WO2023226171A1 - Cod skin collagen peptide-loaded trimethyl chitosan nanoparticle and use thereof - Google Patents

Abstract

A cod skin collagen peptide-loaded trimethyl chitosan nanoparticle and the use thereof. A method for preparing the cod skin collagen peptide-loaded trimethyl chitosan nanoparticle, wherein the method comprises: adding an aqueous solution of sodium tripolyphosphate to a mixed aqueous solution of a cod skin collagen peptide and trimethyl chitosan to obtain the cod skin collagen peptide-loaded trimethyl chitosan nanoparticle. The cod skin collagen peptide-loaded trimethyl chitosan nanoparticle can effectively improve the bioavailability of the cod skin collagen peptide, can be prepared into an oral preparation for oral administration, and can effectively prevent or alleviate the damage caused by oxidative stress to the brain of an organism.

Description

Trimethylchitosan nanoparticles loaded with cod skin collagen peptide and its application Technical field

The invention belongs to the field of biomedicine technology, and specifically relates to trimethylchitosan nanoparticles loaded with cod skin collagen peptide and its application.

Background technique

As the aging of the social population becomes increasingly serious, age-related diseases have become a prominent problem affecting human health, such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, etc. Alzheimer's disease (AD) is the main subtype of Alzheimer's disease. It refers to a neurodegenerative disease that causes impairment of memory and other cognitive functions due to degeneration of the nervous system and is severe enough to interfere with daily life. Fashion trends receive a lot of attention. AD patients will initially experience symptoms such as progressive memory decline, cognitive dysfunction, loss of motor function, and personality changes. Later, they will lose their ability to take care of themselves, such as speaking, moving, and swallowing functions. Their quality of life will be severely reduced, and eventually lead to death, which will bring great harm to the patient and himself. It has brought great mental pressure and financial burden to the family members. At the same time, the increasing incidence of AD has caused serious public health problems. The pathogenesis of AD is complex and is caused by the long-term interaction of multiple factors. Regarding its pathogenesis, many theories have been proposed based on its clinical manifestations, pathological characteristics and related pathogenic factors, including the cholinergic injury theory, β-amyloid protein theory, tau protein theory, oxidative stress theory, calcium homeostasis theory, estrogen reduction theory, etc. Increasing evidence shows that oxidative stress and free radicals play an important role in the development of AD disease. The damage caused by oxidative stress in the brain of AD patients obviously precedes the occurrence of β-amyloid deposition and neurofibrillary tangles. It is considered to be the starting signal of a series of pathological process cascades, thereby accelerating the pathological progression of AD. . Therefore, treating AD based on this mechanism may become an important way to treat Alzheimer's disease and is of great significance.

Cod skin collagen peptides (CSCPs) are small molecule active peptides with high biosafety (molecular weight generally 100-5000 Da) that are different from collagen peptides of terrestrial animals, freshwater fish, and shallow-sea fish. Many modern pharmacological research data show that selective hydrolysis of cod skin collagen peptides can produce peptides with extremely strong antioxidant activity, which can inhibit DPPH free radicals, superoxide anion free radicals, hydroxyl free radicals, etc. Oxidative stress is a potential high-quality raw material for antioxidant drugs. Due to their good selectivity and biological activity, peptide drugs often have significant efficacy, few adverse reactions, high safety, and rarely cause serious immune reactions (except for a few heterologous proteins). They have become the first-line drugs for the treatment of many diseases. .

However, because peptide drugs are water-soluble substances, their large hydrophilicity makes them difficult to be taken up by cells. They will encounter a variety of biological obstacles after the first-pass effect in the gastrointestinal tract, liver, etc., and their oral bioavailability is extremely low and easy to absorb. It is deactivated by the influence of temperature, pH and some physical factors (such as ultrasonic waves, violent vibration). In addition, due to the existence of the blood-brain barrier (BBB), approximately 98% of small molecule drugs cannot be delivered directly into the brain through peripheral administration, hindering their use in the diagnosis and treatment of brain diseases. At present, clinical peptide drugs are mainly in injection form, but the compliance of injection administration is poor.

Contents of the invention

In view of the above problems, one of the purposes of the present invention is to provide trimethyl chitosan nanoparticles loaded with cod skin collagen peptide, which improves the bioavailability of cod skin collagen peptide, can be prepared into oral preparations, and can be administered orally. Effectively prevent or reduce damage caused by oxidative stress in the body's brain.

In order to achieve the above purpose, the following technical solutions can be adopted:

In one aspect, the present invention provides trimethylchitosan nanoparticles loaded with cod skin collagen peptide, and its preparation method includes: adding sodium tripolyphosphate aqueous solution to a mixed aqueous solution of cod skin collagen peptide and trimethyl chitosan to obtain Trimethylchitosan nanoparticles loaded with cod skin collagen peptide.

Specifically, trimethylchitosan (TMC) is a derivative formed by methylation of chitosan (CS) and has a permanent positive charge. TMC is more soluble and has stronger physiological activity than CS. In recent years, TMC is a widely used drug carrier material. For oral drugs, it can enhance drug absorption through a variety of mechanisms (increasing drug absorption in the paracellular pathway, increasing drug penetration in intestinal epithelial cells, etc.), preventing drug Degradation in the body improves bioavailability, thereby enabling lower dosages and providing economic advantages for expensive drugs. The nanodrug-carrying system can be absorbed through the paracellular pathway or the M cell phagocytosis pathway of Peyer's patches in the intestinal mucosa, or directly absorbed by intestinal epithelial cells. As a drug delivery system, it can realize small molecule chemical drugs, protein and peptide drugs, and genes. Delivery of drugs for diagnosis and treatment of diseases. By utilizing nano-effects, long-acting drug release or targeted therapy can be achieved, and nanotechnology can be used in research on drug delivery to the brain. Delivery across the BBB occurs due to the electrostatic interaction between the positively charged portion of the trimethylchitosan nanoparticles and the negatively charged plasma membrane surface region on the brain capillary endothelium that triggers adsorption-mediated endocytosis (AMT) Drugs exhibit brain targeting phenomena, so nanomedicine delivery systems based on trimethylchitosan can be used for drug delivery to treat brain diseases. Research on brain-targeted drug delivery systems is of great significance for the diagnosis and treatment of various brain diseases, including Alzheimer's disease, Parkinson's disease, glioma and HIV infection.

Further, the mass ratio of trimethyl chitosan to cod skin collagen peptide can be 1:1-2.

Furthermore, trimethyl chitosan can be prepared by using dimethyl sulfate as a methylating reagent.

Furthermore, the sodium tripolyphosphate aqueous solution also includes sodium alginate.

Specifically, studies have shown that when trimethylchitosan nanoparticles prepared with sodium tripolyphosphate as a cross-linking agent increase the encapsulation rate of certain drugs, there is a burst release phenomenon in vitro. Using sodium tripolyphosphate Combining sodium alginate as a cross-linking agent to modify the prepared nanoparticles can avoid this phenomenon.

Furthermore, the particle size of trimethylchitosan nanoparticles loaded with cod skin collagen peptide can be ≤200nm.

Specifically, according to literature reports, when the particle size of microparticles is less than 10um, it can be taken up by M cells and enter the portal system through the Peryer's node area in the small intestinal epithelial intercellular space; when the particle size of the microparticles is less than 200nm, it can pass through intracellular It can also be absorbed through the intestinal mucosa through paracellular pathways. Therefore, the present invention controls the particle size of trimethyl chitosan nanoparticles loaded with cod skin collagen peptide to be below 200 nm.

Further, the sodium tripolyphosphate aqueous solution is added dropwise to the mixed aqueous solution of cod skin collagen peptide and trimethyl chitosan.

Further, cod skin collagen peptide-loaded trimethylchitosan nanoparticles are added to the scaffold and then freeze-dried to obtain a cod skin collagen peptide-loaded trimethylchitosan nanoparticle freeze-dried agent.

Specifically, in order to ensure that the solid matter in the freeze-dried powder can maintain its original volume, without collapse, shrinkage, and good redispersibility, it is sometimes necessary to add appropriate scaffolding agents. For freeze-drying of nanoparticles, sugars or alcohols are generally used. Substances act as protective agents.

Another aspect of the present invention provides a medicine for preventing or treating Alzheimer's disease, which includes the above-mentioned cod skin collagen peptide-loaded trimethylchitosan nanoparticles and a pharmaceutically acceptable carrier.

Furthermore, the pharmaceutically acceptable carrier is suitable for oral administration, pulmonary inhalation administration, nasal mucosal administration, microneedle transdermal administration or injection administration.

Specifically, the pharmaceutically acceptable carrier and/or diluent means that the above cod skin collagen peptide-loaded trimethylchitosan nanoparticles can be prepared into various desired dosage forms. For example, tablets, powders, pills, powders, granules, fine granules, soft/hard capsules, film-coated agents, pellets, sublingual tablets, ointments, etc. can be used as oral preparations. As non-oral preparations, they can be As injections, suppositories, transdermal preparations, ointments, plasters, external solutions, etc., those skilled in the art can select appropriate dosage forms according to the route of administration, administration targets, etc. For example, in order to improve compliance, it is preferable to prepare it as an oral dosage form.

Another aspect of the present invention provides the use of the above cod skin collagen peptide-loaded trimethylchitosan nanoparticles in preparing preparations for preventing or treating Alzheimer's disease.

Another aspect of the present invention provides the use of the above cod skin collagen peptide-loaded trimethylchitosan nanoparticles in the preparation of preparations for preventing or reducing damage caused by oxidative stress in the brain of the body.

The beneficial effects of the present invention at least include:

(1) The cod skin collagen peptide-loaded trimethylchitosan nanoparticles provided by the present invention can effectively improve the bioavailability of the cod skin collagen peptide, can be prepared into oral preparations for oral administration, and can effectively prevent or alleviate the symptoms of intracranial infarction in the body's brain. Damage caused by oxidative stress.

(2) The cod skin collagen peptide-loaded trimethylchitosan nanoparticles provided by the present invention are prepared by an ion gelation method, which is simple to operate, does not require the use of organic solvents, is gentle in the process, and can effectively protect the activity of the cod skin collagen peptide.

Description of the drawings

Figure 1 shows the infrared spectrum of TMC.

Figure 2 shows the hydrogen nuclear magnetic spectrum of TMC.

Figure 3 is a macroscopic view of nanoparticle colloids (from left to right, TMC-TPP/SL NPs, TMC-TPP-CSCPs NPs, TMC-TPP/SL-CSCPs NPs and deionized water).

Figure 4 shows the absorption curve of CSCPs.

Figure 5 shows the standard curve of CSCPs.

Figure 6 shows the cumulative release rate of TMC-TPP-CSCPs NPs and TMC-TPP/SL-CSCPs NPs.

Figure 7 shows the electron microscope image of TMC-TPP/SL-CSCPs NPs.

Figure 8 shows the particle size distribution diagram of TMC-TPP/SL-CSCPs NPs.

Figure 9 shows the zeta potential of nanoparticles made from TMC with different levels of quaternary ammonia.

Figure 10 is a macroscopic view of the freeze-dried preparation.

Figure 11 is an electron microscope image of the freeze-dried preparation.

Figure 12 is a particle size distribution diagram of the freeze-dried preparation.

Figure 13 is a macroscopic state diagram of the mouse.

n＝6；*,P<0.05；**,P<0.01)。 Figure 14 shows the results of acquired training in mice (

n=6; *, P<0.05; **, P<0.01).

Figure 15 is a mouse acquisition training path diagram.

Figure 16 is a diagram of the mouse’s spatial exploration path.

Detailed ways

The examples are given to better illustrate the present invention, but the content of the present invention is not limited to the examples. Therefore, those skilled in the art can make non-essential improvements and adjustments to the embodiments based on the above-mentioned content of the invention, which still fall within the protection scope of the present invention.

The terminology used herein is used to describe particular embodiments only and is not intended to limit the disclosure. Expressions in the singular include expressions in the plural unless there is a clearly different meaning in the context. As used herein, it should be understood that terms such as "includes," "has," "comprises" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, assemblies, parts, elements, materials or combinations thereof may be present or may be added. As used herein, "/" may be interpreted as "and" or "or" depending on the context.

In order to better understand the present invention, the content of the present invention is further clarified below with reference to specific examples, but the content of the present invention is not limited only to the following examples.

Example 1 Synthesis and characterization of trimethylchitosan (TMC)

Trimethylchitosan synthesis

(1) Preparation using methyl iodide as methylating reagent (one-step method)

Weigh 2.0g chitosan and 4.8g sodium iodide into a beaker, add 80ml N-methylpyrrolidone, 11ml 15% sodium hydroxide solution, and 11.5ml methyl iodide, and react in a 60°C water bath in the dark for 90 minutes to obtain Brown reaction liquid, use a certain amount of ethanol to precipitate the reaction liquid, pour the precipitate into 10% 40ml sodium chloride solution to exchange Cl ^- for I ^- , centrifuge, then wash the precipitate twice with ethanol and ether, and dry it to obtain As a white powder, dialyze it in deionized water for 3 days and freeze-dry it to obtain TMC1.

(2) Preparation using methyl iodide as methylating reagent (two-step method)

Weigh 2.0g chitosan and 4.8g sodium iodide into a beaker, add 80ml N-methylpyrrolidone, 11ml sodium hydroxide solution and 11.5ml methyl iodide, and react in a water bath at 60°C for 60 minutes in the dark to obtain a brown color. For the reaction solution, use a certain amount of ethanol to precipitate the precipitate, and wash the precipitate twice with ethanol and ether; transfer the precipitate to a beaker, then add 80ml N-methylpyrrolidone, magnetically stir in a 60°C water bath, and evaporate the ether; Then add 4.8g sodium iodide, 11mL sodium hydroxide solution and 7mL methyl iodide, and react in a 60°C water bath for 40 minutes to obtain a brown-red reaction liquid; the reaction product is poured into 10% 40ml sodium chloride solution to exchange Cl- for I- , then use a certain amount of ethanol to precipitate the precipitate; centrifuge, wash the precipitate twice with ethanol and ether each, dry it to obtain a white powder, dialyze it in deionized water for 3 days, and freeze-dry it to obtain TMC2.

(3) Preparation using dimethyl sulfate as methylating reagent

Weigh 0.85g of chitosan and place it in a beaker. Dissolve 0.88g of NaCl in 15ml of deionized water. Place the beaker in a 40°C water bath. Turn on the stirrer and stir for 10 minutes at a constant speed to make the chitosan evenly dispersed in the solution. ; Use a pipette to measure 16ml of dimethyl sulfate into the beaker, weigh 1.2g NaOH and dissolve it in 10ml of deionized water, slowly drop it into the beaker, stir magnetically for 5h, and obtain a yellow viscous reaction liquid. The reaction is completed. Rotate and concentrate the reaction solution to evaporate the excess solvent, then transfer the concentrated solution into a dialysis bag and put it into deionized water for dialysis for 3 days. Change the deionized water every 12 hours; after dialysis, use a rotary evaporator to evaporate excess solvent. The solvent (evaporated to 1/3 of the original volume) was then left to stand in absolute ethanol for 24 hours to obtain a precipitate; the precipitate was filtered, and then washed twice with ethanol and diethyl ether each; the product was freeze-dried to obtain TMC3.

Characterization of trimethylchitosan

(1) Infrared (IR) measurement of trimethylchitosan

TMC1, TMC2 and TMC3 prepared by the above different preparation methods were pressed into tablets with KBr dry powder, and the infrared spectrum of the samples was measured with a Fourier transform infrared spectrometer (scanning wave number range is 4000-400cm ^-1 , resolution 4cm ^-1 ).

The results are shown in Figure 1. The absorption peak at 1595 cm ^-1 caused by NH bending vibration in the chitosan spectrum disappears in the TMC spectrum. In addition, a new absorption peak appears at 1473-1490cm ^-1 in the TMC spectrum, which is attributed to the asymmetric stretching vibration absorption peak of -N(CH ₃ ) ₃ , which indicates the nucleophilic center of the chitosan molecule during the reaction. The two hydrogen atoms attached to the amino group are replaced by methyl groups.

(2) Hydrogen nuclear magnetic resonance spectroscopy ( ¹ HNMR)

TMC1, TMC2 and TMC3 prepared by the above different preparation methods were respectively dissolved in D ₂ O to prepare a 10g/L solution, and analyzed with a 300MHz nuclear magnetic resonance instrument. The detection temperature was 353K, and the nuclear magnetic resonance spectrum parameters were: pulse intensity was 90° , pulse width is 8.2pm; LB=0.3Hz. The results are shown in Figure 2.

(3) Analysis of the degree of quaternization of TMC

Calculation formula for the degree of quaternization of TMC: DQ (%) = [(∫TM/∫H) The integral of the H peak in the quaternary amino group, ∫H: represents the integral of the H peak from 4.7 to 5.7ppm ¹ .

The degree of quaternization was calculated for TMC1, TMC2 and TMC3 prepared by the above different preparation methods. The degree of quaternization was 8.36%, 54.19% and 56.92% respectively.

The different degree of quaternization of TMC determines the amount of positive charge it carries. The higher the degree of quaternization, the more positive charges it carries, and the stronger the interaction with proteins and peptide drugs, so it has a negative impact on nanoparticles. has an important impact on drug loading and drug release. At the same time, the degree of quaternization of TMC plays an important role in drug penetration. Studies have shown that TMC with a high degree of quaternization is an effective absorption accelerator at pH 7.4, while TMC with a low degree of quaternization is ineffective as an absorption accelerator under this condition. Therefore, quaternization TMC at a level of 8.36% is not suitable for use in the present invention. In addition, methyl iodide is highly toxic, volatile and expensive, so it is more economical to synthesize TMC by DMS method. Therefore, TMC with a quaternization degree of 56.92% (ie, TMC3) was selected for further preparation of nanoparticles.

Example 2 Preparation and Characterization of Nanoparticles

Preparation of nanoparticles

(1) Preparation of blank nanoparticles

At room temperature, weigh 10 mg TMC and dissolve it in 5 ml deionized water, stir in a water bath; weigh 1.2 mg sodium tripolyphosphate (TPP) and dissolve it in 2 ml deionized water, and add 0.6 mg sodium alginate (SL). Among them; the obtained solution is added dropwise to the TMC solution at a speed of 1ml/min, and after magnetic stirring for 10 minutes, a blank nanoparticle colloid (TMC-TPP/SL NPs) can be obtained.

(2) Preparation of TMC-TPP-CSCPs NPs

At room temperature, weigh 10 mg TMC and 21 mg CSCPs and dissolve them in 5 ml deionized water and stir in a water bath; weigh 1.2 mg sodium tripolyphosphate (TPP) and dissolve it in 2 ml deionized water and add it dropwise at a speed of 1 ml/min. In the TMC solution, after magnetic stirring for 10 minutes, trimethylchitosan nanoparticles (TMC-TPP-CSCPs NPs) loaded with cod skin collagen peptide can be obtained.

(3) Preparation of TMC-TPP/SL-CSCPs NPs

Weigh 10 mg TMC and 21 mg CSCPs and dissolve them in 5 ml of deionized water, place them in a water bath at 25°C and stir; weigh 1.2 mg of TPP and dissolve them in 2 ml of deionized water, and add 0.6 mg of sodium alginate (SL) to the solution. Add dropwise into the mixed solution of CSCPs and TMC at a speed of 1ml/min, and stir magnetically for 10 minutes to obtain trimethylchitosan nanoparticles (TMC-TPP/SL-CSCPs) modified with sodium alginate and loaded with cod skin collagen peptide. NPs).

Characterization of Nanoparticle Colloids

(1) Color observation

The TMC-TPP/SL NPs, TMC-TPP-CSCPs NPs and TMC-TPP/SL-CSCPs NPs prepared above are shown in Figure 3. They all show white opalescence when observed with the naked eye, and the Tyndall effect appears under the irradiation of the laser pointer. , preliminarily indicating that the nanoparticle colloid was successfully prepared.

(2) Encapsulation efficiency and drug loading capacity

Determine the encapsulation efficiency and drug loading capacity by UV spectrophotometry; centrifuge the prepared colloid for 15 minutes at 10,000*g, 4°C, separate the supernatant (liquid to be tested), and use deionized water as a blank For control, measure the content of free peptide in the supernatant, and weigh the precipitate obtained after centrifugation. After vacuum freeze-drying, the encapsulation efficiency and drug loading capacity are calculated using the following formula:

Encapsulation rate = (dosing amount - free peptide amount)/dosing amount;

Drug loading = (drug dosage - free peptide amount)/dry weight of nanoparticles.

Drawing of the absorption curve: Using deionized water as the reference solution, conduct wavelength scanning measurement of the cod skin collagen peptide solution with a concentration of 0.3 mg/ml to obtain the absorption curve. The results are shown in Figure 4. From the absorption curve, it can be seen that the maximum absorption wavelength of the 0.3mg/mL standard cod skin collagen peptide solution is 212nm, and the absorbance at this wavelength is 2.029.

Drawing of the standard curve: Take six test tubes, number them and use deionized water to prepare 0, 0.3, 0.4, 0.5, 0.6, 0.7 mg/ml cod skin collagen peptide solutions. Measure the absorbance value of the solution in each tube at 212nm, and get standard curve line. The results are shown in Figure 5. The standard curve equation is y=1.8237x+0.0189, R ² =0.9942.

Determination of samples to be tested: Take 2 ml of the TMC-TPP/SL-CSCPs NPs and TMC-TPP-CSCPs NPs test solutions prepared above, dilute to 10 ml with deionized water, and measure the absorbance value at 212 nm according to the above method. Measured in triplicate. The absorbances of TMC-TPP/SL-CSCPs NPs and TMC-TPP-CSCPs NPs are 0.738 and 0.727 respectively. Substituting into the standard curve equation, the free content of TMC-TPP/SL-CSCPs NPs and TMC-TPP-CSCPs NPs can be obtained. According to the above formula calculation of encapsulation rate and drug loading capacity, the encapsulation rates of TMC-TPP/SL-CSCPs NPs and TMC-TPP-CSCPs NPs prepared above are 86.86% and 87.06% respectively, and the drug loading capacity is 72.39%. % and 81.62%, both with high encapsulation efficiency and drug loading capacity.

(3) In vitro release assay

Transfer the TMC-TPP/SL-CSCPs NPs and TMC-TPP-CSCPs NPs prepared above to the dialysis bag respectively, place them in a phosphate buffer solution with a pH of 7.4, stir in a 37°C water bath, and take 2 ml every 15 minutes. Test the external liquid, and add the same amount of release mediator to ensure that the total volume of the release medium remains unchanged. The measured values are averaged three times, and the cumulative drug release rate is calculated according to the following formula:

Q: Cumulative release rate of drug; Ve: replacement volume of PBS; V0: total volume of release medium; Ci: concentration of the release solution during the i-th replacement sampling; m _drug : total mass of drug carried by nanoparticles; n: replacement of PBS frequency.

The in vitro release results are shown in Figure 6. The cumulative release rate of non-SL-modified nanoparticles (TMC-TPP-CSCPs NPs) within 2 hours was 52.73%, and reached 81.57% within 6 hours; while the SL-modified nanoparticles (TMC- The cumulative release rate of TPP/SL-CSCPs NPs) within 2 hours was only 25.52%, reached 58.37% within 6 hours, and reached 79.71% after 24 hours, indicating that as a sustained and controlled release preparation, TMC-TPP/SL-CSCPs NPs is better and can Continuously and slowly releases cod skin collagen peptide to extend the drug's action time.

(3) Morphological observation

The TMC-TPP/SL-CSCPs NPs prepared above were dried with infrared and placed on the stage, sprayed with gold to prepare samples, and then placed the samples in a Hitachi S-4800 scanning electron microscope, and observed the particles at an accelerating voltage of 5kV. Form and take photos. The results are shown in Figure 7. The nanoparticles prepared by the present invention are nearly spherical, but a large number of adhesion and agglomeration phenomena occur. It can be seen that the nanoparticles prepared by the present invention are not suitable for storage in liquid form.

(4) Zeta potential, particle size and distribution measurement

After ultrasonicating the TMC-TPP/SL-CSCPs NPs (TMC, 56.92%) sample prepared above for 5 minutes, remove a certain amount of colloid and add it to the nanoparticle size and Zeta potential analyzer (DLS) for detection, and record the potential and particle size. path. The prepared TMC-TPP/SL NPs (TMC, 8.36%/54.19%/56.92%) sample was ultrasonicated for 5 minutes, and a certain amount of colloid was removed and added to the nanoparticle size and Zeta potential analyzer (DLS) for detection, and the potential was recorded. .

The particle size results are shown in Figure 8. The particle size of the nanoparticles prepared above is around 100 nm, but due to the instability of the prepared colloid, the particle size distribution is very uneven.

The important significance of zeta potential is that its value is related to the stability of colloidal dispersion. Zeta potential is a measure of the strength of mutual repulsion or attraction between particles. The smaller the molecules or dispersed particles, the higher the absolute value (positive or negative) of the Zeta potential, and the more stable the system, that is, dissolution or dispersion can resist aggregation. On the contrary, the lower the Zeta potential (positive or negative), the more likely it is to condense or agglomerate, that is, the attractive force exceeds the repulsive force, and the dispersion is destroyed and coagulation or agglomeration occurs. The potential test results are shown in Figure 9. The potential of nanoparticles prepared from TMC (8.36%) is negative, and the potential of nanoparticles prepared from TMC (54.19%, 56.92%) is positive. The potential has a certain value after loading cod skin collagen peptide. degree of decline. It is generally believed that the colloid is unstable when the zeta potential value is below |30mV|, and the stability is relatively good when it is above |30mV|. Therefore, it can be seen from the zeta potential value that the stability of the colloid after loading the peptide is poor, which is not conducive to storage and use, so it is convenient for subsequent follow-up. For use, make it into a freeze-dried preparation for storage.

Example 3 Preparation and Characterization of Nanoparticle Lyophilized Agent

Preparation of nanoparticle lyophilizer

When freeze-drying the sample, add 5% sucrose as a scaffolding agent to the prepared colloid, shake to dissolve, pre-freeze it in a -40°C ultra-low temperature freezer for 3 hours according to the conventional method, and then freeze-dry it in a vacuum freeze dryer to obtain the result. Freeze-dried product is required. The TMC-TPP/SL-CSCPs NPs lyophilized agent was placed in the refrigerator (4°C, -20°C) and taken out after 1, 2, and 3 months to examine the appearance and redispersibility.

Characterization of Nanoparticle Lyophilized Formulation

(1) Appearance evaluation

The macroscopic view of the freeze-dried preparation prepared above is shown in Figure 10. It can basically maintain the original volume, does not collapse or shrink, has a fine texture, a smooth surface, uniform color, no spots, is loose, and can fall off in one piece but does not fall apart. , has a certain brittleness.

(2) Morphological observation

The freeze-dried preparation was placed on the stage, sprayed with gold to prepare the sample, and then the sample was placed in a Hitachi S-4800 scanning electron microscope. The morphology of the particles was observed and photographed at an accelerating voltage of 5kV.

The electron micrograph of the freeze-dried preparation is shown in Figure 11. The overall size and shape of the prepared nanoparticles are relatively uniform and spherical, with occasional adhesion.

(3) Zeta potential, particle size and distribution measurement

After the TMC-TPP/SL-CSCPs NPs lyophilized agent prepared above was reconstituted with deionized water, it was sonicated for 5 minutes. A certain amount of colloid was removed and added to the nanoparticle size and Zeta potential analyzer (DLS) for detection, and the potential was recorded. and particle size. The particle size distribution of the freeze-dried preparation is shown in Figure 12. The particle size is around 90 nm, which is not significantly different from that before freeze-drying.

(4) Research on resolubility and stability

After 1, 2, and 3 months, take the TMC-TPP/SL-CSCPs NPs freeze-dried agent and observe the appearance. No obvious changes are found. After adding deionized water and shaking, the colloid can be quickly dispersed into a uniform colloid with good resolubility. Good dispersion.

Example 4 Animal Test

(1) Experimental animals

Kunming male mice, 6-8 weeks old, weighing 35-45 grams, SPF grade, animal feeding conditions: (25±2)℃, relative humidity: (55±5)%.

(2) Experimental animal grouping and modeling

The model was created by daily subcutaneous injection of D-galactose in the back of the neck and the corresponding test substance was administered daily. The specific operation is shown in Table 1.

Table 1 Experimental animal grouping and modeling

(3) Observation of general status of mice

Observe the general status of mice in each group every day, such as drinking water, food intake, activity, changes in coat color and luminosity, etc., and compare between each group (take pictures once a week), as shown in Figure 13.

The first week was adaptive feeding. The mice in each group had a normal diet, responded quickly, had white and thick hair, and were in good spirits. After eight weeks of modeling, the mice in the N group had a normal diet, moved more quickly, had bright coat color, and were in good spirits. Good mental state; Mice in group M showed symptoms such as decreased diet, unresponsiveness, dim and bunched coats, and listlessness, indicating that the modeling was successful; mice in group J had similar physical signs to those in group M, with slow activity and decreased activity. , lethargic and likes to curl up, the hair color gradually turns yellow and sparse, and the mental state declines, indicating that the matrix has no obvious improvement effect on the physical signs of D-Gal-induced mice; compared with the M group, the physical signs, mental status, etc. of the T group are somewhat improved The degree of improvement is not obvious, indicating that cod skin collagen peptide can resist D-Gal-induced damage to mice to a certain extent; the behavior, hair condition, mental state, etc. of the mice in the NPs group were significantly improved, almost close to the normal group mice, indicating that the TMC-TPP/SL-CSCPs NPs prepared in this experiment have certain effects on improving the appearance and spirit of D-gal-induced aging mice.

(4)Morris Water Maze

Before the start of the test, the mice were put into the pool (without placing the platform) to swim freely for 1 minute to familiarize them with the maze environment. After that, a five-day training began, with 4 training sessions at a fixed time every day. During the 4 training sessions, the mice were put into the water from four different starting points (different quadrants). At the beginning of training, place the platform in the second quadrant. After the mouse finds the platform or cannot find the platform within 60 seconds (the experimenter takes it to the platform), it rests on the platform for 15 seconds before conducting the next test. The free video recording system records the time it takes for the mouse to find the platform (escape latency) and the swimming path. The mouse cannot find the platform within 60 seconds (the latency is recorded as 60 seconds). The average of the latency of the four training times of the mouse is used every day. As the day’s academic performance. On the 6th day, the original platform was removed for testing, and the mice were put into the water from the center of the fourth quadrant. All mice must enter the water at the same point, and the number of times the mice crossed the original platform within 1 minute was recorded.

Memory acquisition ability experimental results

The water maze test is a very effective method to test the spatial learning and memory ability of rodents. In this example, the escape latency of each group of mice showed a decreasing trend during training (D1-D5), indicating that the spatial learning and memory ability of mice increased with the increase in training times. However, the escape latency of mice in group M decreased very slowly within five days, indicating that their brain damage was obvious. The escape latency of mice in group N decreased relatively fastest. The decrease rate of the other groups was between group N and group M. Group P The decrease was particularly significant in the and NPs groups.

On the first day of acquisition training, no significant differences were found between mice in each group. From the third day on, the escape latency of mice in group M increased significantly compared with group N (P<0.01), indicating that D-gal causes degeneration. The model was successfully established; compared with mice in group M, the escape latency period of mice in group T was shortened, but no significant difference was found (P>0.05); compared with mice in group M, there was no difference. This shows that the matrix has no interfering effect on this injury mouse model; compared with mice in group P and NPs and mice in group M, and mice in NPs group compared with group T, the escape latency was significantly reduced, and there was a significant difference on days 3-4 (P< 0.05), there was a very significant difference (P<0.01) on the 5th day, which was statistically significant. The results show that using nanoparticles as carrier materials is more effective in treating brain damage in mice caused by D-gal than using peptides alone, as shown in Figure 14. In addition, it can be seen from Figure 15 that the movement routes of mice in groups M and J are irregular, while the mice in groups N, P, and NPs have a clear purpose of finding the platform, indicating that the TMC-TPP/SL prepared in this experiment -CSCPs NPs can improve the spatial learning ability of aging model mice.

Space exploration experiment results

The results of mouse space exploration are shown in Table 2 and Figure 16. Compared with N, the time spent by mice in group M in the target quadrant was significantly reduced (P<0.01), and the number of times they crossed the original platform was significantly reduced (P<0.01), indicating continuous injection. D-gal impairs memory in mice. Compared with the mice in the M group, the mice in the P group, T group and NPs group increased their stay time in the target quadrant and the number of times they crossed the platform to varying degrees. Sexual differences, and compared with mice in the T group, the time spent in the target quadrant and the number of times crossing the platform were significantly increased in the mice in the NPs group (P<0.01), which shows that TMC-TPP/SL-CSCPs NPs can improve aging model mice. spatial memory ability.

n＝6) Table 2 Mouse exploration training results (

n=6)

Group Number of target quadrant crossings (times) Residence time in target quadrant (s) N 5.17±2.04 17.22±2.91 M 1.5±2.07 ^## 8.95±4.22 ^## P 4.17±1.17 ^** 14.71±5.02 ^* T 3.5±0.84 ^* 10.73±1.3 J 1.67±1.37 9.2±4.44 NPs 4.83±1.72 ^** 15.22±3.57 ^**,§

Note: #, vs.N; *, vs.M; §, vs.T; ##/**, P<0.01; */§, P<0.05.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified. Modifications or equivalent substitutions without departing from the spirit and scope of the technical solution of the present invention shall be covered by the claims of the present invention.

Claims (10)

A cod skin collagen peptide-loaded trimethyl chitosan nanoparticle is characterized in that its preparation method includes: adding sodium tripolyphosphate aqueous solution to a mixed aqueous solution of cod skin collagen peptide and trimethyl chitosan to obtain cod skin collagen peptide and trimethyl chitosan nanoparticles. Collagen peptide trimethyl chitosan nanoparticles. Trimethylchitosan nanoparticles loaded with cod skin collagen peptide according to claim 1, characterized in that the mass ratio of trimethyl chitosan to cod skin collagen peptide is 1:1-2. The cod skin collagen peptide-loaded trimethylchitosan nanoparticles according to claim 1 is characterized in that the trimethylchitosan is prepared by using dimethyl sulfate as a methylating reagent. The cod skin collagen peptide-loaded trimethylchitosan nanoparticles according to claim 1, wherein the sodium tripolyphosphate aqueous solution further includes sodium alginate. The cod skin collagen peptide-loaded trimethylchitosan nanoparticles according to any one of claims 1 to 4, characterized in that the sodium tripolyphosphate aqueous solution is added to a mixed aqueous solution of cod skin collagen peptide and trimethyl chitosan. The method in is dripping. The cod skin collagen peptide-loaded trimethylchitosan nanoparticles according to any one of claims 1 to 4, characterized in that the cod skin collagen peptide-loaded trimethylchitosan nanoparticles are added to the scaffold and then freeze-dried. DeZai cod skin collagen peptide trimethyl chitosan nanoparticles freeze-dried agent. The medicine for preventing or treating Alzheimer's disease is characterized by comprising the cod skin collagen peptide-loaded trimethyl chitosan nanoparticles described in any one of claims 1 to 6 and a pharmaceutically acceptable carrier. The medicine for preventing or treating Alzheimer's disease according to claim 7, characterized in that the pharmaceutically acceptable carrier is suitable for oral administration, pulmonary inhalation administration, nasal mucosal administration, and microneedle transdermal administration. Any dosage form of administration or injection. Application of the cod skin collagen peptide-loaded trimethylchitosan nanoparticles described in any one of claims 1 to 6 in the preparation of preparations for preventing or treating Alzheimer's disease. Application of the cod skin collagen peptide-loaded trimethylchitosan nanoparticles described in any one of claims 1 to 6 in the preparation of preparations for preventing or alleviating damage caused by oxidative stress in the brain of the body.

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