CN102174080B - Polypeptide with brain targeted medicine delivery characteristic and preparation method thereof - Google Patents

Abstract

The invention belongs to the technical field of protein polypeptides, relates to a polypeptide with a brain targeted medicine delivery characteristic, and provides a molecular structure of the polypeptide, and application of the polypeptide to brain targeted medicine delivery. A phage monoclonal antibody which has the function of penetrating a blood brain barrier and can be enriched in a brain is obtained through repeated in-vivo Sprague Dawley (SD) outbred stock rat screening by a phage display technology; deoxyribonucleic acid (DNA) sequencing proves that the phase display polypeptides comprise a consensus sequence; meanwhile, an immunohistochemical method and in-vivo recovery rate comparison prove that the obtained polypeptide has the brain targeted medicine delivery characteristic. The polypeptide and derivative peptides thereof can be used for modifying medicine-carrying nanoparticles, liposomes, vesicles or micelles to construct a brain targeted medicine delivery system; and the delivery of medicines in the brain can be improved, the treatment effect on brain diseases is enhanced and systemic toxic and side effects are reduced.

Description

There is polypeptide of brain targeted medicine delivery characteristic and preparation method thereof

Technical field

The invention belongs to protein and peptide technical field, relate to the polypeptide with brain targeted medicine delivery characteristic, comprise the molecular structure of described peptide and in the application of preparing in brain targeting drug delivery.

Background technology

Along with the aging of human society, the sickness rate of encephalopathy is just being the trend rising year by year, and the mankind's life and health in positive serious harm.Conventionally encephalopathy comprises the diseases such as central nervous system disease (Parkinson's disease, senile dementia), cerebral tumor, cerebrovascular disease, brain virus and bacterium infection.

It is the barrier system being present between blood and cerebral tissue that prior art discloses hemato encephalic barrier (BBB), outside it can be isolated in material nonessential or harmful cerebral tissue, plays the effect of protection cerebral tissue.Brain capillary endothelial cell on hemato encephalic barrier and spongiocyte have formed tight connection jointly, are the mechanical barriers of one BBB.Studies show that, on brain capillary endothelial cell film, also there are some special enzyme systems, can make some drugs degraded, generate as Dopamine HCL decarboxylase can make L-3,4 dihydroxyphenylalanine and the decarboxylation of L-5-hyroxytrypophan the L-3,4 dihydroxyphenylalanine amine and the L-5-hydroxy-tryptamine that are difficult for seeing through BBB, thereby form the enzyme barrier of BBB; P-glycoprotein on brain capillary endothelial cell film etc. also plays a part efflux pump, and it can optionally pump objectionable impurities in brain or surplus substance outside brain, the brain delivery of restrictive substance.The above-mentioned functions of hemato encephalic barrier has maintained the constant of environment in cerebral tissue under normal physiological state, but in disease treatment process, has limited seeing through of medicine.According to the relevent statistics, after current routine clinical preparation administration, nearly 98% chemicals and almost 100% protein polypeptide or genomic medicine, because the effect of hemato encephalic barrier is difficult to into brain, have limited the treatment of encephalopathy to a great extent.Therefore, increasing medicine especially passs in the brain of protein polypeptide medicine and releases technical study and become the study hotspot of pharmaceutics and cross discipline thereof.

At present, see through in the strategy of hemato encephalic barrier increasing medicine, receptor mediated endocytosis transports that to pass release system into the nanometer of brain of greatest concern.On brain capillary endothelial cell film, there is multiple specific receptors, as TfR, insulin receptor, low density lipoprotein receptor etc.Receptor mediated endocytosis transhipment is exactly the specific binding that utilizes these acceptors and respective ligand or monoclonal antibody, makes drug-loading system (as nanoparticle, liposome etc.) to mediate corresponding medicine in the mode of endocytosis and enters brain.Wherein studying the target function base that more mediation enters brain has: Transferrins,iron complexes, Regular Insulin and monoclonal antibody OX26 etc., they all can mediate nano medicament carrying system and cross over hemato encephalic barrier, but still there is some deficiency, and as: TfR may be saturated by endogenous Transferrins,iron complexes; The Half-life in vivo of Regular Insulin is very short, and may cause hypoglycemia; The immunogenicity of OX26 and animal species selectivity are stronger, are applied to human body and need adopt anti-human Transferrin Receptor Monoclonal Antibody, and technology of preparing is comparatively complicated etc.Therefore, seek novel brain target function base, build more reasonable, effectively brain target is passed release system and is had very high researching value and clinical meaning.

It is a kind of biotechnology of screening function polypeptide that prior art discloses phage display peptide library technology, and it is expressed in rondom polypeptide library on the surface of filobactivirus, by " affine-wash-out-amplification " step of many wheels, realizes the affine screening of high-throughput.There is research to filter out the polypeptide of being combined with endothelial cell specific by display technique of bacteriophage, wherein the most representative be can with the arginine-glycine-aspartic acid of tumor vascular endothelial cell specific combination (RGD) tripeptide sequence, the long circular nanometer drug-loading system of RGD combination shows good tumor-targeting.Pasqualini etc. obtain peptide a kind of and pneumonocyte specific binding, by being connected and the delivery system of target pepx on cytolemma with Anti-adenovirus antibody.Wan Xiaomei etc. also obtain a kind of can via intranasal application olfactory mucosa enter seven peptide sequences of brain.Studies show that, the polypeptide avidity that obtains through phage display screening is high, high specificity, security are good, biological degradation and biocompatibility is good, synthetic convenient, and be easy to modify endways.

Prior art related to the present invention also has: Ito, et al. The effect of RGD peptide-conjugated magnetite cationic liposomes on cell growth and cell sheet harvesting. Biomaterials, 2005,26:6185; Trepel M, et al. Molecular adaptors for vascular-targeted adenoviral gene delivery. Hum Gene Ther, 2000,11:1971; Xiao-Mei Wan et al. Identification of nose-to-brain homing peptide through phage display. Peptides, 2009,30:343.

But so far, there is not yet by display technique of bacteriophage and screen the correlative study and the report that enter the target function base of brain through intravenous injection.

Summary of the invention

The object of this invention is to provide the related polypeptide with hemato encephalic barrier with affinity, and the molecular structure of this related polypeptide is provided.

Another object of the present invention is to provide above-mentioned related polypeptide in the purposes building in brain targeting drug delivery system.

Object of the present invention is achieved by following technical proposals:

First, adopt phage display peptide library to filter out the polypeptide with hemato encephalic barrier affinity by wheel, it comprises step: adopt screening method in rat body, two kinds of peptide storehouses by Niu Yinglun Bioisystech Co., Ltd (NEW ENGLAND Biolabs): the random ring seven peptide of phage display storehouse (Ph.D-C7C ^tMphage Display Peptide Library Kit), the random dodecapeptide of phage display storehouse (Ph.D-12 ^tMphage Display Peptide Library Kit) carry out screening in many wheel bodys, and increase elutriation dynamics by wheel, obtain phage mono-clonal brain to pathoklisis; After order-checking, obtain the aminoacid sequence of the polypeptide of SEQ ID NO:1～20; Confirm that through deoxynucleotide (DNA) order-checking those phage-displayed polypeptides include common sequence, 25% or more of described polypeptide is made up of alkaline amino acid residue (K, H or R);

In the present invention, the sequence of SEQ ID NO:1～10 is obtained by phage display random loops seven peptide library selections, and described polypeptide is made up of 9 amino acid, comprises a cyclic peptide region; The disulfide linkage that this cyclic peptide region is formed by the cysteine residues at polypeptide two ends and by cyclisation; The aminoacid sequence of 10 described cyclic peptide is respectively: CPMKSHTNC, CSTPNLMLC, CTSTSAPYC, CVCCLAGLC, CNKWNLLNC, CTHDYPLVC, CPFGSPALC, CMTRHHLDC, CWANQPATC, CMRTHHMQC;

In the present invention, the sequence of SEQ ID NO:11～20 is obtained by the screening of the random dodecapeptide of phage display storehouse; Described polypeptide is the linear peptides being made up of 12 amino acid, wherein comprise at least one or more alkaline amino acid residue (K, H or R), the aminoacid sequence of 10 described linear peptides is respectively: AHSLKSITNHGL, HHERMRLPQPPV, TGNPHATNILFR, AHSCPMKSHTNC, TPQLPKDRNADR, SISTRFHAYKLK, TGNYKALHPHNG, TGWARSTFYPHP, GPSYLHRLVPAF, DYGRDWLRYRLH;

Secondly, increase, lack or replace after 1～4 amino acid on above-mentioned any its aminoacid sequence of a polypeptide, obtaining 22 derived peptide, their essential property is similar to original peptide, is numbered respectively SEQ ID NO:21～44;

Then, above-mentioned 44 Phage Display Peptides and derived peptide are divided into four groups at random, are connected to respectively four kinds of nano carriers (nanoparticle, liposome, vesicle and micella) surface, be built into the nano-carrier that phage-displayed polypeptides or derived peptide are modified.

Through inside and outside affinity and Evaluation on Its Targeting Performance, result confirm, described polypeptide and derived peptide thereof have with hemato encephalic barrier in the special affine characteristic of brain capillary endothelial cell; Described polypeptide and derived peptide thereof are used for modifying drug-carrying nanometer particle, liposome, vesicle or micella, be built into brain targeting drug delivery system, this type systematic can improve the brain delivery of medicine, strengthens the result for the treatment of for encephalopathy, reduces the toxic side effect of general.

What the present invention obtained through screening has specific target tropism's SEQ ID NO:1～44 polypeptide to brain, show through bioinformatic analysis comparison, with known in center, the U.S. state-run biotechnology dwelling (NCBI) GenBank DNA sequence library and Swiss-Prot albumen database and albumen without homology.

Compared with prior art, the present invention has following beneficial effect:

1, the present invention screens the polypeptide obtaining, and molecular weight is little, high with the avidity of hemato encephalic barrier, high specificity, and good biocompatibility, synthetic convenient, with low cost.

2, the present invention screens the polypeptide obtaining and has good brain targeting, can be as the structure of encephalopathy targeted therapeutic carrier.

Accompanying drawing explanation

Fig. 1 is Phage Display Peptide brain targeting identified by immunofluorescence result,

In figure, shown the distribution in brain after the injection of phage mono-clonal rat tail vein, wherein A～T respectively corresponding displayed polypeptide sequence numbering be 1,5,8,11,14,16,19,20,21,24,25,28,30,31,33,36,37,40,41,44 phage.

Fig. 2 is median size and the size distribution thereof of PLGA nanoparticle.

Fig. 3 is the Zeta potential measurement result of PLGA nanoparticle.

Fig. 4 is the qualitative observation that rat brain capillary endothelial cell (BCEC cell) absorbs peptide modified nano-carrier,

In figure B-J for the sample of cellular uptake respectively: Pep3-NP, Pep6-LS, Pep11-PO, Pep14-LS, Pep21-LS, Pep26-NP, Pep29-MC, Pep31-MC, Pep33-MC;

In figure A, do not connect the nanoparticle (NP) of polypeptide for surface.

Fig. 5 is common nano-carrier, the live body distribution results of phage-displayed polypeptides decorated nanometer carrier after nude mice tail vein injection 1 h,

Figure A is contrast nude mice, tail vein injection be that surface does not connect the liposome (LS) of polypeptide; Nude mice in B-J figure respectively Wei Static arteries and veins feeds: Pep4-PO, Pep8-NP, Pep13-PO, Pep16-PO, Pep27-MC, Pep28-LS, Pep30-MC, Pep38-PO, Pep43-NP.

Embodiment

The present invention is described further by following examples, but not as restriction of the present invention.

screening in the body in the embodiment random ring seven peptide of 1 phage display storehouse

The present invention adopts Phage display random peptide library screening to have the polypeptide of hemato encephalic barrier affinity, and it comprises that step is as follows:

By the Ph.D-C7C of NEW ENGLAND Biolabs ^tMpeptide storehouse is diluted to 1 × 10 ¹⁰pfu titre, diluent is TBS damping fluid (tri methylol amino methane, Tris) (50 mM Tris, 150 mM sodium-chlor), feed in rat body by tail vein injection, after 60 min, put to death rat, physiological saline cardiac perfusion is to remove nonspecific phage in body circulation.In super clean bench, take out rat cerebral tissue, homogenate under aseptic condition adds containing 1 mM phenyl sulfonic acid fluoride (PMSF) TBS of 20 μ g/mL proteinase inhibitor (Aprotin) and 1 μ g/mL leupeptin (Leupeptin) simultaneously; With washings TBST (0.1% tween 20) flushing three times; Add glycine elution buffer (0.2M Glycine-HCl, pH 2.2) that the specific phage wash-out that enters brain is reclaimed, add 150 μ L TBS(pH 9.1) neutralization, collect supernatant, be the phage that first round screening obtains.Get appropriate gained phage, use the titre of determining phage in the ER2738 of logarithmic phase intestinal bacteria by volumetry, after remaining phage amplification, screen for next round.

Second takes turns when screening, and the tween 20 concentration in washings TBST is increased to 0.5%, and washing times is increased to 5 times, and all the other conditions, step are identical with the first round.

In third round when screening,, the tween 20 concentration in washings TBST is increased to 1%, and washing times is increased to 8 times, and all the other conditions, step are identical with the first round.

In fourth round when screening,, condition, step are identical with third round.

Measure each and take turns the phage titre that screening obtains, and taking turns result random picking from last, to go out phage mono-clonal for subsequent use.

screening in the body in the embodiment random dodecapeptide of 2 phage display storehouse

Select the Ph.D-12 of NEW ENGLAND Biolabs ^tMpeptide storehouse.First round screening method is as follows: by the Ph.D-C7C of NEW ENGLAND Biolabs ^tMpeptide storehouse is diluted to 1 × 10 ¹⁰pfu titre, diluent is TBS damping fluid (tri methylol amino methane, Tris; 50 mM Tris, 150 mM sodium-chlor), rat tail vein injection.After 60min, by rat anesthesia, under aseptic condition, extract cerebrospinal fluid 100 μ L.Get the appropriate cerebrospinal fluid titre of determining phage in the ER2738 of logarithmic phase intestinal bacteria by volumetry, after remaining amplification, screen for next round.Carry out four-wheel screening with this, measure each and take turns the phage titre that screening obtains, and taking turns result random picking from last, to go out phage mono-clonal for subsequent use.

In embodiment 1 and embodiment 2, LB substratum used is this area collective media, is the product of OXOID.

the mensuration of embodiment 3 phage titres

Every phage LB substratum of taking turns screening acquisition in embodiment 1 and embodiment 2 is carried out to the dilution of 10 multiple proportions, getting phage after 10 μ L dilutions and the intestinal bacteria ER2738 bacterium liquid of 200 μ L logarithmic phases mixes, join the LB top-agar (top-agar: every liter containing 10 g Tryptoness of 50 ℃ of preheatings, 5 g yeast extracts, 5 g sodium-chlor, 1 g magnesium chloride and 7 g agar powders, for subsequent use after autoclaving) after pour rapidly the LB plate that contains isopropyl-β-D-thiogalactoside(IPTG) and the chloro-3-indoles of the bromo-4-of 5-galactoside (IPTG/Xgal) into, spend the night, count by phage locus coeruleus number.

Titre calculation formula: phage titre (pfu)=100 × plaque number × extension rate, every 1 mL pnagus medius forms unit.

Show the enrichment of brain targeting polypeptide phage: as shown in table 1, table 2, after four-wheel screening, the gyrus yield that is screened the phage obtaining by ring seven peptide storehouse and dodecapeptide storehouse four-wheel has all improved more than 200 times than the first round, show that the phage of reclaiming from brain has obtained obvious enrichment, target phage has brain affinity.

Table 1 screens in four-wheel body for phage random ring seven peptide storehouse, and the phage rate of recovery in each brain of taking turns can calculate brain capture rate by the amount that reclaims phage from brain with the amount of feeding (%ID/g ).

Table 2 screens in four-wheel body for phage random dodecapeptide storehouse, and the phage rate of recovery in each brain of taking turns can calculate uptake ratio by the amount that reclaims phage from brain with the amount of feeding (%ID/g ).

table 1

Screening wheel number	Feed the amount of phage	In brain, reclaim the amount of phage	%ID/g
				One takes turns	1.00E+10	3.67E+04	1.44E-05
Two take turns	1.00E+10	1.51E+04	1.01E-05
				Three-wheel	1.00E+10	4.69E+04	3.18E-04
Four-wheel	1.00E+10	2.05E+05	3.93E-03

table 2

Screening wheel number	Feed the amount of phage	In brain, reclaim the amount of phage	%ID/g
				One takes turns	1.00E+10	2.25E+04	1.13E-05
Two take turns	1.00E+10	2.15E+04	2.15E-05
				Three-wheel	1.00E+10	3.02E+04	6.03E-05
Four-wheel	1.00E+10	1.45E+05	2.90E-03

embodiment 4 phage DNAs extract and order-checking

From the fourth round the selection result of embodiment 1 and embodiment 2, random choose goes out multiple phage mono-clonals respectively, knows the sequence of displayed polypeptide by order-checking after DNA extraction.

Phage mono-clonal is increased separately: after ER2738 overnight culture is diluted by 1:100, be inoculated in LB substratum, separate 1mL in culture tube, the phage mono-clonal that needs are identified is by clone's inoculation of every pipe, and 37 ℃ of shaking tables are cultivated 5 h.After medium centrifugal, 500 μ L supernatants are proceeded in clean centrifuge tube to the phage of must increasing storage liquid.In each pipe, add 200 μ L polyoxyethylene glycol/sodium-chlor (PEG/NaCl), put upside down and mix rear room temperature and place 15 min; Centrifugal 10 min, abandoning supernatant.Throw out is resuspended in to (iodide damping fluid: 10 mM Tris-HCl pH 8.0,1 mM ethylenediamine tetraacetic acid (EDTA)-EDTA, 4M NaI) in 100 μ L iodide damping fluids.Add 250 μ L ethanol; Room temperature incubation 10 min.The room temperature incubation of short period of time makes single stranded phage DNA precipitation, and most of phage albumen can remain in solution.Centrifugal 10 min, abandon supernatant, wash precipitation, of short duration vacuum-drying with 70% ethanol.Precipitation is resuspended in 30 μ L TE (10 Mm Tris-HCl pH 8.0,1 mM EDTA).

Get the above-mentioned template solution of 5 μ L to carry out automated cycle dideoxy sequencing, using-96 primers as automatic sequencing.(96 gIII sequencing primers: 5 '- ^oHcCC TCA TAG TTA GCG TAA CG-3 ', 100 pmol/ μ L, 1 pmol/mL).This primer is apart from object fragment at a distance of 96 bp, and sequencing result is as shown in SEQ ID NO:1～20 in table 3.

Table 3 is the SEQ ID NO that embodiment 1 and embodiment 2 obtain after four-wheel screening: the aminoacid sequence sequencing result of 1～20 Phage Display Peptide.

table 3

SEQ ID NO	Aminoacid sequence
		1	CPMKSHTNC
2	CSTPNLMLC
		3	CTSTSAPYC
4	CVCCLAGLC
		5	CNKWNLLNC
6	CTHDYPLVC
		7	CPFGSPALC
8	CMTRHHLDC
		9	CWANQPATC
10	CMRTHHMQC
		11	AHSLKSITNHGL
12	HHERMRLPQPPV
		13	TGNPHATNILFR
14	AHSCPMKSHTNC
		15	TPQLPKDRNADR
16	SISTRFHAYKLK
		17	TGNYKALHPHNG
18	TGWARSTFYPHP
		19	GPSYLHRLVPAF
20	DYGRDWLRYRLH

the derived peptide of embodiment 5 Phage Display Peptides is synthetic

The polypeptide of SEQ ID NO:1～20, through increasing, reduce or replacing 1～4 amino acid, is synthesized and is numbered with solid-phase synthesis respectively: 21～44 polypeptide (as shown in table 4).

The present invention screens 44 polypeptide of acquisition: SEQ ID NO:1～44, and wherein SEQ ID NO:1～10th, is obtained by the random ring seven peptide of phage display storehouse; SEQ ID NO:11～20th, is obtained by the random dodecapeptide of phage display storehouse; SEQ ID NO:21～44th, is derived and obtains through increase, minimizing or 1～4 amino acid of replacement by the polypeptide of SEQ ID NO:1～20.Show through bioinformatic analysis comparison, in the polypeptide of SEQ ID NO:1～44 and center, the U.S. state-run biotechnology dwelling (NCBI) GenBank DNA sequence library and Swiss-Prot albumen database, known gene and albumen are without homology, and domestic and foreign literature is not reported, show thus SEQ ID NO of the present invention: 1～44 polypeptide is brand-new polypeptide.

Table 4 is the aminoacid sequence of the derived peptide of the Phage Display Peptide of SEQ ID NO:21～44.

table 4

immunohistofluorescence's evaluation-hemato encephalic barrier affinity of embodiment 6 Phage Display Peptides one of is evaluated

From the phage of above-mentioned SEQ ID NO:1～44, arbitrarily choose 20 and increase, be then diluted to same titre with TBS: 1 × 10 ¹⁰pfu.Get rat, the above-mentioned phage of tail vein injection, chloral hydrate anesthesia after 60 min, routinely through left ventricle front end perfusion physiological saline; To be seenly pour into the cold paraformaldehyde of 4 % through the apex of the heart again after bleaching to lung, liver etc. and be fixed; To be seen to stopping perfusion after rat tail, liver etc. present sclerosis; In super clean bench, separate brain, fix 24 h in 4 % paraformaldehydes, 4 ℃; Contrast blank mouse and give 150 l TBS solution, same treatment; Then put into 15% sucrose solution dehydration and spend the night, then put into 30% sucrose solution to sample and be deposited to bottom completely, discard sucrose liquid, the raffinate of exhaustion sample surfaces, near the position of repairing out slice triangle intersects, with OCT embedding 5 min, Ultralow Temperature Freezer is freezing; Freezing microtome section, thick 10 μ m, are bonded on slide glass.

The treatment process of immunofluorescence section is as follows:

1. 0.01 M PBS rinsing, 5 min/ time, rinsing 3 times; 2. normal goats serum sealing, hatches 20 min for 37 ℃; 3. serum deprivation, drips primary antibodie, and primary antibodie is the mouse-anti M13 monoclonal antibody (Phamacia company product) of 1:100 1% BSA dilution, hatches 30 min for 37 ℃; 4. PBS rinsing, 5 min/ time, rinsing 3 times; 5. drip two anti-(goat anti-mouse iggs of Cy3 mark), hatch 1 h for 37 ℃; 6. PBS rinsing, 5 min/ time, rinsing 3 times; 7. 1 g/mL DAPI 8 min that dye; 8. PBS rinsing, 5 min/ time, rinsing 3 times; 9. phosphoric acid-glycerine mounting.

Fluorescence microscope result shows, what blue-fluorescence showed is the nucleus of DAPI institute mark, and red fluorescence is met two anti-upper Cy3 by phage and is sent (as shown in Figure 1), result shows, above-mentioned phage mono-clonal all has the good brain-capacity power that enters, and shows that itself and hemato encephalic barrier have stronger affinity.

What the Immunohistochemical of the present embodiment was identified employing is this area universal method, and reagent and reaction conditions related in test are also that those skilled in the art are in common knowledge.

the amplification of embodiment 7 mono-clonal phages and enter two of gyrus yield-hemato encephalic barrier affinity evaluation

Using the phage mono-clonal of 44 polypeptide of displaying of embodiment 4, embodiment 5 gained as alternative clone, increase respectively.Infectious bacteria liquid 1 mL that gets alternative clone adds in 200 mL LB nutrient solutions, incubated overnight in 37 ℃ of shaking tables.Centrifugal 20 min of 8000 rpm, obtain the supernatant containing phage after precipitation thalline, add the PEG/NaCl of 1/6 volume, and 4 ℃ of precipitations are spent the night.Centrifugal 20 min of 12000 rpm, get precipitation, with the dilution of TBS damping fluid, after centrifugal 15 min of 5000 rpm, get supernatant.Again add the PEG/NaCl of 1/6 volume, ice bath 1 h, centrifugal 20 min of 12000 rpm, precipitate after resuspended with TBS and obtain the monoclonal amplification liquid of phage, measure respectively the titre of phage by the method for embodiment 2.

With TBS mixing 10 ¹⁰the alternative mono-clonal phage of pfu, injects by SD rat tail vein.After 1 h, rat is fixed, carry out cardiac perfusion with physiological saline, until liver is faint yellow, the brain of rat is taken out, homogenate under aseptic condition after weighing, add containing PMSF (1mM) TBS of Aprotin (20 μ g/mL) and Leupeptin (1 μ g/ mL) simultaneously.With washings TBST (0.1% tween 20) flushing three times; Add glycine elution buffer (0.2M Glycine-HCl, pH 2.2) the specific phage wash-out that enters brain is reclaimed, add 150 μ L Tris-HCl (pH 9.1) neutralizations, collect supernatant and to carry out titer determination according to the method for embodiment 3 after 10 times of doubling dilutions.Units of Account weight is organized the rate of recovery (%ID/g) of pnagus medius.Result demonstration, 44 polypeptide of the present invention all have the higher brain volume that enters, and show to have good affinity with hemato encephalic barrier.

Table 5 is the mono-clonal phage rat brain rate of recovery of SEQ ID NO:1～44.

table 5

embodiment 8: the cytotoxicity test of Phage Display Peptide and derived peptide thereof

Select CCK-8 cytoactive detection method to measure the impact of SEQ ID NO:1～44 polypeptide on rat brain hair cell vascular endothelial cell BCEC cell viability, investigate with this cytotoxicity and the biocompatibility that screen gained polypeptide.

BCEC cell is with every hole 5 × 10 ⁴individual cell density is inoculated in overnight incubation in 96 porocyte culture plates.Adding respectively concentration is 0.01 mg/ mL, 0.1 mg/ mL, and the HBSS solution of SEQ ID NO:1～44 polypeptide of 1 mg/mL, hatches respectively 4 h and 24 h for 37 ℃.110 μ L fresh mediums for solution (containing CCK-8 10 μ L) are replaced, and hatch 2 h for 37 ℃; By microplate reader at 450 nm place readings; Compare with nutrient solution, calculate cell viability.Result shows, in the time that the concentration of SEQ ID NO:1～44 polypeptide is between 0.01mg/mL～1mg/mL scope, hatch after 4 h and 24 h with BCEC cell, vigor on cell does not almost affect (p > 0.05), shows extremely low cytotoxicity and good biocompatibility.

Table 6 is the cell survival rate after SEQ ID NO:1～44 polypeptide of different concns (0.01mg/ mL, 0.1mg/ mL, 1mg/mL) and BCEC cell are hatched.

table 6

embodiment 9: build polyethylene glycol-polylactic acid-polyglycolic acid (PLGA) nanoparticle that phage-displayed polypeptides or derived peptide are modified

Selecting biodegradable PLGA is material, prepare maleimide-polyoxyethylene glycol (Maleimide-PEG, MAL-PEG) and the co-modified poly(lactic acid)-polyglycolic acid nanoparticle (NP in methoxy poly (ethylene glycol) (MPEG) surface by emulsification/menstruum method of evaporation _pLGA); And random choose goes out 10 polypeptide (being respectively 1,2,3,7,8,18,26,35,43, No. 44) for decorated nanometer grain (PepX-NP from the phage-displayed polypeptides of SEQ ID NO:1～44 or derived peptide _pLGA, X=1,2,3,7,8,18,26,35,43,44).The median size of preparing gained nanoparticle is about 100 nm(as shown in Figure 2), Zeta potential is about-20 mV(as shown in Figure 3).

embodiment 10: build phage-displayed polypeptides or derived peptide modified liposome

Adopt distearoyl phosphatidylcholine (DSPC), cholesterol (Chol) and distearyl acid phosphatidylethanolamine-Macrogol 2000-carboxyl commissure thing (DSPE-PEG2000-COOH) (amount of substance is than being 6:3.0:0.6) as the film material of preparing liposome.Film material is dissolved in organic solvent (chloroform-methanol: 2:1), adopts film dispersion method to prepare liposome.From the phage-displayed polypeptides of SEQ ID NO:1～44 or derived peptide, random choose goes out 12 polypeptide (being respectively 6,9,10,12,14,15,19,21,24,28,37,41) for decorated nanometer grain.Be connected the peptide modified liposome (PepX-LS, X=6,9,10,12,14,15,19,21,24,28,37,41) of preparation with the amino of Phage Display Peptide by the pendant carboxylic group end on film material DSPE-PEG2000-COOH.The liposome median size making is 60 nm ± 12nm, and Zeta potential is in-20 mV left and right.

embodiment 11: build phage-displayed polypeptides or derived peptide and modify vesicle

Adopt biodegradable polyethylene glycol-caprolactone (PEG-PCL) to prepare polymkeric substance vesicle, from the phage-displayed polypeptides of SEQ ID NO:1～44 or derived peptide, random choose goes out 11 polypeptide, PEG-PCL polymkeric substance vesicle is connected and is prepared peptide modified vesicle (PepX-PO, X=4,5,11,13,16,22,23,25,32,34,38) by the dimaleoyl imino on vesicle surface with the phage-displayed polypeptides (numbering is respectively: 4,5,11,13,16,22,23,25,32,34, No. 38) of sulfhydrylation.Prepared peptide modified vesicle (PepX-PO) median size is 130 ± 10 nm, and Zeta potential is in-24 mV left and right.

embodiment 12: build phage-displayed polypeptides or derived peptide and modify micella

By 11 polypeptide that random choose goes out from the Phage Display Peptide of SEQ ID NO:1-44 or derived peptide, (numbering is respectively: 17, 20, 27, 29, 30, 31, 33, 36, 39, 40 and 42) be connected on PEG2000-DSPE (PEG-DSPE), press the mixture (mol ratio 1:100) of polypeptide-PEG2000-DSPE (PEG-DSPE) and methoxy poly (ethylene glycol)-DSPE (MPEG-DSPE) as solid support material, be prepared into respectively peptide modified polymer micelle (PepX-MC, X=17, 20, 27, 29, 30, 31, 33, 36, 39, 40, 42).The micella median size making is in about 20nm, and Zeta potential is in-15mV left and right.

qualitative observation-the brain targeting of embodiment 13:BCEC cellular uptake Phage Display Peptide or derived peptide decorated nanometer carrier one of is evaluated

By BCEC cell with 10 ⁴density be inoculated on the coated slide glass of poly-lysine, cultivate 24 h and approach 70 ~ 80% to cell degree of converging.Cell is first hatched 15min in advance with HBSS, from embodiment 9, 10, 11, in 12, random choose goes out 9 kinds of nano-carrier: Pep3-NP, Pep6-LS, Pep11-PO, Pep14-LS, Pep21-LS, Pep26-NP, Pep29-MC, Pep31-MC, Pep33-MC, take Coumarin-6 as fluorescent probe, above-mentioned 9 kinds of nano-carriers are added respectively on BCEC cell, the common nanoparticle that does not connect polypeptide with surface compares, at 37 ℃, hatch after 1 h, discard solution, cell washes away the nano-carrier that is adsorbed on cell surface with PBS, taking-up slide is put into 4% paraformaldehyde room temperature and is fixed 20 min, with after PBS rinsing three times, with fluorescence mountant (phospho-glycerol) mounting, fluorescence microscopy Microscopic observation.Result shows, nano-carrier after phage-displayed polypeptides or derived peptide modification is all significantly better than the not modified common nanoparticle in surface by the green fluorescence of cellular uptake, and prompting BCEC will be significantly higher than common nanoparticle (as shown in Figure 4) to the picked-up of the nano-carrier after peptide modified.

two of quantitative analysis-brain targeting evaluation of embodiment 14:BCEC cellular uptake phage-displayed polypeptides or derived peptide decorated nanometer carrier

By BCEC cell with 10 ⁴density be inoculated in 24 well culture plates and cultivate 1 day.Take Coumarin-6 as fluorescent probe, from embodiment 9,10,11,12, random choose goes out 22 kinds of nano-carriers, is respectively: Pep1-NP, Pep2-NP, Pep5-PO, Pep7-NP, Pep10-LS, Pep12-LS, Pep15-LS, Pep17-MC, Pep18-NP, Pep19-LS, Pep20-MC, Pep22-PO, Pep23-PO, Pep24-LS, Pep25-PO, Pep32-PO, Pep36-MC, Pep37-LS, Pep40-MC, Pep41-LS, Pep42-MC, Pep44-NP.Add in culture hole with the concentration of 10 μ g/mL respectively, every hole 1 mL, puts into 37 ℃ of incubators and cultivates 1 h, investigates the picked-up situation of BCEC to various nano-carriers.After experiment finishes, with 1%Triton-X100 400 μ L lysing cell, measure protein concentration in cell pyrolysis liquid by BCA method, divided by corresponding protein concentration, obtain uptake index (Uptake Index, UI, μ g/mg) with the concentration of nano-carrier.Result is as shown in table 7, the amount of nanoparticle, liposome, vesicle and the micella of cellular uptake after Phage Display Peptide or derived peptide are modified, and all the amount of the not modified common nanoparticle of specific surface, liposome, vesicle, micella wants high 2～5 times.

Table 7 is BCEC cellular uptake Phage Display Peptide or derived peptide decorated nanometer carrier quantitation analytical results.

table 7

Nano-carrier	Nano-carrier concentration (ng/mL)	Protein concentration (μ g/mL)	UI(μg/mg)
				NP	710.84±31.13	60.42±4.52	11.76±2.53
LS	722.04±19.42	74.24±4.32	9.73±5.42
				PO	897.3±23.42	70.83±5.42	12.67±4.83
MC	730.24±34.23	83.53±3.45	8.74±4.89
				Pep1-NP	2238.53±35.23	78.97±6.342	28.34±2.94
Pep2-NP	2648.03±18.23	80.35±3.85	32.95±1.20
				Pep5-PO	2353.6±35.23	86.32±2.57	27.26±2.45
Pep7-NP	2436.94±41.22	84.63±6.42	28.79±5.42
				Pep10-LS	1983.64±39.32	68.64±4.32	28.89±3.93
Pep12-LS	2046.35±29.42	83.59±5.42	24.48±4.21
				Pep15-LS	1952.67±31.99	74.08±6.39	26.35±5.32
Pep17-MC	1835.64±15.76	57.35±3.05	32.00±2.94
				Pep18-NP	2458.93±63.32	85.39±4.52	28.79±8.32
Pep19-LS	1750.32±34.08	60.69±3.67	28.84±3.98
				Pep20-MC	2463.98±53.29	63.86±4.67	38.58±1.36
Pep22-PO	2695.74±29.42	70.24±2.08	38.37±4.42
				Pep23-PO	2543.5±35.42	63.74±4.90	39.90±3.84
Pep24-LS	2865.42±52.28	73.67±3.83	38.89±4.42
				Pep25-PO	1962.84±37.23	49.53±3.965	39.62±5.32
Pep32-PO	2046.84±57.32	68.2±5.28	30.01±3.576
				Pep36-MC	1724.62±24.21	70.43±3.67	24.48±2.45
Pep37-LS	1973.24±39.28	59.28±4.52	33.28±3.57
				Pep40-MC	2944.57±45.42	80.42±3.92	36.61±4.2
Pep41-LS	2953.62±30.52	69.4±4.57	42.55±4.52
				Pep42-MC	1856.93±32.08	73.67±6.25	25.20±3.65
Pep44-NP	1974.6±29.34	69.76±7.32	28.30±5.42

embodiment 15: three of the live body distribution observation-brain targeting evaluation of Phage Display Peptide or derived peptide decorated nanometer grain

Utilize nir dye DiR to investigate the DYNAMIC DISTRIBUTION situation of nanoparticle in nude mouse.From embodiment 9,10,11,12, random choose goes out 9 kinds of nano-carriers, is respectively: Pep4-PO, Pep8-NP, Pep13-PO, Pep16-PO, Pep27-MC, Pep28-LS, Pep30-MC, Pep38-PO, Pep43-NP.Choose 1 nude mice in contrast, tail vein injects the liposome that carries DiR.Select 9 nude mices else, tail vein injects above-mentioned 9 kinds of nano-carriers respectively.Inject after 1 h, utilize small animal living body imager observe and take a picture.Select the wavelength of transmitted light of 780 nm as DiR, fixing each exposure time is 60 ms.Result demonstration, the nanoparticle after peptide modified can be distributed into rapidly brain; Fluorescence intensity in its brain is significantly better than common nanoparticle, shows that Phage Display Peptide or derived peptide decorated nanometer grain that the present invention screens acquisition have good brain drug delivery performance (as shown in Figure 5).

embodiment 16: four of the cerebral tissue distribution-brain targeting evaluation of Phage Display Peptide or derived peptide decorated nanometer grain

The present embodiment is from the brain targeting of tissue distribution angle quantitative expedition Phage Display Peptide or derived peptide decorated nanometer carrier.Select Coumarin-6 as fluorescent probe, from embodiment 9,10,11,12, random choose goes out 4 kinds of nano-carriers, that is: Pep9-LS, Pep34-PO, Pep35-NP, Pep39-MC.The four kinds of nano-carriers (LS, PO, NP, MC) that do not connect any polypeptide with surface as a control group.ICR male mice is divided into 8 groups at random, 36 every group.By the dosage of the 300 μ g/kg Coumarin-6s above-mentioned 8 kinds of nano-carriers of tail vein injection respectively, respectively at 0.083,0.25,0.5,1,2,4,8,12 and 24 h after injection, ICR mouse is carried out to cardiac perfusion, take out after cerebral tissue is weighed and carry out tissue sample processing, the fluorescence intensity in high effective liquid chromatography for measuring tissue sample.

Adopt target index D TI to evaluate the brain targeting of above-mentioned nano-carrier.As shown in table 8, compared with conventional liposome, the brain target index of the liposome after peptide modified is 3.7; Compared with common vesicle, the brain target index of the vesicle after peptide modified is 2.4; Compared with common nanoparticle, the brain target index of the nanoparticle after peptide modified is 2.6; With common micellar phase ratio, the brain target index of the micella after peptide modified is 2.2.Reconfirm that the present invention screens the Phage Display Peptide of acquisition or the nano-carrier of derived peptide modification has good brain targeting.

Table 8 is the brain target index of Phage Display Peptide and derived peptide decorated nanometer carrier.

table 8

SEQUENCE LISTING

<110> Fudan University

<120> has polypeptide of brain targeted medicine delivery characteristic and preparation method thereof

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<160> 44

<170> PatentIn version 3.3

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Claims (5)

1. the polypeptide with brain targeted medicine delivery characteristic, is characterized in that, described polypeptide is as shown in SEQ ID NO:17.

2. by the polypeptide with brain targeted medicine delivery characteristic claimed in claim 1, it is characterized in that, the polypeptide of the aminoacid sequence of described SEQ ID NO:17 is obtained by the screening of the random dodecapeptide of phage display storehouse; Described polypeptide is the linear peptides being made up of 12 amino acid.

3. the purposes in the special affinity characteristic preparation of brain capillary endothelial cell of the polypeptide with brain targeted medicine delivery characteristic of claim 1 in preparation and hemato encephalic barrier.

4. the polypeptide with brain targeted medicine delivery characteristic of claim 1 is in the purposes of preparing in brain targeting drug delivery system.

5. by the purposes of claim 4, wherein said brain targeting drug delivery system is that peptide modified drug-carrying nanometer particle, liposome, vesicle or the micella of the aminoacid sequence of SEQ ID NO:17 made.