CN105859832A - Polypeptides using RGD as active site and application thereof to preparation of targeted medicament for treating ischemic stroke - Google Patents
Polypeptides using RGD as active site and application thereof to preparation of targeted medicament for treating ischemic stroke Download PDFInfo
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Abstract
The invention belongs to the field of biological medicine technology, and relates to an application of polypeptides using RGD as an active site to preparation of a targeted medicament for treating ischemic stroke. The RGD peptide is a tripeptide sequence (Arg-Gly-Asp) which is composed of arginine, glycine and aspartic acid; polypeptides which use the RGD tripeptide as the active site comprise pentapeptide, heptapeptide, and cyclopeptide or peptide analogue compounds; tests show that linear peptides and cyclic peptides using RGD as the active site are used for modifying a nanometer drug delivery system, so that drug accumulation of the system on ischemic focus of ischemic stroke is substantially improved, and secondary targeting drug delivery characteristics at the intracerebral lesion location are obvious. The system can be applied to entrapment of antioxidant medicaments, so that antioxidation effects of Edaravone in an ischemia reperfusion model rat are obviously improved, and infarct volume is reduced.
Description
Technical field
The invention belongs to bio-pharmaceutical technical field, it hits at preparation treatment ischemic cerebral apoplexy to relate to the polypeptide with RGD as active center
Application in medicine, the particularly described polypeptide that RGD is active center can build cerebral infarction targeting drug delivery as target head
System.
Inventive technique
Showing according to World Health Organization's statistical data, the mortality rate of China's apoplexy and sickness rate rank first in the world and second respectively
Position.But for the treatment of such disease, due to the existence of blood brain barrier (BBB), make little point of nearly all macromole and 98%
Sub-medicine cannot be introduced into brain and central nervous system, and clinical conventional administering mode is due to inorganizable specificity and diseased region
Targeting thus cause dosage big, easily cause serious toxic and side effects.In recent years, Brain targeting preparation research achieves huge
Achievement, by antibody or ligand modified immunolipid body preparation, not only extends at body circulation time, has targeting function simultaneously,
Increase the brain delivery of medicine.But how targeting drug delivery means realize intracerebral lesion position after being delivered by medicine and crossing over BBB at present
Selectivity is that brain-targeted drug delivery studies the new difficult problem faced further.Existing preparation means, after helping medicine by BBB, lack
Second selecting effect in brain, thus the drug level at intracerebral lesion position is the highest, and generation same to normal cell infringement.
The most how to set up and not only may span across BBB but also can be that apoplexy is controlled further to the brain targeting drug delivery system at intracerebral lesion position
The key treated.
Without leukocyte in normal cerebral tissue, but numerous studies confirm, during Cerebral Ischemia/Reperfusion, leukocyte is (in predominantly
Property granulocyte and mononuclear cell) can be activated under the chemotaxis effect of cytokine modulating, be gathered in cerebral microvascular and by BBB,
Infiltration ischemic tissue of brain.The leukocyte being activated discharges inflammatory mediator and noxious substance (oxygen-derived free radicals etc.) simultaneously, increases the weight of office in brain
Portion's inflammatory reaction and oxidative damage, increase the weight of neuronal damage after ischemia.Therefore, during Cerebral Ischemia/Reperfusion, leukocyte is not only
Can pass through BBB, and the position of neuronal damage in brain can be arrived.In this pathological process, the leukocyte in blood circulation is
Crucial: it passes through BBB and arrive ischemic tissue of brain under the chemotaxis effect of cytokine regulation, if leukocyte can be utilized, makes
It carries drug-loading system and crosses over BBB arrive Nao Nei ischemic tissue, then can build and a kind of very good have multistage Brain targeting function
Transmission system.
RGD peptide (Arg-Gly-Asp) is the tripeptide sequence being made up of arginine-glycine-aspartic acid, for fibronectin
Specific binding site with its receptor.RGD peptide and integrin receptor α v β 3 have high affinity, and α v beta 3 receptor is swollen
There is on endotheliocyte in tumor angiogenic process the expression of height, therefore use the carrier of RGD modification or diagnostic reagent to apply
Treatment and diagnosis in tumor.Additionally, with RGD tripeptides as active center, research synthesized a series of pentapeptide, heptapeptide, cyclic peptide or
Peptidomimetic compound, result cyclisation peptide has more stable structure and higher activity in vivo.In in recent years with RGD tripeptides as activity
The research in terms of cancer target preparation of the serial polypeptide of the heart is extremely active and obtains effect.
Prior art related to the present invention has:
1. Wang Dawei, Fu Yan. the diagnosis of emergency treatment thrombotic disease and treatment. world Acute critical diseases medical journal .2006,3
(4):1385-1389.
2.Tosi G,Costantino L,et al.Polymeric nanoparticles for the drug delivery to the
central nervous system.Expert Opin Drug Deliv.2008Feb；5(2):155-74.
3.Suzuki Y,Matsumoto Y,et al.SM-20220,a Na(+)/H(+)exchanger inhibitor:effects
on ischemic brain damage through edema and neutrophil accumulation in a rat middle
cerebral artery occlusion model.Brain Res.2002,945(2):242-248.
4.Phillips JB,Williams AJ,et al.Proteasome inhibitor PS519reduces infarction and
attenuates leukocyte infiltration in a rat model of focal cerebral ischemia.Stroke.
2000,(7):1686-1693.
5.Auzzas L,Zanardi F,Battistini L,et al.Targeting alphavbeta3integrin:design
and applications of mono-and multifunctional RGD-based peptides and semipeptides.
Curr Med Chem.2010,17(13):1255-1299.
6.Shuang Liu.Radiolabeled Cyclic RGD Peptides as Integrinαvβ3-Targeted
Radiotracers:Maximizing Binding Affinity via Bivalency.Bioconjug Chem.2009,
20(12):2199-2213.
7.Jain S,Mishra V,et al.RGD-anchored magnetic liposomes for monocytes/neutrophils
mediated brain t argeting.Inter J Pharm.2003,261:43-55.
8.Eyal Afergan,Hila Epstein,Rachel Dahan,et al.Delivery of serotonin to the brain
by monocytes following phagocytosis of liposomes.J Control Release,2008,(132)
84-90.。
Summary of the invention
It is an object of the invention to, it is provided that a kind of polypeptide with RGD as active center being different from prior art.
Invention further provides the polypeptide that RGD is active center and build cerebral infarction targeting drug delivery system as target head
In application.
Basis based on prior art, in fact in addition to α v β 3, RGD peptide has height with integrin receptor family as α v β 1
The affinity of degree.α v β 1 be leukocyte surface height express integrin receptor, therefore RGD modify carrier can by with
The high-affinity of α v β 1 is combined with leukocyte, is then carried by leukocyte during inflammatory reaction and is subject to by BBB and in arriving brain
Damage position, it is achieved multistage Brain targeting mesh.
In the present invention, the polypeptide with RGD tripeptides as active center includes pentapeptide, heptapeptide, cyclic peptide or peptidomimetic compound, described RGD
Peptide is the tripeptide sequence (Arg-Gly-Asp) being made up of arginine-glycine-aspartic acid；In embodiments of the invention preferably
Ring-type rgd peptide；
In the present invention, carry out the polypeptide using RGD as active center and build cerebral infarction targeting drug delivery system as target head
Test, result shows, the present invention polypeptide using RGD as active center is as target head, after being applied to different dosing system, can obtain
The significantly effect of brain targeting drug delivery, and significantly improve the curative effect of model drug.
In the present invention, described delivery system includes but not limited to: liposome, nanoparticle, phosphatide complexes, microsphere, polymer,
Any one such as nanosphere, nano-emulsion, self-microemulsion, clathrate is common in the dosage form of clinic；
In the present invention, described RGD peptide is applied to the mode of delivery system and includes but be not limited to: with any one of delivery system
Common functional material coupling, described functional material is: one end active ester Han NHS, and one end is any normal containing sulfydryl etc.
The group that can react with polypeptide seen, in embodiments of the invention, preferred functional material includes, but are not limited to: function
Property phospholipid, functional cholesterol, functional PEG, functional polymer, functional cholesterol etc.
In the present invention, the linear peptides with RGD as active center and modified cyclic peptide administration nano-drug administration system, it is remarkably improved this system and exists
The drug accumulation of cerebral infarction ischemia focus, has two grades of targeting drug delivery features at obvious intracerebral lesion position.Apply this to be
System bag carries anti-oxidation medicine, can significantly improve Edaravone antioxidant effect in ischemia-reperfusion injury model rat body, reduces infarction
Volume.Its mechanism is mainly concerned with the inflammatory reaction in cerebral infarction pathogenic process.
It is only used as the explanation of the present invention below in conjunction with the accompanying drawings with specific embodiment, but should be in no way restrictive.
Accompanying drawing explanation
Figure is known in Fig. 1, RGD and reactant liquor TLC inspection.
Fig. 2, RGDL particle diameter distribution (A) and transmission electron microscope photo (B).
Fig. 3 I/R nude mice gives RGDL-Dir back brain fluorescence photo, wherein,
A:RGDL-Dir；B:PL-Dir.
Fig. 4, DSPE-PEG-NHS (A) and DSPE-PEG-cRGD (B) modi-toff collection of illustrative plates.
Fig. 5, cRGDL and PL particle diameter is distributed.
Fig. 6, cRGDL and PL transmission electron microscope photo.
Fig. 7, laser confocal microscope photo, wherein
A is green: the THP-1 cell of CFSE dyeing；B is red: the liposome of Cy5 labelling；C:AB superposition photo.
Fig. 8, cRGDL-Dir and PL-Dir give living body fluorescent photo after I/R nude mice, wherein
A:cRGDL-Dir；B:PL-Dir.
Fig. 9, I/R nude mice gives PL-Dir or cRGDL-Dir tissues following MCAO in rats section TTC dyeing (A) and fluorescence photo (B).
Figure 10, inflammation mice gives PG solution group, common nanoparticle group PG, tissue distribution after cRGD-NP and cRGD-NP
Result.
Figure 11, I/R rat gives different prescription cerebral infarction volume result respectively at different time after filling again.
Figure 12, I/R nude mice gives living body fluorescent after PL-Dir, RGDL-Dir and cRGDL-Dir.
Detailed description of the invention
Embodiment 1
RGD and the coupling of phospholipid:
Precision weighs appropriate linear RGD tripeptides and DSPE-PEG-NHS (peptide: DSPE-PEG-NHS=1:2, molar ratio), point
It is not dissolved in a small amount of anhydrous DMF without ammonia, stirs to all dissolving.Peptide solution is slowly added dropwise to quickly stirring
In DSPE-PEG-NHS solution, add a small amount of triethylamine regulation pH extremely alkalescence, logical nitrogen protection, reaction 24 hour is stirred at room temperature.
Anti-PL uses TLC method 1,2,3-indantrione monohydrate chromogenic reaction tracing detection during answering, until the speckle of peptide disappears, terminates reaction.TLC developing solvent is
N-butyl alcohol: glacial acetic acid: water=3:1:2.Fig. 1 is shown in by reaction end TLC photo.On the left of result lamellae, RGD clear spot is visible, explanation
This reaction can sensitive detection RGD peptide, can not detect RGD during the reaction end of right side, illustrate to react complete.Polydextran gel
Post G-50 is isolated and purified, and to obtain DSPE-PEG-RGD lyophilizing standby；
The preparation of the liposome (RGDL) that RGD modifies:
Weigh soybean phospholipid 100mg, A cholesterol 20mg, DSPE-PEG-RGD 4mg to be placed in 250mL eggplant-shape bottle, add
10mL chloroform dissolves, and room temperature rotation is steamed into film.Add 3mL absolute ether, immobilized artificial membrane redissolved, adds 1mL distilled water,
Water bath sonicator 5min forms the w/o Emulsion of white.40 DEG C of rotations are steamed, and pump ether, after forming clear viscous gel, add 9mL
Water, continues rotation and washes film, magnetic agitation 1h, Probe Ultrasonic Searching, obtain RGDL；
Prescription is added without DSPE-PEG-RGD, prepares conventional liposome (plain liposome, PL) as stated above；
The preparation of fluorescence labeled fatty acid body:
Dir adding liposome prescription (Dir: phosphatidase 1: 100mol/mol), the most ibid weighs soybean phospholipid 100mg, gallbladder is solid
Alcohol 20mg, DSPE-PEG-RGD 4mg is placed in 250mL eggplant-shape bottle, adds 10mL chloroform and dissolves, and room temperature rotation is steamed into film.
Adding 3mL absolute ether, redissolved by immobilized artificial membrane, add 1mL distilled water, water bath sonicator 5min forms the w/o breast of white
Agent.40 DEG C of rotations are steamed, and pump ether, after forming clear viscous gel, add 9mL water, continue rotation and wash film, magnetic agitation 1h,
Probe Ultrasonic Searching, obtains the RGDL-Dir of Dir labelling；
Prescription is added without DSPE-PEG-RGD, prepares fluorescently-labeled conventional liposome PL-Dir as stated above；
The evaluation of RGDL: suitably diluted by the RGDL of preparation, surveys its particle size distribution and Zeta potential with particle instrument, result such as Fig. 2 A-B
Shown in, RGDL particle diameter is about 115nm, and form is similar round；
Middle cerebral artery occlusion art is used to prepare nude mice ischemia-reperfusion (I/R, Ischemic/Reperfusion) model.By RGDL-Dir
Give I/R model nude mice, adopt after administration 0.5h, 1h, 3h, 6h, 9,12h small animal living body imager observe nude mice brain glimmering
Light intensity, result is shown in Fig. 3 (red boxes in for brain), and result is visible, and after the administration of RGDL-Dir group nude mice brain, 1h i.e. goes out
Now obvious fluorescence, is all continuously detected stronger fluorescence in 3-9 hour.PL-Dir group 1h has week fluorescent, in other times
All it is not detected by fluorescence compared with RGDL-Dir, illustrates that RGDL-Dir can significantly be gathered in brain, there is stronger Brain targeting
Effect.
Embodiment 2
RGD cyclic peptide and the coupling of phospholipid:
Precision weighs appropriate RGD five cyclic peptide (cRGD, Arg-Gly-Asp-D-Phe-Lys) and DSPE-PEG-NHS
(peptide: DSPE-PEG-NHS=2:1, molar ratio), is dissolved separately in a small amount of anhydrous DMF without ammonia, stirs to all dissolving.
Peptide solution is slowly added dropwise in the DSPE-PEG-NHS solution to quickly stirring, adds a small amount of triethylamine regulation pH extremely alkalescence,
Logical nitrogen protection, is stirred at room temperature reaction 24 hours.DSPE-PEG-cRGD is separated by polydextran gel with unreacted little peptide.
Then DSPE-PEG-cRGD and DSPE-PEG-NHS being carried out modi-toff detection, result as shown in Figure 4, can from figure
To find out, the mean molecule quantity of DSPE-PEG-NHS is 4273, and the mean molecule quantity of DSPE-PEG-cRGD is 4633,
The proton peak of DSPE-PEG-cRGD migrates to the right, and cRGD peptide success and DSPE-PEG-NHS coupling are described；
The preparation of the liposome (cRGDL) that cRGD modifies:
Weigh soybean phospholipid 100mg, A cholesterol 20mg, DSPE-PEG-cRGD 6mg to be placed in 250mL eggplant-shape bottle, add
10mL chloroform dissolves, and room temperature rotation is steamed into film.Add 3mL absolute ether, immobilized artificial membrane redissolved, adds 1mL distilled water,
Water bath sonicator 5min forms the w/o Emulsion of white.40 DEG C of rotations are steamed, and pump ether, after forming clear viscous gel, add 9mL
Water, continues rotation and washes film, magnetic agitation 1h, Probe Ultrasonic Searching, obtain cRGDL；
Prescription is added without DSPE-PEG-cRGD, prepares conventional liposome (plain liposome, PL) as stated above；
The preparation of fluorescence labeled fatty acid body:
Double for iodate 1,1'-octadecyl 3,3,3', 3'-tetramethyl Yin tri-carbocyanine (Dir) or 3H--indole cyanine type dye (Cy5) are added
Enter liposome prescription (Dir/Cy5: phosphatidase 1: 100mol/mol), the most ibid weigh soybean phospholipid 100mg, cholesterol 20mg,
DSPE-PEG-cRGD 6mg is placed in 250mL eggplant-shape bottle, adds 10mL chloroform and dissolves, and room temperature rotation is steamed into film.Add 3mL
Absolute ether, redissolves immobilized artificial membrane, adds 1mL distilled water, and water bath sonicator 5min forms the w/o Emulsion of white.40 DEG C of rotations
Steam, pump ether, after forming clear viscous gel, add 9mL water, continue rotation and wash film, magnetic agitation 1h, Probe Ultrasonic Searching,
Obtain the cRGDL-Dir of Dir labelling；The cRGDL-Cy5 of Cy5 labelling；
Prescription is added without DSPE-PEG-cRGD, prepare as stated above fluorescently-labeled conventional liposome PL-Dir or
PL-Cy5；
The evaluation of cRGDL: suitably diluted by cRGDL and PL of preparation, surveys its particle size distribution with particle instrument, and transmission electron microscope is seen
Examining its form, result is shown in Fig. 5-6.CRGDL particle diameter is about 114nm, PL particle diameter and is about 110nm, the two mean diameter and grain
Footpath is distributed without significant difference, cRGDL and PL transmission electron microscope observing is similar round；
Person monocytic cell's strain THP-1 cell strain 1640 cell culture mediums containing 10%FBS, 5%NEAA, 5%PS are cultivated
At 37 DEG C, 5%CO2Incubator in, change liquid every other day, when cell density reaches 1 × 105Individual/cm2Time add CF 5(6)-Carboxyfluorescein two
Acetate succinimide ester (CFSE) cytoplasmic dye is hatched.Then cRGDL-Cy5 liposome hatches 30min jointly, swashs
Light confocal microscopy liposome is by the situation of THP-1 cellular uptake, and result is as it is shown in fig. 7, cRGDL can be by THP-1
Huge uptake enters in Cytoplasm, and THP-1 is the leukocyte strain of people source, illustrates that cRGDL can be combined with leukocyte；
Middle cerebral artery occlusion art is used to prepare nude mice ischemia-reperfusion (I/R, Ischemic/Reperfusion) model.Will
CRGDL-Dir gives I/R model nude mice, adopt after administration 0.5h, 1h, 2h, 4h, 6,8h small animal living body imager observe
Nude mice brain fluorescence intensity, result is shown in Fig. 8.Result is visible, i.e. occurs bright after the administration of cRGDL-Dir group nude mice brain after 30min
Aobvious fluorescence, is all continuously detected stronger fluorescence in 8 hours, PL-Dir group is all not detected by fluorescence, cRGDL-Dir is described
Can significantly be gathered in brain, there is stronger Brain targeting effect；
After 12h, animal is put to death, send out after taking cerebral tissue brain frozen section TTC dyeing and detect in small animal living body imager, result
See Figure 10.In TTC section statining, visible white part is ischemic area (as shown in Figure 9 A), and in living imaging photo, left side is right
Answer obvious fluorescence seen from region to assemble (as shown in Figure 9 B), overlap with TTC dyeing ischemic area, the fat that cRGD modifies is described
Plastid has obvious two grades of Brain targeting effects compared with conventional liposome, can be gathered in ishemic part in brain.
Embodiment 3
Carry preparation and the Evaluation on Its Targeting Performance of propyl ester (PG) nanoparticle
By propyl ester 100mg, monoglyceride 600mg, DSPE-PEG-RGD or DSPE-PEG-cRGD of 2mol%, it is dissolved in
In 15ml ethanol, ultrasonic dissolution, add phosphatidase 1 00mg, slight fever forms organic facies.Separately take poloxamer soluble in water, structure
Becoming poloxamer concentration is the aqueous phase of 0.5%, under agitation injects in 30ml aqueous phase by organic facies syringe needle, and whole process keeps temperature
Degree, at about 75 DEG C, continues stirring, and the translucent system of gained, to original 1/3, is quickly mixed in the 20ml of 0-2 DEG C by concentration volume
In aqueous phase, continue stirring 90min, obtain medicine carrying RGD-NP, cRGD-NP；
Be added without as stated above DSPE-PEG-RGD or DSPE-PEG-cRGD medicine carrying common nanoparticle NP；
Above-mentioned three kinds of nanoparticles and PG drug solution group tail vein are given IL-1 β and causes inflammatory model mice, often group 12, dosage
It is calculated as 12mg/kg respectively with PG；After administration 10,15min puts to death immediately, dissect core, liver, spleen, lung, kidney, brain group
Knit；Tissue sample is with after normal saline flushing surface blood and content, and filter paper is weighed after blotting, and loads in valve bag, in-20
DEG C refrigerator is frozen to be measured, takes each tissue sample and takes 0.1g (inadequate 0.1g takes entirely), adds 1mL30% methanol solution and organizes
Homogenate, measure be administered after each Drug content in tissues, result as shown in Figure 10, compared with solution group PG, NP, RGD-NP,
CRGD-NP is all remarkably improved concentration in medicine brain, and RGD-NP and cRGD-NP effect is better than NP；cRGD-NP
Compared with RGD-NP, in brain, drug level significantly improves, and illustrates that cRGD-NP Brain targeting effect is better than RGD-NP.
Embodiment 4
Carry the preparation of Edaravone liposome
Precision weighs soybean phospholipid 100mg, cholesterol 20mg, the DSPE-PEG-RGD of Edaravone 10mg, 2mol% or
DSPE-PEG-cRGD is placed in 250ml eggplant-shape bottle, adds 10ml dehydrated alcohol, and waters is ultrasonic to be completely dissolved, 25 DEG C of decompressions
Rotation is evaporated film forming, adds distilled water solution 10ml, and 25 DEG C of rotations make film dissolve, Probe Ultrasonic Searching.I.e. can obtain being loaded with Yi Dala
The ER-RGD-L given, ER-cRGD-L.If being added without DSPE-PEG-RGD or DSPE-PEG-cRGD and i.e. obtaining general
Logical liposome ERL, measures the envelop rate of three kinds of liposomees, drug loading, mean diameter and Zeta potential, result such as table 1 respectively
Shown in, three kinds of liposome encapsulations are about 75%, and drug loading is about 5%, about mean diameter 110nm, and Zeta potential-20mV
Left and right, it can be seen that be loaded into medicine or RGD/cRGD target head is modified and do not affected liposome physicochemical property；
1 three kinds of liposome physicochemical properties of table characterize
Carry Edaravone liposome pharmacodynamic evaluation
SD rat 90, is randomly divided into 6 groups, is respectively as follows: sham operated rats, Edaravone solution group (ERS group), ERL
Group, ER-RGD-L group, ER-cRGD-L group, model group (Model group).12h fasting before experiment, freely drinks water, greatly
Brain medium-sized artery obturation art sets up rat I/R model, injects above-mentioned 4 kinds respectively in filling 0h, 3h, 6h, 12h, 24h tail vein again
Different preparations, sham operated rats and model group give PBS aqueous solution.Carrying out Neuroscore after 48h, result is as shown in table 2,
Then with 0.9%NaCl solution by rat heart perfusion, take out cerebral tissue, cut into slices after quick-freezing 20min under the conditions of-20 DEG C, 2%TTC
After dyeing calculate brain infarction area %, result as shown in figure 12,
Brain infarction area %=(area in the area-non-infarction portion, operation side of operation side hemisphere)/
Area × 100% of operation side hemisphere
Neuroscore result
Each group rat carries out Neuroscore to it after being administered 48h, as seen from Table 2, then fills after each time point is administered, mould
Type group rat all cannot walk, function of nervous system's highest scoring (average 4 points), shows modeling success.ERS group removes 0h and 24h
Outside administration, Neuroscore and model group have significant difference, and little (the average 3.4 points) animal of fall shows as permissible
Walking, but the phenomenon toppled over to side occur, after filling again be described, 3-12h gives Edaravone and can promote that I/R animal nerve function is extensive
Multiple, but degree is less,
ERL group 0-24h is administered Neuroscore all has significant difference that the ERL neural merit to I/R rat is described with model group
Can recover that there is obvious facilitation (average 3 points)；Compared with ERS group, 0h and 24h is administered has significance with ERS group
Difference, 3-12h is administered scoring and there was no significant difference with ERS group, illustrates that 3-12h is administered ERL with ERS and promotes that function of nervous system is extensive
Multiple degree zero difference, but ERL can substantially widen the treatment window of ER, and administration time is extended to 24h,
After ER-RGD-L group 0-24h is administered, most animals turns clockwise when only showing walking, Neuroscore and model group,
ERS group and ERL group are compared all significant difference, illustrates ER-RGD-L to have significantly and promotes neurological functional recovery effect,
And recovery extent is significantly higher than ERS and ERL,
After ER-cRGD-L group 0-24h is administered, animal only occurs that right fore cannot stretch, and can walk, walk and topple over or suitable
The phenomenon that hour hands are turn-taked occurs, Neuroscore is minimum and fall is maximum, with model group, ERS, ERL and ER-RGD-L
Compare and be respectively provided with significant difference, illustrate that each time point of ER-cRGD-L is administered and be respectively provided with obvious therapeutic effect, and effect is notable
Being better than ER-RGD-L, ERL, ERS and model group, animal nerve functional rehabilitation level is in optimal level in each group；
Table .2 Neuroscore result (n=3)
Brain infarction area evaluation result
Figure 11 brain infarction area result of calculation shows, model group rats Infarction volume reaches more than 30%, and modeling success is described, false
There is not infarction in operation group, and the interference of not false positive results is described；ERS group compares with model group, infarction after 0-24h administration
Area all has significant difference, and ER can reduce infarct size, but 0-24h different time administering effect zero difference；
ERL group 0-24h is administered, and brain infarction area compares with model group and is respectively provided with significant difference, illustrates that ERL is significantly reduced
The brain infarction area that IS causes；Comparing with ERS group, 0-6h is administered infarct size and there was no significant difference, 12h and 24h is administered stalk
Unleavened dough is long-pending significantly less than ERS group, is administered ER therapeutic effect without potentiation, but 12h and 24h gives in ERL0-6h is described
Medicine is remarkably improved ER therapeutic effect and has widened the treatment window of ERS；
ER-RGD-L group and ER-cRGD-L group 0-24h are administered and compare with model group, ERS group and ERL group, brain infarction area
All it is decreased significantly, and ER-cRGD-L group declines more, illustrate that ER-RGD-L and ER-cRGD-L all can strengthen ER
Therapeutical effect, effect is better than ERL, and ER-cRGD-L action effect is optimum；
The preparation of fluorescently-labeled PL, RGDL and cRGDL and Evaluation on Its Targeting Performance
Weigh soybean phospholipid 300mg, cholesterol 60mg, DSPE-PEG-RGD or DSPE-PEG-cRGD 2mol% to be placed in
In 250mL eggplant-shape bottle, adding 10mL chloroform and dissolve, room temperature rotation is steamed into film.Add 3mL absolute ether, immobilized artificial membrane redissolved,
Adding 1mL distilled water, water bath sonicator 5min forms the w/o Emulsion of white.40 DEG C of rotations are steamed, and pump ether, form thickness saturating
After bright gel, add 9mL water, continue rotation and wash film, magnetic agitation 1h, Probe Ultrasonic Searching, obtain RGDL-Dir and cRGDL-Dir.
If being added without DSPE-PEG-RGD or DSPE-PEG-cRGD, i.e. obtain PL-Dir.I/R is given respectively by three kinds of liposomees
Model nude mice, and be administered after 24h in small animal living body luminoscope detect fluorescence, result as shown in figure 12, result and PL-Dir
Comparing, RGDL-Dir and cRGDL-Dir has obvious Brain targeting effect, and cRGDL-Dir effect is significantly stronger than
RGDL-Dir。
Claims (6)
1. the polypeptide with RGD as active center, it is characterised in that described polypeptide includes pentapeptide, heptapeptide, cyclic peptide or peptidomimetic
Compound, described RGD peptide is the tripeptide sequence (Arg-Gly-Asp) being made up of arginine-glycine-aspartic acid.
2. the RGD described in claim 1 is the polypeptide in active center purposes in preparing cerebral infarction targeting drug delivery system,
Wherein said polypeptide is as target head.
3. the purposes as described in claim 2, it is characterised in that described delivery system is selected from liposome, nanoparticle, phospholipid
Any one of complex, microsphere, polymer, nanosphere, nano-emulsion, self-microemulsion or clathrate dosage form.
4. the purposes as described in claim 2, it is characterised in that described RGD peptide for the mode of delivery system is: with pass
The functional material coupling that any one of medicine system is common.
5. the purposes as described in claim 4, it is characterised in that described functional material is: one end active ester Han NHS,
The one end group that can react with polypeptide containing sulfydryl.
6. the purposes as described in claim 4 or 5, it is characterised in that described functional material is: functional phospholipid, merit
Energy property cholesterol, functional PEG, functional polymer or functional cholesterol etc..
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106349344A (en) * | 2016-08-30 | 2017-01-25 | 苏州普罗达生物科技有限公司 | L-arginine accelerant polypeptide and application thereof |
| CN108186772A (en) * | 2018-03-08 | 2018-06-22 | 南方医科大学 | A kind of method of modifying of lemon excretion body |
| CN109381706A (en) * | 2017-08-11 | 2019-02-26 | 复旦大学 | The purposes in cerebral arterial thrombosis targeting drug delivery system is being prepared by the polypeptide in activated centre of PGP |
| CN110152015A (en) * | 2018-02-11 | 2019-08-23 | 上海市第六人民医院东院 | Load human pluripotent stem cells excretion body of anti-tumor drug and preparation method thereof and purposes |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106349344A (en) * | 2016-08-30 | 2017-01-25 | 苏州普罗达生物科技有限公司 | L-arginine accelerant polypeptide and application thereof |
| CN109381706A (en) * | 2017-08-11 | 2019-02-26 | 复旦大学 | The purposes in cerebral arterial thrombosis targeting drug delivery system is being prepared by the polypeptide in activated centre of PGP |
| CN110152015A (en) * | 2018-02-11 | 2019-08-23 | 上海市第六人民医院东院 | Load human pluripotent stem cells excretion body of anti-tumor drug and preparation method thereof and purposes |
| CN108186772A (en) * | 2018-03-08 | 2018-06-22 | 南方医科大学 | A kind of method of modifying of lemon excretion body |
| CN108186772B (en) * | 2018-03-08 | 2021-02-26 | 南方医科大学 | Modification method of lemon exosomes |
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