CN104491869A - Brain-targeted medicine-carried nano-particles - Google Patents
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Abstract
The invention discloses brain-targeted medicine-carried nano-particles. The brain-targeted medicine-carried nano-particles comprise the following components in parts by weight: 10-20 parts of a carrier, 0.5-1 part of medicine and 15.94-24.05 parts of surface polypeptide, wherein the carrier of the medicine-carried nano-particle is a block copolymer of PLGA and PEG; the medicine comprises a fluorescent tracer, a contrast medium and/or a medicine acting on the brain; the surface polypeptide is THRPPMWSPVWPC and/or TFFYGGSRGKRNNFKTEEYC. A preparation method of the brain-targeted medicine-carried nano-particles is simple and mild in reaction condition; the activity of the medicine is greatly protected; the medicine is delivered by using a nano technology; the capability of the medicine in crossing over the blood-brain barrier and entering the brain is improved; specific molecular peptides are modified by grating on the surfaces of the nano medicine-carried particles; the nano particles are induced by an active targeting effect to enter the brain; the problem that the conventional manner of passively targeting to enter the brain is low in efficiency can be solved.
Description
Technical field
The invention belongs to biomedical sector, relate to Brain targeting drug-loading nanoparticles, particularly relate to a kind of with the block copolymer of PLGA and PEG for carrier and surface has the Brain targeting drug-loading nanoparticles of covalent modification polypeptide.
Background technology
Neurodegenerative diseases (as senile dementia disease, parkinson disease, gradually freezing people's disease etc.) is a class major disease of serious harm human health.But in the Drug therapy of neurodegenerative diseases, the existence of blood brain barrier hinders most medicine and enters its Drug therapy effect of performance in brain.Design and build can specific recognition cross over blood brain and shield dirty medicine-carried system, that improves medicine enters brain efficiency, plays vital effect to the treatment of neurodegenerative diseases.
In various drug system, medicament-carried nano system due to effects such as the small-size effect of nano material, skin effect and surface chemical modifications, for medicine provides possibility through blood brain barrier.PLGA is the degradable high polymer material that U.S. food Drug Administration (FDA) ratifies, and is widely used as the carrier of Nano medication.In order to increase the circulation time in vivo of nano material; available different surfactant modified nano-particle; such as Pegylation nano-particle or polysorbate80 coating modifying nano-particle; nano grain surface hydrophilic can be increased; extend the circulation time of nano-particle in vivo in blood, make more nano-particle arrive brain.
But, how to improve the probability that drug-loading nanoparticles enters brain, namely improve Brain targeting efficiency, be still in the laboratory research stage at present.The brain targeting drug delivery mode of current existence has active targeting and passive target administration.In active targeting, by drug-loading nanoparticles surface connect can with the part of target cell (forming the brain microvessel endothelial cells in vitro of blood brain barrier) specific binding, as protein, antibody, peptide chain etc., making nano-particle send into brain by receptor-mediated transcytosis by Nano medication is technology the most ripe at present.Summary Targeting nanoparticles across the blood-brain barrier with monoclonalantibodies people such as () Loureiro, Joana A describes the PLGA nano-particle being loaded with treatment neurodegenerative diseases medicine and modifies monoclonal antibody outward, monoclonal antibody can with the receptors bind on blood brain barrier surface, thus reach the effect of the targeted delivery of neurodegenerative diseases medicine.
But for protein and antibody modification, peptide molecule causes extensive concern because of advantages such as its molecular weight is little, cost is low, good stability, modification efficiency height.Dual-functional nanoparticlestargeting amyloid plaques in the brains of Alzheimer's disease mice (BIOMATERIALS, the people such as Zhang) describe the peptide modified PLA nano-particle of Brain targeting, the peptide sequence of employing is TGNYKALHPHNG and QSHYRHISPAQV respectively.Wherein TGN polypeptide is used for targeting blood brain barrier, and QSH is used in conjunction with A β in brain
1-42proteinosis.Described Brain targeting peptide T GN utilizes display technique of bacteriophage to screen, but its binding site in conjunction with blood brain barrier and molecule mechanism are still not clear.
Have been reported, peptide T HRPPMWSPVWP in conjunction with the TfR (TfR) on brain capillary endothelial cell, can cross over blood brain barrier after nanogold particle modifies this peptide molecule; And peptide chain angiopep-2 (TFFYGGSRGKRNNFKTEEY) is as the part of the low density lipoprotein receptor (LRP) on brain capillary endothelial cell, there is the ability of the leap blood brain barrier more superior than transferrins.
Summary of the invention
Peptide THRPPMWSPVWPC (TC13) and/or TFFYGGSRGKRNNFKTEEYC (TC20) combines with the carrier based on PLGA by the present invention first, object is to provide the PLGA medicament-carried nano system that a kind of circulation time in vivo is long, specificity active Brain targeting efficiency is high, and that improves Nano medication enters brain efficiency.
For reaching this object, the present invention by the following technical solutions:
A kind of Brain targeting drug-loading nanoparticles, it is by the parts by weight of each component, comprise carrier 10-20 part, medicine 0.5-1 part, surface polypeptide is respectively 15.94-24.05 part, the carrier of wherein said drug-loading nanoparticles is the block copolymer of PLGA and PEG, and described medicine is fluorescence tracer, contrast agent and/or the medicine acting on brain, and described polypeptide is THRPPMWSPVWPC and/or TFFYGGSRGKRNNFKTEEYC.
Wherein, the molecular weight of described PLGA is 30000-60000, and its lactic acid and glycolic acid ratio are 1:1; The molecular weight of described PEG is 3400.
Brain targeting drug-loading nanoparticles of the present invention uses the material PLGA of U.S. FDA approval, forms the block copolymer PLGA-b-PEG of PLGA and PEG as carrier material in its end modified PEG fragment.Because PLGA nano-particle circulation time is in vivo limited, use Surfactant PEG to modify PLGA, nano grain surface hydrophilic can be increased, extend the circulation time of nano-particle in vivo in blood, make more nano-particle arrive brain.Using PLGA-b-PEG as the carrier of drug-loading nanoparticles, and bag carries spike or the treatment that fluorescence tracer, contrast agent and/or medicine are used for experiment in vivo in described drug-loading nanoparticles.Subsequently surface polypeptide grafting and modifying is carried out to nano grain surface, to strengthen the specific brain targeting effect of drug-loading nanoparticles to blood brain barrier, increase the efficiency that medicine enters brain.
On the other hand, the present invention also provides the preparation method of described Brain targeting drug-loading nanoparticles, and it comprises the following steps:
1) synthetic vectors PLGA-b-PEG;
2) described carrier is dissolved in organic solvent, and dissolves fluorescence tracer, contrast agent and/or act on the medicine of brain, nano-precipitation legal system obtains drug-loading nanoparticles;
3) by water-soluble for described drug-loading nanoparticles, surface active is carried out;
4) by water-soluble for the drug-loading nanoparticles of described activation, add peptide T HRPPMWSPVWPC and/or TFFYGGSRGKRNNFKTEEYC and carry out surperficial scion grafting, obtained Brain targeting drug-loading nanoparticles.
Wherein, described step 1) the published method in any this area can be adopted to synthesize the block copolymer of PLGA and PEG, the mass ratio of described PLGA and PEG is 10-20:1.Preferably, described PEG is that end contains NH
2with the NH of COOH
2the molecular weight of-PEG-COOH, described PLGA is 30000-60000, and its lactic acid and glycolic acid ratio are 1:1, and this PLGA molecule has terminal carboxyl group.Utilize terminal carboxyl group and the NH of PLGA
2the amino of-PEG-COOH reacts and prepares the block copolymer of PLGA and PEG.At PLGA and NH
2before-PEG-COOH reaction, 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride EDC and N-hydroxy-succinamide NHS is used to activate the terminal carboxyl group of PLGA.The mass ratio of described EDC, NHS and PLGA is 41:23:2000.In described organic solvent, the concentration of described carrier is 10-20mg/ml, and the concentration of described fluorescence tracer, contrast agent and/or the medicine that acts on brain is 0.5-1mg/ml.
The mechanism of activation is the terminal carboxyl group combination of NHS and PLGA, forms more active PLGA-NHS and NH2-PEG-COOH reaction, prepares carrier PLGA-b-PEG.The type of the solvent that this reaction is used and other materials and materials are techniques well known, and those skilled in the art can select according to practical situation.
A kind of available carrier synthesis step is as follows: get PLGA ultrasonic dissolution in CH
2cl
2in, add EDC and NHS successively under stirring, at room temperature stir 30min.Add ether and make it precipitation, then wash 3 times with the ether of ice and methyl alcohol mixed liquor, wash away unnecessary EDC and NHS.Again solvent is spin-dried for, obtains white-yellowish solid PLGA-NHS.Get PLGA-NHS ultrasonic dissolution in CHCl
3.NH is added under stirring
2-PEG-COOH and catalyst n, N-diisopropylethylamine.At room temperature stirring reaction 48 hours.After having reacted, add ice methanol extraction, and by ice methanol wash three times, wash away unreacted NH
2-PEG-COOH.Finally solvent is spin-dried for, obtains end product PLGA-b-PEG copolymer.Product is white-yellowish solid ,-20 DEG C of storages.
Obtained medicine carrying particle size distribution and surface charge is measured with dynamic light scattering (DLS), and size and the surface topography of nano-particle is observed with transmission electron microscope (TEM), the particle size distribution of carrier prepared by visible the present invention is mainly at about 100nm, ball-type symmetrically, meets the requirement of nanometer formulation.
In described step 2) in, dimethyl formamide (DMF) and tetrafluoroethene (TFE) mixed solvent of described organic solvent to be volume ratio be 65:35, the concentration of described carrier is 20mg/ml, the concentration of described fluorescence tracer, contrast agent and/or the medicine that acts on brain is 0.5mg/ml, described in be dissolved as ultrasonic dissolution.The concrete steps of described nano-precipitation method are: high-speed stirred intermediate water forms whirlpool, dropwise the DMF/TFE solution dissolving carrier and fluorescence tracer, contrast agent and/or the medicine that acts on brain is instilled this whirlpool, after dropwising, form the nano-particle suspension of the micro-blueness of milkiness shape, keep high-speed stirred a period of time with the organic solvent in evaporating solution, filter, the centrifugal floccule formed when synthesis of nano granule with removing, get supernatant ultrafiltration centrifugal, concentrated obtained drug-loading nanoparticles.Preferably, the volume ratio of described intermediate water and described DMF/TFE solution is 3000:2; The rotating speed of described high-speed stirred is 700-800rpm, and mixing time is 5-6h; Described centrifugal be the centrifugal 30min of 5000rpm; Described ultrafiltration is centrifugal for adopting the centrifugal 15min of Millipore ultra-filtration centrifuge tube 5000rpm, repeats 2-3 time; Centrifugal for ultrafiltration gained solution is concentrated into 5ml by described simmer down to.In said process, fluorescence molecule or drug molecule are wrapped in carrier granular, form drug-loading nanoparticles.
In described step 3) in, the concentration of described drug-loading nanoparticles is 2mg/ml, use EDC and NHS to carry out surface active to described drug-loading nanoparticles, and the concentration of EDC and NHS is respectively 100mM and 50mM in water; Soak time is 30min.Therefore drug-loading nanoparticles surface carboxyl groups is activated thus can be reacted with the amino in peptide sequence end.After activation, ultrafiltration is centrifugal and wash with water to remove unreacted EDC and/or NHS.Particularly, carry out centrifugal with Millipore ultra-filtration centrifuge tube, at the centrifugal 15min of 5000rpm ultrafiltration, get the centrifugal product of upper part, add water, ultrasonic disperse, repeat centrifugal 2-3 time of ultrafiltration.
In step 4) in, by water-soluble for the drug-loading nanoparticles of described activation, concentration is 2mg/ml, then in solution, add described peptide T HRPPMWSPVWPC (molecular weight 1594g/mol) and/or TFFYGGSRGKRNNFKTEEYC (molecular weight 2405g/mol), its concentration is respectively 10
-3m, the concentration of two peptide species is respectively 1.594mg/ml and 2.405mg/ml.Preferably, described scion grafting reaction is carried out under room temperature 600rpm stirs, and the response time is 8-12h, and after reaction, ultrafiltration is centrifugal and wash with water, and repeats 2-3 time to remove unreacted peptide molecule, obtains described Brain targeting drug-loading nanoparticles.
The surface that can observe described Brain targeting drug-loading nanoparticles with XPS has element sulphur to exist.There is then explanation polypeptide and be successfully grafted to nano grain surface in element sulphur, otherwise then illustrates that grafting polypeptide is unsuccessful.
The present invention also provides described Brain targeting drug-loading nanoparticles preparing the application in fluorescence tracer, contrast agent and/or the medicine acting on brain.The described Brain targeting drug-loading nanoparticles comprising fluorescence tracer is injected mice, and is inserted imaging in the multispectral small animal living body imaging system of CRI Maestro 2, observe the distribution of nano-particle in Mice Body that bag carries fluorescence molecule.Dissect mice take out its brain subsequently, carry out brain fluorescence imaging, observe the aggregation extent of nanoparticle at mouse brain, it is stronger to can observe fluorescence signal.Peptide modified drug-loading nanoparticles is strengthened at brain enrichment degree, and the specific brain targeting effect of medicine strengthens.
In sum, the present invention adopts nanotechnology to modify with polypeptide surface and combines, and improves Brain targeting effect and the brain enrichment of medicine.Preparation method is simple, reacts and carries out under the temperate condition of room temperature, normal pressure, aqueous phase, pH neutrality, greatly protect the activity of medicine.Nanotechnology is adopted to pass medicine, help and improve medicine to cross over the ability that blood brain barrier enters brain, and at nanoparticle surface grafting and modifying specific polypeptide molecule, utilize active targeting effect to mediate nano-particle and enter brain, solve traditional passive target and enter the inefficient problem of brain.
Accompanying drawing explanation
Fig. 1 is the preparation method route map of shown Brain targeting drug-loading nanoparticles.
Fig. 2 is nuclear magnetic resonance, NMR (NMR) spectrogram of carrier PLGA-b-PEG.
Fig. 3 is the phenogram of described drug-loading nanoparticles, and wherein, a is dynamic light scattering (DLS) test pattern of described drug-loading nanoparticles distribution of sizes, and b, c are transmission electron microscope (TEM) figure of drug-loading nanoparticles in zones of different.
Fig. 4 is the XPS figure of described drug-loading nanoparticles.
Fig. 5 is the fluorescence intensity figure of the brain after the different nano-particle 1h of mouse tail vein injection, and wherein a is brain fluorescence photo, and b is the bar diagram of brain fluorescence signal intensity.
Detailed description of the invention
Technical scheme of the present invention is further illustrated by detailed description of the invention below in conjunction with accompanying drawing.
Embodiment 1: Brain targeting drug-loading nanoparticles and preparation method thereof
Brain targeting drug-loading nanoparticles of the present invention comprises carrier and medicine, and its surface has covalent modification polypeptide, the carrier of wherein said drug-loading nanoparticles is the block copolymer of PLGA and PEG, described medicine is fluorescence tracer, contrast agent and/or the medicine acting on brain, and described polypeptide is THRPPMWSPVWPC and/or TFFYGGSRGKRNNFKTEEYC.The molecular weight of described PLGA is 30000-60000, and its lactic acid and glycolic acid ratio are 1:1; The molecular weight of described PEG is 3400.
Its preparation method as shown in Figure 1, comprises the following steps:
1) synthetic vectors;
Described carrier is dissolved in organic solvent by 2, and dissolve fluorescence tracer, contrast agent and/or act on the medicine of brain, nano-precipitation legal system obtains drug-loading nanoparticles: the concentration of described carrier is 10-20mg/ml, the concentration of described fluorescence tracer, contrast agent and/or the medicine that acts on brain is 0.5-1mg/ml, the dimethyl formamide (DMF) of described organic solvent to be 1ml volume ratio be 65:35: tetrafluoroethene (TFE) mixed solvent;
3) by water-soluble for described drug-loading nanoparticles, surface active is carried out;
4) by water-soluble for the drug-loading nanoparticles of described activation, add peptide T HRPPMWSPVWPC and/or TFFYGGSRGKRNNFKTEEYC and carry out surperficial scion grafting, obtained Brain targeting drug-loading nanoparticles.
Embodiment 2: the synthesis of carrier PLGA-b-PEG
Because PLGA nano-particle circulation time is in vivo limited, the present invention adopts hydrophilic Polyethylene Glycol PEG molecular modification PLGA, improves PLGA hydrophilic, thus improves nano-particle circulation time in vivo.The molecular weight 30000-60000 of described PLGA, its lactic acid: glycolic acid ratio is 1:1.The molecular weight polyethylene glycol adopted is 3400, and end is respectively NH
2with COOH group, i.e. NH
2-PEG
3400-COOH.
Carrier synthesis step: get PLGA 2g, ultrasonic dissolution is at 10ml CH
2cl
2in, add 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) 41mg under stirring, then add N-hydroxy-succinamide (NHS) 23mg, at room temperature stir 30min.Add 5ml ether and make it precipitation, then wash 3 times with the ether of ice and methyl alcohol mixed liquor, wash away unnecessary EDC and NHS.Again solvent is spin-dried for, obtains white-yellowish solid PLGA-NHS.Get PLGA-NHS 1g, be dissolved in 4ml CHCl
3, ultrasonic dissolution.NH is added under stirring
2-PEG-COOH 100mg and DIPEA 10mg.At room temperature stirring reaction 48 hours.After having reacted, add ice methanol extraction, and with 5ml ice methanol wash 3 this, wash away unreacted NH
2-PEG-COOH.Finally solvent is spin-dried for, obtains end product PLGA-b-PEG.Product is white-yellowish solid, and store at-20 DEG C, as shown in Figure 2, visible the method can prepare the higher carrier of purity to its nuclear magnetic resonance map.
Embodiment 3: the synthesis of drug-loading nanoparticles
Dimethyl formamide (DMF) 1ml volume ratio is 65:35: dissolve 20mg PLGA-b-PEG in tetrafluoroethene (TFE) mixed solvent, room temperature is ultrasonic makes it fully dissolve.And dissolve RhodamineB fluorescence tracer or drug molecule wherein.In the boiling flask of 50ml, add 30ml intermediate water, high-speed stirred forms vortex.Draw DMF/TFE solution with 20 μ l liquid-transfering guns, be drop by drop slowly added dropwise in the aqueous solution of high-speed stirred until terminate.After titration completes, form the nano-particle suspension of the micro-blueness of milkiness shape, namely PLGA-b-PEG nano-particle, is labeled as NPs.Keep high-speed stirred 5-6h, the organic solvent in evaporating solution.Filter, and at the centrifugal 30min of 5000rpm, be separated, get supernatant, remove the floccule formed when synthesis of nano granule.Adopt Millipore ultra-filtration centrifuge tube to carry out ultrafiltration afterwards centrifugal, the centrifugal 15min of 5000rpm, gets supernatant, topped up with water, ultrasonic disperse, repeats centrifugal 2-3 time of ultrafiltration, and concentrated nanoparticles solution is to 5ml.In nano-particle forming process, fluorescence molecule or drug molecule are wrapped in the carrier, form drug-loading nanoparticles.By dynamic light scattering (DLS) and particle size distribution and the surface charge of measuring nano-particle, and observe size and the surface topography of nano-particle with transmission electron microscope (TEM), as described in Figure 3.
Embodiment 4: the surface active of described drug-loading nanoparticles
Above-mentioned drug-loading nanoparticles is resuspended in the aqueous solution of 10ml, under agitation adds the carboxylic group that 100mM EDC and 50mM NHS activates nano grain surface.Stirring at room temperature 30min, activation drug-loading nanoparticles surface carboxyl groups, to react with the amino in peptide sequence end afterwards.After having reacted, carry out centrifugal with Millipore ultra-filtration centrifuge tube, at the centrifugal 15min of 5000rpm ultrafiltration, get the centrifugal product of upper part, add water, ultrasonic disperse, repeat centrifugal 2-3 time of ultrafiltration, remove unreacted EDC and NHS.
Embodiment 5: the polypeptide grafted modification of described drug-loading nanoparticles
The nano-particle 20mg of activation is resuspended in the aqueous solution of 10ml, adds end and contain NH
2peptide sequence THRPPMWSPVWPC (being labeled as TC13, molecular weight 1594g/mol) and/or TFFYGGSRGKRNNFKTEEYC (being labeled as TC20, molecular weight 2405g/mol), it is 10 that this polypeptide adds rear concentration
-3m, for above-mentioned two peptide species, its concentration is respectively 1.594mg/ml and 2.405mg/ml, normal-temperature reaction overnight, thus obtains finishing and have the PLGA-PEG-TC13 of polypeptide (being labeled as NPs-TC13) or PLGA-PEG-TC20 (being labeled as NPs-TC20) nano-particle.Remove unreacted polypeptide by above-mentioned ultrafiltration centrifuging, and clean nano-particle 2-3 time.
Measure the chemical composition on the drug-loading nanoparticles NPs surface of nano-particle NPs-TC13, NPs-TC20 and non-scion grafting polypeptide with XPS, see if there is element sulphur and exist.There is then explanation polypeptide and be successfully grafted to nano grain surface in element sulphur, otherwise then illustrates that grafting polypeptide is unsuccessful.As shown in Figure 4, NPs-TC13, NPs-TC20 surface prepared by visible the present invention all has element sulphur, polypeptide scion grafting success, and the drug-loading nanoparticles surface of non-scion grafting polypeptide is not containing element sulphur.
Embodiment 6: the Evaluation on distribution of described drug-loading nanoparticles in Mice Body
Drug-loading nanoparticles that the bag prepared of the present invention carries Rhodamine B fluorescence tracer is used to detect the Brain targeting effect of drug-loading nanoparticles of the present invention.Four kinds of compounds are selected to evaluate: bag carries Rhodamine B fluorescence tracer and the drug-loading nanoparticles (NPs-TC13-RhoB) of scion grafting TC13; Bag carries Rhodamine B fluorescence tracer and the drug-loading nanoparticles (NPs-TC20-RhoB) of scion grafting TC20; Bag carries the drug-loading nanoparticles (NPs-RhoB) of Rhodamine B fluorescence tracer not scion grafting polypeptide; Normal saline (saline).
Above-mentioned four groups of compounds are configured to the solution of 12mg/ml.Getting body weight is 20g Balb/c nude mice, is divided into four groups, often organizes mice tail vein injection administration 100 μ l compound respectively, namely presses the injection of 60mg/kg Mouse Weight.After injection 15min, 30min, 1h, with 3% isoflurane gas anesthetized mice, and inserted imaging in the multispectral small animal living body imaging system of CRI Maestro 2, observed the distribution of nano-particle in Mice Body that bag carries fluorescence molecule.Dissect mice subsequently and take out its brain, carrying out brain fluorescence imaging, observing the aggregation extent of nanoparticle at mouse brain, the fluorescence intensity figure after each group administration 1h as shown in Figure 5.The brain fluorescence intensity of visible NPs-TC13-RhoB and NPs-TC20-RhoB group is obviously better than NPs-RhoB group and saline group, and particularly, in four groups of experiments, corresponding mouse brain average fluorescent strength is respectively (8.47 ± 1.05) x 10
6, (8.66 ± 0.94) x 10
6, (2.27 ± 0.68) x 10
6(1.39 ± 0.51) x 10
6photon/square centimeter/second.
Applicant states, the present invention illustrates method detailed of the present invention by above-described embodiment, but the present invention is not limited to above-mentioned method detailed, does not namely mean that the present invention must rely on above-mentioned method detailed and could implement.Person of ordinary skill in the field should understand, any improvement in the present invention, to equivalence replacement and the interpolation of auxiliary element, the concrete way choice etc. of each raw material of product of the present invention, all drops within protection scope of the present invention and open scope.
Claims (9)
1. a Brain targeting drug-loading nanoparticles, is characterized in that, described Brain targeting drug-loading nanoparticles, by the parts by weight of each component, comprises carrier 10-20 part, medicine 0.5-1 part, surface polypeptide 15.94-24.05 part.The carrier of wherein said drug-loading nanoparticles is the block copolymer of PLGA and PEG, and described medicine is fluorescence tracer, contrast agent and/or the medicine acting on brain, and described polypeptide is THRPPMWSPVWPC and/or TFFYGGSRGKRNNFKTEEYC.
2. Brain targeting drug-loading nanoparticles according to claim 1, is characterized in that, the molecular weight of described PLGA is 30000-60000, and its lactic acid and glycolic acid ratio are 1:1; The molecular weight of described PEG is 3400.
3. the preparation method of Brain targeting drug-loading nanoparticles according to claim 1 and 2, is characterized in that, described preparation method comprises the following steps:
1) synthetic vectors;
2) described carrier is dissolved in organic solvent, and dissolves fluorescence tracer, contrast agent and/or act on the medicine of brain, nano-precipitation legal system obtains drug-loading nanoparticles;
3) by water-soluble for described drug-loading nanoparticles, surface active is carried out;
4) by water-soluble for the drug-loading nanoparticles of described activation, add peptide T HRPPMWSPVWPC and/or TFFYGGSRGKRNNFKTEEYC and carry out surperficial scion grafting, obtained Brain targeting drug-loading nanoparticles.
4. preparation method according to claim 3, is characterized in that, step 1) described in the mass ratio of PLGA and PEG be 10-20:1; Preferably, described PEG is that end contains NH
2with the PEG of COOH group, NH
2-PEG-COOH;
Preferably, at PLGA and NH
2before-PEG-COOH reaction, the terminal carboxyl group of activation PLGA;
Preferably, use EDC and NHS to activate the terminal carboxyl group of PLGA, the mass ratio of described EDC, NHS and PLGA is 41:23:2000.
5. preparation method according to claim 3, is characterized in that, in described step 2, DMF and the TFE mixed solvent of described organic solvent to be volume ratio be 65:35;
Preferably, the concentration of described carrier is 20mg/m, and the concentration of described fluorescence tracer, contrast agent and/or the medicine that acts on brain is 0.5mg/ml;
Preferably, ultrasonic dissolution is dissolved as described in;
Preferably, the step of described nano-precipitation method is: dropwise the DMF/TFE solution dissolving carrier and fluorescence tracer, contrast agent and/or the medicine that acts on brain is added intermediate water under high-speed stirred, filter, centrifugal, it is centrifugal to get supernatant ultrafiltration, concentrated obtained described drug-loading nanoparticles.
6. preparation method according to claim 5, is characterized in that, the volume ratio of described intermediate water volume and described DMF/TFE solution is 3000:2;
Preferably, the rotating speed of described high-speed stirred is 700-800rpm, and mixing time is 5-6h;
Preferably, described centrifugal be the centrifugal 30min of 5000rpm;
Preferably, described ultrafiltration is centrifugal for adopting the centrifugal 15min of Millipore ultra-filtration centrifuge tube 5000rpm, repeats 2-3 time;
Preferably, described centrifugal molecular cut off is 3500;
Preferably, centrifugal for ultrafiltration gained solution is concentrated into 5ml by described simmer down to.
7. preparation method according to claim 3, is characterized in that, in described step 3) in, the concentration of described drug-loading nanoparticles is 2mg/ml;
Preferably, use EDC and NHS to carry out surface active to described drug-loading nanoparticles, and the concentration of EDC and NHS is respectively 100mM and 50mM in water;
Preferably, described soak time is 30min;
Preferably, after activation, remove unreacted EDC and/or NHS, the method for unreacted EDC and/or NHS of described removing is that ultrafiltration is centrifugal and wash with water, and the centrifugal molecular cut off of described ultrafiltration is 3500, washes repetition 2-3 time with water.
8. preparation method according to claim 3, is characterized in that, in step 4) in, the concentration of the drug-loading nanoparticles of described activation is 2mg/ml, and the concentration of described peptide T HRPPMWSPVWPC and/or TFFYGGSRGKRNNFKTEEYC is respectively 10
-3m;
Preferably, described scion grafting reaction is carried out under room temperature 600rmp stirs;
Preferably, the time of described scion grafting reaction is 8-12h;
Preferably, after scion grafting reaction, ultrafiltration is centrifugal and wash with water, and the centrifugal molecular cut off of described ultrafiltration is 6000-8000, and washes repetition 2-3 time with water.
9. Brain targeting drug-loading nanoparticles according to claim 1 and 2 is preparing the application in fluorescence tracer, contrast agent and/or the medicine acting on brain.
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| CN112316142A (en) * | 2020-11-23 | 2021-02-05 | 苏州大学 | Semiconductor polymer nano-particles and preparation method and application thereof |
| CN112741837A (en) * | 2020-12-03 | 2021-05-04 | 暨南大学 | Brain-targeting nano-drug delivery system and preparation method thereof |
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| ROGER PRADES ET AL.: "《Delivery of gold nanoparticles to the brain by conjugation with a peptide that recognizes the transferrin receptor》", 《BIOMATERIALS》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020114390A1 (en) * | 2018-12-03 | 2020-06-11 | Master Dynamic Limited | Nanoparticle Delivery System |
| CN111888333A (en) * | 2020-08-11 | 2020-11-06 | 深圳大学 | Transferrin receptor targeted nano micelle and preparation method and application thereof |
| CN112316142A (en) * | 2020-11-23 | 2021-02-05 | 苏州大学 | Semiconductor polymer nano-particles and preparation method and application thereof |
| CN112316142B (en) * | 2020-11-23 | 2021-06-04 | 苏州大学 | Semiconductor polymer nano-particles and preparation method and application thereof |
| CN112741837A (en) * | 2020-12-03 | 2021-05-04 | 暨南大学 | Brain-targeting nano-drug delivery system and preparation method thereof |
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| CN104491869B (en) | 2018-01-05 |
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