阅读说明：本技术 负载催产素的壳聚糖纳米凝胶在制备阿尔茨海默症早期干预药物中的应用 (Application of oxytocin-loaded chitosan nanogel in preparation of early intervention drug for Alzheimer's disease ) 是由 窦妍 叶才华 汪俊萍 于春水 于 2021-09-15 设计创作，主要内容包括：本发明涉及负载催产素的壳聚糖纳米凝胶在制备阿尔茨海默症早期干预药物中的应用。本发明研究发现负载催产素的壳聚糖纳米凝胶在早期治疗阿尔兹海症方面具有显著的功效。细胞水平实验结果表明,负载催产素的壳聚糖纳米凝胶可以抑制脂多糖所致的小胶质细胞激活,并减少小胶质细胞中炎症因子的表达。动物水平实验结果表明,负载催产素的壳聚糖纳米凝胶可以显著降低阿尔茨海默症早期模型小鼠海马炎症因子的表达水平,显著减少β-淀粉样蛋白沉积及神经元损伤,显著提高阿尔茨海默症早期模型小鼠的学习和记忆能力。且负载催产素的壳聚糖纳米凝胶制备过程简单,反应条件温和,易表面修饰。(The invention relates to application of chitosan nanogel loaded with oxytocin in preparation of an early intervention drug for Alzheimer's disease. The research of the invention finds that the chitosan nano gel loaded with oxytocin has obvious effect on treating Alzheimer's disease in the early stage. Cell level experiment results show that the chitosan nanogel loaded with oxytocin can inhibit the activation of microglia caused by lipopolysaccharide and reduce the expression of inflammatory factors in the microglia. Animal level experiment results show that the oxytocin-loaded chitosan nanogel can obviously reduce the expression level of hippocampal inflammatory factors of early-stage model mice of Alzheimer's disease, obviously reduce beta-amyloid deposition and neuron damage and obviously improve the learning and memory abilities of the early-stage model mice of Alzheimer's disease. And the chitosan nanogel loaded with oxytocin has simple preparation process, mild reaction condition and easy surface modification.)

1. The chitosan nanogel loaded with oxytocin is applied to the preparation of early intervention drugs for Alzheimer's disease.

2. The use of claim 1, wherein the oxytocin-loaded chitosan nanogel is an oxytocin-loaded chitosan nanogel that is functionalized or not functionalized;

the chitosan nanogel loaded with oxytocin is modified by functional molecules crossing the blood brain barrier and loaded with oxytocin;

the oxytocin in the chitosan nanogel loaded with the oxytocin realizes loading by forming a hydrogen bond between amino and hydroxyl of chitosan.

3. The use according to claim 2, wherein the chitosan comprises any one of or a combination of at least two of carboxylated chitosan, pegylated chitosan, polyvinylalcohol chitosan, and polyethyleneoxide chitosan.

4. The use of claim 2, wherein the oxytocin comprises any one or a combination of oxytocin and oxytocin acetate.

5. The use of claim 2, wherein the functional molecule that crosses the blood brain barrier comprises any one of Angiopep-2, TAT peptide, transferrin, lactoferrin, or RVG29, or a combination of at least two thereof.

6. The use according to claim 2, wherein the oxytocin-loaded chitosan nanogel has a particle size of 75-200 nm.

7. The use of any one of claims 1 to 6, wherein the medicament is in a dosage form selected from the group consisting of an injection, a spray, a nasal drop, a drop pill, a solution, a suspension, a granule and a capsule.

8. The use of any one of claims 1 to 6, wherein the route of administration of the medicament comprises intravenous, intramuscular, subcutaneous, intraperitoneal, oral, sublingual or nasal administration.

9. The application of chitosan nanogel loaded with oxytocin in the preparation of an inflammation reaction marker expression inhibitor, wherein the inflammation reaction markers comprise microglia activation markers Iba-1, inflammatory factors TNF-alpha and inflammatory factors IL-1 beta; the chitosan nanogel loaded with oxytocin is the chitosan nanogel loaded with oxytocin for use according to any one of claims 1 to 6.

10. A drug for early intervention of Alzheimer's disease, which comprises chitosan nanogel loaded with oxytocin for the use of any one of claims 1-6, and can also be used for preparing drugs for other nervous system degenerative diseases related to inflammatory pathogenesis.

Technical Field

The invention belongs to the technical field of biological medicines, relates to a new application of chitosan nanogel loaded with oxytocin, and particularly relates to an application of chitosan nanogel loaded with oxytocin for early intervention of Alzheimer's disease through anti-inflammatory effect.

Background

Alzheimer's Disease (AD) is a common neurodegenerative disorder characterized by progressive memory loss and cognitive decline. Beta-amyloid (a β) deposition and tau hyperphosphorylation are considered as the main pathological features of AD, but early clinical intervention for both ends up largely failing. Microglial-mediated chronic inflammation occurs early in AD, preceded by the accumulation of the two pathogenic proteins. Since the innate immune response is constantly triggered to restore tissue homeostasis, it leads to excessive activation of microglia, sustained release of a large number of inflammatory mediators, induction of cascade reactions and vicious circle, and finally to neuronal degeneration. Therefore, inhibition of early neuroinflammation is expected to slow or block early progression of AD.

Oxytocin has been proved to reduce inflammation of macrophage and microglia caused by lipopolysaccharide and to protect mouse from acute lung injury. Recent studies show that oxytocin can promote hippocampal plasticity and reverse A beta-induced long-term potentiation (LTP) injury of mouse hippocampus. The expression of the oxytocin receptor of microglia in the brain is up-regulated with the progress of AD, and a large number of binding sites are provided for the oxytocin to play an anti-inflammatory role. Exogenous oxytocin can be used as a promising anti-inflammatory drug for early intervention of AD due to the extremely low oxytocin content in the central nervous system. However, the short half-life of exogenous oxytocin leads to low effective concentration in the brain, which greatly limits its application in the central nervous system.

As an effective nano-drug carrier, the chitosan nano-gel has the advantages of simple preparation process, good biocompatibility and the like, and is widely applied to the field of biomedicine for many years. For example, CN104817660A discloses a method for preparing modified carboxymethyl chitosan nanogel, which prepares modified carboxymethyl chitosan nanogel by free radical polymerization with N, N-bis (acryloyl) cystamine as a cross-linking agent, and the nanogel successfully loads anticancer drug doxorubicin hydrochloride and has better pH and reduction sensitivity. For example, CN107496984A discloses a preparation method and application of a nano-dicalcium silicate/nano-silver loaded chitosan gel, wherein the nano-dicalcium silicate/nano-silver loaded chitosan gel is obtained by mixing a nano-dicalcium silicate/nano-silver suspension and a chitosan/acetic acid solution under the action of a genipin cross-linking agent; it has the functions of promoting bone formation and resisting bacteria, can guide the regeneration of bone tissue around the implanted bone and can prevent bone absorption and infection around the implanted bone.

However, no related report exists on the application of chitosan nanogel loaded with oxytocin to the research of early intervention of Alzheimer's disease.

Disclosure of Invention

The invention aims to solve the problem that the application of exogenous oxytocin to early intervention of Alzheimer's disease is limited due to low effective action concentration in brain caused by short half-life period of exogenous oxytocin, and provides the application of chitosan nanogel loaded with oxytocin to the preparation of early intervention medicaments of Alzheimer's disease, namely the early intervention of Alzheimer's disease through anti-inflammatory effect.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides application of chitosan nanogel loaded with oxytocin in preparation of an early intervention drug for Alzheimer's disease.

The oxytocin-loaded chitosan nanogel disclosed by the invention performs early intervention on Alzheimer's disease through anti-inflammatory effect. The cell level experiment result of the invention shows that the chitosan nanogel loaded with oxytocin can inhibit the activation of microglia caused by lipopolysaccharide and reduce the expression of inflammatory factors in the microglia. Animal level experiment results show that the chitosan nanogel loaded with oxytocin can obviously reduce the expression level of hippocampal inflammatory factors of an AD early model mouse, obviously reduce Abeta deposition and neuron damage and obviously improve the learning and memory abilities of the AD early model mouse.

The method for loading oxytocin by chitosan nanogel related by the invention can be prepared by the technicians in the field according to the conventional method disclosed in the prior art, and the invention does not specifically limit the preparation method of the chitosan nanogel loaded with oxytocin. Illustratively, the chitosan nanogel loaded with oxytocin can be prepared by a one-step method, and the specific preparation steps comprise the following steps:

(1) dissolving chitosan in deionized water, and adjusting the pH value to 4.8-5.2; then oxytocin is added under magnetic stirring, and the mass ratio of the chitosan to the oxytocin is 2: 1, stirring at normal temperature (850 rpm) for 3-5 minutes;

(2) adding a sodium tripolyphosphate solution, wherein the mass ratio of chitosan to sodium tripolyphosphate is 4.2: and 1, magnetically stirring for 3-5 minutes at normal temperature, and centrifugally washing to obtain the chitosan nanogel which is not subjected to functional modification and is loaded with oxytocin.

The chitosan nanogel loaded with oxytocin is the chitosan nanogel loaded with oxytocin which is subjected to functional modification or is not subjected to functional modification. The oxytocin in the chitosan nanogel loaded with the oxytocin realizes loading by forming a hydrogen bond between amino and hydroxyl of chitosan. The particle size of the chitosan nanogel loaded with oxytocin is 75-200 nm.

The chitosan nanogel loaded with oxytocin has the advantages of simple preparation process, mild reaction conditions and easy surface modification, and a person skilled in the art can perform functional modification on the surface according to the actual application requirement.

Preferably, the chitosan nanogel loaded with oxytocin is the chitosan nanogel loaded with oxytocin and crossing blood brain barrier functional molecules. The functional molecule crossing the blood brain barrier comprises any one of Angiopep-2, TAT peptide, transferrin, lactoferrin or RVG29 or a combination of at least two of the same.

Preferably, the chitosan comprises any one of or a combination of at least two of carboxylated chitosan, pegylated chitosan, polyvinylalcohol chitosan and polyethylene oxide chitosan.

Preferably, the oxytocin comprises any one or a combination of two of oxytocin and oxytocin acetate.

Preferably, the chitosan nanogel loaded with oxytocin is the chitosan nanogel loaded with oxytocin contained in the pharmaceutical composition.

The oxytocin-loaded chitosan nano gel provided by the invention can also be matched with other bioactive components with anti-inflammatory effects according to different proportions to form a pharmaceutical composition, and the chitosan nano gel plays a role together.

Preferably, the dosage form of the medicament comprises injection, spray, nasal drops, dripping pills, solutions, suspensions, granules or capsules.

Preferably, the route of administration of the medicament comprises intravenous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, oral administration, sublingual administration or nasal administration.

The oxytocin-loaded chitosan nanogel provided by the invention can also be applied to the preparation of medicines for treating other nervous system degenerative diseases related to inflammation pathogenesis, and not only is the Alzheimer disease, but also the depression, the multiple sclerosis, the Parkinson disease and the Huntington disease.

The occurrence and development of the degenerative diseases of the nervous system are all related to inflammatory reaction, namely, in the early stage of the diseases, the innate immune response is continuously triggered to recover the tissue homeostasis, so that the excessive activation of microglia is caused; microglial cell over-activation to release large amounts of inflammatory mediators; the resulting persistent inflammatory response can cause pathological cascade and malignant cycle, which results in damage and loss of neurons, and ultimately the above-mentioned diseases.

The invention also provides application of the oxytocin-loaded chitosan nanogel in preparation of an inflammation reaction marker expression inhibitor, wherein the inflammation reaction markers comprise a microglia activation marker Iba-1, an inflammation factor TNF-alpha and an inflammation factor IL-1 beta.

The invention also provides application of the oxytocin-loaded chitosan nanogel in preparation of an A beta deposition marker amyloid protein MOAB-2 and a cell apoptosis TUNEL expression inhibitor.

The invention provides a medicament for early intervention of Alzheimer's disease, which comprises chitosan nanogel loaded with oxytocin and can also be used for preparing medicaments for other nervous system degenerative diseases related to inflammation pathogenesis.

The invention has the advantages and beneficial effects that:

the invention relates to oxytocin-loaded chitosan nanogels which have significant efficacy in early intervention of Alzheimer's disease through anti-inflammatory effects. The cell level experiment result of the invention shows that the chitosan nanogel loaded with oxytocin can inhibit the activation of microglia caused by lipopolysaccharide and reduce the expression of inflammatory factors in the microglia. Animal level experiment results show that the chitosan nanogel loaded with oxytocin can obviously reduce the expression level of hippocampal inflammatory factors of an AD early model mouse, obviously reduce Abeta deposition and neuron damage and obviously improve the learning and memory abilities of the AD early model mouse. And the chitosan nanogel loaded with oxytocin has simple preparation process, mild reaction condition and easy surface modification.

Drawings

FIG. 1 is a transmission electron microscope image of chitosan nanogel loaded with oxytocin;

FIG. 2 is a graph of the particle size characterization of chitosan nanogels loaded with oxytocin;

FIG. 3 is a potential characterization graph of oxytocin-loaded chitosan nanogels;

FIG. 4 is a graph showing the results of cellular immunofluorescence detecting microglial activation;

FIG. 5 is a graph showing the results of measuring the expression level of a cytokine by Western blotting;

FIG. 6 is a graph showing the results of Western blotting for detecting the levels of inflammatory factors and A.beta.deposits in animals;

FIG. 7 is a graph showing the results of detecting the level of neuronal apoptosis in brain slices using the TUNEL kit;

FIG. 8 is a graph showing the results of the water maze test.

Detailed Description

The technical solution of the present invention is further explained by the accompanying drawings and the detailed description. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

The reagents or starting materials mentioned in the following examples are commercially available or may be prepared according to the common general knowledge of those skilled in the art, unless otherwise specified.

Mouse microglial strain BV-2 cells were either donated by general Hospital of Tianjin medical university or purchased from ATCC cell banks, and APP/PS1 transgenic mice and C57BL/6 mice were purchased from Beijing Huafukang Biotech GmbH.

Example 1

This example prepares a nonfunctionalized chitosan nanogel loaded with oxytocin. The preparation method comprises the following steps:

(1)10mg of polyethylene glycol-modified carboxymethyl chitosan was dissolved in 10mL of ultrapure water, and the pH was adjusted to 5.0.

(2) 5mg of oxytocin was added under magnetic stirring (850 rpm), followed by 3.96mL of a 0.6mg/mL TPP solution and stirring for 3 minutes.

(3) And centrifuging the solution at 9000 r/min for 20 min to obtain chitosan nanogel loaded with oxytocin without functional modification, and storing at 4 ℃ in the dark for later use.

The prepared chitosan nanogel loaded with oxytocin is subjected to particle size and potential characterization, and the result is as follows: the dynamic light scattering particle size is 91nm, and the surface potential is about 0 mV.

Example 2

This example prepares a chitosan nanogel loaded with oxytocin modified by functional molecules crossing the blood brain barrier. The preparation method comprises the following steps:

(1)10mg of polyethylene glycol-modified carboxymethyl chitosan was dissolved in 10mL of ultrapure water, and then 400. mu.L of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride having a concentration of 10mg/mL and 200. mu.L of N-hydroxysuccinimide having a concentration of 10mg/mL were added thereto and magnetically stirred in ultrapure water for 1 hour.

(2) 3.5mg of angiopep-2 was added to the above solution, magnetically stirred under dark conditions for 6 hours, and then dialyzed in ultrapure water under dark conditions for 24 hours to obtain an angiopep-2-modified chitosan solution.

(3) The pH of the angiopep-2 modified chitosan solution was adjusted to 5.0, 10mL of the above solution was taken, 5mg of oxytocin was added thereto, followed by 3.96mL of a 0.6mg/mL sodium tripolyphosphate solution, and stirred for 3 minutes.

(4) And centrifuging the solution at 9000 r/min for 20 min to obtain the blood brain barrier crossing functional molecule modified chitosan nanogel loaded with oxytocin, and storing at 4 ℃ in a dark place for later use.

The prepared chitosan nanogel which is modified by blood brain barrier crossing functional molecules and loaded with oxytocin is subjected to the following characterization test:

the transmission electron microscope characterization is carried out, and the results are shown in figure 1: the nanogel is spherical, uniform in particle size and good in dispersity.

(II) particle size and potential characterization, the results are shown in FIG. 2 and FIG. 3: the nanogel has a particle size of about 120nm and a potential of about 8.6 mV.

Example 3

This example evaluates the efficacy of oxytocin-loaded chitosan nanogels prepared in example 2 for early intervention in alzheimer's disease.

(I) cell level assay:

a microglia cell strain BV-2 cell is taken as an experimental object, and an Alzheimer disease cell model with inflammatory response is constructed through lipopolysaccharide stimulation, wherein the method comprises the following steps: BV-2 cells were plated at 2.5X 10 per well⁵The density of individual cells was seeded in 24-well plates and cultured at 37 ℃ for 24h, and when the cells grew to around 80%, 1mL of a medium containing 50. mu.g/mL lipopolysaccharide was added and incubation continued for 24 hours.

The method is characterized in that microglia BV-2 cells are used as an experimental object, and the Alzheimer disease early stage is constructed through chitosan nanogel pretreatment loaded with oxytocinPhase cell model, procedure was as follows: BV-2 cells were plated at 2.5X 10 per well⁵The density of each cell is inoculated in a 24-well plate and cultured for 24h at 37 ℃, when the cells grow to about 80 percent, 1mL of culture medium containing 100 mu g/mL chitosan nanogel loaded with oxytocin is added for incubation for 24h, then 30 mu L of lipopolysaccharide with 50 mu g/mL is added, and the incubation is continued for 24 h. Cells without any treatment, cells incubated alone with oxytocin-loaded chitosan nanogels were used as a control group.

The degree of microglial activation was detected by cellular immunofluorescence: cells were fixed in 4% paraformaldehyde for 15 min, blocked with 5% BSA for 30 min, then incubated with anti-Iba-1 primary antibody (dilution 1: 300) and left overnight at 4 ℃. Subsequently, the cells were washed with PBS and incubated with a secondary antibody containing a red fluorescent label for 1 hour. Finally, the cells were incubated with DAPI (10 μ g/mL) for 5 minutes, washed with PBS, observed using an inverted fluorescence microscope and fluorescence images taken to investigate the inhibitory effect of the oxytocin-loaded chitosan nanogel on microglial activation. The results are shown in FIG. 4: for a blank control group and a chitosan nanogel group which is only loaded with oxytocin, the red fluorescence of two groups of cells is weak; cells in a lipopolysaccharide stimulation group show stronger red fluorescence, and the chitosan nanogel loaded with oxytocin is treated firstly and then is stimulated by lipopolysaccharide, so that the red fluorescence in the cells can hardly be detected, and the fact that the chitosan nanogel loaded with oxytocin can obviously inhibit the activation of microglia induced by lipopolysaccharide is shown.

Then collecting cells, extracting total protein, detecting the expression level of inflammatory factors in microglia by adopting a western blotting method, and researching the influence of the oxytocin-loaded chitosan nanogel on the expression of the inflammatory factors, wherein the result is shown in figure 5: compared with a blank control group, the chitosan nanogel group which is simply loaded with oxytocin can not cause the change of the levels of TNF-alpha and IL-1 beta, and lipopolysaccharide stimulation can obviously increase the expression levels of the TNF-alpha and the IL-1 beta; and through chitosan nanogel treatment firstly loading oxytocin and then lipopolysaccharide stimulation oxytocin gel pretreatment, the levels of TNF-alpha and IL-1 beta in cells are obviously reduced, and the chitosan nanogel loading oxytocin has obvious inhibition effect on the expression of inflammatory factors in lipopolysaccharide induced microglia.

(II) animal level test:

12-week-old (female) APP/PS1 transgenic mice were used as early animal models of Alzheimer's disease, and the same-week-old, female C57BL/6 mice were used as controls, and were grouped as follows: (1) wild type group: c57BL/6 mice; (2) group of alzheimer's disease: APP/PS1 mouse; (3) treatment groups: APP/PS1 mice treated with chitosan nanogel loaded with oxytocin were administered; each group had 20. For the treatment group, oxytocin-loaded chitosan nanogel (1M,100 μ L) was administered into mice by tail vein injection once a week for 12 times. After treatment, each group of mice is respectively drawn and tested for relevant indexes.

Respectively extracting total protein from each group of mouse hippocampus, and detecting the expression level of the pathological feature marker by a western blot method, wherein the expression level comprises the following steps: TNF-alpha, IL-1 beta and MOAB-2 expression. The results are shown in FIG. 6: the levels of TNF-alpha, IL-1 beta and MOAB-2 in the Alzheimer disease group are obviously higher than those of a wild type, and the levels of TNF-alpha, IL-1 beta and MOAB-2 in the treatment group are obviously lower than those of the Alzheimer disease group, so that the early intervention of the oxytocin-loaded chitosan nanogel can obviously inhibit the expression of APP/PS1 mouse hippocampal inflammatory factors, reduce the A beta deposition level and play a role in neuroprotection.

Respectively obtaining all brain tissues of each group of mice, fixing the tissues by paraformaldehyde to prepare all brain paraffin sections, and detecting the apoptosis level of neurons by a TUNEL apoptosis kit. The results are shown in FIG. 7: the TUNEL green fluorescence of the Alzheimer disease group is obviously higher than that of a wild type and mainly distributed in the hippocampus and the cortex, and the TUNEL green fluorescence level in the treatment group is obviously lower than that of the Alzheimer disease group, so that the early intervention of the chitosan nanogel loaded with oxytocin can obviously inhibit the neuronal apoptosis of the hippocampus and the cortex of an APP/PS1 mouse, and the neuroprotective effect is achieved.

After treatment, the learning and memory abilities of the mice are evaluated through a mine field test, a water maze test and a new object recognition test, and the specific method comprises the following steps: (1) and (3) mine field test: observing the autonomous behavior of the mouse, exploring the behavior and the tension. The holding experiment was performed in a quiet environment. The method comprises the following steps of putting animals into the center of the bottom surface in a box, simultaneously carrying out camera shooting and timing, observing the movement condition of mice in 120s, and recording the contents of the movement distance, the movement track and the movement speed (2) of each mouse in a water maze test: the Morris water maze test detects the space learning and memory ability of the mouse, and comprises a positioning navigation test for hiding a platform and a space exploration test for removing the platform. In a positioning navigation test, the mouse is placed into a water pool from four quadrants facing the pool wall every 5 days continuously, the time for the mouse to find a platform is observed and recorded in 60s in an experiment, and the learning ability of the mouse is inspected; and removing the platform in the last day, putting the mouse into the water from the position farthest away from the platform, observing the swimming track of the mouse, and inspecting the memory capacity of the mouse on the original platform. (3) Identification test of new objects: mice were tested for their innate ability to learn and remember to explore new objects. Including training and testing trials. In the training experiment, A, B two objects were placed on the left and right ends of a side wall, and the mouse was placed in the field with the mouse back facing the two objects, and the length of the tip of the mouse nose from the two objects was the same. The mice were placed for 10 minutes and observed to record the contact of the mice with both objects, including the number of times the nose, mouth touched the object and the time of the study within 2-3 cm from the object. And starting the test after the mouse has a rest for 1 hour, replacing the object B in the field with the object C, keeping the mouse back to the two objects, observing the nose tip for 2-5 minutes according to the same distance between the two objects, and observing the indexes as described above.

The water maze test results are shown in fig. 8: the swimming tracks of the wild mice and the treated mice show obvious tropism to the quadrant where the platform is located and the platform position, while the swimming tracks of the mice in the Alzheimer disease group have no purpose and have a circling phenomenon, which indicates that the early intervention of the chitosan nanogel loaded with oxytocin can improve the learning and memory abilities of the APP/PS1 mice.

The applicant states that the preparation and application of the oxytocin-loaded chitosan nanogel in the invention in early intervention of alzheimer disease are illustrated by the above examples, but the invention is not limited by the above examples, that is, the invention does not mean that the invention is implemented only by relying on the above examples. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.