Emotion regulating substance enteric brain targeting delivery system based on nano-liposome and preparation method of emotion regulating substance enteric brain targeting delivery system

Abstract

The invention provides an emotional regulation substance intestinal brain targeting delivery system based on nano-liposome and a preparation method of the emotional regulation substance intestinal brain targeting delivery system. According to the research, a nucleic acid aptamer capable of specifically recognizing the small intestine epithelial cells is obtained through screening by utilizing an SELEX technology, a delivery system with a nano-liposome as a core shell, an emotion regulating substance as a core and the nucleic acid aptamer as a target is constructed on the basis of the nucleic acid aptamer, and the delivery system recognizes the small intestine epithelial cells in a targeted mode by means of the intestinal brain axis; the directional enrichment of emotion regulating substances in the brain position is realized, and scientific significance and application value are provided for related emotion control and treatment.

Description

Technical Field

[0001] The present invention relates to the field of targeted delivery technology, and more specifically, to a nanoliposome-based gut-brain targeted delivery system for mood regulating substances and a preparation method thereof. Background Art

[0002] Mood-regulating substances are chemicals that can influence emotional states. They work in the brain and body, helping us cope with stress and regulate emotions and behavior. In recent years, targeted delivery of mood-regulating substances through delivery systems to treat related diseases has become a hot topic in the industry. However, existing technologies lack specific delivery solutions or effective gut-brain targeted delivery systems. Therefore, there is an urgent need to develop gut-brain targeted delivery systems for mood-regulating substances. Summary of the Invention

[0003] To address the technical issues existing in the prior art, the present invention provides a nanoliposome-based gut-brain targeted delivery system for mood-regulating substances and its preparation method. This study utilized SELEX technology to screen and identify a nucleic acid aptamer that specifically recognizes small intestinal epithelial cells. This aptamer was then used to construct a delivery system based on the nanoliposome as a core-shell, the mood-regulating substance as a core, and the nucleic acid aptamer as a target. This delivery system, by targeting small intestinal epithelial cells and leveraging the gut-brain axis, achieves targeted enrichment of mood-regulating substances in the brain, providing scientific significance and application value for related mood control and treatment.

[0004] One of the objectives of the present invention is to provide a nucleic acid aptamer that can recognize small intestinal epithelial cells with high specificity, obtained by screening using the SELEX technology. The sequence of the nucleic acid aptamer is shown in SEQ ID NO.1.

[0005] Preferably, there is a Cholesterol modification at the 5' end and a dt modification at the 3' end of SEQ ID NO. 1.

[0006] Another object of the present invention is to provide a nanoliposome-based gut-brain targeted delivery system for mood regulating substances, wherein the system has a mood regulating substance as the core, a nanoliposome as the shell, and a nucleic acid aptamer modified on the surface of the shell.

[0007] Preferably, the nanoliposome is a mixture comprising cationic lipid material L319, DSPC, DMG-PEG2000 and cholesterol.

[0008] Preferably, the weight portion of the cationic lipid material is 50-80, the weight portion of DSPC is 5-15, the weight portion of DMG-PEG2000 is 5-10, and the weight portion of cholesterol is 10-25.

[0009] Furthermore, the present invention also provides a method for preparing a nanoliposome-based gut-brain targeted delivery system for mood regulating substances, the method comprising: Weigh L319, DSPC, DMG-PEG2000, and cholesterol according to the stated weight portions, and take out 10-20 mg of the mixture for later use; Take out 0.5-1 mg of the mood-regulating substance and add it to 10-20 mg of the mixture in step 1) and dissolve it in 5-10 mL of dichloromethane. After rotary evaporation to dryness, add 1-5 mL of sterile phosphate-buffered saline (1× PBS) to dissolve it; Add 0.5-1 mg of nucleic acid aptamer to the solution described in step 2), and shake at 300-500 RPM for 5-10 hours to obtain a nanoliposome-based gut-brain targeted delivery system for mood regulating substances.

[0010] Preferably, the mood regulating substance includes but is not limited to at least one of dopamine, serotonin, norepinephrine, γ-aminobutyric acid, glutamate, cortisol, adrenaline, endorphin, oxytocin, melatonin, and cannabinoids.

[0011] Further preferably, the mood regulating substance comprises at least one of dopamine, serotonin or gamma-aminobutyric acid.

[0012] Another object of the present invention is to provide a dopamine gut-brain targeted delivery system based on nanoliposomes, wherein the system has dopamine as the core, the shell is a nanoliposome, and the surface of the shell is modified with a nucleic acid aptamer, and the sequence of the nucleic acid aptamer is shown in SEQ ID NO.1.

[0013] The nanoliposomes are a mixture of cationic lipid material L319, DSPC, DMG-PEG2000 and cholesterol, wherein the weight proportion of the cationic lipid material is 50, the weight proportion of the DSPC is 15, the weight proportion of the DMG-PEG2000 is 10, and the weight proportion of the cholesterol is 25.

[0014] The preparation method of the delivery system comprises: Weigh L319, DSPC, DMG-PEG2000, and cholesterol according to the stated weight portions, and take out 10 mg of the mixture for later use; Take out 0.5 mg of dopamine and add it to the 10 mg mixture in step 1) and dissolve it in 5 mL of dichloromethane. After rotary evaporation to dryness, add 1 mL of sterile phosphate-buffered saline (1×PBS) to dissolve it; 0.5 mg of nucleic acid aptamer was added to the solution described in step 2), and the mixture was shaken at 300 RPM for 5 h to obtain a nanoliposome-based dopamine gut-brain targeted delivery system.

[0015] Another object of the present invention is to provide a nanoliposome-based 5-HT gut-brain targeted delivery system, wherein the system is based on endorphin as the core, the shell is a nanoliposome, and the surface of the shell is modified with a nucleic acid aptamer.

[0016] The nanoliposomes are a mixture of cationic lipid material L319, DSPC, DMG-PEG2000 and cholesterol, wherein the weight proportion of the cationic lipid material is 60, the weight proportion of the DSPC is 10, the weight proportion of the DMG-PEG2000 is 10, and the weight proportion of the cholesterol is 20.

[0017] The preparation method of the delivery system comprises: Weigh L319, DSPC, DMG-PEG2000, and cholesterol according to the stated weight portions, and take out 15 mg of the mixture for later use; Take out 0.8 mg of 5-HT and add it to the 15 mg mixture from step 1) and dissolve it in 8 mL of dichloromethane. After rotary evaporation to dryness, add 2 mL of sterile phosphate-buffered saline (1× PBS) to dissolve it. 0.8 mg of nucleic acid aptamer was added to the solution described in step 2), and the mixture was shaken at 400 RPM for 8 h to obtain a nanoliposome-based 5-HT gut-brain targeted delivery system.

[0018] Another object of the present invention is to provide a nanoliposome-based γ-aminobutyric acid gut-brain targeted delivery system, wherein the system is based on γ-aminobutyric acid as the core, the shell is a nanoliposome, and the shell surface is modified with a nucleic acid aptamer.

[0019] The nanoliposomes are a mixture of cationic lipid material L319, DSPC, DMG-PEG2000 and cholesterol, wherein the weight proportion of the cationic lipid material is 75, the weight proportion of the DSPC is 10, the weight proportion of the DMG-PEG2000 is 5, and the weight proportion of the cholesterol is 10.

[0020] The preparation method of the delivery system comprises: Weigh L319, DSPC, DMG-PEG2000, and cholesterol according to the stated weight portions, and take out 20 mg of the mixture for later use; Take out 1 mg of γ-aminobutyric acid and add it to 20 mg of the mixture in step 1) and dissolve it in 10 mL of dichloromethane. After rotary evaporation to dryness, add 5 mL of sterile phosphate-buffered saline (1× PBS) to dissolve it; Add 1 mg of nucleic acid aptamer to the solution described in step 2), and shake at 500 RPM for 10 h to obtain the nanoliposome-based γ-aminobutyric acid gut-brain targeted delivery system.

[0021] The advantages of the present invention are as follows: This study used SELEX technology to screen a nucleic acid aptamer that specifically recognizes small intestinal epithelial cells, and based on this, constructed a delivery system based on nanoliposomes as the core-shell, emotion-regulating substances as the core, and nucleic acid aptamers as the target. This delivery system targets small intestinal epithelial cells and, with the help of the gut-brain axis, achieves the targeted enrichment of emotion-regulating substances in the brain, providing scientific significance and application value for related emotion control and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 .Analysis of the distribution ratio of mood regulating substances in cerebrospinal fluid / small intestinal fluid. DETAILED DESCRIPTION

[0023] The present invention will be further described in detail below with reference to specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0024] The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work are within the scope of protection of the present invention.

[0025] In the examples of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0026] The present invention obtains a highly specific nucleic acid aptamer for recognizing small intestinal epithelial cells by screening through SELEX technology, and the sequence of the nucleic acid aptamer is shown in SEQ ID NO.1; 5'-Cholesterol-ttttttttttttgttgacacccagaaaatgaggaatggcagtcgttctaattcgccagggacgcccattagtcagtgcgagcacaaccagtgactggagadt-3' (SEQ ID NO. 1), which has a Cholesterol modification at the 5' end and a dt modification at the 3' end.

[0027] The present invention also provides a nanoliposome-based gut-brain targeted delivery system for mood regulating substances, wherein the system has a mood regulating substance as the core, a nanoliposome as the shell, and a nucleic acid aptamer modified on the surface of the shell.

[0028] The nanoliposome is a mixture of cationic lipid material L319, DSPC, DMG-PEG2000 and cholesterol, wherein the weight proportion of the cationic lipid material is 50-80, the weight proportion of the DSPC is 5-15, the weight proportion of the DMG-PEG2000 is 5-10, and the weight proportion of the cholesterol is 10-25.

[0029] The preparation method of the delivery system comprises: Weigh L319, DSPC, DMG-PEG2000, and cholesterol according to the stated weight portions, and take out 10-20 mg of the mixture for later use; Take out 0.5-1 mg of the mood-regulating substance and add it to 10-20 mg of the mixture in step 1) and dissolve it in 5-10 mL of dichloromethane. After rotary evaporation to dryness, add 1-5 mL of sterile phosphate-buffered saline (1× PBS) to dissolve it; Add 0.5-1 mg of nucleic acid aptamer to the solution described in step 2), and shake at 300-500 RPM for 5-10 hours to obtain a nanoliposome-based gut-brain targeted delivery system for mood regulating substances.

[0030] Example 1 The present invention also provides a dopamine gut-brain targeted delivery system based on nanoliposomes. The system has dopamine as the core, the shell is nanoliposomes, and the surface of the shell is modified with a nucleic acid aptamer. The sequence of the nucleic acid aptamer is shown in SEQ ID NO.1.

[0031] The nanoliposomes are a mixture of cationic lipid material L319, DSPC, DMG-PEG2000 and cholesterol, wherein the weight proportion of the cationic lipid material is 50, the weight proportion of the DSPC is 15, the weight proportion of the DMG-PEG2000 is 10, and the weight proportion of the cholesterol is 25.

[0032] The preparation method of the delivery system comprises: Weigh L319, DSPC, DMG-PEG2000, and cholesterol according to the stated weight portions, and take out 10 mg of the mixture for later use; Take out 0.5 mg of dopamine and add it to the 10 mg mixture in step 1) and dissolve it in 5 mL of dichloromethane. After rotary evaporation to dryness, add 1 mL of sterile phosphate-buffered saline (1×PBS) to dissolve it; 0.5 mg of nucleic acid aptamer was added to the solution described in step 2), and the mixture was shaken at 300 RPM for 5 h to obtain a nanoliposome-based dopamine gut-brain targeted delivery system.

[0033] Example 2 The present invention also provides a nanoliposome-based 5-HT gut-brain targeted delivery system, wherein the system is based on endorphin as the core, the shell is the nanoliposome, and the surface of the shell is modified with a nucleic acid aptamer.

[0034] The nanoliposomes are a mixture of cationic lipid material L319, DSPC, DMG-PEG2000 and cholesterol, wherein the weight proportion of the cationic lipid material is 60, the weight proportion of the DSPC is 10, the weight proportion of the DMG-PEG2000 is 10, and the weight proportion of the cholesterol is 20.

[0035] The preparation method of the delivery system comprises: Weigh L319, DSPC, DMG-PEG2000, and cholesterol according to the stated weight portions, and take out 15 mg of the mixture for later use; Take out 0.8 mg of 5-HT and add it to the 15 mg mixture from step 1) and dissolve it in 8 mL of dichloromethane. After rotary evaporation to dryness, add 2 mL of sterile phosphate-buffered saline (1× PBS) to dissolve it. 0.8 mg of nucleic acid aptamer was added to the solution described in step 2), and the mixture was shaken at 400 RPM for 8 h to obtain a nanoliposome-based 5-HT gut-brain targeted delivery system.

[0036] Example 3 The present invention also provides a nanoliposome-based γ-aminobutyric acid gut-brain targeted delivery system, wherein the system is based on γ-aminobutyric acid as the core, the shell is the nanoliposome, and the surface of the shell is modified with a nucleic acid aptamer.

[0037] The nanoliposomes are a mixture of cationic lipid material L319, DSPC, DMG-PEG2000 and cholesterol, wherein the weight proportion of the cationic lipid material is 75, the weight proportion of the DSPC is 10, the weight proportion of the DMG-PEG2000 is 5, and the weight proportion of the cholesterol is 10.

[0038] The preparation method of the delivery system comprises: Weigh L319, DSPC, DMG-PEG2000, and cholesterol according to the stated weight portions, and take out 20 mg of the mixture for later use; Take out 1 mg of γ-aminobutyric acid and add it to 20 mg of the mixture in step 1) and dissolve it in 10 mL of dichloromethane. After rotary evaporation to dryness, add 5 mL of sterile phosphate-buffered saline (1× PBS) to dissolve it; Add 1 mg of nucleic acid aptamer to the solution described in step 2), and shake at 500 RPM for 10 h to obtain the nanoliposome-based γ-aminobutyric acid gut-brain targeted delivery system.

[0039] Comparative Examples 1-3 Compared with Example 1, the nanoliposome shell of the delivery system of Comparative Example 1 is not modified with nucleic acid aptamers.

[0040] Compared with Example 2, the nanoliposome shell of the delivery system of Comparative Example 2 was not modified with nucleic acid aptamers.

[0041] Compared with Example 3, the nanoliposome shell of the delivery system of Comparative Example 3 is not modified with nucleic acid aptamers.

[0042] Verification Example Pharmacokinetics: Anesthetized male SD rats (200 ± 20 g, purchased from the Guangdong Medical Laboratory Animal Center) were administered 200 μL of Examples 1-3 and Comparative Examples 1-3 via enema. A blank control group was administered an equal volume of saline. Cerebrospinal fluid and small intestinal fluid were collected 6 hours after administration, and the concentrations of dopamine, 5-hydroxytryptamine, and γ-aminobutyric acid were measured by HPLC. The distribution ratios of mood-regulating substances in the cerebrospinal fluid / small intestinal fluid were calculated.

[0043] The results show that: Figure 1 As shown, compared with comparative examples 1-3, the distribution of the mood-regulating substances in Examples 1-3 of the present invention in the cerebrospinal fluid is more concentrated than that in the small intestinal fluid, further confirming that the delivery system of Examples 1-3 of the present invention can effectively deliver the mood-regulating substances to the brain by targeting the small intestinal epithelial cells and utilizing the gut-brain axis, and exert their effects.

[0044] It is important to note that the above embodiments are intended only to further illustrate and describe the technical solutions of the present invention and are not intended to further limit the technical solutions of the present invention. The methods of the present invention are merely preferred implementations and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A nucleic acid aptamer that can recognize small intestinal epithelial cells with high specificity obtained by SELEX technology, characterized in that: The sequence of the nucleic acid aptamer is shown in SEQ ID NO.

1.

2. A nanoliposome-based gut-brain targeted delivery system for mood-regulating substances, characterized in that: The system is based on a mood regulating substance as a core, a nanoliposome as an outer shell, and the nucleic acid aptamer according to claim 1 is modified on the surface of the outer shell.

3. The delivery system according to claim 2, wherein The nano liposome is a mixture consisting of cationic lipid material L319, DSPC, DMG-PEG2000 and cholesterol.

4. The delivery system according to claim 3, wherein The weight portion of the cationic lipid material is 50-80, the weight portion of the DSPC is 5-15, the weight portion of the DMG-PEG2000 is 5-10, and the weight portion of the cholesterol is 10-25.

5. The delivery system according to claim 4, wherein The weight portion of the cationic lipid material is 50, the weight portion of the DSPC is 15, the weight portion of the DMG-PEG2000 is 10, and the weight portion of the cholesterol is 25.

6. The delivery system according to claim 4, wherein The weight portion of the cationic lipid material is 60, the weight portion of the DSPC is 10, the weight portion of the DMG-PEG2000 is 10, and the weight portion of the cholesterol is 20.

7. The delivery system according to claim 4, wherein The weight portion of the cationic lipid material is 75, the weight portion of the DSPC is 10, the weight portion of the DMG-PEG2000 is 5, and the weight portion of the cholesterol is 10.

8. The method for preparing the nanoliposome-based gut-brain targeted delivery system for mood regulating substances according to any one of claims 2 to 7, characterized in that: The method comprises: Weigh L319, DSPC, DMG-PEG2000, and cholesterol according to the stated weight portions, and take out 10-20 mg of the mixture for later use; Take out 0.5-1 mg of the mood-regulating substance and add it to 10-20 mg of the mixture from step 1), dissolve it in 5-10 mL of dichloromethane, evaporate it to dryness, and then add 1-5 mL of sterile phosphate-buffered saline to dissolve it; Add 0.5-1 mg of nucleic acid aptamer to the solution described in step 2), and shake at 300-500 RPM for 5-10 hours to obtain a nanoliposome-based gut-brain targeted delivery system for mood regulating substances.

9. The preparation method according to claim 8, wherein The mood regulating substance includes but is not limited to at least one of dopamine, serotonin, norepinephrine, gamma-aminobutyric acid, glutamate, cortisol, adrenaline, endorphin, oxytocin, melatonin, and cannabinoids.

10. The preparation method according to claim 8, characterized in that The mood regulating substance includes at least one of dopamine, 5-hydroxytryptamine or gamma-aminobutyric acid.

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