Lipid nanoparticles and synthesis method thereof

Abstract

According to the lipid nanoparticles and the synthesis method thereof, the lipid nanoparticles comprise lipidosome, curcumin and nicotinamide ribose. The mass ratio of curcumin to nicotinamide ribose ranges from 1: 1 to 1: 10. Adding nicotinamide ribose into water to form a water-phase liquid; adding curcumin into the basic lipid to form an oil-phase liquid; and mixing the oil-phase liquid with the water-phase liquid to form a lipid nanoparticle solution. The medicine can be delivered through the nasal cavity, bypasses the blood brain barrier and directly and quickly enters the brain, has better targeting property on nerve diseases, and can make key components of the medicine directly reach nerves, so that the medicine has a better treatment effect on the nerve diseases.

Description

Technical Field

[0001] The present application belongs to the technical field of pharmaceutical compositions based on lipid nanoparticles and preparation methods thereof, and in particular, to compositions capable of targeted repair of pro-inflammatory microglia and preparation methods thereof. The prepared compositions can be used to treat neuroinflammation of neurological dysfunction and defects. Background Art

[0002] Liposome is a new type of drug preparation with targeted drug delivery function. It is a preparation formed by encapsulating drug molecules in vesicles formed by phospholipid bilayer membranes, and has both hydrophilic and hydrophobic properties. Lipid nanoparticles (LNP) are a type of nanoparticles formed by lipids.

[0003] Liposomes are the most widely studied nano drug carriers in drug delivery systems. Liposomes have a biological membrane-like structure and can encapsulate water-soluble and fat-soluble drugs. They have the characteristics of high selectivity, non-toxicity, non-immunogenicity, and suitability for degradation in vivo. As drug carriers, liposomes have a high degree of targeting, can effectively protect the encapsulated drugs, and can control and slow-release drugs, significantly improve the drug therapeutic index, and reduce the adverse reactions of drugs.

[0004] Liposome drug delivery system plays a very important role in reducing drug toxicity, increasing drug aggregation at target sites and improving drug efficacy.

[0005] Currently, researchers have designed targeted liposomes with or without target recognition molecules on the membrane, including anti-tumor drugs, anti-parasitic drugs, anti-fungal drugs, hormones, peptides, enzyme drugs, and drugs used for vaccines, gene therapy and immunodiagnosis.

[0006] Many liposome drugs have been approved and used in medical practice. There are already 50 nanodrugs on the market, including a variety of nanoformulations, among which lipid nanoparticles are the best.

[0007] Lipid nanoparticles are multicomponent lipid systems that typically contain phospholipids, ionizable lipids, cholesterol, and PEGylated lipids.

[0008] Lipid nanoparticles are able to encapsulate and deliver therapeutic drugs to specific locations in the body and release their contents at specific times, thus providing a valuable specific delivery channel for a variety of drugs.

[0009] Lipid nanoparticles are usually used to directly encapsulate chemical drugs. In the field of gene therapy, lipid nanoparticles are used to encapsulate nucleic acids, such as mRNA, siRNA, pDNA, etc., which are called nucleic acid lipid nanoparticles.

[0010] Neurological diseases, namely CNS (central nervous system) diseases such as Parkinson's disease, Alzheimer's disease, multiple sclerosis and ischemic stroke, are not fully effective due to the existence of the blood-brain barrier.

[0011] The blood-brain barrier refers to the barrier between plasma and brain cells formed by the walls of brain capillaries and glial cells, and the barrier between plasma and cerebrospinal fluid formed by the choroid plexus. These barriers can prevent certain substances (mostly harmful) from entering the brain tissue from the blood. The blood-brain barrier prevents most small molecule drugs and large molecules (for example, gene drugs and peptide and protein drugs) from entering the brain. Summary of the invention

[0012] In order to provide a drug that can cross the blood-brain barrier and better treat neurological diseases, this application designs a lipid nanoparticle and its synthesis method, which can be directly delivered into the brain through the nasal cavity. The cell membrane can reduce immune rejection, while the M2 cell membrane and MG1 promote the uptake of nanoparticles by microglia in the brain, thereby having a better therapeutic effect on neurological diseases. A composition that can target and repair pro-inflammatory microglia and its preparation method, the prepared composition can be used to treat neuroinflammation of neurological dysfunction and defects.

[0013] The technical solution of the present application to solve the above technical problems is a lipid nanoparticle, including: liposomes, curcumin, and nicotinamide riboside.

[0014] The mass ratio of curcumin to nicotinamide riboside may be in the range of 1:1 to 1:10.

[0015] Can be, including M2 microglial cell membrane.

[0016] It may be that the M2 microglial cell membrane is an M2 microglial cell membrane induced by IL-4;

[0017] It may be that the M2 microglial cell membrane surface includes MG1.

[0018] The lipid nanoparticles may be used to treat Parkinson's disease, Alzheimer's disease or neuroinflammation.

[0019] The lipid nanoparticles may have a diameter of less than 200 nanometers.

[0020] The lipid nanoparticles may have a diameter greater than 100 nanometers and less than 180 nanometers; and the lipid nanoparticles are used for delivery into the brain through the nasal cavity.

[0021] The technical solution of the present application to solve the above technical problems can also be a lipid nanoparticle synthesis method, comprising: adding nicotinamide riboside to water to form an aqueous phase liquid; adding curcumin to a base lipid to form an oil phase liquid; mixing the oil phase liquid with the aqueous phase liquid to form a lipid nanoparticle solution; the lipid nanoparticles include: liposomes, curcumin, and nicotinamide riboside.

[0022] Alternatively, the M2 microglial cell membrane may be added to water to form an aqueous phase liquid.

[0023] The M2 type microglial cell membrane can be added into water to form an aqueous liquid; the M2 type microglial cell membrane is obtained by adding MG1 dry powder to obtain the M2 type microglial cell membrane with MG1.

[0024] The oil phase liquid and the water phase liquid may be mixed by precipitation, emulsification, solvent evaporation or ultrasonic mixing.

[0025] The oil phase liquid and the water phase liquid may be mixed in a microfluidic chip.

[0026] Alternatively, the lipid nanoparticle solution may be prepared into lipid nanoparticle dry powder.

[0027] Alternatively, the lipid nanoparticle solution may be freeze-dried to prepare lipid nanoparticle dry powder.

[0028] The oil phase liquid and the water phase liquid may be mixed in a microfluidic chip; and the flow ratio of the oil phase liquid to the water phase liquid may range from 1:9 to 1:1.

[0029] One of the technical effects of the above technical solution is that the two key drug ingredients, curcumin and nicotinamide riboside, are made into lipid nanoparticles through liposomes. The particles are small enough to enter the brain through nasal delivery, thereby crossing the blood-brain barrier.

[0030] One of the technical effects of the above technical solution: During the development of neuroinflammation, excessive degradation of damaged organelles and foreign matter by intracellular lysosomes will lead to insufficient lysosomal acidification and inability to function normally. Nicotinamide riboside (NR) is a derivative of vitamin B3 and a precursor substrate of nicotinamide adenine dinucleotide (NAD+). After ingestion, it can increase NAD+ levels. At the same time, according to the physical and chemical properties of NR itself, its pH value is acidic. After being engulfed by lysosomes, it can lower the lysosomal pH value and restore the lysosomal degradation function.

[0031] One of the technical effects of the above technical solution is that curcumin can not only activate M2 microglia to exert anti-inflammatory effects, but also activate the autophagy level of cells.

[0032] One of the technical effects of the above technical solution is that nanoparticles made of liposomes, curcumin, and nicotinamide riboside can break through brain barriers and enhance the efficacy of traditional drugs.

[0033] One of the technical effects of the above technical solution is that the mass ratio of curcumin and nicotinamide riboside ranges from 1:1 to 1:10, and the mass ratio of the two can be adjusted as needed, such as 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9; so that the ability to restore lysosomal degradation function and activate the autophagy level of cells is balanced.

[0034] One of the technical effects of the above technical solution: M2 microglial cell membrane is also added to the formula of lipid nanoparticles, which greatly enhances the targeting of lipid nanoparticles, makes the drug efficacy more prominent, and helps "resting" microglia to transform into M2 microglia, thereby achieving an anti-inflammatory effect. Curcumin and nicotinamide riboside can promote the efficacy of M2 microglial cell membrane.

[0035] One of the technical effects of the above technical solution is that the surface of the M2 microglia cell membrane includes MG1, which further increases the targeting of lipid nanoparticles. It enables them to target microglia in the brain and promote the transformation of "resting" microglia into M2 microglia; MG1 and the M2 microglia cell membrane form a "dual target" effect, further increasing the efficacy.

[0036] One of the technical effects of the above technical solution is that the M2 microglial cell membrane and MG1 can enable lipid nanoparticles to target microglial cells in the brain more accurately, so that the drug combination of curcumin and nicotinamide riboside at a set ratio can exert its efficacy in the microglial cells in the brain.

[0037] One of the technical effects of the above technical solution is that the diameter of the lipid nanoparticles is less than 200 nanometers, which facilitates the delivery and absorption of drugs.

[0038] One of the technical effects of the above technical solution is that the diameter of the lipid nanoparticles is greater than 100 nanometers and less than 180 nanometers; it is convenient to be delivered into the brain through the nasal cavity and cross the blood-brain barrier.

[0039] One of the technical effects of the above technical solution is that the synthesis method of lipid nanoparticles is simple, reliable and has high preparation efficiency.

[0040] One of the technical effects of the above technical solution is that the flow ratio range of the oil phase liquid and the aqueous phase liquid facilitates the realization of the mass ratio range of curcumin and nicotinamide riboside, and the flow ratio can be adjusted according to the concentration of the corresponding solution so that the drug ratio in the lipid nanoparticles reaches the target range.

[0041] One of the technical effects of the above technical solution is that the lipid nanoparticle solution can be used as a medicine, and can also be prepared into a dry powder, which is easier to store. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 is a schematic diagram of the components of lipid nanoparticles;

[0043] Figure 2 is a schematic diagram of the components of lipid nanoparticles;

[0044] Figure 3 is a schematic diagram of the components of lipid nanoparticles;

[0045] Figure 4 is a schematic diagram of the lipid nanoparticle synthesis method;

[0046] Figure 5 is a schematic diagram of the lipid nanoparticle synthesis method;

[0047] Figure 6 is a schematic diagram of the lipid nanoparticle synthesis method;

[0048] Figure 7 is a schematic diagram of the lipid nanoparticle synthesis method;

[0049] Figure 8 is a schematic diagram of the lipid nanoparticle synthesis method;

[0050] Fig. 9 is a schematic diagram of the microfluidic chip system used in the lipid nanoparticle synthesis method;

[0051] Fig.10 It is the ratio of liposome particle size at different flow rates;

[0052] Fig.11 This is a schematic diagram of the drug concentration curve in the CCK8 assay for BV2 cell activity;

[0053] Fig.12 This is an electron microscope image of nanoparticles;

[0054] Fig.13 is a schematic diagram of nanoparticle size and nanoparticle membrane potential;

[0055] Fig.14 It is a schematic diagram of flow cytometry detection of the uptake level of nanoparticles by BV2 cells;

[0056] Fig.15 It is a schematic diagram of ELISA detection of IL-10 and TNF-α content in BV2 cell supernatant;

[0057] Fig.16is immunofluorescence detection of LC3B and LAMP protein expression levels in BV2 cells;

[0058] Fig.17 It is the in vivo imaging to detect the residence time of nanoparticles in the brain;

[0059] Fig.18 This is a diagram of the results of behavioral experiments. A: Rotation experiment; B: Pole climbing experiment; C: Grip strength experiment;

[0060] Fig.19 This is a schematic diagram of immunohistochemical detection of TH and α-Syn content in mouse brain tissue;

[0061] Fig. 20 This is a schematic diagram of the relative expression levels of TNF-α, IL-6, IL-1β and IL-10 mRNA in mouse brain tissue detected by Q-PCR. DETAILED DESCRIPTION

[0062] The content of this application is further described in detail below in conjunction with the accompanying drawings. It should be noted that the following is a description of the preferred embodiments of the present invention and does not constitute any limitation to the present invention. The description of the preferred embodiments of the present invention is only an illustration of the general principles of the present invention. The numbers such as "first", "second" and "A" and "B" involved in the present invention are only for the convenience of explanation and do not represent a temporal or spatial order relationship.

[0063] Microglia are cells in the nervous tissue. Microglia are characterized by multiple synapses and plasticity. They are intrinsic immune effector cells in the central nervous system and play an extremely important role in the physiological processes of the central nervous system. Microglia are involved in the occurrence of a series of neurodegenerative diseases, and microglial activation and neuroinflammation are the main characteristics of neuropathology. It mediates the endogenous immune response to central nervous system damage and diseases, thereby exerting neuroprotective or neurotoxic effects. Microglia-mediated chronic inflammation is involved in the pathological process of a variety of chronic neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, etc.

[0064] Microglia are important glial cells in the central nervous system and the main regulators of the central nervous system's inflammatory response. They play an important role in tissue repair, injury, regeneration and inflammation regulation in a variety of central nervous system diseases. Microglia are the first cells to respond to central nervous system damage.

[0065] When stimulated by injury and pathological stress, "quiescent" microglia undergo various functional reprogramming, defined as polarization, to achieve adaptive responses of innate immunity. These two polarized phenotypes with completely different functions are called classically activated M1 microglia (pro-inflammatory) and alternatively activated M2 microglia (anti-inflammatory).

[0066] M1 microglia induce neuroinflammatory reactive oxygen species (ROS) by expressing and releasing multiple proinflammatory mediators, such as inducible nitric oxide synthase (iNOS), interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α), which promote neuronal damage and hinder brain repair.

[0067] In contrast, M2 microglia confer neuronal survival and promote brain repair and regeneration by secreting anti-inflammatory cytokines and trophic factors (interleukin-10 (IL-10), brain-derived neurotrophic factor (BDNF), and arginase-1 (Arg-1)).

[0068] There is little exploration in the industry on how to design a drug composition that can accurately target the CNS (central nervous system) and activate M2 microglia.

[0069] like Figures 1 to 3 , a lipid nanoparticle, characterized in that it includes: liposomes, curcumin, and nicotinamide riboside. In some embodiments, the mass ratio of curcumin to nicotinamide riboside ranges from 1:1 to 1:10. The lipid nanoparticle is used to treat Parkinson's disease, Alzheimer's disease or neuroinflammation. The basic lipid in the liposome can be a conventional lipid. Figures 5 to 8 , the basic lipids can include cholesterol and lecithin.

[0070] In some embodiments, the lipid nanoparticles have a diameter of less than 200 nanometers.

[0071] In some embodiments, the diameter of the lipid nanoparticle is greater than 100 nanometers and less than 180 nanometers; and the lipid nanoparticle is used for delivery into the brain through the nasal cavity.

[0072] like Figure 2 , lipid nanoparticles, including M2 microglia cell membranes. In some embodiments, the M2 microglia cell membranes are M2 microglia cell membranes induced by IL-4.

[0073] like Figure 3, the surface of M2 microglia cell membrane includes MG1. MG1 is the MG1 polypeptide sequence, which is a specific amino acid sequence with specific biological activity and targeting. The MG1 sequence is CHHSSSARC. The amino acids include Ala (Alanine) -A; Arg (Arginine) -R; Cys (Cysteine) -C; His (Histidine) -H; Ser (Serine) -S. A represents alanine, C represents cysteine, H represents histidine; S represents serine; R represents arginine.

[0074] like Figure 4 A method for synthesizing lipid nanoparticles, comprising: adding nicotinamide riboside to water to form an aqueous phase liquid; adding curcumin to a base lipid to form an oil phase liquid; mixing the oil phase liquid with the aqueous phase liquid to form a lipid nanoparticle A solution; the lipid nanoparticle A comprises: liposomes, curcumin, and nicotinamide riboside.

[0075] In some embodiments, the oil phase liquid and the water phase liquid are mixed by precipitation, emulsification, solvent evaporation or ultrasonic mixing.

[0076] like Figure 4 The oil phase liquid and the water phase liquid are mixed in a microfluidic chip; the microfluidic chip includes a water phase channel and an oil phase channel; the water phase channel and the oil phase channel are provided with respective microfluidic pumps for separate flow control. The oil phase channel is used for the oil phase liquid to pass through; the water phase channel is used for the water phase liquid to pass through; the water phase channel and the oil phase channel merge into a main channel, and the main channel is used for the mixed liquid to pass through; the flow ratio of the oil phase channel to the water phase channel, i.e., the range of the oil phase liquid and the water phase liquid is 1:9 to 1:1.

[0077] When the flow rate ratio of the oil phase channel to the water phase channel is 1:1, the concentration of curcumin in the oil phase liquid can be 10 mg / ml; the concentration of nicotinamide riboside in the water phase liquid can be 100 mg / ml. The corresponding concentration and flow rate are coordinated to ensure that the mass ratio of curcumin and nicotinamide riboside is controlled in the range of 1:5 to 1:10.

[0078] When the flow rate ratio of the oil phase channel to the water phase channel is 1:3, the concentration of curcumin in the oil phase liquid can be 10 mg / ml; the concentration of nicotinamide riboside in the water phase liquid can be 33 mg / ml. The corresponding concentration and flow rate are coordinated to ensure that the mass ratio of curcumin and nicotinamide riboside is controlled in the range of 1:5 to 1:10.

[0079] When the flow rate ratio of the oil phase channel to the water phase channel is 1:6, the concentration of curcumin in the oil phase liquid can be 10 mg / ml; the concentration of nicotinamide riboside in the water phase liquid can be 16.6 mg / ml. The corresponding concentration and flow rate are coordinated to ensure that the mass ratio of curcumin and nicotinamide riboside is controlled in the range of 1:5 to 1:10.

[0080] When the flow rate ratio of the oil phase channel to the water phase channel is 1:9, the concentration of curcumin in the oil phase liquid can be 10 mg / ml; the concentration of nicotinamide riboside in the water phase liquid can be 11.1 mg / ml. The coordination of the corresponding concentration and flow rate finally ensures that the mass ratio of curcumin and nicotinamide riboside is controlled in the range of 1:5 to 1:10.

[0081] like Figure 5 , a lipid nanoparticle synthesis method, using a chip including three channels, one for water phase channel, and two for water phase channels; including: adding nicotinamide riboside to water to form a water phase liquid, the first water phase channel is used for the water phase liquid to flow through; adding curcumin to the basic lipid to form an oil phase liquid; mixing M2 microglia membrane dry powder and water to form M2 microglia membrane solution, i.e. another water phase liquid, the second water phase channel is used for the water phase liquid to flow through; mixing the oil phase liquid with the first water phase channel, and then mixing with the second water phase liquid to form a lipid nanoparticle B solution containing lipid nanoparticles B; the lipid nanoparticles include: liposomes, curcumin, and nicotinamide riboside. The M2 microglia membrane is obtained by adding MG1 dry powder to obtain the M2 microglia membrane with MG1. After the oil phase channel is mixed with the first water phase channel at a flow ratio of 1:9 to 1:1, the flow ratio with the second channel is 1:1 to 5:1, which can be adjusted and adapted according to the corresponding substance content in the solution.

[0082] like Figure 6 The lipid nanoparticle solution can also be made into lipid nanoparticle dry powder; the lipid nanoparticle solution can be made into lipid nanoparticle dry powder by freeze-drying.

[0083] like Figure 7 A lipid nanoparticle synthesis method includes: adding MG1 dry powder to a lipid nanoparticle B solution, coating the surface of the M2 microglia cell membrane with MG1, and forming a lipid nanoparticle C solution. The lipid nanoparticle C solution can also be made into a lipid nanoparticle dry powder.

[0084] like Figure 8, a lipid nanoparticle synthesis method, comprising: adding nicotinamide riboside to water to form an aqueous phase liquid; adding curcumin to a basic lipid to form an oil phase liquid; mixing the oil phase liquid with the aqueous phase liquid to form a lipid nanoparticle A solution; the lipid nanoparticle A comprises: liposomes, curcumin, and nicotinamide riboside. The lipid nanoparticle A solution is freeze-dried to form a lipid nanoparticle dry powder A. Freeze-drying to form lipid nanoparticle dry powder A can effectively remove the organic solvent in the lipid nanoparticle A solution, thereby improving the stability and shelf life of the liposome. The lipid nanoparticle A solution formed after the lipid nanoparticle dry powder A is re-dissolved and the M2 microglia membrane solution is mixed again by a microfluidic pump of a microfluidic chip to form a lipid nanoparticle B solution. The microfluidic chip is mixed, and the flow ratio of the oil phase channel to the aqueous phase channel ranges from 1:1 to 5:1. The lipid nanoparticle B solution is formed, and then MG1 dry powder is added to form a lipid nanoparticle C solution in which the surface of the M2 microglia membrane is coated with MG1. The lipid nanoparticle C solution can be used to prepare lipid nanoparticle C dry powder.

[0085] Lipid nanoparticles (LNPs) play a key role in effectively protecting mRNA and transporting it into cells. Liposomes, an early version of LNPs, are a versatile nano-drug delivery platform.

[0086] In the present application, curcumin can not only activate M2 microglia to exert anti-inflammatory effects, but also activate autophagy levels.

[0087] In this application, during the development of neuroinflammation, excessive degradation of damaged organelles and foreign matter by intracellular lysosomes will lead to insufficient lysosomal acidification and inability to function normally. Nicotinamide riboside (NR) is a derivative of vitamin B3 and a precursor substrate of nicotinamide adenine dinucleotide (NAD+). Ingestion can increase NAD+ levels. At the same time, according to the physical and chemical properties of NR itself, its pH value is acidic, and after being engulfed by lysosomes, it can reduce the lysosomal pH value and restore the lysosomal degradation function.

[0088] In the present application, a nanoplatform with immunosuppressive properties was developed by adding cell membranes derived from microglia polarized to the M2 type to drug-regulate the ability of microglia polarization.

[0089] Nano-microglia have potent regulatory effects on M1 microglia and substantially reprogram the inflammatory cell content in Parkinson's disease and lipopolysaccharide-induced neuroinflammation models.

[0090] In the present application, by setting a suitable particle size, the drug-carrying nanoparticles can be delivered through the nasal cavity, overcoming the dense blood-brain barrier, directly entering the brain through the olfactory nerve and the trigeminal nerve, and precisely targeting inflammatory microglia.

[0091] At the same time, MG1 was designed on the surface of microglial cell membranes, enabling it to target microglia in the brain, forming a "double-target" effect with the microglial cell membrane. M2 microglial cell membrane-coated nanoparticles (CNM2M) were delivered into the brain through the nasal cavity, and then targeted and bound to microglia under the action of M2 microglial cell membranes and MG1. Inflammatory microglia then took up CNM2M, resulting in the release of curcumin and nicotinamide riboside NR.

[0092] Curcumin promotes the expression of endogenous anti-inflammatory cytokines and the production of two potent antioxidants, CO and bilirubin, by converting microglia from M1 to M2 state. Anti-inflammatory cytokines together with antioxidants significantly improved the inflammatory environment and had excellent therapeutic effects in Parkinson's disease and LPS (Lipopolysaccharides) models.

[0093] At the same time, curcumin can activate the level of cellular autophagy, encapsulate α-Syn in Parkinson's brain tissue and deliver it to lysosomes. After NR is taken up by lysosomes, the pH value is lowered, the degradation function is restored, and it can bind to autophagosomes to degrade the "waste" transported by autophagosomes, forming a closed loop and playing a synergistic therapeutic role. This simple engineered nanoglia provides a new and reliable strategy for the management of overactivated brain immune microenvironment.

[0094] The combination of curcumin and nicotinamide riboside has, on the one hand, medicinal effects: curcumin has anti-inflammatory and antioxidant effects, and can polarize microglia from M1 to M2, and nicotinamide riboside also has the effect of repairing damage; on the other hand, curcumin can activate the level of cell autophagy, and nicotinamide can improve insufficient lysosomal acidification. After the autophagosome engulfs the protein, it further combines with the lysosome to degrade the target protein and treat the disease.

[0095] The nanoliposome system loaded with curcumin and nicotinamide riboside was synthesized through the "harpoon" microfluidic chip. The nanoparticles are suitable for nasal delivery to cross the blood-brain barrier. CNM2M enters the brain lesion area through the olfactory nerve and trigeminal nerve, targets and binds to microglia under the action of MG1, and better fuses with microglia under the action of M2 cell membrane and enters the cell.

[0096] By activating cellular autophagy and improving downstream lysosomal function, it is applicable to a variety of pathological mechanisms in which autophagy levels are inhibited and lysosomes are damaged, including but not limited to: Parkinson's disease, Alzheimer's disease, neuroinflammation, etc.

[0097] CNM2M is a liposome synthesized by microfluidics and loaded with curcumin and nicotinamide riboside. The combination of these two drugs has been reasonably optimized. The flow rate ratio of the oil phase and the water phase ranges from 1:9 to 1:1, and the mass ratio of curcumin and nicotinamide riboside ranges from 1:1 to 1:10. The optimal flow rate ratio is 1:6, and the optimal drug ratio is 1:5. Animal experimental dosing concentration: 1mg / kg-10mg / kg, dosing frequency 1-2 times a week, time 1-2 months.

[0098] The dual targeting of CNM2M consists of IL-4-induced M2 microglia membrane coating and MG1, which has both low immunogenicity and dual targeting.

[0099] like Figure 8 and Fig. 9 , by adjusting the different flow rate ratios of the aqueous phase and the oil phase, liposomes were synthesized and the particle size was detected. Fig. 9 The microfluidic chip has two inlets, inlet 1 and inlet 2, one for water phase liquid to enter and the other for oil phase liquid to enter; after mixing, the liquid is output from the outlet.

[0100] Fig.10 The results showed that when the ratio of oil phase to water phase was 1:6, the particle size was the smallest and the efficiency was the highest, which was suitable for nasal delivery into the brain. Therefore, in subsequent experiments, we chose to synthesize liposomes with a ratio of oil phase to water phase of 1:6.

[0101] Cell activity is detected by CCK8 detection, i.e. Cell Counting Kit-8 cell counting experiment. CCK-8 reagent (Cell Counting Kit-8 cell counting experiment) contains WST-8: Chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonate benzene)-2H-tetrazole monosodium salt, which is reduced to a highly water-soluble yellow formazan product (Formazan) by dehydrogenase in the cell mitochondria under the action of the electron carrier 1-methoxy-5-methylphenazine dimethyl sulfate (1-MethoxyPMS). The amount of formazan generated is proportional to the number of living cells. The light absorption value is measured at a wavelength of 450nm using an enzyme-linked immunosorbent assay, which can indirectly reflect the number of living cells. This method has been widely used in the activity detection of some biologically active factors, large-scale anti-tumor drug screening, cell proliferation tests, cytotoxicity tests, and drug sensitivity tests.

[0102] BV2 is a type of microglia derived from mice. BV2 cells were inoculated into a 96-well plate. When the cell confluence reached about 80%, different concentrations of curcumin and nicotinamide riboside were added for 24 hours, and then CCK8 reagent was added for incubation for 1 hour. Fig.11, indicating that curcumin had no effect on BV2 cell activity at concentrations of 20, 40, and 80 μg / ml. Nicotinamide riboside had no effect on BV2 cell activity at concentrations of 40, 80, and 160 μg / ml.

[0103] like Fig.12 and Fig.13 The applicant detected the particle size and membrane potential of the microfluidic synthesized liposomes and the liposomes coated with polarized cell membranes. After the liposomes were coated with the cell membrane and connected to MG1, the particle size gradually increased and the membrane potential gradually decreased, indicating that BV2 was successfully coated and successfully connected to MG1. Fig.12 The left group is the electron micrograph of CNNPs, with the whole electron micrograph on the top and the partial enlarged image on the bottom; the middle group is the electron micrograph of CN@MNPs, with the whole electron micrograph on the top and the partial enlarged image on the bottom; the right group is the electron micrograph of CN@MMNPs, with the whole electron micrograph on the top and the partial enlarged image on the bottom. C is the first letter of the English word curcumin, N is the first letter of nicotinamide riboside; NPs is the abbreviation of lipid nanoparticles; the first M is the abbreviation of M2 type, and the second M is the abbreviation of MG1.

[0104] like Fig.14 Targeting detection: BV2 cells were inoculated into 96-well plates, and when the cell confluence was about 80%, CNNPs, CN@MNPs, and CN@MMNPs were added for 6 hours, and then the cell supernatant was removed. The fluorescence intensity of nanoparticles bound to BV2 cells was detected by flow cytometry, and it was found that the uptake rate of CNNPs was 77.68%, the uptake rate of CN@MNPs was 82.61%, and the uptake rate of CN@MMNPs was 92.96%. This shows that the M2-type BV2 cell membrane promotes the uptake of nanoparticles by BV2 cells, and MG1 further enhances the targeting.

[0105] The effect of CN@MMNPs on the inflammatory level of BV2 cells was detected by enzyme-linked immunosorbent assay (ELISA). BV2 cells were inoculated into 96-well plates, and CNNPs, CN@MNPs, and CN@MMNPs were added when the cell confluence was about 80%. The cells were intervened for 24 hours, and then the cell supernatant was collected. Fig.15 ,ELISA results showed that CNNPs, CN@MNPs, and CN@MMNPs could significantly reduce the release of pro-inflammatory factor TNF-α and promote the release of anti-inflammatory factor IL-10. Among them, CN@MMNPs had the best anti-inflammatory effect because BV2 took up the most drugs.

[0106] like Fig.16, the effect of CN@MMNPs on the autophagy level of BV2 cells was detected by immunofluorescence. BV2 cells were inoculated into 96-well plates, and CNNPs, CN@MNPs, and CN@MMNPs were added when the cell confluence was about 80%. After 24 hours of intervention, the cell supernatant was collected. The immunofluorescence results showed that CNNPs, CN@MNPs, and CN@MMNPs could significantly promote the expression level of LC3B protein. In addition, CNNPs had little effect on the expression level of LAMP protein. CN@MNPs enhanced the function of lysosomes while activating autophagy, and the autophagy and lysosomal functions of CN@MMNPs were further enhanced.

[0107] like Fig.17 , the residence time of CN@MMNPs in the mouse brain was detected by in vivo imaging. The nanoparticles were first incubated with Cy7 dye at 37°C for 1 hour, and then the unbound dye was removed by ultrafiltration tube. After anesthesia, CNNPs, CN@MNPs, and CN@MMNPs were dripped into the nasal cavity of the mice, and the fluorescence intensity in the brain was detected at different times. The results showed that fluorescence began to appear in the brains of mice in each group 1 hour after the nanoparticles were dripped, and each group reached a peak at the 4h time point. At the same time, the fluorescence intensity in the brain of the mice in the CN@MNPs group was greater than that in the CNNPs group, indicating that the M2 cell membrane was conducive to the binding of nanoparticles to BV2 cells. In addition, the fluorescence intensity in the brain of the mice in the CN@MMNPs group was the largest, indicating that under the dual effects of the M2 cell membrane and the human MG1 targeting peptide, the nanoparticles bound to the BV2 cells the most. Moreover, with the extension of time, the fluorescence in the brain of the CN@MMNPs group decayed the slowest.

[0108] like Fig.18 , the effect of nanoparticles on the behavior of PD mice was detected by behavioral testing. First, the model group and the drug-treated group were given MPTP modeling for 7 days, and then each drug-treated group delivered nanoparticles nasally twice a week for 4 consecutive weeks. Behavioral testing was performed after the experiment. It was found that after MPTP modeling, the time for the model group mice to fall from the rotating rod was shortened, the time for climbing the pole was prolonged, and the grip strength score was reduced. After 4 weeks of intervention with CNNPs, CN@MNPs, and CN@MMNPs, the time for mice to fall from the rotating rod was significantly increased, the time for climbing the pole was reduced, and the grip strength was improved, which suggests that after the administration of CNNPs, CN@MNPs, and CN@MMNPs, the behavioral disorders of mice were significantly improved. At the same time, the CN@MMNPs group had the best therapeutic effect.

[0109] Immunohistochemistry was used to detect the effect of nanoparticles on brain tissue pathology in PD mice. Fig.19As shown in the figure, after MPTP (1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine) induction, TH+ (tyrosine hydroxylase-positive, tyrosine hydroxylase) in the substantia nigra of the model group mice brain tissue was significantly reduced, and α-Syn (α-synuclein, α-synuclein) was significantly increased. After treatment with CNNPs, CN@MNPs, and CN@MMNPs, compared with the model group, the TH+ positive rate of each group increased significantly, and the α-Syn deposition decreased significantly, indicating that CNNPs, CN@MNPs, and CN@MMNPs can improve the pathological damage of PD mice to varying degrees, and the treatment effect of the CN@MMNPs group is the most obvious.

[0110] like Fig. 20 , Q-PCR stands for Quantitative Polymerase Chain Reaction, which is a technology for quantitative detection of DNA or RNA. The effect of nanoparticles on neuroinflammation in PD mice was detected by Q-PCR. After the behavioral experiment, brain tissue was taken for grinding, RNA was extracted, and the relative expression levels of TNF-α, IL-6, IL-1β and IL-10 mRNA in brain tissue were detected by Q-PCR. The experimental results showed that compared with the control group, the relative expression levels of TNF-α, IL-6 and IL-1β mRNA in the brain tissue of mice in the model group increased significantly, and the relative expression level of IL-10 mRNA decreased significantly. After treatment with CNNPs, CN@MNPs, and CN@MMNPs, the relative expression levels of TNF-α, IL-6 and IL-1β mRNA decreased significantly, and the relative expression level of IL-10 mRNA increased significantly, and the CN@MMNPs group was more significant. This shows that CN@MMNPs targeting BV2 cells can better play an anti-inflammatory role.

[0111] Cytokines are mainly synthesized by leukocytes and mainly mediate the interaction between leukocytes. Therefore, these factors are collectively named interleukins (IL) and named with Arabic numerals in the order of discovery, such as IL-1, IL-2, and IL-3. In this application, IL-6 and IL-1β are typical pro-inflammatory factors, which are highly expressed in Parkinson's disease models.

[0112] Although the present invention is illustrated and described according to the preferred embodiment and several alternatives, the invention is not limited by the specific description in this specification. Other additional replacement or equivalent components can also be used to practice the present invention.

Claims

1. A lipid nanoparticle, characterized in that: include: Liposomes, curcumin, nicotinamide riboside.

2. The lipid nanoparticle according to claim 1, characterized in that The mass ratio of curcumin to nicotinamide riboside is in the range of 1:1 to 1:

10.

3. The lipid nanoparticle according to claim 1, characterized in that Includes M2 microglial cell membranes.

4. The lipid nanoparticle according to claim 3, characterized in that Include any of the following technical features: TA20: the M2 microglial cell membrane is the M2 microglial cell membrane induced by IL-4; TA30: The M2 microglial cell membrane surface includes MG1.

5. The lipid nanoparticle according to claim 1, characterized in that The lipid nanoparticles are used to treat Parkinson's disease, Alzheimer's disease or neuroinflammation.

6. The lipid nanoparticle according to claim 1, characterized in that Include any of the following technical features: TB10: The diameter of the lipid nanoparticle is less than 200 nanometers; TB20: The diameter of the lipid nanoparticle is greater than 100 nanometers and less than 180 nanometers; the lipid nanoparticle is used for delivery into the brain through the nasal cavity.

7. A method for synthesizing lipid nanoparticles, characterized in that: include: Adding nicotinamide riboside to water to form an aqueous liquid; Adding curcumin to the base lipid to form an oil phase liquid; Mixing the oil phase liquid with the aqueous phase liquid through microfluidics to form a lipid nanoparticle solution; The lipid nanoparticles include: liposomes, curcumin, and nicotinamide riboside.

8. The method for synthesizing lipid nanoparticles according to claim 7, characterized in that: Include any of the following technical features: TF10: M2 microglial cell membranes were added to water to form an aqueous liquid phase; TF20: M2 microglial cell membrane is added into water to form an aqueous liquid; M2 microglial cell membrane is obtained by adding MG1 dry powder to obtain M2 microglial cell membrane with MG1.

9. The method for synthesizing lipid nanoparticles according to claim 7, wherein Include any of the following technical features: TE10: The oil phase liquid and the water phase liquid are mixed by precipitation, emulsification, solvent evaporation or ultrasonic mixing; TE20: mixing the oil phase liquid and the water phase liquid in a microfluidic chip; TE30: preparing the lipid nanoparticle solution into lipid nanoparticle dry powder; TE40: preparing lipid nanoparticle dry powder by freeze-drying the lipid nanoparticle solution; TE50: The oil phase liquid and the water phase liquid are mixed in a microfluidic chip; the flow ratio of the oil phase liquid to the water phase liquid ranges from 1:9 to 1:1.

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