Preparation method and application of TEMP-coated Lip nano material
By preparing TEMP@Lip nanomaterials with small particle size and uniform distribution, the problem of difficulty in effectively targeting and activating iNKT cells in the prior art is solved, and the immunotherapy effect in tumor treatment is improved, and the raw materials are cheap and stable. high.
Patent Information
- Application Number
- CN202411219710.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-02
- Publication Date
- 2024-12-10
AI Technical Summary
Existing nanoformulations are difficult to effectively target and activate iNKT cells in tumor therapy and immunotherapy, resulting in poor immunotherapy effects.
Using the preparation method of TEMP@Lip nanomaterials, nanocarriers with small particle size and uniform distribution are synthesized through specific solvents and reaction steps, equipped with iNKT cell activator to ensure its stability and biocompatibility in the organisms and achieve iNKT Targeted activation of cells.
It realizes effective activation of iNKT cells, enhances the immune response of organisms, improves the effect of tumor treatment, and has cheap raw materials and high stability in the preparation process, which is suitable for practical applications.
Smart Images
Figure CN119097601A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of biomedical materials, and in particular to a preparation method of a TEMP@Lip nanomaterial and application thereof. Background Art
[0002] In recent years, nano-preparations and nano-drugs have made significant progress in tumor treatment. Some nano-preparations with good biocompatibility can transport tumor drugs with high toxicity and poor biocompatibility into the body for efficient drug treatment. At the same time, in immunotherapy, nano-preparation materials can be used to carry agonists of immune cells. Through targeting, nano-carriers can release agonists to target immune cells, thereby stimulating the immune activity of immune cells and improving the body's own immunity.
[0003] The TEMP@Lip nanomaterial prepared by the present invention is a nanocarrier with small particle size and uniform distribution. It also has the characteristics of good biocompatibility and can carry the activator of iNKT cells and safely transport it into cells, thereby enhancing the immunity of living organisms and achieving the purpose of immunotherapy. Summary of the invention
[0004] The purpose of the present invention is to provide a preparation method of TEMP@Lip nanomaterial and its application to solve the problems raised by the above background technology.
[0005] To achieve the above-mentioned purpose, the present invention provides the following technical scheme: a method for preparing TEMP@Lip nanomaterials, wherein the preparation materials include anhydrous ethanol (CH3CH2OH), chloroform (CHCl3), anhydrous tetrahydrofuran (THF), dichloromethane (CH2Cl2), trimethylamine [N(CH3)3], N-hydroxysuccinimide (NHS), 3-mercaptopropionic acid, 1,3-dicyclohexylcarbodiimide (DCC), 4-amino-2,2,6,6-tetramethylpiperidine (TEMP-NH2), phosphate buffer (PBS), α-GalCer, (2,3-dioleyloxypropyl)trimethylammonium chloride (DOTAP), 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE-PEG2000-MAL), and dioleoylphosphatidylethanolamine (DOPE);
[0006] The steps include:
[0007] S1: Dissolve 12mM NHS in solvent A, then add 10mM 3-mercaptopropionic acid in solvent A, mix well, add 50ml 12mM DCC in solvent A, ice bath for a suitable time, stir at room temperature for 24h, remove solvent A by suction, and obtain a yellow oily product: N-hydroxysuccinyl 3-mercaptopropionic acid;
[0008] S2: Dissolve 0.156 g of TEMP-NH2 in 5 ml of solvent A, place in an ice bath for 60 min, then drop 0.25 g of N-hydroxysuccinyl 3-mercaptopropionic acid, 50 μL of N(CH3)3 and 25 ml of CH2Cl2, stir at room temperature for a certain period of time, wash with an appropriate amount of deionized water, collect the water layer and freeze-dry to obtain the product: 3-mercapto-N-(2,2,6,6-tetramethylpiperidin-4-yl)propionamide;
[0009] S3: Under nitrogen protection, 50 mg DOPE-PEG2000-MAL and 48.8 mg 3-mercapto-N-(2,2,6,6-tetramethylpiperidin-4-yl)propionamide were dispersed in PBS at pH = 6.8, reacted for 12 hours, dialyzed with deionized water, and then freeze-dried to obtain TEMP@Lip.
[0010] As a preferred technical solution of the present invention, the solvent A in S1 is one of THF, CH3CH2OH or DMSO.
[0011] As a preferred technical solution of the present invention, the ice bath time in S1 is 2-3 hours.
[0012] The present invention proposes the application of TEMP@Lip nanomaterials in carrying and transporting iNKT cell agonists.
[0013] The TEMP@Lip nanomaterial proposed in the present invention has good biocompatibility, adapts to the in vivo environment, and can safely transport the iNKT cell activator into the iNKT cells.
[0014] The TEMP@Lip nanomaterial proposed in the present invention has a small particle size and is evenly distributed, has a negative potential and good biological solubility, is relatively stable after entering the body, can safely transport iNKT cell agonists, activate iNKT to enhance the body's own immunity, and achieve the purpose of using the body's own immune system to achieve tumor treatment.
[0015] Compared with the prior art, the present invention has the following beneficial effects: the raw materials involved in the present invention are cheap and have good practicality. The α-GalCer@TEMP@Lip liposome nanomaterial prepared by the present invention has good stability, uniform size distribution, and can target iNKT cells, activate the immune system, and enhance the body's own immunity. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 This is a physical picture of the α-GalCer@TEMP@Lip liposome nanomaterial of the present invention;
[0017] Figure 2This is the UV-visible absorption spectrum of the α-GalCer@TEMP@Lip liposome nanomaterial of the present invention;
[0018] Figure 3 This is a particle size distribution diagram of the α-GalCer@TEMP@Lip liposome nanomaterial of the present invention;
[0019] Figure 4 This is the potential diagram of the α-GalCer@TEMP@Lip liposome nanomaterial of the present invention. DETAILED DESCRIPTION
[0020] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0021] The purpose of the present invention is achieved through the following technical solutions:
[0022] Example 1: A solvent selected is THF.
[0023] Step 1: Synthesis of TEMP@Lip liposome nanomaterials:
[0024] (1) Dissolve 12 mM NHS in THF, then add 10 mM 3-mercaptopropionic acid in THF, mix well, add 50 ml 12 mM DCC in THF, ice bath for an appropriate time, and stir at room temperature for 24 hours. Remove the solvent by suction filtration to obtain a yellow oily product: N-hydroxysuccinyl 3-mercaptopropionic acid.
[0025] (2) Dissolve 0.156 g of TEMP-NH2 in 5 ml of THF and place in an ice bath for 60 min. Then, add dropwise 0.25 g of N-hydroxysuccinyl 3-mercaptopropionic acid, 50 μL of N(CH3)3 and 25 ml of CH2Cl2. Stir at room temperature for a certain period of time, wash with appropriate deionized water, collect the aqueous layer and lyophilize to obtain the product: 3-mercapto-N-(2,2,6,6-tetramethylpiperidin-4-yl)propionamide.
[0026] (3) Under nitrogen protection, 50 mg of DOPE-PEG2000-MAL and 48.8 mg of 3-mercapto-N-(2,2,6,6-tetramethylpiperidin-4-yl)propionamide were dispersed in PBS at pH = 6.8 and reacted overnight. The mixture was then dialyzed with deionized water and freeze-dried to obtain TEMP@Lip.
[0027] Step 2: Synthesis of iNKT cell-targeted liposome α-GalCer@TEMP@Lip: DOPE, TEMP@Lip, and DOTAP were mixed in chloroform in a certain ratio, 20 μg of α-GalCer was added, and after mixing, a lipid film was obtained by rotary evaporation. The obtained lipid film was dispersed in PBS at pH = 7.4, sonicated at 4°C, and then extruded through a liposome extruder to obtain liposome α-GalCer@TEMP@Lip.
[0028] Example 2: A solvent is selected as CH3CH2OH.
[0029] Step 1: Synthesis of TEMP@Lip liposome nanomaterials:
[0030] (1) Dissolve 12 mM NHS in CH3CH2OH, then add 10 mM 3-mercaptopropionic acid in CH3CH2OH. After mixing well, add 50 ml 12 mM DCC in CH3CH2OH. Place in an ice bath for an appropriate period of time and stir at room temperature for 24 h. Remove the solvent by suction filtration to obtain a yellow oily product: N-hydroxysuccinyl 3-mercaptopropionic acid.
[0031] (2) Dissolve 0.156 g of TEMP-NH2 in 5 ml of CH3CH2OH, place in an ice bath for 60 min, then drop 0.25 g of N-hydroxysuccinyl 3-mercaptopropionic acid, 50 μL of N(CH3)3 and 25 ml of CH2Cl2, stir at room temperature for a certain period of time, wash with appropriate deionized water, collect the aqueous layer and lyophilize to obtain the product: 3-mercapto-N-(2,2,6,6-tetramethylpiperidin-4-yl)propionamide.
[0032] (3) Under nitrogen protection, 50 mg of DOPE-PEG2000-MAL and 48.8 mg of 3-mercapto-N-(2,2,6,6-tetramethylpiperidin-4-yl)propionamide were dispersed in PBS at pH = 6.8 and reacted overnight. The mixture was then dialyzed with deionized water and freeze-dried to obtain TEMP@Lip.
[0033] Step 2: Synthesis of iNKT cell-targeted liposome α-GalCer@TEMP@Lip: DOPE, TEMP@Lip, and DOTAP were mixed in chloroform in a certain ratio, 20 μg of α-GalCer was added, and after mixing, a lipid film was obtained by rotary evaporation. The obtained lipid film was dispersed in PBS at pH = 7.4, sonicated at 4°C, and then extruded through a liposome extruder to obtain liposome α-GalCer@TEMP@Lip.
[0034] Example 3: A solvent selection DMSO.
[0035] Step 1: Synthesis of TEMP@Lip liposome nanomaterials:
[0036] (1) Dissolve 12 mM NHS in DMSO, then add 10 mM 3-mercaptopropionic acid in DMSO, mix well, add 50 ml 12 mM DCC in DMSO, place in an ice bath for an appropriate time, and stir at room temperature for 24 hours. Remove the solvent by suction and obtain a yellow oily product: N-hydroxysuccinyl 3-mercaptopropionic acid.
[0037] (2) Dissolve 0.156 g of TEMP-NH2 in 5 ml of DMSO and place in an ice bath for 60 min. Then, add dropwise 0.25 g of N-hydroxysuccinyl 3-mercaptopropionic acid, 50 μL of N(CH3)3 and 25 ml of DMSO. Stir at room temperature for a certain period of time, wash with appropriate deionized water, collect the aqueous layer and lyophilize to obtain the product: 3-mercapto-N-(2,2,6,6-tetramethylpiperidin-4-yl)propionamide.
[0038] (3) Under nitrogen protection, 50 mg of DOPE-PEG2000-MAL and 48.8 mg of 3-mercapto-N-(2,2,6,6-tetramethylpiperidin-4-yl)propionamide were dispersed in PBS at pH = 6.8 and reacted overnight. The mixture was then dialyzed with deionized water and freeze-dried to obtain TEMP@Lip.
[0039] Step 2: Synthesis of iNKT cell-targeted liposome α-GalCer@TEMP@Lip: DOPE, TEMP@Lip, and DOTAP were mixed in DMSO in a certain ratio, and 20 μg of α-GalCer was added. After mixing, a lipid film was obtained by rotary evaporation. The obtained lipid film was dispersed in PBS at pH = 7.4, sonicated at 4°C, and then extruded through a liposome extruder to obtain liposome α-GalCer@TEMP@Lip.
[0040] Example 4:
[0041] (1) Dissolve 12 mM NHS, 10 mM 3-mercaptopropionic acid, and 12 mM DCC in DMSO, mix well, place in an ice bath for an appropriate period of time, and stir at room temperature for 24 h. Remove the solvent by suction filtration to obtain a yellow oily product: N-hydroxysuccinyl 3-mercaptopropionic acid.
[0042] (2) Dissolve 0.156 g of TEMP-NH2 in 5 ml of DMSO and place in an ice bath for 60 min. Then, add dropwise 0.25 g of N-hydroxysuccinyl 3-mercaptopropionic acid, 50 μL of N(CH3)3 and 25 ml of DMSO. Stir at room temperature for a certain period of time, wash with appropriate deionized water, collect the aqueous layer and lyophilize to obtain the product: 3-mercapto-N-(2,2,6,6-tetramethylpiperidin-4-yl)propionamide.
[0043] (3) Under nitrogen protection, 50 mg of DOPE-PEG2000-MAL and 48.8 mg of 3-mercapto-N-(2,2,6,6-tetramethylpiperidin-4-yl)propionamide were dispersed in PBS at pH = 6.8 and reacted overnight. The mixture was then dialyzed with deionized water and freeze-dried to obtain TEMP@Lip.
[0044] Step 2: Synthesis of iNKT cell-targeted liposome α-GalCer@TEMP@Lip: DOPE, TEMP@Lip, and DOTAP were mixed in DMSO in a certain ratio, and 20 μg of α-GalCer was added. After mixing, a lipid film was obtained by rotary evaporation. The obtained lipid film was dispersed in PBS at pH = 7.4, sonicated at 4°C, and then extruded through a liposome extruder to obtain liposome α-GalCer@TEMP@Lip.
[0045] Example 5:
[0046] (1) Dissolve 12 mM NHS, 10 mM 3-mercaptopropionic acid, and 12 mM DCC in CHCl3, mix well, place in an ice bath for an appropriate period of time, and stir at room temperature for 24 h. Remove the solvent by suction filtration to obtain a yellow oily product: N-hydroxysuccinyl 3-mercaptopropionic acid.
[0047] (2) Dissolve 0.156 g of TEMP-NH2 in 5 ml of CHCl3, place in an ice bath for 60 min, then drop 0.25 g of N-hydroxysuccinyl 3-mercaptopropionic acid, 50 μL of N(CH3)3 and 25 ml of CHCl3, stir at room temperature for a certain period of time, wash with appropriate deionized water, collect the aqueous layer and lyophilize to obtain the product: 3-mercapto-N-(2,2,6,6-tetramethylpiperidin-4-yl)propionamide.
[0048] (3) Under nitrogen protection, 50 mg of DOPE-PEG2000-MAL and 48.8 mg of 3-mercapto-N-(2,2,6,6-tetramethylpiperidin-4-yl)propionamide were dispersed in PBS at pH = 6.8 and reacted overnight. The mixture was then dialyzed with deionized water and freeze-dried to obtain TEMP@Lip.
[0049] Step 2: Synthesis of iNKT cell-targeted liposome α-GalCer@TEMP@Lip: DOPE, TEMP@Lip, and DOTAP were mixed in CHCl3 in a certain ratio, and 20 μg of α-GalCer was added. After mixing, the lipid film was obtained by rotary evaporation. The obtained lipid film was dispersed in PBS at pH = 7.4, sonicated at 4°C, and then extruded through a liposome extruder to obtain liposome α-GalCer@TEMP@Lip.
[0050] Example 6:
[0051] (1) Dissolve 12 mM NHS, 10 mM 3-mercaptopropionic acid, and 12 mM DCC in DMSO, mix well, place in an ice bath for an appropriate period of time, and stir at room temperature for 24 h. Remove the solvent by suction filtration to obtain a yellow oily product: N-hydroxysuccinyl 3-mercaptopropionic acid.
[0052] (2) Dissolve 0.156 g of TEMP-NH2 in 5 ml of CHCl3, place in an ice bath for 60 min, then drop 0.25 g of N-hydroxysuccinyl 3-mercaptopropionic acid, 50 μL of N(CH3)3 and 25 ml of CHCl3, stir at room temperature for a certain period of time, wash with appropriate deionized water, collect the aqueous layer and lyophilize to obtain the product: 3-mercapto-N-(2,2,6,6-tetramethylpiperidin-4-yl)propionamide.
[0053] (3) Under nitrogen protection, 50 mg of DOPE-PEG2000-MAL and 48.8 mg of 3-mercapto-N-(2,2,6,6-tetramethylpiperidin-4-yl)propionamide were dispersed in PBS at pH = 6.8 and reacted overnight. The mixture was then dialyzed with deionized water and freeze-dried to obtain TEMP@Lip.
[0054] Step 2: Synthesis of iNKT cell-targeted liposome α-GalCer@TEMP@Lip: DOPE, TEMP@Lip, and DOTAP were mixed in CHCl3 in a certain ratio, and 20 μg of α-GalCer was added. After mixing, the lipid film was obtained by rotary evaporation. The obtained lipid film was dispersed in PBS at pH = 7.4, sonicated at 4°C, and then extruded through a liposome extruder to obtain liposome α-GalCer@TEMP@Lip.
[0055] The contents not described in detail in this specification belong to the prior art known to professional and technical personnel in the field. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.
Claims
1. The method for preparing TEMP@Lip nanomaterial according to claim 1, characterized in that: The solvent A in S1 is one of THF, CH3CH2OH or DMSO.
2. The method for preparing TEMP@Lip nanomaterial according to claim 1, characterized in that: The ice bath time in S1 is 2-3 h.
3. Application of the TEMP@Lip nanomaterial prepared by the preparation method of the TEMP@Lip nanomaterial according to claims 1-3 in carrying and transporting iNKT cell agonists.
4. The TEMP@Lip nanomaterial prepared by the preparation method of the TEMP@Lip nanomaterial according to claims 1-3 has good biocompatibility, adapts to the in vivo environment, and can safely transport the iNKT cell activator into the iNKT cells.
5. The TEMP@Lip nanomaterial prepared by the preparation method of the TEMP@Lip nanomaterial according to claims 1-3 has a small particle size and is evenly distributed, has a negative potential and good biological solubility, is relatively stable after entering the body, can safely transport iNKT cell agonists, activate iNKT to enhance the body's own immunity, and achieve the purpose of using the body's own immune system to achieve tumor treatment.