CN109730966B - Chitosan oligosaccharide modified self-carried carrier-free nasal cavity nano preparation brain targeting delivery system and preparation method thereof - Google Patents
Chitosan oligosaccharide modified self-carried carrier-free nasal cavity nano preparation brain targeting delivery system and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a chitosan oligosaccharide modified self-carried non-carrier nasal cavity nano preparation brain targeting delivery system and a preparation method thereof. Comprises hydrophobic micromolecular drugs with neuroprotective effect, polyethylene glycol derivatives and chitosan oligosaccharide. The invention also provides a preparation method of the nasal nano preparation brain targeting delivery system. Firstly, preparing nano-particle freeze-dried powder, and secondly, stirring the freeze-dried powder and chitosan oligosaccharide in isotonic normal saline before use to obtain the nasal preparation with good membrane permeability. The system has simple preparation method, can improve the hydrophobicity of the small molecular drug, reduce the toxicity and enhance the neuroprotective effect; no carrier, no biodegradation problem and accumulative toxicity, high drug loading rate up to more than 25%, good transdermal absorption after chitosan oligosaccharide modification, and high-targeting delivery of the drug into brain. The dosage form has the advantages of nasal drop, spray, etc., simple operation, convenient administration for patients taking medicine for a long time, and good application prospect in the treatment of nervous system diseases.
Description
Technical Field
The invention belongs to the field of biological medicines, and relates to a brain-targeted delivery system of a nasal nano preparation and a preparation method thereof. In particular to a chitosan oligosaccharide modified self-carried type non-carrier nasal cavity nano preparation brain targeting delivery system and a preparation method thereof.
Background
In recent years, the incidence of nervous system diseases has increased year by year, and therapeutic drugs are in serious shortage. Most of small molecules with neuroprotective activity, which are screened in drug research and development, are hydrophobic organic small molecules, and the problems of difficult water solubility, low bioavailability in vivo, short blood circulation time, instability and the like greatly hinder the clinical application of the small molecules.
For example, curcumin has been reported to have significant neuroprotective pharmacological activity in vitro, but its in vivo use has been limited by problems such as poor water solubility. In addition, in a conventional oral administration mode, after the medicine passes through intestinal mucosa and liver and is subjected to inactivation and metabolism, the dosage entering systemic circulation is reduced, and the medicine effect is reduced. And due to the existence of blood brain barrier, the content of the medicine capable of entering the brain is extremely low, so that the application of the medicine in nervous system diseases is limited.
With the rapid development of nanotechnology, the nano-preparation has been widely used in the medical and biological fields and gradually applied to nervous system diseases due to its advantages of protecting the drug from being destroyed, prolonging the effective drug retention time, controlling the drug release, and reducing the toxic and side effects of the drug. Many of the reported nano-preparations are drug-loaded nanoparticles carried by carriers such as liposomes and polyester copolymers (patent nos. CN107029247A, CN101897669B, CN 102283812B, etc.). However, the nano-particles have three disadvantages: firstly, with long-term administration, the enrichment of the polymer and other carriers in the brain can bring potential toxic and side effects; secondly, the drug is wrapped in the carrier and may lose the original self-targeting recognition function; thirdly, the drug loading rate of the existing polymer-based nano drug loading system is usually lower than 10%, which is a troublesome problem that further application of nano particles to clinic is hindered. Therefore, there is a need to develop a nano delivery system with high drug loading, safety, non-toxicity, simplicity, easy implementation, and general applicability to the existing large amount of hydrophobic drugs, and the nano delivery system is applied to the treatment of nervous system diseases.
In recent years, it has become highly desirable to develop alternative self-contained nano-drug delivery strategies without the use of any carrier. In 2012, KasaiI et al successfully applied the strategy for the first time by forming a self-contained carrier-free pure nano-drug with a particle size of 30-50nm by a reprecipitation method after two drug molecules are linked into a dimer. However, the nano-drugs can not be successfully applied to the drugs for treating brain diseases so far, and the main reason is that under the condition of conventional preparations, the nano-drugs can only circulate in blood for a long time and can not enter the brain through the blood brain barrier (the pore diameter of brain capillary endothelial cells is only 14-18nm), so that the delivery of the drugs for treating the brain can not be realized.
Nasal administration is a novel safe and non-invasive administration method at present, and the drug can reach cerebrospinal fluid or brain along the connecting tissue surrounding the olfactory nerve bundle or the axon of the olfactory neuron through the olfactory mucosa, and the drug including macromolecular proteins and nanoparticles can bypass the blood brain barrier through the route and directly enter the central nervous system to play a role, so that the delivery of the brain therapeutic drug is realized. In addition, after nasal administration, the drug is not metabolized through gastrointestinal tract and degraded by liver, and a small amount of drug can reach higher drug concentration in brain, so that the drug administration dose and frequency can be reduced, and the side effect of dose dependence can be reduced. Compared with intravenous injection, nasal administration only needs a nasal drip and spray mode, the operation is simpler and safer, especially for neurodegenerative disease patients taking medicines for a long time, the pain of the patients can be relieved, the patients can easily accept the medicine, the medicine taking by themselves is convenient, and the risk brought by long-term medicine taking is reduced.
However, the nasal cavity has many careful requirements for formulation due to its unique environment. The first requirement is the membrane-permeable absorption properties of the drug. The serous fluid and mucus secreted by the glands in the nasal cavity contain rich proteolytic enzymes, and are one of the factors influencing the nasal absorption of the medicine. The pH value of nasal cavity mucus is 5.5-6.5, which is the most suitable pH value of proteolytic enzyme, and in addition, the nasal cavity mucus has the function of clearing away nasal mucociliary. The above-mentioned varieties of development of nasal formulations present challenges. The use of the absorption enhancer can enhance the nasal mucosa absorption of the medicine to a certain extent. Currently, commonly used absorption enhancers are anionic surfactants such as stearic acid, lauric acid, sodium lauryl sulfate, sulfonates, and the like, and nonionic surfactants such as polysorbate, brij, and the like. However, cholates have certain adverse effects on nasal mucosa, such as burning sensation, pain, etc., and produce strong irritation to nasal mucosa at a lower concentration (2%), a high concentration (5%) can destroy the epithelial structure of nasal mucosa, and a higher concentration can completely shed nasal cilia or epithelial cells. Thus, an effective and non-toxic absorption enhancer is key to nasal formulations.
Patents CN105617395A, CN105582545A and CN105617396A respectively relate to a preparation method of a nasal administration brain-targeted nano drug-loading system, a nasal administration nano brain-targeted drug of lycoramine and a preparation method thereof, a nasal administration nano brain-targeted drug of galanthamine and a preparation method thereof. The three patents aim at obtaining the targeted drug by using the esterification reaction of the hydrophilic group of the hydrophilic micromolecular drug and the carboxylated chitosan; the invention develops the preparation and the application of a nasal cavity nano preparation brain targeting delivery system aiming at hydrophobic micromolecule drugs specifically.
Experiments show that after puerarin is nasally administrated, the peak concentration and bioavailability of the medicine at the olfactory bulb part are 1.72 times and 3.05 times of those of intravenous injection, the brain targeting index is up to 14 percent and is 7.5 times of those of the intravenous injection, so that the nasal cavity approach has very wide prospect for improving the curative effect of the puerarin. The patent CN107184554 discloses a preparation method of puerarin liquid crystal nanoparticles, however, the loading rate of nano-drugs prepared by a film method is generally not more than 10 percent, the drug effect is limited, and the poloxamer 407 auxiliary material used is a polymer which can not be biodegraded, the proportion of the polymer in the preparation is four times of that of the drugs, and the potential toxic and side effects can be caused due to long-term administration and accumulation. In addition, there is a report of nasal delivery of danshensu nanoparticles into brain via polymer carrier (patent CN107029247A), but the polymer carrier still has three inherent defects as mentioned above, which limits its clinical transformation application. If the hydrophobic micromolecules are prepared, the defects are overcome through a more optimized nano preparation brain targeting delivery system, the drug treatment effect is expected to be further improved, and the hydrophobic micromolecules have very important significance for promoting the treatment application of the hydrophobic micromolecules in nervous system diseases.
Disclosure of Invention
The invention mainly aims to provide a brain-targeted delivery system of a nasal nano-preparation aiming at the problems that the hydrophobic organic micromolecules with neuroprotective effect have low bioavailability due to poor drug-forming property, the drugs cannot cross blood brain barriers in the conventional administration route and the like. In particular to a chitosan oligosaccharide modified and self-carried type non-carrier nasal cavity nano preparation brain targeting delivery system.
The technical scheme of the invention is as follows:
a chitosan oligosaccharide modified self-carried non-carrier nasal cavity nano preparation brain targeting delivery system comprises chitosan oligosaccharide, hydrophobic micromolecular drug with nerve protection effect and polyethylene glycol derivative; firstly, preparing a good solvent solution of a hydrophobic micromolecule drug with the concentration of 1-10mg/mL and 0.5-5mg/mL of polyethylene glycol derivatives, and then dropwise adding the good solvent solution into deionized water, wherein the volume ratio of the good solvent solution to the deionized water is (0.5-5): 50, blowing nitrogen while dropwise adding to assist in volatilizing a good solvent; preparing self-carried carrier-free nanoparticle suspension emulsion with particle size of 50-200nm by reprecipitation, and freeze drying to obtain lyophilized powder; before use, the freeze-dried powder and the chitosan oligosaccharide react for 0.5 to 2 hours in isotonic normal saline by utilizing physical adsorption, wherein the concentration of the chitosan oligosaccharide in the isotonic normal saline is 0.01 to 0.2 percent (w/v), reactants are removed, and a product is purified to obtain the self-carrying type carrier-free nano-drug nasal cavity preparation modified by the chitosan oligosaccharide.
In the invention, the chitosan oligosaccharide is used for the synergistic action of nasal mucosa penetration promotion and neuroprotection.
Preferably, the surface potential of the self-carrying unsupported nanoparticles is from-10 to-60 mV.
Preferably, the hydrophobic small molecule drug with neuroprotective effect is one or more of curcumin or curcumin analogues.
Further preferably, the hydrophobic small molecule is a curcumin analogue of the following structural formula, a mixture of cis-isomers thereof:
further preferably, the weight ratio of the cis-isomer in the mixture is 25-35% of the total mixture.
Further preferably, the mixture of cis-isomers in a weight ratio of 25-35% of the total mixture is prepared by subjecting a methanol solution of curcumin analogs to ultraviolet irradiation for 1.5-2.5 h.
Preferably, the concentration of the methanol solution of the curcumin analog upon ultraviolet irradiation is 0.5 to 5mg/ml, and more preferably 0.5 to 1.5 mg/ml. If the time of the ultraviolet irradiation is shorter than 1.5 hours, the cis-isomer cannot be formed in a sufficient amount, and if it is longer than 2.5 hours, the by-production of the by-product starts, and the ultraviolet irradiation is particularly preferably 2 hours.
Preferably, the molecular weight range of the polyethylene glycol derivative is less than 5000, and more preferably less than 2000.
More preferably, it is a carboxyl polyethylene glycol or a polymaleic anhydride 18 carbene-polyethylene glycol.
Preferably, the gas is nitrogen or an inert gas, preferably nitrogen. And (3) volatilizing the good solvent to ensure the formation of the nano particles and prevent potential safety hazards caused by solvent residue.
Preferably, the reprecipitation method is to drop the good solvent solution into deionized water, stir for 2-10min at a temperature of 20-30 ℃, and stand for 3-30min to obtain a nanoparticle suspension.
The brain-targeted delivery system of the nasal nano preparation has no carrier, no biodegradation problem and accumulated toxicity, the drug-loading rate is up to more than 25 percent, the brain-targeted delivery system can slowly release micromolecule drugs in a pH-responsive manner, and the binding capacity of the micromolecules and targeted receptors is highly reserved; the polyethylene glycol derivative can enhance the water dispersibility and stability of the particles; the chitosan oligosaccharide is an absorption enhancer, and negative nano-drug particles are modified by positive and negative charge adsorption, so that the chitosan oligosaccharide nasal mucosa transdermal patch has nasal mucosa permeation-promoting performance and has a neuroprotective synergistic effect.
The chitosan is a macromolecule obtained by deacetylating chitin, has a molecular weight of hundreds of thousands to millions of Da, and is insoluble in water. The chitosan is degraded by special biological enzyme technology and the like to obtain an oligosaccharide product with the polymerization degree of 2-20, wherein the oligosaccharide product is chitosan oligosaccharide, namely chitosan oligosaccharide and oligomeric chitosan, has the molecular weight of less than or equal to 3200Da, and has a plurality of unique functions of higher solubility, full solubility in water, easy absorption and utilization by organisms and the like which are not possessed by the chitosan. The chitosan oligosaccharide is nontoxic functional low molecular weight amino sugar and has a polycation structure, and can be modified outside nanoparticles to prevent irritation of drugs to the environment in nasal cavity; the chitosan oligosaccharide can easily act with negatively charged groups on the surface of a mucosal cell to change the fluidity and permeability of a cell membrane and increase the absorption of nanoparticles, and in addition, the chitosan oligosaccharide has certain immunoregulation and neuroprotection effects, and the effect of the chitosan oligosaccharide is 14 times that of chitosan.
The chitosan oligosaccharide used in the invention has a polymerization degree of 2-20 or a molecular weight of less than or equal to 3200 Da.
The chitosan oligosaccharide of the invention has the advantages that the concentration of isotonic physiological saline solution is 0.01-0.2% (w/v), if the concentration is less than 0.01%, the chitosan oligosaccharide has difficulty in playing roles in increasing absorption, preventing stimulation and the like, and if the concentration is more than 0.2%, negatively charged nanoparticles are easy to aggregate.
Re-suspending the freeze-dried powder in isotonic physiological saline, wherein the concentration of the freeze-dried powder can be prepared according to the requirement. Preferably 3-7 mg/ml.
Preferably, the hydrophobic drug nanoparticles have an average particle size of 50 to 150nm, more preferably 50 to 120 nm.
Preferably, the surface potential of the hydrophobic drug nanoparticles is-30 to-60 mV.
Preferably, the drug loading rate of the hydrophobic drug nanoparticles is 25% or more.
The formation of self-assembled nano-particles is influenced by the molecular structure of hydrophobic small molecules, and non-covalent binding force exists among molecules, so that the difference of the nano-structures can be caused due to the difference of molecular configurations. In order to enhance the stability of the nano-particles, the invention adds the polyethylene glycol derivative before solvent exchange, uniformly mixes the polyethylene glycol derivative and hydrophobic micromolecules in an organic solvent according to a certain proportion, then carries out solvent exchange, and obtains the nano-particles without carrier coating by a reprecipitation method.
The hydrophobic drug nano-particles of the invention have no other carrier components, so the drug loading is high, the toxicity is low, the safety is good, the particle size is small and uniform, the stability is high, and the circulation time in vivo is long.
Preferably, the hydrophobic drug molecule is a natural product having neuroprotective effect and a modification thereof.
More preferably, the natural product with neuroprotective effect and its modification is one or more of curcumin or its analogues.
Furthermore, other pharmaceutically effective auxiliary materials, such as bacteriostatic agents, isotonic regulators and the like can be added into the brain targeting delivery system of the nasal nano preparation, and the dosage of the brain targeting delivery system is the conventional dosage specified in pharmaceutics.
The invention can also add antioxidant, the antioxidant can be one or more of sodium metabisulfite, sodium bisulfite, sodium sulfite, sodium thiosulfate, cysteine hydrochloride, vitamin C, vitamin E and thiourea, and the dosage is the conventional dosage specified in pharmacy.
The invention can also be added with preservative which can be one or more of methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate, butyl p-hydroxybenzoate, benzalkonium bromide, benzalkonium chloride, chlorobutanol, phenethyl alcohol, thimerosal, phenylmercuric nitrate, sorbic acid and chlorhexidine, and the dosage of the preservative is conventional dosage specified in pharmaceutics.
The invention can also be added with an osmotic pressure regulator, wherein the osmotic pressure regulator can be one or more of sodium chloride, glucose, lactose and mannitol, and the dosage of the osmotic pressure regulator is the conventional dosage specified in pharmaceutics.
In a second aspect, the present invention provides a method for preparing a chitosan oligosaccharide modified self-contained unsupported nasal nanoparticle formulation as described in the first aspect, said method comprising the steps of:
1) firstly, preparing good solvent solution of 1-10mg/mL and 0.5-5mg/mL of hydrophobic micromolecule drug of polyethylene glycol derivative, and then dropwise adding the good solvent solution into deionized water: the volume ratio of the good solvent solution to the deionized water is (0.5-5): 50, dripping while blowing gas and volatilizing good solvent, 2) preparing self-carried carrier-free nano particle suspension emulsion with the particle size of 50-200nm by a reprecipitation method, and freeze-drying to prepare freeze-dried powder;
3) before use, the freeze-dried powder and the chitosan oligosaccharide react for 0.5 to 2 hours in isotonic normal saline by physical adsorption, reactants are removed, and the product is purified to obtain the chitosan oligosaccharide modified self-carried carrier-free nano-drug nasal cavity preparation.
The method for preparing the nano-particles is carried out by a reprecipitation method, when a good solvent is converted into water (a poor solvent), hydrophobic drug molecules are separated out to form the nano-particles, and the stability and the water dispersibility of the nano-particles can be further enhanced by adding the polyethylene glycol derivative. The method is simple and easy to implement, does not need complex operation and conditions, and can be carried out at room temperature.
It should be emphasized that, in the present invention, the time for adding the polyethylene glycol derivative should be before the nanoparticles are formed, i.e. dissolving the polyethylene glycol derivative in the good solvent together with the small molecules, mixing them uniformly, and then adding the aqueous phase dropwise to prepare the nanoparticles. When the organic solvent is dropped, a gas (preferably nitrogen gas) is blown to remove the organic solvent. The method is different from the method of firstly forming nano particles and then adding the amphiphilic surfactant for surface modification, and the product is also different.
The freeze-dried powder and the chitosan oligosaccharide are mixed in isotonic normal saline just before use, so that aggregation and precipitation generated after long-term placement are avoided, and the nasal absorption is ensured during use.
In the method for preparing the hydrophobic drug nanoparticles, the good solvent is mutually soluble with water. According to the Flory-Krigboum dilute solution theory, a good solvent refers to a solvent with a solute interaction parameter of less than 0.5. Preferably, the good solvent is one or more of acetone, methanol, ethanol and tetrahydrofuran, and more preferably tetrahydrofuran.
In the method for preparing hydrophobic drug nanoparticles of the present invention, the water may be deionized water, distilled water, double distilled water, or the like, preferably deionized water.
In the method for preparing hydrophobic drug nanoparticles, the concentration of the hydrophobic drug molecules dissolved in the good solvent in the step (1) is 0.5-5mg/mL, preferably 1 mg/mL.
In the method for preparing hydrophobic drug nanoparticles, the volume ratio of the good solvent to water in the step (1) is preferably (1-3): 50, preferably 2: 50. preferably, the reaction temperature in step (1) is 20 to 30 ℃, more preferably 25 ℃.
In the method for preparing hydrophobic drug nanoparticles according to the present invention, the concentration of the polyethylene glycol derivative added in the step (2) in the good solvent may be 1-10mg/mL, for example, 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL, 5mg/mL, 6mg/mL, 7mg/mL, 8mg/mL, 9mg/mL or 10mg/mL, preferably 2 mg/mL.
Preferably, the time for ultrasonic dispersion in the step (2) is 3-30min, and more preferably 5 min. Preferably, the concentration of the chitosan oligosaccharide isotonic physiological saline solution in the step (3) is 0.05-0.2w/v, such as 0.05% w/v, 0.1% w/v or 0.2% w/v, preferably 0.1% w/v. Preferably, the stirring time in step (3) is 0.5 to 2h, such as 0.5h, 1h, 1.5h or 2h, preferably 1 h. Preferably, the centrifugation conditions in step (3) are 10000-.
The self-assembled nano particles can be aggregated when being stored in an aqueous solution for a long time, and the stability of the self-assembled nano particles can be obviously improved by freeze-drying the self-assembled nano particles, wherein the freezing temperature is 10-20 ℃ lower than the eutectic point of the nano particles and water, and the freeze-drying is carried out for 24-90h, preferably 48h, under the pressure of 10 Pa.
In order to avoid aggregation and particle size change of the freeze-dried nanoparticles, a cryoprotectant, such as glucose, mannitol, lactose, NaCl and the like, is added firstly, so that a large amount of micro ice crystals are promoted to form during freezing, or a freeze-dried product is in a loose state, and the nanoparticles can be favorably kept in the original shape and can be easily re-dispersed in water.
In a third aspect, the invention provides the use of a chitosan oligosaccharide modified self-contained carrier-free nasal nano-formulation brain-targeted delivery system as described in the first aspect. It is used for brain targeted delivery. Can be made into nasal spray or nasal drop. In one embodiment, the chitosan oligosaccharide modified curcumin analogue M1 has an improvement effect on MPTP-induced behavior of Parkinson model mice such as exploration motor activity, anxiety, gait and the like after being nasally administered from a self-contained carrier-free nasal cavity nano preparation system.
The brain targeting delivery system of the invention is administered by nasal route in the form of spray or nasal drops.
The invention has the beneficial effects that:
the invention relates to a nano preparation brain-targeted delivery drug system, aiming at hydrophobic small molecules with neuroprotective effect, the nano preparation brain-targeted delivery drug system is prepared into a chitosan oligosaccharide modified self-carried type carrier-free nasal cavity nano preparation brain-targeted delivery system, and can be applied to the treatment of a series of nervous system diseases. After the nasal preparation is prepared, gastrointestinal degradation and liver first-pass effect can be avoided, and the nasal preparation has the characteristics of high bioavailability, quick response, good patient compliance and the like. Compared with the conventional preparation, the nasal preparation has the advantages that the nasal preparation bypasses blood brain barriers and is directly delivered into the brain along olfactory nerves and other ways through nasal-brain passages, so that the brain targeting property can be obviously enhanced, the visceral organ enrichment of a peripheral circulatory system is reduced, and the potential side effect of long-term administration is reduced. Compared with injection, the nasal preparation can reduce drug accumulation in peripheral circulatory organs such as liver; compared with oral medicine, the nasal preparation has no first-pass effect and reduced medicine loss. Convenient use, no wound and high patient compliance. Compared with intravenous injection, nasal administration only needs modes of nasal dripping, spraying and the like, is simpler and safer to operate, can relieve the pain of patients, has good patient compliance, is convenient for self-administration, reduces the risk brought by long-term administration and has good application prospect, particularly for patients suffering from neurodegenerative diseases who take medicines for a long time. In addition, compared with intravenous injection, the nasal drops, spray and other modes are simple and safe to operate, and particularly for neurodegenerative disease patients taking medicines for a long time, the medicine is convenient for self-administration, and has good application prospect in the aspect of treatment of nervous system diseases.
The chitosan oligosaccharide modified self-carried non-carrier nasal cavity nano preparation is simple to operate, wide in application range and strong in universality. Compared with a hydrophobic organic micromolecule prodrug, the prodrug has the advantages of remarkably improving water dispersibility and drug forming property, enhancing bioavailability, reducing administration frequency, reducing toxic and side effects and the like, and is safer to take for a long time. Compared with the traditional polymer nano drug-carrying system, the nano drug-carrying system has no carrier, no biodegradation problem and accumulative toxicity, the drug-carrying rate is up to more than 25 percent, and the chitosan oligosaccharide modified drug-carrying system has good transdermal absorption and can enter the brain through olfactory nerves as a nasal preparation with extremely high targeting. The polyethylene glycol derivative not only enhances the stability, but also reduces the irritation of mucosa and prolongs the circulation time in vivo.
The nanometer preparation highly retains the binding ability of targeting receptors of original molecules, has pH-responsive drug release characteristics, degrades at specific pH (5.5) of lysosome after being taken by cells, and releases small molecules into cytoplasm to exert drug effect. At the same time, the drug of the present invention is stable during delivery. Fig. 7 illustrates that the invention can lead the drug small molecule to circulate in the cerebral medullary fluid and blood for a long time and release slowly (more than 64h), thereby reducing the administration frequency and the administration dosage.
Therefore, the invention has high safety, high drug loading rate and high brain targeting property; the administration dosage is small, the administration frequency is low (slow release and long effect), and the side effect of the whole body is low (the content of liver and kidney is low); non-invasive administration, etc.
Compared with the original hydrophobic micromolecule drug, the delivery system has good water dispersibility, greatly enhances the micromolecule druggability, reduces the toxicity of the micromolecule drug and enhances the neuroprotection effect; compared with other nanometer preparations, the delivery system has no carrier, no biodegradation problem and accumulative toxicity, the drug loading rate is up to more than 25 percent, the binding capacity of the targeted receptor of the original molecule is highly retained, the chitosan oligosaccharide modified permeable membrane has good absorption, and the chitosan oligosaccharide modified permeable membrane can enter the brain through olfactory nerves with extremely high targeting as a nasal preparation. Has pH response performance, can be slowly released in cells and can maintain effective concentration for a long time.
Drawings
The invention is further illustrated by the following figures and examples.
FIG. 1 is a Scanning Electron Microscope (SEM) image of self-carried non-carrier nanoparticles of hydrophobic small molecule drugs, wherein a is curcumin nanoparticles; b is M1 nanoparticles; c is M1 nanoparticles loaded with TPAAQ probe.
FIG. 2 is a particle size distribution diagram of the self-carried non-carrier nanoparticles of the hydrophobic small molecule drug, wherein a is curcumin nanoparticles; b is M1 nanoparticles; c is M1 nanoparticles loaded with TPAAQ probe.
FIG. 3 is a potential distribution diagram of self-carried non-carrier nanoparticles of hydrophobic small molecule drugs, wherein a is curcumin nanoparticles; b is M1 nanoparticles.
FIG. 4 is a Tyndall effect optical characterization diagram of hydrophobic small molecule drug self-carried carrier-free nanoparticles, wherein a is curcumin nanoparticles; b is M1 nanoparticles.
FIG. 5 is a graph of the loading rate test of the self-carried unsupported nanoparticles of hydrophobic small molecule drugs: wherein a is curcumin nanoparticles; b is M1 nanoparticles, c is M1 nanoparticles loaded with TPAAQ probes.
FIG. 6 shows the effect of cell uptake of curcumin nanoparticles modified with chitosan oligosaccharide; wherein a is a cellular uptake map of M1 NPs which are not modified by chitosan oligosaccharide; b is the cellular uptake map of M1 NPs after chitosan oligosaccharide modification.
FIG. 8 is a cytotoxicity plot of M1 nanoparticles versus small molecule M1 drug.
Figure 9, graph of the in vitro neuroprotective effect of M1 nanoparticles.
FIG. 10 is a graph showing the binding effect of M1 nanoparticles to a target protein.
FIG. 11 is a cell uptake map of M1 nanoparticles loaded with TPAAQ fluorescent probes.
Fig. 12 transmission electron microscope image of brain section of mouse after nasal administration of curcumin nanoparticles.
FIG. 13 is a graph of M1 drug content in mouse brain and plasma after nasal administration of M1 nanoparticles.
FIG. 14 is a fluorescence micrograph of the brain and organs of a mouse to which M1 nanoparticles carrying TPAAQ fluorescent probes were nasally administered.
FIG. 15 is a graph of the open field behavior of Parkinson model mice after nasal administration of M1 nasal nano-formulation of example 2.
FIG. 16 is a gait pattern of Parkinson model mice after nasal administration of M1 nasal cavity nano-formulation of example 2.
FIG. 17 Mass Spectrum of example 2 shows a compound having a molecular weight of 294.34
Fig. 18 is the conversion of curcumin analogs under different conditions in example 2.
Detailed Description
Embodiments of the present invention will be described in detail with reference to examples. It will be appreciated by those skilled in the art that the following examples are only preferred embodiments of the invention to facilitate a better understanding of the invention and should not be taken as limiting the scope of the invention. Various modifications and changes may be made by those skilled in the art, and any modification, equivalent replacement or improvement made without departing from the spirit and principle of the present invention should be covered within the protection scope of the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified; the experimental materials used, unless otherwise specified, were purchased from conventional biochemical manufacturers.
Example 1: preparation of curcumin nasal nano preparation
The curcumin used in the present embodiment has the following structure:
preparing 10mL of tetrahydrofuran solution containing 1.5mM curcumin drug molecules and 2mg/mL polymaleic anhydride 18 carbene-polyethylene glycol, dropwise adding 200 mu L of the curcumin solution into 4mL deionized water, simultaneously blowing nitrogen while dropwise adding, violently stirring the aqueous solution to remove the organic solvent, stirring for 10min, standing to obtain a self-carried carrier-free curcumin nanoparticle suspension, and freeze-drying to form freeze-dried powder. Before use, the freeze-dried powder is re-dispersed in isotonic physiological saline, added into chitosan oligosaccharide (0.1w/v) physiological saline solution drop by drop, stirred for 0.5h, reacted for 1h by physical adsorption, centrifuged for 10min at 10000 g of rotation speed, the supernatant is discarded, the reactant is removed, and the product is purified to obtain the chitosan oligosaccharide modified self-carried carrier-free curcumin nasal nano preparation.
Results
(1) Determination of curcumin nanoparticle form, particle size and potential distribution
The curcumin nanoparticles prepared in example 1 were observed using a scanning electron microscope (FEI Quanta200, the netherlands) according to the method in the specification thereof, and the scanning electron microscope image thereof is shown in fig. 1 a. The curcumin nanoparticles prepared in example 1 were subjected to dynamic light scattering measurement using a laser particle sizer (malvern, uk) according to the method in the specification thereof, and the mean particle size of the curcumin nanoparticles prepared in example 1 was measured to be 64.57nm, and the particle size distribution graph is shown in fig. 2 a. The curcumin nanoparticles prepared in example 1 were subjected to Zeta-potential analysis using a laser particle size analyzer according to the method described in the specification thereof, and the average charge of the curcumin nanoparticles prepared in example 1 was measured to be-10.5 mV, indicating that they have a weak negative charge, and the distribution thereof is shown in fig. 3 a.
(2) Determination of curcumin nanoparticle optical characterization
The curcumin molecule and the curcumin nanoparticles prepared in example 1 are respectively dissolved in water and an organic solvent, as shown in fig. 4a, it can be seen that the curcumin small molecule is difficult to dissolve in water and can be dissolved in tetrahydrofuran, and the curcumin nanoparticles can be dispersed in water and have the tyndall effect under laser.
Determination of curcumin nanoparticle drug loading rate
Curcumin molecules are dissolved in acetonitrile, curcumin acetonitrile solutions (6.25, 12.5, 25, 50 and 100ug/mL) with series concentrations are prepared in a gradient manner, and absorbance is measured at 428nm by using Ultra Performance Liquid Chromatography (UPLC) to make a standard curve. Dissolving three batches of 100ug/mL curcumin nanoparticles in acetonitrile respectively, performing ultrasonic treatment for 5min, measuring by the same method, and calculating curcumin content in the nanoparticles by using a standard curve. As shown in FIG. 5a, the drug loading rate of curcumin nanoparticles was (25.12. + -. 2.50)%
(3) Determination of chitosan oligosaccharide modification effect of curcumin nanoparticles
Respectively modifying the fluorescent FITC probe, curcumin nanoparticles which are not modified by chitosan oligosaccharide and curcumin nanoparticles which are modified by chitosan oligosaccharide, then respectively incubating and culturing the modified curcumin nanoparticles and N2a cells for 6h, washing the cells for three times by PBS (phosphate buffer solution), exciting the cells under a laser confocal microscope by using a wavelength of 488nm, measuring the fluorescence intensity by using an emission wavelength of 190-540nm, and performing comparative analysis. As shown in fig. 5, cellular uptake of curcumin nanoparticles was significantly enhanced after chitosan oligosaccharide modification.
Example 2: preparation of curcumin analogue M1 nasal cavity nano preparation
The curcumin analog M1 used in this example was an isomeric mixture of curcumin analogs of the following structural formula:
according to the results of the computer simulations of the applicant, the higher the proportion of cis-isomers in the mixture, the greater the biological activity of the product. In practice, however, the higher the product, the higher the yield of by-products.
In this example, a process was provided in which an isomer product was obtained at a conversion of 30%, the production process was simple, and no by-product was produced.
According to the hydrophobic property of the curcumin analogue, the curcumin analogue is dissolved in a good solvent, and different degrees of isomer conversion can occur under different irradiation conditions, so that cis-trans isomer mixtures with different proportions are obtained. Wherein the conversion rate of sunlight irradiation is the highest, but byproducts are generated; the conversion rate is ultraviolet irradiation and radioactive iodine irradiation, and the temperature has no influence on the structure of the curcumin analogue under the condition of keeping out of the sun.
Further, the good solvent is preferably acetonitrile, methanol, ethanol, acetone, or tetrahydrofuran.
Furthermore, the isomer product with 30 percent of conversion rate can be obtained by UV irradiation for 2h, the preparation method is simple, and no by-product is generated.
Method for preparing cis-trans isomer Mixture M1(mix 1) of curcumin analogue
Preparing methanol solution containing 1mg/mL curcumin analogue, sealing with aluminum foil paper, and standing at 4 deg.C, 25 deg.C and 50 deg.C for 8 hr to obtain reaction product 1-3; preparing methanol solution of curcumin analogs with the same concentration, and respectively performing sunlight irradiation for 2h, sunlight irradiation for 24h, ultraviolet irradiation for 2h and radioactive iodine 131 irradiation for 2h at room temperature to obtain reaction products 4-7 so as to obtain cis-trans isomer mixtures of curcumin analogs with various ratios.
Results
(1) Molecular weight identification of the conversion products
The molecular weight of the product was determined by using a high performance liquid chromatography-time of flight mass spectrometer, as can be seen from fig. 17, the molecular weights of the curcumin analogues and the converted products thereof were 294.34, and both were confirmed to be isomers, and further, as can be seen from the structures, they were cis-trans isomers.
(2) Determination of the conversion of cis-trans isomers:
the cis-trans isomer conversion of curcumin analogs in sample solutions 1-7 was determined by High Performance Liquid Chromatography (HPLC) at a maximum absorption wavelength of 384 nm. As shown in FIG. 2, no new substance was produced in any of the reaction products 1 to 3, indicating that no isomer conversion of curcumin analogues occurred (FIGS. 18a to c). In the reaction product 4, after being irradiated by sunlight for 2 hours, 73.91 percent of the curcumin analogue content is converted into an isomer (figure 18 d); and after 24h of sunlight irradiation, the reaction product 5 has a plurality of complex products besides curcumin analogue isomers (FIG. 18 e). The conversion rate of the isomer of curcumin analogue of reaction product 6 after 2h of ultraviolet irradiation was 29.59% (fig. 18 f). The conversion rate of the isomer of the curcumin analogue of the reaction product 7 under the condition of radioactive iodine 131 radiation for 2h is 27.91% (figure 18/g).
5mL of tetrahydrofuran solution containing 1mg/mL of M1 and 2mg/mL of carboxyl polyethylene glycol is prepared and mixed evenly, 200 mu L of M1 molecular solution is dropwise added into 5mL of deionized water, and nitrogen is blown while dropwise adding to remove the organic solvent. Magnetically stirring at 25 deg.C for 10min, standing to obtain M1 self-carried unsupported nanoparticle suspension, and freeze drying to obtain lyophilized powder. Before use, the freeze-dried powder is dropwise added into an isotonic normal saline solution containing chitosan oligosaccharide (0.1w/v), stirred for 0.5-2h, reacted for 1h by physical adsorption, centrifuged for 5-30min at 10000-150000 g, the supernatant is discarded, the reactant is removed, and the product is purified to obtain the chitosan oligosaccharide modified self-carried carrier-free M1 nasal nano preparation.
Results
(1) Determination of M1 nanoparticle morphology, particle size and potential distribution
The M1 nanoparticles prepared in example 2 were observed using a scanning electron microscope (FEI Quanta200, the netherlands) as described in the specification thereof, which is shown in fig. 1 b. The M1 nanoparticles prepared in example 2 were subjected to dynamic light scattering measurements using a laser particle sizer (malvern, uk) according to the method described in the specification, and the mean particle size of the M1 nanoparticles prepared in example 2 was found to be 62.73nm, the particle size distribution map being shown in figure 2 b. Zeta-potential analysis of the M1 nanoparticles prepared in example 2 using a laser particle sizer according to the protocol indicated in the specification gave M1 nanoparticles prepared in example 2 having an average charge of-56.5 mV, indicating a weakly negative charge, which is distributed as shown in FIG. 3 b.
(2) Determination of the optical characterization of the M1 particles
When M1 small molecule and M1 nanoparticle prepared in example 2 were dissolved in water and organic solvent, respectively, as shown in fig. 4b, it can be seen that M1 small molecule is difficult to dissolve in water and soluble in tetrahydrofuran, while M1 nanoparticle can be dispersed in water and has tyndall effect under laser.
3) Determination of M1 nanoparticle drug loading rate
M1 molecules were dissolved in acetonitrile, and a series of M1 acetonitrile solutions (6.25, 12.5, 25, 50 and 100ug/mL) were prepared in a gradient, and absorbance was measured at 428nm using Ultra Performance Liquid Chromatography (UPLC) to prepare a standard curve. And (3) taking three batches of 100ug/mL M1 nanoparticles, respectively dissolving in acetonitrile, performing ultrasonic treatment for 5min, performing the same method for determination, and calculating the content of M1 in the nanoparticles by using a standard curve. As shown in FIG. 5b, the drug loading rate of M1 nanoparticles was (31.49. + -. 2.03)%
(4) M1 nanoparticle drug release profile determination
The M1 nanoparticles prepared in example 2 were equally divided into six parts, 3 parts were added to the artificial nasal solution, 3 parts were added to 5% plasma, separately filled into dialysis bags, dispersed and diluted, separately added to dialysis bags (3500 molecular weight,
usa) followed by soaking in 200 ml of buffer of the same pH with constant stirring at 37 ℃ and collecting 2ml of solution from the solution at a certain time point. During dialysis, 2ml PBS was added after each sampling to keep the solution volume constant. And (4) measuring the absorbance by adopting a UV-VIS method, and calculating the drug release amount. Each sample was tested 3 times, averaged, and statistically analyzed, with the results shown in fig. 7. It can be seen that the M1 nanoparticles prepared in example 2 have a slow release property, do not show an initial explosive drug release, but release slowly and stably, which is crucial for the application of M1 nanoparticles in vivo, and can reduce drug toxicity, drug leakage, etc.
(5) Cytotoxicity assays
The neuroma blast N2a cells were cultured according to the method described in the literature (cell culture, Sedrin Town, world book publishing Co., 1996), then the M1 nanoparticles prepared in example 2 were added to the cells and the cells were cultured, and after 24 hours of addition, the survival rate of the cells was measured according to the method described in the literature (MTT method), which is the M1 nanoparticle group. A group of N2a cells treated in the same manner with the same concentration of free M1 as that of the M1 nanoparticle group was used as a positive control group; the negative control group was N2a cells cultured in a blank medium without the hydrophobic drug, wherein the survival rate of the cells in the negative control group was calculated as 100%. As shown in fig. 8, as the concentration increases, the free M1 can exhibit dose-dependent cytotoxicity, while the M1 nanoparticles in example 2 have no toxic effect on N2a cells at the same concentration, and may inhibit the cumulative toxicity of M1 small-molecule drugs at higher concentrations due to the sustained release effect of the M1 nanoparticles.
(6) Neuroprotective assay for M1 nanoparticles
The neural cell line PC12 cells were treated with MPP + neurotoxin to create a model of neurotoxic cells. Adding M1 nanoparticles prepared in example 2 for pretreatment 6h before molding to obtain a M1 nanoparticle group; model control group was obtained without drug treatment, and normal control group was obtained without MPP + neurotoxin. After modeling, the cells are continuously cultured for 48h, the absorbance is measured according to a literature method, and the result is shown in fig. 9, the cell survival rate of the M1 nanoparticle group is obviously higher than that of the MPP + model group, and the M1 nanoparticles prepared in example 2 can protect PC12 nerve cells in a dose-dependent manner and reduce cell damage induced by MPP + neurotoxin.
(7) Binding effect assay of M1 nanoparticles to target proteins
The target protein of free M1 molecule for neuroprotection is TFEB protein in cytoplasm, and M1 can promote the dephosphorylation of TFEB protein into nucleus and up-regulate the expression of autophagy related gene, thereby playing the role of neuroprotection. In this experiment, M1 nanoparticles prepared in example 2 were added to MF7 cells overexpressing a fluorescent-labeled TFEB protein, and after 24h of treatment, TFEB nuclear entry was observed, and as a result, as shown in fig. 9, M1 nanoparticles prepared in example 2 can promote TFEB nuclear entry in a dose-dependent manner, confirming that M1 nanoparticles retain the targeting property of the original molecule.
Example 3: m1 nasal cavity nano preparation brain targeting delivery system carrying fluorescent probe TPAAQ
TPAAQ is a hydrophobic micromolecule fluorescent probe excited by 473nm wavelength and emitting by 650nm wavelength, and can be used for monitoring in-vivo fluorescence distribution of nano materials. Because it is also a hydrophobic small molecule, similar to the preparation process of the M1 nanoparticles of example 2, M1 nasal cavity nano preparation carrying TPAAQ can be obtained by the same method.
5mL of tetrahydrofuran solution containing 1mg/mL of M1 and 2mg/mL of TPAAQ is prepared and mixed evenly, 200 mu L of the M1 molecular solution is added into 5mL of deionized water dropwise, and nitrogen is blown at the same time to remove the organic solvent. Magnetically stirring the mixture for 10 minutes at the temperature of 25 ℃, standing the mixture to obtain M1 self-carried carrier-free nanoparticle suspension emulsion carrying the fluorescent probe TPAAQ, and freeze-drying the suspension emulsion to form freeze-dried powder. Before use, the freeze-dried powder is re-dispersed in isotonic normal saline, added into chitosan oligosaccharide (0.1w/v) normal saline solution drop by drop, stirred for 0.5-2h, reacted for 1h by physical adsorption, centrifuged for 5-30min at 10000-.
Example 3 results
(1) Determination of morphology and particle size distribution of M1 nanoparticles carrying fluorescent probe TPAAQ
The fluorescent probe TPAAQ-loaded M1 nanoparticles prepared in example 3 were observed using a scanning electron microscope (FEI Quanta200, the Netherlands) according to the method described in the specification, and the scanning electron microscope image thereof is shown in FIG. 1 c. The M1 nanoparticles carrying the fluorescent probe TPAAQ prepared in example 3 were subjected to dynamic light scattering measurement using a laser particle sizer (Malvern, UK) according to the method described in the specification, and the M1 nanoparticles carrying the fluorescent probe TPAAQ prepared in example 3 were found to have an average particle diameter of 178.2 nm, and the particle diameter distribution chart is shown in FIG. 2 c.
(2) Determination of M1 nanoparticle drug loading rate of fluorescent probe TPAAQ
Using the standard curve of M1 acetonitrile solution prepared in the test (3) of example 2, three batches of 100ug/mL M1 nanoparticles carrying a fluorescent probe TPAAQ were dissolved in acetonitrile, subjected to ultrasonic treatment for 5min, and subjected to the same method, and the curcumin content in the nanoparticles was calculated using the standard curve. As shown in FIG. 5c, the drug loading rate of the M1 nanoparticles loaded with the fluorescent probe TPAAQ was (26.95. + -. 1.50)%.
(2) Cell uptake assay
Nerve cells are normally cultured, the M1 nasal nano preparation carrying the fluorescent probe TPAAQ prepared in example 3 is added, after 3 hours of culture, the cell uptake condition is observed under a laser confocal scanning microscope at a specific wavelength, as shown in figure 11, and as can be seen from a fluorescent signal, the M1 nasal nano preparation carrying the fluorescent probe TPAAQ prepared in example 3 can be greatly taken by the cells.
Example 4: application of curcumin nanoparticle to nasal brain targeted delivery system
6 male C57BL/6J mice of strain 25g were selected and acclimatized for 3 days. The nano preparation of the curcuminoid nasal cavity prepared in the example 1 is dispersed in isotonic physiological saline with the concentration of 5mg/ml, the nasal cavity of a mouse is given with 15ul, brain tissues are dissected and taken out after 24 hours, the section is fixed, and the brain distribution of the nano particles is observed under a transmission electron microscope. As shown in fig. 12, the distribution of curculin nanoparticles in olfactory bulb and cortex of brain can be clearly seen.
Example 5: application of M1 nanoparticles to nasal brain targeting delivery system
6 male C57BL/6J mice of strain 25g were selected and acclimatized for 3 days. The M1 nasal cavity nano preparation prepared in example 2 is dispersed in isotonic physiological saline with the concentration of 5mg/ml, 15ul of the nano preparation is given to a mouse nasal cavity, brain tissue, cerebrospinal fluid and plasma are dissected and taken out after 24h, the brain tissue is divided into an olfactory bulb part and the rest part of the brain, all samples are respectively added with methanol to remove protein, and the content of M1 drugs in the samples is analyzed by using triple quadrupole liquid chromatography-mass spectrometry. The results are shown in fig. 13, the M1 nasal nano-formulation brain-targeted delivery system delivers M1 drug into olfactory bulb with very high targeting, and has a distribution in cerebrospinal fluid that is three times higher than the plasma content, and a distribution in other parts of brain that is twice the plasma content. It was confirmed that the absorption pathway was through the olfactory bulb to the brain and was transmitted to other parts of the brain. Its delivery may be time-dependent and will continue to be delivered via the cerebrospinal fluid after 24 h.
Example 6: application of M1 nanoparticles loaded with TPAAQ fluorescent probe to nose-brain targeted delivery system
9 male C57BL/6J mice of strain 25g were selected and acclimatized for 3 days. The M1 nasal cavity nano preparation carrying the fluorescent probe TPAAQ prepared in the example 3 is dispersed in physiological saline with the concentration of 5mg/ml, 15ul of the nano preparation is given to a mouse nasal cavity, a small animal fluorescence imaging system is applied after 24h and 48h respectively, the in-vivo fluorescence of the brain of the mouse and fluorescence signals in organs and blood such as a brain, a heart, a liver, a spleen, a lung, a kidney and the like are detected, and the result is shown in fig. 14, the brain signals are remarkably stronger than other parts and tissues of a body, which indicates that the brain targeting delivery system in the example 3 can successfully deliver the M1 nasal cavity nano preparation into the brain with high targeting, and the distribution of the medicine in peripheral tissues is reduced.
Example 7: therapeutic application of self-carried carrier-free M1 nasal nano-preparation in Parkinson model mice
30 male C57BL/6J strain mice weighing 25g were divided into three groups, a first wild type group (WT group), a second model group (MPTP group), and a third model-administered group (M1 NPs), each of which was 10 mice. The second and third groups of mice were injected intraperitoneally with MPTP neurotoxin for five days at a dose of 20mg/kg following literature procedures to create a model of Parkinson's disease. The model is manufactured and treated simultaneously, normal saline is given to the nasal cavities of mice in WT group and MPTP group, self-carried carrier-free M1 nasal cavity nano preparation is given to the nasal cavities of M1 NPs group, namely, the M1 nasal cavity nano preparation prepared in the example 2 is dispersed in isotonic normal saline and is prepared newly for clinical application, the concentration is 1mg/ml, and the nasal cavity is given to the mice for 15 ul. The medicine is administrated at intervals of one day, four times, and the effect is observed after two weeks after the molding is finished.
Example 7 results
(1) Detection of behavior of Parkinson model mouse by open field test
The MPTP Parkinson mouse model has the symptoms of exploration dyskinesia, obvious anxiety and the like, and can be detected by an open field test. The behavioral performance of the parkinson model mice in example 7 was examined according to literature methods. The results are shown in fig. 15a, compared with the wild type mice in the control group, the movement track of the model mice is significantly changed, and the movement track of the model mice is approximately normal after the treatment of the M1 nasal cavity nano preparation. Statistics data show that compared with wild mice, the movement time, the average speed (figure 15b) and the regional shuttling times (figure 15c) of model mice in a mine field are all remarkably reduced, and after the self-contained carrier-free M1 nasal nano-preparation is used for treatment, the pathological conditions are all remarkably improved, and the M1 nasal nano-preparation is proved to be capable of effectively relieving the behavioral symptoms of the Parkinson disease model.
Gait test for detecting behavior expression of Parkinson model mouse
The clinical manifestations of Parkinson's disease mainly include resting tremor, bradykinesia, muscular rigidity, gait disorder of posture and the like. A DigiGait imaging system is adopted on an animal, the animal is imaged under a transparent running belt, and software quantifies characteristics such as gait mechanics, posture indexes and the like, so that the behavioral characteristics of the Parkinson model mouse can be detected. The results are shown in fig. 16, compared with the wild type mouse, the gait signal of the parkinson model mouse is disordered, the coordination is reduced, the sole contact area is obviously reduced, and after the treatment of the self-carrying type carrier-free M1 nasal cavity nano preparation, the pathological conditions are all obviously improved, which proves that the M1 nasal cavity nano preparation can effectively improve the behavioral symptoms of parkinson disease.
The applicant declares that the present invention is described by the above embodiments as the detailed features and the detailed methods of the present invention, but the present invention is not limited to the above detailed features and the detailed methods, that is, it is not meant that the present invention must be implemented by relying on the above detailed features and the detailed methods. It will be apparent to those skilled in the art that any modification of the present invention, equivalent substitutions of selected components of the present invention, additions of auxiliary components, selection of specific modes and the like, are within the scope and disclosure of the present invention.
Claims (6)
1. A brain-targeted chitosan oligosaccharide modified self-contained carrier-free nasal nano preparation is characterized in that:
the chitosan oligosaccharide-polyethylene glycol composite comprises chitosan oligosaccharide, hydrophobic micromolecular drugs with neuroprotective effect and polyethylene glycol derivatives with negative electricity, wherein the polyethylene glycol derivatives with negative electricity are carboxyl polyethylene glycol or polymaleic anhydride 18 carbene-polyethylene glycol; firstly, preparing a good solvent solution of carboxyl polyethylene glycol or polymaleic anhydride 18 carbene-polyethylene glycol 1-10mg/mL and 0.5-5mg/mL of hydrophobic micromolecule drug with neuroprotective effect, and then dropwise adding the good solvent solution into deionized water, wherein the volume ratio of the good solvent solution to the deionized water is (0.5-5): 50, blowing gas while dropwise adding to assist in volatilizing a good solvent; preparing self-carried carrier-free nano particle suspension emulsion with the particle size of 50-200nm by a reprecipitation method, and freeze-drying to prepare freeze-dried powder;
before use, stirring and reacting the freeze-dried powder and chitosan oligosaccharide in isotonic normal saline for 0.5-2 hours by utilizing physical adsorption, wherein the concentration of the chitosan oligosaccharide in the isotonic normal saline is 0.01-0.2% (w/v), removing reactants, and purifying a product to obtain the brain-targeted chitosan oligosaccharide modified self-carried carrier-free nasal nano preparation;
the hydrophobic micromolecule drug with neuroprotective effect is curcumin analogue with the following structural formula and a mixture of light conversion isomers thereof, wherein the isomers account for 25-35% of the total amount of the mixture:
the mixture is prepared by subjecting a methanol solution of the curcumin analogue to ultraviolet irradiation for 1.5-2.5 h.
2. The brain-targeted chitosan oligosaccharide-modified self-contained non-carrier nasal nano-preparation according to claim 1, which is characterized in that: the polymerization degree of the chitosan oligosaccharide is 2-20, or the molecular weight is less than or equal to 3200 Da.
3. The preparation method of the brain-targeted chitosan oligosaccharide-modified self-contained carrier-free nasal nano-preparation as claimed in claim 1, comprising the following steps:
1) firstly, preparing good solvent solution of carboxyl polyethylene glycol or polymaleic anhydride 18 carbene-polyethylene glycol 1-10mg/mL and 0.5-5mg/mL of hydrophobic micromolecule drug with neuroprotective effect, and then dripping the good solvent solution into deionized water: the volume ratio of the good solvent solution to the deionized water is (0.5-5): 50, blowing gas while dropwise adding to assist in volatilizing a good solvent;
2) preparing self-carried carrier-free nano particle suspension emulsion with the particle size of 50-200nm by a reprecipitation method, and freeze-drying to prepare freeze-dried powder;
3) before use, the freeze-dried powder and the chitosan oligosaccharide are reacted for 0.5 to 2 hours in isotonic normal saline by physical adsorption, reactants are removed, and the product is purified to obtain the brain-targeted chitosan oligosaccharide modified self-carried non-carrier nasal nano preparation.
4. The use of the brain-targeted chitosan oligosaccharide-modified self-contained, carrier-free nasal nano-formulation of any one of claims 1 or 2 in the preparation of a nasal spray or nasal drops.
5. The use of the brain-targeted chitosan oligosaccharide-modified self-contained, carrier-free nasal nano-formulation of any one of claims 1 or 2 in the preparation of a medicament for the prevention and treatment of neurological diseases.
6. The use of claim 5, wherein: the nervous system disease is Parkinson disease.
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| CN103251555A (en) * | 2012-12-25 | 2013-08-21 | 苏州大学 | Preparation method of high drug loading rod-shaped nanometer drug for cancer treatment |
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