CN106512021A

CN106512021A - Paclitaxel-loading asymmetric dendrimer nanometer drug carrier system and preparation method thereof

Info

Publication number: CN106512021A
Application number: CN201611152662.6A
Authority: CN
Inventors: 罗奎; 顾忠伟; 李宁
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2016-12-14
Filing date: 2016-12-14
Publication date: 2017-03-22

Abstract

The invention provides a paclitaxel-loading asymmetric dendrimer nanometer drug carrier system. The drug carrier system is a conjugate formed by the antitumor drug, namely, paclitaxel, the tumour endoenzyme sensitive oligopeptide GFLG and PEGylated asymmetric peptide dendrimers. The system satisfies all the SAIR requirements as the efficient drug delivery system, and by combining with the asymmetric peptide dendrimers, the PEG modification, the nanoscale system and the features of enzyme-sensitive connecting bonds, the nano particles show the anti-tumor treatment index and biosecurity obviously enhanced compared with those of free PTX during the in-vivo treatment.

Description

Paclitaxel loaded asymmetric dendrimer nano medicament carrying system and preparation method thereof

Technical field

The invention belongs to the preparation field of macromolecule targeted nano carrier, and in particular to a kind of paclitaxel loaded asymmetric Dendrimer nano medicament carrying system and preparation method thereof.

Background technology

At present, chemotherapy faces many challenges, and such as medicine is distributed so as to the toxic and side effect for causing in the high of normal structure, And tumor tissues low enrichment and caused anticancer effect for making us less being satisfied with etc..Many tactful studied applications are carrying Effect of high anticarcinogen, these strategies can be abbreviated as altogether SAIR (Stability, Accumulation, Internalization and Release), that is, improve stability (Stability) of the medicine in cyclic process, by master Dynamic or passive target strengthen medicine tumor tissues Enriching accumulation (Accumulation), promote medicine to rapidly enter tumour Cell is realized internalization (Internalization) and accelerates medicine rate of release (Release) in the cell.These strategies In some reached the purpose for strengthening some specific drugs characteristics, but successfully do not realize that anticancer effect is omnibearing and carry Rise.

Taxol (PTX) is a kind of cytotoxic efficient cancer therapy drug of tool.However, which can be fast in the circulatory system Remove and cause the toxic and side effects such as bone marrow toxicity fastly, these unfavorable factors limit the application of taxol.Additionally, one Individual critically important aspect is that the fracture of PTX itself ester bonds can cause which to be degraded in the circulatory system.

Peptides dendrimer is the macromolecular that a class is made up of amino acid, the accurate cladodification of the structure which has class tree-like Structure.They have many premium properties, such as structure controllable precise, the structure of highly -branched, highdensity functional end-group, tool There are water solubility and good biological safety, these rush can become potential drug delivery vehicle.Additionally, by two kinds Chemical functional concentrates on its kernel respectively and the asymmetric dendrimer on top layer can undertake different task and goals.Due to Different functions is concentrated in an independent molecule, asymmetric dendrimer is compared with common fan-shaped or spherical tree-shaped big point Son can provide more features and wider purposes.However, the carrier of the dendrimer of low algebraically its particle diameter is less than 10nm, its particle diameter are not big enough, it is impossible to which meeting strengthens the standard of infiltration and retention effect (EPR effects).Although in addition, high algebraically Dendrimer has relatively large size, and its pole in cyclic process is difficult to be degraded and increased induction cytotoxicity Risk.

The intravenous injection (i.v.) of nano-particle would generally occur plasma protein suction-operated, and the process is that everybody institute is ripe The opsonification known.The method for reducing or reducing opsonification at present is concentrated mainly on introducing hydrophilic radical or to grain Son is modified modification and makes its surface charge into electroneutral or electronegativity.Polyethylene glycol (PEG) can reinforcing material water-soluble and The stability of plasma protein, while reducing immunogenicity.Using polyethylene glycol come modification nano-carrier, so as to reduce monokaryon Macrophage system (MPS) is swallowed to the non-specific cellular of nano-carrier, at the same time can also adjust the circulation of nano-particle Half-life.Research shows that the dendrimer drug delivery vehicle of PEG modifications possesses effective anticancer function.However, carrying The highdensity PEG in body surface face has interfered with intake of the cell to it.

Cathepsin B is a kind of lysosomal cysteine protease, and which is in the major part tumour cell such as breast cancer and swells Overexpression in knurl endothelial cell.These provide more thinkings for the design of enzyme response type drug delivery vehicle.Glycine-benzene Four fragments of peptides (Gly-Phe-Leu-Gly of Ala-Leu-glycine；GFLG), be cathepsin B a kind of preferable bottom Thing；Which has been used for connecting anticarcinogen in macromolecular, and these macromolecular-drug conjugates shown in blood plasma and Characteristic with good stability during serum transport, while in rapidly realizing intracellular lysosome after encytosis The release of medicine.Such as patent of invention CN201310511453.6, there is provided a kind of PEGization peptides dendrimer based on GFLG The preparation method of delivery system.The delivery system is PEGization peptides dendrimer, targeted functional factor GFLG and resists swollen Knurl treats the conjugate of the factor, will be treated after the factor is connected with PEGization peptides dendrimer by GFLG and forms functionalization Dendrimer, while good biocompatibility is obtained, makes Isodose or the medicine of low dosage reach significantly anti-swelling Knurl curative effect.But the delivery system is hydrophily dendrimer, and biocompatibility is poor.

The content of the invention

Based on above technical problem, the invention provides a kind of paclitaxel loaded asymmetric dendrimer nano drug-carrying System.The system meets all SAIR requirements as efficient drug delivery system, by tree-shaped big with reference to asymmetric peptides Molecule, PEG are modified, the feature of nano-scale systems and enzyme sensitivity connecting key, and nano-particle is presented in treating in vivo than trip The antineoplaston index significantly increased from PTX and biological safety.

In order to realize foregoing invention purpose, the present invention is adopted the following technical scheme that：

Paclitaxel loaded asymmetric dendrimer nano medicament carrying system, the drug-loading system are antineoplastic Japanese yew The conjugate of alcohol, tumour endocellular enzyme sensitivity small peptide GFLG and the asymmetric peptides dendrimer of PEGization；Concrete structure is as follows：

A_n-B

Wherein, A is：

B is the asymmetric lysine peptides dendrimer of PEGization with ethylenediamine as core, the macromolecular be G1L-G2L, The asymmetric dendrimer of G2L-G3L or G3L-G4L, PEG modifications occur less in the asymmetric dendrimer algebraically One end, A instead of the terminal amino group of the other end, n=1-16.

Asymmetric dendrimer of the present invention is G2L-G3L dendrimers.Maximize drugloading rate and most appropriate Hydrophilic group modification.

The preparation method of paclitaxel loaded asymmetric dendrimer nano medicament carrying system, is first respectively synthesized Azide The PEGization dendrimer of GFLG-PTX and alkynyl, then by the click-reaction of copper catalysis synthesize end-product dendrimer- Taxol conjugate.

Preferably, the synthesis of Azide GFLG-PTX is as follows：

Using CH₃ONa is by N₃- GFLG-OMe sloughs methyl esters protection, by product N₃- GFLG-OH is placed under blanket of nitrogen, DCM is used as solvent for reaction system, adds DMAP and dicyclohexylcarbodiimide (DCC), after reaction half an hour, adds PTX, instead Answer 48 hours；Insoluble matter is filtered to remove, is spin-dried for removing solvent；Ethyl acetate lysate is used again, and organic phase is used saturation respectively NaHCO₃After the washing of solution, watery hydrochloric acid and saturation NaCl solution, then drying, filtration, vacuum rotary steam concentration, recrystallization are obtained final product.

Preferably, the synthesis of the asymmetric dendrimer nano medicament carrying system is as follows：

The synthesis of the asymmetric dendrimer of A, alkynyl

With one end as Cbz radical protections, the other end is raw material for the asymmetric dendrimer of Boc radical protections, in hydrogen Atmosphere is enclosed lower and Pb/C reactions and sloughs Cbz protection groups, after reaction terminates, in N₂Under atmosphere protection, by product, HBTU and HOBT It is dissolved in dry DMF, with DIPEA and 5- hexin acid reactions under ice bath；

The synthesis of B, PEGization dendrimer

PEG of the product of step A with Azide is dissolved in containing CuSO₄·5H₂In the water of O and sodium ascorbate, reaction terminates Afterwards, solvent and impurity are removed using bag filter dialysis, the solution stayed in after dialysis in bag filter is collected into lyophilized that mPEG is modified Dendrimer；

The alkynyl of C, PEGization dendrimer

In N₂Under atmosphere protection, PEGization dendrimer is dissolved in anhydrous methylene chloride, trifluoroacetic acid is then added dropwise to, After stirring 4 hours, solvent is removed, sample after vacuum drying, is collected；In N₂Atmosphere protection under, by the sample for obtaining, HATU, HOAt with 5- hexynic acids are dissolved in dry DMF, under ice bath, add DIPEA reactions；

The synthesis of D, dendrimer-taxol conjugate

Under the protection of Ar atmosphere, the product that step C is obtained is dissolved in containing CuSO with the GFLG-PTX of Azide₄·5H₂O and anti- In the water of bad hematic acid sodium, after reaction terminates, solvent and impurity are removed using bag filter dialysis, will be stayed in bag filter after dialysis Solution is collected to freeze and obtains final product dendrimer-taxol conjugate.

It is further preferred that described step A and step C, after the completion of reaction, vacuum distillation removes solvent, and adds EtOAc, organic phase is used saturation NaHCO respectively₃Respectively washing three times of solution, watery hydrochloric acid and saturation NaCl solution；Use anhydrous slufuric acid Magnesium is dried, and is filtered to remove solution decompression concentrated by rotary evaporation after magnesium sulfate, is recrystallized using ethylacetate/ether, obtained final product.

It is further preferred that in described step B and D steps, the molecular cut off of bag filter is 3500.

It is further preferred that in described step B and D steps, ethylenediamine tetra-acetic acid two is added in the deionized water of dialysis Sodium salt, help are dialysed except the copper ion in dereaction.

It is further preferred that described step B and D steps, the solution stayed in bag filter are collected freeze after dialysis The solid dissolving for arriving, using exclusion chromatography separating-purifying product, lucifuge collects product, then freezes Jing after pure water dialysis and obtain final product product Thing.

The beneficial effects of the present invention is：

1st, the present invention has successfully constructed and has integrated the PEGylated dendrimer-GFLG-PTX of SAIR functions and receive Rice corpuscles, has carried out comprehensive structure design and excellent to asymmetric PEGylated dendrimer-GFLF-PTX nano-particles Change, be a kind of safely and efficiently drug delivery system.Intelligent macromolecular carrier is used for into the delivery of dewatering medicament taxol so as to Maximum stable state is kept in cyclic process and the quick insoluble drug release of intracellular can be realized after reaching tumour.Comprehensively effectively Improve taxol antitumous effect and have good biological safety.

2nd, using the drug-loading system of amphipathic asymmetric dendrimer, for high algebraically dendrimer in cyclic process Middle pole is difficult the risk that be degraded and increased induction cytotoxicity, therefore adopts low algebraically dendrimer；Simultaneously for low Algebraically particle diameter is not big enough, it is impossible to which meeting strengthens the standard of infiltration and retention effect (EPR effects), is modified using PEG and from group Solving, the dendrimer for specially modifying low algebraically using PEG forms amphipathic asymmetric tree-shaped big point to the means of dress Son, hydrophobic group concentrate on its kernel, and hydrophilic radical concentrates on its top layer, and the property for recycling which amphipathic realizes material The self assembly of material, is self-assembled into as the suitable nano-particle of size.

3rd, drug-loading system of the invention meets all SAIR requirements as efficient drug delivery system.As medicine is It is covalently attached on carrier by an enzyme sensitivity small peptide, and asymmetric dendrimer was optimized using PEG modifications, this resists Cancer medicine taxol rapidly can be discharged from nano-particle in the presence of enzyme, and remains original structure, should Nano-particle can effectively induce the apoptosis of breast cancer cell, compare using free drug taxol, its poison to normal cell Property is much lower.By with reference to asymmetric peptides dendrimer, PEG modified, nano-scale systems and enzyme sensitivity connecting key Feature, nano-particle present antineoplaston index and the biological safety that specific ionization PTX is significantly increased in treating in vivo.

4th, preparation method of the present invention has synthesized the asymmetric dendrimer of the accurate alkynyl of structure by liquid phase method, profit Reacted with two steps " click " and mPEG is efficiently accurately connected on dendrimer with the sensitive short peptide-drug conjugates of enzyme.Adopt Dendrimer with Cbz radical protections is raw material, can optionally deprotection as needed, optionally to asymmetric big point The high algebraically end of son is modified, and otherwise just cannot get asymmetric dendrimer, but spherical dendrimer.

Description of the drawings

Fig. 1 is the mass spectral characteristi figure of asymmetric dendrimer PEG-G2L-G3L.

Mass spectral characteristi figures of the Fig. 2 for PEGylated dendrimer-GFLF-PTX.

DLS water phase particle diameter (A), AFM (B) of the Fig. 3 for PEGylated dendrimer-GFLF-PTX nano-particles, and Zeta current potentials (C) are characterized.

Fig. 4 is to detect insoluble drug release by HPLC.PTX parenteral solutions (A) and it is incubated 48 hours through papain The HPLC of PEGylated dendrimer-GFLF-PTX (B) goes out peak figure.

Fig. 5 is incubated 24 for PEGylated dendrimer-GFLF-PTX nano-particles in the cushioning liquid that pH is 5.4 The water phase change of size of hour.

Fig. 6 changes for the particle diameter pattern of PEGylated dendrimer-GFLF-PTX nano-particles.

Internal anti-tumor experiment evaluations of the Fig. 7 for nano-particle.Tumor-bearing mice has injected physiological saline, taxol respectively Parenteral solution and PEGylated dendrimer-GFLF-PTX nano-particles.(A) Relative tumor size variation curve.(B) mouse Changes of weight curve.(C) knurl weight, (D) tumour inhibiting rate.

Fig. 8 is the Mouse Weight change curve of 19 days treatment cycles.

Fig. 9 is that mouse takes blood and carries out routine blood test detection and analysis after the treatment cycle of 19 days.

Specific embodiment

The essentiality content of the present invention is described in further detail with reference to specific embodiment.

Embodiment 1

Paclitaxel loaded asymmetric dendrimer nano medicament carrying system, the drug-loading system are antineoplastic Japanese yew The conjugate of alcohol, tumour endocellular enzyme sensitivity small peptide GFLG and the asymmetric peptides dendrimer of PEGization；

Concrete structure is as follows：

A_n-B

Wherein, A is：

B is the asymmetric lysine peptides dendrimer of PEGization with ethylenediamine as core, the macromolecular be G1L-G2L, The asymmetric dendrimer of G2L-G3L or G3L-G4L, PEG modifications occur less in the asymmetric dendrimer algebraically One end, A instead of the terminal amino group of the other end.

Couplet I：N=1, B are the G1L-G2L of PEGization for asymmetric dendrimer.

Couplet II：N=4, B are the G1L-G2L of PEGization for asymmetric dendrimer.

Couplet III：N=5, B are the G2L-G3L of PEGization for asymmetric dendrimer.

Couplet IV：N=8, B are the G2L-G3L of PEGization for asymmetric dendrimer.

Couplet V：N=16, B are the G3L-G4L of PEGization for asymmetric dendrimer.

Embodiment 2

Embodiment 3

The present embodiment is on the basis of embodiment 2：

The synthesis of Azide GFLG-PTX is as follows：

Using CH₃ONa is by N₃- GFLG-OMe sloughs methyl esters protection, and reacted N3-GFLG-OH is placed under blanket of nitrogen, DCM is used as solvent for reaction system；Add after being dissolved in the DMAP and DCC reaction half an hour of DCM, add PTX.Low temperature stirring is anti- After answering 48h, stop reaction.Insoluble matter is filtered to remove, is spin-dried for removing solvent；Ethyl acetate lysate is used again, by organic phase point Yong not respectively washing three times of saturation NaHCO3 solution, watery hydrochloric acid and saturation NaCl solution；It is dried with anhydrous magnesium sulfate, is filtered to remove sulphur By solution decompression concentrated by rotary evaporation after sour magnesium, recrystallized using ethylacetate/ether, obtained final product, MALDI-TOF MS test results are m/z:1733.8([M+H]⁺)。

Embodiment 4

The preparation method of paclitaxel loaded asymmetric dendrimer nano medicament carrying system, comprises the following steps：

The synthesis of the asymmetric dendrimer of A, alkynyl

Dendrimer with Cbz radical protections sloughs Cbz protection groups with Pb/C reactions under an atmosphere of hydrogen as raw material, After reaction terminates, in N₂Atmosphere protection under, product, HBTU and HOBT are dissolved in dry DMF, under ice bath with DIPEA and 5- hexin acid reactions；

The synthesis of B, PEGization dendrimer

The alkynyl of C, PEGization dendrimer

The synthesis of D, dendrimer-taxol conjugate

Embodiment 5

The present embodiment is on the basis of embodiment 4：

In described step B and D steps, the molecular cut off of bag filter is 3500.

Embodiment 6

The present embodiment is on the basis of embodiment 4：

In described step B and D steps, the molecular cut off of bag filter is 3500.

In described step B and D steps, in the deionized water of dialysis, disodium EDTA is added.

Embodiment 7

The present embodiment is on the basis of embodiment 4：

In described step B and D steps, the molecular cut off of bag filter is 3500.

The solution stayed in after dialysis in bag filter is collected the lyophilized solid dissolving for obtaining by described step B and D steps, Using exclusion chromatography separating-purifying product, lucifuge collects product, then freezes Jing after pure water dialysis and obtain final product product.

Embodiment 8

The present embodiment is on the basis of embodiment 4：

In described step B and D steps, the molecular cut off of bag filter is 3500.

Described step A and step C, after the completion of reaction, vacuum distillation removes solvent, and adds EtOAc, by organic phase Saturation NaHCO is used respectively₃Respectively washing three times of solution, watery hydrochloric acid and saturation NaCl solution；It is dried with anhydrous magnesium sulfate, is filtered to remove By solution decompression concentrated by rotary evaporation after magnesium sulfate, recrystallized using ethylacetate/ether, obtained final product.

Embodiment 9

The synthetic route of the asymmetric dendrimer nano medicament carrying systems of G2L-G3L is as follows：

The synthesis of compound Alkyne-G2L-G3L

In N₂Under atmosphere protection, using Pd/C in high pressure H₂The Cbz protection groups of G2L-G3L (4.7g, 2mmol) are sloughed under atmosphere. N₂Under atmosphere protection, by sloughing, white solid, HBTU (10.6g, 12mmol) that protection group obtains are molten with HOBT (4.4g, 12mmol) In 100mL dry DMFs.Under ice bath, be slowly dropwise added dropwise to 6mLDIPEA (39mmol) and 5- hexynic acids (1.4mL, 12mmol).Which under ice bath is continued to stir half an hour, is then reacted 72 hours at room temperature.After reaction terminates, vacuum distillation Solvent is removed, and adds 300mL EtOAc, organic phase is used saturation NaHCO respectively₃Solution, watery hydrochloric acid (1M) and saturation NaCl Solution respectively washing three times.It is dried with anhydrous magnesium sulfate, is filtered to remove solution decompression concentrated by rotary evaporation to 15mL after magnesium sulfate.Utilize Ethylacetate/ether is recrystallized, and obtains white solid 4.8g, yield 96%.1H NMR(400MHz,CDCl₃), δ=5.30 (s, 4H,CHCCH₂),4.11(m,10H,COCH(NH)-CH₂),3.07-3.73(t,32H,CH₂CH₂-NH and CHCCH₂CH₂CH₂CO),2.04(t,8H,CHCCH₂CH₂),1.26-1.73(m,68H,CH₂-Lys,CHCCH₂CH₂CH₂CO and s,72H,CH₃-Boc)。MALDI-TOF MS:m/z 2542[(M+Na)⁺,C1₂₆H₂₁₆N₂₂O₃₀Na⁺]。

The synthesis of compound mPEG-G2L-G3L

Compound a lkyne-G2L-G3L (0.5g, 0.2mmol) is dissolved in into 40mL water with the PEG (2g, 1mmol) of Azide (contain 5mol%CuSO₄·5H₂O and 10mol% sodium ascorbates) in.Stirring reaction 24 hours at 50 DEG C.Reaction terminates Afterwards, using bag filter, (molecular weight cute off (MwCO=3500) dialysis removes solvent and impurity, and period exists Substantial amounts of disodium EDTA (EDTA-Na is added in the deionized water of dialysis₂) help dialyse except the copper in dereaction Ion.The solution stayed in after dialysis in bag filter is collected and freezes to obtain yellow solid.Exclusion chromatography will be utilized after the solid dissolving Method, using Superose 12HR/10/300GL chromatographic columns, passes throughFPLC systems (GE Healthcare) are carried to separate Pure products (by the use of the aqueous sodium acetate solution containing 30% acetonitrile that pH is 6.5 as the mobile phase of pillar).To receive under the conditions of lucifuge The product section for collecting using pure water dialysis after freeze yellow mPEG modification dendrimer product (mPEG-G2L-G3L) 2.0g, yield 92.7%.MALDI-TOF MS:m/z 10710,[M+Na]⁺.The conjunction of compound mPEG-G2L-G3L-alkyne Into

In N₂Under atmosphere protection, compound mPEG-G2L-G3L (480mg, 0.05mmol) is dissolved in the anhydrous DCM of 2mL.Will The solution is stirred 5 minutes under ice bath, is then added dropwise to 2mL TFA.After stirring 4 hours, remove solvent and to be vacuum dried 1 little When collect sample.In N₂Under atmosphere protection, by the sample for obtaining before, HATU (760mg, 2mmol), HOAt (272mg, 2mmol) It is dissolved in 30mL dry DMFs with 5- hexynic acids (0.22mL, 2mmol).Under ice bath, it is slowly added dropwise into quantitative DIPEA (1.3mL,8mmol).Which under ice bath is continued to stir half an hour, is then reacted 72 hours at room temperature.After reaction terminates, subtract Pressure distillation removes solvent, and adds 300mL EtOAc, and organic phase is used saturation NaHCO respectively₃Solution, watery hydrochloric acid (1M) with it is full With NaCl solution respectively washing three times.It is dried with anhydrous magnesium sulfate, is filtered to remove solution decompression concentrated by rotary evaporation after magnesium sulfate extremely 15mL.Recrystallized using ethylacetate/ether, obtain brown solid (mPEGylated G2L-G3L-alkyne) 470mg, yield 88.2%.MALDI-TOF MS:m/z 10651[M+Na]⁺。

The synthesis of compound PEGylated Peptide Dendrimer-GFLG-PTX

Under the protection of Ar atmosphere, by compound PEG-G2L-G3L-alkyne (540mg, 50 μm of ol) and the GFLG- of Azide PTX (680mg, 0.50mmol) is dissolved in 100mL water (containing 5mol%CuSO₄·5H₂O and 10mol% sodium ascorbates) in. Stirring reaction 24 hours at 50 DEG C.After reaction terminates, using bag filter (molecular weight cute off (MwCO= 3500) dialysis removes solvent and impurity, and period adds substantial amounts of disodium EDTA in the deionized water of dialysis (EDTA-Na₂) help dialyse except the copper ion in dereaction.The solution stayed in after dialysis in bag filter is collected into yellowishly lyophilized Color solid.Exclusion chromatography will be utilized after the solid dissolving, using 12 HR/10/300GL chromatographic columns of Superose, passed throughFPLC systems (GE Healthcare) carry out separating-purifying product (using the sodium acetate water containing 30% acetonitrile that pH is 6.5 Mobile phase of the solution as pillar).The product section collected is obtained into faint yellow product 846mg using lyophilized after pure water dialysis, Yield 91.2%.

Under the protection of Ar atmosphere, will faint yellow product (500mg, 28 μm of ol) obtained above and Azide Cy5.5 (5mg, 5 μm of ol) 160mL water is dissolved in (containing 5mol%CuSO₄·5H₂O and 10mol% sodium ascorbates) in.The stirring reaction at 50 DEG C 24 hours.After reaction terminates, using bag filter, (molecular weight cute off (MwCO=3500) dialysis removes molten Agent and impurity, period add substantial amounts of disodium EDTA (EDTA-2Na) to help dialyse in the deionized water of dialysis Except the copper ion in dereaction.The solution stayed in after dialysis in bag filter is collected and freezes to obtain green solid.By the solid dissolving It is (water-soluble using the sodium acetate containing 30% acetonitrile that pH is 6.5 by exclusion chromatography purified product using such as previous product afterwards Mobile phase of the liquid as pillar).The product section collected is obtained into green product using lyophilized after pure water dialysis under the conditions of lucifuge (PEGylated dendrimer-GFLG-PTX) 420mg, yield 83.3%.MALDI-TOF MS:m/z 18472,[M+H]⁺。 Detected by fluorescence spectrum, the content of Cy5.5 is close to 0.5%, is detected in the conjugate containing about 20.9% by HPLC Medicine (taxol).

As the graft numbers of GFLG-PTX in above-mentioned synthesis are uncontrollable processes, the terminal amino group of macromolecular has been grafted It is optimal state entirely, but actually due to steric effect and yield issues, it may appear that partially grafted situation, experimental result It is 5 to obtain average number of grafts.

In order to these necessary functions of SAIR are integrated in a single Arboraceous support, present invention design has synthesized two Dendrimer-the GFLG-PTX of parent's property.In order to be efficiently synthesized the tree-shaped conjugate of this functional form, using click-reaction come Synthesis PEGylated dendrimer-GFLF-PTX.Synthesize dendrimer (the dendrimer PEG- of PEG modifications first G2L-G3L-alkyne).Which passes through MALDI-TOF mass spectrums and draws maximum abundance peak for m/z=10, and 673, it is labeled as [M+H]⁺ (as shown in Figure 1), this shows have 4 mPEG segments to be covalently attached on Arboraceous support.Secondly, there is tissue in cancer cell in a large number Cathepsin B, by the N sensitive to the enzyme₃Dendrimer (the PEG-G2L- of-GFLG-PTX molecules and pegylation G3L-alkyne) it is coupled.In addition, Cy5.5 has also been covalently attached on dendrimer by click-reaction.Using dilute The micro copper ion that toxicity may be caused in reaction is removed clean by EDTA solution by the method for dialysis.Finally, what is obtained is first Product passes throughFPLC systems are further purified using exclusion chromatography post (12 HR/16/30 of Superose).To divide Carry out dialysing, freeze from the liquid for getting off, obtain amphipathic PEGylated dendrimer-GFLF-PTX conjugates.Pass through MALDI-TOF flight mass spectrums detect its molecular weight, obtain maximum abundance peak for m/z=18,472, which is product PEGylated Dendrimer-GFLF-PTX, and it is labeled as [M+H]⁺(as shown in Figure 2).This shows about PEGylated dendron Cell-average is connected with 5 GFLG-PTX.This is more less slightly than theoretical 8, the result may the reason for be chemical reaction process Exist larger sterically hindered.Drugloading rate is drawn for 20.9wt%, the knot obtained with MALDI-TOF mass spectrums by HPLC detections Fruit matches.As medicine is covalently attached on dendrimer, the tree-shaped conjugate of the pegylation in PBS or It is stable in person's blood circulation.

Embodiment 10

With reference to embodiment 9, macromolecular raw material is G1L-G2L for tree-shaped big point to the synthetic method of couplet I and couplet II Son.

With reference to embodiment 10, macromolecular raw material is G3L-G4L for dendrimer to the synthetic method of couplet V.

Embodiment 11

The synthetic route of asymmetric dendrimer is as follows：

The synthesis of compound G1L

In N₂Under atmosphere protection, Z-Lys (Z)-OH (14.5g, 35mmol) and N-Boc-ethylenediamine (7.9g, 42mmol) it is dissolved in 100mL dry DMFs.The solution is stirred 5 minutes under ice bath, be subsequently adding DIPEA (29mL, 175mmol).Again HBTU (16.1g, 47mmol) and HOBT (6.7g, 47mmol) is added in solution system.By which under ice bath Stirring half an hour, then react 24 hours at room temperature.After reaction terminates, vacuum distillation removes solvent, and adds 300mL EtOAc, organic phase is used saturation NaHCO respectively₃Solution, watery hydrochloric acid (1M) and respectively washing three times of saturation NaCl solution.With anhydrous Magnesium sulfate is dried, and is filtered to remove solution decompression concentrated by rotary evaporation to 15mL after magnesium sulfate.Recrystallized using ethylacetate/ether, Obtain white solid 17.8g, yield 91.4%.1H NMR(400MHz,CDCl₃), δ=7.34 (s, 10H, NHCOOCH₂Ph-H), 5.09(s,4H,OCH₂-Ph),4.08(m,1H,CH(NHCbz)-CH₂),3.33-3.19(t,6H,CH₂CH₂-NH),1.25- 1.83(m,6H,CH₂-Lys and s,9H,CH₃-Boc).MALDI-TOF MS:M/z=559 is [(M+H)⁺,C₂₉H₄₁N₄O₇ ⁺]。

The synthesis of compound G1L-G1L

In N₂Under atmosphere protection, G1L (14.4g, 26mmol) is dissolved in the anhydrous DCM of 20mL.The solution is stirred into 5 under ice bath Minute, then it is added dropwise to 20mL TFA (260mmol).After stirring 4 hours, solvent is removed.Absolute ether is added, white is produced solid Body powder.Separate and collect white solid powder as G1L and slough product after Boc groups.In N₂Under atmosphere protection, by what is collected before White solid, Boc-Lys (Boc)-OH (10.9g, 31mmol), HBTU (12.1g, 36mmol) and HOBT (5.0g, 36mmol) it is dissolved in 100mL DMF.Under ice bath, it is slowly added dropwise into 22mL DIPEA (130mmol).By its under ice bath after Continuous stirring half an hour, then react 24 hours at room temperature.After reaction terminates, vacuum distillation removes solvent, and adds 300mL EtOAc, organic phase is used saturation NaHCO respectively₃Solution, watery hydrochloric acid (1M) and respectively washing three times of saturation NaCl solution.With anhydrous Magnesium sulfate is dried, and is filtered to remove solution decompression concentrated by rotary evaporation to 15mL after magnesium sulfate.Recrystallized using ethylacetate/ether, Obtain white solid 17.7g, yield 86.8%.1H NMR(400MHz,CDCl₃), δ=7.34 (s, 10H, NHCOOCH₂Ph-H), 5.09(s,4H,OCH₂-Ph),4.08(m,2H,CH(NHCbz)-CH₂),3.33-3.19(t,8H,CH₂CH₂-NH),1.25- 1.83(m,12H,CH₂-Lys and s,18H,CH₃-Boc)。MALDI-TOF MS:m/z 785[(M+H)⁺,C₄₀H₆₁N_6O10⁺], 807[(M+Na⁾⁺,C₄₀H₆₀N_6O1ONa⁺]。

The synthesis of compound G1L-G2L

In N₂Under atmosphere protection, G1L-G1L is sloughed with reference to the method that Boc protection groups are taken off in " compound G1L-G1L " synthesis The Boc protection groups of (10.6g, 14mmol).In N₂Under atmosphere protection, white solid, Boc-Lys that protection group is obtained will be sloughed (Boc)-OH (11.3g, 32mmol), HBTU (14.4g, 38mmol) are dissolved in 100mL dry DMFs with HOBT (5.2g, 38mmol) In.Under ice bath, it is slowly added dropwise into 23mL DIPEA (135mmol).Which is continued to stir half an hour, then in room under ice bath The lower reaction of temperature 24 hours.After reaction terminates, vacuum distillation removes solvent, and adds 300mL EtOAc, and organic phase is used full respectively And NaHCO₃Solution, watery hydrochloric acid (1M) and respectively washing three times of saturation NaCl solution.It is dried with anhydrous magnesium sulfate, is filtered to remove sulfuric acid By solution decompression concentrated by rotary evaporation to 15mL after magnesium.Recrystallized using ethylacetate/ether, obtain white solid 16.1g, yield 92.4%.1H NMR (400MHz, CDCl3), δ=7.33 (s, 10H, NHCOOCH₂Ph-H),5.08(s,4H,OCH₂-Ph), 4.08(m,4H,COCH(NH)-CH₂),3.00-3.20(t,12H,CH₂CH₂-NH),1.25-1.80(m,24H,CH₂-Lys and s,36H,CH₃-Boc)。MALDI-TOF MS:m/z 1264[(M+Na)⁺,C₆₂H₁₀₀N₁₀O₁₆Na⁺]。

The synthesis of compound G2L-G2L

G1L-G2L (14.5g, 12mmol) is dissolved in 60mL methyl alcohol (HPLC levels)；Then Pd/C (6g) is added into solution In.In H₂Under atmosphere, keep autoclave in pressure be 0.8MPa, stirring reaction 48 hours.After reaction terminates, it is filtered to remove Pd/C, revolving remove the product that solvent is sloughed " Cbz protection groups ".In N₂Under atmosphere protection, add what is sloughed in side tube flask The product of " Cbz protection groups ", Z-Lys (Z)-OH (11.5g, 28mmol), HBTU (12.1g, 32mmol) and HOBT (4.4g, 32mmol), and 100mL dry DMFs are added to dissolve which.Under ice bath, it is slowly added dropwise into 19mL DIPEA (115mmol).Will Which continues to stir half an hour under ice bath, then reacts 24 hours at room temperature.After reaction terminates, vacuum distillation removes solvent, And 300mL EtOAc are added, organic phase is used saturation NaHCO respectively₃Solution, watery hydrochloric acid (1M) are respectively washed with saturation NaCl solution Three times.It is dried with anhydrous magnesium sulfate, is filtered to remove solution decompression concentrated by rotary evaporation to 15mL after magnesium sulfate.Using ethyl acetate/ Diethyl ether recrystallization, obtains white solid 16.6g, yield 78.2%.1H NMR(400MHz,CDCl₃), δ=7.33 (s, 20H, NHCOOCH₂Ph-H),5.06(s,8H,OCH₂-Ph),4.08(m,6H,COCH(NH)-CH₂),2.95-3.25(t,16H, CH₂CH₂-NH),1.25-1.73(m,36H,CH₂-Lys and s,36H,CH₃-Boc)。MALDI-TOF MS:m/z 1789[(M +Na)⁺,C₉₀H₁₃₆N₁₄O₂₂Na⁺]。

The synthesis of compound G2L-G3L

In N₂Under atmosphere protection, G2L-G2L is sloughed with reference to the method that Boc protection groups are taken off in " compound G1L-G1L " synthesis The Boc protection groups of (13.2g, 7.5mmol).In N₂Under atmosphere protection, white solid, Boc-Lys that protection group is obtained will be sloughed (Boc)-OH (13.0g, 38mmol), HBTU (16.0g, 42mmol) are dissolved in 100mL dry DMFs with HOBT (5.7g, 42mmol) In.Under ice bath, it is slowly added dropwise into 25mL DIPEA (150mmol).Which is continued to stir half an hour, then in room under ice bath The lower reaction of temperature 24 hours.After reaction terminates, vacuum distillation removes solvent, and adds 300mL EtOAc, and organic phase is used full respectively And NaHCO₃Solution, watery hydrochloric acid (1M) and respectively washing three times of saturation NaCl solution.It is dried with anhydrous magnesium sulfate, is filtered to remove sulfuric acid By solution decompression concentrated by rotary evaporation to 15mL after magnesium.Recrystallized using ethylacetate/ether, obtain white solid 17.6g, yield 87.7%.1H NMR (400MHz, DMSO), δ=7.33 (s, 20H, NHCOOCH₂Ph-H),5.07(s,8H,OCH₂-Ph),4.08 (m,10H,COCH(NH)-CH₂),3.07-3.73(t,24H,CH₂CH₂-NH),1.26-1.73(m,60H,CH₂-Lys and s, 72H,CH₃-Boc)。MALDI-TOF MS:m/z 2701[(M+Na)⁺,C₁₃₄H₂₁₆N₂₂O₃₄Na⁺]。

The synthesis of compound G3L-G4L

Synthesis with initial reference to compound G2L-G2L obtains compound G3L-G3L, refers again to the synthesis of compound G2L-G3L Obtain compound G3L-G4L.

The present invention carries out experiment detection to G2L-G3L dendrimers-taxol conjugate：

First, the sign of the particle diameter of nano-particle, pattern, zeta current potentials and stability

Using DLS and SEM (SEM) come real-time monitored conjugate particle diameter under different conditions with Pattern.First, deionized water prepares PBS (pH 7.4) and McIlvaine ' s buffer solutions (pH 5.4).By the idol Connection thing is dissolved in both cushioning liquid respectively, and forms nano-particle.Take portion of material (1mg/mL) again to be dissolved in In McIlvaine ' s buffer solutions, it is incubated 6 hours with papain (2.0 μM, pH=5.4) altogether at 37 DEG C.At the same time, exist Under the conditions of 37 DEG C without papain, nano-particle is incubated in (1mg/mL respectively in two kinds of different cushioning liquid；PH= 7.4 and pH=5.4) 24 hours.In different time points, its particle diameter and zeta current potentials are detected using DLS.Specific Time point such as 0h, 1h, 4h and 6h, take out part solution and are diluted preparation SEM samples.

Fig. 3-A show that sample is gathered into nano-particle in water, and average water phase particle diameter is about 69nm, and particle diameter distribution It is narrow, PDI=0.230.Meanwhile, morphology analysis are carried out to sample using AFM, it was observed that the uniform-spherical knot of particle diameter about 74nm Structure (Fig. 3-B), the result is with DLS testing results closely.Result that grain diameter measurement draws is carried out also with DLS's by AFM As a result it is consistent.The nano-grade size and narrow particle diameter distribution that are drawn by DLS and AFM show the amphipathic peptides that we prepare Dendrimer-PTX conjugates can be self-assembled into the nano-particle of size uniformity.Various strategies such as self assembly are designed to structure Build nano carrier.However, the hydrophilic material such as dendrimer individually chemically or physically cannot be interacted.Originally grinding In studying carefully, self assembly is mediated by PEGylated dendrimer-GFLF-PTX itself.The driving force of self assembly may attribution In the reduction of interface energy, which is subject to flat between flexible PEG hydrophilic interactions and dendrimer-GFLF-PTX fragment hydrophobic effects Weighing apparatus affects.Additionally, the factor such as other such as hydrogen bonds, π-π stackings and dipole effect is likely to have influence on the self assembly row of conjugate For.Additionally, little particle can penetrate into tumor tissues faster than big particle, but minimum particle can be clear by liver kidney easily Remove.Strengthening to the penetrating power of tumor tissues (by reducing Nano medication size) and the toxicity for reducing normal tissue and It is a huge challenge that balance is found between MPS scavenging actions (by increasing Nano medication size).Therefore, it is of the invention to receive Rice corpuscles size is about 60-80nm, is with long circulating and higher tumor infiltrating carrier.

Preferably drug delivery system requires tumor tissues are being enriched to after blood long circulating.The table of nano-particle Surface charge plays pivotal player during which experiences blood circulation.PEGylated dendrimer- be have detected using DLS The zeta current potentials of GFLF-PTX nano-particles, display surface electric charge are electronegativity.Intravenous injection positive charge nano-particle can be huge Phagocyte or reticuloendothelial system (RES) are quickly removed from the circulatory system.Conversely, electronegativity or electroneutral nanoparticle Son will reduce abnormal protein combination in theory, and therefore low probability stimulate to immune system, ultimately result in longer following The ring half-life.So, the PEGylated dendrimer-GFLF-PTX conjugates have suitable nano-grade size with weak table Face negative electrical charge, which can correspondingly have chance to be enriched to tumour by EPR passive target effects as a drug delivery system Tissue, and avoid quickly being removed, ultimately result in anticancer therapeutic enhancing.

2nd, drug release test

Insoluble drug release slowly limits the anti-cancer effectiveness of drug delivery system.Once nano-particle drug delivery system internalization Into after cancer cell, the present invention devises the nano-particle based on conjugate of an intelligence, and which can be by cancer cell in intracellular Cathepsin B's triggering of overexpression.Therefore, these carriers are evaluated most important is just seemed to the ability of stimuli responsive.Pawpaw With the activity similar to lyase in-vivo tissue Cathepsin B, which is selected for evaluating enzyme sensitive medicaments release conditions protease.It is logical Cross enzyme/experiment without enzyme (papain) different condition to detect its vitro drug release, and using RP-HPLC testing As a result.

By mPEGylated dendrimer-GFLG-PTX are respectively placed in containing papain and without pawpaw egg Drug release studies are carried out in the solution of white enzyme.Final cushioning liquid is configured with reference to chapter 3 3.2.4.Nano-particle is added 48 hours are incubated in the cushioning liquid (enzyme concentration is 2.0 μM, pH=5.4) and at 37 DEG C, wherein nanoparticle concentration is 3mg/mL.At the same time, the nano-particle (3mg/mL) is added into (pH=7.4 in the cushioning liquid without enzyme under the same terms PBS cushioning liquid and pH=5.4 McIlvaine cushioning liquid), 48 hours are incubated equally at 37 DEG C.Specific Sample is taken out and is utilized high performance liquid chromatograph analysis (liquid phase analysis post 4.6 × 150mm of C8column) by time point, wherein Mobile phase is selected as (mobile phase A:Deionized water, Mobile phase B:Acetonitrile), the wash-out of 20 minutes is carried out with the flow velocity of 1.5mL/min (Mobile phase B content is 50%), Detection wavelength is 227nm.The sample that release is ruptured is separated and collected and profit through HPLC Use ESI-MS phenetic analysis.

Fig. 4 shows PTX standard samples (such as Fig. 4-A, peak:5.6min) rupture with release sample (such as Fig. 4-B, peak:5.6min) with same elution time, it means that the product released from nano-particle fracture is active compound Japanese yew Alcohol.Additionally, rupture, peak value is characterized for the collection of products of 5.6min and with ESI-MS.These discoveries show in pawpaw Ammonolysis reaction in the presence of protease has not significant impact the structure of PTX.

The concentration of the PTX that different time points are discharged is detected using RP-HPLC, as a result shows PEGylated There is the characteristic of enzyme response in the insoluble drug release of dendrimer-GFLF-PTX nano-particles.In the experiment that papain is present In, quick insoluble drug release is observed, after being incubated 8 hours with papain, about 50% PTX is released, and About 90% PTX has been discharged after 24 hours.Conversely, in the experiment without enzyme incubation, in pH=7.4 or 5.4 after 48 hours Buffer solution in detect only a small amount of PTX be broken off release (such as Fig. 4-C).Therebetween, the PBS (pH=7.4) of hyclone Buffer solution is chosen as a template to evaluate nano-particle insoluble drug release vigor in cyclic process in blood.These results show There is high Papain enzyme selectivity and high stability in PEGylated dendrimer-GFLF-PTX.Although additionally, PTX The hydrolysis of the ester bond of molecule is inevitable, the nano-particle based on conjugate show high stability and PTX has obtained the effective protection of carrier in the circulatory system.These lower opsonification rate, high stability and circulations During minimal amount of medicine reveal minimumization of the reduction and anti-cancer effectiveness loss for contributing to toxic and side effect.

Test result indicate that the nano-particle of PEG protections can keep stable in the circulatory system, and the sensitive nanometer of enzyme Particle can discharge dewatering medicament PTX in the presence of cathepsin B.DLS and SEM are used to real-time monitored nano-particle Under condition of different pH and whether there is enzyme be incubated in the case of particle size and pattern situation of change.The fracture of hydrophobic units is released The decrease that result in self assembly effect is put, this causes nano particle structure no longer closely to cave in accumulation process in turn. Under without enzyme incubation, particle keeps about 60 140nm (as shown in Figure 5) under buffer solution in 24 hours, shows significantly not tie Structure caves in.Conversely, the particle through the incubation of papain enzyme is found loosely organized, such as DLS detections are found with big And wide PDIs (as shown in Figure 6).And by SEM carry out topography analysis show similar result.After being incubated 4 hours i.e. It was observed that the particle diameter of particle increases and caves in.These results show that these particles can be stable in cyclic process.By group Knit the change on the particle diameter and pattern that Cathepsin B causes further demonstrate enzyme sensitive property and enzyme induction particle cave in Be conducive to rapidly insoluble drug release.

3rd, internal antitumous effect evaluation

The sensitive PEGylated of enzyme is carried out on the BALB/c mouse of inoculated 4T1 mouse source breast cancer cell The antitumous effect and toxicity research of dendrimer-GFLF-PTX nano-particles and free PTX.Tumor-bearing mice is randomly assigned Into three groups (7 per group) and it is marked, then by tail vein injection to injected in mice medicament (200 μ L), is Japanese yew respectively Nano-particle and physiological saline as a control group that alcohol injection, PEGylated dendrimer-GLFG-PTX are formed, It is 5mg/kg PTX that each of which time is administered to dose unification, daily the co-injection 10 days of injection one time.Detected per two days once little The gross tumor volume and body weight of mouse, and the gross tumor volume that first day is determined is respectively set as 100% with Mouse Weight.At the 19th day When, euthanasia is implemented to all mouse, tumour is peeled off and weighed, then according to tumour inhibiting rate formula calculates tumour inhibiting rate (TGI).New vessels, propagation and the Apoptosis feelings of tumour are studied further by the immunohistochemical analysis of tumor tissues Condition.

Daily clinical symptoms of detection mouse, the gross tumor volume and body weight that first day is determined are set as 100% and every Measure every two days once.Fig. 7-A show that gross tumor volume is changed over and increased, and start all medicines from third time treatment and control The tumour for the treatment of group mouse presents significant growth compared to control group and blocks.Taxol and saline therapy group are compared, Nano-particle treatment group restrained effectively the growth of tumour.When Fig. 7-A are displayed in the 19th day, each group mouse Relative tumor Volume size is respectively 972 ± 134% (control groups), 902 ± 152% (PTX treatment groups) and 383 ± 71% (NPs treatment groups). Particularly, after the treatment of 19 days, such as tumor growth curve figure (Fig. 7-A, p<0.001 relative to physiological saline and p<0.002 Relative to clinical injection medicine PTX) and macroscopic solid tumor size, nano-particle treatment group ratio uses identical administration The paclitaxel treatment group of scheme shows the antitumor efficacy for significantly increasing.The tumour conspicuousness ground ratio of nano-particle treatment group (control group and PTX treatment groups) tumour of other treatment group is less.After treatment end, tumour inhibiting rate (TGI) and knurl weight are measured simultaneously Calculate.Fig. 7-C and Fig. 7-D show the knurl weight of PTX injection for treating group and nano-particle treatment group be respectively 994.2 ± 158.1mg and 465.3 ± 84.8mg (p<0.0001 relative to physiological saline group, p<0.001 notes relative to free drug taxol Penetrate liquid group).Additionally, respective TGIs is respectively 16.6% (PTX) and 61.0% (NPs), it is higher that this shows that nano-particle has Antitumor curative effect.These results have benefited from the design for being intended to improve antitumous effect (SAIR) scheme comprehensively.Can specifically return Because improving in characteristics such as the electronegative surface of material, suitable nano-scale dimension (Fig. 7-A) and lower opsonification rates Stability of the medicine in cyclic process.Simultaneously these features result in its pass through EPR effects rapidly Enriching accumulation to tumour Tissue, realizes the efficient quick release of taxol in intracellular lysosome Jing after cancer cell endocytosis and intake.It is antitumor in vivo Research shows that the dendrimer nano-particle of the PEG modifications meets the standard of SAIR, therefore it is anti-swollen to improve which in all directions Knurl curative effect.

4th, toxicity in vivo evaluation

By 6 to 8 week big Healthy female BALB/c mouses (body weight about 20 ± 2g) be randomly divided into three groups, 7 per group and right Every mouse is marked.Then by tail vein injection to injected in mice medicament (200 μ L), be respectively paclitaxel injection, Nano-particle and physiological saline as a control group that PEGylated dendrimer-GFLF-PTX are formed, wherein being administered to Dose unification is 5mg/kg PTX, daily the co-injection 10 times of injection one time.Body weight and the observation of a mouse were recorded per two days The behavior of mouse and the body weight of first day is set as into 100%.After 19 days, mouse is carried out euthanasia, collects its blood and enters Row analysis.

In the research process of whole treatment, the universal well-tolerated of duplicate injection of nano-particle, and mouse does not have table Revealing any significant body weight reduces (Fig. 8).After treatment cycle terminates, euthanasia is implemented to mouse, its blood collection is entered Row conventional analysis.

In order to further evaluate PTX parenteral solutions and PEGylated dendrimer-GFLF-PTX nano-particles to blood Biocompatibility, the present invention have carried out analysis of Hematology Changes and routine blood test detection.The level major part of these hematology labels exists In normal range (NR) (Fig. 9).Therebetween, the neutral grain of PEGylated dendrimer-GFLF-PTX nano-particles treatment group mouse is thin Born of the same parents (GR#) and platelet count (PLT) are similar to the mouse of physiological saline group.Conversely, clinical medicine PTX injection for treating group mouse The level of these labels there is significant reduction.Observe the haematics toxicity (PTX dose limiting toxicities) of nano-particle It is extremely low even without toxicity, and this contributes to the lifting of dosage in future.These results show by being injected intravenously administration PEGylated dendrimer-GFLF-PTX are a kind of potential with high biocompatibility and low hematotoxicity Drug delivery system.

Following table is present invention experiment primary drug used.

Reagent

N, N-dimethylformamide add anhydrous CaH₂After being dried 24h, steam again, midbarrel is collected in decompression.

Dichloromethane adds calcium hydride overnight, air-distillation, discards front-end volatiles, collects midbarrel, and drier is put in sealing Temporarily preserve.Existing use is steamed now, storage time is less than 2 weeks.

It is pure for analysis when other reagents are not specified, process is not further purified, is directly used.

Instrument

Magnetic force heating stirrer (German IKA companies).

BP211D type electronic balances (German Sartorius companies).

Vacuum drying chamber (one permanent Science and Technology Ltd. of Shanghai, DZF-6050 types).

Electric drying oven with forced convection (one permanent Science and Technology Ltd. of Shanghai, DHG-9140 types).

Supercentrifuge (Germany, eppendorf)

HIGH RESOLUTION SUPERCONDUCTING nuclear magnetic resonance chemical analyser (Bruker companies of Switzerland, Bruker AV II-400MHz).

Flight mass spectrometer (Brooker dalton company of the U.S., Autoflex MALDI-TOF/TOF).

Liquid chromatography mass combined instrument (American AB I company, API3000LC-MS/MS).

Mass spectrograph (Waters, US, Q-TOF Premier).

(Thermo companies of the U.S., FT-IR, Nicolet is 6700) for Fourier infrared spectrograph.

Fast protein liquid chromatography (Sweden, GE Healthcare,FPLC system)。

Ultraviolet spectrometer (U.S. Perkin-Elmer).

Sepectrophotofluorometer (Japanese HITACHI companies, F700).

(Malvern companies of Britain, DTS software version are 3.32) for nano particle size and potentiometric analyzer.

AFM (Digital Instruments companies of the U.S., Asylum Research MFP-3D-Bio).

High performance liquid chromatograph (U.S. Agilent, 1260LC, Zorbax 4.6 × 150mm of C8column).

Laser confocal microscope (German Leica, TCS SP5).

Flow cytometer (U.S., BD companies).

PH meter (Switzerland, plum Teller-Tuo Duoli).

Living imaging instrument (U.S., Cri, Maestro In-Vivo Imaging System).

Claims

1. paclitaxel loaded asymmetric dendrimer nano medicament carrying system, it is characterised in that the drug-loading system is antitumor The conjugate of drug taxol, tumour endocellular enzyme sensitivity small peptide GFLG and the asymmetric peptides dendrimer of PEGization；Concrete structure It is as follows：

A_n-B

Wherein, A is：

B is the asymmetric lysine peptides dendrimer of PEGization with ethylenediamine as core, and the macromolecular is G1L-G2L, G2L- The asymmetric dendrimer of G3L or G3L-G4L, PEG modifications occur in the asymmetric dendrimer algebraically less one End, A instead of the terminal amino group of the other end, n=1～16.

2. paclitaxel loaded asymmetric dendrimer nano medicament carrying system according to claim 1, it is characterised in that Described asymmetric dendrimer is G2L-G3L dendrimers.

3. the preparation method of paclitaxel loaded asymmetric dendrimer nano medicament carrying system according to claim 1, Characterized in that, the PEGization dendrimer of the GFLG-PTX and alkynyl of Azide is first respectively synthesized, then by copper catalysis Click-reaction synthesizes end-product dendrimer-taxol conjugate.

4. the preparation method of paclitaxel loaded asymmetric dendrimer nano medicament carrying system according to claim 3, It is characterized in that, the synthesis of Azide GFLG-PTX is as follows：

Using CH₃ONa is by N₃- GFLG-OMe sloughs methyl esters protection, by product N₃- GFLG-OH is placed under blanket of nitrogen, reaction DCM is used as solvent for system, adds DMAP and dicyclohexylcarbodiimide, after reaction half an hour, adds PTX, reacts 48 hours； Insoluble matter is filtered to remove, is spin-dried for removing solvent；Ethyl acetate lysate is used again, and organic phase is used saturation NaHCO respectively₃It is molten After the washing of liquid, watery hydrochloric acid and saturation NaCl solution, then drying, filtration, vacuum rotary steam concentration, recrystallization are obtained final product.

5. the preparation method of paclitaxel loaded asymmetric dendrimer nano medicament carrying system according to claim 3, It is characterized in that, comprise the following steps：

The synthesis of the asymmetric dendrimer of A, alkynyl

With one end as Cbz radical protections, the other end is raw material for the asymmetric dendrimer of Boc radical protections, in nitrogen atmosphere Enclose lower and Pb/C reactions and slough Cbz protection groups, after reaction terminates, in N₂Under atmosphere protection, product, HBTU and HOBT are dissolved in In dry DMF, with DIPEA and 5- hexin acid reactions under ice bath；

The synthesis of B, PEGization dendrimer

PEG of the product of step A with Azide is dissolved in containing CuSO₄·5H₂In the water of O and sodium ascorbate, after reaction terminates, Solvent and impurity are removed using bag filter dialysis, the solution stayed in after dialysis in bag filter is collected into the tree of lyophilized mPEG modifications Shape macromolecular；

The alkynyl of C, PEGization dendrimer

In N₂Under atmosphere protection, PEGization dendrimer is dissolved in anhydrous methylene chloride, is then added dropwise to trifluoroacetic acid, stirring 4 After hour, solvent is removed, sample after vacuum drying, is collected；In N₂Atmosphere protection under, by the sample for obtaining, HATU, HOAt and 5- oneself Acetylenic acid is dissolved in dry DMF, under ice bath, adds DIPEA reactions；

The synthesis of D, dendrimer-taxol conjugate

Under the protection of Ar atmosphere, the product that step C is obtained is dissolved in containing CuSO with the GFLG-PTX of Azide₄·5H₂O and Vitamin C In the water of sour sodium, after reaction terminates, solvent and impurity, the solution that will be stayed in after dialysis in bag filter are removed using bag filter dialysis Collect to freeze and obtain final product dendrimer-taxol conjugate.

6. the preparation method of paclitaxel loaded asymmetric dendrimer nano medicament carrying system according to claim 5, Characterized in that, in described step B and D steps, the molecular cut off of bag filter is 3500.

7. the preparation method of paclitaxel loaded asymmetric dendrimer nano medicament carrying system according to claim 5, Characterized in that, in described step B and D steps, adding disodium EDTA in the deionized water of dialysis.

8. the preparation method of paclitaxel loaded asymmetric dendrimer nano medicament carrying system according to claim 5, Characterized in that, the solution stayed in after dialysis in bag filter is collected the lyophilized solid for obtaining molten by described step B and D steps Solution, using exclusion chromatography separating-purifying product, lucifuge collects product, then freezes Jing after pure water dialysis and obtain final product product.

9. the preparation method of paclitaxel loaded asymmetric dendrimer nano medicament carrying system according to claim 5, Characterized in that, described step A and step C, after the completion of reaction, vacuum distillation removes solvent, and adds EtOAc, will have Machine mutually uses saturation NaHCO respectively₃Respectively washing three times of solution, watery hydrochloric acid and saturation NaCl solution；It is dried with anhydrous magnesium sulfate, is filtered By solution decompression concentrated by rotary evaporation after removing magnesium sulfate, recrystallized using ethylacetate/ether, obtained final product.