CN107789632A - A kind of active Brain targeting nanoscale medicine delivery system of T7 peptides modification and preparation method thereof - Google Patents
A kind of active Brain targeting nanoscale medicine delivery system of T7 peptides modification and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to active Brain targeting nanoscale medicine delivery system of a kind of T7 peptides modification and preparation method thereof.The present invention uses T7 peptides（Sequence C HAIYPRH）As specific Brain targeting part, carrier using PEG PLGA diblock copolymers as nanoscale medicine delivery system, insoluble drug molecule is contained using emulsion-solvent evaporation method, then T7 peptides are covalently attached by difunctional NHS PEG MAL, builds active Brain targeting nanoscale medicine delivery system.Preparation technology of the present invention is simple, and reaction condition is gentle, and the organic solvent of introducing is few, can effectively reduce the cytotoxicity of nano carrier material, extends circulation time in vivo, improves biocompatibility.T7 peptides modification Brain targeting nanoscale medicine delivery system active targeting involved in the present invention is strong, greatly improve the efficiency that nano-drug transporter crosses over blood-brain barrier, make drug-rich in lesion tissue, improve the bioavilability of medicine, effective Drug controlled release, there is fabulous application prospect in terms for the treatment of with diagnosing ischemia cerebral apoplexy and brain tumor.
Description
Technical field
The present invention relates to pharmaceutical technology field, is related to a kind of the active Brain targeting nanoscale medicine delivery system and its system of the modification of T7 peptides
Preparation Method.
Background technology
Ischemia apoplexy and brain tumor are the principal disease for causing population in the world dead all the time, especially in person in middle and old age
It is more common in colony, seriously endanger the life and health of the mankind.With the fast development of economy and carrying for people's living standard
The trend that becomes younger is presented in the patient groups of height, cerebral infarction and brain tumor, is brought to entire society and family heavy
Financial burden.Studies have found that during treatment ischemia apoplexy and brain tumor, due to blood-brain barrier（BBB）'s
Hinder, about 98% hydrophilic small molecules and most macromoleculars all can not be by making drug molecule be difficult to reach focus
Place, limits the clinical efficacy to ischemia apoplexy and brain tumor.Therefore, research and design can carry medicine and efficiently pass through
BBB, the nano-medicament carrier of selectively targeted Cerebral Region nerve cell or brain tumor diseased region, there is great innovation valency
Value and scientific meaning.
With the fast development of nanometer technology, nano material is widely used in biomedical sector, especially
In the field such as medical imaging, medical diagnosis on disease, drug targeting is delivered, cancer is precisely treated, gene transfection.Degradable nano carrier by
In excellent biocompatibility and modifiability, it has also become one of focus of active brain targeting drug delivery system research.
Poly(D,L-lactide-co-glycolide (poly (lactic-co-glycolic acid), PLGA) is by lactic acid
A kind of function that (lactic acid, LA) and hydroxyacetic acid (glycolic acid, GA) are polymerized according to different proportion
Property macromolecule organic material, passes through food and medicine Surveillance Authority (FDA) certification in the U.S..At present, have been widely used for
Prepare artificial conduit, slow releasing carrier of medication (microballoon, nanoparticle, micropill), in the preparation such as implants and film.But due to
PLGA block crystallinity is stronger, and has hydrophobicity, so easily by reticuloendothelial system（RES）Remove, polyethylene glycol (PEG) tool
There is excellent hydrophily, and nontoxic, non-immunogenicity.Using hydrophilic bifunctional polyethylene glycol（NH2-PEG-MAL）It is right
PLGA carries out surface modification, can not only reduce PLGA cytotoxicity, and can further improve PLGA carrier systems
Hydrophily, increase the drugloading rate to insoluble drug, significantly reduce cytotoxicity, effectively delay RES removing, extend nanometer medicine
The body circulation time of thing carrier, the long-time sustained release of medicine can be realized（Several weeks or several months）, hence it is evident that reduce medicine frequency；And
And research is found, PEG-PLGA surfaces by it is selectively targeted it is ligand modified after, can carrying medicaments molecule pass through blood-brain barrier, it is aobvious
The valid density for improving medicine at cerebral tissue change position is write, reaches the purpose of precisely treatment brain diseases.Reduce other non-diseases
Become the drug concentration at position, reduce the toxic side effect to body
Research finds that BBB permeability studies table depending on the close connection between brain capillary endothelial cell (BCECs)
Bright, forming on BBB BCECs has many acceptors, including TfR, insulin receptor, LDL receptor
GAP-associated protein GAP (LRP) acceptor etc., wherein transferrins (transferrin, Tf) are the ligands specific of TfR
(transferrin receptor TfR), targeting BBB Targeting Effects can be reached in nano-carrier surface by being modified.So
And the nanoparticle of endogenic Tf energy Reverse transcriptases Tf modifications is specifically bound with TfR【Han L, Li JF, Huang
SX, et al. Peptide-conjugated polyamidoamine dendrimer as a nanoscale tumor-
targeted T1 magnetic resonance imaging contrast agent [J]. Biomaterials,
2011, 32:2989-2998】, and the change of protein conformation easily causes the forfeiture of its function, so as to be unfavorable for playing targeting
Effect【Mahmoudi M, Shokrgozar MA, Sardari S, et al. Irreversible changes in
protein conformation due to interaction with superparamagnetic iron oxide
nanoparticles [J]. Nanoscale,2011, 3: 1127-1138】.A kind of novel polypeptide of recent study discovery-
T7 peptides (sequence C HAIYPRH), it can be specifically bound with TfR, binding constant is up to 10 nmolL-1, and due to
The difference of binding site, the nanoparticle that endogenous Tf will not suppress that T7 can be promoted to modify on the contrary are specifically bound with TfR【Yuan Duan
Cutting edge of a knife or a sword, Zong Taili, Gao Huile, the preparation of the double modified liposomes of what diligent cell-penetrating peptides TAT and brain tumor targeting peptides T7 and targeting
Property evaluation [J] Acta Pharmaceutica Sinicas, 2015, (01):104-110】.In addition, T7 is micromolecule polypeptide, chemically synthesize, stability
It is good, it is small as active Brain targeting head base steric hindrance, efficiency high is targetted, potential applicability in clinical practice is good.
The content of the invention
For above-mentioned during treatment ischemia apoplexy or brain tumor, there is that drug molecule BBB transmitances are low and brain
The technical problems such as targeting deficiency, it is an object of the invention to provide a kind of active Brain targeting nanoscale medicine delivery system of T7 peptides modification
Preparation method.
The present invention uses novel polypeptide-T7 peptides as specific Brain targeting part, functional high-polymer organic material PLGA
Based on carrier, contain insoluble drug tanshinone IIA (Tanshinone IIA, TSIIA) and the brain target of efficient active be made
To nanoscale medicine delivery system.The active Brain targeting nanoscale medicine delivery system of the T7 peptides modification of preparation is to pass through BBB with receptor-mediated manner,
Medicine is improved to cerebral ischemia or the targeting ability at brain tumor pathological tissues position, reaches accurate treatment ischemia apoplexy or brain and swells
The purpose of knurl.
The present invention has the advantages of transferrins is as Brain targeting head base, and effectively avoids the dry of endogenous transferrins
Disturb, targeting and therapeutic efficiency height, preparation method are simple, and can apply to other central nervous system diseases（Such as：A Erci
Sea silent disease, senile dementia etc.）Accurate targeted therapy.
To reach above-mentioned purpose, present invention employs following technical scheme：
The present invention provides a kind of active Brain targeting nanoscale medicine delivery system of T7 peptides modification, mainly by high-molecular copolymer PLGA,
Polyethylene glycol（PEG）, T7 peptides and insoluble drug composition, be using T7 peptides as efficient Brain targeting part, be total to PLGA macromolecules
Nano-medicament carrier material based on polymers, insoluble drug molecule is contained using emulsion-solvent evaporation method, then by difunctional
NHS-PEG-MAL is covalently attached T7 peptides, builds active Brain targeting nanoscale medicine delivery system.
Heretofore described high-molecular copolymer PLGA end groups are respectively carboxyl（-COOH）And hydroxyl（-OH）, its
Molecular weight is 10000~30000；Wherein, the ratio of glycolide and lactide is 1/100~99/100 during synthesizing PLGA.
Heretofore described polyethylene glycol（PEG）Molecular weight 1000~5000, end group are respectively amino（-NH2)
And maleimide（MAL）.
Heretofore described peptide sequence is CHAIYPRH T7 peptides, the scope of described nanometer particle size for 100~
400 nm, described insoluble drug are tanshinone IIA, and drugloading rate is 1%~40% in described Brain targeting nanoscale medicine delivery system,
Envelop rate 50%~95%.
The present invention prepares the active Brain targeting nanoscale medicine delivery system of T7 peptides modification as follows:
PEG-PLGA diblock copolymers are prepared first with DCC condensation methods；Weigh a certain amount of PEG-PLGA polymer and medicine
Thing tanshinone IIA is dissolved in mixed organic solvents, by the way of being added dropwise under liquid, is added to certain density emulsifier solution
In, it is ultrasonic under ice-water bath, the organic solvent contained in evaporating solution is stirred, high speed centrifugation is cleaned three times, gone repeatedly with ultra-pure water
Except the emulsifying agent in suspension solution, with being freezed after filtering with microporous membrane, the PEG-PLGA nanoparticles of blank are prepared as ibid
State method；Weigh the PEG-PLGA nanoparticles PBS for carrying tanshinone IIA（PH=7.0）Solution redissolves, and adds the stirring of T7 peptides lucifuge
24 h are reacted, unreacted T7 peptides pass through bag filter（MWCO：5KDa）Remove, will dialyse in PBS (pH 7.0) buffer solution
Solution afterwards freezes, you can obtains the active Brain targeting nanoscale medicine delivery system of T7 peptides modification.
Heretofore described high-molecular copolymer PLGA and polyethylene glycol（PEG）Mol ratio be 1:1~1:40, T7
The mol ratio of peptide and PEG-PLGA diblock copolymers is 1:1~1:5, nano-medicament carrier rubs with insoluble drug molecule
You are than being 1:1~1: 20.
In heretofore described emulsion-solvent evaporation method mixed organic solvents used be dichloromethane, chloroform,
Any two kinds of mixing in acetone, ethyl acetate, methanol, absolute ethyl alcohol；Emulsifier type used is Tween-80, polyvinyl alcohol
（PVA）, PLURONICS F87, soybean lecithin or lecithin one or more of mixture therein, the mass fraction of emulsifying agent are
0.1%~10%.
The mode that heretofore described T7 peptides are connected with PEG-PLGA copolymers, it is thin base and PEG chains by T7 peptides
The maleimide base group reaction forming of one end is made.
The present invention carries out Structural Identification, knot using hydrogen nuclear magnetic resonance hydrogen spectrum to the T7-PEG-PLGA Brain targetings carrier of synthesis
Fruit shows that described high-molecular copolymer PLGA is with containing after difunctional NHS-PEG-MAL reacts in δ=3.646ppm
With occur PEG characteristic absorption peak at δ=7.002ppm, show MAL-PEG-NH2Successfully it has been connected to PLGA strands
End；PEG-PLGA further with after T7 reactive polypeptides, in δ=6.589ppm, 6.617ppm and δ=8.077ppm ~ 8.945ppm
There is the diagnostic protons peak of T7 peptides, and at δ=7.002ppm PEG ends maleimide base group（MAL）Diagnostic protons peak
Disappear, fully demonstrate and base and the MAL radical reactions of MAL-PEG-PLGA ends are dredged in T7 peptides, be successfully prepared T7-
PEG-PLGA nano-carriers.
The present invention also provides the fluorescence probe tracer that the T7 peptides modification Brain targeting nanoscale medicine delivery system acts on brain
Application.By the T7 peptides modification Brain targeting drug-loading nanoparticles injection ICR mouse comprising fluorescence probe tracer DiR, and by its
Insert in multispectral living imaging system and be imaged, observation contains distribution of the nano particle of fluorescence molecule in Mice Body.Then
Dissection mouse simultaneously takes out its brain, carries out brain fluorescence imaging, aggregation extent of the observation nanoparticle in mouse brain.Its
In can be observed T7 peptides modification Brain targeting drug-loading nanoparticles brain enrichment degree strengthen.
Advantages of the present invention and it is characterized in：
The present invention, which selects, has low toxicity, hydrophily and the preferable PEG-PLGA diblock copolymers of biological degradability as nanometer
Pharmaceutical carrier.Wherein, when PEG-PLGA copolymers are degraded in human body, final catabolite is carbon dioxide (CO2) and water
(H2), O will not assemble in vivo.Therefore, good biocompatibility, non-immunogenicity are safe.And PEG hydrophilic radical
Opsonic action of the blood to nanoparticle can be reduced, so as to extend the blood halflife of medicine；And secondary solvent need not be used,
Normal tissue organ can be avoided to produce side effect, increase patient adaptability.
Use of the present invention can specifically bind the novel polypeptide T7 peptides and PEG- of blood-brain barrier surface TfR
PLGA diblock copolymers are combined, and prepare Brain targeting nanoscale medicine delivery system, and that improves medicine BBB penetrates efficiency, is greatly improved
Nano-drug transporter crosses over the efficiency of blood-brain barrier, makes drug-rich in lesion tissue, improves the bioavilability of medicine,
The toxic side effect of medicine and effective Drug controlled release are reduced, in treatment and diagnosing ischemia cerebral apoplexy and brain tumor disease side
Face has fabulous application prospect.
Brief description of the drawings
Fig. 1 is the synthetic schemes that T7 peptides modify PEG-PLGA Brain targeting nanoscale medicine delivery systems.
Fig. 2 is the hydrogen nuclear magnetic resonance spectrogram for the PEG-PLGA copolymers that embodiment 1 synthesizes.
Fig. 3 is nano-carrier T7-PEG-PLGA prepared by embodiment 3 hydrogen nuclear magnetic resonance spectrogram.
Fig. 4 is the active Brain targeting nanoscale medicine delivery system grain size distribution of T7 peptides modification.
Fig. 5 is the active Brain targeting nanoscale medicine delivery system potential image of T7 peptides modification.
Fig. 6 is the scanning electron microscope (SEM) photograph of the active Brain targeting nanoscale medicine delivery system of T7 peptides modification.
Fig. 7 is the active Brain targeting nanoscale medicine delivery system drug release patterns in vitro figure of T7 peptides modification.
Fig. 8 is that the active Brain targeting nanoscale medicine delivery system fluorescent vital of T7 peptides modification is imaged distribution map, and wherein a lives for mouse
Body fluorescence distribution figure, b are intracorporeal organ fluorescence distribution figure.
Embodiment
The invention will be further described with reference to embodiments, but the invention is not limited in these embodiments.
The carrier PEG-PLGA of embodiment 1 preparation
Weigh 250 mg PLGA-COOH to be dissolved in 2 ml dichloromethane, then add 10.78 mg DMAP (PLGA:DMAP
1:6, mol ratio), 10.15 mg NHS（PLGA:NHS 1：6, mol ratio）With 18.2 mg DCC（PLGA:DCC 1：6, rub
That ratio）, in nitrogen N224 h are stirred at room temperature under protection.After reaction terminates, dicyclohexylurea (DCU) is filtered to remove（DCU）, filtrate is delayed
Slowly it is added drop-wise in 10 ml ice absolute ethers, the precipitation PLGA-NHS of precipitation is washed 3 times with the mixed liquor of ice ether and methanol, is removed
Unreacted NHS and DCC.It is again molten with 2 ml dichloromethane to obtain polymer 4 hours under temperature 60 C vacuum drying for solvent
Solution, then adding 33.2 mg MAL-PEG-NH2（1：1.3 PLGA:PEG mol ratios）With 3 drop N, N- diisopropyl ethyl amines
（DPIEA）, in nitrogen N2Stirring reaction 12h under the conditions of the insured, solution is slowly dropped in the absolute ether of 10 ml ice, analysed
The white flock precipitate gone out is washed with cold methanol, removes unreacted MAL-PEG-NH2, last product MAL-PEG-PLGA
With ether Precipitation, then it is dried in vacuo 4 hours and carries out proton nmr spectra sign, as a result as shown in Figure 2.
Embodiment 2 contains the preparation of tanshinone IIA PEG-PLGA nanoparticles
Weigh 30 mgPEG-PLGA and 2mg tanshinone IIAs and be dissolved in 2.0 mL dichloromethane and acetone（Acetone：Dichloromethane
= 1:9 v/v）Mixing organic phase in, by the way of being added dropwise under liquid, be added to 20ml mass fractions be 0.5%PVA it is water-soluble
In liquid, 250W ultrasounds 10s intervals 10s under ice-water bath, ultrasonic 15min, 600 r/min stirring 5h evaporating solutions in contain it is organic
Solvent, then 14000 r/min high speed centrifugations 30min
Cleaned repeatedly with ultra-pure water three times, the emulsifying agent in nanoparticle suspension solution is removed, after 0.22 μm of filtering with microporous membrane
Freezed, be made and contain tanshinone IIA PEG-PLGA nanoparticles, the PEG-PLGA nanoparticles (PEG-PLGA-NPs of blank）
Prepare such as the above method.
The T7 peptides of embodiment 3 modify the preparation of PEG-PLGA Brain targeting nano-carriers
By the PEG-PLGA nanoparticles PBS of 20mg blank（PH=7.0）Solution redissolves, and adds 1mgT7 peptides, T7 peptides and PEG-
PLGA mol ratio is 1:1, lucifuge stirring reaction 24h, unreacted T7 peptides pass through bag filter（MWCO：5KDa）In PBS
Removed in (pH 7.0) buffer solution, the solution after dialysis is freezed, you can T7-PEG-PLGA Brain targeting nano-carriers are made, enter
Row proton nmr spectra characterizes, as a result as shown in Figure 3.
The preparation of the active Brain targeting nanoscale medicine delivery system of the T7 peptides of embodiment 4 modification
25mg is carried into tanshinone IIA PEG-PLGA nanoparticles PBS（PH=7.0）Solution redissolve, add 1mgT7 peptides, T7 peptides with
PEG-PLGA mol ratio is 1:1, lucifuge stirring reaction 24h, unreacted T7 peptides pass through bag filter（MWCO：5KDa）
Removed in PBS (pH 7.0) buffer solution, the solution after dialysis is freezed, you can the active Brain targeting nanometer of T7 peptides modification is made
Delivery system.
The active Brain targeting nanoscale medicine delivery system nanometer particle size of the T7 peptides of embodiment 5 modification and the measure of current potential.
The PEG-PLGA tanshinone IIA nanoparticles (T7-PEG-PLGA-TSIIA that T7 peptides prepared by embodiment 4 are modified
NPs）Freeze-dried powder is dissolved in distilled water, is prepared into 1 mg/mL solution, after 0.22 u m filtering with microporous membrane, is placed in
Particle diameter distribution is detected in Zeta-sizer current potential Particle Size Analyzers and Potential distribution, each sample test in triplicate, are averaged
Value, the particle diameter for measuring T7-PEG-PLGA-TSIIA NPs is 158.6 ± 0.263nm, and zeta current potentials are -35.6 ± 0.82mV,
As a result as shown in Figure 4 and Figure 5.
The sign of the active Brain targeting nanoscale medicine delivery system ESEM of the T7 peptides of embodiment 6 modification.
T7-PEG-PLGA-TSIIA NPs prepared by embodiment 4, it is fixed on sheet metal with conducting resinl, is plated under vacuum
Gold, the exterior appearance and dispersiveness of nanoparticle are observed using scanning electron microscope sem, as a result as shown in figure 5, as can be seen from the figure making
Standby nanoparticle, has more regular spherical, and size is homogeneous, favorable dispersibility.
The active Brain targeting nanoscale medicine delivery system drugloading rate of the T7 peptides of embodiment 7 modification and the measure of envelop rate.
Using the content of high performance liquid chromatography measure tanshinone IIA:Chromatographic column：Waters C18 chromatographic columns（250 mm
× 4.6 mm, 5 μm）, mobile phase：Methanol：Water=75: 25（v/v）Column temperature：25 DEG C of flow velocitys：1.0 ml/min ultraviolet detection ripples
It is long：268~270 nm, sample size：20 μL.
The tanshinone IIA standard solution that concentration is 1,5,10.0,25.0,50,80,100 μ g/ml is taken respectively, according to color
Spectral condition is tested, and tanshinone IIA concentration is fitted with peak area, establishes regression equation.Precision weighs 2 freezed
Mg T7-PEG-PLGA-TSIIA-NPs, it is placed in 10 ml volumetric flasks, with shaking up constant volume after methanol dilution；0.22 μm of micropore filter
After membrane filtration, the 20 μ L every time of sample introduction 3 times；Detected with HPLC, using peak area A as ordinate, tanshinone IIA concentration C is horizontal seat
Mark, carries out linear regression, and equation of linear regression is：A = 1.5178C + 4.8852 (R2=0.9998).According to standard curve
Regression equation, calculate the content of tanshinone IIA.According to below equation, the PEG-PLGA tanshinone IIAs that can be modified in the hope of T7 peptides
Drugloading rate and envelop rate in nanoparticle (tanshinone IIA high-efficient liquid phase chromatogram is as shown in Figure 6)；Envelop rate %=（In nano-carrier
Total dose of contained dose/input）× 100%, drugloading rate %=（Total dose of contained dose/input in nano-carrier）×
100%, the drugloading rate for finally measuring T7-PEG-PLGA-TSIIA NPs is 14.3% ± 1.02, and envelop rate is 82.7% ± 1.34.
The measure of the active Brain targeting nanoscale medicine delivery system tablets in vitro of the T7 peptides of embodiment 8 modification.
Each 6mg of T7-PEG-PLGA-TSIIA NPs after precision weighing tanshinone IIA and freeze-drying, are respectively placed in 5
In the PBS of the mol/L of mL 0.01 pH=7.4,50 mL centrifuge tube is placed into, is immediately placed on 37 ± 1 DEG C of waters bath with thermostatic control
In shaking table, 100 r/min are at the uniform velocity stirred, and keep sink conditions.Respectively at 0,15 min, 30 min, 1 h, 2 h, 4 h, 8
H, 12 h, 24 h, 48 h, the mL of point in time sampling 1 as defined in 72 h, while supplement the fresh medium of equivalent isothermal.Sample is through 15
After 000 r/min centrifuges 25 min, supernatant is taken out with after 0.22 μm of filtering with microporous membrane, the methanol dilution for the amount of doubling is laggard
The μ L of sample 20, peak area is recorded, substitute into standard curve, calculate the concentration liquid of tanshinone IIA in solution, the medicine in the computational rules time
Thing adds up release rate.Each 3 parts of sample operation repetitive, it is bent that vitro cumulative release is drawn to the time with the average value of preparation
Line（As shown in Figure 7）.Drug molecule is generally contained by polymer carrier, it discharges the speed of medicine mainly by macromolecule material
The speed of material degraded influences, it can be seen that the extension of drug-carrying nanometer particle over time can progressively release medicine from drug release profiles
Release, tanshinone IIA（TSIIA）In 72h cumulative release amounts it is respectively 90. 46% Hes with T7-PEG-PLGA-TSIIA NPs
70. 31%。
The evaluation of the active Brain targeting nanoscale medicine delivery system fluorescent vital imaging distribution of the T7 peptides of embodiment 9 modification.
Blank PLGA NPs, PEG-PLGA NPs and T7-PEG-PLGA NPs are prepared using the method for the present invention, then
Prepare DiR DMSO solutions（The mg/ml of concentration 1）.The μ l DiR of accurate measuring 100 DMSO solution, is placed in 3 brown bottles
In, it is then respectively adding PLGA, PLGA-PEG and T7-PEG-PLGA PBS（PH=7.0）In solution, lucifuge is stirred overnight, warp
PD-10 post purified nanotubes grains, the DiR of unentrapped is removed, be made and contain PLGA NPs of DiR fluorescence probes, contain DiR
PEG-PLGA NPs, contain DiR T7-PEG-PLGA NPs.
Male ICR mouse 9 is chosen, the g of body weight 20 ± 2, is randomly divided into three groups, i.e. PLGA NPs groups, PEG-PLGA
The PLGA NPs PEG-PLGA of NPs groups and T7-PEG-PLG NPs groups, respectively tail vein injection load DiR fluorescence probes
NPs and T7-PEG-PLG NPs, the mg/Kg of dosage 100, upon administration 30 min, 1 h, 3 h three time points, point
One is randomly selected in other every group of clump, 0.2 ml 10% of intraperitoneal injection chloraldurate solution is anaesthetized, and then puts live body into
In imager, distribution situation (as shown in Figure 8) of the observation load fluorescence probe nano-carrier in Mice Body.
Fig. 8 is ICR mouse living body fluorescent images, and wherein A and a are respectively the small of injection load DiR PLGA NPs groups
Mouse live body and intracorporeal organ fluorescence distribution figure, B and b be respectively inject load DiR PEG-PLGA NPs groups mouse live body with
Intracorporeal organ fluorescence distribution figure, C and c are respectively T7-PEG-PLGA NPs mouse and the intracorporeal organ fluorescence point for injecting load DiR
Butut, it can be seen that the fluorescence intensity that T7-PEG-PLGA NPs are entered in ICR Mice brain tissues is significantly higher than PLGA
NPs and PEG-PLGA NPs, should test result indicates that, by T7 peptides modify active Brain targeting nanoscale medicine delivery system, can significantly
The ability that nanoparticle passes through blood-brain barrier is improved, there is excellent active brain targeting.
Claims (8)
1. a kind of preparation method of the active Brain targeting nanoscale medicine delivery system of T7 peptides modification, it is characterised in that using T7 peptide conducts
Efficient Brain targeting part, the nano-medicament carrier material based on PLGA high-molecular copolymers, using emulsion-solvent evaporation method
Insoluble drug molecule is contained, then T7 peptides are covalently attached by difunctional NHS-PEG-MAL, structure active Brain targeting nanometer passs medicine
System.
2. the active Brain targeting nanoscale medicine delivery system of a kind of T7 peptides modification according to claim 1, it is characterised in that described
High-molecular copolymer PLGA end groups be respectively carboxyl（-COOH）And hydroxyl（-OH）, its molecular weight be 10000~
30000；Wherein, the ratio of glycolide and lactide is 1/100~99/100 during synthesizing PLGA.
3. the active Brain targeting nanoscale medicine delivery system of a kind of T7 peptides modification according to claim 1, it is characterised in that described
Polyethylene glycol（PEG）Molecular weight 1000~5000, end group are respectively amino（-NH2) and maleimide（MAL）.
4. the active Brain targeting nanoscale medicine delivery system of a kind of T7 peptides modification according to claim 1, it is characterised in that described
Peptide sequence be CHAIYPRH T7 peptides, the scope of described nanometer particle size is 100~400 nm；Described slightly solubility medicine
Thing is tanshinone IIA；Drugloading rate is 1%~40% in described active Brain targeting nanoscale medicine delivery system, envelop rate 50%~95%.
A kind of 5. preparation method of T7 peptides modification Brain targeting nanoscale medicine delivery system according to claim 1, it is characterised in that
Particular by following steps:PEG-PLGA diblock copolymers are prepared using DCC condensation methods；Weigh a certain amount of PEG-
PLGA polymer and medicine tanshinone IIA are dissolved in mixed organic solvents, by the way of being added dropwise under liquid, are added to finite concentration
Emulsifier solution in, it is ultrasonic under ice-water bath, stir the organic solvent contained in evaporating solution, high speed centrifugation with ultra-pure water repeatedly
Cleaning three times, removes the emulsifying agent in suspension solution, with being freezed after filtering with microporous membrane, the PEG-PLGA nanoparticles of blank
Prepare such as the above method；Weigh the PEG-PLGA nanoparticles PBS for carrying tanshinone IIA（PH=7.0）Solution redissolves, and adds T7
The h of peptide lucifuge stirring reaction 24, unreacted T7 peptides pass through bag filter（MWCO：5KDa）Removed in PBS (pH 7.0) buffer solution
Go, the solution after dialysis is freezed, you can obtain the active Brain targeting nanoscale medicine delivery system of T7 peptides modification.
6. the preparation method of the active Brain targeting nanoscale medicine delivery system of T7 peptides modification according to claim 5, its feature exist
In described high-molecular copolymer PLGA and polyethylene glycol（PEG）Mol ratio be 1:1~1:40, T7 peptides and PEG-PLGA
The mol ratio of diblock copolymer is 1:1~1:The mol ratio of 5, PEG-PLGA nano-carriers and insoluble drug molecule is 1:
1~1: 20.
7. the preparation method of the active Brain targeting nanoscale medicine delivery system of T7 peptides modification according to claim 5, its feature exist
In described mixed organic solvents are any two in dichloromethane, chloroform, acetone, ethyl acetate, methanol, absolute ethyl alcohol
Kind mixing；Emulsifier type used is Tween-80, polyvinyl alcohol（PVA）, PLURONICS F87, soybean lecithin or lecithin its
In one or more of mixtures, the mass fraction of emulsifying agent is 0.1%~10%.
A kind of 8. active Brain targeting nanoscale medicine delivery system of T7 peptides modification according to claim 5, it is characterised in that T7 peptides
The mode being connected with PEG-PLGA copolymers is the thin base by T7 peptides and the maleimide base group reaction forming of PEG chains one end
It is made.
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