Nonionic gene vector on basis of hydrogen bond donor group, and preparation method therefor and use thereof

Abstract

The present application relates to the technical field of medicine, and in particular to a nonionic gene vector on the basis of a hydrogen bond donor group, and a preparation method therefor and the use thereof. The gene vector loads a gene drug via a group that functions as a hydrogen bond and is formed by a hydrogen bond donor gene and a gene nucleoside or phosphate, and comprises a hydrogen bond donor group being a nonionic lipid and a hydrogen bond donor nonionic polymer. The hydrogen bond donor group mainly comprises thiourea, polyphenol, hydroxyl, urea, thymine, uracil, cytosine, adenine and guanine. The delivery system of the present application can be easily prepared, and loading can be achieved by means of a simple method of blending with mRNA at room temperature, which avoids the previous problems, such as the complex preparation process. In addition, the delivery system of the present invention has the characteristic of being degradable and has good biosafety.

Description

A nonionic gene carrier based on hydrogen bond donor groups and its preparation method and application Technical Field

[0001] The present application relates to the field of pharmaceutical technology, and more specifically, to a non-ionic gene carrier based on a hydrogen bond donor group, and a preparation method and application thereof. Background Art

[0002] Gene therapy, a novel therapeutic approach targeting proteins, RNA, and the genome, is rapidly developing and attracting significant attention. Two mRNA vaccines for COVID-19 have been approved by the FDA, a significant breakthrough that has spurred global interest in gene therapy-based therapies. Gene therapy is a type of electronegative drug that rapidly produces a short-term therapeutic effect upon entry into cells. Currently, gene therapy-based therapies for cancer, genetic diseases, protein replacement therapies, and vaccines have entered clinical trials.

[0003] Despite the obvious advantages of gene drugs, many challenges remain. First, gene drugs are unstable and more susceptible to degradation by nucleases in the body. Second, gene drugs are negatively charged, making it difficult for them to enter the target cells. Genes are foreign molecules that can be recognized by the immune system, inducing adverse immune responses. Finally, after entering the target cell, if the gene does not reach the cytoplasm in time, it will be quickly degraded by lysosomes. Therefore, overcoming the gene delivery barrier requires the use of effective gene transport vectors.

[0004] Traditional gene delivery vehicles include viral and non-viral vectors. Although viral gene delivery vehicles have achieved successful clinical results, the effectiveness of these approaches may be limited by factors such as innate immunity, viral-induced immunogenicity, deleterious genomic integration, gene payload limitations, the inability to repeat administration, complications associated with dose scaling, and expensive vector production costs. As alternatives to viral vectors, non-viral vectors, such as polymers, liposomes, and lipid nanoparticles (LNPs), have garnered widespread attention from researchers, spurring the development of non-viral-based delivery systems. Although non-viral vectors have achieved certain breakthroughs in gene delivery efficiency, many challenges remain. Commonly used polymeric gene delivery vehicles are mostly cationic polymers, and their high positive charge can potentially lead to hemolysis and inflammatory responses in vivo. Currently, LNPs remain the clinically approved gene delivery vehicle. However, traditional LNPs use cationic lipid components to deliver genes, which can cause severe inflammatory responses in vivo. Therefore, the design of gene delivery vehicles still faces significant challenges.

[0005] Existing mRNA delivery mainly relies on liposome nanoparticles, which have the following disadvantages:

[0006] 1. The preparation process is cumbersome and requires the use of microfluidics, which results in a high amount of mRNA required for a single preparation;

[0007] 2. Citric acid buffer solution and ethanol are used in the preparation process, which need to be removed by dialysis later. The dialysis process has high environmental requirements and can also cause mRNA degradation;

[0008] 3. Existing delivery systems cannot be degraded and are toxic in vivo;

[0009] 4. Existing delivery systems have low endosomal escape efficiency;

[0010] 5. Existing delivery systems are prone to cause inflammatory responses in the body. Summary of the Invention

[0011] The present disclosure provides a non-ionic gene carrier based on a hydrogen bond donor group, a preparation method and application thereof. The delivery system of the present application is simple to prepare and can be loaded by a simple method of mixing with mRNA at room temperature, avoiding problems such as the previous complex preparation process. At the same time, the delivery system involved in the present invention is degradable and has good biosafety.

[0012] In the first aspect, the present disclosure provides a non-ionic gene carrier based on a hydrogen bond donor group, which carries a gene drug through a group that forms a hydrogen bond with a hydrogen bond donor gene and a gene nucleoside or phosphate group, including a hydrogen bond donor group that is a non-ionic lipid and a hydrogen bond donor non-ionic polymer, and the hydrogen bond donor group mainly includes thiourea, polyphenol, hydroxyl, urea, thymine, uracil, cytosine, adenine and guanine.

[0013] The purpose of this application is to solve the inflammatory response caused by cationic gene delivery and to develop a non-ionic carrier for in vitro and in vivo gene delivery. This carrier can encapsulate gene drugs through hydrogen bonding groups such as thiourea, polyphenols, hydroxyl groups, urea, thymine, uracil, cytosine, adenine and guanine, thereby avoiding the in vivo inflammatory response caused by the positive charge of traditional cationic gene delivery systems.

[0014] Preferably, the hydrogen bond donor group non-ionic lipid includes a hydrogen bond donor head group and a hydrophobic tail, the hydrogen bond donor group non-ionic lipid is a saturated alkyl chain or an unsaturated alkyl chain, and the hydrogen bond donor group non-ionic polymer carrier includes a polymer main chain and a hydrogen bond donor group side group.

[0015] Provided is a non-ionic nano-delivery system capable of encapsulating gene drugs. This system primarily comprises hydrogen-bond donor groups, such as thiourea, polyphenols, hydroxyl groups, urea, thymine, uracil, cytosine, adenine, and guanine. These hydrogen-bond donor groups can form hydrogen bonds with nucleosides or phosphate groups in genes to effectively load gene drugs. These non-ionic gene delivery systems can be categorized into two main types: non-ionic lipids and non-ionic polymers. A characteristic of these non-ionic hydrogen-bond donor delivery systems is their ability to encapsulate gene drugs and achieve effective in vitro and in vivo delivery. Furthermore, compared to cationic / ionizable cationic gene delivery systems, these non-ionic hydrogen-bond donor gene delivery vectors do not induce inflammatory responses in vivo.

[0016] Preferably, the nanomicelles formed by the hydrogen bond donor group non-ionic nanocarrier and the gene drug exist in the form of an aqueous solution or a lyophilized powder.

[0017] The preparation method of the gene-carrying nano-micelle aqueous solution and the freeze-dried powder thereof is as follows: freeze-drying the prepared gene-carrying nano-micelle solution to obtain the gene-carrying nano-micelle freeze-dried powder.

[0018] Preferably, the nanomicelles formed by the hydrogen bond donor group non-ionic nanocarrier carrying the gene drug are prepared by microfluidics and dialysis to obtain the corresponding nanoparticle aqueous solution, and the nanolipid aqueous solution is freeze-dried to obtain the nanomicelle freeze-dried powder.

[0019] Preferably, the hydrogen bond donor group non-ionic nanocarrier in the nanomicelle aqueous solution system compresses the gene drug through hydrogen bonding and is dispersed in the aqueous phase in the form of a nanocomplex, and the concentration of the nanolipid is 1-2 mg / mL.

[0020] In a second aspect, the present disclosure provides a method for preparing a non-ionic gene carrier based on a hydrogen bond donor group, comprising the following steps:

[0021] (1) N-Boc-glutamic acid was dissolved in dichloromethane to obtain a mixed solution, carbonyldiimidazole was added to the mixed solution, and the mixture was stirred for 1 hour until the solution became clear. An alcohol organic compound was added and stirred for 12 hours. The mixture was washed three times with a saturated saline solution, and the organic phase was collected, dried over anhydrous magnesium sulfate, and rotary evaporated to obtain intermediate 1;

[0022] (2) The intermediate 1 was dissolved in DCM / TFA and deprotected at room temperature for 6 h. The solvent was removed by rotary evaporation. DCM was added again, TEA was added, and CS2 was added. The mixture was stirred at room temperature for 3 h. P-toluenesulfonyl chloride DCM solution was slowly added dropwise in an ice-water bath. The mixture was fully reacted for 48 h. The mixture was washed three times with saturated brine, dried over anhydrous magnesium sulfate, and rotary evaporated to obtain intermediate 2.

[0023] (3) The intermediate 2 was dissolved in DMF, 3-amino-1,2-propanediol was added, the reaction was carried out at room temperature for 2 h, and then at 80°C for 24 h. DCM was added, and the mixture was washed with saturated brine to remove DMF and unreacted 3-amino-1,2-propanediol. The mixture was dried over anhydrous magnesium sulfate and rotary evaporated to obtain the final product.

[0024] Preferably, in step (1), the alcohol substances include stearyl alcohol, lauryl alcohol and n-hexanol.

[0025] In a third aspect, the present disclosure provides an application of a non-ionic gene carrier based on a hydrogen bond donor group, wherein the gene-carrying nanocomplex is injected into the subcutaneous, intramuscular or lungs of mice by subcutaneous, intramuscular or spray administration. The non-ionic nanocarrier with a hydrogen bond donor group delivers the gene drug to APC cells in the lymph nodes or specific cells in the body, thereby expressing corresponding cytokines or major histocompatibility complexes or target proteins or inhibiting the expression of certain specific proteins.

[0026] The present application's series of hydrogen-bond donor group nonionic nanocarriers and their gene-carrying nanomicelle aqueous solutions or lyophilized powders are all redispersible. The mechanism by which these hydrogen-bond donor group nonionic nanocarriers deliver genes is through hydrogen bonding between terminal or side hydrogen-bond donor groups, such as thiourea, polyphenols, hydroxyl groups, urea, thymine, uracil, cytosine, adenine, and guanine, in the hydrogen-bond donor group nonionic nanocarrier components and the gene drug. This gene-carrying method replaces the traditional method of loading gene drugs with cationic carrier components through positive and negative electrical interactions. This avoids the in vivo inflammatory response caused by cationic gene carrier components.

[0027] The series of hydrogen bond donor group non-ionic nanocarriers of the present application are specifically implemented using non-ionic lipids as an example, wherein the hydrogen bond donor group is selected as a thiourea group and the lipid carbon chain is selected as a saturated carbon chain.

[0028] In summary, this application has the following beneficial effects:

[0029] 1. Since the carrier in this application can encapsulate gene drugs through hydrogen bonding with groups such as thiourea, polyphenols, hydroxyl groups, urea, thymine, uracil, cytosine, adenine and guanine, it avoids the in vivo inflammatory response caused by positive charges in traditional cationic gene delivery systems;

[0030] 2. The non-ionic gene delivery systems in this application can be divided into two categories: non-ionic lipids and non-ionic polymers. The characteristic feature of this series of hydrogen bond donor group non-ionic delivery systems is that they can achieve the encapsulation of gene drugs and achieve effective delivery of gene drugs in vitro and in vivo;

[0031] 3. The delivery system of the present application is simple to prepare and can be loaded by a simple method of mixing with mRNA at room temperature, avoiding the problems of complex preparation processes in the past. At the same time, the delivery system involved in the present invention is degradable and has good biosafety.

[0032] It should be understood that the foregoing general description and the following detailed description are merely exemplary and explanatory and are not intended to limit the scope of protection of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] 1. Figure 1 is a synthetic route diagram of DHECG prepared in Example 1 of the present application;

[0034] 2. FIG2 is a nuclear magnetic resonance spectrum of the intermediate of DHECG prepared in Example 1 of the present application. The nuclear magnetic peak positions of various hydrogen protons in the figure prove the structural composition of the intermediate;

[0035] 3. FIG. 3 is a nuclear magnetic resonance spectrum of the intermediate of DHECG prepared in Example 1 of the present application. The nuclear magnetic peak positions of various hydrogen protons in the figure prove the structural composition of the intermediate;

[0036] 4. FIG. 4 is a nuclear magnetic resonance spectrum of the intermediate of DHECG prepared in Example 1 of the present application. The nuclear magnetic peak positions of various hydrogen protons in the figure prove the structural composition of DHECG;

[0037] 5. Figure 5 is a synthetic route diagram of DDCCG prepared in Example 2 of the present application;

[0038] 6. FIG6 is an NMR spectrum of the intermediate of DDCCG prepared in Example 2 of the present application. The positions of the NMR peaks of various hydrogen protons in the figure prove the structural composition of the intermediate;

[0039] 7. FIG. 7 is an NMR spectrum of the intermediate of DDCCG prepared in Example 2 of the present application. The positions of the NMR peaks of various hydrogen protons in the figure prove the structural composition of the intermediate;

[0040] 8. FIG8 is a nuclear magnetic resonance spectrum of DDCCG prepared in Example 2 of the present application. The nuclear magnetic peak positions of various hydrogen protons in the figure prove the structural composition of DDCCG;

[0041] 9. Figure 9 is a synthetic route diagram of DOCCG prepared in Example 3 of the present application;

[0042] 10. FIG. 10 is an NMR spectrum of the intermediate of DOCCG prepared in Example 3 of the present application. The positions of the NMR peaks of various hydrogen protons in the figure prove the structural composition of the intermediate;

[0043] 11. FIG. 11 is an NMR spectrum of the intermediate of DOCCG prepared in Example 3 of the present application. The positions of the NMR peaks of various hydrogen protons in the figure prove the structural composition of the intermediate;

[0044] 12. FIG. 12 is a nuclear magnetic resonance spectrum of DOCCG prepared in Example 3 of the present application. The positions of the nuclear magnetic peaks of various hydrogen protons in the figure prove the structural composition of DOCCG;

[0045] 13. FIG. 13 shows the particle size and potential of the nonionic lipid nanoparticles prepared in Example 4 of the present application. The results in the figure demonstrate that all nonionic lipid components can be prepared into lipid nanoparticles using microfluidic technology, and the potentials of all nonionic lipid nanoparticles are close to neutrality.

[0046] 14. Figure 14 shows the gene-carrying capacity test of the non-ionic lipid nanoparticles prepared in Example 4 of the present application. The results in the figure prove that all non-ionic nanoparticles can effectively carry genes. DETAILED DESCRIPTION

[0047] The present application is further described in detail below with reference to the examples. It is particularly noted that if no specific conditions are specified in the following examples, the reactions are carried out according to conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, the raw materials used in the following examples can be obtained from common commercial sources.

[0048] Example

[0049] Example 1

[0050] Synthesis of DHECG

[0051] N-Boc-glutamic acid (2 g, 8.09 mmol) was dissolved in dichloromethane (100 mL). Carbonyldiimidazole (CDI, 2.624 g, 16.18 mmol) was then added to the mixture and stirred for 1 h until the solution became clear. Hexanol (1.653 g, 16.18 mmol) was added and the reaction was stirred for 12 h. The mixture was washed three times with saturated saline solution. The organic phase was collected, dried over anhydrous magnesium sulfate, and rotary evaporated to obtain Intermediate 1.

[0052] Intermediate 1 (3.011 g, 7.25 mmol) was dissolved in DCM / TFA (10 mL, V / V = 1 / 1) and deprotected at room temperature for 6 h. The solvent was removed by rotary evaporation, and DCM (5 mL) was added again. TEA (1.5 mL, 10.821 mmol) was added, and CS2 (0.90 mL, 14.50 mmol) was added. The mixture was stirred at room temperature for 3 h. In an ice-water bath, p-toluenesulfonyl chloride (1.7 g, 8.70 mmol) DCM solution (5 mL) was slowly added dropwise. The mixture was fully reacted for 48 h. The mixture was washed three times with saturated brine, dried over anhydrous magnesium sulfate, and rotary evaporated to obtain intermediate 2. Subsequently, product 2 was dissolved in DMF (10 mL), 3-amino-1,2-propanediol (1.32 g, 14.50 mmol) was added, the reaction was carried out at room temperature for 2 h and at 80 ° C for 24 h, DCM was added, and the mixture was washed with saturated brine to remove DMF and unreacted 3-amino-1,2-propanediol. The mixture was dried over anhydrous magnesium sulfate and rotary evaporated to obtain the final product DHECG (2.5 g, yield 77%).

[0053] Example 2

[0054] Synthesis of DDDCG

[0055] N-Boc-glutamic acid (2.00 g, 8.09 mmol) was dissolved in dichloromethane (100 mL). Carbonyldiimidazole (CDI, 2.624 g, 16.18 mmol) was then added to the mixture and stirred for 1 h until the solution became clear. Lauryl alcohol (3.015 g, 16.18 mmol) was added and the reaction was stirred for 12 h. The mixture was washed three times with saturated saline solution. The organic phase was collected, dried over anhydrous magnesium sulfate, and rotary evaporated to obtain Intermediate 1.

[0056] Intermediate 1 (4.188 g, 7.18 mmol) was dissolved in DCM / TFA (10 mL, V / V = 1 / 1) and deprotected at room temperature for 6 h. The solvent was removed by rotary evaporation, and DCM (5 mL) was added again. TEA (1.51 mL, 10.893 mmol) was added, and CS2 (0.88 mL, 14.50 mmol) was added. The mixture was stirred at room temperature for 3 h. In an ice-water bath, p-toluenesulfonyl chloride (1.66 g, 8.70 mmol) DCM solution (5 mL) was slowly added dropwise. The mixture was fully reacted for 48 h. The mixture was washed three times with saturated brine, dried over anhydrous magnesium sulfate, and rotary evaporated to obtain intermediate 2. Subsequently, the product 2 was dissolved in DMF (10 mL), 3-amino-1,2-propanediol (1.31 g, 14.36 mmol) was added, the reaction was carried out at room temperature for 2 h, and at 80 ° C for 24 h, DCM was added, and the mixture was washed with saturated brine to remove DMF and unreacted 3-amino-1,2-propanediol. The mixture was dried over anhydrous magnesium sulfate and rotary evaporated to obtain the final product DDDCG (5.8 g, yield 76%).

[0057] Example 3

[0058] Synthesis of DOCCG

[0059] N-Boc-glutamic acid (2.00 g, 8.09 mmol) was dissolved in dichloromethane (100 mL), and carbonyldiimidazole (CDI, 2.624 g, 16.18 mmol) was added to the mixture and stirred for 1 h until the solution became clear. Stearyl alcohol (4.377 g, 16.18 mmol) was added and the reaction was stirred for 12 h. The mixture was washed three times with saturated saline solution, and the organic phase was collected, dried over anhydrous magnesium sulfate, and rotary evaporated to obtain Intermediate 1.

[0060] Intermediate 1 (3.40 g, 5.22 mmol) was dissolved in DCM / TFA (10 mL, V / V = 1 / 1) and deprotected at room temperature for 6 h. The solvent was removed by rotary evaporation, and DCM (5 mL) was added again. TEA (1.20 mL, 7.83 mmol) was added, and CS2 (0.67 mL, 10.44 mmol) was added. The mixture was stirred at room temperature for 3 h. In an ice-water bath, p-toluenesulfonyl chloride (1.20 g, 6.264 mmol) DCM solution (5 mL) was slowly added dropwise. The mixture was fully reacted for 48 h. The mixture was washed three times with saturated brine, dried over anhydrous magnesium sulfate, and rotary evaporated to obtain intermediate 2. Subsequently, product 2 was dissolved in DMF (10 mL), 3-amino-1,2-propanediol (0.95 g, 10.44 mmol) was added, the reaction was carried out at room temperature for 2 h and at 80 °C for 24 h, DCM was added, and the mixture was washed with saturated brine to remove DMF and unreacted 3-amino-1,2-propanediol. The mixture was dried over anhydrous magnesium sulfate and rotary evaporated to obtain the final product DOCCG (2.72 g, yield 67%).

[0061] Example 4

[0062] Preparation and Characterization of Nonionic Lipid Nanoparticles

[0063] Nonionic lipids and cholesterol were dissolved in anhydrous ethanol, extruded into a centrifuge tube with a certain volume of deionized water through a microfluidic device, dialyzed to remove ethanol, and fixed to volume to obtain nonionic lipid nanoparticles with a concentration of 1 mg / mL, which were stored at 4°C for future use.

[0064] The nonionic nanoparticle solution was transferred to a sample cell, and the particle size and potential of DHECG, DDDCG, and DOCCG nanoparticles were detected by dynamic light scattering (DLS) at a temperature of 25°C and an angle of 173°.

[0065] Example 5

[0066] Detection of gene encapsulation ability of non-ionic nanoparticles

[0067] The gene-enhanced aqueous solution and nonionic lipid ethanol solution were extruded into a centrifuge tube using a microfluidic device. The solution was dialyzed against DEPC water, fixed to a certain concentration of gene-enhanced nonionic lipid nanoparticles, and stored at 4°C. A certain volume of the gene-enhanced nonionic lipid nanoparticle aqueous solution was mixed evenly with the loading buffer and transferred to the sample wells of the agarose plate. The agarose gel plate was transferred to an electrophoresis tank for gel running, and the gene loading capacity was detected by UV light.

[0068] The above description is merely an exemplary embodiment of the present disclosure, but the scope of protection of the present disclosure is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present disclosure should be included in the scope of protection of the present disclosure. Therefore, the scope of protection of the present disclosure should be based on the scope of protection of the claims.

Claims

1. A nonionic gene carrier based on a hydrogen bond donor group, characterized in that, The gene vector loads gene drugs through groups that form hydrogen bonds with hydrogen bond donor genes and gene nucleosides or phosphate groups, including hydrogen bond donor groups being nonionic lipids and hydrogen bond donor nonionic polymers. The hydrogen bond donor groups mainly include thiourea, polyphenol, hydroxyl group, urea, thymine, uracil, cytosine, adenine, and guanine.

2. The non-ionic gene carrier based on a hydrogen bond donor group according to claim 1, wherein The nonionic lipid of the hydrogen bond donor group includes a hydrogen bond donor head group and a hydrophobic tail. The nonionic lipid of the hydrogen bond donor group is a saturated alkyl chain or an unsaturated alkyl chain. The nonionic polymer carrier of the hydrogen bond donor group includes a polymer backbone and a hydrogen bond donor group side chain.

3. The nonionic gene vector based on a hydrogen bond donor group according to claim 1, wherein The nanomicelles formed by the nonionic nanocarrier of the hydrogen bond donor group and the gene drug exist in the form of an aqueous solution or a lyophilized powder.

4. The nonionic gene carrier based on a hydrogen bond donor group according to claim 1, characterized in that, The nanomicelles formed by the nonionic nanocarrier of the hydrogen bond donor group carrying gene drugs are prepared into corresponding aqueous solutions of nanoparticles through microfluidics technology and dialysis, and the aqueous solution of nanolipids is freeze-dried to obtain the lyophilized powder of nanomicelles.

5. The nonionic gene carrier based on a hydrogen bond donor group according to claim 3, wherein In the aqueous solution system of the nanomicelles, the nonionic nanocarrier of the hydrogen bond donor group compresses the gene drug through hydrogen bonding and is dispersed in the aqueous phase in the form of a nanocomplex. The concentration of the nanolipid is 1 - 2 mg / mL.

6. The preparation method of the nonionic gene carrier based on a hydrogen bond donor group according to any one of claims 1-5, characterized in that, It includes the following steps: (1) Dissolve N-Boc-glutamic acid in dichloromethane to obtain a mixed solution. Add carbonyldiimidazole to the mixed solution and stir for 1 h until the solution becomes clear. Add an alcohol organic substance and stir for reaction for 12 h. Wash three times with saturated saline solution, collect the organic phase, dry with anhydrous magnesium sulfate, and rotary evaporate to obtain intermediate 1. (2) Take intermediate 1 and dissolve it in DCM / TFA. Deprotect at room temperature for 6 h, rotary evaporate to remove the solvent, add DCM again, add TEA, add CS2, stir at room temperature for 3 h, slowly dropwise add a DCM solution of p-toluenesulfonyl chloride under an ice-water bath condition, react fully for 48 h, wash three times with saturated saline, dry with anhydrous magnesium sulfate, and rotary evaporate to obtain intermediate 2. (3) Dissolve intermediate 2 in DMF, add 3-amino-1,2-propanediol, react at room temperature for 2 h, react at 80 °C for 24 h, add DCM, wash with saturated saline to remove DMF and unreacted 3-amino-1,2-propanediol, dry with anhydrous magnesium sulfate, and rotary evaporate to obtain the final product.

7. The preparation method of a nonionic gene carrier based on a hydrogen bond donor group according to claim 6, wherein, In step (1), the alcohol substances include stearyl alcohol, lauryl alcohol, and n-hexanol.

8. Use of the nonionic gene carrier based on a hydrogen bond donor group according to any one of claims 1-7, characterized in that, By means of subcutaneous, intramuscular, or spray administration, the gene-loaded nanocomplex is injected subcutaneously, intramuscularly, or into the lungs of mice. The nonionic nanocarrier of the hydrogen bond donor group delivers the gene drug to APC cells in the lymph nodes or specific cells in the body, and then expresses corresponding cytokines, major histocompatibility complex, or target proteins, or inhibits the expression of certain specific proteins.

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