Hydroxyapatite nanoparticles encapsulating beta-nicotinamide mononucleotide, and methods of making and using the same

Abstract

The present application relates to a kind of β-nicotinamide mononucleotide encapsulated hydroxyapatite nanoparticles (NMN-HAP) and its preparation method and application. Specifically, the nanoparticles include β-nicotinamide mononucleotide and hydroxyapatite. The preparation method of the NMN-HAP includes the following steps: hydroxyapatite is mixed with pure water and dispersed, then pure water in the suspension is replaced with ethanol, and β-nicotinamide mononucleotide aqueous solution is added;After vortex incubation of the mixture, remove the supernatant. The NMN-HAP provided in the present application has the potential to improve the bioavailability of NMN and in vivo NAD + anti-aging potential while maintaining bone health, providing potential for dual health benefits.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to a kind of β-nicotinamide mononucleotide (NMN) loaded hydroxyapatite (HAP) nanoparticles and its preparation method and application. BACKGROUND

[0002] Nicotinamide adenine dinucleotide (NAD + ) is a coenzyme widely present in cells, which plays a key role in promoting redox reactions in metabolic pathways, including citric acid cycle, glycolysis, fatty acid and steroid biosynthesis, etc. In addition, NAD + also acts as an essential coenzyme for a variety of NAD + -consuming enzymes, playing an important role in many biological processes. Aging is closely related to a significant decrease in intracellular NAD + levels, and NAD + imbalance is closely related to a variety of aging-related diseases such as neurodegenerative diseases and diabetes. Therefore, maintaining NAD + levels has become a potential therapeutic strategy for prolonging healthy lifespan.

[0003] Due to the inherent instability of NAD + and limited bioavailability to most cells, direct supplementation of exogenous NAD + is not the best way to restore NAD + levels in the body. Instead, by supplementing NAD + biosynthesis precursors such as nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR), it can promote the production of intracellular NAD + , which is considered a more effective method. In addition, it was found that the transporter Slc12a8 exists in the intestine, which helps to specifically absorb NMN, making it more effective in increasing NAD + levels relative to other precursors.

[0004] One of the problems commonly existing in the current market oral NMN products is that they are rapidly metabolized and excreted in the gastrointestinal tract and liver. Therefore, there is an urgent need to develop an innovative NMN formulation to improve its bioavailability.

[0005] WO2020113811A1 discloses an NMN-containing biological macromolecular nanosphere, its pharmaceutical preparation or functional food, and a preparation method and application thereof. The biological macromolecular nanosphere comprises a biological macromolecular carrier and NMN dispersed on the biological macromolecular carrier, and the biological macromolecular carrier has a three-dimensional network structure with a flat diameter particle size of 200-1000 nm, which can be used for preparing drugs and functional foods for preventing and treating sub-health and tumors.

[0006] CN109350611B discloses a NMN nanomicrosphere coated with konjac glucomannan (KGM), which comprises a NMN nanosphere, and the outside of the NMN nanosphere is coated with a KGM nanosphere, the particle size of the NMN nanosphere is 500-1000 nm, and the particle size of the KGM nanosphere is 200-1000 nm. The KGM nanoparticles are coated outside the unstable NMN, the KGM nanoparticles protect the NMN inside, preventing the NMN from being unstable and easily decomposed after encountering light or oxygen. After the NMN is prepared into a coated composite with KGM, the NMN can be released slowly within a certain period of time, avoiding immediate decomposition by gastric acid. The composite nanomicrosphere can be used for producing oral preparations and for resisting cancer. The preparation process and application of the NMN nanomicrosphere coated with KGM are also disclosed, the preparation process has simple steps and mild conditions, and the prepared nanomicrosphere can be widely used in the production of anticancer drugs, health care drugs and pet drugs.

[0007] The nanospheres described in the above two patent applications have a particle size of more than 200 nm. However, studies have shown that particles with a particle size in the range of 10-100 nm are considered to be the most ideal, because they can exhibit a prolonged circulation time in the body, effectively avoid the clearance of the reticuloendothelial system, and have the ability to penetrate narrow capillaries.

[0008] CN112891241B relates to the field of cosmetics, in particular to a skin anti-aging nanocomposition targeting mitochondria, comprising β-nicotinamide mononucleotide (NMN), other anti-aging active ingredients and a nanocarrier; the NMN accounts for 0.1-10% of the total mass of the nanocomposition; the other anti-aging active ingredients include at least one of an antioxidant, a protein synthesis promoter, an anti-photoaging agent and a moisturizer; the raw materials of the nanocarrier include a cell penetration enhancer, an emulsifier, a co-emulsifier, a liquid lipid and water. The application reasonably matches different mechanism anti-aging active ingredients, and synergistically enhances the effect; the anti-aging active ingredients are wrapped by the nanocarrier, improving the stability of the anti-aging ingredients; the active ingredients efficiently enter the deep tissue of the skin and the target cells and mitochondria of the skin, realizing dual targeting of the tissue and the cells, improving the bioavailability and increasing the anti-aging effect. The skin is mild and non-irritating, and can be widely used in cosmetics.

[0009] CN115282117A discloses a kind of oral mucosa administration β-nicotinamide mononucleotide nano-suspension, wherein the β-nicotinamide mononucleotide nano-suspension is oil-in-water type nanodroplet, the β-nicotinamide mononucleotide nano-suspension includes aqueous phase component and oil phase component, wherein the oil phase component includes glycerol, the aqueous phase component includes pure water and β-nicotinamide mononucleotide. Correspondingly, the application also discloses a manufacturing method of β-nicotinamide mononucleotide nano-suspension. The application makes β-nicotinamide mononucleotide directly penetrate through oral mucosa absorption into blood, increases the absorption rate of β-nicotinamide mononucleotide, avoids the degradation of β-nicotinamide mononucleotide by gastrointestinal enzymes.

[0010] The particle size of the nano-composition and nano-suspension described in the above two patent applications is 10-300nm and 50-200nm respectively, which is ideal. However, the preparation steps of the two patent applications are relatively complicated, involving complex ingredients, including emulsifiers, co-emulsifiers and oil phases. At the same time, the biocompatibility of these ingredients may not be ideal.

[0011] CN113712987A relates to a composition for improving NAD + levels and its use, in particular to a composition comprising nicotinamide mononucleotide, resveratrol and ginsenoside and its use. The three active ingredients in the composition provided by the application show good synergistic effect, which can significantly improve the NAD + levels in brain, liver and muscle tissue. However, since the composition is not a nano-drug delivery system, it is not clear whether it has an advantage in NMN bioavailability in vivo.

[0012] Hydroxyapatite (HAP) is the main inorganic component of human bone, and is widely used as a dietary supplement to promote bone health. In recent years, due to its microporous nanostructure, non-toxicity and excellent biocompatibility and bioactivity, HAP has become an ideal drug carrier for delivering various drugs (from small molecules to proteins and nucleic acids and other macromolecules). However, there is no nano-preparation using HAP as a carrier to encapsulate NMN.

[0013] In addition, studies have shown that HAP can also enhance the production of NAD + in the mitochondria of macrophages. Therefore, the present application aims to provide a HAP-based nano-drug delivery system, called NMN-HAP, which will more effectively improve NMN bioavailability and NAD + , NR levels in vivo, and at the same time maintain bone health, providing potential for achieving dual health benefits. SUMMARY

[0014] The present application provides a β-nicotinamide mononucleotide-loaded hydroxyapatite nanoparticle (NMN-HAP) comprising β-nicotinamide mononucleotide and hydroxyapatite.

[0015] In an embodiment of the present application, the weight ratio of β-nicotinamide mononucleotide to hydroxyapatite is 2-5:1, preferably 3:1.

[0016] In an embodiment of the present application, the β-nicotinamide mononucleotide-loaded hydroxyapatite nanoparticle is a rod-shaped structure with a particle size of 50-150 nm, preferably 100 nm.

[0017] In an embodiment of the present application, the encapsulation efficiency of the β-nicotinamide mononucleotide-loaded hydroxyapatite nanoparticle is 45.15±1.57%.

[0018] In an embodiment of the present application, the drug loading capacity of the β-nicotinamide mononucleotide-loaded hydroxyapatite nanoparticle is 42.42±0.71%.

[0019] The present application also provides a method for preparing a β-nicotinamide mononucleotide-loaded hydroxyapatite nanoparticle, comprising the following steps: mixing and dispersing hydroxyapatite with pure water, then replacing the pure water in the suspension with ethanol, and adding a β-nicotinamide mononucleotide aqueous solution; after vortex incubation of the mixture, removing the supernatant.

[0020] Further, the hydroxyapatite is a rod-shaped structure with a particle size of 50-150 nm, preferably 100 nm. The weight ratio of hydroxyapatite to pure water is 1:5-10, preferably 1:7. The specific step of mixing and dispersing is that after mixing hydroxyapatite with pure water, the suspension is dispersed using ultrasonic waves, preferably using a 960-watt probe ultrasonic, preferably 5 seconds of ultrasonic and 3 seconds of rest, preferably for 1.5 hours. The final weight ratio of β-nicotinamide mononucleotide to hydroxyapatite is 2-5:1, preferably 3:1. The vortex incubation of the mixture is incubation at room temperature for 12 hours. The supernatant is removed by centrifugation, specifically 3000 revolutions per minute for 5 minutes.

[0021] In an embodiment of the present application, the preparation method of the hydroxyapatite nanoparticles loaded with β-nicotinamide mononucleotide comprises the following steps: first, mix rod-shaped HAP with a particle size of about 100 nm and pure water at a weight ratio of 1:7, and use a 960-watt probe ultrasonic wave to perform suspension dispersion after 5 seconds of ultrasonic and 3 seconds of rest, for 1.5 hours. Subsequently, replace the pure water in the suspension with ethanol, and add an NMN aqueous solution. The final weight ratio of NMN to HAP is 3:1, and the final volume ratio of ethanol to pure water is 7:3. After the mixture is vortexed at room temperature for 12 hours, centrifugation is performed at a speed of 3,000 revolutions per minute for 5 minutes, and the supernatant is removed.

[0022] The hydroxyapatite nanoparticles loaded with β-nicotinamide mononucleotide prepared in the present application have a rod-shaped structure, a particle size of 50-150 nm, and preferably 100 nm; an encapsulation efficiency of 45.15±1.57%; and a drug loading capacity of 42.42±0.71%.

[0023] The present application also provides the use of the hydroxyapatite nanoparticles loaded with β-nicotinamide mononucleotide for the preparation of a medicament for preventing or treating tumors, aging-related diseases, or promoting bone health; the aging-related diseases include neurodegenerative diseases, diabetes.

[0024] The present application also provides the use of the hydroxyapatite nanoparticles loaded with β-nicotinamide mononucleotide for anti-aging and promoting bone health for non-disease treatment purposes; the anti-aging includes skin anti-aging.

[0025] Through research, the present application has at least the following beneficial effects compared with the prior art:

[0026] 1. The NMN-HAP nanoparticles provided by the present application have a particle size of about 100 nm, can exhibit a prolonged circulation time in vivo, can effectively escape the clearance of the reticuloendothelial system, and have the ability to penetrate narrow capillaries. Experimental results prove that the NMN-HAP nanoparticles have sustained release characteristics compared with free NMN, thereby prolonging the circulation time of NMN and improving the bioavailability thereof.

[0027] 2. The NMN-HAP nanoparticles provided by the present application have a persistent and enhanced effect on improving the NAD + level in blood plasma and some tissues, and enhance the ability to improve the NR level and promote the absorption of tissues as a precursor of NAD + . This effect is tissue-specific, and NMN mainly accumulates in the liver, and significantly improves the targeting effect on the brain, and the targeting efficiency of NMN-HAP in the brain is increased by more than 10 times.

[0028] 3、The NMN-HAP nanoparticles provided by the application have a simpler preparation procedure, the HAP has excellent biocompatibility, only releases calcium and phosphorus ions after metabolism, and can be used in vivo; and the HAP also has the effect of maintaining bone health.

[0029] Therefore, the NMN-HAP nanoparticles provided by the application effectively improve the bioavailability and in vivo NAD + level of NMN, have anti-aging potential, and simultaneously maintain bone health, thereby providing potential possibilities for achieving double health benefits. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 The electron micrographs of NMN-HAP (the scale shown on the left side is 500 nm, and the scale shown on the right side is 100 nm).

[0031] Figure 2 The average plasma concentration-time curve of NMN in rats after oral administration of NMN-HAP and free NMN (equivalent to a dose of 500 mg / kg of NMN) (n=6; mean ± standard deviation).

[0032] Figure 3 The average plasma concentration-time curves of (A) NAD + and (B) NR in rats after oral administration of NMN-HAP, free NMN and free HAP (equivalent to a dose of 500 mg / kg of NMN) (n=6; mean ± standard deviation).

[0033] Figure 4 The NAD + level in each tissue of rats after oral administration of NMN-HAP, free NMN and free HAP (equivalent to a dose of 500 mg / kg of NMN) (n=6; mean ± standard deviation).

[0034] Figure 5 The NR level in each tissue of rats after oral administration of NMN-HAP, free NMN and free HAP (equivalent to a dose of 500 mg / kg of NMN) (n=6; mean ± standard deviation). DETAILED DESCRIPTION

[0035] The application will be further described below in combination with examples; however, the examples do not limit the scope of the application. Unless otherwise stated, all reactants used in the examples are obtained from commercial channels; the instruments and equipment used in the preparation experiments and product analysis and detection are all conventional instruments and equipment commonly used.

[0036] Example 1: Preparation of NMN-HAP

[0037] First, rod-like HAP with a particle size of about 100 nm was mixed with pure water at a weight ratio of 1:7, and a 960-watt probe ultrasonic wave (5 seconds of ultrasonic followed by 3 seconds of rest) was used for suspension dispersion for 1.5 hours. Subsequently, the pure water in the suspension was replaced with ethanol, and an NMN aqueous solution was added. The final weight ratio of NMN to HAP was 3:1, and the final volume ratio of ethanol to pure water was 7:3. After the mixture was vortexed at room temperature for 12 hours, the supernatant was removed by centrifugation at 3,000 rpm for 5 minutes. Finally, the NMN-HAP sample (NMN content of about 65 mg / mL) was resuspended by adding pure water.

[0038] Example 2: Characterization of NMN-HAP

[0039] Morphological analysis: Transmission electron microscopy (TEM) was used to analyze the particle size and morphology of NMN-HAP. After the sample was suspended in pure water, it was deposited on a carbon-coated copper TEM grid and observed using an FEI Tecnai Spirit 120kv transmission electron microscope. As shown in FIG. 1, the prepared NMN-HAP exhibited a rod-like structure with a particle size of about 100 nm. Figure 1

[0040] Encapsulation efficiency (EE%) detection: The encapsulation efficiency of NMN-HAP was detected using high-speed centrifugation. First, 50 μL of the NMN-HAP sample was centrifuged at 14,000 rpm for 5 minutes. Subsequently, the concentration of NMN in the supernatant was measured using the ultra-high performance liquid chromatography (UHPLC) conditions described later. The concentration of NMN in the supernatant was defined as the concentration of free NMN (C1). Then, the precipitate was dissolved in 50 μL of 0.1N hydrochloric acid and treated with ultrasonic for 10 minutes to measure the concentration of encapsulated NMN (C2). The calculation formula of encapsulation efficiency is: EE% = C2 / (C1+C2) x 100%. The experimental results showed that the encapsulation efficiency of NMN-HAP was 45.15 ± 1.57%.

[0041] Drug loading capacity (DL%) detection: Drug loading capacity (DL%) represents the amount of NMN loaded per unit weight of HAP in NMN-HAP. First, 50 μL of the NMN-HAP sample was centrifuged at 14,000 rpm for 5 minutes. Subsequently, the supernatant was removed, and the precipitate was speed vacuum dried. The dried powder was collected and weighed (W1). Then, the amount of NMN present in the powder (W2) was measured. The calculation formula of DL% is: DL% = W2 / W1 x 100%. The experimental results showed that the drug loading capacity of NMN-HAP was 42.42 ± 0.71%.

[0042] ​UHPLC conditions: Agilent 1290 Infinity UHPLC system was used for analysis, and Waters Acquity BEH C 18 column (2.1 x 100 mm, 1.7 pm). The mobile phase was composed of solvent A (pure water containing 0.1% formic acid) and solvent B (acetonitrile containing 0.1% formic acid). A gradient elution program was used with a flow rate of 0.35 mL / min, and the specific settings were as follows: 0-3 min, 2-10% B; 3-5 min, 10-100% B; 5-6 min, 100-100% B; 6-6.1 min, 100-2% B; 6.1-8 min, 2-2% B. The column temperature was set at 40 °C, and the injection volume was 1 pL. The UV detection wavelength was set at 266 nm. The NMN stock solution was prepared in pure water at a concentration of 5 mg / mL. A series of standards were prepared by 2-fold dilution method, with a concentration range of 0.0625-2 mg / mL. The standard curve was constructed by plotting the peak area (y) against the corresponding concentration (x).

[0043] Example 3: Comparison of pharmacokinetics of NMN-HAP and free NMN

[0044] Experimental animals: Healthy C57 / BL6 mice, male, aged 12-14 weeks (body weight 23-28 grams) were selected.

[0045] Grouping and administration: A total of 99 mice were randomly divided into four groups: a control group (n = 9) and three experimental groups (NMN-HAP group, free NMN group, and free HAP group, n = 30 in each group). In the NMN-HAP group, the mice were orally administered with NMN-HAP suspension (containing 67.94 mg / mL of NMN and 40.38 mg / mL of HAP), with a dose equivalent to 500 mg / kg of NMN. The free NMN group was orally administered with NMN solution (prepared by dissolving NMN at a concentration of 50 mg / mL in pure water) at a dose of 500 mg / kg. The free HAP group was orally administered with HAP suspension at the same dose of HAP as in the NMN-HAP group. At specific time intervals (1, 4, 8, 12, and 24 hours) after administration, 6 mice were randomly selected from each group for blood collection through intraorbital bleeding and subsequent cervical dislocation. All mice in the control group were bled at time point 0 and subsequently sacrificed.

[0046] Plasma sample processing: Blood samples were collected using heparinized tubes and immediately centrifuged at 3,000 rpm for 10 minutes to separate the plasma. To extract the analytes (NMN, NAD +and precipitate the proteins, 50 μL of plasma sample was added to 200 μL of pre-chilled mixed solvent (ACN and MeOH in a volume ratio of 50:50). The mixture was shaken well and then incubated on ice for 20 minutes. Subsequently, the sample was centrifuged at 14,000 rpm for 10 minutes at 4°C. The obtained supernatant was transferred to a new tube and dried using speed vacuum. The obtained residue was re-dissolved with 50 μL of pure water, followed by analysis using UHPLC-MRM-MS.

[0047] UHPLC-MRM-MS conditions: The samples were analyzed for the content of NMN, NAD + and NR using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-QQQ-MS) analysis technique. A Zorbax Eclipse AAA column (4.6 x 150 mm, 3.5 μm) was selected, and the mobile phase system consisted of water containing 5 mM ammonium formate and 0.05% formic acid (pH 3.0) as the aqueous phase and methanol as the organic phase. The flow rate was 0.3 mL / min. The following gradient elution program was used: 0-8 minutes, 2-5% B; 8-15 minutes, 5-15% B; 15-18 minutes, 15-80% B; 18-18.1 minutes, 80-100% B. The electrospray ion source (ESI) was used in the positive ion mode, and the multiple reaction monitoring mode (MRM) was selected for scanning detection.

[0048] Results: Using the obtained data, the NMN plasma concentration-time curves for the NMN-HAP group and the free NMN group were constructed, as shown in Figure 2 In addition, we calculated and listed the pharmacokinetic parameters in Table 1. The analysis results showed that there were significant differences between NMN-HAP and free NMN. Compared with free NMN, NMN-HAP showed a longer time to reach maximum plasma concentration (T max ), a higher maximum plasma concentration (C max ), a longer half-life (t1 / 2), and a lower clearance rate (CL). In particular, the area under the curve (AUC 0-t ) of NMN-HAP was significantly increased compared with free NMN. These findings suggest that NMN-HAP may have sustained release characteristics, thereby prolonging the circulation time of NMN and improving its bioavailability.

[0049] Table 1. Plasma pharmacokinetic parameters of male C57 / BL6 mice after oral administration of NMN-HAP and free NMN (equivalent to a dose of 500 mg / kg of NMN); represented as mean ± standard deviation (n = 6)

[0050]

[0051] C max : maximum plasma concentration; T max : time to reach maximum plasma concentration; t 1 / 2 : half-life; AUC 0-t : area under the curve from time 0 to the last time point; CL: clearance.

[0052] *P value < 0.05 indicates a significant difference between the two groups

[0053] Example 4: Comparison of in vivo tissue distribution of NMN-HAP and free NMN

[0054] Tissue sampling and processing: After the mice were sacrificed, their brain, heart, liver, kidney, lung, skeletal muscle, spleen, and thymus were collected immediately, washed with PBS, and weighed. Then, the tissues were homogenized with phosphate buffered saline (PBS) at a volume 5 times the weight of the tissue. 100 μL of the tissue sample was taken for analyte extraction, and another 50 μL of the tissue sample was taken for protein extraction. All operations were performed quickly, and all samples were stored at -80°C until further processing. For analyte extraction, 100 μL of the tissue sample was added to 400 μL of pre-cooled ACN / MeOH mixed solvent (volume ratio 50 / 50). After the mixture was shaken well, it was incubated on ice for 20 minutes. Subsequently, it was centrifuged at 14,000 rpm for 10 minutes at 4°C, and the supernatant was transferred to a new tube and dried using a speed vacuum. The residue was re-dissolved with 50 μL of pure water and analyzed by UHPLC-MRM-MS. For protein extraction, 50 μL of the tissue sample was mixed with 50 μL of 2-fold concentrated RIPA buffer. The mixture was shaken well and incubated on ice for 30 minutes. Subsequently, it was centrifuged at 14,000 rpm for 10 minutes at 4°C, and the supernatant was collected and the protein concentration of each sample was determined using the Bradford method with bovine serum albumin (BSA) as the standard.

[0055] Results: After oral administration of NMN-HAP or free NMN, the distribution of NMN in various tissues was studied, and the T max and AUC 0-t were determined. Based on these parameters, the peak concentration ratio (C e ), the relative uptake rate (R e ), and the targeting efficiency (T e ) were calculated using the following equations: C e = C max of NMN-HAP / C max of free NMN, R e = AUC of NMN-HAP / AUC of free NMN, T e= AUC of a single tissue / Sum of AUCs of all tissues. Results are shown in Table 2. The study results indicate that T in the liver... e A value exceeding 90% indicates that NMN primarily accumulates in the liver. Additionally, C-values ​​are found in the brain, lungs, skeletal muscles, spleen, and thymus. e and R e The values ​​all exceeded 1, indicating enhanced distribution of NMN-HAP in these tissues. Of particular note is the increased T0.05 concentration of NMN-HAP in the brain compared to free NMN. e The value increased more than 10 times, and its C e and R e The values ​​were 5.29 and 13.72, respectively. These findings indicate that the NMN-HAP nanoparticles significantly improved the targeting effect on the brain. Conversely, NMN-HAP showed reduced T in the heart. e Its C value is 2 times lower than that of free NMN. e and R e The values ​​were 0.67 and 0.54, respectively, indicating a reduced targeting effect in the heart. Therefore, the effect of NMN-HAP on the distribution of NMN appears to be tissue-specific.

[0056] Table 2. Targeting parameters of NMN-HAP and free NMN in mouse tissues (n=6). Immediately collected samples from the heart, liver, kidneys, lungs, skeletal muscle, spleen, and thymus.

[0057]

[0058] C e Peak concentration ratio, C e =C of NMN-HAP max / C of free NMN max .

[0059] R e : Relative uptake rate, R e = AUC of NMN-HAP / AUC of free NMN.

[0060] T e Targeting efficiency, T e = AUC of a single organization / Sum of AUCs of all organizations.

[0061] Example 5: NAD+ levels in plasma and tissues after oral administration of NMN-HAP, free NMN, and free HAP + Comparison of abundance

[0062] After oral administration of NMN-HAP, free NMN, and free HAP, NAD + The plasma concentration-time curve is as follows: Figure 3 As shown in Figure A, different NAD values ​​are displayed.+ NMN-HAP administration. NAD + levels rapidly increased within the first 4 hours and then gradually decreased until 24 hours. In contrast, free NMN administration led to NAD + concentrations rapidly increased within the first hour and then increased at a slower rate for the next 8 hours, followed by a sharp decrease in the next 4 hours. These findings suggest that the cumulative NAD + content in plasma was higher after NMN-HAP administration compared to free NMN. On the other hand, free HAP administration led to NAD + levels in plasma slightly increased within 4 hours and returned to baseline levels after 8 hours. These results suggest that the HAP-based NMN nanomedicine delivery system has a stronger therapeutic effect in elevating plasma NAD + levels and exhibits a lower degradation rate, thereby having a superior ability to restore NAD + levels in vivo compared to free NMN.

[0063] In addition, NAD + levels in tissues were also examined at different time intervals after oral administration of NMN-HAP, free NMN, or free HAP, as Figure 4 shown. In the brain, liver, kidney, and lung, NAD + levels after NMN-HAP administration were initially lower than those of free NMN. However, after 8 hours, NAD + levels after NMN-HAP administration were significantly higher than those of free NMN. Furthermore, in skeletal muscle and thymus, NAD + levels after NMN-HAP administration were initially lower than those of free NMN, but reversed the trend at 24 hours. In contrast, in the heart and spleen, NAD + levels after NMN-HAP administration were consistently lower than those after free NMN administration. Interestingly, the administration of HAP appeared to increase NAD + levels in tissues, particularly in the brain, heart, kidney, and skeletal muscle. These results are consistent with the plasma results, suggesting that the HAP-based NMN nanomedicine delivery system has a persistent and enhanced therapeutic effect in elevating plasma NAD + levels. However, it is important to note that this effect is tissue-specific. In addition, the effect of HAP administration can also contribute to the higher tissue NAD + levels observed after NMN-HAP administration.

[0064] Example 6: Comparison of NR abundance in plasma and tissues after oral administration of NMN-HAP, free NMN, and free HAP

[0065] NMN can be converted to NR in the blood by the action of extracellular nucleotidases, which is subsequently taken up by tissues and used as a precursor of NAD + Thus, after oral administration of NMN-HAP, free NMN or free HAP, we evaluated the levels of NR in plasma and tissues. Figure 3 B shows the concentration-time profile of NR in plasma. It was observed that both NMN-HAP and free NMN administration significantly increased the levels of NR in plasma. However, there were differences between the two administration modalities. After NMN-HAP administration, the levels of NR increased within 12 hours and then decreased, while after free NMN administration, the levels of NR reached a peak within 8 hours and then decreased. In addition, NMN-HAP administration resulted in higher accumulation of plasma NR compared to free NMN. These findings suggest that the HAP-based NMN nanodrug delivery system enhances the ability to elevate plasma NR levels and facilitates its uptake by tissues as a precursor of NAD + On the other hand, administration of free HAP did not seem to have an effect on the levels of NR in plasma.

[0066] Figure 5 The levels of NR in tissues at different time intervals after oral administration of NMN-HAP, free NMN or free HAP are shown. In the brain and liver, the initial levels of NR after NMN-HAP administration were lower than those of free NMN. However, after 8 hours, the levels of NR after NMN-HAP administration were significantly higher than those of free NMN. Similarly, in the kidney and skeletal muscle, the initial levels of NR after NMN-HAP administration were lower than those of free NMN, but the trend was reversed at 24 hours. Conversely, in the heart, the levels of NR were consistently lower than those after free NMN administration. In addition, administration of HAP seemed to be able to increase the levels of NR in tissues, particularly in the heart, kidney and skeletal muscle. These trends in NR levels in tissues are consistent with the observed results for NAD + levels. These findings further emphasize the importance of the conversion of NMN to NR and its further utilization as a precursor of NAD+ for maintaining NAD + levels in the body.

[0067] It can be seen that the embodiments of the present application demonstrate that the provided NMN-HAP nanoparticles have a particle size of about 100 nm and have sustained release characteristics, thereby prolonging the circulation time of NMN and improving its bioavailability. Compared with free NMN, the distribution of NMN-HAP in tissues such as the brain, lung, skeletal muscle, spleen and thymus is increased, and the targeting efficiency in the brain is increased by more than 10 times. NMN-HAP significantly increases the levels of NAD + and NR (NAD +levels. Thus, the NMN-HAP has anti-aging potential to improve NMN bioavailability and in vivo NAD + levels, and can simultaneously exert a role in maintaining bone health.

[0068] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details of the above-described embodiments, and various simple modifications can be made to the technical solutions of the present application within the technical concept of the present application, and these simple modifications all belong to the protection scope of the present application.

[0069] In addition, it should be noted that each specific technical feature described in the above specific embodiments can be combined in any appropriate manner without contradiction, and in order to avoid unnecessary repetition, various possible combinations are not described again by the present application.

[0070] Furthermore, various different embodiments of the present application can also be combined in any manner, as long as it does not deviate from the idea of the present application, and it should be considered as disclosed by the present application.

Claims

1. A hydroxyapatite nanoparticle encapsulating β nicotinamide mononucleotide, comprising β nicotinamide mononucleotide and hydroxyapatite, β nicotinamide mononucleotide and hydroxyapatite in a weight ratio of 2-5:1; the nanoparticle is a rod-like structure with a particle size of 50-150 nm.

2. The package of claim 1, wherein the β The hydroxyapatite nanoparticles of nicotinamide mononucleotide, the β The weight of nicotinamide mononucleotide to hydroxyapatite is 3:

1.

3. The package of claim 1, wherein the β Hydroxyapatite nanoparticles of nicotinamide mononucleotide, the nanoparticles having a particle size of 100 nm.

4. The β Hydroxyapatite nanoparticles of nicotinamide mononucleotide, the encapsulation efficiency of the nanoparticles being 45.15 ± 1.57%.

5. The entrapment of β Hydroxyapatite nanoparticles of nicotinamide mononucleotide, the drug loading capacity of the nanoparticles being 42.42 ± 0.71%.

6. A method of preparing a hydroxyapatite nanoparticle encapsulating a β nicotinamide mononucleotide, comprising the steps of: dispersing hydroxyapatite in pure water, subsequently replacing the pure water in the suspension with ethanol, and adding β nicotinamide mononucleotide in water; after vortex incubation of the resulting mixture, removing the supernatant; wherein said beta The final weight ratio of the nicotinamide mononucleotide to the hydroxyapatite is 2-5:

1. The hydroxyapatite is rod-shaped structure with a particle size of 50-150 nm.

7. The method of preparing nanoparticles according to claim 6, wherein the β The final weight ratio of nicotinamide mononucleotide to hydroxyapatite is 3:

1.

8. The method of claim 6, wherein the hydroxyapatite has a particle size of 100 nm.

9. The method of any one of claims 6-8, wherein the weight ratio of the hydroxyapatite to pure water is 1:5-10.

10. The method of claim 9, wherein the weight ratio of the hydroxyapatite to pure water is 1:

7.

11. The method of any one of claims 6-8, wherein the step of mixing and dispersing comprises using ultrasonic wave to disperse the suspension after mixing the hydroxyapatite with pure water.

12. The method of claim 11, wherein the ultrasonic wave is 960 watts, 5 seconds on and 3 seconds off, for 1.5 hours.

13. The method of any one of claims 6-8, wherein the vortex incubation of the mixture is at room temperature for 12 hours.

14. The method of any one of claims 6-8, wherein the supernatant is removed by centrifugation.

15. The method of claim 14, wherein the centrifugation is at 3,000 rpm for 5 minutes.

16. The method of claim 6, comprising the following steps: first, mixing the rod-shaped HAP with a particle size of 100 nm with pure water at a weight ratio of 1:7, and using an ultrasonic wave of 960 watts, 5 seconds on and 3 seconds off, to disperse the suspension for 1.5 hours; then, replacing the pure water in the suspension with ethanol, and adding an aqueous solution of NMN, with a final weight ratio of NMN to HAP of 3:1 and a final volume ratio of ethanol to pure water of 7:3; vortex incubating the mixture at room temperature for 12 hours, and then centrifuging at 3,000 rpm for 5 minutes to remove the supernatant.

17. The method of claim 16, wherein the obtained nanoparticles have an encapsulation efficiency of 45.15±1.57% and a drug loading capacity of 42.42±0.71%.

18. The nanocapsules of claim 1-5, wherein the β The nanocapsules of claim 1-5, wherein the β Use of the nanocapsules of claim 1-5, wherein the β nucleotides for the preparation of a medicament for the prevention or treatment of diabetes.

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