Brain-targeted NMN liposome and application thereof in preparation of drugs for treating neurodegenerative diseases

Abstract

The invention discloses a brain-targeted NMN liposome and application of the brain-targeted NMN liposome in preparation of drugs for treating neurodegenerative diseases. According to the targeted liposome, targeted modification is carried out through a chemical reaction between an amino group contained in transferrin and a carboxyl group of the liposome. According to the invention, the active ingredient NMN of the medicine is entrapped in the liposome, and the medicine-carrying liposome is subjected to transferrin modification, so that brain tissue targeting is realized, and the blood brain barrier permeability of the medicine is enhanced, thereby promoting the release and enrichment of the medicine at a focus part and improving the medicine effect. The drug-loaded liposome can be used for preparing a Parkinson's disease treatment drug, and has an important clinical value.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a brain-targeted NMN liposome and an application thereof in preparing a neurodegenerative disease drug. Background Art

[0002] Neurodegenerative diseases are caused by the loss of neurons and / or their myelin sheaths, which deteriorate over time and lead to neurological dysfunction. The pathogenic mechanism is complex, and common causes include oxidative stress, mitochondrial dysfunction, excitotoxins, and immune inflammation. According to the occurrence and progression of the disease, neurodegenerative diseases can be divided into acute and chronic. The former mainly includes cerebral ischemia, brain injury, and epilepsy; the latter includes Alzheimer's disease, Parkinson's disease, Huntington's disease, Pick's disease, amyotrophic lateral sclerosis, and different types of spinocerebellar ataxia. Among them, Parkinson's disease (PD) is a progressive central nervous system disease that affects the motor system. With the acceleration of the aging process of the social population, the incidence of PD has risen sharply. At present, the number of PD patients in my country accounts for about half of the global total. The prevalence rate of people over 65 years old is 1.7%, and it is expected to exceed 5 million by 2030. It has become a serious global health problem, causing a heavy social and economic burden. Since surgery cannot cure PD, clinical treatment for PD is mainly drug therapy, and there is an urgent need to develop safe and efficient PD treatment drugs. Even levodopa, which is currently known as the "gold standard" for the treatment of PD, can only improve clinical symptoms and cannot block its pathogenic mutations. Therefore, using the advantages of multiple pathways and multiple targets to comprehensively regulate brain function and thus improve patients' neurological dysfunction will become an important direction for the development of PD treatment drugs, and the prospects are broad. Summary of the invention

[0003] The purpose of the present invention is to overcome the defects of the prior art and provide a brain-targeted NMN liposome and its use in preparing neurodegenerative disease drugs, specifically a brain-targeted NMN liposome and its use in preparing PD therapeutic drugs.

[0004] The present invention increases the ability of liposomes to penetrate the blood-brain barrier by modifying the functional ligand transferrin with the ability to penetrate the blood-brain barrier onto the surface of liposomes; at the same time, the present invention encapsulates NMN into targeted liposomes to promote the effect of NMN in the brain.

[0005] The object of the present invention is to provide a brain-targeted NMN liposome.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned brain-targeted liposomes loaded with NMN.

[0007] Another object of the present invention is to provide an application of the above-mentioned brain-targeted liposome loaded with NMN.

[0008] A brain-targeted NMN liposome, the brain-targeted NMN liposome consisting of a drug-loaded liposome and transferrin, wherein the mass ratio of the transferrin to the liposome is 1:35-45;

[0009] The drug-loaded liposomes are composed of drug NMN and liposomes, and the mass ratio of the drug to the liposomes is 1:10-50.

[0010] Wherein, the drug NMN is β-nicotinamide mononucleotide and / or its salt,

[0011] The liposome comprises lecithin, cholesterol, DSPE-PEG2000 (distearoylphosphatidylethanolamine-polyethylene glycol), DSPE-PEG2000-COOH (distearoylphosphatidylethanolamine-polyethylene glycol-carboxyl), and, by mole fraction, lecithin comprises 76 to 82 parts, cholesterol comprises 6 to 9 parts, DSPE-PEG2000 comprises 5 to 8 parts, and DSPE-PEG2000-COOH comprises 0.5 to 0.8 parts.

[0012] Preferably, the lecithin for preparing liposomes is soybean lecithin or egg yolk lecithin, and the transferrin is lactoferrin (Lf).

[0013] The present invention provides a specific drug-loaded liposome through specific experiments, which is obtained by encapsulating the drug in the targeted liposome. Among them, one of the preferred embodiments is as follows:

[0014] Preferably, the molar fractions of the raw materials for preparing liposomes are specifically:

[0015] 79 parts of lecithin;

[0016] 8 parts of cholesterol;

[0017] 20007 parts of the DSPE-PEG;

[0018] The DSPE-PEG2000-COOH is 0.7 parts.

[0019] Preferably, the mass ratio of the transferrin to the liposome to the liposome is 1:35-45, specifically 1:40.

[0020] In the drug-loaded liposome, the drug may specifically be β-nicotinamide mononucleotide and / or a salt thereof.

[0021] The drug-to-lipid ratio of the drug-loaded liposome is 1:15-1:30, specifically 1:22.

[0022] The drug-to-lipid ratio refers to the ratio of the mass of the drug to the sum of the masses of the phosphatidylcholine and the cholesterol in the targeted liposome.

[0023] The particle size of the drug-loaded liposome is (100-250) nm, specifically 160 nm.

[0024] The polydispersity index of the drug-loaded liposome is 0.1-0.3, specifically 0.2.

[0025] The zeta potential of the drug-loaded liposome is -(7-12) mV, specifically -9 mV.

[0026] The drug carrier with the targeted liposome as the active ingredient also belongs to the protection scope of the present invention.

[0027] The present invention further provides a method for preparing the targeted liposome, comprising the following steps: reacting overnight at a certain temperature in the presence of EDCl (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide), wherein the transferrin is connected to the carboxyl group of the liposome to obtain the targeted liposome.

[0028] A method for preparing brain-targeted NMN liposomes comprises the following steps:

[0029] (1) dissolving NMN in water, dissolving lecithin, cholesterol, DSPE-PEG2000, and DSPE-PEG2000-COOH in an organic solvent, and mixing the two to obtain solution A;

[0030] (2) subjecting the solution A obtained in step (1) to a first ultrasonic emulsification using a cell disruptor, and then adding water and subjecting it to a second ultrasonic emulsification to obtain a solution B;

[0031] (3) at a temperature of 32° C., the organic solvent was removed from the solution B obtained in step (2) under reduced pressure to obtain emulsion C;

[0032] (4) The emulsion obtained in step (3) was repeatedly squeezed through a microporous filter membrane (0.22 μm), and then mixed with EDCl (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), NHS (N-hydroxysuccinimide) and transferrin, and reacted overnight to obtain emulsion D;

[0033] (5) The emulsion D obtained in step (4) is dialyzed to remove free small molecules, thereby obtaining brain-targeted NMN liposomes.

[0034] In the above preparation method, the molar ratio of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to the transferrin is 1:1 to 350:450, specifically 1:400;

[0035] The molar ratio of the N-hydroxysuccinimide is 1:350 to 1:450, and specifically 1:400.

[0036] The present invention provides a preferred method for preparing transferrin-modified NMN liposomes, wherein one of the preferred embodiments is as follows:

[0037] Includes steps:

[0038] (1) fully dissolving the liposome preparation material in an organic solvent, and mixing it with an aqueous solution of NMN at a specific concentration in a certain proportion to obtain solution A;

[0039] (2) using a cell disruptor to sonicate solution A at a certain power, then adding a certain proportion of water and sonicating again at the same power to obtain solution B;

[0040] (3) evaporating solution B under reduced pressure at a specific temperature for a period of time to remove the organic solvent to obtain an emulsion;

[0041] (4) After filtering the emulsion through a microporous filter membrane, the emulsion is mixed with a certain proportion of EDCl (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), NHS (N-hydroxysuccinimide), and lactoferrin, and reacted at room temperature overnight;

[0042] (5) The emulsion obtained after the reaction is dialyzed to remove free small molecules, thereby obtaining the NMN liposome nanoparticles.

[0043] In a preferred experimental scheme of the present invention, the mass ratio of soybean lecithin, cholesterol, DSPE-PEG2000, DSPE-PEG2000-COOH, and lactoferrin is 20:2.1:1.67:0.186:0.13;

[0044] In a preferred experimental scheme of the present invention, the particle size of the NMN liposome nanoparticles is 160 nm;

[0045] In a preferred experimental scheme of the present invention, the organic solvent is chloroform;

[0046] In a preferred experimental scheme of the present invention, the concentration of the NMN aqueous solution is 4 mg / ml;

[0047] In a preferred experimental scheme of the present invention, the power of ultrasonic emulsification is 200W;

[0048] In a preferred experimental scheme of the present invention, the volume ratio of organic solvent to water in the first ultrasonic emulsification is 4.3:1;

[0049] In a preferred experimental scheme of the present invention, the volume ratio of the external aqueous phase to the internal aqueous phase in the second ultrasonic emulsification is 9:1;

[0050] In a preferred experimental scheme of the present invention, the time of the first phacoemulsification is 4.5 minutes;

[0051] In a preferred experimental scheme of the present invention, the temperature of reduced pressure evaporation is 32°C;

[0052] In a preferred experimental scheme of the present invention, the time of reduced pressure evaporation is 10 min;

[0053] In a preferred embodiment of the present invention, DSPE-PEG 2000 -COOH and DSPE-PEG 2000 The mass ratio is 1:9;

[0054] In a preferred experimental scheme of the present invention, the dialysis bag used for dialysis is MW3414000D.

[0055] Application of brain-targeted NMN liposomes in the preparation of drugs for the treatment of Parkinson's disease.

[0056] The beneficial effect of the present invention is that the NMN brain-targeted liposomes composed of a specific ratio provided by the present invention have good blood-brain barrier penetration ability.

[0057] The present invention encapsulates the active ingredient NMN into liposomes, and performs transferrin modification on the drug-loaded liposomes, thereby achieving brain tissue targeting and enhancing the blood-brain barrier permeability of the drug, thereby promoting the release and enrichment of the drug at the lesion site and improving the pharmacodynamic effect. The drug-loaded liposomes can be used to prepare drugs for the treatment of Parkinson's disease, and have important clinical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 This is a scanning electron microscope photograph of the NMN brain-targeted liposomes in Example 1 of the present invention.

[0059] Figure 2 This is the particle size distribution result detected by dynamic light scattering method of NMN brain-targeted liposomes in Example 1 of the present invention. DETAILED DESCRIPTION

[0060] Unless otherwise specified, the experimental methods used in the examples of the present invention are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0061] Experimental materials: Soybean lecithin (LPC) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; cholesterol was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; DSPE-PEG2000 was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.; DSPE-PEG2000-COOH was purchased from Shanghai Pengshuo Biotechnology Co., Ltd.; lactoferrin (Lf) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCl) and N-hydroxysuccinimide (NHS) were purchased from Shanghai MacLean Biotechnology Co., Ltd.; DMEM high glucose medium, fetal bovine serum, and 0.25% trypsin were purchased from Wuhan Pronocell Life Science Co., Ltd.; b.End3 cells were purchased from Wuhan Pronocell Life Science Co., Ltd., and C57BL / 6 mice were provided by Shanghai Lingchang Biotechnology Co., Ltd.

[0062] Experimental instruments: Rotary evaporator SB-2000 (Shanghai Airo Instrument Co., Ltd.); high performance liquid chromatograph (Waters, USA); IKAminiG centrifuge (IKA, Germany); JEM 1400 PLus transmission electron microscope (Japan Co., Ltd.); Baxter laser particle size analyzer (Dandong Baxter Instrument Co., Ltd.); ultrasonic pulverizer (Kunshan Jielimei Ultrasonic Instrument Co., Ltd.).

[0063] Example 1. Preparation of NMN brain-targeted liposomes

[0064] Soy lecithin, cholesterol, DSPE-PEG2000, and DSPE-PEG2000-COOH were fully dissolved in chloroform and mixed with 4 mg / ml NMN aqueous solution at a ratio of 4.5:1 to obtain solution A. The obtained solution A was sonicated for 10 min at a power of 200 W using a cell disruptor, and then water was added at a ratio of 9:1 between the external aqueous phase and the internal aqueous phase and sonicated again at the same power to obtain solution B. Solution B was evaporated under reduced pressure at 32°C for 10 min to remove the organic solvent to obtain an emulsion; the emulsion was filtered through a microporous filter membrane (0.22 μm), and EDCl (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), NHS (N-hydroxysuccinimide), DSPE-PEG2000-COOH, and Lf (lactoferrin) were added in a molar ratio of 400:400:10:1, respectively, mixed, and reacted at room temperature overnight; the free NMN, EDCI, and NHS were removed from the emulsion after the reaction by dialysis to obtain the NMN brain-targeted liposomes.

[0065] Example 2. Characterization of basic properties of NMN brain-targeted liposomes

[0066] (1) Drug loading and encapsulation efficiency

[0067] The determination was carried out by high performance liquid chromatography. Chromatographic conditions: Agilent TC-C18 column (4.6×150 mm, 5μm), column temperature 30°C, mobile phase of methanol-0.1% formic acid aqueous solution 20:80 (v / v), detection wavelength 265nm, injection volume 10μL, flow rate 1mL / min.

[0068] 0.1 mL of liposome nanosuspension was taken, 0.9 mL of methanol was added to break the emulsion, and the NMN content was determined under the above conditions. The drug loading and encapsulation efficiency of the prepared NMN brain-targeted liposomes were calculated to be (5.02±0.34)% and (25.1±1.7)%, respectively (n=3).

[0069] (2) Morphology observation and particle size analysis

[0070] Take the freshly prepared liposome solution and drip it onto the copper grid in small amounts several times. Stain it with 2% phosphotungstic acid for 5 minutes. After it is dried naturally, observe the morphology of the liposomes under a transmission electron microscope. Figure 1 The test results showed that the prepared liposomes were spherical particles with an average particle size of 160 nm and uniform size.

[0071] Take 1 mL of liposome solution in a quartz cuvette and place it in a laser particle size analyzer at 25°C for 120 seconds to measure the particle size, PDI, and Zeta potential. Repeat 3 times. Figure 2 As shown, the particle size of the prepared liposomes measured by dynamic light scattering was (159.85±0.78) nm, the PDI was (0.25±0.04), and the Zeta potential was -(10.4±0.41) mV.

[0072] (3) In vitro drug release characteristics

[0073] Take 2 mL of NMN and NMN brain-targeted liposome (Lf-NMN-Lip) solution (drug content is 1 mg each), place them in dialysis bags (MW 14000), put them in centrifuge tubes containing 15 mL of phosphate buffer (pH 7.4), and incubate them with shaking (37°C, 50 rpm). After sampling 0.2 mL each time, add fresh isothermal release medium of equal volume, measure the collected samples, and calculate the cumulative release rate.

[0074] The results showed that the cumulative release rate of NMN solution within 2 hours was (91.67±2.57)%, indicating that the release was basically complete and had no sustained-release characteristics; the cumulative drug release rate of Lf-NMN-Lip within 8 hours was (52.09±2.33)%, showing obvious drug sustained-release characteristics.

[0075] (4) Blood-brain barrier permeability

[0076] An in vitro blood-brain barrier model was constructed by b.End3 cells to compare the blood-brain barrier penetration ability of different preparations. After the model was successfully constructed, the culture medium solution containing the drug to be evaluated (150 μg / mL in terms of NMN) was added to the upper chamber of the transwell plate chamber, and the drug content in the lower chamber solution was determined after 24 hours of incubation, and the blood-brain barrier permeability was calculated, and the test was repeated 3 times. The results showed that the blood-brain barrier permeabilities of NMN raw material and Lf-NMN-Lip were (10.37±1.97)% and (62.03±2.30)%, respectively.

[0077] The results showed that the drug permeability of Lf-NMN-Lip of the present invention was increased by more than 5 times compared with NMN (p<0.001), which can significantly enhance the blood-brain barrier penetration ability of the encapsulated water-soluble drug NMN and has good brain targeting potential.

[0078] Example 3: Treatment of Parkinson's disease model animals with NMN brain-targeted liposomes

[0079] Parkinson's disease is also known as tremor paralysis, which is mainly manifested by external symptoms such as paralysis and slow movement. The present invention uses the classic MPTP Parkinson's model to verify the efficacy of the drug.

[0080] (1) Construction and grouping of mouse Parkinson's disease models

[0081] Healthy male C57BL / 6 mice (10 weeks old) were used as experimental subjects, and Parkinson's disease symptoms were induced by injection of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). After one week of pre-adaptation, 40 experimental mice were randomly divided into 4 groups (n=10). Except for the normal control group (group I), the mice in the other groups were given MPTP saline solution at a dose of 20 mg / kg by intraperitoneal injection (group II). On this basis, the animals in the NMN drug treatment groups (groups III and IV) continued to be given NMN and NMN brain-targeted liposomes (both 50 mg / kg in terms of NMN) by intraperitoneal injection; the normal control group was also given an equal volume of saline by intraperitoneal injection as the treatment group.

[0082] (2) Behavioral examination of mouse spontaneous activity

[0083] 30 minutes after drug treatment, mice in each group were placed in an open plexiglass box (25 cm × 25 cm × 10 cm) with grids (6 cm × 6 cm) on the bottom, and placed in a dark and quiet room, and then the number of grids moved by the mice in the box was counted. The counting was repeated for 5 days, and the mean number of grids moved by animals in each group and the sum of the examination results for 5 days were calculated. The results are shown in Table 1.

[0084] Table 1. Results of behavioral examination of spontaneous activity of mice in each treatment group during the experiment (Mean±SD, n=10)

[0085]

[0086] Note: Compared with the normal group (group I), * p<0.05, ** p<0.01; compared with the model group (group II), # p<0.05, ## p<0.01.

[0087] The experimental data showed that the mean number of grids moved by animals in group IV within one week was 445±87, 136±32, 144±46, 157±40, and 269±63 (n=8), respectively. Further comparison between the groups showed that the number of grids moved by animals in group II (MPTP model group) was significantly lower than that in the normal group (group I, p<0.01), indicating that the MPTP-induced PD model of slow movement in mice was successfully established;

[0088] The average number of grids moved by mice in the NMN free drug (Group III) within one week was similar to that in the model group, and there was no significant difference (p>0.05), indicating that the NMN free drug had no significant effect on alleviating the slow movement of mice induced by MPTP;

[0089] Only the mice in the NMN brain-targeted liposome treatment group (IV group) of the present invention showed a nearly 1-fold increase in the number of grids moved within one week compared with the model group, and the difference between the groups was significant (p<0.01), indicating that the NMN brain-targeted liposomes of the present invention have a relieving and inhibitory effect on the symptoms of Parkinson's disease, and that the NMN brain-targeted liposomes of the present invention have brain-targeting properties.

[0090] In summary, the above experimental data firstly prove that the present invention successfully synthesized NMN brain-targeted liposomes; moreover, the experimental data further show that the NMN brain-targeted liposomes provided by the present invention can significantly alleviate the MPTP-induced slow movement of mice, thereby inhibiting the progression of Parkinson's related diseases. Therefore, the NMN brain-targeted liposomes of the present invention show good PD treatment potential and can be used to prepare Parkinson's disease treatment drugs.

[0091] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A brain-targeted NMN liposome, characterized in that: The brain-targeted NMN liposomes are composed of drug-loaded liposomes and transferrin, and the mass ratio of transferrin to liposomes is 1:35-45; The drug-loaded liposomes are composed of drug NMN and liposomes, and the mass ratio of the drug to the liposomes is 1:10-50. Wherein, the drug NMN is β-nicotinamide mononucleotide and / or its salt, The liposome comprises lecithin, cholesterol, DSPE-PEG2000 (distearoylphosphatidylethanolamine-polyethylene glycol), and DSPE-PEG2000-COOH (distearoylphosphatidylethanolamine-polyethylene glycol-carboxyl), and, by mole fraction, lecithin comprises 76-82 parts, cholesterol comprises 6-9 parts, DSPE-PEG2000 comprises 5-8 parts, and DSPE-PEG2000 comprises 0.5-0.8 parts.

2. The brain-targeted NMN liposome according to claim 1, characterized in that: The lecithin used for preparing liposomes is soybean lecithin or egg yolk lecithin, and the transferrin is lactoferrin (Lf).

3. The method for preparing a brain-targeted NMN liposome according to claim 1, characterized in that: The following steps are involved: (1) Dissolve NMN in water, dissolve lecithin, cholesterol, DSPE-PEG2000, and DSPE-PEG2000-COOH in an organic solvent, and then mix them to obtain solution A; (2) subjecting the solution A obtained in step (1) to a first ultrasonic emulsification using a cell disruptor, and then adding water and subjecting it to a second ultrasonic emulsification to obtain a solution B; (3) at a temperature of 32° C., the organic solvent was removed from the solution B obtained in step (2) under reduced pressure to obtain emulsion C; (4) The emulsion obtained in step (3) is repeatedly extruded through a microporous filter membrane, and then mixed with EDCl, NHS (N-hydroxysuccinimide) and transferrin, and reacted overnight to obtain emulsion D; (5) The emulsion D obtained in step (4) is dialyzed to remove free small molecules, thereby obtaining brain-targeted NMN liposomes.

4. Use of the brain-targeted NMN liposome according to claim 1 to prepare a drug for treating Parkinson's disease.

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