the technical scope disclosed by the present invention and within the spirit and principles of the present invention should be covered by the scope of protection of the present invention.

Claims

1. A target-mediated nucleic acid nanoparticle preparation, characterized in that: include: Cationic lipid: C6A1 lipid, accounting for 20% to 30% of the total weight; Polymer: poly(lactic-co-glycolic acid) (PLGA), accounting for 40% to 50% of the total weight; Modified lipids: 1,2-distearoyl sn glycerol 3-phosphoethanolamine N [polyethylene glycol 2000] (DSPEPEG2000), accounting for 5% to 10% of the total weight; Targeting agent: specific antibody or peptide, accounting for 5% to 10% of the total weight. The above-mentioned nanoformulation adopts a composite structure of cationic lipids and polymer nanoparticles, and modifies specific antibodies, peptides or small molecule ligands on the surface of the nanoformulation to ensure that PEG modification on the surface of liposomes and nanoparticles effectively improves stability and targeting.

2. The targeting agent-mediated nucleic acid nanoparticle preparation according to claim 1, characterized in that: The target is a HER2-specific antibody, the cationic lipid is a C6A1 lipid, the polymer is PLGA, the modified lipid is DSPEPEG2000, and the nucleic acid nanoparticle preparation is prepared by connecting the HER2-specific antibody to DSPEPEG2000 through a chemical coupling method to form an antibody-PEG lipid complex, and inserting the complex into the surface of PLGA nanoparticles.

3. The targeting agent-mediated nucleic acid nanoparticle preparation according to claim 1, characterized in that: The target is RGD peptide, the cationic lipid is C6A1 lipid, the polymer is PLGA, the modified lipid is DSPEPEG2000, and the nucleic acid nanoparticle preparation is prepared by connecting the RGD peptide to DSPEPEG2000 through a chemical coupling method to form an RGD peptide PEG lipid complex, and inserting the complex into the surface of PLGA nanoparticles.

4. A method for preparing a targeting agent-mediated nucleic acid nanoparticle preparation as claimed in claim 1, characterized in that: The following steps are involved: Step 1, preparing the raw material polymer PLGA, the modified lipid DSPEPEG2000, synthesizing the cationic lipid C6A1 containing a six-carbon chain, and preparing specific antibodies or peptides by chemical synthesis or biotechnology methods; Step 2, dissolving PLGA in an organic solvent, adding it dropwise to the aqueous phase for emulsification, and evaporating the organic solvent to form nanoparticles; Step 3, mixing the nanoliposomes and polymer nanoparticles, and combining them by electrostatic interaction; modifying the target on the surface of the composite nanoparticles by chemical covalent bonding or adsorption; Step 4: Centrifuge the prepared composite nanoformulation in an ultracentrifuge to remove unbound components and obtain a purified targeted nucleic acid nanoformulation.

5. The method for preparing a targeting agent-mediated nucleic acid nanoparticle preparation according to claim 4, characterized in that: The preparation of liposomes includes: (1) C6A1, phosphatidylcholine PC, Chol and cholesterol DSPEPEG2000 are mixed in proportion and dissolved in chloroform to form a lipid solution; (2) placing the lipid solution in a rotary evaporator, evaporating chloroform under reduced pressure to form a uniform lipid film; (3) Hydrate the lipid film in an appropriate amount of PBS buffer at pH 7.4 and allow the lipid film to stand for 30 minutes to fully hydrate the lipid film; use an ultrasonic processor to disperse the hydrated lipid film into nano-sized liposome particles.

6. The method for preparing a targeting agent-mediated nucleic acid nanoparticle preparation according to claim 5, characterized in that: Mass ratio: C6A1:PC:Chol:DSPEPEG2000=20:50:20:

10.

7. The method for preparing a targeting agent-mediated nucleic acid nanoparticle preparation according to claim 5, characterized in that: In the step (2), the rotation speed is 80 rpm and the water bath temperature is 45° C.; in the step (3), the ultrasonic frequency is 22 kHz, the ultrasonic power is 250 W, and the treatment time is 10 minutes. Intermittent treatment is performed, with each ultrasonic treatment lasting 30 seconds and an interval of 30 seconds.

8. The method for preparing a target-mediated nucleic acid nanoparticle preparation according to claim 4, characterized in that: The preparation of polymer nanoparticles in step 2 includes: 1) preparing a PLGA solution: dissolving PLGA in an appropriate amount of an organic solvent (such as ethyl acetate or dichloromethane) to form a uniform PLGA solution; 2) Emulsification and evaporation: the PLGA solution is added dropwise to the aqueous phase containing the stabilizer PVA for emulsification; a high-speed homogenizer is used for emulsification until a nano-scale emulsion is formed; after emulsification, the organic solvent of the emulsion is evaporated in a rotary evaporator to form PLGA nanoparticles.

9. The method for preparing a targeting agent-mediated nucleic acid nanoparticle preparation according to claim 6, characterized in that: In the step 2), the rotation speed is 80 rpm and the water bath temperature is 40°C.

10. The method for preparing a targeting agent-mediated nucleic acid nanoparticle preparation according to claim 4, characterized in that: In step 3, the prepared nanoliposomes and PLGA nanoparticles are mixed in a certain volume ratio, the volume ratio of nanoliposomes: PLGA nanoparticles = 1:1; the target is connected to the surface of the composite nanoparticles by chemical covalent bonding or adsorption; the linker: 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide EDC, hydroxysuccinimide NHS; the connection reaction: the reaction is stirred in an ice bath for 2 hours, and then the unbound target is removed by dialysis or ultrafiltration; In step 4, the composite nanoformulation suspension is centrifuged in an ultracentrifuge at a centrifugal speed of 15,000 rpm and a centrifugal time of 30 minutes; after centrifugation, the supernatant is collected to obtain a purified targeted nucleic acid nanoformulation, and unbound components and small molecule impurities are further removed by ultrafiltration or dialysis.

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