CN104491869B - A kind of Brain targeting drug-loading nanoparticles - Google Patents
A kind of Brain targeting drug-loading nanoparticles Download PDFInfo
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- CN104491869B CN104491869B CN201410748001.4A CN201410748001A CN104491869B CN 104491869 B CN104491869 B CN 104491869B CN 201410748001 A CN201410748001 A CN 201410748001A CN 104491869 B CN104491869 B CN 104491869B
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Abstract
The invention discloses a kind of Brain targeting drug-loading nanoparticles, based on its parts by weight by each component, comprising 10 20 parts of carrier, 0.5 1 parts of medicine, surface polypeptide is respectively 15.94 24.05 parts.The carrier of wherein described drug-loading nanoparticles is PLGA and PEG block copolymer, and the medicine is fluorescence tracer, contrast agent and/or the medicine for acting on brain, and the polypeptide is THRPPMWSPVWPC and/or TFFYGGSRGKRNNFKTEEYC.Preparation method of the present invention is simple; reaction condition is gentle; greatly protect the activity of medicine; medicine is passed using nanometer technology; improve the ability that medicine enters brain across blood-brain barrier; and in nanoparticle surface grafting and modifying specific polypeptide molecule, enter brain using active targeting effect mediation nano particle, it is low to solve the problems, such as that traditional passive target enters brain efficiency.
Description
Technical field
The invention belongs to biomedical sector, is related to Brain targeting drug-loading nanoparticles, more particularly to one kind with PLGA and PEG
Block copolymer there are the Brain targeting drug-loading nanoparticles of covalent modification polypeptide for carrier and surface.
Background technology
Nerve degenerative diseases (such as senile dementia disease, Parkinson's, amyotrophic lateral sclerosis disease) are to seriously endanger human health
A kind of major disease.But in the drug therapy of nerve degenerative diseases, the presence of blood-brain barrier hinders the overwhelming majority and controlled
Treat medicine and play the effect of its drug therapy into intracerebral.Design and structure being capable of specific recognitions and across the dirty load medicine of blood brain screen
System, that improves medicine enters brain efficiency, and the treatment to nerve degenerative diseases plays vital effect.
In various drug systems, medicament-carried nano system is due to the small-size effect, skin effect and surface of nano material
The effects such as chemical modification, possibility is provided through blood-brain barrier for medicine.PLGA is food and medicine Surveillance Authority of the U.S. (FDA) batch
Accurate degradable high polymer material, it is widely used as the carrier of Nano medication.In order to increase circulation time inside nano material,
Different surfactant modified nano particles, such as Pegylation nano particle or the modification of polysorbate80 coating can be used
Nano particle, can increase nano grain surface hydrophily, extend circulation time of the nano particle in vivo in blood, make more
Nano particle reach brain.
However, how to improve the probability that drug-loading nanoparticles enter brain, that is, Brain targeting efficiency is improved, at present still in real
Test room conceptual phase.The brain targeting drug delivery mode that presently, there are has active targeting and passive target administration., will in active targeting
The connection of drug-loading nanoparticles surface can be with target cell (brain microvessel endothelial cells in vitro for forming blood-brain barrier) specific binding
Part, such as protein, antibody, peptide chain, nano particle is set to deliver Nano medication by receptor-mediated transcytosis
Enter the technology that brain is presently the most ripe.Summarize Targeting nanoparticles across the blood-brain
Barrier with monoclonal antibodies (Loureiro, Joana A et al.) describe be loaded with treatment nerve move back
Monoclonal antibody is modified outside the PLGA nano particles of row disease medicament, monoclonal antibody can be with the acceptor knot on blood-brain barrier surface
Close, so as to reach the effect of the targeted delivery of nerve degenerative diseases medicine.
But for protein and antibody modification, peptide molecule is because its molecular weight is small, cost is low, stability
Well, the advantages such as efficiency height are modified and causes extensive concern.Dual-functional nanoparticles targeting
Amyloid plaques in the brains of Alzheimer's disease mice (BIOMATERIALS, Zhang
Et al.) describe the peptide modified PLA nano particles of Brain targeting, the peptide sequence of use be respectively TGNYKALHPHNG and
QSHYRHISPAQV.Wherein TGN polypeptides are used for targetting blood-brain barrier, and QSH is used for combining intracerebral A β1-42Proteinosis.The brain
Target polypeptide TGN is screened using display technique of bacteriophage, but it combines the binding site and molecule machine of blood-brain barrier
Reason is still not clear.
It has been reported that peptide T HRPPMWSPVWP can combine the TfR on brain capillary endothelial cell
(TfR), nanogold particle can cross over blood-brain barrier after modifying the peptide molecule；And peptide chain angiopep-2
(TFFYGGSRGKRNNFKTEEY) part as the LDL receptor (LRP) on brain capillary endothelial cell, tool
There is the ability across blood-brain barrier more superior than transferrins.
The content of the invention
The present invention is first by peptide THRPPMWSPVWPC (TC13) and/or TFFYGGSRGKRNNFKTEEYC (TC20) and base
It is combined in PLGA carrier, and it is an object of the present invention to provide a kind of circulation time in vivo long, specific active Brain targeting efficiency high
PLGA medicament-carried nano systems, that improves Nano medication enters brain efficiency.
To use following technical scheme up to this purpose, the present invention：
A kind of Brain targeting drug-loading nanoparticles, based on its parts by weight by each component, include carrier 10-20 parts, medicine 0.5-1
Part, surface polypeptide is respectively 15.94-24.05 parts, wherein the block that the carrier of the drug-loading nanoparticles is PLGA and PEG is total to
Polymers, the medicine are fluorescence tracer, contrast agent and/or the medicine for acting on brain, and the polypeptide is THRPPMWSPVWPC
And/or TFFYGGSRGKRNNFKTEEYC.
Wherein, the molecular weight of the PLGA is 30000-60000, and its lactic acid is 1 with glycolic acid ratio:1；The PEG
Molecular weight be 3400.
The material PLGA that Brain targeting drug-loading nanoparticles of the present invention are ratified using U.S. FDA, it is end modified at its
PEG fragments form PLGA and PEG block copolymer PLGA-b-PEG as carrier material.Due to PLGA nano particles in vivo
Circulation time it is limited, PLGA is modified using Surfactant PEG, nano grain surface hydrophily can be increased, prolonged
Long nano particle circulation time in blood in vivo, more nano particles are made to reach brains.Load is used as using PLGA-b-PEG
The carrier of medicine nano particle, and contain fluorescence tracer, contrast agent, and/or medicine in the drug-loading nanoparticles and be used for body
The tracer or treatment of interior experiment.Surface polypeptide grafting and modifying then is carried out to nano grain surface, to strengthen drug-loading nanoparticles
To the special Brain targeting effect of blood-brain barrier, increase medicine enters the efficiency of brain.
On the other hand, the present invention also provides the preparation method of the Brain targeting drug-loading nanoparticles, and it includes following step
Suddenly：
1) synthetic vectors PLGA-b-PEG；
2) carrier is dissolved in organic solvent, and dissolves fluorescence tracer, contrast agent and/or the medicine for acting on brain
Thing, nano-precipitation legal system obtain drug-loading nanoparticles；
3) drug-loading nanoparticles are dissolved in water, carry out surface active；
4) drug-loading nanoparticles of the activation are dissolved in water, add peptide T HRPPMWSPVWPC and/or
TFFYGGSRGKRNNFKTEEYC carries out surface scion grafting, and Brain targeting drug-loading nanoparticles are made.
Wherein, the step 1) can use the published method synthesis PLGA and PEG in any this area block copolymer,
The mass ratio of the PLGA and PEG are 10-20:1.Preferably, the PEG is that NH is contained in end2With COOH NH2-PEG-
COOH, the PLGA molecular weight are 30000-60000, and its lactic acid is 1 with glycolic acid ratio:1, the PLGA molecules have end
End carboxyl.Utilize PLGA terminal carboxyl group and NH2- PEG-COOH amino, which reacts, prepares PLGA and PEG block copolymerization
Thing.In PLGA and NH2Before-PEG-COOH reactions, 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides are used
EDC and n-hydroxysuccinimide NHS activation PLGA terminal carboxyl group.Described EDC, NHS and PLGA mass ratio are 41：23：
2000.In the organic solvent, the concentration of the carrier is 10-20mg/ml, fluorescence tracer, contrast agent and/or the work
Concentration for the medicine of brain is 0.5-1mg/ml.
The mechanism of activation is that NHS is combined with PLGA terminal carboxyl group, forms more active PLGA-NHS and NH2-PEG-
COOH reacts, and prepares carrier PLGA-b-PEG.The type and materials of solvent and other materials used in the reaction are that this area is public
Know technology, those skilled in the art can be selected according to actual conditions.
A kind of available carrier synthesis step is as follows：PLGA ultrasonic dissolutions are taken in CH2Cl2In, sequentially add EDC under stirring
And NHS, 30min is stirred at room temperature.Add ether to be allowed to precipitate, then washed 3 times with the ether and methyl alcohol mixed liquor of ice, washed away
Unnecessary EDC and NHS.Solvent is spin-dried for again, obtains white-yellowish solid PLGA-NHS.PLGA-NHS ultrasonic dissolutions are taken in CHCl3。
Stirring is lower to add NH2- PEG-COOH and catalyst n, N- diisopropylethylamine.Reaction 48 hours is stirred at room temperature.React
Cheng Hou, ice methanol extraction is added, and washed three times with ice methanol, wash away unreacted NH2-PEG-COOH.Finally solvent is revolved
It is dry, obtain final product PLGA-b-PEG copolymers.Product is white-yellowish solid, in -20 DEG C of storages.
Obtained load medicine particle diameter distribution and surface charge are measured with dynamic light scattering (DLS), and uses transmission electron microscope
(TEM) size and surface topography of nano particle are observed, it is seen that the particle diameter distribution of carrier prepared by the present invention is mainly left in 100nm
The right side, in symmetrical ball-type, meet the requirement of nanometer formulation.
In the step 2), the organic solvent is that volume ratio is 65：35 dimethylformamide (DMF) and tetrafluoro second
Alkene (TFE) mixed solvent, the concentration of the carrier are 20mg/ml, the fluorescence tracer, contrast agent and/or act on brain
The concentration of medicine be 0.5mg/ml, it is described to be dissolved as ultrasonic dissolution.The nano-precipitation method concretely comprises the following steps：High-speed stirred
Secondary water forms whirlpool, dropwise by dissolve carrier and fluorescence tracer, contrast agent and/or act on brain medicine DMF/
TFE solution instills the whirlpool, after being added dropwise, forms the micro- blue nano particle suspension of milkiness shape, keeps high-speed stirred one
The section time with the organic solvent in evaporating solution, filtering, centrifuges to remove the floccule formed when synthesizing nano particle, taken
Clear liquid ultrafiltration centrifuges, and concentrates and drug-loading nanoparticles are made.Preferably, the secondary water and the volume ratio of the DMF/TFE solution
For 3000：2；The rotating speed of the high-speed stirred is 700-800rpm, mixing time 5-6h；The centrifugation centrifuges for 5000rpm
30min；The ultrafiltration centrifugation is to centrifuge 15min using Millipore ultra-filtration centrifuge tubes 5000rpm, is repeated 2-3 times；It is described dense
It is condensed to ultrafiltration centrifugation resulting solution being concentrated into 5ml.In said process, fluorescence molecule or drug molecule are wrapped in carrier granular
In, form drug-loading nanoparticles.
In the step 3), the concentration of the drug-loading nanoparticles is 2mg/ml, using EDC and NHS to the load medicine
Nano particle carries out surface active, and EDC and NHS concentration is respectively 100mM and 50mM in water；Soak time is 30min.
Therefore drug-loading nanoparticles surface carboxyl groups are activated so as to be reacted with the amino in peptide sequence end.After activation,
Ultrafiltration is centrifuged and is washed with water to remove unreacted EDC and/or NHS.Specifically, entered with Millipore ultra-filtration centrifuge tubes
Row centrifugation, 15min is centrifuged in 5000rpm ultrafiltration, takes upper part to centrifuge product, added water, ultrasonic disperse, repeat ultrafiltration centrifugation 2-
3 times.
In step 4), the drug-loading nanoparticles of the activation are dissolved in water, concentration 2mg/ml, then added into solution
Enter the peptide T HRPPMWSPVWPC (molecular weight 1594g/mol) and/or TFFYGGSRGKRNNFKTEEYC (molecular weight
2405g/mol), its concentration is respectively 10-3M, the concentration of two kinds of polypeptides is respectively 1.594mg/ml and 2.405mg/ml.Preferably,
Scion grafting reaction is carried out under normal temperature 600rpm stirrings, reaction time 8-12h, after reaction ultrafiltration centrifuge and be washed with water
Wash, and repeat 2-3 times to remove unreacted peptide molecule, the Brain targeting drug-loading nanoparticles are made.
The surface of the Brain targeting drug-loading nanoparticles is can observe with the presence of element sulphur with XPS.Element sulphur, which exists, then to be said
Bright polypeptide is successfully grafted to nano grain surface, and otherwise then explanation grafting polypeptide is unsuccessful.
The present invention also provides fluorescence tracer, the contrast agent that the Brain targeting drug-loading nanoparticles act on brain in preparation
And/or the application in medicine.The Brain targeting drug-loading nanoparticles comprising fluorescence tracer are injected into mouse, and are put into
It is imaged in the multispectral small animal living body imaging systems of CRI Maestro 2, observation contains the nano particle of fluorescence molecule in mouse
Internal distribution.Then dissect mouse and take out its brain, carry out brain fluorescence imaging, observation nanoparticle is in mouse brain
The aggregation extent in portion, it is stronger to can observe fluorescence signal.Peptide modified drug-loading nanoparticles are strengthened in brain enrichment degree, medicine
The special Brain targeting effect enhancing of thing.
In summary, the present invention is combined using nanometer technology with polypeptide surface modification, improves the Brain targeting effect of medicine
With brain enrichment.Preparation method is simple, reacts and is carried out under the neutral temperate condition of normal temperature, normal pressure, aqueous phase, pH, greatly
Protect the activity of medicine.Medicine is passed using nanometer technology, helps and improve the ability that medicine enters brain across blood-brain barrier, and
In nanoparticle surface grafting and modifying specific polypeptide molecule, entered using active targeting effect mediation nano particle big
Brain, it is low to solve the problems, such as that traditional passive target enters brain efficiency.
Brief description of the drawings
Fig. 1 is the preparation method route map of shown Brain targeting drug-loading nanoparticles.
Fig. 2 is carrier PLGA-b-PEG nuclear magnetic resonance (NMR) spectrogram.
Fig. 3 is the phenogram of the drug-loading nanoparticles, wherein, a is the dynamic of the drug-loading nanoparticles Size Distribution
Light scattering apparatus (DLS) test chart, b, c are transmission electron microscope (TEM) figure of drug-loading nanoparticles in different zones.
Fig. 4 is that the XPS of the drug-loading nanoparticles schemes.
Fig. 5 is the fluorescence intensity figure of the brain after mouse tail vein injection difference nano particle 1h, and wherein a is brain fluorescence
Photo, b are brain fluorescence signal intensity column diagram.
Embodiment
Further illustrate technical scheme below in conjunction with the accompanying drawings and by embodiment.
Embodiment 1：Brain targeting drug-loading nanoparticles and preparation method thereof
The Brain targeting drug-loading nanoparticles of the present invention include carrier and medicine, and its surface has covalent modification polypeptide, its
Described in drug-loading nanoparticles carrier be PLGA and PEG block copolymer, the medicine be fluorescence tracer, contrast agent
And/or the medicine of brain is acted on, the polypeptide is THRPPMWSPVWPC and/or TFFYGGSRGKRNNFKTEEYC.It is described
PLGA molecular weight is 30000-60000, and its lactic acid is 1 with glycolic acid ratio:1；The molecular weight of the PEG is 3400.
Its preparation method is as shown in figure 1, comprise the following steps：
1) synthetic vectors；
The carrier is dissolved in organic solvent by 2, and dissolves fluorescence tracer, contrast agent and/or the medicine for acting on brain,
Nano-precipitation legal system obtains drug-loading nanoparticles：The concentration of the carrier is 10-20mg/ml, the fluorescence tracer, contrast agent
And/or it is 0.5-1mg/ml to act on the concentration of the medicine of brain, the organic solvent is that 1ml volume ratios are 65：35 diformazan
Base formamide (DMF)：Tetrafluoroethene (TFE) mixed solvent；
3) drug-loading nanoparticles are dissolved in water, carry out surface active；
4) drug-loading nanoparticles of the activation are dissolved in water, add peptide T HRPPMWSPVWPC and/or
TFFYGGSRGKRNNFKTEEYC carries out surface scion grafting, and Brain targeting drug-loading nanoparticles are made.
Embodiment 2：Carrier PLGA-b-PEG synthesis
Because the circulation time of PLGA nano particles in vivo is limited, the present invention is using hydrophilic polyethylene glycol PEG points
Son modification PLGA, improves PLGA hydrophilies, so as to improve the circulation time of nano particle in vivo.The molecular weight of the PLGA
30000-60000, its lactic acid：Glycolic acid ratio is 1：1.Used molecular weight polyethylene glycol is 3400, and end is respectively
NH2And COOH group, i.e. NH2-PEG3400-COOH。
Carrier synthesis step：PLGA 2g are taken, ultrasonic dissolution is in 10ml CH2Cl2In, stir lower addition 1- (3- dimethylaminos
Propyl group) -3- ethyl-carbodiimide hydrochlorides (EDC) 41mg, n-hydroxysuccinimide (NHS) 23mg is added, at room temperature
Stir 30min.Add 5ml ether to be allowed to precipitate, then wash 3 times with the ether and methyl alcohol mixed liquor of ice, wash away unnecessary EDC with
NHS.Solvent is spin-dried for again, obtains white-yellowish solid PLGA-NHS.PLGA-NHS 1g are taken, are dissolved in 4ml CHCl3, ultrasonic dissolution.
Stirring is lower to add NH2- PEG-COOH 100mg and N, N- diisopropylethylamine 10mg.Reaction 48 hours is stirred at room temperature.Instead
Should after the completion of, add ice methanol extraction, and with 5ml ice methanol wash 3 this, wash away unreacted NH2-PEG-COOH.Finally will
Solvent is spin-dried for, and obtains final product PLGA-b-PEG.Product is white-yellowish solid, is stored at -20 DEG C, its nuclear magnetic resonance map
As shown in Figure 2, it is seen that the method can prepare the higher carrier of purity.
Embodiment 3：The synthesis of drug-loading nanoparticles
It is 65 in 1ml volume ratios：35 dimethylformamide (DMF)：Tetrafluoroethene (TFE) in the mixed solvent dissolves 20mg
PLGA-b-PEG, normal temperature ultrasound make it fully dissolve.And RhodamineB fluorescence tracer or drug molecule are dissolved wherein.
30ml secondary waters are added in 50ml boiling flask, high-speed stirred, which is formed, to be vortexed.DMF/TFE solution is drawn with 20 μ l liquid-transfering guns,
Drop by drop it is slowly dropped into the aqueous solution of high-speed stirred until terminating.After the completion of titration, form that milkiness shape is micro- blue receives
Rice grain suspension, i.e. PLGA-b-PEG nano particles, labeled as NPs,.High-speed stirred 5-6h is kept, having in evaporating solution
Solvent.Filtering, and 5000rpm centrifuge 30min, separation, take supernatant, remove formed when synthesizing nano particle it is cotton-shaped
Thing.Ultrafiltration centrifugation is carried out using Millipore ultra-filtration centrifuge tubes afterwards, 5000rpm centrifugation 15min, takes supernatant, topped up with water,
Ultrasonic disperse, repeat ultrafiltration and centrifuge 2-3 times, and concentrate nanoparticles solution to 5ml.In nano particle forming process, fluorescence
Molecule or drug molecule are wrapped in the carrier, form drug-loading nanoparticles.With dynamic light scattering (DLS) and measurement nano particle
Particle diameter distribution and surface charge, and with transmission electron microscope (TEM) observe nano particle size and surface topography, such as scheme
Described in 3.
Embodiment 4：The surface active of the drug-loading nanoparticles
Above-mentioned drug-loading nanoparticles are resuspended in the 10ml aqueous solution, add 100mM EDC and 50mM under agitation
NHS activates the carboxylic group of nano grain surface.30min is stirred at room temperature, drug-loading nanoparticles surface carboxyl groups are activated, so as to afterwards
Reacted with the amino in peptide sequence end.After the completion of reaction, centrifuged with Millipore ultra-filtration centrifuge tubes,
5000rpm ultrafiltration centrifuges 15min, takes upper part to centrifuge product, adds water, ultrasonic disperse, repeats ultrafiltration and centrifuge 2-3 times, remove
Unreacted EDC and NHS.
Embodiment 5：The polypeptide grafted modification of the drug-loading nanoparticles
The nano particle 20mg of activation is resuspended in the 10ml aqueous solution, end is added and contains NH2Peptide sequence
THRPPMWSPVWPC (being labeled as TC13, molecular weight 1594g/mol) and/or TFFYGGSRGKRNNFKTEEYC (are labeled as
TC20, molecular weight 2405g/mol), concentration is 10 after the polypeptide adds-3M, for above two polypeptide, its concentration is respectively
1.594mg/ml and 2.405mg/ml, normal-temperature reaction overnight, there are the PLGA-PEG-TC13 of polypeptide (marks so as to obtain surface modification
It is designated as NPs-TC13) or PLGA-PEG-TC20 (being labeled as NPs-TC20) nano particle.Removed with above-mentioned ultrafiltration centrifugal process not anti-
The polypeptide answered, and clean nano particle 2-3 times.
The drug-loading nanoparticles NPs surfaces of nano particle NPs-TC13, NPs-TC20 and non-scion grafting polypeptide are determined with XPS
Chemical composition, see if there is element sulphur presence.Element sulphur, which exists, then illustrates that polypeptide is successfully grafted to nano grain surface, otherwise then
Illustrate that grafting polypeptide is unsuccessful.As shown in Figure 4, it is seen that NPs-TC13, NPs-TC20 surface prepared by the present invention all has sulphur member
Element, polypeptide scion grafting success, and the drug-loading nanoparticles surface of non-scion grafting polypeptide is free of element sulphur.
Embodiment 6：Evaluation on distribution of the drug-loading nanoparticles in Mice Body
The load of the drug-loading nanoparticles for the containing Rhodamine B fluorescence tracers detection present invention prepared using the present invention
The Brain targeting effect of medicine nano particle.Evaluated from four kinds of compounds：Contain Rhodamine B fluorescence tracers and scion grafting
TC13 drug-loading nanoparticles (NPs-TC13-RhoB)；The load medicine for containing Rhodamine B fluorescence tracers and scion grafting TC20 is received
Rice grain (NPs-TC20-RhoB)；Contain the drug-loading nanoparticles (NPs- of Rhodamine B fluorescence tracer not scion grafting polypeptides
RhoB)；Physiological saline (saline).
Above-mentioned four groups of compounds are configured to 12mg/ml solution.It is 20g Balb/c nude mouse to take body weight, is divided into
Four groups, 100 μ l compounds are administered in every group of mouse difference tail vein injection, i.e., are injected by 60mg/kg mouse weights.Injection 15min,
After 30min, 1h, with 3% isoflurane gas anesthetized mice, and be put into 2 multispectral small animal living bodies of CRI Maestro into
As being imaged in system, observation contains distribution of the nano particle of fluorescence molecule in Mice Body.Then dissect mouse and take out it
Brain, brain fluorescence imaging is carried out, observe aggregation extent of the nanoparticle in mouse brain, the fluorescence after 1h is administered in each group
Intensity map is as shown in Figure 5.It can be seen that the brain fluorescence intensity of NPs-TC13-RhoB and NPs-TC20-RhoB groups is significantly stronger than NPs-
RhoB groups and saline groups, specifically, in four groups of experiments corresponding mouse brain average fluorescent strength be respectively (8.47 ±
1.05)x 106, (8.66 ± 0.94) x 106,(2.27±0.68)x 106(1.39 ± 0.51) x 106Photon/square li
Meter per second.
Applicant states that the present invention illustrates the method detailed of the present invention, but not office of the invention by above-described embodiment
It is limited to above-mentioned method detailed, that is, does not mean that the present invention has to rely on above-mentioned method detailed and could implemented.Art
Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present invention
Addition, selection of concrete mode etc., within the scope of all falling within protection scope of the present invention and disclosing.
Claims (24)
1. a kind of Brain targeting drug-loading nanoparticles, it is characterised in that the Brain targeting drug-loading nanoparticles press the weight of each component
Part meter, includes carrier 10-20 parts, medicine 0.5-1 parts, surface polypeptide 15.94-24.05 parts；Wherein described drug-loading nanoparticles
Carrier is PLGA and PEG block copolymer, and the medicine is fluorescence tracer, contrast agent and/or the medicine for acting on brain,
The polypeptide is THRPPMWSPVWPC and/or TFFYGGSRGKRNNFKTEEYC；
The Brain targeting drug-loading nanoparticles are prepared with the following method, and the preparation method comprises the following steps：
1) synthetic vectors；
2) carrier is dissolved in organic solvent, and dissolves fluorescence tracer, contrast agent and/or the medicine for acting on brain, received
Drug-loading nanoparticles are made in rice sedimentation；
3) drug-loading nanoparticles are dissolved in water, carry out surface active；
4) drug-loading nanoparticles of the activation are dissolved in water, add peptide T HRPPMWSPVWPC and/or
TFFYGGSRGKRNNFKTEEYC carries out surface scion grafting, and Brain targeting drug-loading nanoparticles are made；
Wherein, the molecular weight of the PLGA is 30000-60000, and its lactic acid is 1 with glycolic acid ratio:1；Point of the PEG
Son amount is 3400；The mass ratio of the PLGA and PEG are 10-20:1.
2. the preparation method of Brain targeting drug-loading nanoparticles according to claim 1, it is characterised in that the preparation method
Comprise the following steps：
1) synthetic vectors；
2) carrier is dissolved in organic solvent, and dissolves fluorescence tracer, contrast agent and/or the medicine for acting on brain, received
Drug-loading nanoparticles are made in rice sedimentation；
3) drug-loading nanoparticles are dissolved in water, carry out surface active；
4) drug-loading nanoparticles of the activation are dissolved in water, add peptide T HRPPMWSPVWPC and/or
TFFYGGSRGKRNNFKTEEYC carries out surface scion grafting, and Brain targeting drug-loading nanoparticles are made.
3. preparation method according to claim 1, it is characterised in that the step 1) PEG is that NH is contained in end2And COOH
The PEG of group, NH2-PEG-COOH。
4. preparation method according to claim 1, it is characterised in that in PLGA and NH2Before-PEG-COOH reactions, activation
PLGA terminal carboxyl group.
5. preparation method according to claim 1, it is characterised in that PLGA terminal carboxyl group is activated using EDC and NHS,
Described EDC, NHS and PLGA mass ratio are 41：23：2000.
6. preparation method according to claim 2, it is characterised in that in the step 2), the organic solvent is body
Product is than being 65：35 DMF and TFE mixed solvents.
7. preparation method according to claim 2, it is characterised in that the concentration of the carrier is 20mg/ml, the fluorescence
Tracer, contrast agent and/or to act on the concentration of medicine of brain be 0.5mg/ml.
8. preparation method according to claim 7, it is characterised in that step 2) is described to be dissolved as ultrasonic dissolution.
9. preparation method according to claim 2, it is characterised in that be the step of step 2) the nano-precipitation method：It is high
The DMF/TFE solution of carrier and fluorescence tracer, contrast agent and/or the medicine for acting on brain will be dissolved under fast stirring dropwise
Secondary water is added, filtering, centrifugation, takes supernatant ultrafiltration to centrifuge, concentrates and the drug-loading nanoparticles are made.
10. preparation method according to claim 9, it is characterised in that the secondary water volume and the DMF/TFE solution
Volume ratio be 3000：2.
11. preparation method according to claim 9, it is characterised in that the rotating speed of the high-speed stirred is 700-800rpm,
Mixing time is 5-6h.
12. preparation method according to claim 9, it is characterised in that the centrifugation centrifuges 30min for 5000rpm.
13. preparation method according to claim 9, it is characterised in that the ultrafiltration centrifugation is using Millipore ultrafiltration
Centrifuge tube 5000rpm centrifuges 15min, repeats 2-3 times.
14. preparation method according to claim 13, it is characterised in that the molecular cut off of the centrifugation is 3500.
15. preparation method according to claim 9, it is characterised in that the concentration is that ultrafiltration centrifugation resulting solution is dense
It is reduced to 5ml.
16. preparation method according to claim 2, it is characterised in that in the step 3), the drug-loading nanoparticles
Concentration be 2mg/ml.
17. preparation method according to claim 2, it is characterised in that using EDC and NHS to the drug-loading nanoparticles
Surface active is carried out, and EDC and NHS concentration is respectively 100mM and 50mM in water.
18. preparation method according to claim 2, it is characterised in that the soak time is 30min.
19. preparation method according to claim 2, it is characterised in that after activation, unreacted EDC and/or NHS are removed,
The method for removing unreacted EDC and/or NHS is centrifuged and is washed with water for ultrafiltration, the retention point of the ultrafiltration centrifugation
Son amount is 3500, and repetition 2-3 times is washed with water.
20. preparation method according to claim 2, it is characterised in that in step 4), the medicament-carried nano of the activation
The concentration of grain is 2mg/ml, and the concentration of the peptide T HRPPMWSPVWPC and/or TFFYGGSRGKRNNFKTEEYC is respectively 10- 3M。
21. preparation method according to claim 2, it is characterised in that the scion grafting reaction is under normal temperature 600rmp stirrings
Carry out.
22. preparation method according to claim 2, it is characterised in that the time of the scion grafting reaction is 8-12h.
23. preparation method according to claim 2, it is characterised in that ultrafiltration is centrifuged and is washed with water after scion grafting reaction,
The molecular cut off of the ultrafiltration centrifugation is 6000-8000, and repetition 2-3 times is washed with water.
24. Brain targeting drug-loading nanoparticles according to claim 1 act on fluorescence tracer, the radiography of brain preparing
Application in agent and/or medicine.
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| CN102151336A (en) * | 2010-02-12 | 2011-08-17 | 复旦大学 | Active targeting drug conveying system for crossing blood brain barrier through mediation of acetylcholine receptor |
| CN103655517A (en) * | 2013-11-19 | 2014-03-26 | 南京医科大学 | Pep-1 peptide modified gliomas targeted nano drug delivery system and preparation method thereof |
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| CN102151336A (en) * | 2010-02-12 | 2011-08-17 | 复旦大学 | Active targeting drug conveying system for crossing blood brain barrier through mediation of acetylcholine receptor |
| CN103655517A (en) * | 2013-11-19 | 2014-03-26 | 南京医科大学 | Pep-1 peptide modified gliomas targeted nano drug delivery system and preparation method thereof |
Non-Patent Citations (2)
| Title |
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| 《Angiopep-conjugated poly(ethylene glycol)-co-poly(e-caprolactone) nanoparticles as dual-targeting drug delivery system for brain glioma》;Hongliang Xin et al.;《Biomaterials》;20110321;第32卷;摘要，2.1 material * |
| 《Delivery of gold nanoparticles to the brain by conjugation with a peptide that recognizes the transferrin receptor》;Roger Prades et al.;《Biomaterials》;20120615;第33卷;摘要 * |
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