CN109730966A - A kind of self-carrying carrier-free nasal cavity nanometer formulation Brain targeting delivery system and preparation method thereof of chitosan oligosaccharide modification - Google Patents
A kind of self-carrying carrier-free nasal cavity nanometer formulation Brain targeting delivery system and preparation method thereof of chitosan oligosaccharide modification Download PDFInfo
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Abstract
The invention discloses a kind of self-carrying carrier-free nasal cavity nanometer formulation Brain targeting delivery systems and preparation method thereof of chitosan oligosaccharide modification.Including hydrophobic small molecules drug, polyethyleneglycol derivative and chitosan oligosaccharide with neuroprotection.The present invention also provides the preparation methods of above-mentioned nasal cavity nanometer formulation Brain targeting delivery system.The first step, prepares nano particle freeze-dried powder, and freeze-dried powder and chitosan oligosaccharide are stirred in isotonic physiological saline before use, become the good nasal cavity preparation of permeable membrane by second step.Present system preparation method is simple, can improve small-molecule drug hydrophobicity, reduce toxicity, enhancing neuroprotection；Carrier-free, inanimate object degradation problem and cumulative toxicity, carrying drug ratio are up to 25% or more, and permeable membrane absorbs good after chitosan oligosaccharide is modified, and the high targeting of drug is delivered into brain.The dosage form administration mode is collunarium, spraying etc., easy to operate, convenient for the patient medication of Long-term taking medicine, is had a good application prospect in terms of the treatment of the nervous system disease.
Description
Technical field
The invention belongs to biomedicine field, it is related to a kind of nasal cavity nanometer formulation Brain targeting delivery system and its preparation side
Method.Specifically a kind of modification of chitosan oligosaccharide, self-carrying carrier-free nasal cavity nanometer formulation Brain targeting delivery system and its preparation
Method.
Background technique
In recent years, the nervous system disease disease incidence increases year by year, and therapeutic agent is seriously in short supply.What medicament research and development filtered out
Small molecule with neuroprotective activity, most of is hydrophobicity small organic molecule, itself is insoluble in water, life in vivo
The problems such as object availability is low, blood circulation time is short and unstability greatly hinders its application clinically.
For example, curcumin, which is reported, significant ex vivo nerve protection pharmacological activity, but because the problems such as its poorly water-soluble limits
Its vivo applications is made.And under traditional oral mode, drug enters after being subjected to inactivation metabolism by intestinal mucosa and liver
The dose of body circulation is reduced, drug effect reduces.And due to the presence of blood-brain barrier, the content that drug is able to enter intracerebral is extremely low, limit
Its application in the nervous system disease is made.
With the fast development of nanotechnology, nanometer formulation because have protect drug not to be destroyed, extend active drug tie up
It holds the time, the release for controlling drug, reduce the advantages that toxic side effect of drug, obtained extensively in medicine and field of biology
Application, and gradually start be applied to the nervous system disease in.Report that more nanometer formulation is liposome, polyesters copolymerization
The carriers such as object contain the nano particle (patent CN107029247A, CN101897669B, CN 102283812B etc.) of drug.But
The type nano granular has three big disadvantages: one, with long-term use, the carriers such as the polymer may bring latent in the enrichment of brain
Toxic side effect；Two, drug is wrapped in carrier, may lost its itself original targeting identification function；Three, mesh
The preceding nanometer medicine-carried system based on polymer, usual drugloading rate are lower than 10%, become and nano particle is hindered to further apply and face
The thorny problem of bed.Therefore, need to develop a kind of high drug load, safe and non-toxic, simple and easy, pervasive in existing a large amount of hydrophobic
The nano-delivery system of property drug, the treatment applied to its nervous system disease.
In recent years, alternative self-carrying Nano medication delivery strategies right and wrong are developed in the case where not using any carrier
Often desirable.In 2012, KasaI etc. by forming partial size 30- through reprecipitation method after two drug molecules to be connected into dimer
The pure Nano medication of self-carrying carrier-free of 50nm is the successful application for the first time of the strategy.But so far, fail application
In brain diseases therapeutic agent, under the conditions of main cause is conventional formulation, such Nano medication is only capable of long-acting following in blood
Ring but can not enter brain by blood-brain barrier (brain capillary endothelial cell aperture is only 14-18nm), so can not be real
The delivering of existing brain therapeutic agent.
Nasal-cavity administration is a kind of current safety, the novel medication of Noninvasive, drug can by regio olfactoria mucous membrane along
The aixs cylinder for surrounding the connection tissue around olfactory nerve beam or olfactory nerve member reaches cerebrospinal fluid or brain, including high molecular weight protein
And nano particle etc. can bypass blood-brain barrier by this approach, be directly entered central nervous system and play a role, and realize brain
The delivering of portion's therapeutic agent.In addition, degrade after nasal-cavity administration without gastrointestinal tract and liver, minimal amount of drug reach compared with
High brain drug concentration, so dosage, the frequency can be reduced and reduce dose-dependent side effect.It is infused compared to vein
It penetrates, nasal-cavity administration only needs collunarium, spraying mode, and operation is more simple and safe, especially for the nervus retrogression of Long-term taking medicine
Disease can mitigate the pain of patient, be easy to be accepted by patients, and be convenient for self-medication, and reduce long-term administration bring
Risk.
However to preparation, there are many careful requirements due to its unique environment for nasal cavity.The permeable membrane absorbability of drug is required first
Energy.Slurries and the mucus proteolytic enzyme rich in of glandular secretion in nasal cavity are the factors for influencing drug nasal absorption
One of.The pH value of nasal cavity mucus is 5.5~6.5, is the optimum pH of proteolytic enzyme, in addition there are the clear of schneiderian membrance cilium
Except effect.The research and development of above-mentioned various pairs of nasal cavity preparations propose challenge.The use of sorbefacient can increase to a certain extent
The Nasal Mucosa Absorption of strong drug.Currently used sorbefacient is anionic surfactant such as stearic acid, lauric acid, the moon
Lauryl sulfate, sulphonic acid compound etc. and nonionic surfactant such as polysorbate, Brij etc..However, cholic acid salt is to nose
Mucous membrane has certain adverse reaction, such as burning sensation, pain, and strong schneiderian membrane thorn will be generated at low concentration (2%)
Swash, high concentration (5%) can destroy nasal epithelial structure, and higher concentration can be such that nose cilium or epithelial cell completely falls off.Cause
This, effective and nontoxic sorbefacient is the key that nasal cavity preparation.
Patent CN105617395A, CN105582545A and CN105617396A relate separately to a kind of nose administration Brain targeting
The preparation method of nanometer medicine-carried system, lycoramine nose administration nanometer Brain targeting drug and preparation method thereof, galanthamine warp
Nasal administration nanometer Brain targeting drug and preparation method thereof.Three patents are directed to hydrophilic small molecule drugs, utilize its hydrophilic group
Group carries out esterification with carboxylated chitosan and obtains targeted drug；And the present invention is specifically to be directed to hydrophobic small molecules
Drug develops the preparation and application of its nasal cavity nanometer formulation Brain targeting delivery system.
After having experiment to show Puerarin nose administration, the peak drug levels and bioavilability at olfactory bulb position are vein note
1.72 times penetrated and 3.05 times, Brain targeting index is up to 14%, is 7.5 times of intravenous injection, so nasal is for pueraria lobata
Element, which improves curative effect, has boundless prospect.Patent " CN107184554 " discloses a kind of system of Puerarin liquid crystal nanoparticle
Preparation Method, however membrane process prepares Nano medication charging ratio and is usually no more than 10%, limits the performance of its drug effect, and used
Poloxamer188 auxiliary material is the polymer that cannot be biodegradable, and ratio is four times of drug in the formulation, because of long term administration
Accumulation may cause potential toxic side effect.Danshensu nano particle nasal delivery is entered by brain by polymer support in addition, having
Report (patent CN107029247A), but polymer support limits it and faces there are still foregoing three big inherent shortcomings
Bed Transformation Application.If such hydrophobic small molecules, the nanometer formulation Brain targeting delivery system more optimized by one kind, gram
Disadvantages described above is taken, is expected to further increase medication effect, it is non-to promoting its treatment use in the nervous system disease to have
Often important meaning.
Summary of the invention
It is a primary object of the present invention to be directed to the hydrophobicity small organic molecule with neuroprotection, druggability
The disadvantage and conventional administration route drug that difference causes bioavilability low are difficult to the problems such as crossing over blood-brain barrier, provide a kind of nose
Chamber nanometer formulation Brain targeting delivery system.Specifically a kind of modification of chitosan oligosaccharide, self-carrying carrier-free nasal cavity nanometer formulation
Brain targeting delivery system.
Technical scheme is as follows:
A kind of modification of chitosan oligosaccharide, self-carrying carrier-free nasal cavity nanometer formulation Brain targeting delivery system, including chitosan oligosaccharide, tool
There are the hydrophobic small molecules drug and polyethyleneglycol derivative of neuroprotection；Polyethyleneglycol derivative 1- is configured first
The good-solvent solution of 10mg/mL and 0.5-5mg/mL hydrophobic small molecules drug, then by the good-solvent solution to go from
It is added dropwise in sub- water, the volume ratio of the good-solvent solution and deionized water is (0.5-5): 50, dropwise addition while, is aided with nitrogen
It blows, auxiliary good solvent volatilization；The outstanding cream of self-carrying carrier-free nano particle that partial size is 50-200nm is prepared by reprecipitation method
Liquid, freeze-drying are prepared into freeze-dried powder；Before use, freeze-dried powder and chitosan oligosaccharide are utilized to physical absorption in isotonic physiological saline
Action-reaction 0.5-2 hours, wherein chitosan oligosaccharide is 0.01-0.2% (w/v) in isotonic physiological saline solution concentration, is removed anti-
Object is answered, the self-carrying carrier-free Nano medication nasal cavity preparation that product purification is modified up to chitosan oligosaccharide.
In the present invention, chitosan oligosaccharide promotees permeability for nasal membrane can be with neuroprotection synergistic effect.
Preferably, the surface potential of self-carrying carrier-free nano particle is -10~-60mV.
Preferably, the hydrophobic small molecules drug with neuroprotection is curcumin or curcumin analogue
It is one or more.
Further preferably, the hydrophobic small molecules are the curcumin analogue of following structural formula, its cis-isomer
Mixture:
Further preferably, the weight ratio of the cis-isomer in mixture accounts for the 25-35% of total mixture amount.
Further preferably, the weight ratio of the cis-isomer accounts for the mixture of the 25-35% of total mixture amount, by such as
Lower section method is made, and by the methanol solution of curcumin analogue, is subject to ultraviolet irradiation 1.5-2.5h.
Preferably, in ultraviolet irradiation, the concentration of methanol solution of curcumin analogue is 0.5-5mg/ml, further preferably
For 0.5-1.5mg/ml.If the time of ultraviolet irradiation shorter than 1.5h, cannot generate the cis-isomer of sufficient amount, if
Extra 2.5h can then start to generate by-product, particularly preferably ultraviolet irradiation 2h.
Preferably, polyethyleneglycol derivative molecular weight ranges are further preferably to be lower than 2000 lower than 5000.
It further preferably, is carboxy polyethylene glycol or polymaleic anhydride 18 carbenes-polyethylene glycol.
Preferably, the gas is nitrogen or inert gas, preferably nitrogen.Auxiliary good solvent volatilizes, to guarantee
The formation of nano particle, while preventing safety risks caused by dissolvent residual.
Preferably, the reprecipitation method is that the good-solvent solution is added dropwise into deionized water, is 20-30 DEG C in temperature
Under, 2-10min is stirred, 3-30min is stood, obtains nano particle suspended emulsion.
Nasal cavity nanometer formulation Brain targeting delivery system of the invention, carrier-free, inanimate object degradation problem and cumulative toxicity carry
Dose is up to 25% or more, can pH responsively sustained release small molecule drug, height retains the combination energy of small molecule and receptor targeted
Power；Polyethyleneglycol derivative can enhance pellet moisture and dissipate property and stability；Chitosan oligosaccharide is sorbefacient, passes through positive and negative charge
Electronegative Nano medication particle is modified in suction-operated, and there is nasal membrane to promote permeability and can and have both neuroprotection synergistic effect.
Chitosan is the macromolecular that chitin passes through that deacetylation obtains, and molecular weight is in hundreds of thousands to millions of Da, no
It is dissolved in water.Chitosan is degraded through special biological enzyme technology etc., a kind of oligosaccharides of the degree of polymerization between 2~20 can be obtained and produce
Product are chitosan oligosaccharide, are called Chitosan poly oligosaccharide, chitosan oligomer, and molecular weight≤3200Da has chitosan unexistent compared with Gao Rong
Xie Du, it can be dissolved in water entirely, be easy that many unique functions are absorbed and utilized etc. by organism.Chitosan oligosaccharide is a kind of nontoxic functionality
Low molecular weight amino sugar, is polycation structure, and the present invention is modified outside nano particle, drug can be prevented to nasal cavity
The stimulation of interior environment；It is easily acted on the negatively charged group in mucomembranous cell surface, to change the mobility of cell membrane and penetrating
Property, increase the absorption of nano particle, in addition, chitosan oligosaccharide itself also has certain immunological regulation and neuroprotection, effect
Fruit is 14 times of chitosan.
Chitosan oligosaccharide used in the present invention, degree of polymerization 2-20 or molecular weight≤3200Da.
Chitosan oligosaccharide of the present invention is 0.01-0.2% (w/v) in isotonic physiological saline solution concentration, if being less than 0.01%,
It is difficult to play the role of increasing absorption, prevents from if it is greater than 0.2%, being then easy to cause electronegative receive in stimulation etc.
Rice grain aggregation.
Freeze-dried powder is hanged again in isotonic physiological saline, freeze-dried powder concentration can configure as needed.Preferably 3-7mg/
ml。
Preferably, the average grain diameter of the hydrophobic drug nano particle is 50-150nm, more preferable 50-120nm.
Preferably, the surface potential of the hydrophobic drug nano particle is -30~-60mV.
Preferably, the carrying drug ratio of the hydrophobic drug nano particle is 25% or more.
The formation of self-assembling nanoparticles, the molecular structure by hydrophobic small molecules are influenced, and intermolecular is non-covalent knot
With joint efforts, because to can lead to nanostructure variant for molecular configuration difference.For the stability for enhancing nano particle, the present invention is in solvent
Before exchange, polyethyleneglycol derivative is added, after being mixed in organic solvent with hydrophobic small molecules with certain proportion, then carries out
Exchange of solvent obtains the nano particle of carrier-free package by reprecipitation method.
Hydrophobic drug nano particle of the invention is without other carrier components, therefore the high and low poison of its drugloading rate, safety
Good, partial size is small and uniform, and stability is high, circulation time is long in vivo.
Preferably, the hydrophobic drug is the natural products and its modifier with neuroprotection.
It is highly preferred that the natural products and its modifier with neuroprotection is in curcumin or its analog
It is one or more.
Further, the nasal cavity nanometer formulation Brain targeting delivery system of invention can also be added in other pharmacies effective auxiliary
Material, such as bacteriostatic agent, isotonic regulator, dosage are the conventional amount used of defined in pharmacy.
Antioxidant can also be added in the present invention, and the antioxidant can be sodium metabisulfite, sodium hydrogensulfite, sulfurous
One of sour sodium, sodium thiosulfate, cysteine hydrochloride, vitamin C, vitamin E, thiocarbamide are a variety of, and dosage is medicine
The conventional amount used of the upper defined of agent.
Preservative can also be added in the present invention, and the preservative can be methyl p-hydroxybenzoate, para hydroxybenzene first
Acetoacetic ester, propylparaben, butyl p-hydroxybenzoate, benzalkonium bromide, benzalkonium chloride, anesin, benzene second
One of alcohol, thimerosal, phenylmercuric nitrate, sorbic acid, chlorohexidene are a variety of, and dosage is the routine of defined in pharmacy
Dosage.
Osmotic pressure regulator can also be added in the present invention, and the osmotic pressure regulator can be sodium chloride, glucose, cream
One of sugar, mannitol are a variety of, and dosage is the conventional amount used of defined in pharmacy.
In second aspect, the present invention provides a kind of self-carrying carrier-free nasal cavity of chitosan oligosaccharide modification as described in relation to the first aspect
The preparation method of nanometer formulation, described method includes following steps:
1) the good molten of polyethyleneglycol derivative 1-10mg/mL and 0.5-5mg/mL hydrophobic small molecules drug is configured first
Then agent solution the good-solvent solution is added dropwise dropwise into deionized water: the body of the good-solvent solution and deionized water
Product is than being (0.5-5): 50, dropwise addition while, is aided with gas and blows, auxiliary good solvent volatilization, 2) granulating is prepared by reprecipitation method
Diameter is the self-carrying carrier-free nano particle suspended emulsion of 50-200nm, and freeze-drying is prepared into freeze-dried powder；
3) before use, that freeze-dried powder and chitosan oligosaccharide using physisorption are reacted 0.5-2 in isotonic physiological saline is small
When, remove reactant, the self-carrying carrier-free Nano medication nasal cavity preparation that product purification is modified up to chitosan oligosaccharide.
The method that the present invention prepares nano particle is carried out by reprecipitation method, and good solvent is transferred into water (poor solvent)
When middle, hydrophobic drug is precipitated to form nano particle, and polyethyleneglycol derivative is being added, can further increased
Its strong stability and water dispersible.This method is simple and easy, does not need complicated operation and condition, can carry out at room temperature.
It is emphasized that the present invention in, the addition time of polyethyleneglycol derivative, should before nano particle is formed,
It is dissolved in good solvent together with small molecule, after mixing, adds dropwise into water phase, prepare nano particle.It is added dropwise
When, it is aided with gas (preferably nitrogen) and blows, to cleared organic solvent.The method is different from being initially formed nano particle, adds two afterwards
The method that parent's property surfactant makees surface modification, product be not also identical.
The present invention just mixes freeze-dried powder with chitosan oligosaccharide just before use in isotonic physiological saline, avoid placement long it
After generate aggregate and precipitate, the absorption of nasal cavity when guaranteeing to use.
The present invention is prepared in the method for hydrophobic drug nano particle, and the good solvent is miscible with water.According to Flory-
Krigboum dilute solution theory, good solvent refer to the solvent with solute interaction parameter less than 0.5.Preferably, described good molten
Agent is mixing one or more in acetone, methanol, ethyl alcohol and tetrahydrofuran, more preferably tetrahydrofuran.
The present invention is prepared in the method for hydrophobic drug nano particle, and the water can be deionized water, distilled water or double
Steam water etc., preferably deionized water.
The present invention is prepared in the method for hydrophobic drug nano particle, and hydrophobic drug is dissolved in the step (1)
Concentration in good solvent is 0.5-5mg/mL, preferably 1mg/mL.
The present invention is prepared in the method for hydrophobic drug nano particle, the volume ratio of good solvent and water in the step (1)
Preferably (1-3): 50, preferably 2:50.Preferably, in the step (1) reaction temperature be 20-30 DEG C, more preferably 25
℃。
The present invention is prepared in the method for hydrophobic drug nano particle, and the polyethylene glycol being added in the step (2) is derivative
Object can be 1-10mg/mL, such as 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL, 5mg/mL, 6mg/ in the concentration of good solvent
ML, 7mg/mL, 8mg/mL, 9mg/mL or 10mg/mL, preferably 2mg/mL.
Preferably, the time of ultrasonic disperse is 3-30min, more preferably 5min in the step (2).Preferably, described
In step (3) chitosan oligosaccharide isotonic physiological saline solution concentration be 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, in the step (3) mixing time be 0.5-2h, such as 0.5h, 1h,
1.5h or 2h, preferably 1h.Preferably, centrifugal condition is revolving speed 10000-150000*g in the step (3), preferably
150000*g is centrifuged 30min.
Self-assembling nanoparticles may be assembled in aqueous solution stored for extended periods, be freeze-dried, and can significantly improve
Its stability, cryogenic temperature is lower than being freeze-dried 24- under 10-20 DEG C of the eutectic point that nano particle and water coexist, 10Pa pressure
90h, preferably 48h.
For avoid freeze-drying after nanoparticle aggregate and change of size, cryoprotector is first added, as glucose, mannitol,
Lactose, NaCl etc. promote a large amount of tiny ice crystals to be formed, or make dried frozen aquatic products in rarefaction in freezing, in favor of nano particle
The state that holds its shape simultaneously is easy to redisperse in water.
In the third aspect, the present invention provides the self-carrying carrier-free nasal cavity nanometer of chitosan oligosaccharide modification as described in relation to the first aspect
The application of preparation brain targeting delivery system.It is used for brain targeting delivering.Nasal mist or nasal drop can be made.
In one embodiment, the curcumin analogue M1 self-carrying carrier-free nasal cavity nanometer formulation system nose administration of chitosan oligosaccharide modification
Afterwards, exploring the behaviors such as locomotor activity, anxiety, gait to the mouse model of Parkinson's disease of MPTP induction has improvement result.
Brain targeting delivery system of the present invention passes through nasal delivery using spraying or collunarium form.
The invention has the benefit that
A kind of nanometer formulation Brain targeting of the present invention delivers drug system, for the hydrophobic of neuroprotection
Property small molecule, be prepared into chitosan oligosaccharide modification self-carrying carrier-free nasal cavity nanometer formulation Brain targeting delivery system, can apply
In a series for the treatment of of the nervous system diseases.It is made after nasal cavity preparation, can avoid gastrointestinal tract degradation and liver first-pass effect,
Have the characteristics that bioavilability is high, rapid-action, good patient compliance.Compared with regular dosage form, nasal cavity preparation is logical by nose brain
Drug is directly delivered around blood-brain barrier into brain along approach such as olfactory nerves, can significantly increase brain targeting, reduce periphery by road
The enrichment of circulatory system internal organs, reduces the potential side effect of long-term use.Compared with injection, nasal cavity preparation can reduce liver etc.
The drug accumulation of peripheral circulation organ；Compared with oral medicine, nasal cavity preparation reduces drug loss without first pass effect.It is easy to use
Noninvasive, patient compliance improves.Compared to intravenous injection, nasal-cavity administration only needs the modes such as collunarium, spraying, the simpler peace of operation
Entirely, especially for the neurodegenerative disease patient of Long-term taking medicine, pain, the good patient compliance of patient can be mitigated, just
In self-medication, and long-term administration bring risk is reduced, there is applications well prospect.In addition, compared with intravenous injection, drop
The modes safety easy to operate such as nose, spraying is convenient for self-medication particularly with the neurodegenerative disease patient of Long-term taking medicine,
It has a good application prospect in terms of the treatment of the nervous system disease.
The self-carrying carrier-free nasal cavity nanometer formulation of chitosan oligosaccharide modification in the present invention is easy to operate, applied widely and general
Adaptive is strong.Compared to hydrophobicity small organic molecule prodrug, with more being obviously improved water dispersible and druggability, enhancing are given birth to
Object availability reduces dosage rate, reduces the advantages such as toxic side effect, takes for a long time safer.Compared to traditional polymer nano
Rice drug-loading system, nanosystems carrier-free of the present invention, inanimate object degradation problem and cumulative toxicity, carrying drug ratio are up to 25% or more,
And after chitosan oligosaccharide modification permeable membrane absorb it is good, as nasal cavity preparation can high targeting brain entered by olfactory nerve.Poly- second two
01 derivatives also reduce mucosal irritation in addition to enhancing stability, extend circulation time in vivo.
The nanometer formulation height retains the receptor targeted binding ability of original molecule, and the drug release with pH responsiveness is special
Property, it degrades under the specific pH of lysosome (5.5) after cellular uptake, discharges small molecule into cytoplasm to play drug effect.Together
When, drug of the present invention is stable in delivery process.Fig. 7 explanation, the present invention can make Medicine small molecule in Cerebrospinal fluid and blood
Long-acting circulation and slow release (more than 64h) in liquid so as to reduce dosage rate, and reduce dosage.
To which the present invention is with high security, carrying drug ratio is high, brain targeting is high；It is (slow that dosage is few, dosage rate is few
Release long-acting), systemic side effects it is few (liver kidney content is low)；The advantages that non-invasive is administered.
Compared with former hydrophobic small molecules drug, delivery system of the present invention has good water dispersible, greatly strengthens
Small molecule druggability, and reduce small-molecule drug toxicity, enhancing neuroprotection；Compared with other nanometer formulations, the present invention
Delivery system carrier-free, inanimate object degradation problem and cumulative toxicity, carrying drug ratio are up to 25% or more, and height retains original molecule
Receptor targeted binding ability, and after chitosan oligosaccharide modification permeable membrane absorb it is good, as nasal cavity preparation can high targeting by smelling mind
Through entering brain.There is pH response performance, it can be in intracellular slow release, long-acting maintenance effective concentration.
Detailed description of the invention
Present invention will be further explained below with reference to the attached drawings and examples.
Scanning electron microscope (SEM) figure of Fig. 1 hydrophobic small molecules drug self-carrying carrier-free nano particle: wherein a is turmeric
Plain nano particle；B is M1 nano particle；C is the M1 nano particle for being loaded with TPAAQ probe.
The grain size distribution of Fig. 2 hydrophobic small molecules drug self-carrying carrier-free nano particle: wherein a receives for curcumin
Rice grain；B is M1 nano particle；C is the M1 nano particle for being loaded with TPAAQ probe.
The potential image of Fig. 3 hydrophobic small molecules drug self-carrying carrier-free nano particle: wherein a receives for curcumin
Rice grain；B is M1 nano particle.
The Tyndall effect optical characterisation figure of Fig. 4 hydrophobic small molecules drug self-carrying carrier-free nano particle: wherein a
For curcumin nano particle；B is M1 nano particle.
The charging ratio test chart of Fig. 5 hydrophobic small molecules drug self-carrying carrier-free nano particle: wherein a is curcumin
Nano particle；B is M1 nano particle, and c is the M1 nano particle for being loaded with TPAAQ probe.
Fig. 6 curcumin nano particle cellular uptake effect after chitosan oligosaccharide is modified；Wherein a is to modify without chitosan oligosaccharide
M1 NPs cellular uptake figure；B is the M1 NPs cellular uptake figure after chitosan oligosaccharide modification.
Fig. 7 .M1 nano particle pH response medicine releasing curve diagram.
The cytotoxicity figure of Fig. 8 .M1 nano particle and small molecule M1 drug.
The ex vivo nerve protective effect figure of Fig. 9 .M1 nano particle.
Figure 10 .M1 nano particle compliance test result figure in conjunction with target protein.
Figure 11 is loaded with the M1 nano particle cellular uptake figure of TPAAQ fluorescence probe.
The brain section transmission electron microscope picture of mouse after Figure 12 curcumin nano particle nose administration.
M1 medicament contg figure in mouse brain and blood plasma after the administration of Figure 13 .M1 nano particle intranasal brain.
Figure 14 is loaded with the brain and internal organs biological of the mouse after the M1 nano particle nose administration of TPAAQ fluorescence probe
Figure.
Open-field activities figure after the M1 nasal cavity nanometer formulation of Figure 15 mouse model of Parkinson's disease nose administration embodiment 2.
Gait behavior figure after the M1 nasal cavity nanometer formulation of Figure 16 mouse model of Parkinson's disease nose administration embodiment 2.
The compound map that mass spectrogram middle-molecular-weihydroxyethyl is 294.34 in Figure 17 embodiment 2
Figure 18 is the conversion ratio of the curcumin analogue in embodiment 2 under different condition.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment.Those skilled in the art will manage
Solution, once embodiment is only the preferred embodiment invented, and in order to more fully understand the present invention, thus should not be regarded as limiting this
The range of invention.For those skilled in the art, the invention may be variously modified and varied, all of the invention
Within spirit and principle, made any modification, equivalent replacement or improvement etc., should be included in protection scope of the present invention it
It is interior.Experimental method in following embodiments is unless otherwise specified conventional method；Experimental material used, such as without special theory
It is bright, it is purchased from conventional biochemical reagent manufacturers.
Embodiment 1: the preparation of curcumin nasal cavity nanometer formulation
Curcumin structure used is as follows in this implementation:
Configure 18 carbenes of polymaleic anhydride-polyethylene glycol of curcumin drug molecule and 2mg/mL containing 1.5mM concentration
- tetrahydrofuran solution 10mL, take curcumin solution described in 200 μ L to be added dropwise into 4mL deionized water, dropwise addition while is aided with
Nitrogen is blown, while being vigorously stirred aqueous solution, with cleared organic solvent, is stood after stirring 10min, is obtained self-carrying carrier-free turmeric
Plain nano particle suspended emulsion, freeze-drying form freeze-dried powder.Before use, disperse freeze-dried powder in again isotonic physiological saline
In, it is added dropwise in chitosan oligosaccharide (0.1w/v) normal saline solution, stirs 0.5h, it is small using physisorption reaction 1
When, 10min is centrifuged with the revolving speed of 10000*g, throws aside supernatant, reactant is removed, product purification is modified up to chitosan oligosaccharide
Self-carrying carrier-free curcumin nasal cavity nanometer formulation.
As a result
(1) measurement of curcumin nano particle shape, partial size and Potential distribution
Using scanning electron microscope (FEI Quanta200, Holland) according to the method in its specification, observes and made in embodiment 1
Standby curcumin nano particle, scanning electron microscope (SEM) photograph are as shown in Figure 1a.Using laser particle analyzer (Malvern, Britain) according to it
Method in specification carries out dynamic light scattering measurement to the curcumin nano particle prepared in embodiment 1, measures embodiment 1
The average grain diameter of the curcumin nano particle of middle preparation is 64.57nm, and grain size distribution is as shown in Figure 2 a.Use laser particle size
Instrument carries out Zeta- potentiometric analysis according to the method in its specification, to the curcumin nano particle prepared in embodiment 1, measures
It is -10.5mV that curcumin nano particle mean charge is prepared in embodiment 1, shows it with faint negative electrical charge, distribution is such as
Shown in Fig. 3 a.
(2) measurement of curcumin nano particle optical characterization
The curcumin nano particle prepared in curcumin molecule and embodiment 1 is dissolved separately in water and organic solvent,
As shown in Fig. 4 a, it is seen that curcumin small molecule is difficult to be dissolved in water, dissolves in tetrahydrofuran, and curcumin nano particle can
It is dispersed in water, there is Tyndall effect under laser.
The measurement of curcumin nano particle carrying drug ratio
By curcumin molecular melting in acetonitrile, the curcumin acetonitrile solution of gradient configuration series of concentrations (6.25,12.5,
25,50 and 100ug/mL), absorbance is measured in 428nm using ultra performance liquid chromatography (UPLC), makes standard curve.Take three
The curcumin nano particle of batch 100ug/mL, is dissolved separately in acetonitrile, and ultrasonic 5min is measured in the same method, utilizes standard curve
Calculate the turmeric cellulose content in nano particle.As shown in Figure 5 a, the carrying drug ratio of curcumin nano particle is (25.12 ± 2.50) %
(3) the chitosan oligosaccharide modification effect measurement of curcumin nano particle
By fluorescence FITC probe with modified without chitosan oligosaccharide curcumin nano particle, chitosan oligosaccharide modification after curcumin receive
After rice grain is modified respectively, it is respectively incubated for culture 6h altogether with N2a cell, cell is cleaned three times with PBS, aobvious in laser co-focusing
It is excited under micro mirror with 488nm wavelength, 190-540nm launch wavelength, measures fluorescence intensity, and compare analysis.As shown in figure 5,
After chitosan oligosaccharide modification, the cellular uptake of curcumin nano particle is significantly increased.
Embodiment 2: the preparation of curcumin analogue M1 nasal cavity nanometer formulation
Curcumin analogue M1 used in the present embodiment is the isomers mixing of the curcumin analogue of following structural formula
Object:
According to the Computer simulation results of applicant, in the mixture, the ratio of syn-isomerism body is higher, product
Bioactivity is stronger.But in practice, product is higher, and the yield of by-product is also higher.
In the present embodiment, a kind of isomer products that can get 30% conversion ratio are provided, preparation method is simple, and nothing
The method that by-product generates.
It according to the hydrophobic property of curcumin analogue, is dissolved in good solvent, gives different radiation conditions, can occur not
Isomers with degree converts, and obtains the cis-trans-isomer mixture of different proportion.Wherein, the conversion ratio highest of solar radiation,
But there is by-product generation；Secondly conversion ratio is ultraviolet irradiation, radioiodine radiation, and under the conditions of being protected from light, temperature is to curcumin
Like object structure without influence.
Further, good solvent is preferably acetonitrile, methanol, ethyl alcohol, acetone, tetrahydrofuran.
Further, UV, which irradiates 2h, can get the isomer products of 30% conversion ratio, and preparation method is simple, and no coupling product is raw
At.
Cis-trans-isomer mixture M 1 (Mixture 1) preparation method of curcumin analogue
The methanol solution for configuring the curcumin analogue containing 1mg/mL, after being sealed with aluminium-foil paper, respectively at 4 DEG C, 25 DEG C
And 8h is placed in the environment of 50 DEG C, obtain reaction product 1-3；Methanol of the another configuration equally with the curcumin analogue of concentration is molten
Liquid, at room temperature, be subject to respectively solar radiation 2h, solar radiation for 24 hours, ultraviolet irradiation 2h, radioactivity iodine 131 radiation 2h, obtain anti-
Product 4-7 is answered, to obtain the curcumin analogue cis-trans-isomer mixture of various ratios.
As a result
(1) molecular weight identification of converted product
Using the molecular weight of efficient liquid phase combination time of-flight mass spectrometer measurement product, as shown in Figure 17, curcumin is seemingly
The molecular weight of object and its converted product is 294.34, and both confirmations are isomer, further by structure it is found that being suitable
Trans isomer.
(2) measurement of cis-trans isomerism transformation rate:
Using high performance liquid chromatography (HPLC), under 384nm maximum absorption wavelength, measure in sample solution 1-7, turmeric
The cis-trans isomerism transformation rate of plain analog.As a result as shown in Fig. 2, nothing has novel substance generation, explanation in reaction product 1-3
Curcumin analogue isomers conversion (Figure 18 a-c) does not occur.Reaction product 4, after solar radiation 2h, curcumin analogue contains
Amount has 73.91% to be converted into its isomers (Figure 18 d)；And solar radiation is for 24 hours, reaction product 5 removes curcumin analogue isomers
Except, there are many more complicated products to generate (Figure 18 e).Reaction product 6, ultraviolet irradiation 2h, the conversion of curcumin analogue isomers
Rate is 29.59% (Figure 18 f).Reaction product 7, under radioactivity iodine 131 radiation condition 2h, the conversion of curcumin analogue isomers
Rate is 27.91% (Figure 18/g).
Using reaction product 6 as M1 below:
The tetrahydrofuran solution 5mL of the carboxy polyethylene glycol of the M1 containing 1mg/mL and 2mg/mL is configured, mixes, takes 200
M1 molecular solution described in μ L, is added dropwise into 5mL deionized water dropwise, and dropwise addition while is aided with nitrogen and blows, with cleared organic solvent.
After magnetic stirring for 10 minutes are stood at 25 DEG C, obtain M1 self-carrying carrier-free nano particle suspended emulsion, and freeze-drying forms freeze-drying
Powder.Before use, freeze-dried powder is added dropwise in the isotonic physiological saline solution containing chitosan oligosaccharide (0.1w/v), stirs 0.5-2h,
It is reacted 1 hour using physisorption, 5-30min is centrifuged with the revolving speed of 10000-150000*g, throws aside supernatant, is removed
Reactant, the self-carrying carrier-free M1 nasal cavity nanometer formulation that product purification is modified up to chitosan oligosaccharide.
As a result
(1) measurement of M1 nano particle form, partial size and Potential distribution
Using scanning electron microscope (FEI Quanta200, Holland) according to the method in its specification, observes and made in embodiment 2
Standby M1 nano particle, scanning electron microscope are as shown in Figure 1 b.Using laser particle analyzer (Malvern, Britain) according to its specification
In method, dynamic light scattering measurement is carried out to the M1 nano particle for preparing in embodiment 2, measures the M1 prepared in embodiment 2
The average grain diameter of nano particle is 62.73nm, and grain size distribution is as shown in Figure 2 b.Using laser particle analyzer according to its specification
In method, Zeta- potentiometric analysis is carried out to the M1 nano particle for preparing in embodiment 2, measures the M1 prepared in embodiment 2
Nano particle mean charge is -56.5mV, shows it with faint negative electrical charge, distribution is as shown in Figure 3b.
(2) measurement of M1 particle optical characterization
The M1 nano particle prepared in M1 small molecule and embodiment 2 is dissolved separately in water and organic solvent, such as Fig. 4 b
It is shown, it is seen that M1 small molecule is difficult to be dissolved in water, dissolves in tetrahydrofuran, and M1 nano particle is dispersed in water, is swashing
There is Tyndall effect under light.
3) measurement of M1 nano particle carrying drug ratio
By M1 molecular melting in acetonitrile, the acetonitrile solution of the M1 of gradient configuration series of concentrations (6.25,12.5,25,50 and
100ug/mL), absorbance is measured in 428nm using ultra performance liquid chromatography (UPLC), makes standard curve,.Take three batches
The M1 nano particle of 100ug/mL, is dissolved separately in acetonitrile, and ultrasonic 5min is measured in the same method, and calculates nanometer using standard curve
M1 content in particle.As shown in Figure 5 b, the carrying drug ratio of M1 nano particle is (31.49 ± 2.03) %
(4) M1 nanoparticulate drug release profiles measure
M1 nano particle prepared by embodiment 2 is divided into six parts, in 3 parts of addition artificial nose liquid, 3 parts of 5% blood plasma of addition,
Be respectively charged into bag filter, disperse and dilute, be separately added into bag filter (3500 molecular weight,The U.S.) in,
Then it is immersed in 200 milliliters of the buffer of identical pH, 37 DEG C are stirred continuously, and collect from solution in regular hour point
2ml solution.In dialysis procedure, 2ml PBS is supplemented after every sub-sampling, keeps constant liquor capacity.It is surveyed using UV-VIS method
Determine absorbance, calculates release amount of medicine.Each sample carries out 3 tests, is averaged, and statisticallys analyze, as a result as shown in Fig. 7.
As it can be seen that M1 nano particle prepared by embodiment 2 has the property of slow release, initial burst drug release is not shown, and
Be slow and steadily discharge, this be for the application of M1 nano particle in vivo it is vital, drug toxicity can be reduced,
Reduce drug leakage etc..
(5) cell toxicity test
According to the method culture nerve in document (" cell culture ", Si Tuzhenqiang, world book publishing company, 1996)
Then tumor mother cell N2a cell is added M1 nano particle prepared by embodiment 2 and continues to cultivate, according to document after dosing 24 hours
Method (mtt assay) in (" cell culture ", Si Tuzhenqiang, world book publishing company, 1996) detects cell survival rate,
This is M1 nano particle group.With the free M1 with M1 nano particle group containing same concentrations by the group of same procedure processing N2a cell
For positive controls；The N2a cell of blank cultures culture without hydrophobic drug is as negative control group, wherein with yin
Property control group in cell survival rate by 100% calculate.As a result as shown in figure 8, with concentration raising, free M1 can embody
Dose-dependent cytotoxicity out, and the M1 nano particle in embodiment 2 makees N2a cytotoxic under same concentrations
With the cumulative toxicity of M1 small-molecule drug may be inhibited in higher concentration due to the slow releasing function of M1 nano particle.
(6) the neuroprotection measurement of M1 nano particle
Nerve cell strain PC12 cell is handled using MPP+ neurotoxin, causes neurotoxicity cell model.Before modeling
6h is added the M1 nano particle pretreatment prepared in embodiment 2, is M1 nano particle group；It is model pair that drug-treated, which is not added,
According to group, not plus MPP+ neurotoxin is Normal group.Continue to cultivate 48h after modeling, survey absorbance according to literature method,
As a result as shown in figure 9, the cell survival rate ratio MPP+ model group of M1 nano particle group significantly increases, the M1 prepared in embodiment 2
Nano particle can dose dependent protect PC12 nerve cell, reduce the cellular damage that induces it of MPP+ neurotoxin.
(7) the combination effect of M1 nano particle and target protein measures
The target protein of free M1 molecule neuroprotection is the TFEB albumen in cytoplasm, and M1 is by promoting TFEB albumen
Dephosphorylation enters in nucleus, and raises the expression of autophagy related gene, to play the role of neuroprotection.This experiment
In, the M1 nano particle prepared in embodiment 2 is added in the MF7 cell for being overexpressed fluorescent marker TFEB albumen, processing is for 24 hours
Observation TFEB enters core situation afterwards, as a result as shown in figure 9, the M1 nano particle prepared in embodiment 2 can dose-dependently promote
TFEB enters core, it was demonstrated that M1 nano particle remains the targeting characteristic of original molecule.
Embodiment 3: the M1 nasal cavity nanometer formulation brain targeting delivery system of fluorescence probe TPAAQ is carried
TPAAQ is the hydrophobic small molecules fluorescence probe of a kind of excitation of 473nm wavelength, the transmitting of 650nm wavelength, can be used for receiving
The internal fluorescence distribution monitoring of rice material.Because it is also hydrophobic small molecules, therefore prepared with the M1 nano particle of embodiment 2
Journey is similar, and the M1 nasal cavity nanometer formulation for being loaded with TPAAQ can be obtained with method.
The tetrahydrofuran solution 5mL of the TPAAQ of the M1 containing 1mg/mL and 2mg/mL is configured, mixes, takes described in 200 μ L
M1 molecular solution is added dropwise in 5mL deionized water, while being aided with nitrogen and being blown, with cleared organic solvent.Magnetic force stirs at 25 DEG C
It mixes and stands after ten minutes, obtain the M1 self-carrying carrier-free nano particle suspended emulsion for carrying fluorescence probe TPAAQ, be freeze-dried shape
At freeze-dried powder.Before use, it disperses freeze-dried powder in isotonic physiological saline again, is added dropwise to chitosan oligosaccharide (0.1w/v)
In normal saline solution, stir 0.5-2h, using physisorption react 1 hour, with the revolving speed of 10000-150000*g from
Heart 5-30min throws aside supernatant, removes reactant, and product purification is loaded with taking certainly for TPAAQ probe up to chitosan oligosaccharide modification
Formula carrier-free M1 nasal cavity nanometer formulation.
3 result of embodiment
(1) measurement of M1 the nano particle form, particle diameter distribution of fluorescence probe TPAAQ is carried
Using scanning electron microscope (FEI Quanta200, Holland) according to the method in its specification, observes and made in embodiment 3
The M1 nano particle of standby load fluorescence probe TPAAQ, scanning electron microscope (SEM) photograph is as illustrated in figure 1 c.Use laser particle analyzer (Ma Er
Text, Britain) according to the method in its specification, to the M1 nano particle of the load fluorescence probe TPAAQ prepared in embodiment 3 into
Mobile state determination of light scattering, the average grain diameter for measuring the M1 nano particle of the load fluorescence probe TPAAQ prepared in embodiment 3 are
178.2 nm, grain size distribution are as shown in Figure 2 c.
(2) measurement of the M1 nano particle carrying drug ratio of fluorescence probe TPAAQ is carried
Using the done M1 acetonitrile solution standard curve of test (3) of embodiment 2, take the load fluorescence of three batch 100ug/mL
The M1 nano particle of probe TPAAQ, is dissolved separately in acetonitrile, and ultrasonic 5min is measured in the same method, and is received using standard curve calculating
Turmeric cellulose content in rice grain.As shown in Figure 5 c, the carrying drug ratio for carrying the M1 nano particle of fluorescence probe TPAAQ is (26.95
± 1.50) %.
(2) cellular uptake is tested
Nerve cell is normally cultivated, and the M1 nasal cavity nanometer formulation of the load fluorescence probe TPAAQ prepared in embodiment 3 is added,
After cultivating 3h, cellular uptake situation is observed under specific wavelength with laser confocal scanning microscope, as shown in figure 11, from glimmering
Optical signal is as it can be seen that the M1 nasal cavity nanometer formulation of the load fluorescence probe TPAAQ prepared in embodiment 3 can be by cell huge uptake.
Embodiment 4: the application of curcumin nano particle intranasal Brain targeting delivery system
Taking weight is male C57BL/6J Strains of Mouse 6, adaptive feeding 3 days of 25g.By what is prepared in embodiment 1
Curcumin nasal cavity nanometer formulation is scattered in isotonic physiological saline, concentration 5mg/ml, is given mouse nasal cavity 15ul, is dissected afterwards for 24 hours
Brain tissue is taken out, fixed slice observes the brain distribution of nano particle under transmission electron microscope.As shown in figure 12, it is apparent that ginger
Distribution of the flavine nano particle in brain olfactory bulb, cortical sites.
The application of embodiment 5:M1 nano particle intranasal Brain targeting delivery system
Taking weight is male C57BL/6J Strains of Mouse 6, adaptive feeding 3 days of 25g.By what is prepared in embodiment 2
M1 nasal cavity nanometer formulation is scattered in isotonic physiological saline, concentration 5mg/ml, gives mouse nasal cavity 15ul, and dissection is taken out afterwards for 24 hours
Brain tissue, cerebrospinal fluid and blood plasma, and brain tissue is divided into olfactory bulb part and brain rest part, all samples add methanol to remove respectively
After albumen, using liquid chromatography-tandedm mass spectro-metry chromatography, M1 medicament contg in sample is analyzed.As a result as shown in figure 13, M1 nasal cavity
M1 drug delivery is entered into the high targeting of nanometer formulation Brain targeting delivery system olfactory bulb, and is had in cerebrospinal fluid higher than in blood plasma
The distribution of content three times, and have the drug distribution of blood plasma doubling dose at the other positions of brain.Confirm its absorption features for by smelling
Ball and reach brain, and the other positions of brain can be transmitted to.It, which is transmitted, to be time dependence, will continue to afterwards via brain for 24 hours
Spinal fluid transmits backward.
Embodiment 6: it is loaded with the application of the M1 nano particle intranasal Brain targeting delivery system of TPAAQ fluorescence probe
Taking weight is male C57BL/6J Strains of Mouse 9, adaptive feeding 3 days of 25g.By what is prepared in embodiment 3
The M1 nasal cavity nanometer formulation for carrying fluorescence probe TPAAQ is scattered in physiological saline, and concentration 5mg/ml gives mouse nasal cavity 15ul,
Respectively at for 24 hours, apply toy fluoroscopic imaging systems after 48h, detect mouse brain in body fluorescence, and in vitro brain, the heart, liver,
Fluorescence signal in the internal organs and blood such as spleen, lung, kidney, as a result as shown in figure 14, brain signal is significantly stronger than other body parts
And tissue, it prompts 3 brain targeting delivery system of embodiment that can successfully deliver the high targeting of M1 nasal cavity nanometer formulation into brain, reduces
Distribution of the drug in peripheral tissues.
Embodiment 7: treatment use of the self-carrying carrier-free M1 nasal cavity nanometer formulation in mouse model of Parkinson's disease
Taking weight is male C57BL/6J Strains of Mouse 30 of 25g, points three groups, first group of wild type group (WT group), the
Two group model groups (MPTP group), third group model administration group (M1 NPs), every group of 10 mouse.According to literature method, by
Two, 20mg/kg dosage MPTP is persistently injected intraperitoneally neurotoxin five days in three groups of mouse, causes Parkinson disease model.Modeling is same
Phase does drug treatment, and WT group, MPTP group mouse nasal cavity give physiological saline, and M1 NPs group nasal cavity gives self-carrying carrier-free M1
Nasal cavity nanometer formulation, that is, it disperses the M1 nasal cavity nanometer formulation prepared in embodiment 2 in isotonic physiological saline, faces with brand-new,
Concentration 1mg/ml, nasal cavity give mouse 15ul.Between be administered every two days, be administered four times altogether, modeling terminates observation effect after two weeks.
7 result of embodiment
(1) open field test detection mouse model of Parkinson's disease behaviouristics performance
MPTP Parkinson's mouse model, which has, explores the symptoms such as dyskinesia and significant anxiety, can be detected by open field test.Root
According to literature method, the behaviouristics performance of mouse model of Parkinson's disease in embodiment 7 is detected.As a result it as shown in figure Figure 15 a, and compares
Group wild-type mice is compared, and model mice motion profile significantly changes, and after the treatment of M1 nasal cavity nanometer formulation, track tends to
Normally.Statistical data shows, compares wild-type mice, run duration of the model mice in mining site, average speed (Figure 15 b)
It is substantially reduced with region shuttle number (Figure 15 c) etc., and after the treatment of self-carrying carrier-free M1 nasal cavity nanometer formulation, on
It states lesion situation to significantly improve, it was demonstrated that the behaviouristics symptom of Parkinson disease model can be effectively relieved in M1 nasal cavity nanometer formulation.
Gait test detects the performance of mouse model of Parkinson's disease behaviouristics
The clinical manifestation of Parkinson's disease mainly includes static tremor, bradykinesia, myotonia and posture gait disorder etc..
DigiGait imaging system is used on animal, by below transparent treadbelt to animal imaging, software quantification gait mechanics and
The features such as posture index can detect the Behavioral feature of mouse model of Parkinson's disease.As a result as shown in figure 16, comparison wild type is small
Mouse, the gait signal disorder of mouse model of Parkinson's disease, harmony reduce, sole lands, and area is substantially reduced, and pass through self-carrying
After the treatment of carrier-free M1 nasal cavity nanometer formulation, above-mentioned lesion situation is significantly improved, it was demonstrated that M1 nasal cavity nanometer formulation can effectively change
The behaviouristics symptom of kind Parkinson disease.
The Applicant declares that the present invention is explained by the above embodiments detailed features and method detailed of the invention, but
The invention is not limited to above-mentioned detailed features and method detaileds, that is, do not mean that the present invention must rely on above-mentioned detailed spy
Sign and method detailed could be implemented.It should be clear to those skilled in the art, any improvement in the present invention, right
The present invention selects equivalence replacement and addition, selection of concrete mode of auxiliary element of component etc., all falls within protection of the invention
Within range and the open scope.
Claims (10)
1. a kind of self-carrying carrier-free nasal cavity nanometer formulation Brain targeting delivery system of chitosan oligosaccharide modification, it is characterised in that:
Hydrophobic small molecules drug and polyethyleneglycol derivative including chitosan oligosaccharide, with neuroprotection；It configures first
The good-solvent solution of polyethyleneglycol derivative 1-10mg/mL and 0.5-5mg/mL hydrophobic small molecules drug, then will be described
Good-solvent solution is added dropwise into deionized water, and the volume ratio of the good-solvent solution and deionized water is (0.5-5): 50, it is added dropwise
While be aided with gas and blow, auxiliary good solvent volatilization；The self-carrying no-load that partial size is 50-200nm is prepared by reprecipitation method
Body nano particle suspended emulsion, freeze-drying are prepared into freeze-dried powder；
Before use, freeze-dried powder and chitosan oligosaccharide are stirred to react 0.5-2 hours in isotonic physiological saline using physisorption,
Wherein, chitosan oligosaccharide is 0.01-0.2% (w/v) in isotonic physiological saline solution concentration, removes reactant, by product purification to obtain the final product
The self-carrying carrier-free Nano medication nasal cavity preparation of chitosan oligosaccharide modification.
2. the self-carrying carrier-free nasal cavity nanometer formulation Brain targeting delivery system of chitosan oligosaccharide modification according to claim 1,
It is characterized in that, the hydrophobic small molecules drug of the neuroprotection is one kind or more of curcumin or curcumin analogue
Kind.
3. the self-carrying carrier-free nasal cavity nanometer formulation Brain targeting delivery system of chitosan oligosaccharide modification according to claim 1,
It is characterized in that, the hydrophobic small molecules drug of the neuroprotection is the curcumin analogue and its light of following structural formula
The mixture of isomers is converted, isomers accounts for the 25-35% of amount of the mixture:
4. the self-carrying carrier-free nasal cavity nanometer formulation Brain targeting delivery system of chitosan oligosaccharide modification according to claim 3,
It is characterized by: the mixture that the weight ratio of the cis-isomer accounts for the 25-35% of total mixture amount is made by the following method
, by the methanol solution of curcumin analogue, it is subject to ultraviolet irradiation 1.5-2.5h.
5. a kind of self-carrying carrier-free nasal cavity nanometer formulation Brain targeting of chitosan oligosaccharide modification according to claim 1 delivers system
System, it is characterised in that: the polyethyleneglycol derivative is electronegative polyethyleneglycol derivative, preferably with the poly- of carboxyl
Ethylene glycol derivative or polymaleic anhydride 18 carbenes-polyethylene glycol.
6. a kind of self-carrying carrier-free nasal cavity nanometer formulation Brain targeting of chitosan oligosaccharide modification according to claim 1 delivers system
System, it is characterised in that: the chitosan oligosaccharide degree of polymerization is 2-20 or molecular weight≤3200Da.
7. a kind of preparation method of the self-carrying carrier-free nasal cavity nanometer formulation Brain targeting delivery system of chitosan oligosaccharide modification, including such as
Lower step:
1) good solvent for configuring polyethyleneglycol derivative 1-10mg/mL and 0.5-5mg/mL hydrophobic small molecules drug first is molten
Then liquid the good-solvent solution is added dropwise into deionized water: the volume ratio of the good-solvent solution and deionized water is
(0.5-5): 50, dropwise addition while, is aided with gas and blows, auxiliary good solvent volatilization；
2) the self-carrying carrier-free nano particle suspended emulsion that partial size is 50-200nm, freeze-drying system are prepared by reprecipitation method
For at freeze-dried powder；
3) freeze-dried powder is reacted 0.5-2 hours in isotonic physiological saline using physisorption with chitosan oligosaccharide before use, is removed
Dereaction object, the self-carrying carrier-free Nano medication nasal cavity preparation that product purification is modified up to chitosan oligosaccharide.
8. such as a kind of self-carrying carrier-free nasal cavity nanometer formulation brain target of chitosan oligosaccharide modification as claimed in any one of claims 1 to 6
Application to delivery system, in nasal mist or nasal drop.
9. such as a kind of self-carrying carrier-free nasal cavity nanometer formulation brain target of chitosan oligosaccharide modification as claimed in any one of claims 1 to 6
To delivery system, in the application for preventing and treating nervous system disease agent, the nervous system disease agent is preferably pa gold
Gloomy drug.
10. a kind of hydrophobic small molecules cis-trans-isomer mixture with neuroprotection, which is characterized in that described hydrophobic
Property small molecule be following structural formula curcumin analogue, by daylight, ultraviolet or radioactive radiation irradiation generates has
The cis-trans-isomer mixture of neuroprotection:
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102512404A (en) * | 2011-11-18 | 2012-06-27 | 上海交通大学 | Lung targeting preparation of curcumin class compound as well as preparation method and application thereof |
| 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 |
| CN103550776A (en) * | 2013-10-31 | 2014-02-05 | 国家纳米科学中心 | Hydrophobic drug nanoparticles as well as preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102512404A (en) * | 2011-11-18 | 2012-06-27 | 上海交通大学 | Lung targeting preparation of curcumin class compound as well as preparation method and application thereof |
| 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 |
| CN103550776A (en) * | 2013-10-31 | 2014-02-05 | 国家纳米科学中心 | Hydrophobic drug nanoparticles as well as preparation method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| JU-XIAN SONG ET AL.: ""A novel curcumin analog binds to and activates TFEB in vitro and in vivo independent of MTOR inhibition"", 《AUTOPHAGY》 * |
| 万丽卿等: ""A549 细胞对壳寡糖及其纳米粒的摄取作用"", 《药学学报》 * |
| 陈溪等: ""温敏型姜黄素鼻用原位凝胶增强脑靶向性"", 《药学与临床研究》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020143662A1 (en) * | 2019-01-09 | 2020-07-16 | 纳菲(深圳)制药科技有限公司 | Brain-targeted delivery system for carrier-free nasal nano preparation modified by chitosan oligosaccharide and preparation method therefor |
| CN114920639A (en) * | 2020-11-24 | 2022-08-19 | 纳菲(深圳)制药科技有限公司 | Novel method for synthesizing curcumin analogue |
| CN114920639B (en) * | 2020-11-24 | 2024-01-19 | 纳菲(深圳)制药科技有限公司 | Novel method for synthesizing curcumin analogues |
Also Published As
| Publication number | Publication date |
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| WO2020143662A1 (en) | 2020-07-16 |
| CN109730966B (en) | 2021-06-25 |
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