NAD liposome and preparation method thereof

Abstract

The invention belongs to the field of pharmaceutical preparations, and particularly relates to an NAD liposome and a preparation method thereof. The NAD liposome comprises NAD, phospholipid, cholesterol, DSPE-PEG 2000, a stabilizing agent and a proper amount of water. By optimizing the dosage proportion of the membrane material in the liposome, the ratio of the membrane material to the NAD, the variety and dosage of the stabilizer and the prescription composition of the liposome, the prepared NAD liposome is high in encapsulation efficiency, high in drug loading capacity and good in stability.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical preparations and relates to a NAD liposome and a preparation method thereof. Background Art

[0002] NAD (nicotinamide adenine dinucleotide), also known as coenzyme I, is formally known as nicotinamide adenine dinucleotide. It is an essential metabolite of the human body. + NAD and its phosphorylated and reduced form NADP are present in all living cells. + NADH and NADPH, as coenzymes of hydride acceptors and donors, play a central role in cell metabolism and energy production. As we age, the level of NAD in the body gradually decreases, and the occurrence of age-related diseases such as aging, hearing impairment, and heart damage is accompanied by this process. + and its related metabolites, including several nucleosides and nucleotides (NAD + Metabolome) is a powerful indicator for measuring biochemical reactions such as glycolysis, gluconeogenesis, and fatty acid oxidation in cells or tissues. + The state of the metabolome can be used as an indicator of nutrition, health, and disease. Studies have shown that exogenous supplementation can effectively increase the level of NAD in the body, which is expected to improve the functional defects caused by aging. Existing NAD products include coenzyme I for injection and compound coenzyme. There are reports of cases of adverse reactions to intravenous infusion of compound coenzyme, and this variety has the highest incidence of adverse reactions among enzyme and coenzyme drugs.

[0003] Liposomes are an ideal drug carrier system used for drug delivery due to their unique properties. Their morphology is similar to that of cell membranes and they have received much attention as delivery systems for bioactive substances in vitro and in vivo. Liposomes consist of single or multiple lipid bilayers formed with an aqueous phase by hydrophilic and hydrophobic interactions. Liposomes are mainly composed of phospholipids, amphiphilic molecules with a hydrophilic head and two non-polar hydrophobic chains. When phospholipids are dispersed in aqueous solution, they have a strong tendency to form membranes due to their amphiphilic nature, which can retain lipophilic and hydrophilic agents in the lipid membrane and aqueous core, respectively. Typically, liposomes used for medical purposes range between 50nm and 450nm.

[0004] Liposomes have many advantages, such as low immunogenicity, extended drug half-life, passive targeting, and protection of drug active groups by lipid shell encapsulation. However, liposomes are easily taken up by the mononuclear phagocyte system (MPS) and are cleared from the blood quickly, resulting in a rapid decrease in blood drug concentration. In order to achieve better drug delivery effects, structural and surface modifications have been carried out based on classic lipid molecules. Most of the research focuses on the modification of the liposome bilayer and surface chemical composition, such as long-circulating liposomes, which further expands the application of liposomes in biomedicine.

[0005] Long-circulating liposomes are liposomes that have been modified by coating their surface with inert polymer molecules to alter their physicochemical properties. Among these hydrophilic molecules, polyethylene glycol (PEG) polymers are a successful material that has been used to stabilize liposomes. The components of liposomes are phospholipids and cholesterol. Phospholipids are used together with cholesterol to support the formation and stability of liposomes, while the incorporation of PEGylated lipids (DSPE-PEG 2000) can improve the stability of the formed liposomes.

[0006] In view of the advantages of liposome delivery system, it is necessary to develop NAD liposomes with high encapsulation efficiency, high drug loading and good stability. Summary of the invention

[0007] The main purpose of the present invention is to provide a NAD liposome and a preparation method thereof. The prepared NAD liposome has high encapsulation efficiency, high drug loading capacity and good stability.

[0008] The invention provides a NAD liposome. The liposome comprises NAD, a membrane material, a stabilizer and an appropriate amount of water. The membrane material comprises phospholipids, cholesterol and DSPE-PEG 2000.

[0009] Preferably, the phospholipid is selected from one or more of hydrogenated phospholipids, egg yolk lecithin, soybean lecithin, cephalin and distearoylphosphatidylcholine.

[0010] Preferably, the stabilizer is selected from chitosan, carboxymethyl chitosan, hyaluronic acid, sodium alginate, mannitol, HPMC, Tween-80. More preferably, the stabilizer is selected from carboxymethyl chitosan.

[0011] Preferably, the concentration of NAD is 10-80 mg / ml.

[0012] Preferably, the concentration of the membrane material is 10-40 mg / ml.

[0013] Preferably, the concentration of the stabilizer is 5-20 mg / ml.

[0014] Preferably, in the membrane material, the ratio of phospholipid, cholesterol, and DSPE-PEG 2000 is (15-5): (1-4): (1-3). More preferably, in the membrane material, the ratio of phospholipid, cholesterol, and DSPE-PEG 2000 is (10-6): (2-3): (1-2).

[0015] Preferably, the ratio of the membrane material to NAD is 1:(1-4). More preferably, the ratio of the membrane material to NAD is 1:(1-2).

[0016] The present invention also provides a method for preparing NAD liposomes, comprising the following steps: weighing a membrane material, adding an organic solvent, ultrasonically dissolving the membrane material completely, rotary evaporating to remove the organic solvent, forming a liposome membrane with uniform texture, and drying; adding NAD, then adding an appropriate amount of water, stirring, and extruding using a liposome extruder to obtain NAD liposomes.

[0017] Preferably, the organic solvent is selected from one or more of ethanol, propylene glycol, glycerol, ethylene glycol, isopropanol, butylene glycol, and pentanediol.

[0018] Preferably, rotary evaporation is performed at 40-60°C.

[0019] Preferably, the mixture is dried in a vacuum drying oven for 24-48 hours.

[0020] Preferably, the liposome extruder lining membrane is 100-200 nm.

[0021] Preferably, the prepared NAD liposomes are prepared as lyophilized powder.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention prepares a NAD liposome, and optimizes the dosage ratio of membrane materials, the ratio of membrane materials to NAD, the type and dosage of stabilizers in the liposomes, and optimizes the prescription composition of the liposomes. The prepared NAD liposomes have high encapsulation rate, high drug loading capacity and good stability.

[0024] The NAD liposome prepared by the present invention is a drug delivery system that improves the stability of the drug by encapsulating the drug in a lipid bilayer membrane. The liposome can reduce the influence of external environmental factors on the drug NAD contained therein, ensuring that its active ingredients remain stable during storage and transportation.

[0025] NAD liposomes are composed of phospholipids and other materials to form a double-layer membrane structure, which simulates the characteristics of biological membranes and makes it easy to penetrate into cells. The membrane structure of liposomes helps protect drug molecules from being damaged by enzymatic hydrolysis or other degradation pathways, and to a certain extent prolongs the duration of drug action.

[0026] The main function of liposomes is to act as drug carriers for carrying various types of drug molecules, including small molecule drugs and macromolecule drugs. By encapsulating NAD inside liposomes, the drug release rate can be slowed down, the duration of action can be prolonged, and the bioavailability of NAD can be improved. At the same time, liposomes have good biocompatibility, which enables them to run smoothly in the body and promote the effective absorption of drugs. DETAILED DESCRIPTION

[0027] In view of the defects of the prior art, the inventor has proposed the technical solution of the present invention after long-term research and extensive practice. The present invention is further illustrated by embodiments below, but these embodiments do not constitute a limitation of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0028] Example 1 Liposome Prescription Dosage Screening

[0029]

[0030]

[0031] The membrane material is egg yolk lecithin, cholesterol and DSPE-PEG 2000, and the proportion of the membrane material is egg yolk lecithin, cholesterol and DSPE-PEG 2000.

[0032] Preparation method: Weigh egg yolk lecithin, cholesterol, and DSPE-PEG 2000 membrane material and place them in an eggplant-shaped bottle, add 15 mL of anhydrous ethanol, and dissolve the membrane material completely by ultrasound. Place it on a rotary evaporator and remove the anhydrous ethanol at 45°C to make a liposome membrane with uniform texture. Place it in a vacuum drying oven at 25°C and dry it for 24 hours. Add NAD and then add 10 mL of pure water, stir for 3 hours, and extrude it using a liposome extruder (lined with a 200 nm membrane) to obtain NAD liposomes.

[0033] Encapsulation efficiency and drug loading determination method:

[0034] Place 5 mg of NAD in 50 ml of pure water to prepare a solution with a concentration of 100 μg / ml; take 0.5, 1, 2, 3, 4, 6, 7, and 8 ml respectively and dilute to 10 ml. Perform absorbance measurement, perform linear regression of absorbance against mass concentration, and draw a standard curve.

[0035] Add 1 ml of liposomes to an ultrafiltration centrifuge tube and centrifuge at 3000 rpm for 20 min. Collect the lower filtrate, dilute it 200 times, and measure the absorbance. Calculate the free drug concentration based on the standard curve, and then calculate the free drug mass of 10 ml of the preparation, and then measure the drug loading and encapsulation efficiency.

[0036] Encapsulation efficiency: Encapsulation efficiency = (drug encapsulated in liposomes / total amount of drug in liposomes) × 100%

[0037] Drug loading: drug loading = [drug amount in liposomes / (drug amount in liposomes + total amount of carrier)] × 100%

[0038] From the results in the above table, it can be seen that the ratio of lecithin, cholesterol, and DSPE-PEG 2000 is (10-6): (2-3): (1-2), and the ratio of membrane material to NAD is 1: (1-2). The average particle size of the prepared liposomes is below 200nm, and the encapsulation efficiency and drug loading are relatively high. When the ratio of lecithin, cholesterol, and DSPE-PEG 2000 is 3:1:1, the ratio of membrane material to NAD is 1:2, the amount of membrane material is 20mg / ml, and the amount of NAD is 40mg / ml, the average particle size of prescription 1 is the smallest, and the encapsulation efficiency and drug loading are the highest.

[0039] Example 2 Screening of stabilizer types and dosages

[0040]

[0041]

[0042] The membrane material is egg yolk lecithin, cholesterol and DSPE-PEG 2000, and the proportion of the membrane material is egg yolk lecithin, cholesterol and DSPE-PEG 2000.

[0043] Preparation method: Weigh egg yolk lecithin, cholesterol, and DSPE-PEG 2000 membrane material and place them in an eggplant-shaped bottle, add 15 mL of anhydrous ethanol, and dissolve the membrane material completely by ultrasound. Place it on a rotary evaporator and remove the anhydrous ethanol at 45°C to make a liposome membrane with uniform texture. Place it in a vacuum drying oven at 25°C and dry it for 24 hours. Add NAD and stabilizer, then add 10 mL of pure water, stir for 3 hours, and extrude it using a liposome extruder (lined with a 200 nm membrane) to obtain NAD liposomes.

[0044] As can be seen from the results in the above table, adding hyaluronic acid, sodium alginate, mannitol, HPMC or Tween-80 did not significantly improve the average particle size, encapsulation efficiency and drug loading of liposomes. Adding chitosan improved the average particle size, encapsulation efficiency and drug loading of liposomes, but the effect was not obvious. Adding carboxymethyl chitosan can significantly reduce the average particle size of liposomes, increase the encapsulation efficiency and drug loading of liposomes, and the encapsulation efficiency reaches 95% and the drug loading reaches 70%. Therefore, carboxymethyl chitosan was selected as a stabilizer to prepare NAD liposomes.

[0045] Example 3 Pharmacokinetic Study

[0046] Twenty-four SD rats were randomly divided into three groups (free NAD group, DSPE-NAD-LIP group (prescription 11), NAD-LIP group (the difference from prescription 11 is that DSPE-PEG 2000 is not added)), with 8 rats in each group, and adaptive feeding and water supply for 7 days. SD rats were injected with tail vein, the first group was injected with NAD-Sol, the second group was injected with DSPE-NAD-LIP, and the third group was injected with NAD-LIP, all at a dose of 80 mg / kg. Fasting began 12 hours before administration, and water was available freely. At 0.08, 0.167, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours after administration, 0.5 ml of blood was collected from the rat eye sockets and placed in a sodium heparin-treated centrifuge tube, centrifuged at 12000 r / min for 5 minutes, and the supernatant was removed and frozen at -80°C for testing. The measured data were analyzed using DAS2.0 pharmacokinetic software to analyze blood drug concentrations and calculate pharmacokinetic parameters.

[0047] Pharmacokinetic parameters NAD-Sol NAD-LIP NAD-DSPE-LIP <![CDATA[AUC 0-t (mg / L*h)]]> 455.825±21.798 458.112±32.383 458.357±35.824 <![CDATA[MRT 0-t (h)]]> 3.923±0.186 6.094±0.317 11.135±0.199 <![CDATA[t 1 / 2 (h)]]> 1.431±0.164 4.916±3.114 7.614±1.967

[0048] From the above results, it can be seen that making NAD into a liposome drug delivery system can prolong the circulation time of NAD in the body, and making it into long-circulation liposomes can further prolong the circulation time of NAD in the body.

[0049] It should be understood that the technical solution of the present invention is not limited to the above-mentioned specific implementation cases. Any technical deformation made according to the technical solution of the present invention without departing from the scope of protection of the purpose of the present invention and the claims shall fall within the protection scope of the present invention.

Claims

1. A NAD liposome, characterized in that The liposome comprises NAD, a membrane material, a stabilizer and an appropriate amount of water, wherein the membrane material comprises phospholipids, cholesterol and DSPE-PEG 2000, and the stabilizer is carboxymethyl chitosan. In the membrane material, the ratio of phospholipids, cholesterol and DSPE-PEG 2000 is (10-6):(2-3):(1-2), and the ratio of the membrane material to NAD is 1:(1-2).

2. The NAD liposome according to claim 1, characterized in that The phospholipid is selected from one or more of hydrogenated phospholipids, egg yolk lecithin, soybean lecithin, cephalin and distearoylphosphatidylcholine.

3. The NAD liposome according to claim 2, characterized in that The concentration of NAD is 10-80 mg / ml.

4. The NAD liposome according to claim 2, characterized in that The concentration of the membrane material is 10-40 mg / ml.

5. The NAD liposome according to claim 2, characterized in that The concentration of the stabilizer is 5-20 mg / ml.

6. The method for preparing NAD liposomes according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: weighing a membrane material, adding an organic solvent, dissolving the membrane material completely by ultrasound, removing the organic solvent by rotary evaporation, forming a liposome membrane with uniform texture, and drying; adding NAD and then adding an appropriate amount of water, stirring, and extruding by a liposome extruder to obtain NAD liposomes.

7. The method for preparing NAD liposomes according to claim 6, characterized in that: The organic solvent is selected from one or more of ethanol, propylene glycol, glycerol, ethylene glycol, isopropanol, butylene glycol, and pentanediol.

8. The method for preparing NAD liposomes according to claim 6, characterized in that: The liposomes were evaporated by rotary evaporation at 40-60°C and dried in a vacuum oven for 24-48 hours. The lining membrane of the liposome extruder was 100-200 nm.

9. NAD liposomes prepared according to the preparation method according to any one of claims 6 to 8.

10. The NAD liposome according to claim 9, characterized in that The NAD liposomes were prepared as lyophilized powder.

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