Tumor vaccine of targeted delivery system and preparation process of tumor vaccine
By self-assemblying nanocarriers combined with a targeted delivery system with a variety of targeted molecules and immune enhancers, the problems of low delivery efficiency and high production cost in the prior art are solved, and efficient delivery of tumor vaccines and enhanced immune responses are achieved, and the prospect of industrial application is good.
Patent Information
- Application Number
- CN202510508610.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-22
- Publication Date
- 2025-08-01
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
The existing targeted delivery system has low efficiency in tumor vaccine delivery, is susceptible to tumor microenvironment interference, has complex production processes and is expensive, making it difficult to apply on a large scale.
Self-assembled nanocarriers are used to combine a variety of targeted molecules, immune enhancers and functional molecules to prepare targeted delivery systems through ultrasonic assisted solvent volatilization method and microfluidic liquid-liquid phase distribution method. Low-cost materials and optimized preparation processes are used to achieve precise targeted delivery and immune enhancement.
It significantly improves the delivery efficiency and immune response intensity of tumor vaccines, simplifies production processes, reduces costs, and has good industrial prospects.
Smart Images
Figure CN120392987A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedical technologies, and particularly to tumor vaccines of a targeted delivery system and their preparation processes. Background Art
[0002] In the field of tumor immunotherapy, tumor vaccines of a targeted delivery system have great potential. However, there are many problems in the prior art. The low delivery efficiency makes it difficult for tumor vaccines to effectively reach the tumor site and fully stimulate the immune response. This is because the targeting of traditional delivery systems is limited, vulnerable to interference from the tumor microenvironment, and has poor uptake efficiency for tumor cells. For example, some liposome-based delivery systems, although having certain targeting properties, are easily cleared in the blood circulation, resulting in insufficient vaccine dosage reaching the tumor tissue.
[0003] In terms of production processes, existing methods are complex and costly. Multiple-step synthesis, modification, and dependence on expensive materials increase the production difficulty and cost. For example, the preparation of certain nanoparticles requires complex chemical synthesis steps and the use of rare metal catalysts, which not only makes the operation cumbersome but also significantly increases the production cost, limiting the large-scale application of tumor vaccines;
[0004] Therefore, there is a need for a tumor vaccine of a targeted delivery system and its preparation process to significantly improve the delivery efficiency, simplify the production process, reduce the production cost, and overcome the defects of the prior art. Summary of the Invention
[0005] Aiming at the deficiencies of the prior art, the present invention provides a tumor vaccine of a targeted delivery system and its preparation process, solving the problems raised in the above background art.
[0006] Technical Solution: To solve the above technical problems, according to one aspect of the present invention, more specifically, a tumor vaccine of a targeted delivery system includes a self-assembled nanocarrier, multiple targeting molecules, an immune enhancer, and a functional molecule. The self-assembled nanocarrier is formed by self-assembly of a biodegradable polymer and an amphiphilic block copolymer, and the self-assembled nanocarrier can spontaneously assemble into a nanostructure according to environmental conditions in an aqueous solution;
[0007] The multiple targeting molecules are bound to the self-assembled nanocarrier, and the targeting molecules include at least one of a tumor-specific antibody, a tumor vascular endothelial growth factor receptor ligand, and an aptamer of an overexpressed protein on the tumor cell surface;
[0008] The immune enhancer is integrated into the self-assembled nanocarrier, and the immune enhancer includes at least one of a Toll-like receptor agonist and a cytokine;
[0009] The functional molecules are pH-responsive, temperature-responsive, and enzyme-responsive functional molecules, which are introduced into the self-assembled nanocarriers for controlling the loading and release of vaccines.
[0010] Furthermore, the raw materials of the self-assembled nanocarriers are poly(lactic-co-glycolic acid) copolymer and amphiphilic block copolymer, and the ratio of the hydrophilic segment to the hydrophobic segment of the amphiphilic block copolymer is 3:2.
[0011] Furthermore, the multiple targeting molecules are introduced with active groups capable of binding to the nanocarriers through chemical modification, and the active groups are at least one of maleimide group, amino group, and carboxyl group to achieve binding to the self-assembled nanocarriers.
[0012] Furthermore, the immunopotentiator is integrated into the self-assembled nanocarriers by physical encapsulation or chemical conjugation, and the chemical conjugation is connected by a degradable chemical bond.
[0013] Furthermore, the pH-responsive, temperature-responsive, and enzyme-responsive functional molecules are introduced into the self-assembled nanocarriers by copolymerization, grafting, or coupling. The pH-responsive functional molecule is a polymer containing pH-sensitive chemical bonds, the temperature-responsive functional molecule is a polymer with lower critical solution temperature characteristics, and the enzyme-responsive functional molecule is a compound containing chemical bonds that can be cleaved by tumor-related enzymes.
[0014] According to another aspect of the present invention, more specifically, the preparation process of the tumor vaccine of the targeted delivery system includes the tumor vaccine of the targeted delivery system, and the process steps are as follows:
[0015] S1. Preparation of self-assembled nanocarriers:
[0016] Prepare raw materials: Weigh an appropriate amount of poly(lactic-co-glycolic acid) copolymer and amphiphilic block copolymer, dissolve them in dichloromethane, and prepare a solution with a concentration of 50 mg / mL.
[0017] Ultrasonic treatment: Place the above solution in an ultrasonic instrument and perform ultrasonic treatment for 15 minutes under the conditions of a power of 200 W and a frequency of 40 kHz to promote molecular dispersion.
[0018] Solvent evaporation: Transfer the ultrasonicated solution to a rotary evaporator and perform rotary evaporation at a temperature of 40 °C and a vacuum of 30 mbar to volatilize dichloromethane, and self-assembly of nanoparticles is formed.
[0019] Purification: Dialyze the self-assembled product through a dialysis bag (cut-off molecular weight of 3000 Da) in deionized water for 24 hours to remove unreacted raw materials and impurities, and obtain purified self-assembled nanocarriers;
[0020] S2. Binding of Multifunctional Targeting Molecules
[0021] Targeting molecule modification: Chemically modify targeting molecules such as tumor-specific antibodies and aptamers, and introduce active groups that can bind to the nanocarrier.
[0022] Binding reaction: Mix the modified targeting molecule and the self-assembled nanocarrier at a molar ratio of 5:1 in phosphate buffer solution (pH = 7.2), and incubate the reaction for 6 hours at 37 °C.
[0023] Separation and purification: Separate and purify the binding product by gel filtration chromatography to remove the unbound targeting molecules, and obtain the self-assembled nanocarrier bound with multifunctional targeting molecules.
[0024] S3. Implementation of High-Efficiency Production Process
[0025] Microfluidic chip preparation: Clean and assemble the microfluidic chip to ensure unobstructed channels.
[0026] Solution preparation: Prepare an organic phase solution (such as dissolved in dichloromethane) and an aqueous phase solution (such as phosphate buffer solution containing surfactant) containing nanoparticle precursors, vaccines, and immune enhancers respectively.
[0027] Microfluidic mixing and sonication: Inject the organic phase and the aqueous phase into the mixing area of the microfluidic chip through different inlets at a flow rate ratio of 1:3, and apply sonication outside the chip, with a sonication power of 150 W and a frequency of 35 kHz.
[0028] Collect the product: Collect the formed nanoparticle solution at the outlet of the microfluidic chip, and perform post-treatment by methods such as centrifugation and freeze-drying to obtain nanoparticles with high purity and good uniformity loaded with vaccines and immune enhancers;
[0029] S4. Verify Vaccine Loading and Release Control:
[0030] Load the vaccine onto the self-assembled nanocarrier by physical adsorption method. Mix the nanocarrier loaded with the vaccine with the vaccine solution, and incubate with shaking at 37 °C and 150 rpm for 4 hours to allow the vaccine to adsorb on the surface of the nanocarrier;
[0031] In buffer solutions with a simulated tumor microenvironment pH of 6.8, 7.0, a temperature of 37 °C, 40 °C, and containing a specific concentration of matrix metalloproteinase, regularly detect the concentration of the vaccine in the solution by high performance liquid chromatography, plot the vaccine release curve, and verify the responsive release performance of the vaccine;
[0032] S5. Verify the Integration and Synergistic Effect of Immune Enhancers:
[0033] Verification of the Integrated and Synergistic Effects of Immunopotentiators
[0034] Integration of immunopotentiators: Dissolve Toll-like receptor agonists in an organic solvent, mix with a solution containing nanoparticle precursors, and integrate the immunopotentiators into self-assembled nanocarriers through co-assembly.
[0035] Cell experiment: Co-culture mouse macrophages with nanocarriers loaded with vaccines and immunopotentiators, and use enzyme-linked immunosorbent assay to detect the secretion levels of cytokines (such as tumor necrosis factor-α, interleukin-6) in the cell culture supernatant to evaluate the activation effect of immunopotentiators on immune cells.
[0036] Animal experiment: Select a tumor-bearing mouse model and divide it into an experimental group (treated with the tumor vaccine of the present invention), a control group 1 (treated with a tumor vaccine without added immunopotentiator), and a control group 2 (treated with normal saline). Regularly measure the tumor volume of the mice, draw a tumor growth curve, compare the tumor growth inhibition rates and survival rates of the mice in each group, and verify the synergistic treatment effect of the immunopotentiator and the tumor vaccine.
[0037] Furthermore, in the step of preparing the self-assembled nanocarrier, the organic solvent is dichloromethane, the ultrasonic power is 200 W, the frequency is 40 kHz, the rotary evaporation temperature is 40 °C, the vacuum degree is 30 mbar, and the molecular weight cut-off of the dialysis bag is 3000 Da.
[0038] Furthermore, in the step of binding multiple targeting molecules, the molar ratio of the targeting molecule to the self-assembled nanocarrier is 5:1, the incubation reaction temperature is 37 °C, and the pH value of the buffer solution is 7.2.
[0039] Furthermore, in the implementation step of the high-efficiency production process, the flow rate ratio of the organic phase to the aqueous phase is 1:3, the ultrasonic power is 150 W, the frequency is 35 kHz, and the post-treatment method includes centrifugation and freeze-drying.
[0040] Furthermore, in the step of verifying vaccine loading and release control, the oscillation incubation temperature for vaccine loading is 37 °C, the rotation speed is 150 rpm, the incubation time is 4 hours, and the pH values of the simulated tumor microenvironment are 6.8 and 7.0, and the temperatures are 37 °C and 40 °C.
[0041] The beneficial effects of the tumor vaccine and its preparation process of the targeted delivery system of the present invention are as follows:
[0042] (1) Compared with the prior art, the multiple targeting mechanism and optimized nanocarriers of the present invention significantly improve the delivery efficiency of the tumor vaccine; effectively enhance the intensity of the immune response.
[0043] Through the preparation process combining the "ultrasonic-assisted solvent evaporation method" and the "microfluidic liquid-liquid phase separation method", the traditional multi-step synthesis is simplified to a one-step rapid synthesis, shortening the production time.
[0044] Meanwhile, the use of low-cost materials reduces the production cost, presenting good prospects for industrial production; and through the synergistic effect of the immune enhancer and the vaccine, the tumor-specific immune response is enhanced. Brief Description of the Drawings
[0045] The present invention will be further described in detail below with reference to the drawings and specific implementation methods.
[0046] Figure 1 It is a schematic structural diagram of the present invention. Detailed Description of the Invention
[0047] The present invention will be described in detail below with reference to the drawings and embodiments. It should be noted that, without conflict, the embodiments in the present application and the features in the embodiments may be combined with each other.
[0048] To make the technical solution of the present invention clearer, the present invention will be further described in detail below with reference to the drawings and specific embodiments.
[0049] Refer to Figure 1 , the tumor vaccine of the targeted delivery system includes a self-assembled nanocarrier, multiple targeting molecules, an immune enhancer, and a functional molecule;
[0050] The self-assembled nanocarrier is formed by self-assembly of a biodegradable polymer and an amphiphilic block copolymer, and the self-assembled nanocarrier can spontaneously assemble into a nanostructure according to environmental conditions in an aqueous solution;
[0051] The multiple targeting molecules are bound to the self-assembled nanocarrier, and the targeting molecules include at least one of a tumor-specific antibody, a tumor vascular endothelial growth factor receptor ligand, and an aptamer of an overexpressed protein on the tumor cell surface;
[0052] The immune enhancer is integrated into the self-assembled nanocarrier, and the immune enhancer includes at least one of a Toll-like receptor agonist and a cytokine;
[0053] The functional molecule is a pH-responsive, temperature-responsive, and enzyme-responsive functional molecule, and the functional molecule is introduced into the self-assembled nanocarrier for controlling the loading and release of the vaccine.
[0054] Preferably, the raw materials of the self-assembled nanocarrier are poly(lactic-co-glycolic acid) and an amphiphilic block copolymer, and the ratio of the hydrophilic segment to the hydrophobic segment of the amphiphilic block copolymer is 3:2.
[0055] Preferably, the multiple targeting molecules are introduced with reactive groups capable of binding to the nanocarrier through chemical modification, and the reactive groups are at least one of maleimide groups, amino groups, and carboxyl groups, so as to achieve binding to the self-assembled nanocarrier.
[0056] Preferably, the immunopotentiator is integrated into the self-assembled nanocarrier by physical encapsulation or chemical conjugation, and the chemical conjugation is connected by a degradable chemical bond.
[0057] Preferably, the pH-responsive, temperature-responsive, and enzyme-responsive functional molecules are introduced into the self-assembled nanocarrier by copolymerization, grafting, or conjugation. The pH-responsive functional molecule is a polymer containing pH-sensitive chemical bonds, the temperature-responsive functional molecule is a polymer with a lower critical solution temperature property, and the enzyme-responsive functional molecule is a compound containing chemical bonds that can be cleaved by tumor-related enzymes.
[0058] Refer to Figure 1 , the preparation process of the tumor vaccine of the targeted delivery system, including the tumor vaccine of the targeted delivery system, comprises the following steps:
[0059] S1. Preparation of the self-assembled nanocarrier:
[0060] Prepare raw materials: Weigh appropriate amounts of poly(lactic-co-glycolic acid) copolymer and amphiphilic block copolymer, dissolve them in dichloromethane, and prepare a solution with a concentration of 50 mg / mL.
[0061] Ultrasonic treatment: Place the above solution in an ultrasonic instrument and perform ultrasonic treatment for 15 minutes under the conditions of a power of 200 W and a frequency of 40 kHz to promote molecular dispersion.
[0062] Solvent evaporation: Transfer the ultrasonicated solution to a rotary evaporator and perform rotary evaporation under the conditions of a temperature of 40 °C and a vacuum degree of 30 mbar to volatilize dichloromethane, and self-assembly of nanoparticles is formed.
[0063] Purification: Dialyze the self-assembled product through a dialysis bag (cut-off molecular weight of 3000 Da) in deionized water for 24 hours to remove unreacted raw materials and impurities, and obtain a purified self-assembled nanocarrier;
[0064] S2. Binding of multiple targeting molecules
[0065] Targeting molecule modification: Chemically modify targeting molecules such as tumor-specific antibodies and aptamers to introduce reactive groups capable of binding to the nanocarrier.
[0066] Conjugation reaction: The modified targeting molecule and the self-assembled nanocarrier were mixed in a phosphate buffer solution (pH = 7.2) at a molar ratio of 5:1 and incubated at 37 °C for 6 hours.
[0067] Separation and purification: The conjugated product was separated and purified by gel filtration chromatography to remove the unbound targeting molecules, and the self-assembled nanocarrier conjugated with multiple targeting molecules was obtained.
[0068] S3. Implementation of the high-efficiency production process
[0069] Microfluidic chip preparation: The microfluidic chip was cleaned and assembled to ensure unobstructed channels.
[0070] Solution preparation: An organic phase solution (such as dissolved in dichloromethane) and an aqueous phase solution (such as a phosphate buffer solution containing a surfactant) containing nanoparticle precursors, vaccines, and immune enhancers were prepared respectively.
[0071] Microfluidic mixing and ultrasonication: The organic phase and the aqueous phase were injected into the mixing area of the microfluidic chip through different inlets at a flow rate ratio of 1:3, and ultrasonication was applied outside the chip with an ultrasonic power of 150 W and a frequency of 35 kHz.
[0072] Product collection: The formed nanoparticle solution was collected at the outlet of the microfluidic chip and post-treated by methods such as centrifugation and freeze-drying to obtain nanoparticles with high purity and good homogeneity loaded with vaccines and immune enhancers;
[0073] S4. Verification of vaccine loading and release control:
[0074] The vaccine was loaded onto the self-assembled nanocarrier by physical adsorption. The nanocarrier loaded with the vaccine was mixed with the vaccine solution and incubated with shaking at 37 °C and 150 rpm for 4 hours to allow the vaccine to adsorb on the surface of the nanocarrier;
[0075] In buffer solutions with a simulated tumor microenvironment pH of 6.8, 7.0, a temperature of 37 °C, 40 °C, and containing a specific concentration of matrix metalloproteinase, the concentration of the vaccine in the solution was regularly detected by high-performance liquid chromatography, and the vaccine release curve was plotted to verify the responsive release performance of the vaccine;
[0076] S5. Verification of the integration and synergistic effect of the immune enhancer:
[0077] Verification of the integration and synergistic effect of the immune enhancer
[0078] Integration of the immune enhancer: The Toll-like receptor agonist was dissolved in an organic solvent and mixed with the solution containing nanoparticle precursors, and the immune enhancer was integrated into the self-assembled nanocarrier by co-assembly.
[0079] Cell experiment: Mouse macrophages were co - cultured with nanocarriers loaded with vaccines and immune enhancers, and the enzyme - linked immunosorbent assay was used to detect the secretion levels of cytokines (such as tumor necrosis factor - α, interleukin - 6) in the cell culture supernatant to evaluate the activation effect of immune enhancers on immune cells.
[0080] Animal experiment: A tumor - bearing mouse model was selected and divided into an experimental group (treated with the tumor vaccine of the present invention), a control group 1 (treated with a tumor vaccine without an immune enhancer), and a control group 2 (treated with normal saline). The tumor volume of the mice was measured regularly, the tumor growth curve was plotted, and the tumor growth inhibition rate and survival rate of the mice in each group were compared to verify the synergistic therapeutic effect of the immune enhancer and the tumor vaccine.
[0081] Preferably, in the step of preparing the self - assembled nanocarrier, the organic solvent is dichloromethane, the ultrasonic power is 200 W, the frequency is 40 kHz, the rotary evaporation temperature is 40 °C, the vacuum degree is 30 mbar, and the molecular weight cut - off of the dialysis bag is 3000 Da.
[0082] Preferably, in the step of binding multiple targeting molecules, the molar ratio of the targeting molecule to the self - assembled nanocarrier is 5:1, the incubation reaction temperature is 37 °C, and the pH value of the buffer solution is 7.2.
[0083] Preferably, in the implementation step of the high - efficiency production process, the flow rate ratio of the organic phase to the aqueous phase is 1:3, the ultrasonic power is 150 W, the frequency is 35 kHz, and the post - treatment method includes centrifugation and freeze - drying.
[0084] Preferably, in the step of verifying vaccine loading and release control, the oscillation incubation temperature for vaccine loading is 37 °C, the rotation speed is 150 rpm, the incubation time is 4 hours, and the pH values of the simulated tumor microenvironment are 6.8 and 7.0, and the temperatures are 37 °C and 40 °C.
[0085] Working principle: Innovative targeted delivery system
[0086] Self - assembled nanocarrier: Amphiphilic block copolymers and other biodegradable polymers are selected as raw materials, and the ratio of the hydrophilic segment to the hydrophobic segment is specific (such as 3:2). In an aqueous solution, according to conditions such as environmental pH and temperature, these molecules spontaneously arrange and combine to form stable nanostructures, such as nanomicelles. Compared with traditional nanoparticles, this carrier has good biodegradability and biocompatibility, and can precisely control the size, morphology, and surface properties of the carrier by adjusting the composition and ratio of the raw materials, enhancing the uptake by tumor cells.
[0087] Multiple targeting mechanisms: Multiple targeting molecules, such as tumor-specific antibodies, tumor vascular endothelial growth factor receptor ligands, and aptamers of overexpressed proteins on the surface of tumor cells, are chemically modified to introduce active groups (such as maleimide groups, etc.) and combined with self-assembled nanocarriers. pH-responsive and enzyme-responsive targeting linkers are designed for the low pH value and high expression of certain enzymes in tumor tissues. In the tumor microenvironment, these linkers are activated, prompting the targeting molecules to precisely act on tumor cells and achieve precise targeting.
[0088] Optimizing the preparation process
[0089] Efficient synthesis method: Based on the self-assembly characteristics of nanoparticles, a process combining "ultrasonic-assisted solvent evaporation method" and "microfluidic liquid-liquid phase separation method" is used. The organic phase containing nanoparticle precursors, vaccines, and immune adjuvants is injected into the aqueous phase through a microfluidic chip at a specific flow rate ratio (such as 1:3), while applying ultrasound (such as power 150W, frequency 35kHz). The ultrasound accelerates the solvent evaporation and makes it more uniform, and the microfluidics precisely controls the mixing of the two phases, promoting the rapid self-assembly of nanoparticles, and a large number of nanoparticles with uniform size can be obtained in a short time.
[0090] Low-cost material selection: Common and inexpensive biodegradable polymers (polylactic-co-glycolic acid copolymer), natural polysaccharides (chitosan), etc. are used as the basic materials of nanocarriers. These materials have wide sources, low costs, high biosafety, and good modifiability.
[0091] Vaccine loading and release control
[0092] Intelligent responsive release: pH-responsive, temperature-responsive, and enzyme-responsive functional molecules are introduced into the self-assembled nanocarriers. For example, a pH-sensitive chemical bond is used to connect the vaccine and the nanocarrier. When the nanocarrier enters the tumor microenvironment (pH value about 6.5 - 7.0), the chemical bond breaks, and the vaccine is rapidly released. By adjusting the structure and proportion of the responsive molecules, precise release of the vaccine under different tumor microenvironments can be achieved.
[0093] Long-acting sustained release design: The nanocarrier adopts a multi-layer wrapping structure, and the outer layer is wrapped with a polymer membrane that can slowly degrade. The vaccine is first rapidly released from the inner layer to initiate an immune response, and then the outer membrane slowly degrades, continuously releasing the vaccine and prolonging the immune effect.
[0094] Immune adjuvant synergy
[0095] Integrated immune adjuvants: Immune adjuvants such as Toll-like receptor agonists and cytokines are integrated into the self-assembled nanocarriers through physical encapsulation or chemical conjugation (such as connecting with biodegradable chemical bonds).
[0096] Enhanced immune response: Immunopotentiators activate immune system-related pathways, enhancing the ability of immune cells to recognize and respond to tumor vaccines. Nanocarriers precisely deliver immunopotentiators and vaccines to the tumor microenvironment, increasing the intensity of local immune responses and overcoming tumor immune escape.
[0097] The above-described embodiments merely represent several implementation manners of the present invention. Their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent for the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention shall be subject to the appended claims.
Claims
1. A tumor vaccine with a targeted delivery system, comprising a self-assembled nanocarrier, multiple targeting molecules, an immune enhancer, and a functional molecule, characterized in that: The self-assembled nanocarrier is formed by self-assembly of a biodegradable polymer and an amphiphilic block copolymer, and the self-assembled nanocarrier can spontaneously assemble into a nanostructure according to environmental conditions in an aqueous solution; The multiple targeting molecules are bound to the self-assembled nanocarrier, and the targeting molecules include at least one of a tumor-specific antibody, a tumor vascular endothelial growth factor receptor ligand, and an aptamer of an overexpressed protein on the surface of tumor cells; The immune enhancer is integrated into the self-assembled nanocarrier, and the immune enhancer includes at least one of a Toll-like receptor agonist and a cytokine; The functional molecule is a pH-responsive, temperature-responsive, and enzyme-responsive functional molecule, and the functional molecule is introduced into the self-assembled nanocarrier to control the loading and release of the vaccine.
2. The tumor vaccine of the targeted delivery system according to claim 1, characterized in that: The raw materials of the self-assembled nanocarrier are poly(lactic-co-glycolic acid) copolymer and an amphiphilic block copolymer, and the ratio of the hydrophilic segment to the hydrophobic segment of the amphiphilic block copolymer is 3:
2.
3. The tumor vaccine of the targeted delivery system according to claim 2, characterized in that: The multiple targeting molecules introduce active groups capable of binding to the nanocarrier through chemical modification, and the active groups are at least one of a maleimide group, an amino group, and a carboxyl group to achieve binding to the self-assembled nanocarrier.
4. The tumor vaccine of the targeted delivery system according to claim 3, characterized in that: The immune enhancer is integrated into the self-assembled nanocarrier by physical encapsulation or chemical conjugation, and the chemical conjugation uses a degradable chemical bond connection.
5. The tumor vaccine of the targeted delivery system according to claim 4, wherein: The pH-responsive, temperature-responsive, and enzyme-responsive functional molecules are introduced into the self-assembled nanocarrier by copolymerization, grafting, or coupling. The pH-responsive functional molecule is a polymer containing a pH-sensitive chemical bond, the temperature-responsive functional molecule is a polymer with a lower critical solution temperature property, and the enzyme-responsive functional molecule is a compound containing a chemical bond that can be cleaved by tumor-related enzymes.
6. The preparation process of the tumor vaccine of the targeted delivery system, including the tumor vaccine of the targeted delivery system according to any one of claims 5, characterized in that, The process steps are as follows: S1. Preparation of the self-assembled nanocarrier: Prepare raw materials: Weigh an appropriate amount of poly(lactic-co-glycolic acid) copolymer and an amphiphilic block copolymer, dissolve them in dichloromethane, and prepare a solution with a concentration of 50 mg / mL; Ultrasonic treatment: Place the above solution in an ultrasonic instrument and ultrasonically treat it for 15 minutes under the conditions of a power of 200 W and a frequency of 40 kHz to promote molecular dispersion; Solvent evaporation: Transfer the ultrasonically treated solution to a rotary evaporator and rotate and evaporate it under the conditions of a temperature of 40 °C and a vacuum degree of 30 mbar to volatilize dichloromethane, and nanoparticles self-assemble to form; Purification: Dialyze the self-assembled product through a dialysis bag in deionized water for 24 hours to remove unreacted raw materials and impurities to obtain a purified self-assembled nanocarrier; S2. Binding of multiple targeting molecules Targeting molecule modification: Chemically modify targeting molecules such as tumor-specific antibodies and aptamers to introduce active groups capable of binding to the nanocarrier; Binding reaction: Mix the modified targeting molecules and the self-assembled nanocarrier in a phosphate buffer solution at a molar ratio of 5:1 and incubate and react at a temperature of 37 °C for 6 hours; Separation and purification: The binding product was separated and purified by gel filtration chromatography to remove unbound targeting molecules, and self-assembled nanocarriers conjugated with multiple targeting molecules were obtained. S3. Implementation of high-efficiency production process Microfluidic chip preparation: The microfluidic chip was cleaned and assembled to ensure unobstructed channels. Solution preparation: Organic phase solutions and aqueous phase solutions containing nanoparticle precursors, vaccines, and immune enhancers were prepared separately. Microfluidic mixing and sonication: The organic phase and the aqueous phase were injected into the mixing area of the microfluidic chip through different inlets at a flow rate ratio of 1:3, and sonication was applied outside the chip with a sonication power of 150 W and a frequency of 35 kHz. Product collection: The formed nanoparticle solution was collected at the outlet of the microfluidic chip and post-treated by methods such as centrifugation and freeze-drying to obtain nanoparticles with high purity and good uniformity loaded with vaccines and immune enhancers. S4. Verification of vaccine loading and release control: The vaccine was loaded onto the self-assembled nanocarriers by physical adsorption. The nanocarriers loaded with the vaccine were mixed with the vaccine solution and incubated with shaking at 37 °C and 150 rpm for 4 hours to allow the vaccine to adsorb on the surface of the nanocarriers. In buffer solutions simulating the pH values of the tumor microenvironment of 6.8 and 7.0, temperatures of 37 °C and 40 °C, and containing a specific concentration of matrix metalloproteinase, the concentration of the vaccine in the solution was regularly detected by high-performance liquid chromatography, and the vaccine release curve was plotted to verify the responsive release performance of the vaccine. S5. Verification of the integration and synergistic effect of the immune enhancer: Verification of the integration and synergistic effect of the immune enhancer Integration of the immune enhancer: The Toll-like receptor agonist was dissolved in an organic solvent and mixed with the solution containing nanoparticle precursors, and the immune enhancer was integrated into the self-assembled nanocarriers by co-assembly. Cell experiment: Mouse macrophages were co-cultured with the nanocarriers loaded with vaccines and immune enhancers, and the secretion level of cytokines in the cell culture supernatant was detected by enzyme-linked immunosorbent assay to evaluate the activation effect of the immune enhancer on immune cells. Animal experiment: Tumor-bearing mouse models were selected and divided into an experimental group, control group 1, and control group 2. The tumor volume of the mice was measured regularly, the tumor growth curve was plotted, and the tumor growth inhibition rate and survival rate of each group of mice were compared to verify the synergistic therapeutic effect of the immune enhancer and the tumor vaccine.
7. The preparation process of the tumor vaccine of the targeted delivery system according to claim 6, characterized in that: In the step of preparing the self-assembled nanocarriers, the organic solvent is dichloromethane, the sonication power is 200 W, the frequency is 40 kHz, the rotary evaporation temperature is 40 °C, the vacuum degree is 30 mbar, and the molecular weight cut-off of the dialysis bag is 3000 Da.
8. The preparation process of the tumor vaccine of the targeted delivery system according to claim 6, characterized in that: In the step of conjugating multiple targeting molecules, the molar ratio of the targeting molecule to the self-assembled nanocarrier is 5:1, the incubation reaction temperature is 37 °C, and the pH value of the buffer solution is 7.
2.
9. The preparation process of the tumor vaccine of the targeted delivery system according to claim 6, characterized in that: In the step of implementing the high-efficiency production process, the flow rate ratio of the organic phase to the aqueous phase is 1:3, the sonication power is 150 W, the frequency is 35 kHz, and the post-treatment methods include centrifugation and freeze-drying.
10. The preparation process of the tumor vaccine of the targeted delivery system according to claim 6, characterized in that: In the step of verifying vaccine loading and release control, the oscillating incubation temperature of the vaccine loading is 37°C, the rotation speed is 150 rpm, the incubation time is 4 hours, and the pH values simulating the tumor microenvironment are 6.8 and 7.0, and the temperatures are 37°C and 40°C.