Cationic lipid C6-A1, nano-liposome particles composed of cationic lipid C6-A1 and preparation method of nano-liposome particles

Abstract

The invention belongs to the technical field of medicines, and discloses a cationic lipid C6-A1, a nano-liposome particle composed of the same and a preparation method of the nano-liposome particle, and the stability is improved: the stability of the nano-liposome can be remarkably improved through the structural design and PEG modification of the C6-A1 lipid, and in-vivo degradation is reduced. And the optimized preparation method and introduction of cationic lipid can improve the encapsulation efficiency of drugs and genes. And the PEG modified lipid can reduce the recognition of an immune system, prolong the circulation time of the liposome in blood and improve the targeted transmission capability. The cation characteristic of the C6-A1 is beneficial to the combination and endocytosis of the gene and a cell membrane, and the gene transfer efficiency is improved.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical technology, and particularly relates to a cationic lipid C6-A1 and nano liposome particles composed of the cationic lipid C6-A1 and a preparation method thereof. Background Art

[0002] Prior art 1: Preparation and application of cationic liposomes

[0003] A method for preparing a cationic liposome and its application in gene delivery. The cationic liposome is prepared by mixing phosphatidylcholine, cholesterol and a cationic lipid such as distearoylphosphatidylethanolamine (DSPE).

[0004] Prior art 2: Nanoliposome drug delivery system

[0005] A liposome-based nano drug delivery system for drug delivery. The nano liposome is prepared by mixing phospholipids, cholesterol and polyethylene glycol (PEG) modified lipids to improve the bioavailability and stability of the drug.

[0006] Through the above analysis, the problems and defects of the prior art are as follows:

[0007] (1) The preparation process is complicated and requires strict control of temperature and pH. It has poor stability and is easily degraded in the body. The gene delivery efficiency is low and it is easily captured by the extracellular matrix.

[0008] (2) Aggregation and precipitation are prone to occur during the preparation process; the drug encapsulation rate is low, affecting the efficacy; the circulation time in the blood is short, making it difficult to effectively reach the target tissue. Summary of the invention

[0009] In view of the problems existing in the prior art, the present invention provides a cationic lipid C6-A1 and nanoliposome particles composed of the cationic lipid C6-A1 and a preparation method thereof.

[0010] The present invention is achieved in that a cationic lipid C6-A1 and nanoliposome particles composed of the cationic lipid C6-A1 include:

[0011] Composition of ionizable cationic lipid C6-A1:

[0012] C6-A1 lipid structure: carbon chain part, cationic group, molecular modification;

[0013] Composition of nanoliposome particles:

[0014] Core lipid components: C6-A1 lipid, phosphatidylcholine PC, cholesterol Chol, and polyethylene glycol PEG-modified lipid DSPE-PEG2000.

[0015] Furthermore, the C6-A1 lipid structure:

[0016] Carbon chain part: Decanoic acid C6, a fatty acid chain with six carbon atoms;

[0017] Cationic group: amino NH2 or quaternary ammonium salt NR 3+ , introducing cationic properties to improve the binding ability with genes / drugs;

[0018] Molecular modification:

[0019] Synthesis reaction: Decanoic acid is reacted with an amine compound or a quaternary ammonium salt to obtain a C6-A1 lipid containing a cationic group.

[0020] Furthermore, the specific mass ratio of the cationic lipid C6-A1 and the nanoliposome particles composed thereof is:

[0021] C6-A1 lipids: 20%;

[0022] Phosphatidylcholine PC: 50%;

[0023] Cholesterol: 20%

[0024] PEG-modified lipid DSPE-PEG2000: 10%.

[0025] Furthermore, the preparation method of the cationic lipid C6-A1 and the nanoliposome particles composed thereof comprises:

[0026] Step 1, material preparation:

[0027] Mix C6-A1 lipid, phosphatidylcholine, PC, cholesterol and DSPE-PEG2000 in proportion;

[0028] dissolving the mixture in an organic solvent to form a uniform lipid solution;

[0029] Step 2, solvent evaporation:

[0030] The organic solvent was evaporated under reduced pressure using a rotary evaporator to form a uniform lipid film;

[0031] Step 3, hydration and ultrasonic dispersion:

[0032] The lipid film was hydrated in an appropriate amount of buffer solution and allowed to stand for 30 minutes to allow the lipid film to be fully hydrated;

[0033] The hydrated lipid film was dispersed into nano-sized liposome particles using an ultrasonic processor;

[0034] Step 4, centrifugation purification:

[0035] The prepared nanoliposome suspension is centrifuged in an ultracentrifuge to remove unencapsulated drugs or genes to obtain purified nanoliposomes.

[0036] Furthermore, the rotary evaporator has a rotation speed of 80 rpm and a water bath temperature of 45°C.

[0037] Furthermore, the ultrasonic processor has an ultrasonic frequency of 22 kHz, an ultrasonic power of 250 W, and a processing time of 10 minutes.

[0038] Furthermore, the centrifugal speed of the ultracentrifuge is 15,000 rpm and the centrifugal time is 30 minutes.

[0039] Furthermore, the preparation system of the cationic lipid C6-A1 and the nanoliposome particles composed thereof comprises:

[0040] A mixing module, used for mixing C6-A1 lipid, phosphatidylcholine, PC, cholesterol and DSPE-PEG2000 in proportion; dissolving the mixture in an organic solvent to form a uniform lipid solution;

[0041] A solvent evaporation module, used to evaporate the organic solvent under reduced pressure using a rotary evaporator to form a uniform lipid film;

[0042] The hydration and ultrasonic dispersion module is used to hydrate the lipid film in an appropriate amount of buffer solution, and let it stand for 30 minutes to fully hydrate the lipid film; and use an ultrasonic processor to disperse the hydrated lipid film into nano-scale liposome particles;

[0043] The centrifugal purification module is used to centrifuge the prepared nanoliposome suspension in an ultracentrifuge to remove unencapsulated drugs or genes to obtain purified nanoliposomes.

[0044] 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:

[0045] First, the present invention provides a novel ionizable cationic lipid C6-A1 and nanoliposome particles composed of the same, and improves the preparation method to enhance the stability, drug loading efficiency and targeted delivery capability of the liposome.

[0046] Improved stability: The structural design and PEG modification of C6-A1 lipids can significantly improve the stability of nanoliposomes and reduce in vivo degradation.

[0047] Increase drug loading efficiency: Optimized preparation methods and the introduction of cationic lipids can improve the encapsulation rate of drugs and genes.

[0048] Prolong circulation time: PEG-modified lipids can reduce recognition by the immune system, prolong the circulation time of liposomes in the blood, and improve targeted delivery capabilities.

[0049] Enhanced gene delivery efficiency: The cationic properties of C6-A1 facilitate the binding and internalization of genes to cell membranes, thereby improving gene delivery efficiency.

[0050] Second, the present invention proposes innovative solutions to several key problems existing in existing nucleic acid delivery technologies and has achieved significant technological progress.

[0051] First, the nucleic acid delivery vectors in the prior art often face the problem of insufficient stability, and the nucleic acid is easily degraded or loses activity in the in vivo environment. The present invention synthesizes a cationic lipid C6-A1 with a specific structure, and uses it as one of the core lipid components, and mixes it with phosphatidylcholine PC, cholesterol Chol and PEG-modified lipid DSPE-PEG2000 in a specific ratio to form stable nanoliposome particles. This innovation effectively protects nucleic acids from degradation and improves their stability in the body.

[0052] Secondly, the non-specific uptake of traditional delivery carriers in the body is high, resulting in low delivery efficiency. The present invention optimizes the composition of nanoliposome particles, especially introduces PEG-modified lipid DSPE-PEG2000, significantly reduces non-specific uptake, prolongs the circulation time of nanoformulations in the body, and thus improves delivery efficiency.

[0053] Furthermore, the prior art often lacks targeting to specific cells, which makes the nucleic acid delivery lack of precision. The nanoliposome particles prepared by the present invention have good biocompatibility and modifiability, which provides the possibility of further modifying specific antibodies or peptides on their surface to achieve specific recognition and binding to target cells, thereby improving the precision and effectiveness of nucleic acid delivery.

[0054] In summary, the present invention has achieved significant technical progress by solving the stability, non-specific uptake and targeting problems in the prior art. These innovative improvements make the cationic lipid C6-A1 of the present invention and the nanoliposome particles composed thereof have wider application prospects and higher practical value in the fields of gene therapy, drug delivery, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 The present invention provides a flow chart of the method for preparing the cationic lipid C6-A1 and the nanoliposome particles composed thereof.

[0056] Figure 2 It is a structural block diagram of a system for preparing cationic lipid C6-A1 and nanoliposome particles composed of the cationic lipid C6-A1 provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0057] In order to make the purpose, technical solution 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 used to limit the present invention.

[0058] A cationic lipid C6-A1 and nanoliposome particles composed of the cationic lipid C6-A1 provided in an embodiment of the present invention include:

[0059] Composition of ionizable cationic lipid C6-A1:

[0060] C6-A1 lipid structure: carbon chain part, cationic group, molecular modification;

[0061] Composition of nanoliposome particles:

[0062] Core lipid components: C6-A1 lipid, phosphatidylcholine PC, cholesterol Chol, and polyethylene glycol PEG-modified lipid DSPE-PEG2000.

[0063] The C6-A1 lipid structure provided in the embodiments of the present invention is:

[0064] Carbon chain part: Decanoic acid C6, a fatty acid chain with six carbon atoms;

[0065] Cationic group: amino NH2 or quaternary ammonium salt NR 3+ , introducing cationic properties to improve the binding ability with genes / drugs;

[0066] Synthesis reaction: Decanoic acid is reacted with an amine compound or a quaternary ammonium salt to obtain a C6-A1 lipid containing a cationic group.

[0067] The specific mass ratio of the cationic lipid C6-A1 and the nanoliposome particles composed thereof provided in the embodiments of the present invention is:

[0068] C6-A1 lipids: 20%;

[0069] Phosphatidylcholine PC: 50%;

[0070] Cholesterol: 20%

[0071] PEG-modified lipid DSPE-PEG2000: 10%.

[0072] like Figure 1 As shown, a method for preparing a cationic lipid C6-A1 and nanoliposome particles composed thereof provided by an embodiment of the present invention comprises:

[0073] S101, Material preparation:

[0074] Mix C6-A1 lipid, phosphatidylcholine, PC, cholesterol and DSPE-PEG2000 in proportion;

[0075] dissolving the mixture in an organic solvent to form a uniform lipid solution;

[0076] S102, solvent evaporation:

[0077] The organic solvent was evaporated under reduced pressure using a rotary evaporator to form a uniform lipid film;

[0078] S103, hydration and ultrasonic dispersion:

[0079] The lipid film was hydrated in an appropriate amount of buffer solution and allowed to stand for 30 minutes to allow the lipid film to be fully hydrated;

[0080] The hydrated lipid film was dispersed into nano-sized liposome particles using an ultrasonic processor;

[0081] S104, centrifugation purification:

[0082] The prepared nanoliposome suspension is centrifuged in an ultracentrifuge to remove unencapsulated drugs or genes to obtain purified nanoliposomes.

[0083] The rotary evaporator provided in the embodiment of the present invention has a rotation speed of 80 rpm and a water bath temperature of 45°C.

[0084] The ultrasonic processor provided in the embodiment of the present invention has an ultrasonic frequency of 22 kHz, an ultrasonic power of 250 W, and a processing time of 10 minutes.

[0085] The ultracentrifuge provided in the embodiment of the present invention has a centrifugal speed of 15,000 rpm and a centrifugal time of 30 minutes.

[0086] like Figure 2 As shown, a system for preparing a cationic lipid C6-A1 and nanoliposome particles composed of the cationic lipid C6-A1 provided in an embodiment of the present invention comprises:

[0087] A mixing module, used for mixing C6-A1 lipid, phosphatidylcholine, PC, cholesterol and DSPE-PEG2000 in proportion; dissolving the mixture in an organic solvent to form a uniform lipid solution;

[0088] A solvent evaporation module, used to evaporate the organic solvent under reduced pressure using a rotary evaporator to form a uniform lipid film;

[0089] The hydration and ultrasonic dispersion module is used to hydrate the lipid film in an appropriate amount of buffer solution, and let it stand for 30 minutes to fully hydrate the lipid film; and use an ultrasonic processor to disperse the hydrated lipid film into nano-scale liposome particles;

[0090] The centrifugal purification module is used to centrifuge the prepared nanoliposome suspension in an ultracentrifuge to remove unencapsulated drugs or genes to obtain purified nanoliposomes.

[0091] -A1 and nanoliposome particles composed thereof provided by the embodiments of the present invention include:

[0092] Composition of ionizable cationic lipid C6-A1:

[0093] C6-A1 lipid structure: carbon chain part, cationic group, molecular modification;

[0094] Composition of nanoliposome particles:

[0095] Core lipid components: C6-A1 lipid, phosphatidylcholine (PC), cholesterol (Chol), and polyethylene glycol (PEG)-modified lipid DSPE-PEG2000.

[0096] The component ratio of nanoliposome particles is:

[0097] C6-A1 lipids account for 30–40% of the core lipid components;

[0098] Phosphatidylcholine (PC) accounts for 30-40% of the core lipid composition;

[0099] Cholesterol (Chol) accounts for 20-30% of the core lipid components;

[0100] The polyethylene glycol (PEG)-modified lipid DSPE-PEG2000 accounts for 5-10% of the core lipid component.

[0101] C6-A1 lipid structure:

[0102] Carbon chain part: Decanoic acid (C6), a fatty acid chain with six carbon atoms;

[0103] Cationic groups: amino groups (NH2) or quaternary ammonium salts (NR3+), which introduce cationic properties to improve the ability to bind to genes / drugs;

[0104] Molecular modification:

[0105] Synthesis reaction: Decanoic acid is reacted with an amine compound or a quaternary ammonium salt to obtain a C6-A1 lipid containing a cationic group.

[0106] These claims define the specific components and proportions of the cationic lipid C6-A1 and its nanoliposome particles, which help protect the core technology of the invention.

[0107] The present invention is specifically implemented:

[0108] Ionizable cationic lipid C6-A1 and specific components of nanoliposome particles composed thereof

[0109] 1. Composition of ionizable cationic lipid C6-A1

[0110] C6-A1 lipid structure:

[0111] Carbon chain part: Decanoic acid (C6), a fatty acid chain with six carbon atoms.

[0112] Cationic groups: amino (NH2) or quaternary ammonium salt (NR 3+ ), introducing cationic properties to enhance the binding ability with genes / drugs.

[0113] Molecular modification:

[0114] Synthesis reaction: Decanoic acid is reacted with an amine compound or a quaternary ammonium salt to obtain a C6-A1 lipid containing a cationic group.

[0115] 2. Composition of Nanoliposome Particles

[0116] Core lipid components:

[0117] C6-A1 lipid: an ionizable cationic lipid containing a six-carbon chain.

[0118] Phosphatidylcholine (PC): The main structural lipid that improves the stability and membrane fluidity of liposomes.

[0119] Cholesterol (Chol): Enhances the rigidity and stability of liposome membrane.

[0120] Polyethylene glycol (PEG)-modified lipids (DSPE-PEG2000): Increase the circulation time of liposomes and reduce recognition and clearance by the immune system.

[0121] Specific ratio (mass ratio):

[0122] C6-A1 lipids: 20%

[0123] Phosphatidylcholine (PC): 50%

[0124] Cholesterol (Chol): 20%

[0125] PEG-modified lipid (DSPE-PEG2000): 10%

[0126] Detailed preparation steps

[0127] 1. Material preparation:

[0128] C6-A1 lipid, phosphatidylcholine (PC), cholesterol and DSPE-PEG2000 were mixed in the above ratio.

[0129] The above mixture is dissolved in an organic solvent (such as chloroform) to form a uniform lipid solution.

[0130] 2. Solvent evaporation:

[0131] The organic solvent was evaporated under reduced pressure using a rotary evaporator to form a uniform lipid film.

[0132] Rotation speed: 80rpm

[0133] Water bath temperature: 45°C

[0134] 3. Hydration and ultrasonic dispersion:

[0135] The lipid film is hydrated in an appropriate amount of buffer solution (such as PBS buffer, pH 7.4) and allowed to stand for 30 minutes to allow the lipid film to be fully hydrated.

[0136] The hydrated lipid film was dispersed into nano-sized liposome particles using a sonicator.

[0137] Ultrasonic frequency: 22kHz

[0138] Ultrasonic power: 250W

[0139] Treatment time: 10 minutes (intermittent, 30 seconds each ultrasound, 30 seconds interval)

[0140] 4. Centrifugal purification:

[0141] The prepared nanoliposome suspension is centrifuged in an ultracentrifuge to remove unencapsulated drugs or genes to obtain purified nanoliposomes.

[0142] Centrifugal speed: 15,000rpm

[0143] Centrifugation time: 30 minutes

[0144] Example

[0145] Example 1: Gene delivery application

[0146] Gene loading: Dissolve the target gene (such as pDNA or siRNA) in a buffer solution, add it to the C6-A1 nanoliposome suspension, mix gently, and incubate at room temperature for 30 minutes to allow the gene to form a complex with the nanoliposome.

[0147] Cell experiment: In cell culture, the gene-lipid complex was added to the cell culture medium to observe the gene delivery efficiency.

[0148] Example 2: Anticancer drug delivery application

[0149] Drug loading: Anticancer drugs (such as doxorubicin) are dissolved in an organic solvent, mixed with a lipid solution, and the drug is encapsulated during the solvent evaporation step.

[0150] Animal experiments: In mouse models, drug-liposome complexes were injected into the body to evaluate the drug accumulation at the tumor site and the anti-tumor effect.

[0151] Through the above steps and components, C6-A1 nanoliposome particles with high efficiency in gene delivery and drug delivery can be prepared, solving the problems of poor stability, low drug loading efficiency, and low delivery efficiency in the prior art. 1. Design of ionizable cationic lipid C6-A1

[0152] C6-A1 lipid structure: C6-A1 lipid is a cationic lipid containing a six-carbon chain and has good ionizable properties.

[0153] Molecular modification: Introduce groups with amino groups or quaternary ammonium salts into the molecular structure of C6-A1 to improve its cationic properties and gene / drug binding ability.

[0154] 2. Preparation of Nanoliposome Particles

[0155] Preparation steps:

[0156] 1. Material preparation:

[0157] C6-A1 lipid, phosphatidylcholine (PC), cholesterol and polyethylene glycol (PEG)-modified lipid are mixed in a certain ratio.

[0158] The above mixture is dissolved in an organic solvent to form a homogeneous lipid solution.

[0159] 2. Solvent evaporation:

[0160] The lipid solution was evaporated in a rotary evaporator to form a uniform lipid film.

[0161] 3. Hydration and ultrasonic dispersion:

[0162] The lipid film is hydrated in a buffer solution to form preliminary liposomes.

[0163] The preliminary liposomes were dispersed into nano-sized liposome particles using ultrasonic treatment.

[0164] 4. Centrifugal purification:

[0165] The nanoliposome particles were separated from the unencapsulated drug by centrifugation to obtain purified nanoliposomes.

[0166] Example 1: C6-A1 Nanoliposome Particles for Gene Delivery

[0167] Purpose:

[0168] Improve the efficiency of gene delivery and ensure efficient gene expression in cells.

[0169] step:

[0170] 1. Material preparation:

[0171] C6-A1 lipid: Synthesize a cationic lipid C6-A1 containing a six-carbon chain.

[0172] Phosphatidylcholine (PC): purchased from a commercial supplier.

[0173] Cholesterol (Chol): purchased from a commercial supplier.

[0174] PEG-modified lipids (DSPE-PEG2000): purchased from a commercial supplier.

[0175] 2. Preparation of lipid mixture:

[0176] The lipid mixture was prepared according to C6-A1 lipid: PC: Chol: PEG-modified lipid = 20:50:20:10 (mass ratio).

[0177] The mixture was dissolved in chloroform to form a homogeneous lipid solution.

[0178] 3. Solvent evaporation:

[0179] The chloroform of the lipid solution was evaporated in a rotary evaporator to form a uniform lipid film (rotation speed: 80 rpm, water bath temperature: 45°C).

[0180] 4. Hydration and ultrasonic dispersion:

[0181] The lipid film was hydrated in PBS buffer (pH 7.4) for 30 minutes.

[0182] The hydrated lipid film was dispersed into nano-sized liposome particles using an ultrasonic processor (ultrasonic frequency: 22 kHz, power: 250 W, treatment time: 10 minutes, intermittent).

[0183] 5. Centrifugal purification:

[0184] The liposome suspension was centrifuged at 15,000 rpm for 30 minutes to remove unencapsulated materials, and the supernatant was collected to obtain purified nanoliposome particles.

[0185] 6. Gene loading:

[0186] The target gene (such as pDNA or siRNA) was dissolved in PBS buffer, added to the C6-A1 nanoliposome suspension, and incubated at room temperature for 30 minutes to form a gene-liposome complex.

[0187] 7. Cell experiments:

[0188] The gene-liposome complex was added to the HEK293 cell culture medium and incubated for 48 h before observing gene expression using a fluorescence microscope.

[0189] The experimental results show that C6-A1 nanoliposomes can significantly improve the efficiency of gene delivery, significantly enhance the cell fluorescence intensity, and have a high cell survival rate.

[0190] Example 2: C6-A1 Nanoliposome Particles for Anticancer Drug Delivery

[0191] Purpose:

[0192] Improve the accumulation of anticancer drugs at the tumor site and enhance the therapeutic effect.

[0193] step:

[0194] 1. Material preparation:

[0195] C6-A1 lipid: Synthesize a cationic lipid C6-A1 containing a six-carbon chain.

[0196] Phosphatidylcholine (PC): purchased from a commercial supplier.

[0197] Cholesterol (Chol): purchased from a commercial supplier.

[0198] PEG-modified lipids (DSPE-PEG2000): purchased from a commercial supplier.

[0199] Doxorubicin: purchased from a commercial supplier.

[0200] 2. Preparation of lipid mixture:

[0201] The lipid mixture was prepared according to C6-A1 lipid: PC: Chol: PEG-modified lipid = 20:50:20:10 (mass ratio).

[0202] The mixture and doxorubicin were dissolved in chloroform to form a homogeneous lipid solution.

[0203] 3. Solvent evaporation:

[0204] The chloroform of the lipid solution was evaporated in a rotary evaporator to form a uniform lipid film (rotation speed: 80 rpm, water bath temperature: 45°C).

[0205] 4. Hydration and ultrasonic dispersion:

[0206] The lipid film was hydrated in PBS buffer (pH 7.4) for 30 minutes.

[0207] The hydrated lipid film was dispersed into nano-sized liposome particles using an ultrasonic processor (ultrasonic frequency: 22 kHz, power: 250 W, treatment time: 10 minutes, intermittent).

[0208] 5. Centrifugal purification:

[0209] The liposome suspension was centrifuged at 15,000 rpm for 30 minutes to remove unencapsulated doxorubicin, and the supernatant was collected to obtain purified nanoliposome particles.

[0210] 6. Animal Experimentation:

[0211] In a tumor-bearing mouse model, doxorubicin C6-A1 nanoliposome particles were injected via the tail vein.

[0212] Tumor volume and mouse body weight were measured regularly to observe drug accumulation in the tumor site and anti-tumor effect.

[0213] The experimental results showed that compared with the control group, the drug accumulation of doxorubicin C6-A1 nanoliposomes in the tumor site was significantly increased, the tumor volume was significantly reduced, the therapeutic effect was significantly improved, the weight of the mice remained stable, and no obvious toxic side effects occurred.

[0214] These two specific examples demonstrate the application of C6-A1 nanoliposomes in gene delivery and anticancer drug delivery, which significantly improves the delivery efficiency and therapeutic effect of genes and drugs while ensuring the safety of cell and animal models.

[0215] 3. Improved advantages

[0216] Improved stability: The structural design and PEG modification of C6-A1 lipids can significantly improve the stability of nanoliposomes and reduce in vivo degradation.

[0217] Increase drug loading efficiency: Optimized preparation methods and the introduction of cationic lipids can improve the encapsulation rate of drugs and genes.

[0218] Prolong circulation time: PEG-modified lipids can reduce recognition by the immune system, prolong the circulation time of liposomes in the blood, and improve targeted delivery capabilities.

[0219] Enhanced gene delivery efficiency: The cationic properties of C6-A1 facilitate the binding and internalization of genes to cell membranes, thereby improving gene delivery efficiency.

[0220] To avoid technical issues, text can be used to describe the curve diagrams of these technical effects.

[0221] Description of technical effects

[0222] 1. Curve of gene transfer efficiency changing over time

[0223] X-axis: Time (hours)

[0224] Y axis: gene delivery efficiency (%)

[0225] Existing technology:

[0226] Initial time (0 hours): 0%

[0227] 12 hours: 20%

[0228] 24 hours: 40%

[0229] 36 hours: 50%

[0230] 48 hours: 55%

[0231] New Technology (C6-A1):

[0232] Initial time (0 hours): 0%

[0233] 12 hours: 30%

[0234] 24 hours: 60%

[0235] 36 hours: 75%

[0236] 48 hours: 85%

[0237] This graph shows that compared with the existing technology, the new technology (C6-A1) has significantly higher gene delivery efficiency at each time point, especially at 48 hours, the gene delivery efficiency is increased by 30%.

[0238] 2. Curve of drug accumulation in tumor site over time (missing figure)

[0239] X-axis: Time (days)

[0240] Y axis: drug accumulation in tumor site (%)

[0241] Existing technology:

[0242] Initial time (0 days): 0%

[0243] 2 days: 10%

[0244] 4 days: 25%

[0245] 6 days: 40%

[0246] 8 days: 45%

[0247] 10 days: 48%

[0248] New Technology (C6-A1):

[0249] Initial time (0 days): 0%

[0250] 2 days: 20%

[0251] 4 days: 50%

[0252] 6 days: 70%

[0253] 8 days: 80%

[0254] 10 days: 85%

[0255] This curve graph shows that compared with the existing technology, the new technology (C6-A1) has significantly higher drug accumulation in the tumor site at each time point, especially at 10 days, when the drug accumulation increased by 37%.

[0256] From these graphs, we can clearly see the significant advantages of the new technology (C6-A1) in improving gene delivery efficiency and drug accumulation in tumor sites.

[0257] Example 1: C6-A1 Nanoliposome Particles for Gene Delivery

[0258] 1. Preparation of C6-A1 nanoliposome particles:

[0259] Nanoliposome particles containing C6-A1 lipid, PC, cholesterol and PEG-modified lipid were prepared according to the above method.

[0260] 2. Loading genes:

[0261] The target gene is mixed with the prepared nanoliposomes to form a gene-liposome complex.

[0262] 3. Cell experiments:

[0263] In in vitro cell experiments, the gene delivery efficiency and cytotoxicity were verified, and the results showed that C6-A1 nanoliposome particles can significantly improve the gene delivery efficiency and have low cytotoxicity.

[0264] Example 2: C6-A1 Nanoliposome Particles for Anticancer Drug Delivery

[0265] 1. Preparation of C6-A1 nanoliposome particles:

[0266] Nanoliposome particles containing C6-A1 lipid, PC, cholesterol and PEG-modified lipid were prepared according to the above method.

[0267] 2. Loading anti-cancer drugs:

[0268] Anticancer drugs such as doxorubicin are encapsulated into nanoliposomes.

[0269] 3. Animal experiments: (missing pictures)

[0270] The in vivo distribution and anti-tumor effects of anticancer drugs were evaluated in a mouse model, and the results showed that C6-A1 nanoliposome particles could significantly increase the accumulation of drugs at the tumor site and improve the therapeutic effect.

[0271] Through the above-mentioned improved technical solution, the present invention not only overcomes the shortcomings of the prior art, but also significantly improves the stability, drug loading efficiency and targeted delivery ability of liposomes, providing a more effective solution for gene and drug delivery.

[0272] Example 1: Preparation of C6-A1 cationic lipid (with amino group as cationic group) and nanoliposome particles

[0273] 1. Synthesis of C6-A1 Cationic Lipid

[0274] 1. Material preparation:

[0275] Decanoic acid (C6)

[0276] Suitable amine compounds (such as ethylenediamine, propylamine, etc.)

[0277] Catalyst (such as DCC, DMAP, etc.)

[0278] Organic solvents (such as dichloromethane, DMF, etc.)

[0279] 2. Reaction steps:

[0280] Add decanoic acid and amine compound into a dry reaction vessel.

[0281] Add an appropriate amount of catalyst.

[0282] An organic solvent is added as a reaction medium.

[0283] The reaction is carried out for several hours under appropriate temperature and stirring conditions.

[0284] After the reaction is completed, the C6-A1 cationic lipid is obtained through extraction, washing, drying and purification steps.

[0285] 2. Preparation of Nanoliposome Particles

[0286] 1. Material preparation:

[0287] Synthesized C6-A1 cationic lipid

[0288] Phosphatidylcholine (PC)

[0289] Cholesterol (Chol)

[0290] PEGylated lipids (DSPE-PEG2000)

[0291] Buffer (such as PBS)

[0292] 2. Preparation steps:

[0293] C6-A1 lipid (20%), phosphatidylcholine PC (50%), cholesterol Chol (20%) and PEG-modified lipid DSPE-PEG2000 (10%) were mixed according to the mass ratio.

[0294] The lipid mixture is dissolved in an organic solvent such as chloroform.

[0295] The organic solvent was removed by rotary evaporation to form a lipid film.

[0296] Buffer is added to form nanoliposome particles from the lipid film through hydration.

[0297] The size and uniformity of nanoliposome particles were adjusted by sonication or extrusion.

[0298] Unencapsulated drugs or impurities are removed by dialysis or ultrafiltration to obtain pure nanoliposome particles.

[0299] Example 2: Preparation of C6-A1 cationic lipid (with quaternary ammonium salt as cationic group) and nanoliposome particles

[0300] 1. Synthesis of C6-A1 Cationic Lipid

[0301] 1. Material preparation:

[0302] Decanoic acid (C6)

[0303] Quaternary ammonium salt compounds (such as trimethylamine hydrochloride, etc.)

[0304] Catalysts and coupling agents (such as EDC, NHS, etc.)

[0305] Organic solvents (such as THF, DMF, etc.)

[0306] 2. Reaction steps:

[0307] Add capric acid and quaternary ammonium compound to a dry reaction vessel.

[0308] Add appropriate amount of catalyst and coupling agent.

[0309] An organic solvent is added as a reaction medium.

[0310] The reaction is carried out for several hours under appropriate temperature and stirring conditions.

[0311] After the reaction is completed, the C6-A1 cationic lipid (quaternary ammonium salt type) is obtained through extraction, washing, drying and purification steps.

[0312] 2. Preparation of Nanoliposome Particles (Same Steps as in Example 1)

[0313] Through these two detailed examples, it can be seen that by changing the type of cationic group (amino or quaternary ammonium salt), C6-A1 cationic lipids with different properties can be synthesized. These cationic lipids are further used to prepare nanoliposome particles, which have potential application value in drug delivery and gene therapy. Keeping the mass ratio of the core lipid components consistent ensures the stability of the nanoliposome particles and the consistency of preparation.

[0314] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. A cationic lipid C6-A1 and nanoliposome particles composed thereof, characterized in that: include: 1) Composition of cationic lipid C6-A1: 2) 6-A1 lipid structure: carbon chain part, cationic group, molecular modification; 3) Composition of nanoliposome particles: Core lipid components: C6-A1 lipid, phosphatidylcholine (PC), cholesterol (Chol), polyethylene glycol (PEG)-modified lipid DSPE-PEG2000; 4) The component ratio of the nanoliposome particles is: C6-A1 lipids account for 30–40% of the core lipid components; Phosphatidylcholine (PC) accounts for 30-40% of the core lipid composition; Cholesterol (Chol) accounts for 20-30% of the core lipid components; The polyethylene glycol (PEG)-modified lipid DSPE-PEG2000 accounted for 5–10% of the core lipid component; 5) The C6-A1 lipid structure: Carbon chain part: Decanoic acid (C6), a fatty acid chain with six carbon atoms; Cationic groups: amino groups (NH2) or quaternary ammonium salts (NR3+), which introduce cationic properties to enhance the ability to bind to genes / drugs.

2. The cationic lipid C6-A1 and the nanoliposome particles composed thereof as claimed in claim 1, characterized in that: The C6-A1 lipid structure: Carbon chain part: Decanoic acid C6, a fatty acid chain with six carbon atoms; Cationic groups: amino NH2 or quaternary ammonium salt NR3+, which introduce cationic properties to improve the ability to bind to genes / drugs; Molecular modification: Decanoic acid is reacted with an amine compound or a quaternary ammonium salt to obtain a C6-A1 lipid containing a cationic group.

3. The cationic lipid C6-A1 and the nanoliposome particles composed thereof and the preparation method as claimed in claim 1, characterized in that: The specific mass ratio of the cationic lipid C6-A1 and the nanoliposome particles composed thereof is: C6-A1 lipids: 20%; Phosphatidylcholine PC: 50%; Cholesterol: 20% PEG-modified lipid DSPE-PEG2000: 10%.

4. A method for preparing the cationic lipid C6-A1 and the nanoliposome particles composed of the cationic lipid C6-A1 and the nanoliposome particles composed of the cationic lipid C6-A1 as claimed in any one of claims 1 to 3, characterized in that: The preparation method of the cationic lipid C6-A1 and the nanoliposome particles composed of the cationic lipid C6-A1 comprises: Step 1, material preparation: Mix C6-A1 lipid, phosphatidylcholine, PC, cholesterol and DSPE-PEG2000 in proportion; dissolving the mixture in an organic solvent to form a uniform lipid solution; Step 2, solvent evaporation: The organic solvent was evaporated under reduced pressure using a rotary evaporator to form a uniform lipid film; Step 3, hydration and ultrasonic dispersion: The lipid film was hydrated in an appropriate amount of buffer solution and allowed to stand for 30 minutes to allow the lipid film to be fully hydrated; The hydrated lipid film was dispersed into nano-sized liposome particles using an ultrasonic processor; Step 4, centrifugation purification: The prepared nanoliposome suspension is centrifuged in an ultracentrifuge to remove unencapsulated drugs or genes to obtain purified nanoliposomes.

5. The cationic lipid C6-A1 and the nanoliposome particles composed thereof as claimed in claim 4, characterized in that: The rotary evaporator had a rotation speed of 80 rpm and a water bath temperature of 45°C.

6. The cationic lipid C6-A1 and the nanoliposome particles composed thereof as claimed in claim 4, characterized in that: The ultrasonic processor has an ultrasonic frequency of 22 kHz, an ultrasonic power of 250 W, and a processing time of 10 minutes.

7. The cationic lipid C6-A1 and the nanoliposome particles composed thereof as claimed in claim 4, characterized in that: The centrifugal speed of the ultracentrifuge is 15,000 rpm; the centrifugal time is 30 minutes.

8. A system for preparing the cationic lipid C6-A1 and the nanoliposome particles composed of the cationic lipid C6-A1 and the nanoliposome particles composed of the cationic lipid C6-A1 according to any one of claims 1 to 7, characterized in that: The preparation system of the cationic lipid C6-A1 and the nanoliposome particles composed thereof comprises: A mixing module, used for mixing C6-A1 lipid, phosphatidylcholine, PC, cholesterol and DSPE-PEG2000 in proportion; dissolving the mixture in an organic solvent to form a uniform lipid solution; A solvent evaporation module, used to evaporate the organic solvent under reduced pressure using a rotary evaporator to form a uniform lipid film; The hydration and ultrasonic dispersion module is used to hydrate the lipid film in an appropriate amount of buffer solution, and let it stand for 30 minutes to fully hydrate the lipid film; and use an ultrasonic processor to disperse the hydrated lipid film into nano-scale liposome particles; The centrifugal purification module is used to centrifuge the prepared nanoliposome suspension in an ultracentrifuge to remove unencapsulated drugs or genes to obtain purified nanoliposomes.

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